identifying new therapeutics in experimental subarachnoid ... · serendipitously identified the...

Identifying New Therapeutics in Experimental

Subarachnoid Hemorrhage

By

Michael K. Tso

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy (PhD)

Institute of Medical Science

University of Toronto

© Copyright by Michael K. Tso, 2018

ii

Identifying New Therapeutics in Experimental Subarachnoid

Hemorrhage

Michael K. Tso

Doctor of Philosophy

Institute of Medical Science

University of Toronto

2018

Abstract

Aneurysmal subarachnoid hemorrhage (SAH) can result in substantial neurologic injury

and mortality. Drug clinical trials in SAH over the last several decades have failed to

improve clinical outcomes. New therapeutic targets need to be identified and new drug

therapies are needed in this patient population.

In this thesis, we identified therapeutic agents in SAH by two methods. First, we

serendipitously identified the anticonvulsant valproic acid (VPA) as a potential drug

treatment in SAH after learning about its beneficial effects in various animal models of

neurologic disease. We used VPA treatment in a mouse model of SAH. VPA treatment

resulted in improved neurological outcome, decreased brain injury, decreased

microthrombi burden, and decreased blood-brain barrier (BBB) disruption. However,

retrospective analysis of clinical trial data of SAH patients demonstrated no significant

effect of VPA on clinical outcomes. Randomized clinical trials would be needed to

determine safety and efficacy of VPA in patients with SAH.

iii

Secondly, we focused on one aspect of experimental SAH pathophysiology, BBB

disruption, and identified that the time-point with the greatest increase in permeability

was at 24 hours after experimental SAH. We subsequently developed and optimized

two distinct methods of isolating brain endothelial cells from mice and performed whole-

genome expression profiling of these cells. In doing so, we identified potential

therapeutic targets and pathways relevant to SAH. Because ‘prostaglandin synthesis

and regulation’ was one of the top pathways upregulated and cyclooxygenase-2 (COX-

2) was one of the top upregulated genes, we used the selective COX-2 inhibitor,

celecoxib, as drug treatment in our SAH model. Celecoxib administration resulted in

improved neurological outcome and decreased BBB disruption. Randomized clinical

trials with celecoxib are needed.

In summary, we have identified two potential therapeutic agents, VPA and celecoxib,

that could be investigated further with clinical trials in patients with SAH. Repurposing

drugs could provide an important opportunity to improve the outcomes from this

devastating disease.

iv

Acknowledgments

Work produced in this thesis was made possible by salary support from the Vanier

Canada Graduate Scholarship (Canadian Institutes of Health Research, CIHR) and

grant support from the Neurosurgery Research and Education Foundation (NREF)

AANS/CNS Cerebrovascular Section Research Fellowship.

Dr. R. Loch Macdonald has been a wonderful mentor and supervisor. He has allowed

me the opportunity to think critically and independently. His academic career

achievements are a constant reminder to me of what is possible with dedicated,

sustained effort.

Dr. Phil Marsden, my program advisory committee member and co-supervisor has

taught me to be a skeptical scientist and has been very supportive during this PhD. I am

thankful to him for allowing me to participate in the always-enlightening weekly Marsden

lab journal clubs.

Dr. Andrew Baker, my program advisory committee member, has also been very

supportive during this PhD. He has allowed me to see the bigger picture when it comes

to the scientific process.

I would like to acknowledge the chairs and examiners at my PhD qualifying exam (Dr.

Darren Yuen, Dr. Sunit Das, Dr. Andrew Advani) and at my PhD defense (Dr. Sunit Das,

Dr. Issam Awad, Dr. Giles Santyr, Dr. Gregory Hare, Dr. Richard Gilbert).

Dr. Jinglu Ai, as the Macdonald lab manager and senior research associate, has been

so helpful and patient. Brent Steer, as the Marsden lab manager and senior research

v

associate, has also been looking out for me. Angie Donato, Umberta Bottoni, and

Victoria Paz have all been essential in scheduling the numerous meetings throughout

the PhD.

I would like to thank my fellow lab members in both the Macdonald Lab (Josephine

D’Abbondanza, Bert Bosche, Manabu Sumiyoshi, Hoyee Wan, Shakira Brathwaite,

Elliot Lass, Charles Lee, Tian Nie, Mo Sabri) and Marsden Lab (Paul Turgeon, Jeff

Man, Matthew Yan, Aravin Sukumar, Kay Ku, Sarah Park, Lucy Chen, Michelle

Dubinsky, Tiana Downs, John Lee, Lawrence Park, Olga Vexelshtein, David JJ Ho,

Apurva Shirodkar, Maria Chalsev) for their ongoing support.

I would like to acknowledge the Princess Margaret Genomics Centre, for their

assistance with the microarray hybridization and statistical analysis. The St. Michael’s

Hospital core facility staff have also been super helpful all these years (Xiaofeng Lu, Cat

Di Ciano, Pam Plante, and Chris Spring). Youdong Wang, research technician in Dr.

Xiao-Yan Wen’s lab, has also kindly provided technical assistance for fluorescent

microscopy.

Pursuing this PhD degree at the University of Toronto was also made possible by the

ongoing support of the academic leadership of the Division of Neurosurgery at the

University of Calgary: Dr. John Wong (Head, Division of Neurosurgery), Dr. Rajiv Midha

(Head, Department of Clinical Neurosciences), and Dr. R. John Hurlbert (former

Program Director, Division of Neurosurgery). Upon my return to clinical neurosurgery

service in Calgary in the summer of 2016, current Program Director Dr. Jay Riva-

Cambrin has provided support for me to defend my PhD.

vi

Most importantly, I would like to thank my wife Karina, my parents Ann and Lawrence,

my brother David and his wife Pam, my sister Grace and her husband Matt, and

Karina’s parents Trudy and Ray and the rest of her family for being so supportive and

patient throughout this long academic journey. It is truly a team effort! Although maybe

too young to understand why Dad has been so busy, my two young children, Landon

and Hayley, have inspired me to continue chasing my dreams and be present in the

moment.

vii

Contributions

Michael Tso – Conceptualization, planning, and execution of experiments, analysis,

writing thesis

R. Loch Macdonald – Supervision and mentorship for entire PhD thesis

Phil Marsden – Supervision and mentorship for entire PhD thesis

Jinglu Ai – Trouble-shooting and general assistance for experiments in Chapter 5/6

Paul Turgeon – Assistance with endothelial cell isolations and RT-PCR for Chapter 6

Josephine D’Abbondanza – Assistance with SAH model and histology in Chapter 5

Hoyee Wan – Assistance with histology in Chapter 5

Bert Bosche – Assistance with BBB permeability experiments in Chapter 6

Charles Lee – Assistance with neurobehavioural assessments and histology in Chapter 5/6

Tian Nie – Assistance with neurobehavioural assessments and TEM in Chapter 6

Elliot Lass – Assistance with neurobehavioural assessments and histology in Chapter 5

viii

Abbreviations

Aβ: Amyloid beta

ABCB1: ATP-binding cassette b1

ABO: Transferase A, alpha 1-3-N-acetylgalactosaminyltransferase, and transferase B,

alpha 1-3-galactosyltransferase

AcH3: Acetylated histone H3

ADAMTS13: A disintegrin and metalloproteinase with a thrombospondin type 1 motif,

member 13

ADPKD: Autosomal dominant polycystic kidney disease

AED: Antiepileptic drug

ALDH1L1: Aldehyde dehydrogenase 1 family, member L1

ANGPT1: Angiopoietin-1

ANGPT2: Angiopoietin-2

ANPEP: Alanyl (membrane) aminopeptidase

ANOVA: Analysis of variance

ANRIL: Antisense noncoding RNA in the INK4 locus

AP1: Activator protein 1

APC: Allophycocyanin

AQP4: Aquaporin 4

ASA: Acetylsalicylic acid

AT1: Angiotensin 1 receptor

AT2: Angiotensin 2 receptor

AVM: Arteriovenous malformation

ix

aVSP: Angiographic vasospasm

BBB: Blood-brain barrier

BCA: Bicinchoninic acid

BCL-2: B-cell lymphoma 2

BCL6B: B-cell CLL/lymphoma 6 member B protein

BEC: Brain endothelial cell

bFGF: Basic fibroblast growth factor

BK channels: Big potassium channels

BSA: Bovine serum albumin

CBF: Cerebral blood flow

CCL5: Chemokine ligand 5

CD11B: Cluster of differentiation 11B

CD31: Cluster of differentiation 31




CDH5: Cadherin 5

cDNA: Complementary deoxyribonucleic acid

C/EBP: CCAAT-enhancer-binding protein

CLDN5: Claudin 5

cGMP-PKG: Cyclic guanosine monophosphate-dependent protein kinase G

CNS: Central nervous system

CONSCIOUS: Clazosentan to Overcome Neurological Ischemia and Infarction

Occurring after Subarachnoid Hemorrhage

x

COX-1: Cyclooxygenase-1

COX-2: Cyclooxygenase-2

CSF: Cerebrospinal fluid

CSPG4: Chondroitin sulfate proteoglycan 4

CT: Computed tomography

CTA: Computed tomography angiography

CTP: Computed tomography perfusion

CXCL2: Chemokine C-X-C motif ligand 2

DAPI: 4’,6-diamidino-2-phenylindole

DCI: Delayed cerebral ischemia

DHA: Docosahexaenoic acid

DMSO: Dimethyl sulfoxide

EBI: Early brain injury

EC: Endothelial cell

EDTA: Ethylenediaminetetraacetic acid

EFNB2: Ephrin-B2

ELISA: Enzyme-linked immunosorbent assay

ENG: Endoglin

eNOS: Endothelial nitric oxide synthase

EPHB4: Ephrin type-B receptor 4

ERK: Extracellular signal-regulated kinase

ETB: Endothelin B receptor

FACS: Fluorescence-activated cell sorting

FDA: Food and Drug Administration

xi

FDR: False discovery rate

FITC: Fluorescein isothiocyanate

FJB: Fluoro-jade b

FLT1: Fms-related tyrosine kinase 1

FLT4: Fms-related tyrosine kinase 4

FOXL2: Forkhead box protein L2

FSC-A: Forward scatter area

FSC-H = Forward scatter height

GABA: Gamma-aminobutyric acid

GAD1: Glutamate decarboxylase 1

GADD45: Growth arrest and DNA damage-inducible protein

GFAP: Glial fibrillary acidic protein

GFP: Green fluorescent protein

GLUT1: Glucose transporter 1

GO: Gene ontology

GP130: Glycoprotein 130

GSEA: Gene set enrichment analysis

GWAS: Genome-wide association study

H&E: Hematoxylin & Eosin

HBA-A2/HBA-A1: Hemoglobin alpha, adult chain 1 / hemoglobin alpha, adult chain 2

HBSS-/-: Hank’s Balanced Salt Solution without calcium or magnesium

HDAC: Histone deacetylase

HDAC9: Histone deacetylase 9

HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A

xii

HRP: Horseradish peroxidase

HSP70: Heat shock protein 70

HUVEC: Human umbilical vein endothelial cell

ICAM1: Intercellular adhesion molecule 1

ICAM2: Intercellular adhesion molecule 2

ICH: Intracerebral hemorrhage

ICP: Intracranial pressure

ICU: Intensive care unit

IgG: Immunoglobulin G

IgM: Immunoglobulin M

IHRP: Inter-alpha-trypsin inhibitor family heavy chain-related protein

IL1R1: Interleukin 1 receptor 1

IL6RA: Interleukin 6 receptor antagonist

iNOS: Inducible nitric oxide synthase

IP: Intraperitoneal

ITGAM: Integrin α M

JAK: Janus kinase

KLF2: Krüppel-like factor 2

KDR: Kinase insert domain receptor

LCM: Laser capture microdissection

LEFTY2: Left-right determination factor 2

LGALS3: Lectin, galactose binding, soluble 3

lncRNA: Long non-coding ribonucleic acid

LPS: Lipopolysaccharide

xiii

LRP1: Low-density lipoprotein receptor-related protein-1

LY6C1: Lymphocyte antigen 6C1

LYVE1: Lymphatic vessel endothelial hyaluronan receptor 1

MACS: Magnetic-activated cell sorting

MAPK: Mitogen-activated protein kinase

MASH-2: Magnesium for Aneurysmal Subarachnoid Hemorrhage

MBP: Myelin basic protein

MCA: Middle cerebral artery

MCAO: Middle cerebral artery occlusion

MCP-1: Monocyte chemotactic protein-1

MDR1A: Multi-drug resistance transporter 1a

MEOX1: Homeobox protein MOX-1

MFSD2A: Major facilitator superfamily domain containing 2a

MGS: Modified Garcia score

miRNA: MicroRNA

MMP9: Matrix metalloproteinase 9

MMP13: Matrix metalloproteinase 13

MOG: Myelin oligodendrocyte glycoprotein

mPGES-1: Microsomal prostaglandin E synthase 1

MRI: Magnetic resonance imaging

mRNA: Messenger ribonucleic acid

mRS: Modified Rankin scale

MTT: Mean transit time

MYH2: Myosin, heavy polypeptide 2

xiv

MYOCD: Myocardin

ND: Not detected

NES: Normalized enrichment score

NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells

NMDA: N-methyl-D-aspartate

NMI: N-myc-interactor

NOS3: Nitric oxide synthase 3

NSAID: Non-steroidal anti-inflammatory drug

NVU: Neurovascular unit

OCLN: Occludin

OLIG2: Oligodendrocyte transcription factor 2

OR: Odds ratio

pAKT: Phosphorylated AKT

PBS: Phosphate buffered saline

PCA: Principal-component analysis

PDGFRβ: Platelet-derived growth factor receptor beta

PECAM1: Platelet endothelial cell adhesion molecule 1

PFA: Paraformaldehyde

PGE2: Prostaglandin E2

PGES: Prostaglandin E Synthase

PGI2: Prostaglandin I2

PGP: P-glycoprotein

PI: Propidium Iodide

PI3K: Phosphoinositide 3-kinase

xv

PPARγ: Peroxisome proliferator-activated receptor gamma

PPIA: Peptidylprolyl isomerase A

PRDM1: PR domain zinc finger protein 1

PRECISION: Prospective Randomized Evaluation of Celecoxib Integrated Safety

versus Ibuprofen Or Naproxen

PSGL-1: P-selectin glycoprotein ligand-1

PTGS1: Prostaglandin-endoperoxide synthase 1

PTGS2: Prostaglandin-endoperoxide synthase 2

PTPRC: Protein tyrosine phosphatase, receptor type, C

PVC: Perivascular cell

RES: Running enrichment score

RIN: Ribonucleic acid integrity number

RIPA: Radioimmunoprecipitation assay

RMA: Robust multi-array average

RNA: Ribonucleic acid

RNAseq: Ribonucleic acid sequencing

RT-PCR: Real-time polymerase chain reaction

RXFP1: Relaxin/insulin-like family peptide receptor 1

SAH: Subarachnoid hemorrhage

SCA1: Stem cells antigen 1

SEM: Standard error of the mean

SHH: Sonic hedgehog

SLC: Solute carrier

SLC2A1: Solute carrier 2A1

xvi

α-SMA: Alpha-smooth muscle actin

SNP: Single nucleotide polymorphism

Sp1: Specificity protein 1

SPTA1: Spectrin alpha, erythrocytic 1

SSC-W: Side scatter width

STAIR: Stroke Therapy Academic Industry Roundtable

STASH: Simvastatin in Aneurysmal Subarachnoid Hemorrhage

STAT: Signal transducer and activator of transcription

STAT3: Signal transducer and activator of transcription 3

SYP: Synaptophysin

TAGLN3: Transgelin 3

TBI: Traumatic brain injury

TEM: Transmission electron microscopy

TfR: Transferrin receptor

TGFβ: Transforming growth factor beta

TH: Tyrosine hydroxylase

THSD1: Thrombospondin type 1 domain containing protein 1

TIE1: Tyrosine kinase with immunoglobulin and epidermal growth factor homology

domains 1

TIE2: Tyrosine kinase with immunoglobulin and epidermal growth factor homology

domains 2

TIMP1: Tissue inhibitor of metalloproteinase 1

TJP1: Tight junction protein 1

TNFα: Tumour necrosis factor alpha

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TNFRSF1A: Tumour necrosis factor receptor 1

TNFRSF1B: Tumour necrosis factor receptor 2

tPA: Tissue plasminogen activator

TRAP: Translating ribosome affinity purification

TXA2: Thromboxane A2

VCAM1: Vascular cell adhesion molecule 1

VE-cadherin: Vascular endothelial cadherin

VEGF: Vascular endothelial growth factor

VEGFR1: Vascular endothelial growth factor receptor 1



VIGOR: Vioxx Gastrointestinal Outcomes Research

VPA: Valproic acid

VWF: von Willebrand factor

WFNS: World Federation of Neurological Surgeons

ZIC3: Zinc finger protein 3

ZO-1: Zonula occludens 1

ZO-2: Zonula occludens 2

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Table of Contents

Acknowledgments.......................................................................................................................... iv

Contributions................................................................................................................................. vii

Abbreviations ............................................................................................................................... viii

Table of Contents ....................................................................................................................... xviii

List of Tables ............................................................................................................................. xxiv

List of Figures ..............................................................................................................................xxv

Chapter 1 Introduction to Subarachnoid Hemorrhage (SAH) and the Blood-brain Barrier

(BBB) ..........................................................................................................................................1

Introduction to SAH and BBB ....................................................................................................2

1.1 Definition of SAH ................................................................................................................2

1.2 SAH History.........................................................................................................................2

1.3 Epidemiology of SAH..........................................................................................................3

1.3.1 Risk factors ..............................................................................................................4

1.4 Etiology of SAH ..................................................................................................................5

1.4.1 Intracranial aneurysm...............................................................................................5

1.4.2 Perimesencepahlic SAH...........................................................................................6

1.4.3 Other ........................................................................................................................7

1.5 Pathophysiology of SAH .....................................................................................................7

1.5.1 Early brain injury .....................................................................................................7

1.5.2 Large artery vasospasm............................................................................................9

1.5.3 Delayed cerebral ischemia .....................................................................................10

1.6 Diagnosis of SAH ..............................................................................................................17

1.7 Clinical management of SAH ............................................................................................18

1.8 Recent drug clinical trials in SAH .....................................................................................20

xix

1.9 Animal models of SAH ......................................................................................................23

1.9.1 Prechiasmatic injection rodent model ....................................................................24

1.9.2 Endovascular perforation rodent model .................................................................25

1.9.3 Cisterna magna injection rodent model .................................................................26

1.10 Gene expression studies in SAH ........................................................................................27

1.10.1 Early gene expression studies in animal models of SAH ......................................27

1.10.2 Gene expression studies of cerebral arteries in animal models of SAH ................28

1.10.3 Gene expression studies of brain tissue in animal models of SAH .......................29

1.10.4 Gene expression studies involving clinical aneurysm fundus samples .................30

1.10.5 Gene expression studies involving human serum and CSF samples .....................32

1.11 Introduction to Blood-Brain Barrier ..................................................................................33

1.11.1 Definition of Blood-Brain Barrier .........................................................................33

1.11.2 History of BBB ......................................................................................................35

1.11.3 Components of BBB ..............................................................................................36

1.12 BBB dysfunction in neurological disease ..........................................................................42

1.13 BBB permeability assays ...................................................................................................43

1.13.1 Blood protein measurement ...................................................................................43

1.13.2 Intravascular exogenous dye measurement ...........................................................43

1.13.3 In vivo assays .........................................................................................................44

1.13.4 Clinical detection of BBB disruption with MRI and CT .......................................44

1.14 Brain endothelial cell isolation techniques ........................................................................45

1.14.1 Brain microvessel isolation ....................................................................................45

1.14.2 Laser capture microdissection................................................................................46

1.14.3 Transgenic mice with fluorescent endothelial cells ...............................................47

1.14.4 Fluorescent antibodies and cell sorting ..................................................................47

1.14.5 Magnetic antibodies ...............................................................................................49

xx

1.15 Brain endothelial cell whole genome expression studies ..................................................50

Chapter 2 Research Questions and Hypotheses .............................................................................56

Research Questions and Hypotheses .........................................................................................57

2.1 Hypothesis 1: Valproic acid treatment in a mouse model of SAH results in improved

neurological outcomes .......................................................................................................58

2.2 Hypothesis 2: Valproic acid treatment in patients with SAH results in improved

clinical outcomes ...............................................................................................................58

2.3 Hypothesis 3: Pure and viable brain endothelial cells can be efficiently isolated from

mouse brain tissue ..............................................................................................................58

2.4 Hypothesis 4: Whole genome expression profiling of freshly isolated endothelial cells

can identify new potential therapeutic targets in SAH ......................................................58

2.5 Hypothesis 5: Celecoxib treatment in a mouse model of SAH results in improved

neurological outcomes .......................................................................................................58

Chapter 3 Valproic Acid Treatment in Experimental and Clinical SAH .......................................59

Valproic Acid Treatment in Experimental and Clinical SAH ..................................................60

3.1 Background ........................................................................................................................60

3.2 Methods..............................................................................................................................61

3.2.1 Prechiasmatic injection SAH mouse model ...........................................................61

3.2.2 VPA treatment .......................................................................................................62

3.2.3 Neurological assessment ........................................................................................63

3.2.4 Histological assessment after SAH ........................................................................64

3.2.5 Blood brain barrier assessment ..............................................................................67

3.2.6 Brain protein expression ........................................................................................68

3.2.7 Statistics .................................................................................................................69

3.2.8 Clinical trial data ....................................................................................................70

3.3 Results ................................................................................................................................71

3.3.1 Valproic acid improved neurological outcomes after experimental SAH .............71

3.3.2 Valproic acid limited brain injury after experimental SAH ...................................81

xxi

3.3.3 Valproic acid did not affect large artery vasospasm after experimental SAH .......85

3.3.4 Valproic acid decreased microthrombi burden after experimental SAH ...............87

3.3.5 Valproic acid decreased blood-brain barrier disruption after experimental

SAH........................................................................................................................89

3.3.6 Valproic acid increased acetylation of histones .....................................................91

3.3.7 Valproic acid treatment in SAH patients did not significantly affect clinical

outcome ..................................................................................................................93

3.4 Discussion ..........................................................................................................................98

3.4.1 Alternative SAH models treated with valproic acid ..............................................98

3.4.2 Other models of brain injury treated with valproic acid ......................................100

3.4.3 Valproic acid in the context of seizure treatment and prophylaxis ......................100

3.4.4 Valproic acid in the context of coagulopathy risk ...............................................102

3.4.5 Study limitations ..................................................................................................103

3.5 Conclusions ......................................................................................................................104

Chapter 4 Brain Endothelial Gene Expression after SAH ...........................................................105

Brain Endothelial Gene Expression after SAH .......................................................................106

4.1 Background ......................................................................................................................106

4.2 Methods............................................................................................................................107

4.2.1 SAH model...........................................................................................................107

4.2.2 Neurobehavioural Assessment .............................................................................110

4.2.3 Celecoxib Treatments ..........................................................................................110

4.2.4 BBB permeability assay .......................................................................................110

4.2.5 Transmission electron microscopy ......................................................................112

4.2.6 Brain endothelial cell isolation protocol #1: AutoMACS Method (Figure 13) ...113

4.2.7 Brain endothelial cell isolation protocol #2: FACS Method (Figure 13) ............119

4.2.8 Microarray............................................................................................................121

4.2.9 RT-PCR Validations ............................................................................................122

xxii

4.2.10 Immunofluorescence ............................................................................................124

4.2.11 Fluoro-jade b staining ..........................................................................................125

4.2.12 Enzyme-linked immunosorbent assay (ELISA) ..................................................125

4.3 Results ..............................................................................................................................126

4.3.1 Experimental SAH caused neurobehavioural deficits and histological brain

injury ....................................................................................................................126

4.3.2 Experimental SAH caused blood-brain barrier disruption...................................131

4.3.3 Mouse brain endothelial cell isolation by magnetic-based and fluorescence-

activated cell sorting ............................................................................................133

4.3.4 Gene expression patterns distinguish SAH from sham brain endothelial cells ...142

4.3.5 Gene expression changes demonstrate enrichment in inflammatory response

genes ....................................................................................................................146

4.3.6 Brain endothelial cells show increased Ptgs2 (COX-2) and Angpt2 expression

after SAH .............................................................................................................153

4.3.7 Treatment with selective COX-2 inhibitor celecoxib blunted brain endothelial

Ptgs2 upregulation after SAH ..............................................................................157

4.4 Discussion ........................................................................................................................161

4.4.1 COX-2 as a therapeutic target ..............................................................................163

4.4.2 Angiopoietin-2 as a therapeutic target .................................................................168

4.4.3 Limitations ...........................................................................................................170

4.5 Conclusions ......................................................................................................................171

Chapter 5 Future Directions .........................................................................................................172

Future Directions .....................................................................................................................173

5.1 Brain endothelial cell gene expression studies ................................................................173

5.2 COX-2 studies in experimental SAH ...............................................................................174

5.3 Clinical trial design in SAH .............................................................................................175

5.3.1 Consideration of a clinical trial with VPA in patients with SAH ........................175

5.3.2 Consideration of a clinical trial with celecoxib in patients with SAH .................177

xxiii

Chapter 6 Concluding Remarks ...................................................................................................179

Concluding Remarks ...............................................................................................................180

6.1 Summary of results ..........................................................................................................180

6.2 Final comments ................................................................................................................181

References ....................................................................................................................................182

xxiv

List of Tables

Table 1: Base-line characteristics of CONSCIOUS-1 patient population (n=413).

Table 2: Univariate and multivariate analyses determining predictors of poor clinical

outcome at 12 weeks (mRS 4-6).

Table 3: Effect of VPA treatment.

Table 4: VPA use.

Table 5. Forward and reverse primer sequences used for RT-PCR.

Table 6: Gene Set Enrichment Analysis (GSEA) with Gene Ontology (GO) data sets.

Table 7: Gene Set Enrichment Analysis (GSEA) with Pathway Analysis data sets.

xxv

List of Figures

Figure 1. Prechiasmatic injection SAH mouse model.

Figure 2. Activity level.

Figure 3. Morris water maze.

Figure 4. Morris Water Maze supplemental information.

Figure 5. Open field test.

Figure 6. Fluoro-jade b staining at 48h.

Figure 7. Neuronal apoptosis at 48h.

Figure 8. Middle cerebral artery (MCA) vasospasm at 48h.

Figure 9. Microthrombi burden at 48h.

Figure 10. Blood-brain barrier integrity.

Figure 11. Protein quantification of left cerebral hemisphere at 24h.

Figure 12. Tie2-GFP mouse.

Figure 13. Overview of brain endothelial cell isolation protocols.

Figure 14. SAH model characteristics.

Figure 15. SAH model neurobehavioural and histological outcomes.

Figure 16. Blood-brain barrier (BBB) disruption after SAH.

Figure 17. Brain endothelial cell isolation.

Figure 18. Brain endothelial cell viability.

Figure 19. Expression levels of prototypical genes of various cell types in isolated

CD45-CD31+ cells.

Figure 20. Effect of RNA amplification on gene expression.

xxvi

Figure 21. Clustering of SAH and sham samples based on brain endothelial cells

expression patterns.

Figure 22. Top differentially-expressed genes.

Figure 23. Expression of BBB intercellular junction genes in brain endothelial cells after

SAH.

Figure 24. Expression of BBB transporter genes in brain endothelial cells after SAH.

Figure 25. Gene set enrichment analysis (GSEA) demonstrating enrichment of genes

relevant to prostaglandin synthesis and regulation in SAH brain endothelial cells

samples.

Figure 26. Microarray validation studies.

Figure 27. Serum protein expression of ANGPT1, ANGPT2, and TIE2

Figure 28. Effect of selective COX-2 inhibitor celecoxib in SAH.

Figure 29. Effect of selective COX-2 inhibitor celecoxib in SAH.

1

Chapter 1

Introduction to Subarachnoid Hemorrhage (SAH) and the Blood-

brain Barrier (BBB)

2

Introduction to SAH and BBB

Here we provide a concise but comprehensive introduction to subarachnoid

hemorrhage (SAH). Although trauma is the most common cause of SAH, the term SAH

in this manuscript refers to spontaneous SAH. An authoritative review of SAH with an

extensive appraisal of the literature has already been published [Macdonald 2017].

1.1 Definition of SAH

SAH refers to hemorrhage within the subarachnoid space, the layer between the

arachnoid superficially and the pia mater more deeply. This space normally contains

cerebrospinal fluid (CSF) which surrounds and bathes the brain parenchyma. Also, in

this space lie the major intracranial arteries. By far the most common cause of SAH is a

ruptured intracranial aneurysm.

1.2 SAH History

A review of the history of SAH and vasospasm has been published previously

[Macdonald 2016]. It is commonly thought that the Greek physician Hippocrates may

have described the first case of SAH when he wrote around 400 B.C.: “when persons in

good health are suddenly seized with pains in the head, and straightway are laid down

speechless, and breathe with stertor, they die in seven days” [Macdonald 2016]. The

delayed mortality, may have been caused by rebleeding of a ruptured aneurysm or

delayed cerebral ischemia (DCI), although this is just speculation. The Roman

encyclopedist A. Cornelius Celsus later described in 30 A.D. a person likely suffering

from SAH presenting with “strong shivering, nervous relaxation, dimness of sight,

3

delirium, vomiting together with a suppression of voice… besides these symptoms there

is a violent pain chiefly about the temples or occiput” [Macdonald 2016].

The classic pathological description of SAH was made by Bramwell, who described the

“spontaneous meningeal hemorrhage” in 1886 [Straus 1932]. The US neurosurgeon

Arthur Ecker in 1951 described angiographic vasospasm in relationship with ruptured

saccular intracranial aneurysms [Ecker 1982]. The 1950s to 1980 encompassed the

“dark age of delayed aneurysm surgery” in which SAH patients were dehydrated and

underwent induced hypotension [Macdonald 2016]. The timing of the vasospasm,

occurring from days 3 to 12 after SAH and maximal at 7-10 days, was then clarified by

neurosurgeon Bryce Weir in his manuscript “Time course of vasospasm in man” [Weir

1978]. The neurologist/neuropathologist C. Miller Fisher identified the association of

SAH clot burden with development of vasospasm and published his Fisher grading

scale that is still used today [Fisher 1980]. Further refinements in SAH care over the last

several decades include the use of induced hypertension for DCI, the use of balloon

angioplasty for symptomatic large artery vasospasm and the use of nimodipine, a

dihydropyridine calcium channel antagonist [Macdonald 2016]. Other notable

advancements include the use of the operative microscope for microsurgical clipping of

aneuryms, endovascular coiling to treat aneurysms, treating ruptured aneurysms

acutely and routine neurocritical care.

1.3 Epidemiology of SAH

The best current estimate of spontaneous SAH incidence worldwide is 9 per 100,000

people per year [de Rooij 2007]. Overall, the occurrence of SAH has declined over

4

several decades [de Rooij 2007]. The cause of this decline is unclear but may be

related to treatment of unruptured intracranial aneurysms to prevent SAH, better

detection of unruptured aneurysms with improved diagnostic imaging and screening,

better control of hypertension and decline in smoking. SAH is more common in females

and the most common age of occurrence is between 50 and 60, although it can also

occur in pediatric and geriatric populations [de Rooij 2007]. Although SAH represents

approximately 5% of all strokes, the patients with SAH are generally younger than the

intracerebral hemorrhage (ICH) and ischemic stroke patients, resulting in

disproportionate number of years of life lost [Johnston 1998]. Eight percent of patients

die before reaching the hospital, and with an additional 26% mortality rate for patients

surviving to hospital admission [Macdonald 2017]. Many surviving patients will continue

to have longstanding neurological or cognitive issues preventing return to work

[Macdonald 2017].

1.3.1 Risk factors

Non-modifiable risk factors for SAH include increasing age, female sex, larger

aneurysm size, aneurysm location (posterior circulation and anterior communicating

artery), irregular aneurysm shape, family history of SAH and personal history of SAH

[Macdonald 2017]. Specific ethnicities have a predisposition to present with SAH

including the Japanese and Finnish populations based on published epidemiological

data [Backes 2015]. Certain medical conditions predispose to aneurysm formation such

as autosomal dominant polycystic kidney disease (ADPKD) and connective tissue

disorders such as Ehlers-Danlos type IV and Marfan syndrome [Macdonald 2017].

Modifiable risk factors include cigarette smoking, excessive alcohol consumption, and

5

hypertension [Macdonald 2017]. Many of the risk factors for SAH overlap with risk

factors for aneurysm formation and aneurysm growth [Macdonald 2017].

1.4 Etiology of SAH

1.4.1 Intracranial aneurysm

The most common cause of spontaneous SAH is a ruptured intracranial aneurysm,

which occurs in 85% of cases [Macdonald 2017]. The saccular aneurysm is typically

found at arterial bifurcation points in the region of the circle of Willis, presumably

secondary to hemodynamic stress such as disturbed non-laminar flow at arterial branch

points. The most common location of aneurysm rupture is at the anterior communicating

artery. Other aneurysm types include dissecting aneurysms, fusiform aneurysms,

mycotic aneurysms, blood blister-like aneurysms, and flow-related aneurysms

associated with arteriovenous malformations (AVMs). Intracranial aneurysms are

thought to affect 3% of the population, meaning that most aneurysms do not rupture

[Macdonald 2017].

1.4.1.1 Screening for intracranial aneurysms

Screening for intracranial aneurysms is generally recommended if a person has at least

2 first degree relatives with aneurysms and/or SAH [Connolly 2012]. Patients with

ADPKD are also recommended to be screened given that approximately 10% will

harbor an intracranial aneurysm [Perrone 2015]. The interval between screening

examinations and the upper age limit beyond which to not screen for de novo

6

aneurysms after SAH is more controversial and no consensus exists currently, with

some advocating life-long screening.

1.4.1.2 Genetics of intracranial aneurysms

Foroud and colleagues used genome-wide association studies (GWAS) to identify

SOX17 and ANRIL (antisense noncoding RNA in the INK4 locus), a long noncoding

RNA, to be associated with formation of intracranial aneurysms, although smoking

appeared to have a greater risk for developing aneurysms than having these specific

single nucleotide polymorphisms (SNPs) [Foroud 2012]. In a meta-analysis of GWAS

studies, the authors found ANRIL and HDAC9 (histone deacetylase 9), a gene

associated with ischemic stroke on prior GWAS studies, to be associated with

intracranial aneurysm formation [Foroud 2014]. In a familial aneurysm study, mutation of

the THSD1 (thrombospondin type 1 domain containing protein 1) gene, which is

involved with focal adhesion of endothelial cells to the extracellular matrix of the

basement membrane, was associated with aneurysm formation [Santiago-Sim 2016].

THSD1 loss of function resulted in spontaneous intracerebral bleeding in both mice and

zebrafish [Santiago-Sim 2016].

1.4.2 Perimesencepahlic SAH

Approximately 10% of spontaneous SAH have a perimesencephalic distribution around

the basal cisterns and prepontine region but no aneurysm identified on vascular imaging

[Macdonald 2017]. Generally, this pattern of hemorrhage is associated with a better

prognosis. The etiology of this type of hemorrhage is unknown.

7

1.4.3 Other

About 5% of spontaneous SAH is caused by a heterogeneous group of conditions

including AVM rupture, cortical venous sinus thrombosis, reversible cerebral

vasoconstriction syndrome (a disorder with severe headaches and narrowing of

intracranial arteries which may have associated SAH), Moyamoya disease, CNS

(central nervous system) vasculitis, and anticoagulation, among many other conditions.

1.5 Pathophysiology of SAH

1.5.1 Early brain injury

Early brain injury (EBI) refers to the brain damage occurring during the acute period,

loosely defined as within 72 hours of ictus, prior to the onset of angiographic vasospasm

(aVSP). There are two components to EBI: 1) Transient global cerebral ischemia due to

the initial rupture of the aneurysm and relative lack of blood flow to the brain due to the

increased intracranial pressure (ICP), decreased cerebral blood flow (CBF) and

decreased cerebral perfusion pressure, and 2) neurotoxic effects of the subarachnoid

blood itself. If applicable, damage secondary to intracerebral hematoma extension and

herniations would also contribute to early brain injury. Clinically, EBI manifests as loss

of consciousness at time of aneurysm rupture and poor WFNS (World Federation of

Neurological Surgeons) grade, which are predictors of poor clinical outcome

[Suwatcharangkoon 2015].

There are number of mechanisms involved with EBI including decreased vascular

integrity (endothelial cell apoptosis, intercellular adhesion disruption, basement

8

membrane degradation by increased matrix metalloproteinases), dysfunctional nitric

oxide uncoupling, oxidative stress, thrombin activation, excitotoxicity, inflammation with

cytokine release and increased expression of cell adhesion molecules, lipid

peroxidation, ionic disturbances (increased calcium influx, decreased magnesium,

increased potassium efflux), and cortical spreading ischemia [Fuji 2013, Geraghty

2017]. Cellular apoptosis appears more important in SAH pathophysiology than cellular

necrosis, with involvement of AKT/PI3K (protein kinase B / phosphoinositide 3-kinase),

MAPK/ERK (mitogen-activated protein kinase / extracellular signal-regulated kinase),

JNK (c-Jun N-terminal kinase), p38, and p53 pathways, all of which may contribute to

neuronal apoptosis [Fuji 2013]. Microglia can be activated by SAH and contribute to

brain injury [Geraghty 2017]. However, microglial may also limit brain injury by removing

the toxic heme component of blood breakdown products with the enzyme heme

oxygenase-1 [Schallner 2015]. Reactive astrocytes have both protective and deleterious

effects after SAH, including preserving blood-brain barrier (BBB), limiting oxidative

stress, and decreasing edema, but at the same time also contributing to scar formation,

increasing cytokine release, and limiting axonal regeneration [Zheng 2017].

EBI appears to result from a complex dynamic of impaired CBF, ongoing ischemia, toxic

effects of subarachnoid blood, coagulopathy, and inflammation, with much interest in

identifying prognostic biomarkers [Al-Mufti 2017]. Many studies have looked at various

pathways involved in EBI pathophysiology, although it is unclear which pathway(s) to

pursue based on these pre-clinical models. Most phase III clinical trials in aneurysmal

SAH have supposedly studied aVSP, although since mechanisms overlap with EBI, the

distinction is somewhat semantic.

9

There is also a genetic component contributing to the degree of brain injury based on

experimental SAH models [D’Abbondanza 2016]. Mice of different strains have variable

degrees of brain injury from least severe (C57BL/6J, FVB) to most severe (A/J),

although the specific gene(s) determining this variability have yet to be determined

[D’Abbondanza 2016].

1.5.2 Large artery vasospasm

Large artery vasospasm or aVSP occurs in a delayed manner after SAH,

characteristically starting as early as day 3 and typically resolving by day 12 [Weir

1978]. aVSP may occur in almost two thirds of SAH patients but may not necessarily be

associated with symptoms [Macdonald 2014]. The burden of subarachnoid blood

predisposes to developing aVSP, and can be classified based on the Fisher grade

(grade 1 – no SAH, grade 2 – thin < 1mm SAH, grade 3 – thick > 1 mm SAH, grade 4 –

ICH and/or intraventricular hemorrhage (IVH)) or the more predictive modified Fisher

scale (grade 0 - no SAH, grade 1 – thin < 1mm SAH, grade 2 – thin < 1mm SAH and

ICH/IVH, grade 3 – thick > 1mm SAH, grade 4 – thick > 1mm SAH and ICH/IVH) [Fisher

1980, Frontera 2006]. Vasospasm involves both arterial structural changes (initimal

hyperplasia, smooth muscle proliferation, fibrosis, collagen deposition, and endothelial

and vascular smooth muscle necrosis) and dynamic changes such as enhanced

myogenic constriction, resulting in vessel wall thickening and decreased compliance

[Smith 1985, Mayberg 1990, Yagi 2015]. The etiology of aVSP is unclear but is likely

related to degradation products released by the lysis of subarachnoid blood. aVSP has

been an active area of research for many decades. In a meta-analysis of GWAS studies

10

for aVSP after SAH, SNPs in the haptoglobin gene was associated with aVSP (OR 3.9)

[Rosalind Lai 2015].

1.5.3 Delayed cerebral ischemia

DCI refers to delayed neurological injury not identified from the initial aneurysmal

rupture or after the aneurysm-securing procedure and not attributed to a specific cause

such as hydrocephalus, seizure, and metabolic or systemic derangements. DCI results

in a neurologic deficit in association with aVSP or may be related to new delayed

infarcts even in the absence of neurologic deficit or aVSP. DCI has been declining over

last several decades due to improved overall SAH management and may be detected in

up to 29% of cases, with nimodipine treatment decreasing DCI to 22% [Macdonald

2014]. In a meta-analysis of GWAS studies for DCI, a SNP in the endothelial nitric oxide

(eNOS) gene was associated with DCI (Odds Ratio, OR 1.9) [Man 2016].

1.5.3.1 Cortical spreading depression

Waves of depolarization, called cortical spreading depression, can spread along the

cortex after SAH at a rate of 2-5mm per minute [Terpolilli 2015]. In a normal brain, this

depolarization wave would result in increased blood flow and hyperemia, secondary to

functioning neurovascular coupling [Terpolilli 2015]. However, in SAH, there is

impairment in autoregulation in which depolarization waves can result in paradoxical

vasoconstriction resulting in ischemia [Terpolilli 2015]. Winkler and colleagues have

found BBB disruption in patients with SAH to be associated with areas of paradoxical

hypoemic response in regions of spreading depolarizations or seizures [Winkler 2012].

11

1.5.3.2 Microvascular disturbances

SAH pathophysiology includes disturbances in blood vessels at the microcirculation

level, arbitrarily defined here as vessel diameter less than 100μm [Tso 2014]. The first

study to investigate SAH microvascular changes was probably by Herz and colleagues

in 1975, who observed dorsal pial arterial constriction in a vascular micropuncture

model of SAH in guinea pigs [Herz 1975]. Since then, several studies looking at the

microcirculation after SAH have now found altered vasoreactivity, microvascular

constriction, vascular inflammation, BBB impairment, and inversion of neurovascular

coupling, discussed in more detail below, and represent potential targets for therapeutic

intervention. These microvascular changes may contribute to elevated capillary transit

time heterogeneity, resulting in metabolic disturbances [Ostergaard 2013]. Here, we

review preclinical studies investigating microvascular pathophysiology after

experimental SAH.

1.5.3.2.1 Altered vasoreactivity

After experimental SAH, the brain microvasculature has altered responses to vasoactive

agents. Agents that cause vasoconstriction, such as endothelin-1 and elevated

extracellular potassium, resulted in exaggerated vasoconstriction after SAH compared

to controls, whereas agents that generally cause vasodilation, such as adenosine,

adenosine diphosphate (ADP), adenosine triphosphate (ATP), cyclic guanosine

monophosphate (cGMP), vasopressin, and sodium nitroprusside, resulted in modest

vasodilation after SAH compared to controls [Britz 2007, Kajita 1996, Katusic 1993,

Park 2001, Park 2002, Park 2009, Vollmer 1992]. These alterations may be seen within

12

1 hour of SAH induction to as much as 14 days after SAH. Also, SAH resulted in

impairment of normal signal propagation from the penetrating arteries along the

cerebrovascular tree to the parenchymal arterioles [Kajita 1996]. More studies are

needed to understand how subarachnoid blood bathing and surrounding the large

conducting arteries at the base of the brain may affect dynamic control and

autoregulation of the distal arteries and even whether it does.

1.5.3.2.2 Microvascular vasoconstriction

Experimental SAH is associated with vasoconstriction of larger conducting arteries, but

also smaller resistance arteries including the pial arteries and intraparenchymal

arterioles, and observed immediately to as late as 14 days after SAH induction [Kniesel

2000, Wiernsperger 1981, Friedrich 2012, Sun 2009, Nystoriak 2011, Koide 2013, Cach

1987, Zubkov 2000, Ohkuma 1997, Asano 1977, Ohkuma 1999, Ohkuma 2003, Sabri

2012, Johshita 1990, Sehba 2007, Friedrich 2010A]. Such microvascular constriction

may be contributory to capillary transit time heterogeneity, resulting in discrete brain

regions with relative lack of CBF [Ostergaard 2013]. Despite the brain capillaries being

devoid of vascular smooth muscle, it has been established that hemoglobin can induce

α-SMA (alpha-smooth muscle actin) expression in pericytes, resulting in reduction in

brain microvessel diameter after SAH [Li 2016]. The pial microvessel vasoconstriction

seemed to be more severe in smaller vessels compared to larger vessels, and arterioles

rather than venules [Friedrich 2012, Sun 2009]. It is suggested that TNFα (Tumour

necrosis factor alpha) is involved with the pathologic increased myogenic tone after

SAH [Yagi 2015]. Nimodipine was effective in limiting parenchymal arteriolar

vasoconstriction after SAH, perhaps suggesting that its beneficial effect in human SAH

13

is on a microcirculation level as the drug does not have a discernable effect on aVSP

[Nystoriak 2011, Dorhout Mees 2007].

1.5.3.2.3 Inflammation

Several inflammatory pathways are activated in the microvasculature after

experimental SAH, including phosphorylation of MAPK and increased expression of

inflammatory cytokines by TNFα signaling and NF-κB (Nuclear factor kappa-light-chain-

enhancer of activated B cells) activation and nuclear translocation [Zhou 2013, Ansar

2008]. SAH resulted in increased luminal expression of inflammatory cell adhesion

molecules in brain endothelial cells such as p-selectin, e-selectin, VCAM1 (Vascular cell

adhesion molecule 1), and ICAM1 (Intercellular adhesion molecule 1) [Sabri 2012,

Atangana 2017]. These cell adhesion molecules increase leukocyte-endothelial

interactions resulting in rolling and adherence of systemic leukocytes and subsequent

diapedesis and extravasation into the brain observed as early as 10min after SAH

[Sebha 2011, Friedrich 2011]. These adherent neutrophils appear to obstruct flow

through microvessels, with neutrophil depletion or inhibition resulting in decreased

microvascular damage [Ishikawa 2009, Friedrich 2011]. ICAM1 and P-selectin

glycoprotein ligand-1 (PSGL-1) knockout mice had decreased leukocyte-endothelial

interactions in the first 7 days with concomitant reduction of microglia aggregation and

brain injury [Atangana 2017]. The authors suggest that brain inflammation is initiated

from the blood vessels [Atangana 2017]. Using 2-photon microscopy to visualize cortical

microcirculation in mice undergoing the endovascular perforation SAH model, there was

a significant decrease in blood flow 1h after SAH induction with rolling and adherent

leukocyte obstructing flow in the capillaries, which was reversed with pre-treatment with

14

p-selectin monoclonal antibodies [Ishikawa 2016]. Targeting leukocyte-endothelial

interactions after SAH has not been investigated clinically.

1.5.3.2.4 Increased microthrombi burden

Experimental SAH can result in platelet aggregation and microthrombi accumulation in

the brain microvasculature, like the no-reflow phenomenon observed in animal models

of transient global cerebral ischemia [Tso 2013]. Microthrombi can appear as early as

10min after SAH induction and observed on the abluminal side of microvessels

[Friedrich 2010B]. Platelets can be observed rolling and adhering to the luminal surface

of microvessels although not completely occluding the vessel [Ishikawa 2009].

Microthrombi burden was correlated with degree of microvascular vasoconstriction and

neuronal apoptosis [Friedrich 2012, Sabri 2012]. Microthrombi burden appeared to

decrease after urokinase-type plasminogen activator but increased after clazosentan

treatment, an endothelin receptor antagonist [Pisapia 2012]. ADAMTS13 (A disintegrin

and metalloproteinase with a thrombospondin type 1 motif, member 13) may be

involved in preventing microthrombi formation, with recombinant ADAMTS13 treatment

reducing microthrombi burden and brain injury in a prechiasmatic injection mouse model

of SAH [Vergouwen 2014].

Parenchymal microthrombi burden appeared to correlate with DCI in autopsy studies of

patients with SAH [Stein 2006]. Clinically, intraventricular tPA (Tissue plasminogen

activator) was associated with decreased aVSP and need for rescue balloon

angioplasty, but prospective studies are needed to confirm this finding [Ramakrishna

15

2010]. ASA (Acetylsalicylic acid) was not effective in improving outcomes in a

randomized controlled trial with SAH patients [van den Bergh 2006].

1.5.3.2.5 Blood-brain barrier impairment

BBB disruption has been observed after experimental SAH as early as 10min, with

maximum disruption at 24h and persisting up to 1 week after SAH induction [Friedrich

2010A, Imperatore 2000, Suzuki 2010, Germano 2000, Gules 2003]. Techniques

demonstrating increased barrier permeability after SAH included Evans Blue dye which

adheres to albumin, various fluorescent tracers, and presence of extraluminal

immunoglobulins [Kusaka 2004, Park 2002, Johshita 1990, Zhou 2013, Yatsushige

2007, Erdo 1998, Germano 2002, Imperatore 2000, Suzuki 2010, Yan 2013, Doczi

1986A, Doczi 1986B, Germano 2000, Wang 2011, Smith 1996, Yatsushige 2006,

Scholler 2007, Yan 2008, Yan 2011, Gules 2003].

The causes and results of this impairment may include disruption of the interendothelial

junctions (tight junctions, adherens junctions), degradation of extracellular matrix

proteins that form the basal lamina, dysfunctional astrocyte end feet, and dysfunctional

or loss of pericytes [Sehba 2007, Friedrich 2010A, Zhou 2013, Friedrich 2011,

Yatsushige 2007, Yan 2008, Yan 2011, Gules 2003, Sehba 2013, Sehba 2004, Sehba

2010, Friedrich 2012, Prunell 2005]. The effect is an increase in BBB permeability, but it

is unclear if this is based on increased paracellular transport, increased transcytosis or

a combination of the two processes. Many BBB stabilizing agents have been studied in

pre-clinical models of SAH. Some drugs studied in human randomized clinical trials

16

might prevent BBB injury, such as the lipid peroxidation inhibitor tirilazad but none of

them have been shown to improve functional outcome [Zhang 2010].

Clinically, CT perfusion (CTP) may be used evaluate for evidence of BBB disruption.

Ivanidze and colleagues found that in some patients with SAH, an increased

permeability surface area product locally could be an indicator of BBB disruption despite

normal CBF and mean transit time (MTT), and these patients progressed to DCI

[Ivanidze 2015]. Other have found BBB permeability on CTP to be associated with

increased MTT, higher CBV and more severe hydrocephalus [Kishore 2012]. Currently,

CTP is used clinically to make the diagnosis of DCI but there is not enough evidence to

show that it can predict DCI [Cremers 2014]. In a prospective series, Murphy and

colleagues did not find BBB permeability on CTP to be associated with DCI or poor

clinical outcome after SAH [Murphy 2015]. More clinical studies are needed, including

optimizing the modality of measuring BBB and the identifying the ideal contrast agent

based on size, charge, and detection sensitivity.

1.5.3.2.6 Inversion of neurovascular coupling

Under healthy physiological conditions, increased synaptic activity with release of

excitatory neurotransmitter glutamate causes increased intracellular calcium in

astrocytes as part of the tripartite synapse and results in modest opening of BK (Big

potassium) channels, increasing the extracellular potassium (<20mM) and causing

hyperpolarization and relaxation of vascular smooth muscle at the level of parenchymal

arterioles [Koide 2012]. However, in SAH, this increased intracellular calcium signal

transmits through the astrocyte soma to the astrocyte end feet, with excessive opening

17

of BK channels, excessive efflux of potassium into the extracellular space (>20mM)

causing depolarization and vasoconstriction of vascular smooth muscle [Koide 2012]. In

a rat endovascular perforation SAH model, the authors showed significant decrease in

tissue oxygen levels in hippocampus in response to synaptic train stimulation,

suggesting inversion of neurovascular coupling [Galeffi 2016]. This paradoxical

vasoconstriction in response to increased neuronal activity is referred to as inversion of

neurovascular coupling and may be associated with spreading depression [Dreier

2011].

1.6 Diagnosis of SAH

The classic clinical presentation of SAH is a sudden, severe headache that can be

described as the “worst headache of my life” with maximal severity achieved within 1

minute of onset. In addition to headache, patients may also have nausea, vomiting,

photophobia, neck stiffness, cranial neuropathy, altered level of consciousness,

neurological deficit, and coma. Patients with SAH generally present to the Emergency

Department but may also initially be seen in a primary care clinic.

With the appropriate history, there is a low threshold to obtain a non-contrast CT head

scan to rule out SAH, due to the potentially life-threatening etiology. A CT head

demonstrating SAH leads to a subsequent vascular imaging with a CT angiogram (CTA)

to identify a potential aneurysm. In the setting of a negative CT head scan and history

that is particularly suspicious, a lumbar puncture can be performed to rule out SAH

based on presence of red blood cells and xanthochromia. Depending on the location

and morphology of the aneurysm as well as the management decision of the clinical

18

team, a more detailed vascular imaging called digital subtraction angiography may be

obtained.

1.7 Clinical management of SAH

After the patient is stabilized from an airway and hemodynamic standpoint, one of the

first goals is to secure the aneurysm to prevent rebleeding. This can be achieved either

with microsurgical clipping or endovascular coiling. A randomized controlled trial

showed superiority in clinical outcome for patients treated with endovascular coiling, so

this is most often the first option for aneurysm repair, although there are many nuances

in choosing the treatment selection for a particular aneurysm, which is beyond the

scope of this background [Molyneux 2002]. Some patients may develop hydrocephalus

requiring external ventricular drain insertion to remove CSF to relieve the increase in

ICP. In one multivariate analysis, the presence of an external ventricular drain and

larger daily CSF output were significant predictors of needing delayed permanent

ventriculoperitoneal shunt insertion [Tso 2016]. Nimodipine, as mentioned previously, is

indicated to be initiated within 96h to improve outcomes, although the drug is usually

started at the time of hospital admission, and may need to be administered in lower,

more frequent doses or suspended if low blood pressures are encountered. Admission

to the intensive care unit (ICU) or a neurological step-down unit for at least 2 weeks is

generally performed, with goals being to maintain euvolemia and normothermia, as well

as monitor serum sodium levels.

Seizures may occur at the time of aneurysm rupture, acutely during hospital admission,

or in a delayed manner after hospital discharge, with occurrence in 4-26% of patients

19

with aneurysmal SAH [Lanzino 2011]. Seizures are associated with higher mortality

[Rush 2016]. Predictors of seizure during hospital admission include older age, seizure

at time of aneurysm rupture, hydrocephalus, and anterior circulation aneurysm [Jaja

2017]. Subdural hematoma and subarachnoid clot burden were also associated with

development of seizures [Ibrahim 2013]. It is unclear if prophylactic anti-epileptic drugs

are beneficial and may in fact cause more complications [Rosengart 2007]. Phenytoin

has been associated with poor outcome in a cumulative dose-dependent manner

[Naidech 2005].

Monitoring for aVSP and DCI typically begins on admission with daily transcranial

Doppler measurements to detect rising intracranial artery velocities, indicative of aVSP.

Frequent neurologic exams are also performed to detect DCI. If there is a new

neurologic deficit, a repeat CT and CTA are performed to assess for aVSP and infarcts

and rule out other causes of neurologic deficits. CTA and CT perfusion may also be

performed on admission, after aneurysm repair and if there is neurologic deterioration to

assess for areas of CBF/cerebral blood volume mismatch, suggesting potentially

salvageable regions of ischemia. Active regular surveillance of neurologic status is the

most important method in detecting DCI [Findlay 2016]. Other than nimodipine

administration, there is no level I evidence for any specific management of DCI in the

setting of aVSP, although options include induced hypertension, intra-arterial

pharmacologic vasodilators such as verapamil, nicardipine and milrinone, and balloon

angioplasty.

20

Patients with SAH also have a significant systemic inflammatory response, which can

result in respiratory distress resulting in neurogenic pulmonary edema, acute respiratory

syndrome, and pneumonia, as well as cardiac abnormalities such as neurogenic

stunned myocardium [Kerro 2016].

Once the patient has recovered from the initial SAH and has been observed in hospital

for 2 weeks, the patient can be safely discharged home. Some patients may need

additional rehabilitation due to neurologic deficits as a result of the SAH, and others

may have sustained substantial brain injury requiring transfer to a skilled nursing facility

or long-term care.

1.8 Recent drug clinical trials in SAH

A special note should be made regarding aneurysm treatment. Although not a drug

treatment, which is the topic of discussion here, the ISAT (International Subarachnoid

Aneurysm Trial, n=2143) was a randomized clinical trial that found that endovascular

coiling of aneurysm resulted in improved 1-year clinical outcomes compared with

microsurgical clipping in patients with aneurysmal SAH and clinical equipoise [Molyneux

2002]. This treatment has dramatically changed clinical practice, in which now the clear

majority of ruptured and unruptured aneurysms are treated with endovascular

techniques.

Currently the only drug treatment shown to improve outcomes in SAH is nimodipine, a

voltage-gated calcium channel antagonist, which was investigated in the large British

aneurysm nimodipine trial (n=554) [Pickard 1989]. In this trial, enteral nimodipine 60mg

21

every 4h for 21 days and initiated within 4 days, resulted in decreased cerebral

infarction and improved clinical outcomes despite no significant effect on aVSP [Pickard

1989]. Currently, the exact mechanism resulting in improved outcomes with nimodipine

treatment has not been elucidated, although the drug is currently standard of care for

SAH management.

Since the publication of several negative phase III clinical trials investigating the use of

the lipid peroxidase inhibitor tirilazad in 1999, the next 20 years have only produced 3

additional phase III randomized drug clinical trials in SAH [Macdonald 2014]. The first

investigated the use of clazosentan, an endothelin receptor antagonist, started within

56h of aneurysmal SAH onset in patients with aneurysms repaired by microsurgical

clipping in the CONSCIOUS-2 trial (Clazosentan to Overcome Neurological Ischemia

and Infarction Occurring after Subarachnoid Hemorrhage, n=1157) [Macdonald 2011].

This trial had much initial optimism due to the promising results of the phase II

CONSCIOUS-1 trial (n=413), in which the drug was able to decrease aVSP [Macdonald

2008]. Unfortunately, the CONSCIOUS-2 trial results demonstrated no significant

improvement in mortality or clinical outcome after clazosentan treatment, despite the

decrease in utilization of rescue treatment for aVSP [Macdonald 2011]. A concurrent

randomized trial (CONSCIOUS-3) investigating clazosentan use in aneurysmal SAH

patients treated with endovascular coiling was halted prematurely due to the

CONSCIOUS-2 trial results and provided a similar negative result (n=571) [Macdonald

2012]. These trials were pivotal, even in the absence of scientific data, in reshaping the

direction of the global SAH research community, which subsequently shifted their focus

22

toward investigating the pathophysiology and treatment strategies for EBI and brain

microcirculation [Tso 2014].

Magnesium had also been investigated extensively in SAH with mixed results in phase

II clinical trials due to its putative neuroprotective effects by blocking NMDA (N-methyl-

D-aspartate) receptors and voltage-gated calcium channels [Dorhout Mees 2012,

Macdonald 2014]. In the pivotal phase III trial MASH-2 (Magnesium for Aneurysmal

Subarachnoid Hemorrhage, n=1204), Dorhout Mees and colleagues found that

magnesium treatment started within 4 days of SAH did not significantly affect clinical

outcomes [Dorhout Mees 2012]. In terms of clinical management of SAH regarding

magnesium levels, recommendations include maintaining a normal serum magnesium

level.

Statins also presented a promising picture through their potential neuroprotective effects

via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and mixed results

in phase II clinical trials [Kirkpatrick 2014, Macdonald 2014]. Simvastatin 40mg per day

administered with 4 days of SAH ictus was investigated in the phase III STASH

randomized clinical trial (Simvastatin in Aneurysmal Subarachnoid Hemorrhage,

n=803), but resulted in no improvement in clinical outcomes [Kirkpatrick 2014]. A

smaller randomized clinical trial compared 40mg simvastatin per day with a higher dose

of 80mg of simvastatin administered within 4 days of SAH ictus also resulted in no

improvement in clinical outcomes [Wong 2015]. A meta-analysis of randomized trials

using statins in SAH also confirmed no significant effect in clinical outcomes [Akhigbe

2017].

23

There are number of drugs and treatments that are potential candidates for further

investigation with clinical trials [James 2016]. When considering a drug candidate, one

must ensure that the drug is able to penetrate the BBB and enter the CNS [Pardridge

2017]. There are limitations in taking preclinical studies of drugs and applying to

humans. Some of the reasons why there has been failure of translation of animal drug

studies including methodological flaws in the preclinical studies, inappropriate animal

models with unclear similarity to the human condition, and publication bias of positive

studies [van der Worp 2010]. Systematic reviews and meta-analysis of animal studies

may be helpful. The Stroke Therapy Academic Industry Roundtable (STAIR) updated

guidelines from 2009 provide some direction, although the focus has been

predominantly for translation of ischemic stroke treatments [Fisher 2009].

1.9 Animal models of SAH

There is an extensive history of using various types of animal models to study SAH,

especially to investigate vasospasm, with an excellent review comprehensively covering

the breadth of these models already published [Titova 2009]. Animals used include cats,

dogs, non-human primates, rabbits, rats, and mice. Due to cost and ethical issues,

much of the experimental SAH research has shifted away from large animal models to

rodent models. Mouse models have been especially popular due to the availability of

various transgenic strains to study SAH pathophysiology and the ability to include larger

number of biological replicates to power molecular studies. The three most commonly-

used rodent models of SAH include the prechiasmatic injection model, the endovascular

perforation model, and the cisterna magna injection model, which will all be discussed in

24

more detail below. Although in vitro and ex vivo SAH models may be helpful for

mechanistic studies, they will not be discussed here as overall, they have limited

applicability to the clinical condition.

1.9.1 Prechiasmatic injection rodent model

Prunell and colleagues described the prechiasmatic blood injection model in rats via

needle insertion to the anterior skull base and injection of autologous blood [Prunell

2002]. In this model, the mortality rate was directly dependent on the volume of blood

injection, with 200μL, 250μL, and 300μL injections causing 25%, 50%, and 100%

mortality, respectively [Prunell 2002]. This model was then adapted for mice by Sabri

and colleagues and using 100μL of autologous blood with a 10% mortality [Sabri 2009].

These mice were larger CD-1 strain weighing 33-40g, but for smaller mice weighing 20-

25g, 60 to 80 μL of blood was injected [D’Abbondanza 2016, Tso 2015]. Autologous

blood injection may cause lower circulating blood volume, which would be avoided in

the clinical situation, so blood injection was derived from littermate mice [D’Abbondanza

2016, Tso 2015]. The strengths of this model include the fact that the procedure is quite

straightforward technically with short procedure time and resulting in a blood distribution

at the base of the brain that is highly reproducible. In this mouse model, it is critical to

avoid the major veins at the sagittal midline dorsal surface and avoid major vessels in

the fissure between the olfactory bulb and cerebral cortex, to limit blood loss from the

procedure as a confounding factor. Also, it is possible for blood products to decompress

through the burr hole created, so a small amount of bone wax is applied after the needle

is withdrawn to retain the potentially increased ICP created by the blood injection and

limit blood efflux. The model results in MCA vasospasm at 48h and detectable brain

25

injury on histology [Sabri 2009]. Weaknesses of the model include that the brain injury is

relatively modest compared to other models. Also, occasionally, the injected blood goes

into the ventricular system. However, despite the relative technical ease of this model, it

is still important for a study to have the same person performing the procedures to limit

inter-operator variability.

1.9.2 Endovascular perforation rodent model

The endovascular perforation model in rats was developed independently and

simultaneous by Bederson et al. and Veelken et al. in 1995 [Bederson 1995, Veelken

1995]. In this model, a filament is inserted retrograde into the external carotid artery and

fed anterograde to the internal carotid artery and then allowed to perforate an

intracranial vascular bifurcation, causing SAH, acute increase in ICP and a 50%

mortality [Bederson 1995]. Kamii and colleagues adapted the endovascular perforation

model to the mouse, identifying a 30% mortality rate and peak vasospasm at 3 days

that resolved by 7 days [Kamii 1999]. Strengths of this model include maintaining the

physiological environment of a totally-encased skull and modelling a rise in ICP from a

tear in an intracranial artery that mimics the clinical situation of a ruptured intracranial

aneurysm. Drawbacks of this model include the steep learning curve, the high mortality

and the variability in the neurologic injury which may have implications for gene

expression studies. Having the same operator to perform all SAH inductions is also

important with this model.

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1.9.3 Cisterna magna injection rodent model

In 1985, both Solomon et al. and Delgado et al. described a rat SAH model with blood

injection into the cisterna magna with a mortality rate of 11.5% and vasospasm detected

as early as 10min as well as at 48h [Solomon 1985, Delgado 1985]. Later this model

was adjusted by adding a second cisterna magna blood injection 48h later, resulting in

more robust basilar artery vasospasm and surprisingly low mortality 4-9% [Suzuki

1999]. Lin and colleagues adapted this model in the mouse and found very low mortality

(<5%) and no focal neurological deficit [Lin 2003]. The cisterna magna injection model

strength is the relative simplicity of the procedure and reliable creation of basilar artery

vasospasm to study its mechanism. However, this model has very modest neurologic

injury or mortality, making it a challenging model to use for drug studies using

behavioural outcome scales. Also, the distribution of the blood does not mimic the

clinical situation, in which many ruptured aneurysms occur in the anterior circulation,

spreading subarachnoid blood at the base of the brain, and not as much in the posterior

fossa as in this cisterna magna injection model. Like the other models, it is best to have

the same operator performing the SAH induction for a research investigation.

Each of the three most commonly-used rodent models of SAH have their strengths and

weaknesses. There is no perfect model, and currently none of these models mimic the

human SAH condition with the development of delayed neurologic deficits. Each

research group will develop expertise in one model vs. the other. It is essential that the

same operator performs the SAH experiments within one study.

27

1.10 Gene expression studies in SAH

1.10.1 Early gene expression studies in animal models of SAH

With advances in molecular biology, it became possible to investigate whole genome

expression profiling in animal models of SAH. Onda and colleagues were likely the first

to use limited cDNA library expression arrays to perform gene expression analysis in a

cisterna magna double injection SAH model in canines and characterized the gene

expression profile of vasospastic basilar arteries at day 2 and day 7 [Onda 1999]. The

authors identified upregulation of 5 known inflammatory genes (monocyte chemotactic

protein-1 (Mcp-1), cystatin B, serum amyloid A protein, glycoprotein 130 (Gp130), and

inter-alpha-trypsin inhibitor family heavy chain-related protein (Ihrp)) and upregulation of

3 genes associated with cellular stress (Vascular endothelial growth factor (Vegf), BiP

protein, and growth arrest and DNA damage-inducible protein (Gadd45)) [Onda 1999].

Macdonald and colleagues used an autologous clot placement non-human primate

model of SAH and looked at gene expression changes of ipsilateral middle cerebral

artery (MCA) relative to the contralateral MCA at 3 days, 7 days, and 14 days, using an

array containing 5184 genes [Macdonald 2002]. The authors found significant

upregulation of genes relevant to inflammation, cell proliferation, membrane proteins

and receptors, kinases, phosphatases, and regulation of gene expression [Macdonald

2002]. There was a progressive increase in gene expression of parathyroid hormone

and its associated receptor, which was confirmed at a protein level with western blot

assay [Macdonald 2002].

28

1.10.2 Gene expression studies of cerebral arteries in animal models of

SAH

Subsequent gene expression studies in SAH used whole genome expression

microarrays. Several studies continued to look specifically at gene expression changes

of the large arteries [Vikman 2006, Sasahara 2008, Kikkawa 2014, Kurogi 2015]. Using

a blood injection SAH model in rats, Vikman and colleagues isolated the large cerebral

arteries (basilar artery, circle of Willis arteries, MCA) 24h after SAH induction and found

an upregulation of genes relevant to angiogenesis, extracellular matrix remodeling, and

inflammation [Vikman 2006]. The investigators used RT-PCR (real-time polymerase

chain reaction) to confirm upregulation of the genes Nos2/iNOS (Nitric oxide synthase

2/inducible nitric oxide synthase), Mmp9 (matrix metalloproteinase 9), Mmp13, Cxcl2

(Chemokine C-X-C motif ligand 2), endothelin B receptor (ETB), angiotensin 1 receptor

(AT1), and angiotensin 2 receptor (AT2) [Vikman 2006]. Sasahara and colleagues used

the canine cisterna magna double injection model of SAH and isolated the basilar artery

7d after SAH induction and found an upregulation of genes relevant to cell

communication, host-pathogen interaction, defense-immunity protein activity, and

calcium-related cell signaling [Sasahara 2008]. Kikkawa and colleagues used a rabbit

cisterna magna injection model and isolated the basilar artery at day 0, day 3, day 5,

and day 7 after SAH induction [Kikkawa 2014]. The authors found downregulation of

relaxin/insulin-like family peptide receptor 1 (Rxfp1) at day 3 and upregulation of relaxin

at day 7, with this pathway important for vasodilation [Kikkawa 2014]. This same group

also found an upregulation of tissue inhibitor of metalloproteinase 1 (Timp1) at day 3

and then returned to base-line levels at day 5 and day 7, which was mirrored by

29

decreased elastin, laminin, fibronectin, and collagen type I/III/IV at day 3 and return to

baseline at day 7 [Kurogi 2015]. Limitations of these studies include the inclusion of

multiple cell types including endothelial cells, pericytes, vascular smooth muscles, and

perivascular macrophages, which may dampen the signal of specific genes relevant to

specific cell types. Regarding studies pursuing these findings, most have not been

studied further using molecular methods. There is a large body of work, however, using

drugs to modulate the various putative pathways involved such as oxidative stress,

inflammation, and others.

1.10.3 Gene expression studies of brain tissue in animal models of SAH

Others have looked at gene expression changes in brain tissue [Lee 2012, Zheng 2015,

Peng 2017]. The first whole genome expression profiling study using brain tissue in an

SAH model was performed by Lee and colleagues in 2012, in which a rat cisterna

magna injection model was used and whole brain tissue was isolated 2h from SAH

induction [Lee 2012]. The investigators identified 18 upregulated and 18 downregulated

genes, including upregulation of prostaglandin E synthase (Pges), CD14, andTimp1,

which were confirmed with RT-PCR but the magnitude of changes were modest (1.6-2.6

fold change) [Lee 2012]. Zheng and colleagues looked at both messenger RNA (mRNA)

and long non-coding RNA (lncRNA) expression changes in a rat prechiasmatic injection

model of SAH, specifically looking at the temporal lobe 24h after SAH induction [Zheng

2015]. The investigators found 221 upregulated and 181 downregulated mRNA

transcripts, and 64 upregulated and 144 downregulated lncRNA transcripts, with

pathway analysis revealing many transcripts associated with inflammatory pathways

[Zheng 2015]. Peng and colleagues moved away from older microarray technology and

30

used RNA sequencing (RNAseq) in a mouse endovascular perforation model of SAH,

analyzing gene expression changes from cerebral cortex 24h after SAH induction [Peng

2017]. The investigators identified 441 mRNA and 617 lncRNA transcripts that were

differentially expressed, and many of these transcripts were also associated with

inflammation [Peng 2017]. However, these studies are limited by use of whole brain

tissue with its multitude of cell types (e.g. neuron, astrocyte, oligodendrocyte, microglia,

endothelial cell, etc.) which may cloud the signature gene expression patterns of

individual cell types. Studies are needed in SAH that look at specific cell types and then

interrogating their gene expression patterns.

1.10.4 Gene expression studies involving clinical aneurysm fundus

samples

Pera and colleagues examined aneurysm fundus samples from unruptured and

ruptured aneurysms along with unmatched control arteries (middle meningeal artery) by

microarray analysis [Pera 2010]. The investigators found that compared to controls,

aneurysm fundus samples had differentially-expressed genes relevant to muscle

system (downregulation), cell-adhesion (downregulation), and immune system and

inflammatory response (upregulation) [Pera 2010]. Surprisingly, ruptured aneurysm

fundus had a downregulation of genes relevant to immune and inflammatory responses

compared to unruptured aneurysms [Pera 2010]. Using microarrays, Kurki and

colleagues found that aneurysm fundus samples from ruptured aneurysms compared

with unruptured aneurysms, had upregulation of pathways involved in turbulent blood

flow, leukocyte migration, chemotaxis, extracellular matrix degradation, oxidative stress,

and vascular remodeling [Kurki 2012]. Using the more advanced RNAseq methodology,

31

Kleinloog and colleagues compared gene expression analysis of ruptured and

unruptured aneurysm fundi samples as well as unmatched intracranial artery specimens

derived from epilepsy surgery [Kleinloog 2016]. The authors identified enrichment of

extracellular matrix pathway and immunoglobulin genes in aneurysms compared to

control, and upregulation of immune response and lysosome pathways in ruptured

aneurysm compared with unruptured aneurysms [Kleinloog 2016]. Liu and colleagues

looked at microRNA (miRNA) expression in ruptured aneurysm fundus samples

compared to unmatched superficial temporal artery samples, identifying several

endothelial- and vascular smooth muscle-enriched miRNAs, including those relevant to

proliferation, apoptosis, and phenotype shift [Liu 2014]. Nakaoka and colleagues also

found similar results regarding gene expression patterns in aneurysm fundus samples

[Nakaoka 2014]. Pathways that were enriched included inflammation, extracellular

matrix dysregulation, oxidative stress response, smooth muscle proliferation, and

apoptosis [Liu 2014]. Wang and colleagues looked at gene expression analysis of both

mRNA and lncRNA in both ruptured and unruptured aneurysm fundus samples

compared to matched superficial temporal artery samples [Wang 2017]. Among mRNA

differentially-expressed transcripts, aneurysms showed upregulation of pathways

involved with immune response, inflammatory response, and lysosomes and

downregulation of pathways involved with smooth muscle contraction and cGMP-PKG

(cyclic guanosine monophosphate-dependent protein kinase G) signaling [Wang 2017].

lncRNA and mRNA co-expression analysis identified enrichment of pathways relevant

to vascular smooth muscle contraction and inflammatory response [Wang 2017]. All the

above studies were obtained from open surgical procedures. Cooke and colleagues

used an innovative endovascular approach to obtaining aneurysm samples by isolating

32

endothelial cells from the initial temporary coil, although only less than 500 cells were

obtained with results being quite preliminary to make any firm conclusions [Cooke

2017].

1.10.5 Gene expression studies involving human serum and CSF

samples

In one study, serum samples were collected at day 7 from patients with aneurysmal

SAH and microarray analysis was performed regarding miRNA expression, with

comparison to healthy controls [Su 2015]. The investigators identified miRNAs relevant

to axon guidance and TGFβ (Transforming growth factor beta) signaling to be

upregulated after SAH, although there were no miRNAs that differentiated patients with

and without DCI [Su 2015]. Lai and colleagues also collected serum samples but on day

3 after SAH and performed miRNA microarray analysis, identifying several upregulated

miRNAs relative to healthy controls, several of which were associated with worse

WFNS grade and modified Rankin scale (mRS) clinical outcome at 9 months,

suggesting that serum miRNA may be candidate biomarkers for prognosis [Lai 2017].

Using CSF samples collected at heterogeneous time-points from 1 to 18 days from

patients with SAH and a miRNA assay kit, Stylli and colleagues identified several

miRNAs that were differentially-expressed compared to controls, including a few

miRNAs that could distinguish SAH patients with aVSP from those without aVSP, again

suggesting potential candidate biomarkers [Stylli 2017]. Similarly, Bache and colleagues

interrogated CSF miRNA expression 5 days after SAH and found 2 miRNAs that were

differentially expressed in patients with DCI compared to patients without DCI [Bache

2017]. Kikkawa and colleagues examined serum and CSF samples on day 3 after SAH

33

and performed miRNA microarray analysis compared with controls and found several

differentially-expressed miRNAs which had mirrored responses in both the serum and

CSF [Kikkawa 2017].

Collectively, the gene expression studies in animal models of SAH have provided some

insight into the disease pathophysiology, but more work needs to be done, particularly

in identifying patterns in specific cell types. The clinical samples using serum, CSF, and

aneurysm fundi have also provided some insight into aneurysm formation and SAH

pathophysiology, although none of the findings have resulted in a clear advancement in

SAH clinical care. Noticeably, the samples sizes are all quite small – the largest study

had 44 patients. Because of the significant heterogeneity in clinical practice, it is of

utmost importance to have good controls, ideally within the same patient, which was not

possible in most of the clinical studies mentioned above. Meta-analyses of these results

may also provide further insight.

1.11 Introduction to Blood-Brain Barrier

1.11.1 Definition of Blood-Brain Barrier

The BBB is a term describing the highly-selective interface between the systemic

circulation and the relatively distinct environment of the intracerebral milieu. The cellular

components include endothelial cells, pericytes, astrocytes, and perivascular

macrophages [Daneman 2012]. Each cell type must work together to maintain the

barrier, otherwise the barrier breaks down [Obermeier 2013]. The endothelial cells are

interconnected with specialized tight junctions along with adherens junctions as well as

34

a basement membrane along the basal surface of the cells [Hawkins 2005]. BBB

generally refers to the capillary segment of the vascular tree, devoid of vascular smooth

muscle cells, to be distinguished from conducting arteries, resistance arteries and the

venous system. The relatively selective vascular barrier in the brain is distinct from the

systemic circulation, which generally allows free-flowing exchange of many solutes at

the capillary stage [Aird 2007A]. For the brain to function optimally and neurons to fire

appropriately, a very precise ionic environment needs to be maintained. The BBB

restricts many substances based on lipophilicity, size, and charge. Substances that are

nonpolar with smaller molecular weight (< 500Da) can generally cross the barrier

[Gabathuler 2010]. However, most proteins, especially with molecular weight greater

than 1kDa, do not easily cross the BBB, and thus require transporters or receptors on

the luminal surface to cross into the CNS [Pardridge 2007]. When the BBB fails, it is

possible for neuronal dysfunction and neurological disease to occur.

The BBB is thought develop in utero [Ben-Zvi 2014]. Based on fluorescent tracer

(10kDa) studies in embryonic mice, the BBB appears to develop at embryonic day 15.5,

in which the typical gestational period of a mouse is around 19-21 days [Ben-Zvi 2014].

In controversial experiments, Grontoft injected trypan blue in human fetuses delivered

by cesarean section between 18 weeks and 31 weeks gestation and found an intact

BBB when the injection was performed 10 minutes after delivery but intense blue

staining when the injection was performed 30 minutes after delivery, indicating BBB

breakdown after ischemia and hypoxia [Saunders 2014]. The development of BBB in

utero occurs in a ventral to dorsal direction, and a hindbrain to forebrain direction [Ben-

Zvi 2014]. There are several key signaling pathways during development that induce

35

barrier genesis including the WNT/β-catenin and sonic hedgehog (SHH) pathways

[Liebner 2008, Chow 2015, Tran 2016]. As the brain matures, there appears to be an

increased overall expression of genes relevant to extracellular matrix, cell adhesion,

intercellular junction pathways, suggesting a gradual delayed strengthening of the BBB

[Porte 2016]. Although the BBB is unique, it is now recognized that other vascular beds

can have their own selective barrier, such as the gut-vascular barrier that prevents entry

of various antigens and microbiota from entering the systemic circulation [Spadoni

2015].

It is unclear when the BBB developed from an evolutionary perspective. The worm

Caenorhabditis elegans does not have a BBB and its neurons do not need glia to

survive, whereas sharks and insects have a BBB composed of glial cells [Bundgaard

2008]. The current best guess for the formation of the BBB was during the development

of jawless vertebrates, although present evolutionary theories have suggested that

endothelial-based BBB originated at multiple discrete time-points in evolutionary history

[Bundgaard 2008].

1.11.2 History of BBB

The German physician and scientist Paul Ehrlich was likely the first to demonstrate the

BBB in 1885 when after systemic injection of various water-soluble dyes, he found no

obvious staining of the brain or CSF despite the choroid plexus being heavily stained

[Saunders 2014]. However, at the time, Ehrlich attributed this finding to the brain being

repulsed by the dye itself. In 1909, Ehrlich’s student Edwin Goldmann then performed

experiments confirming the BBB by injecting dye into the subarachnoid space and

36

identifying staining of the brain and CSF but not the systemic circulation [Saunders

2014]. In 1900, the German neurologist Max Lewandowsky is credited with coining the

term “blut-hirn-schranke,” German for “blood-brain barrier,” after observing lack of

staining in the brain after systemic injection of potassium ferrocyanide [Saunders 2014].

The BBB continues to be an area of significant clinical interest and active research.

1.11.3 Components of BBB

As mentioned above, the BBB is composed several components including endothelial

cells, pericytes, and astrocytes as well as the basement membrane. Neuronal

processes may also be present.

1.11.3.1 Endothelial cells

Endothelial cells form the luminal endothelium or internal lining of the blood vessel and

represent the initial barrier contact between the circulating blood and the brain. In

general terms, endothelial cells of the BBB are thought to be specialized and distinct

relative to endothelial cells of the systemic circulation due to: 1) presence of tight

junctions, 2) relative lack of transcytosis or pinocytosis, and 3) specialized transporters

with distinct spatial orientation [Hawkins 2005]. These brain endothelial cells, like

endothelial cells found elsewhere in the body, contain Weibel-Palade bodies, the

ellipsoid organelles containing hemostatic agent von Willebrand factor (VWF), the

leukocyte cell adhesion molecule p-selectin, the tetraspanin CD63, RAB27A, and

ANGPT2 (Angiopoietin-2) [Michaux 2004, Fiedler 2004]. A comprehensive review of

endothelial cell gene expression and its regulation has been published [Minami 2005].

37

The brain endothelium, with its BBB, can be distinguished from the continuous,

fenestrated endothelium of the kidneys, endocrine glands, and GI mucosa, and the

discontinuous, sinusoidal endothelium of the liver [Aird 2007A, Aird 2007B]. There is

significant vascular heterogeneity among different tissue beds. Chi and colleagues were

the first to identify distinct and heterogenous gene expression patterns from cultured

endothelial cells derived from various tissues, although brain endothelial cells were not

included in this analysis [Chi 2003]. Daneman and colleagues demonstrated this

heterogeneous gene expression pattern of freshly-isolated endothelial cells derived

from mouse brain, lung, and liver tissue [Daneman 2010]. Nolan and colleagues

expanded the number of vascular tissue beds examined to 9, including brain, and

subsequently confirmed heterogenous endothelial gene expression patterns [Nolan

2013]. There are several endothelial genes that have been identified as common to

virtually all vascular beds including Kdr (Kinase insert domain receptor, also known as

Vegfr2, vascular endothelial growth factor receptor 2), Flt1 (Fms-related tyrosine kinase

1, also known as Vegfr1, vascular endothelial growth factor receptor 1), Tie1 (Tyrosine

kinase with immunoglobulin-like and epidermal growth factor-like domains 1), Tie2

(Tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2),

Cdh5 (Cadherin 5), and Eng (Endoglin) [Wallgard 2008].

1.11.3.1.1 Tight junctions

Tight junctions are composed of several proteins connecting adjacent brain endothelial

cells in the apical half of their interface [Hawkins 2005]. Unique proteins include the

junctional proteins claudins and occludin as well as cytosolic adaptor proteins such as

zonula occludens 1 and 2 (ZO-1, ZO-2) [Hawkins 2005]. The transmembrane proteins

38

claudin-5 and claudin-12 are the main claudins involved in brain endothelial tight

junctions [Tsukita 2003]. Surprisingly, claudin-5 knockout mice had morphologically

normal tight junctions without spontaneous bleeding but caused increased permeability

of small molecules less than 800Da but not larger molecules [Tsukita 2003]. Occludin

provides additional tight junction support, although this protein is not essential as mouse

knockouts of this gene show no BBB impairment [Saitou 2000]. ZO-1 and ZO-2 anchor

the tight junctions to the cytoskeleton. These tight junctions are more apical than the

more widespread adherens junctions, composed of VE-cadherin (Vascular endothelial

cadherin) transmembrane proteins linked together near the basolateral surface of

endothelial cells away from the lumen, and linked to β-catenin and the actin

cytoskeleton on the cytoplasmic side [Lampugnani 2010]. These adherens junctions

contribute to vascular integrity and function in intracellular signaling [Lampugnani 2010].

The interendothelial junctions, including both tight junctions and adherens junctions, can

limit paracellular flow of solutes, especially polar substances [Hawkins 2005].

1.11.3.1.2 Relative lack of transcytosis

Compared to systemic endothelial cells, brain endothelial cells have a relative lack of

caveolae or transport vesicles at the apical surface and a general decrease in the rate

of transcytosis [Tuma 2003]. For example, brain endothelial cells may have 2 vesicles

identified per μm2 whereas lung endothelial cells may have up to 200 vesicles per μm2

[Tuma 2003, Ben-Zvi 2014]. The orphan receptor MFSD2A (Major facilitator superfamily

domain containing 2a) is thought to be a key negative regulator of brain endothelial

transcytosis and its expression is dependent, in part, on the presence of pericytes [Ben-

Zvi 2014]. The authors found that Mfsd2a knockout mice had a dramatic increase in

39

brain endothelial cell transcytosis but no change in vascular organization and normal

tight junction morphology [Ben-Zvi 2014]. MFSD2A was also found to be a key uptake

transporter of the essential fatty acid docosahexaenoic acid (DHA), and its knockout in

mice resulted in neuronal loss, cognitive deficits, anxiety, and microcephaly [Nguyen

2014].

1.11.3.1.3 Specialized transporters

The brain endothelial cells have specialized transporters lining the apical (or luminal)

and basal surfaces. The apical side have many transporters called solute carriers (SLC)

that are unique to the brain and are important for intake of specific nutrients [Geier

2013]. Glucose transporter 1 (GLUT1, also called SLC2A1) is a specialized brain

endothelial cell transporter important for uptake of glucose from the circulating blood

[Daneman 2012]. Surprisingly, GLUT1, like MFSD2A mentioned earlier, aside from

being a key specialized transporter is also important for barrier formation [Zhao 2014].

There are uptake transporters for most major essential nutrients including for many

amino acids, monocarboxylates (lactate, pyruvate), zinc, transferrin, and various organic

anions [Daneman 2012].

The apical side also have several exporters such as p-glycoprotein (PGP, also called

MDR1A (Multi-drug resistance protein 1a), and ABCB1 (ATP-binding cassette b1)),

which is known to efflux many substances from the brain into the circulation [Daneman

2012]. Such efflux transporters have implications for drug development in CNS diseases

as drugs that are actively transported out of the brain may affect the drug’s efficacy.

LRP1 (low-density lipoprotein receptor-related protein-1) is an efflux transporter for Aβ

40

(amyloid beta), and its dysfunction has been linked to Alzheimer’s disease [Storck

2015].

Chun and colleagues used mass spectrometry of freshly isolated mouse brain

microvessels to identify a list of highly-expressed endothelial transporters including

GLUT1 and ABCB1, to support earlier gene expression studies [Chun 2011]. In a similar

study using human brain microvessels, Geier and colleagues identified several unique

ABC and SLC transporters, distinct from kidney and liver endothelial cells [Geier 2013].

Zuchero and colleagues used mouse brain endothelial cells (CD45-CD31+, [Cluster of

differentiation 45, Cluster of differentiation 31]) isolated with fluorescent antibodies and

FACS (fluorescence-activated cell sorting) and performed mass spectrometry to confirm

increased protein expression of several BBB transporters including GLUT1 [Zuchero

2016].

1.11.3.1.4 Basement membrane

The basal lamina or basement membrane contains extracellular matrix proteins

including collagen type IV, laminin, fibronectin, heparan sulfate, and chondroitin sulfate

proteoglycans [Blanchette 2015]. Endothelial cells, pericytes and astrocyte end feet

have adherent connections to the extracellular matrix composing the basement

membrane [Hawkins 2005]. Extracellular matrix proteins are not static and are an

underappreciated regulator of BBB maintenance [Blanchette 2015].

41

1.11.3.2 Pericytes

Pericytes partially line the abluminal side of endothelial cells and are partially embedded

in the basement membrane [Sweeney 2016]. They are essential for BBB integrity,

hemodynamic regulation, angiogenesis, neuroinflammation, toxic metabolite clearance,

and have potential stem cell activity [Sweeney 2016]. Pericytes help limit BBB disruption

by tonic inhibition of transcytosis in endothelial cells and by polarization of astrocytic

end feet [Arrmulik 2010]. In fact, during neurovascular coupling, pericytes are thought to

mediate the blood flow increase by dilation of capillaries, with pericyte loss resulting in

capillary constriction [Hall 2014]. In regions of pericyte injury, the capillary diameter can

increase, with neighbouring pericytes attempting to replace regions lacking pericyte

coverage [Berthiaume 2018].

1.11.3.3 Astrocytes

Astrocytes do more than just cover the clear majority of the endothelial abluminal lining

at the brain capillary level with their end feet [Hawkins 2005]. Astrocytes have been

shown to secrete SHH and activate Hedgehog receptors on brain endothelial cells,

which is important in BBB formation during development but also maintaining immune

system quiescence by suppressing leukocyte adhesion and migration [Alvarez 2011].

Astrocytes participate in neurovascular coupling but may also augment blood flow via

calcium signaling independent of neuronal activity [Iadecola 2007, Takano 2006].

42

1.11.3.4 Neurons

Traditionally, neurons are described as a cellular component of the neurovascular unit

(NVU), in addition to endothelial cells, astrocytes, as well as pericytes and vascular

smooth muscle cells [Koide 2012]. The NVU can dynamically alter local blood flow

based on neuronal activity and metabolic needs [Koide 2012]. However, there is also

evidence of neurons contributing neuronal processes directly on intraparenchymal

vessels with potential role in maintenance of the BBB [Tso 2014].

1.12 BBB dysfunction in neurological disease

BBB is important in maintaining relative homeostasis of the brain interstitial fluid. A

dysfunctional BBB may initiate neurological disease or can be caused by neurological

disease or both [Neuwelt 2011]. With inflammation, the brain endothelial cells undergo

temporal and spatial remodeling of junctional proteins [Reglero-Real 2016]. BBB

disruption may be focal or global and can have varying degrees of disruption from

allowing passage of small molecules to large proteins such as immunoglobulins. We

had earlier outlined the presence of BBB disruption in SAH. In addition, BBB disruption

has been associated with other neurological diseases including ischemic stroke, ICH,

traumatic brain injury (TBI), brain tumours, epilepsy, multiple sclerosis,

neurodegenerative diseases such as Alzheimer’s disease, CNS infections such as

intracerebral abscess, metabolic diseases such as diabetic ketoacidosis, and congenital

diseases such as Alexander’s disease [Obermeier 2013].

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1.13 BBB permeability assays

There are many ways to assess BBB permeability with an excellent review by Kassner

and Merali covering different experimental and clinical methods [Kassner 2015].

1.13.1 Blood protein measurement

Blood proteins such as albumin and immunoglobulins can be measured in the brain

parenchyma using immunohistochemistry and immunofluorescence [Schmidt-Kastner

1993]. This is a straightforward experimental approach in which the animal model must

have the intravascular system perfused to eliminate circulating blood proteins.

Limitations include that the blood protein markers signaling extravasation are quite large

and so this approach may miss subtler changes in BBB disruption. Also, this approach

detects the cumulative leakage of blood proteins into the brain parenchyma and not

from a specific time-point.

1.13.2 Intravascular exogenous dye measurement

Exogenous dyes and tracers can be administered into the vascular system of animal

models and the leakage of dye or tracer can be measured in the brain. Evans blue dye,

a 1kDa azo dye which can reversibly bind to albumin, is a commonly used BBB assay,

also referred to as the Miles assay [Radu 2013]. Evans Blue dye extravasation into the

brain can be measured by direct visualization (bluish discolouration of the brain),

spectrophotometer methods of homogenized brain tissue, and optical imaging [Kassner

2015]. Other options include using fluorescent dyes of various molecular sizes and

horseradish peroxidase (HRP) injections to assess BBB disruption, which can result in

44

variable findings depending on the specific size of the tracer [Kassner 2015].

Quantitative autoradiography using radioactive tracers can provide real-time kinetics of

BBB leakage but are difficult to execute [Knight 2005].

1.13.3 In vivo assays

Real-time imaging of brain vasculature in response to intravascular fluorescent dyes

can be performed using 2-photon microscopy, although the imaging is limited to

relatively superficial cortical vessels and the BBB dynamics may be affected by the

anesthetic process [Kassner 2015]. Creation of a closed cranial window allow multiple

time-point assessments within the same animal in vivo [Schoknecht 2014].

1.13.4 Clinical detection of BBB disruption with MRI and CT

Contrast-enhanced MRI can be used to determine if there is BBB impairment by

leakage of gadolinium-based dyes in the brain [Montagne 2016]. There are many

different types of gadolinium-based dyes available including the linear-based dyes

(Gadobenate dimeglumine, gadopentetate dimeglumine, gadodiamide) and the

macrocyclic dyes (Gadoterate meglumine, gadoteridol), which are less likely to deposit

in brain tissue as seen in future MRI scans [Montagne 2016]. Dynamic contrast-

enhanced MRI allow for creation of a pharmacokinetic profile of BBB disruption

[Kassner 2015]. A similar pharmacokinetic profile can be performed with CTP [Kassner

2015]. Moreover, MR and CT techniques to assess BBB disruption can be applied to

both animal models and the clinical situation, important for translational studies.

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1.14 Brain endothelial cell isolation techniques

Brain endothelial cell isolation techniques have evolved with time. Earlier techniques

involved optimizing isolation of whole brain microvessels [Siakatos 1969]. Later

techniques included laser capture microdissection (LCM) and sorting based on

fluorescent antibodies or magnetic beads or the use of transgenic mice with fluorescent

endothelial cells [Daneman 2010].

One of the challenges in isolating endothelial cells from brain tissue is the large amount

of myelin debris created during brain tissue dissociation. Some studies have avoided

adult animal models, and instead preferred neonatal models, due to the relative lack of

myelination present at birth [Vasudevan 2008]. From personal experience, myelin debris

can cause an increase in the cell sort time. While this may not be a significant issue in

identifying gene expression in naïve mice, for animal models of disease, a prolonged

sort time may cause more stress to the cells than the actual neurological disease

model.

1.14.1 Brain microvessel isolation

Brain microvessel isolation was likely first performed by Siakatos and colleagues in

1969 using density gradient centrifugation and glass bead columns in human and

bovine brain tissue [Siakatos 1969]. Goldstein in 1975, then used various sized meshes

and glass beads to obtain rat brain microvessels [Goldstein 1975]. This technique was

then refined and advanced by Pardridge with further modifications made by Chun and

colleagues [Pardridge 1985, Chun 2011]. Enerson and Drewses isolated rat brain

46

microvessels by passing brain homogenates through 350μm mesh and then 110μm

mesh twice, followed by centrifugation [Enerson 2006]. After resuspending the pellets,

and centrifugation in a swinging bucket rotor, the pellets were resuspended and

microvessels were obtained on a 20μm mesh [Enerson 2006]. Yousif and colleagues

used various methods to isolate rat brain microvessels, employing combinations of

homogenization, various size meshes (20μm vs. 100μm) and glass beads, but found

less purity than what is typically achieved by LCM [Yousif 2007]. The concern with this

technique is the unavoidable presence of contaminating parenchymal cells as well as

adherent vascular smooth muscle cells and pericytes to endothelial cells within the

vessel wall.

1.14.2 Laser capture microdissection

LCM is another method of isolating endothelial cells from brain tissue, using a

microscope with a laser to remove specific regions of tissue at the cellular level

[Kinnecom 2005]. Although cell contamination is possible, the procedure is also

extremely tedious, and the quality and quantity of RNA extracted may not justify whole

genome expression analysis [Macdonald 2010]. Macdonald and colleagues, using the

LCM technique, isolated brain capillary endothelial cells, distinct from brain venule

endothelial cells. Using specific primers, the investigators found increased solute

transporter gene expression in the former and increased inflammatory gene expression

in the latter [Macdonald 2010]. Sufficient quantities of cells are difficult to obtain with this

technique, necessitating pooling of samples [Kinnecom 2005].

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1.14.3 Transgenic mice with fluorescent endothelial cells

Daneman and colleagues were the first to freshly isolate relatively pure and viable brain

endothelial cells [Daneman 2010]. This group used the Tie2-GFP mice that have green

fluorescent protein (GFP) driven by the Tie2 promoter, a receptor tyrosine kinase that is

relatively specific for endothelial cells although may also be found in pericytes and

hematopoietic stem cells [Motoike 2000]. Using two rounds of FACS to isolate Tie2-

GFP+ cells and deplete cells expressing platelet derived growth factor receptor beta

(PDGFRβ, a pericyte marker) using a fluorescent antibody, the authors isolated

endothelial cells (Tie2+PDGFRβ-) with high purity (~ 99%) [Daneman 2010].

Vasudevan and colleagues had previously used the Tie2-GFP mice to freshly isolate

endothelial cells for RNA extraction but did not perform a pericyte depletion step or have

the explicit quality control data published [Vasudevan 2008]. However, the same group

used the Tie2-GFP mice to isolate GFP+ cells while also simultaneously using

fluorescent antibodies for CD31 (also known as a PECAM1, platelet endothelial cell

adhesion molecule 1) to isolate endothelial cells, although the exact purity was not

described [Won 2013]. CD31 is an endothelial cell marker, but also may be expressed

on platelets and leukocytes.

1.14.4 Fluorescent antibodies and cell sorting

Nolan and colleagues used double intravital labelling of endothelial cells with

fluorescent antibodies followed by sorting of endothelial cells [Nolan 2013]. In their

protocol, they retro-orbitally injected anesthetized mice with fluorescent antibodies to

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CD34 (Cluster of differentiation 34) and VE-cadherin, 8min prior to sacrifice. CD34 is

expressed in endothelial cells and hematopoietic stem cells. The authors then

dissociated brain tissue into a single cell suspension with collagenase, dispase and

DNase, followed by sorting to select endothelial cells with depletion of hematopoietic

and erythroid cells with CD45 and TER119 antibodies, and finally straining through

40μm filter [Nolan 2013]. CD45 is a protein tyrosine phosphatase, receptor type C, that

is commonly expressed on leukocytes. The authors achieved cell purity routinely

greater than 95%.

Vasilache and colleagues used a model of inflammation (mouse with intraperitoneal

injection of LPS, lipopolysaccharide), and isolated endothelial cells (CD45-CD31+),

pericytes (CD45-CD31+PDGFRβ+), and perivascular macrophages (CD45+CD206+)

using two rounds of sorting with fluorescent antibodies without a myelin depletion step

[Vasilache 2015]. CD206 (Cluster of differentiation 206) is a mannose receptor

expressed on the surface of macrophages. Although explicit purity and viability numbers

were not described, the representative flow cytometry graphs showed relatively good

isolation, although only about 5000 endothelial cells were isolated per mouse [Vasilache

2015]. The authors found that the dissociation and FACS procedure did not appear to

cause activation of endothelial cells or pericytes but did cause activation of perivascular

macrophages [Vasilache 2015].

Wylot and colleagues investigated various techniques of brain tissue dissociation and

found that the greatest cell viability (~ 80%) was obtained using controlled physical

dissociation with the rotatory GentleMACS system (Miltenyi Biotec, Auburn, CA)

49

compared with repeated pipetting or flushing through a nylon sieve [Wylot 2015]. This

same group isolated mouse brain endothelial cells with myelin depletion using magnetic

beads, followed by CD11B (Cluster of differentiation 11B, also called ITGAM, integrin α

M) magnetic depletion of microglia, and using fluorescent antibodies to select CD45-

SCA1+ endothelial cells with greater than 80% purity [Wylot 2015]. SCA1 (Stem cells

antigen 1) is a common marker for hematopoietic stem cells. Guez-Barber isolated rat

brain endothelial cells with fluorescent antibodies against the transferrin receptor (TfR),

a receptor enriched in brain endothelial cells [Guez-Barber 2012].

Overall, FACS is a relatively versatile technique, in which fluorescent antibodies to

cellular surface markers of interest can be used to isolate relatively pure populations of

cell with as many rounds of sorting as required. Most of the studies focused on isolating

normal brain endothelial cells with FACS. Currently, no study has pursued isolating

brain endothelial cells in an SAH animal model.

1.14.5 Magnetic antibodies

Wu and colleagues were one of the first to isolate brain endothelial cells using CD31

antibodies and secondary magnetic beads followed by magnetic sorting [Wu 2003].

Ohtsuki and colleagues used CD31 magnetic beads to isolate brain endothelial cells

with a mean purity of 86% and viability of 86% [Ohtsuki 2007, Ohtsuki 2008]. Guo and

colleagues used CD31 magnetic beads (Dynabeads) and a magnetic separator to

isolate brain endothelial cells [Guo 2012]. Quality control was not available here in

terms of purity or viability, although indirect evidence from gene expression profiles

showed lack of contaminating cells [Guo 2012]. Kumar and colleagues also used CD31

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magnetic antibodies for isolating brain endothelial cells for the purposes of growing the

cells in culture [Kumar 2014].

Magnetic sorting can be a quicker procedure than FACS and may be a less stressful

procedure for the cell itself, which may be important for studies of gene expression.

1.15 Brain endothelial cell whole genome expression studies

Earlier, we had discussed all the whole genome expression studies performed in SAH

animal models. Currently, there is no such study looking specifically at endothelial cells

in SAH. Here we summarize the studies using whole genome expression profiling of

freshly isolated endothelial cells in naïve mice as well as animal models of other

neurological diseases. Much of the pioneering work of identifying gene expression

patterns in brain endothelial cells were based on older limited suppression subtractive

hybridization techniques and will not be discussed further [Li 2001, Li 2002, Shusta

2002].

Gene expression studies rely heavily on the immediate environment and context from

which the cells were isolated. It is difficult to make definitive conclusions based on gene

expression studies of cultured cells. For example, when compared to freshly isolated

brain endothelial cells, immortalized human endothelial cells in culture have reduced

expression of typical BBB transporters such as GLUT1 and PGP and reduced

expression of tight junction components CLAUDIN-5 and OCCLUDIN [Urich 2012].

Calabria and colleagues compared freshly-isolated rat brain microvessels and cultured

brain endothelial cells and found a downregulation of prototypical BBB genes in cultured

51

cells including GLUT1, MDR1A, and TfR [Calabria 2008]. These cultured cells have lost

some of their expression pattern probably due to removal from their native environment.

There are many in vitro BBB models that have been described, which have been

extensively reviewed [He 2014]. Progress in refining the in vitro models include co-

culture models combining astrocytes with endothelial cells and preferable use, if

possible, of primary, human brain endothelial cells. This emphasizes the need for gene

expression data sets derived from freshly-isolated endothelial cells when possible.

Although ideal for mechanistic studies, such models can also have significant

limitations, and are not discussed below.

In earlier studies, Enerson and Drewes isolated rat brain microvessels described

previously and used the older serial analysis of gene expression (SAGE) technique to

characterize the BBB transcriptome and confirmed the presence of prototypical BBB

transporters and tight junction genes [Enerson 2006]. Daneman and colleagues used

their freshly isolated brain endothelial cells described earlier and then performed whole

genome expression profiling of these cells, with comparison to gene expression profiles

of endothelial cells freshly isolated from lung and liver [Daneman 2010]. This study

provided the first whole genome expression data set of brain endothelial cells

[Daneman 2010]. The same group then used this technique to isolate endothelial cells

and other specific brain cell types including neurons, astrocytes, oligodendrocytes,

microglia, and pericytes, and then generated whole genome expression profiles using

RNAseq in naïve mice [Zhang 2014].

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Nolan and colleagues used intravital labeling of endothelial cells described earlier and

then performed whole genome expression profiling of these cells, comparing 9 different

tissue beds including brain, liver, bone marrow, kidney, heart, lung, muscle, spleen, and

testis [Nolan 2013]. From their dataset, they identified Wnt5a as an angiocrine factor

unique to brain endothelial cells and Prominin1 as a surface marker specific to brain

and testis endothelial cells [Nolan 2013]. Among transcription factors, the brain

endothelial cells had a decrease in expression of Pparγ (Peroxisome proliferator-

activated receptor gamma), a nuclear receptor involved in glucose and lipid metabolism

and a target of thiazolidinediones such as the diabetes drug pioglitazone [Tyagi 2011].

Guo and colleagues isolated mouse brain endothelial cells with CD31 magnetic beads

and compared the gene expression profiles to endothelial cells derived from heart and

kidney glomeruli [Guo 2012]. Whole genome expression profiling revealed functionally

active networks linked to angiogenesis, WNT signaling pathway, leukocyte

transmigration, plasma membrane, transporter activity, and cell adhesion [Guo 2012].

Using naïve juvenile mouse somatosensory cortex and hippocampus, Zeisel and

colleagues performed single cell RNAseq to characterize nine major classes of cells,

including endothelial cells, and 47 subclasses of cells within the brain [Zeisel 2015]. In

this study, the most specific gene expression marker of endothelial cells was Ly6c1

(Lymphocyte antigen 6C1), although it is known to be expressed in peripheral

monocytes [Zeisel 2015]. There were distinct clusters of transcription factors suggesting

endothelial cell origin including Prdm1 (PR domain zinc finger protein 1), Zic3 (Zinc

finger protein 3), Ocln (Occludin, a tight junction component but also transcription co-

factor), Bcl6b (B-cell CLL/lymphoma 6 member B protein), Meox1 (Homeobox protein

MOX-1), Nmi (N-myc-interactor), Sox7, and Foxl2 (Forkhead box protein L2) [Zeisel

53

2015]. Mural cells, including pericytes and vascular smooth muscle cells, were a

separate category and were found to express Pdgfrβ [Zeisel 2015]. Tam and colleagues

isolated mouse brain endothelial cells (CD45-CD31+) using fluorescent antibodies and

FACS and looked at the transcriptome via microarray studies of embryonic, early

postnatal and adult mice [Tam 2012]. The authors found consistent expression of Glut1

over these 3 periods but progressive increased expression of Pgp with aging [Tam

2012].

Hupe and colleagues used an alternative technique to obtain mouse brain endothelial

cell RNA from polysomes called translating ribosome affinity purification (TRAP) without

the need to isolate brain endothelial cells [Hupe 2017]. The TRAP technique was driven

by the endothelial-specific Cdh5 promoter. Translationally-active RNA from embryonic

mouse brain endothelial cells (E11.5 to E17.5) was interrogated with RNAseq, which

resulted in identification of key transcription factors for BBB maturation downstream of

Wnt/β-catenin signaling pathway [Hupe 2017].

Some studies have looked at expression of specific genes in brain endothelial cells

derived from various animal models but have not provided the quality control data to

ensure relatively pure and viable endothelial cells have indeed been isolated. For

example, in an inducible mouse model of cavernous malformation, the investigators

isolated brain endothelial cells by a combination of enzymatic dissociation, straining

through 70 μm filter and then using CD31+ magnetic beads to positively select for

endothelial cells, but did not provide quality control data to confirm the identity of these

cells [Maddaluno 2013]. Ben-Zvi and colleagues used a FACS protocol to isolate brain

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endothelial cells from Tie2-GFP mouse embryos at 13.5 days gestation and perform

whole genome expression profiling, but also did not provide quality control data to

confirm the identity of these cells [Ben-Zvi 2014]. Using only a focused expression array

of 48 genes, Vasilache and colleagues found that systemic LPS caused several

significant gene expression changes in brain endothelial cells including downregulation

of tight junction Cldn5 (Claudin 5), induction of cytokine signaling receptors (Il1r1

[Interleukin 1 receptor 1], Il6ra [Interleukin 6 receptor, alpha subunit], Tnfrsf1a [Tumour

necrosis factor receptor 1], Tnfrsf1b [Tumour necrosis factor receptor 2]), as well as

upregulation of PGE2 (Prostaglandin E2) synthesis (COX-2 [Cyclooxygenase-2],

mPGES-1 [Microsomal prostaglandin E synthase 1]) [Vasilache 2015].

Fernandez-Lopez and colleagues used fluorescent antibody-based selection of brain

endothelial cells in a rat transient MCAO (Middle cerebral artery occlusion) model

comparing adults with neonates at 24h and found surprisingly very small overlap in

genes differentially-expressed, with the adult model having more striking gene

expression changes [Fernandez-Lopez 2012]. They found an increased endothelial

expression of Angpt2 in adult but not in neonate rats [Fernandez-Lopez 2012]. Also, for

both adult and neonate rats, MCAO caused an increase in endothelial expression of cell

adhesion molecules but unsynchronized and limited changes in tight junction genes

[Fernandez-Lopez 2012]. A limitation of this study includes the lack of information

regarding endothelial viability and purity after isolation. Hoffmann and colleagues used a

transient MCAO mouse model and isolated brain endothelial cells 4 days later using

magnetic bead selection for CD31+ cells [Hoffmann 2015]. The authors describe some

contamination and therefore discuss the gene expression changes in these cells as

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referring to the ‘neurovascular niche’ [Hoffmann 2015]. They find that induced

endothelial genetic ablation of Stat3 (Signal transducer and activator of transcription 3)

resulted in a gene expression signature of reduced angiogenesis and reduced axonal

growth, reflected by increased infarct size at 4 weeks compared to controls [Hoffman

2015].

Currently, there is a lack of studies looking at gene expression changes in the individual

cellular components from an SAH animal model. Performing whole genome expression

profiling in specific cell populations may provide more opportunities to better under the

disease pathophysiology and identify potential therapeutic targets.

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Chapter 2

Research Questions and Hypotheses

57

Research Questions and Hypotheses

This thesis project goal was to identify new therapeutics in the treatment for SAH. This

was accomplished by 2 methods. In the first approach, we serendipitously identified

valproic acid (VPA) as a therapeutic agent for EBI after SAH, based on perusal of drugs

investigated in pre-clinical models of neurological diseases published in the literature.

The thought process here was that if VPA was beneficial in other animal models of

neurological diseases, perhaps the drug may also be successful in a SAH animal

model. Also, VPA was found to affect the gene expression patterns of human umbilical

vein endothelial cells (HUVECs), and so there is potential for this treatment to affect the

BBB (Unpublished data, Marsden lab).

In the second approach, a specific aspect of SAH pathophysiology was examined,

namely BBB disruption. Using genome-wide expression profiling of brain endothelial

cells derived from a mouse model of SAH, potential therapeutic targets were identified

and validated. As proof of principle, a specific target, COX-2, was chosen, and a

selective COX-2 inhibitor, celecoxib, was investigated as a treatment option using the

same SAH animal model. In both approaches, the drugs in question were repurposed,

potentially cutting down the time from target identification to standard of clinical practice.

The following hypotheses were investigated in the subsequent chapters of this thesis:

58

2.1 Hypothesis 1: Valproic acid treatment in a mouse model of

SAH results in improved neurological outcomes

2.2 Hypothesis 2: Valproic acid treatment in patients with SAH

results in improved clinical outcomes

2.3 Hypothesis 3: Pure and viable brain endothelial cells can be

efficiently isolated from mouse brain tissue

2.4 Hypothesis 4: Whole genome expression profiling of freshly

isolated endothelial cells can identify new potential

therapeutic targets in SAH

2.5 Hypothesis 5: Celecoxib treatment in a mouse model of SAH

results in improved neurological outcomes

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Chapter 3

Valproic Acid Treatment in Experimental and Clinical SAH

This chapter was modified from the following:

Tso MK, Ai J, Lee CK, Wan H, Nie T, Lass E, D’Abbondanza J, Marsden PA,

Macdonald RL. Valproic acid in experimental and clinical SAH: A potential therapeutic

agent. (In Preparation).

Tso MK, Lass E, Ai J, Macdonald RL. Valproic acid treatment after experimental

subarachnoid hemorrhage. Acta Neurochir Suppl. 2015; 120:81-85.

60

Valproic Acid Treatment in Experimental and Clinical

SAH

3.1 Background

The cost of developing a new drug is by most standards, enormous. Although the exact

cost of developing a new drug is unknown to the public, some estimates range from $92

million US to $883.6 million US [Morgan 2011]. Translational research takes a long-

time. For FDA (Food and Drug Administration) drugs or biologics approved between

2010-2014, the median time interval between pre-clinical research initiation and

initiation of clinical trials was 29 years [McNamee 2017]. The median time interval

between pre-clinical research initiation and FDA approval was 36 years [McNamee

2017]. It is estimated that only 10% of new drugs undergoing clinical trials end up being

successful and FDA-approved [Akhondzadeh 2016]. The time-line from initiation of

clinical trials to approval is estimated to be 10-13 years and is also the costliest aspect

of drug development [Akhondzadeh 2016]. Drug repurposing is becoming an attractive

option, as usually all the required studies have been completed up to a phase I or II

clinical trial, which potentially reduces the number of years required for drug approval

[Ashburn 2004].

VPA is a candidate drug that may be repurposed for the treatment of SAH patients. VPA

is a carboxylic acid that can cross the BBB and has a known safety profile in humans

[Gram 1985, Kang 1990]. VPA is commonly used clinically as an antiepileptic drug

(AED) for seizure control and as a mood stabilizer for bipolar affective disorder

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[Chateauvieux 2010]. VPA has multiple mechanisms of action, including inhibiting the

metabolism of the inhibitory neurotransmitter GABA (γ-aminobutyric acid), limiting

NMDA receptor-induced excitotoxicity, and directly altering cellular gene expression via

inhibition of histone deacetylases (HDACs) resulting in increased histone acetylation

[Chen 2014, Chuang 2005, Costa 2006].

Over the last decade, several studies have demonstrated neuroprotective effects of

VPA in various animal models of TBI, ischemic stroke and ICH [Dash 2010, Kim 2007,

Ren 2004, Sinn 2007]. We had previously published our initial findings that VPA

treatment may improve neurologic and histologic outcomes in a mouse model of SAH

[Tso 2015]. Others have since found neuroprotective effects of VPA treatment in other

models of SAH [Chang 2015, Ying 2016, Hamming 2017]. The goals of this study were

to explore the mechanism of neuroprotection of VPA in experimental SAH and then

utilize clinical trial data to determine the clinical outcomes of SAH patients who were

treated with VPA while in hospital.

3.2 Methods

This study was approved by the Animal Care Committee of St. Michael’s Hospital,

Toronto, ON.

3.2.1 Prechiasmatic injection SAH mouse model

The prechiasmatic injection SAH mouse model was used and has been described

previously [Sabri 2009]. Briefly, male C57BL/6J mice (Jackson Laboratory, Bar Harbor,

ME), 8-10 weeks old and weight 20-25g, were anesthetized with inhaled isoflurane and

62

placed prone with the head fixed in a stereotactic apparatus. Although C57BL/6J mice

have relatively modest brain injury after SAH, we chose this strain due to potential

availability of various transgenic strains with a C57BL/6J background [D’Abbondanza

2016]. A midline incision was created over the dorsal skull. CBF was monitored with

laser flow doppler (Transonics Systems, New York City, NY) placed on the skull 8min

prior to and 10min after SAH induction. Body temperature was maintained with a

homeothermic heating pad (Harvard Apparatus, Holliston, MA) placed under the mouse.

A burr hole was created 1mm to the left of midline and 1mm in front of the fissure

between the olfactory bulb and cerebral hemisphere. Using a 27G spinal needle

inserted at a 37.5o angle from the vertical plane, 80μL of donor (litter-mate) blood was

injected into the prechiasmatic cistern. Sham mice had needle placement without

injection of blood. Skin was re-approximated with interrupted silk sutures. All mice

received subcutaneous injections of buprenorphine (Sigma-Aldrich, St Louis, MO),

0.1mg/kg, at time of SAH induction or sham procedure, and then every 12h for 48h.

Mice were housed in an incubator warmed to 28oC for 48h post-procedure, and then

subsequently transferred back to regular cage rack. Mice received water and standard

chow ad libitum, with 2-5 mice per cage, and standard 12h/12h light/dark cycles.

3.2.2 VPA treatment

Mice were randomly allocated to 4 experimental groups: Sham+vehicle (n=30),

Sham+VPA (n=21), SAH+vehicle (n=35), and SAH+VPA (n=34). Mice were treated, in a

blinded manner, with 400mg/kg of VPA (Sigma-Aldrich, St. Louis, MO) or normal saline

(vehicle) via intraperitoneal (IP) injection within 30min post-procedure and every 12h

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thereafter for 48h, and then daily up to 7 days. The mouse dose was equivalent to the

high-end clinical human dose based on body surface area scaling [Reagan-Shaw 2007].

3.2.3 Neurological assessment

Mice were assessed, blinded to experimental group, at 24h and 48h with the modified

Garcia Score (MGS), an acute neurobehavioural assessment that consists of 6 domains

– spontaneous activity, spontaneous movement of all 4 limbs, forepaw outstretching,

climbing, body proprioception, and response to vibrissae touch [Sherchan 2011]. The

maximum score is 18, indicative of normal mouse behaviour.

Spatial memory was assessed by Morris Water Maze with modifications on days 3-7

[Vorhees 2006]. Briefly, the water bath consisted of a large 2.2m diameter tub filled with

water heated to 22oC and coloured white with dissolved tempera powder paint. Distinct

patterns were placed on the wall on the north, east, south, and west positions. A 12cm

by 12cm platform was in the middle of the southeast quadrant and submerged 1cm

below water level. The swimming paths of mice were tracked with a video camera

mounted to the ceiling. On day 3 (Cue Trial) after SAH induction or sham procedure,

mice underwent training with a cued platform, consisting of a 10cm tall flag attached to

the platform located in the southeast quadrant. Mice were placed in each quadrant

(northwest, northeast, southeast, southwest) twice and allowed to swim for 1min. If the

mouse failed to reach the platform, the mouse was guided to the platform and allowed

to rest for 15s. On days 4-6 (Acquisition Trials), mice underwent acquisition training,

consisting of 4 consecutive trials (once in each quadrant) per day with the cue removed

and the platform submerged. On day 7 (Probe Trial), the platform was removed, and the

64

mouse was allowed to swim freely for 1min after being placed in the northwest

quadrant. Measurements included escape latency (time to reach the platform), swim

speed, and percent time spent in the target quadrant. Thigmotaxis was defined as

swimming with 10cm from the wall.

The open field test is a neurobehavioural test that assesses emotionality, anxiety, and

fear [Seibenhener 2015]. On day 7, mice were placed in a large open cage (72cm x

44cm) with the floor marked by a grid of 10cm squares. A video camera mounted to the

ceiling tracked the mouse movements. Measurements included episodes of defecation

and urination, rearing (positioned on hindfeet as an indicator for curiosity), sniffing (neck

extension as an indicator of fear), grooming, centre crossing, grid-line crossing,

immobility time and speed.

3.2.4 Histological assessment after SAH

At 48h, mice were anesthetized with ketamine (10mg/kg) and xylazine (4mg/kg) and

underwent transcardial perfusion with 4% paraformaldehyde (PFA) in phosphate buffer

at physiological pressures by sphygmomanometer (100mmHg). Brain specimens were

extracted and subsequently fixed in 4% PFA for 24h, followed by specific cuts

previously described, and then processed and embedded with paraffin [Sabri 2009].

Five μm thick sections were created by microtome (Leica, Wetzlar, Germany) slicing

and transferred to microscope slides.

Fluoro-jade b (FJB) staining was used to identify degenerating neurons [Schmued

2000]. Briefly, paraffin-embedded coronal brain slices on microscope slides were

65

rehydrated (xylene 15min, 100% ethanol 3min, 95% ethanol 1min, 80% ethanol 1min,

deionized water 1min) and then placed in 0.06% potassium permanganate solution for

10-15min under gentle agitation. After quick rinse, slides were incubated in the dark with

0.001% FJB (Histo-Chem Inc., Jefferson, AR) working solution with 0.1% acetic acid for

30min. After several rinses, slides were dried, dehydrated (xylene x 2min), and then

cover-slipped with DPX mounting media (Sigma-Aldrich, St. Louis, MO). Slides were

examined and representative images were taken using an upright fluorescent

microscope (Olympus, Center Valley, PA). FJB positive cells were counted from 5

discrete areas along the cortex over each hemisphere and 3 discrete areas around

each hippocampus. Counts were performed by 2 observers, blinded to experimental

group.

Caspase3/NeuN co-immunostaining was used to identify apoptotic neurons. Briefly,

paraffin-embedded coronal brain slices on microscope slides were rehydrated (xylene

15min, 100% ethanol 10min, 95% ethanol 10min, 80% ethanol 10min, deionized water

5min) and heated to 98oC with 1:100 antigen retrieval solution (Vector Laboratories,

Burlingame, CA) for 30min. Slides were then permeabilized with 0.3% Triton X-100

(Sigma-Aldrich, St. Louis, MO) for 1h under gentle agitation. Specimens were then

blocked with 10% goat serum in 1% bovine serum albumin (BSA) dissolved in

phosphate buffered saline (PBS) for 30min. Rabbit anti-active Caspase-3 (1:300, BD

Pharmingen, Franklin Lakes, NJ) incubation overnight at 4oC followed by goat anti-

rabbit Alexa Fluor 488 (1:1,000, Invitrogen, Carlsbad, CA) for 1h. After washes, mouse

anti-NeuN (1:200, Invitrogen, Carlsbad, CA) incubation for 1h was followed by goat anti-

mouse Alexa Fluor 568 (1:1,000, Invitrogen, Carlsbad, CA) for 1h. After washes, DAPI

66

(4’,6-diamidino-2-phenylindole, Sigma-Aldrich) staining for nuclei was performed and

then slides were cover slipped with CC/Mount (Sigma-Aldrich, St. Louis, MO) and

sealed with nail polish. Slides were examined and representative images were taken

using an upright fluorescent microscope (Olympus, Center Valley, PA).

Caspase3+NeuN+ cells were counted from 5 discrete areas along the cortex over each

hemisphere and 3 discrete areas around the hippocampus from each side. Counts were

performed by 2 observers, blinded to experimental group.

Hematoxylin & Eosin (H&E) staining was used to assess vasospasm. Briefly, paraffin-

embedded brain slices on glass slides were rehydrated (xylene 9min, 100% ethanol

9min, 95% ethanol 3min, 80% ethanol 3min, deionized water 5min), followed by

hematoxylin (Poly Scientific, Bayshore, NY) staining (hematoxylin 3min, rinse, tap water

5min, quick acid ethanol dip, rinse with tap water and deionized water). Eosin (Poly

Scientific, Bayshore, NY) staining for 30s was then performed followed by dehydration

(95% ethanol 15min, 100% ethanol 15min, xylene 45min) and cover slipping with

Permount mounting medium (Sigma-Aldrich, St. Louis, MO). Vasospasm was assessed

on parasagittal cortical brain cuts by calculating the ratio of the MCA branch internal

lumen diameter to the MCA branch vessel wall thickness, with a smaller internal lumen

diameter to wall thickness ratio indicative of increased vasospasm. Slides were

examined and representative images were taken using an upright fluorescent

microscope (Olympus, Center Valley, PA). Measurements of diameter and luminal

thickness were performed using Image J (NIH, Bethesda, MD), in a blinded manner.

67

Fibrinogen staining to detect microthrombi has been previously described [Sabri 2011].

Briefly, paraffin-embedded coronal brain slices on microscope slides were rehydrated

(xylene 15min, 100% ethanol 10min, 95% ethanol 10min, 80% ethanol 10min, deionized

water 5min) and then heated to 96oC for 25min in antigen retrieval solution. Slides were

incubated in 0.3% hydrogen peroxide for 30min followed by blocking with 10% goat

serum. Slides were incubated with primary chicken anti-rat fibrinogen antibody (1:200,

Immunology Consultants Laboratory, Newberg, OR) for 60min followed by secondary

goat anti-chicken biotinylated antibody (1:200, Millipore, Billerica, MA) for 30min. The

Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used to obtain fibrinogen

staining as per kit instructions. Slides were then counterstained with 0.5% methyl green

solution for 5min and then dehydrated (95% ethanol, 100% ethanol, xylene), and cover

slipped with Permount mounting media. Slides were examined and representative

images were taken using an upright fluorescent microscope (Olympus, Center Valley,

PA). Microthrombi were counted throughout the entire coronal brain slice (5 discrete

cortical areas and 3 discrete hippocampus areas for each side of the brain) in a blinded

manner by 2 observers.

3.2.5 Blood brain barrier assessment

Briefly, 5cc/kg of 2% Evans Blue dye (Sigma-Aldrich, St Louis, MO) was dissolved in

normal saline and administered via IP injection 1h prior to transcardial perfusion with

PBS at physiological pressures by sphygmomanometer (100mmHg) [Manaenko 2011].

Evans Blue dye binds to albumin and can be a marker of blood vessel permeability

[Radu 2013]. Brain (divided into left and right hemispheres with cerebellum and

brainstem dissected away) and four peripheral organs (heart, lungs, liver, and kidneys)

68

were extracted, weighed, and dried in an oven overnight. After dry weight

measurement, tissue was homogenized with formamide (Sigma-Aldrich, St. Louis, MO),

heated to 55oC for 24h and then centrifuged (15000g x 30min). The absorbance of the

supernatant fluid was measured at 620nm by a spectrophotometer (SpectraMax M5e,

Molecular Devices, Sunnyvale, CA) with reference to standard curve samples.

3.2.6 Brain protein expression

Mice were perfused with PBS at physiological pressures by sphygmomanometer

(100mmHg) and brains were extracted. The cerebellum and brainstem were dissected

away and cerebral hemispheres were divided down the midline with only left cerebral

hemisphere used for brain protein expression, which is the side of the needle insertion

and blood injection. Brain tissue was homogenized in radioimmunoprecipitation assay

(RIPA) buffer (Cell Signaling Technology, Danvers, MA) containing phosphatase

inhibitor PhosStop (Roche, Basel, CH) and protease inhibitor (Roche, Basel, CH) and

subsequently centrifuged at 4oC (16000g x 20min). Supernatant protein concentration

was then quantified with the Pierce BCA (Bicinchoninic acid) assay (Thermo Fisher

Scientific, Waltham, MA) as per manufacturer’s instructions. Samples were then stored

at -80oC prior to western blot experiments.

Equal amounts of total protein samples were loaded into multi-well mini-gels placed in a

Bolt mini-gel tank (Thermo Fisher Scientific, Waltham, MA) filled with running buffer.

The tank was then connected to a PowerPac high-current power supply (Bio-Rad,

Hercules, CA) and set to constant 165V for 45min. Gels were then rinsed in deionized

water and then transferred to nitrocellulose membranes using the iBlot apparatus

69

(Thermo Fisher Scientific, Waltham, MA) as per manufacturer’s instructions. These

blots were then incubated in 5% BSA solution followed by incubation with primary

antibody at 4oC for 18h with gentle agitation. Primary antibodies included rabbit anti-

mouse β-tubulin (1:1,000, Cell Signaling Technology, Danvers MA), rabbit anti-mouse

pAKT (Serine 473 phosphorylated Akt, 1:1,000, Cell Signaling Technology, Danvers,

MA), rabbit anti-mouse pan-AKT (AKT1/AKT2/AKT3, 1:1,000, Cell Signaling

Technology, Danvers, MA), rabbit anti-mouse acetylated histone H3 (Lysine 9/Lysine

14, 1:1,000, Cell Signaling Technology, Danvers, MA), and rabbit anti-mouse total

histone H3 (1:1,000, Cell Signaling Technology, Danvers, MA). After washes, the blots

were incubated with secondary goat anti-rabbit IgG HRP-linked antibody (1:10,000, Cell

Signaling Technology, Danvers, MA) for 1h. After further washes, the blots used the

Amersham ECL Western Prime (GE Healthcare Life Sciences, Marlborough, MA) kit to

develop the signal as per the manufacturer’s instructions. The blot signal was

transferred to x-ray film and then read on a Versadoc Imaging System (Bio-Rad,

Hercules, CA). Images were than analyzed with Image J (NIH, Bethesda, MD). Protein

expression was normalized to β-tubulin expression and then normalized to sham

experimental group.

3.2.7 Statistics

Continuous variables were compared with one-way or two-way ANOVA (Analysis of

variance) with Holm-Sidak post hoc test. Non-parametric variables were compared with

Kruskal-Wallis test with Dunn’s post hoc test. Statistics and graphs were generated with

Prism 6 (GraphPad Software, La Jolla, CA).

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3.2.8 Clinical trial data

Anonymized individual patient data was derived from the CONSCIOUS-1 trial, a

randomized, double-blind, placebo-controlled trial studying the effects of clazosentan on

aVSP after SAH [Macdonald 2008]. This clinical trial was registered (NCT00111085,

https://clinicaltrials.gov). Poor clinical outcome at 12 weeks post-SAH was defined as a

mRS of 4-6. Univariate analysis was performed to identify predictors of poor clinical

outcome with the following input variables: Age, gender, WFNS grade (Dichotomized

variable with poor grade IV/V), surgery, seizure, DCI, and VPA treatment. Predictors

with p<0.15 on univariate analysis were used as input variables for the multivariate

analysis, with significance set at p<0.05. Propensity scores for VPA treatment were

created for all patients using input variables significant for predicting poor outcome on

multivariate analysis: Age, WFNS grade, seizure, and DCI. Propensity score matching

was then performed using the nearest neighbour algorithm to identify a control group of

patients not treated with VPA [Rubin 1997]. Comparisons between patients treated or

not treated with VPA used 2-tailed t-test for continuous variables and chi-square test for

dichotomous variables, with significance set at p<0.05. Statistics were performed with

Stata 12.0 (StataCorp LLC, College Station, TX).

https://clinicaltrials.gov/

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3.3 Results

3.3.1 Valproic acid improved neurological outcomes after experimental

SAH

The brain specimens demonstrated obvious gross swelling at 48h after SAH induction

(Figure 1A). SAH induction resulted in a decrease in CBF to 5-10% of base-line with

gradual return to 60-70% of baseline at 10min post-SAH (Figure 1B). Mortality was

14.3% (5/35) and 2.9% (1/34) in the SAH and SAH+VPA groups respectively, with

death typically occurring shortly after SAH induction. No mortality was found in the

sham groups. SAH caused a significant decrease in weight at 48h but not at 7 days

(Figure 1C). However, VPA treatment also resulted in significant decrease in body

weight at 48h and 7 days for both sham and SAH groups (Figure 1C). SAH caused

neurobehavioural deficits as assessed by the MGS at 24h and 48h, which was

improved with VPA treatment (Figure 1D-E). SAH mice had reduced activity level at

24h and 48h with significant improvement after VPA treatment after 24h but not after

48h (Figure 2). Also, within each experimental group, only SAH mice demonstrated

improvement in MGS at 48h compared with 24h (p<0.05).

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Figure 1. Prechiasmatic injection SAH mouse model. A: Representative brain

specimens removed at 48h after SAH. B: CBF measurements during SAH/sham

induction. n=14-16 per group. Data represent means. Error bars represent standard

errors of the mean (SEM). Two-way ANOVA with Holm-Sidak post hoc correction.

***p<0.001 Sham or Sham+VPA vs. SAH or SAH+VPA. C: Weight change at 48h and 7

days after SAH. Two-way ANOVA with Holm-Sidak post hoc correction. Horizontal lines

represent means. *p<0.01 vs. Sham+VPA, SAH+VPA, and SAH; #p<0.05 vs.

SAH+VPA; @p<0.01 vs. Sham+VPA and SAH+VPA; &p<0.001 vs. Sham+VPA and

SAH+VPA. n=11-13 per group (48h), n=7-8 per group (7 days). D and E: Modified

Garcia Score (MGS) at 24h and 48h after SAH respectively. Kruskal-Wallis Test with

Dunn’s post hoc correction. Horizontal lines represent means. **p<0.01, ***p<0.001.

n=15-16 per group.

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Figure 2. Activity level. One-way ANOVA with Holm-Sidak post hoc correction. n=13-15

per group. Data represent mean +/- SEM. #p<0.01 relative to Sham, Sham+VPA, and

SAH+VPA. @p<0.01 relative to Sham and Sham+VPA. &p<0.01 relative to Sham.

All 4 experimental groups showed ability to be trained to identify the hidden platform in

the Morris Water Maze based on escape latencies measured during the cue trial (day 3)

and acquisition trials (days 4-6) (Figure 3A). VPA treatment appeared to decrease this

ability in sham mice during the cue and first acquisition trials, with no significant

differences in the second and third acquisition trials (Figure 3A). Thigmotaxis, defined

as swimming along within 10cm of the wall, as a measure of the inability to focus on the

task at hand, was not significantly different among the experimental groups (Figure 3B).

Overall, there were no significant differences in maximum and average speed and total

distance traveled among the 4 experimental groups (Figure 4). SAH caused impairment

of spatial memory as determined by decreased time spent in the target quadrant on the

probe trial on day 7, which was improved by VPA treatment (Figure 3C). Other than

increased stretching as an indication of fear after SAH, all the other components of

anxiety as measured by the open field test were not significantly affected in this model

of SAH (Figure 5).

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Figure 3. Morris water maze. A: Escape latency based on the cue trial (day 3) and

acquisition trials (days 4-6). One-way ANOVA, Holm-Sidak post hoc correction. *p<0.05,

***p<0.001. B: Thigmotaxis (swimming along the wall) based on the cue trial (day 3) and

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acquisition trials (days 4-6). C: Time spent in target quadrant in the probe trial (day 7).

Representative mouse swim paths depicted below. One-way ANOVA with Holm-Sidak

post hoc correction. *p<0.05 vs. Sham and SAH+VPA. Data represent means +/- SEM.

n=11-12 per group

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Figure 4. Morris Water Maze supplemental information. A and B: Maximum and mean

speed during cue (day 3), acquisition 1-3 (day 4-6), and probe (day 7) trials. C: Total

distance traveled during cue and acquisition 1-3 trials. n=11-12 per group. Data

represent mean +/- SEM. One-way ANOVA with Holm-Sidak post hoc correction.

*p<0.05.

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Figure 5. Open field test. A-J: On day 7 after SAH, number of episodes of defecation,

urination, rearing, stretching, grooming, crossing the centre, line crossing, immobility,

and maximum and mean speed. n=7-8 per group. Data represent means +/- SEM. One-

way ANOVA, Holm-Sidak post hoc correction. *p<0.05 vs. Sham+VPA and SAH+VPA.

3.3.2 Valproic acid limited brain injury after experimental SAH

SAH resulted in increased neuronal degeneration at 48h as indicated by increased

number of FJB positive cells in both the cortex and hippocampus but was limited by

VPA treatment (Figure 6). Similarly, SAH resulted in increased neuronal apoptosis at

48h as indicated by increased number of Caspase3+NeuN+ cells in both the cortex and

hippocampus but was also limited by VPA treatment (Figure 7).

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Figure 6. Fluoro-jade b (FJB) staining 48h after SAH. Upper: Number of FJB positive

cells in the cerebral cortex, hippocampus and overall. n=7-8 per group. Data represent

means +/- SEM. One-way ANOVA with Holm-Sidak post hoc correction. *p<0.05 vs.

Sham; ***p<0.001 vs. Sham, Sham+VPA; ****p<0.0001 vs. Sham, Sham+VPA,

SAH+VPA; #p<0.05 vs. SAH+VPA. Lower: Representative images of left cerebral

cortex in SAH and SAH+VPA mice. White arrows indicate FJB positive cells. Scale

bar=50μm.

85

Figure 7. Neuronal apoptosis 48h after SAH. A: Number of Caspase3+NeuN+ cells in

the cerebral cortex, hippocampus and overall. n=7-8 per group. Data represent means

+/- SEM. One-way ANOVA with Holm-Sidak post hoc correction. *p<0.05, **p<0.01 vs.

Sham, Sham+VPA, and SAH+VPA. B and C: Representative images of SAH and

SAH+VPA in the cerebral cortex and hippocampus. White arrows indicate

Caspase3+NeuN+ cells. Scale bar=50μm.

3.3.3 Valproic acid did not affect large artery vasospasm after experimental

SAH

SAH and SAH+VPA mice had more significant MCA vasospasm on histology compared

with Sham mice (Figure 8). However, there was no significant difference in vasospasm

between SAH mice treated with VPA and SAH mice treated with vehicle.

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Figure 8. Middle cerebral artery (MCA) vasospasm 48h after SAH. Above: Degree of

vasospasm, determined by the ratio of lumen diameter over the wall thickness with

lower ratio values representing increased vasospasm. Below: Representative images of

the degree of MCA vasospasm in Sham, Sham+VPA, SAH, and SAH+VPA groups.

Scale bar=50μm. n=7-8 per group. Data represent means +/- SEM. One-way ANOVA

with Holm-Sidak post hoc correction. **p<0.01 vs. Sham.

3.3.4 Valproic acid decreased microthrombi burden after experimental SAH

SAH resulted in increased microthrombi at 48h as indicated by fibrinogen staining in

both the cortex and hippocampus (Figure 9). However, VPA treatment decreased the

microthrombi burden overall, especially in the cerebral cortex.

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Figure 9. Microthrombi burden 48h after SAH. A: Number of fibrinogen-positive

microthrombi in the cerebral cortex, hippocampus and overall. n=3-8 per group. Data

represent means +/- SEM. One-way ANOVA with Holm-Sidak post hoc correction.

#p<0.05 vs. Sham+VPA; *p<0.05 vs. Sham, Sham+VPA, SAH+VPA; **p<0.01 vs.

Sham, Sham+VPA. B: Coronal brain slice demonstrating areas where images were

taken (black boxes). Scale bar=1mm. C and D: Representative images of Sham,

Sham+VPA, SAH and SAH+VPA in the cerebral cortex (C) and hippocampus (D). Black

arrows indicate fibrinogen-positive microthrombi. Scale bar=100μm.

3.3.5 Valproic acid decreased blood-brain barrier disruption after

experimental SAH

Evans Blue assay at 24h showed a trend toward BBB disruption in the left cerebral

hemisphere after SAH, with improvement after VPA treatment (p=0.10, Figure 10). The

left hemisphere was the site of blood injection with the blood tending to distribute more

on the left basal subarachnoid space. The 24h time-point was chosen given that prior

published studies showed this time-point to have maximal BBB disruption [Suzuki

2010]. There were no significant differences in BBB disruption in the right cerebral

hemisphere and in any of the peripheral organs after SAH among the 4 experimental

groups (Figure 10).

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Figure 10. Blood-brain barrier integrity. Measurement of Evans Blue dye extravasation

at 24h after SAH in the left and right cerebral hemispheres (Above) and in the heart,

lung, liver, and kidney (Below). n=6 per group. Data represent means +/- SEM. One-

way ANOVA with Holm-Sidak post hoc correction.

3.3.6 Valproic acid increased acetylation of histones

VPA treatment resulted in significantly increased acetylation of histone H3 (Lysine 9 /

Lysine 14) in both sham and SAH mice (Figure 11). However, phosphorylated AKT was

not significantly different between experimental groups (p=0.61, Figure 11). The earlier

24h time-point was chosen given that post-translational modifications tend to decrease

with time [Endo 2006].

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Figure 11. Protein quantification of left cerebral hemisphere 24h after SAH. Above:

Relative protein expression of acetylated histone H3 (AcH3, Lysine 9/Lysine 14) and

total histone H3, normalized to β-tubulin. Ratio of AcH3 over total H3 was then

normalized to sham group average. n=3 per group. Data represent means +/- SEM.

One-way ANOVA with Holm-Sidak post-hoc correction. *p<0.05. Below: Relative

protein expression of phosphorylated AKT (pAKT, Serine 473) and total AKT (AKT1,

AKT2, AKT3), normalized to β-tubulin. Ratio of pAKT over total AKT was then

normalized to sham group average. n=5-6 per group. Data represent means +/- SEM

3.3.7 Valproic acid treatment in SAH patients did not significantly affect

clinical outcome

Base-line characteristics of SAH patients enrolled in CONSCIOUS-1 are found in Table

1. Univariate analysis revealed that older age, female sex, poor WFNS grade, seizure,

DCI, and VPA treatment to be significantly associated with poor clinical outcome at 12

weeks (mRS 4-6, Table 2). On multivariate analysis, age (OR 1.04, 95% CI 1.01-1.07),

poor WFNS grade (IV/V, OR 5.16, 95% CI 2.89-9.24), seizure (OR 2.27, 95% CI 1.08-

4.78), and DCI (OR 6.40, 95% CI 3.47-11.80) were predictive of poor outcome (Table

2). There was a trend toward poor outcome in patients who were treated with VPA on

multivariate analysis (OR 3.07, 95% CI 0.93-10.09, p=0.07).

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Table 1: Base-line characteristics of CONSCIOUS-1 SAH patient population (n=413).

Clinical characteristics Summary

Age (years, mean, SD, range) 51.0 (10.8, 18-71)

Gender (female, %) 292 (70.7%)

WFNS grade (poor grade IV/V, %) 100 (24.2%)

Surgery (%) 201 (48.7%)

Seizure (%) 57 (13.8%)

DCI (%) 78 (18.9%)

DCI: Delayed cerebral ischemia; SD: Standard deviation; WFNS: World Federation of

Neurological Surgeons.

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Table 2: Univariate and multivariate analyses determining predictors of poor clinical

outcome at 12 weeks (mRS 4-6) after SAH.

Variable Univariate

OR

Univariate

95% CI

Univariate

p-value

Multivariate

OR

Multivariate

95% CI

Multivariate

p-value

Age 1.03 1.01-1.05 0.01 1.04 1.01-1.07 <0.01

Gender

(female)

1.80 1.02-3.18 0.04 1.69 0.86-3.29 0.13

WFNS grade

(poor grade

IV/V)

6.30 3.77-

10.53

<0.01 5.16 2.89-9.24 <0.01

Surgery 1.61 1.00-2.59 0.05 1.58 0.89-2.80 0.12

Seizure 2.76 1.52-5.01 <0.01 2.27 1.08-4.78 0.03

DCI 7.92 4.59-

13.66

<0.01 6.40 3.47-11.80 <0.01

VPA 2.85 1.11-7.33 0.03 3.07 0.93-10.09 0.07

CI: Confidence interval; DCI: Delayed cerebral ischemia; mRS: Modified Rankin scale;

OR: Odds ratio; VPA: Valproic acid; WFNS: World Federation Neurological Surgeons.

There were 19 patients who were treated with VPA during hospital admission for SAH.

A propensity-score matched group of patients (n=19) who were not treated with VPA

was created using the significant covariates for poor outcome as input variables (age,

poor WFNS grade, seizure, DCI). VPA treatment did not significant affect mortality or

poor clinical outcome at 12 weeks (mRS 4-6, Table 3). Among patients with available

data, only 35% (6/17) of patients who were treated with VPA, received the drug within 1

day of SAH ictus, and 40% (4/10) were treated for 24h or less (Table 4). Among 13

patients who had platelet counts available before and after VPA treatment in hospital,

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there was a trend toward increasing mean platelet count after VPA treatment (262 vs.

349, x109/L, p=0.06). There were no bleeding complications attributed to VPA use.

Table 3: Effect of VPA treatment.

Variable No VPA (n=19) VPA (n=19) p-value

Age (years, mean, SD,

range)

51.9 (11.0, 33-

70)

50.1 (12.9, 29-

69)

0.64

Gender (female %) 14 (73.7%) 13 (68.4%) 0.72

WFNS grade (poor

grade IV/V, %)

8 (42.1%) 7 (36.8%) 0.74

Surgery (%) 8 (42.1%) 10 (52.6%) 0.52

Seizure (%) 3 (15.8%) 4 (21.1%) 1.00

DCI (%) 3 (15.8%) 4 (21.1%) 1.00

Poor outcome (mRS 4-

6) at 12 weeks (%)

5 (26.3%) 8 (42.1%) 0.31

Death (%) 1 (5.3%) 2 (10.5%) 1.00

CI: Confidence interval; DCI: Delayed cerebral ischemia; IQR: Interquartile range; mRS:

Modified Rankin scale; OR: Odds ratio; SD: Standard deviation; VPA: Valproic acid;

WFNS: World Federation Neurological Surgeons.

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Table 4: VPA use.

Patient

Number

VPA initiation

day*

VPA treatment duration

(days)

1 14 -

2 11 -

3 1 1

4 13 1

5 0 1

6 0 -

7 - -

8 - -

9 6 6

10 14 3

11 15 -

12 14 -

13 0 -

14 10 6

15 0 1

16 9 2

17 5 -

18 11 4

19 0 11

*Relative to SAH ictus. Data not available (-).

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3.4 Discussion

In a mouse model of SAH, VPA treatment improved neurological outcomes, decreased

brain injury and microthrombi burden, but did not affect vasospasm. BBB disruption may

have been attenuated by VPA. The mechanism for decreased brain injury in this SAH

animal model appears to be independent of AKT phosphorylation, part of the cell pro-

survival signaling cascade [Franke 2003]. There was a trend toward poor clinical

outcome after VPA treatment on multivariate analysis of SAH clinical trial data.

However, when compared to a propensity score-matched control group, VPA treatment

had no significant effect on mortality or poor clinical outcome at 12 weeks. We have

demonstrated that VPA is beneficial in a mouse model of SAH but its effect on clinical

SAH is less certain.

3.4.1 Alternative SAH models treated with valproic acid

VPA treatment has also been effective in other animal models of SAH. In a rat

endovascular perforation model of SAH, Ying and colleagues found that VPA treatment

(300mg/kg) immediately after SAH induction and a repeat dose 12h later, decreased

BBB disruption, attenuated brain injury and improved neurobehavioural outcomes at

24h after SAH [Ying 2016]. In this model, VPA appeared to act by decreasing

expression and activity of matrix metallopeptidase 9 (MMP9), limiting degeneration of

tight junction components claudin-5 and occludin, upregulating the molecular chaperone

heat shock protein 70 (HSP70), and increasing phosphorylation of pro-survival signals

AKT and BCL-2 (B-cell lymphoma 2) [Ying 2016]. The neuroprotective effect of VPA

was diminished after co-administration of an HSP70 inhibitor [Ying 2016]. With our

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prechiasmatic injection mouse model, we did not observe the same upregulation of

phosphorylated AKT, which may be due to the differing SAH models. Also, based on

body surface area adjustments, their VPA dose was 50% higher than the dose used in

our study, although the scheduling of VPA treatments were quite similar [Reagan-Shaw

2007].

Chang and colleagues used a cisterna magna double injection rat model of SAH with

VPA treatment initiated 1h after SAH with a continuous infusion using an osmotic mini-

pump (10-40mg/kg/d) for 5 days [Chang 2015]. In this study, VPA decreased basilar

artery vasospasm at 5 days via inhibition of chemokine ligand 5 (CCL5), decreasing cell

adhesion molecule expression (VCAM1, ICAM1, E-selectin) and decreasing leukocyte

extravasation, which was reversed by administration of recombinant CCL5 [Chang

2015]. Again, the differing SAH model and unique VPA dosing may have contributed to

VPA affecting the degree of vasospasm, although the rat cisterna magna double

injection model is known to generate greater vasospasm [Raslan 2012].

Finally, Hamming and colleagues used chronic VPA (200mg/kg/d x 4 weeks) treatment

prior to SAH induction using the rat endovascular perforation model followed by brain

cortex application of KCl (1mol/L) 24h later to induce spreading depolarizations

[Hamming 2017]. Cortical spreading depolarization, a wave of grey matter excitation

followed by loss of ion homeostasis, altered vascular response, and depression of

electrical activity, is thought to contribute to the pathophysiology of SAH [Dreier 2011].

The authors found that VPA prevented brain lesion growth between MRI scans

performed at 24h and 72h and decreased histological brain injury, although VPA did not

100

affect sensorimotor function as measured by the inclination test at both time-points

[Hamming 2017]. These results are less comparable to our study, given that VPA was

administered prior to SAH induction, and would be less applicable in the clinical

situation of a patient presenting with spontaneous SAH.

3.4.2 Other models of brain injury treated with valproic acid

VPA treatment has been studied in various models of brain injury, including permanent

and transient ischemic stroke, transient global ischemia, neonatal hypoxic-ischemic

brain injury, ICH, TBI, and epilepsy [Lee 2013A, Dash 2010, Dekker 2017, Brandt 2006,

Kabakus 2005, Kim 2007, Ren 2004, Sinn 2007]. The mechanism of neuroprotection

with VPA treatment is thought to be a combination of anti-apoptosis, anti-inflammation,

and neurotrophic effects [Chen 2014, Chuang 2005, Dekker 2014, Liu 2012]. VPA can

downregulate the expression of inflammatory cell adhesion molecules VCAM1 and

ICAM1 [Larsson 2012]. In addition, VPA may also limit neurotoxicity of hemoglobin in

models of ICH by downregulating heme oxygenase-1 [Kwon 2013]. Specific efforts have

been made investigating the effect of VPA on gene expression as an HDAC inhibitor

[Bambakidis 2016, Larsson 2012].

3.4.3 Valproic acid in the context of seizure treatment and prophylaxis

There are currently no randomized control trials to provide recommendations on the

prophylaxis and treatment of seizures after SAH [Marigold 2013]. Current guidelines

suggest that prophylactic antiepileptic drugs may be considered in the acute period after

SAH [Connolly 2012]. The reported seizure rate in SAH patients varies widely from 4 to

26% [Lanzino 2011]. Retrospective studies have suggested that SAH patients treated

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with phenytoin for routine seizure prophylaxis had worse clinical outcomes [Naidech

2005, Rosengart 2007]. This may occur, in part, due to the ability of phenytoin to induce

CYP3A4, which increases the metabolism of nimodipine [Tartara 1991]. Nimodipine is a

calcium channel antagonist that has been shown to improve outcomes modestly after

SAH and its use clinically is currently standard of care [Dorhout Mees 2007].

Decreasing the bioavailability of nimodipine by administration of phenytoin may negate

the beneficial effects of nimodipine and thus contribute to poor outcome [Koch 2005,

Wong 2005]. However, VPA use in SAH would not be affected by this issue [Tartara

1991].

VPA as seizure prophylaxis has been investigated in patients with TBI and ICH. In one

randomized trial, patients were treated within 24h of TBI with either 1 week of phenytoin

or 1- or 6-month course of VPA [Temkin 1999]. Patients treated with VPA did not have

improved early or late seizure control, and there was a trend toward increased mortality

with VPA treatment [Temkin 1999]. In a small randomized trial of patients with

spontaneous supratentorial ICH treated with 1-month VPA vs. placebo, the investigators

found no effect on early or late seizure control [Gilad 2011]. There was also no

difference in overall mortality but there was improved neurologic outcomes at 12 months

with VPA treatment [Gilad 2011]. These 2 trials suggest that there is potential for benefit

with VPA treatment in acute neurological disease, but the duration of treatment needs to

be shortened, which in the case of SAH, can be 2 weeks or less.

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3.4.4 Valproic acid in the context of coagulopathy risk

In our study, the reduction of brain injury by VPA treatment may have been directly

related to decreasing microthrombi burden, which has been found to be a component of

SAH pathophysiology [Tso 2014]. The mechanism for the decreased microthrombi in

our study is unclear, although may be related to increased endothelial-derived

expression of tPA [Larsson 2012]. Larsson and colleagues showed that VPA treatment

of cultured HUVECs showed increased tPA mRNA and protein expression as well as

increased acetylation of histones near the tPA promoter [Larsson 2012]. They confirmed

the importance of the HDAC inhibitor activity of VPA in this study, as a structural amide

analogue valpromide (2-propylpentanamide) which lacks HDAC inhibitor activity was not

able to reproduce a similar effect as VPA on tPA expression [Larsson 2012]. Despite

this increased tPA synthesis in response to VPA treatment, there was no associated

increased risk of bleeding in response to vascular injury [Larsson 2016].

Another possible mechanism explaining decreased microthrombi burden in our study

may be related to thrombocytopenia and platelet dysfunction, which are well-described

adverse effects of VPA [Chateauvieux 2010]. In the previously mentioned randomized

controlled trial of TBI patients, patients treated with VPA had platelet counts decrease

by 13% from base-line at day 4, but this did not affect clot strength or cause clinically-

significant bleeding [Anderson 2003].

Thrombocytopenia may occur in 12-18% receiving chronic VPA treatment, and more

frequently found in women and elderly patients and patients with lower baseline platelet

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count [Buoli 2017]. This suggests that VPA treatment in SAH may best be limited to the

acute period and perhaps only after the aneurysm has been secured by coiling or

clipping. Importantly, in this study, we did not observe any bleeding-related

complications attributed to VPA in SAH patients.

3.4.5 Study limitations

Limitations to this study include the lack of serum VPA measurements to ensure

therapeutic levels. Also, platelet counts were not measured to potentially explain the

decrease in microthrombi after VPA treatment. It is unclear how much of the brain injury

was a result of the transient global cerebral ischemia from the initial increased ICP and

how much of the injury was a result of the effect of blood in the subarachnoid space.

Measurement of vasospasm by H&E histology limits the ability to appreciate the

dynamic nature of vasospasm and may overestimate the degree of vasospasm in a

collapsed non-pressurized artery. The SAH model used does not replicate the DCI seen

in SAH patients and so may only be useful as a model for EBI. The precise molecular

mechanism of VPA benefit was not elicited in this study, although other studies using

models of neurological diseases have found several relevant neuroprotective pathways

affected. The retrospective clinical analysis was not powered to identify a significant

difference after VPA treatment. Future complementary mechanistic studies may look at

cultured endothelial cells treated with VPA and identify significantly affected genes and

pathways.

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3.5 Conclusions

This study showed that VPA treatment improved neurological outcomes and decreased

brain injury after experimental SAH. The mechanism is unclear but may be related to

decreasing microthrombi burden and limiting BBB disruption. VPA did not appear to

impact clinical outcomes in patients with SAH on retrospective analysis of a relatively

small number of patients from a clinical trial. Nonetheless, VPA is an attractive drug

treatment to investigate further with clinical trials in SAH patients with its potential

benefits beyond preventing seizures.

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Chapter 4

Brain Endothelial Gene Expression after SAH

This chapter was modified from the following:

Tso MK, Turgeon P, Bosche B, Lee CK, Nie T, D’Abbondanza J, Ai J, Marsden PA,

Macdonald RL. Whole genome expression profiling of brain endothelial cells after

experimental subarachnoid hemorrhage. (In Preparation)

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Brain Endothelial Gene Expression after SAH

4.1 Background

Better understanding of the endothelial component of the BBB is needed, including

more in-depth studies of brain endothelial gene expression, especially in the setting of

neurological disease [Huntley 2014]. With advancements in molecular biology, there

have been several published studies of whole genome expression profiling in SAH

animal models examining whole brain tissue, circle of Willis arteries or brain

microvessels [Peng 2017, Zheng 2015, Kikkawa 2014, Kurogi 2015, Lee 2012,

Sasahara 2008, Vikman 2006]. However, due to inclusion of cells from multiple

lineages, the gene expression signatures of specific cell types may be blunted, which

may erroneously include or exclude pathways important to SAH pathophysiology. The

gene expression patterns of brain endothelial cells in SAH patients can only be

minimally inferred from serum, CSF, aneurysm fundus, and endovascularly-obtained

endothelial samples, emphasizing the importance of a preclinical approach [Su 2015,

Lai 2017, Kikkawa 2017, Stylli 2017, Bache 2017, Pera 2010, Kurki 2012, Kleinloog

2016, Liu 2014, Wang 2017, Nakaoka 2014, Cooke 2017]. In a mouse model of SAH

with evidence of BBB disruption, we freshly isolated brain endothelial cells by two

distinct methods and then interrogated the gene expression profiles of these cells. With

this approach, we identified Ptgs2 (prostaglandin-endoperoxide synthase 2), also known

as COX-2, as a potential therapeutic target that warrants further investigation in SAH

preclinical studies and potentially clinical trials.

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4.2 Methods

4.2.1 SAH model

Mouse strains used included 12-week-old male FVB/NJ mice (Jackson Laboratory) and

12-15-month-old male and female Tg(Tie2-GFP)287Sato/J mice (Jackson Laboratory).

These latter mice have an FVB background but also express GFP driven by the

endothelial cell-specific Tie2 promoter, allowing endothelial cells to be visualized

(Figure 12). Although FVB mice are known to have relatively modest brain injury after

SAH, we chose this strain to match the background strain of the Tie2-GFP mice

[D’Abbondanza 2016]. Mice are housed up to 5 per cage with standard chow and water

ad libitum and 12h /12h light/dark cycles. Anesthesia was induced with inhaled 3%

isoflurane (Fresnius Kabi) with 1L oxygen flow. Hair over the dorsal skull was shaved

and prepped with 10% povidone-iodine topical solution (Laboratoire Atlas Inc.). Mice

also receive subcutaneous injection of buprenorphine (0.2mg/kg) and lubricating

ophthalmic ointment (Refresh Lacri-Lube, Allergan) over the eyes. Mice were placed

prone and the skull fixated using a stereotactic frame. The prechiasmatic injection

mouse model was used, which reliably creates a blood clot along the ventral brain

surface and basal cisterns like what is seen in ruptured anterior circulation aneurysms

[Sabri 2009]. After a midline skin incision over the dorsal skull, a burr hole was created

with a high-speed drill (WE243 rechargeable mini engraver 2, Wecheer Industrial Co.,

Ltd.) with a 1mm round stainless-steel bur (1RF 007, 330 104 001 001, Hager &

Meisinger GmbH) 1mm to the left of midline and 1mm rostral to the fissure separating

the olfactory bulbs and cerebral cortex, to avoid major sinus bleeding. Using a 27G

Whitacre pencil point spinal needle (BD Medical) oriented at 37.5o from the vertical

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plane, 80µL of littermate arterial blood was injected over 45s. After injection, the spinal

needle was left in place for 2min to allow the blood to clot and to minimize reflux through

the burr hole. Upon removal of the spinal needle, bone wax (Ethicon) was used to

occlude the burr hole. Sham procedure involved burr hole creation and needle insertion

without blood injection. Intraoperative CBF was measured by laser flow doppler flow

(BLF 21, Transonic Systems Inc.) placed over the contralateral right parietal skull with

measurements made 10min prior to and post-SAH induction at 2min intervals. Mice

were placed on a homeothermic blanket (Harvard Apparatus). Postoperative

subcutaneous buprenorphine (0.2mg/kg) was administered twice daily. Weights were

measured preoperatively and at 24h and at 48h, if applicable, after SAH/sham

procedure.

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Figure 12. Tie2-GFP mouse. (A) Representative picture of the Tie2-GFP mouse. (B)

Schematic showing GFP incorporated within the Tie2 promoter, exon1, and intronic

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enhancer. (C) Representative brain slice derived from naïve Tie2-GFP mouse showing

intraparenchymal blood vessels expressing GFP. GFP: Green fluorescent protein.

4.2.2 Neurobehavioural Assessment

Global neurobehavioural assessments were performed at 24h and 48h after SAH/sham

procedure using the MGS performed by 2 blinded observers [Sherchan 2011]. As a

measure of activity level, the time to touch 3 separate walls with 2 paws simultaneously

was recorded for up to 5min (the first component of the MGS). If the mouse failed to

touch 3 separate walls, then a time of 5min was recorded. For comparison between 2

groups, the non-parametric Mann-Whitney U test was used. For comparison between

more than 2 groups, the non-parametric Kruskal-Wallis test was used.

4.2.3 Celecoxib Treatments

Mice received IP injections of celecoxib (PZ0008, Sigma-Aldrich Inc., St. Louis, MO) at

a dose of 10mg/kg, dissolved in a 100μL of 1:1 dimethyl sulfoxide (DMSO):normal

saline. Dosing is equivalent to adult human dose of 100mg twice daily, based on body

surface area dosing conversion [Reagan-Shaw 2007]. Doses of celecoxib or vehicle

were administered 30min and 12 h after SAH/sham induction. Vehicle injection

consisted of 100μL of 1:1 DMSO:normal saline.

4.2.4 BBB permeability assay

BBB permeability studies were performed in 12-15-month-old, male and female

Tg(Tie2-GFP)287Sato/J mice (SAH 24h/48h vs. Sham 24h/48h, n=4 per group) and 12-

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week-old male FVB/NJ mice (SAH +/- vehicle/celecoxib vs. sham +/- vehicle/celecoxib,

n=3 per group). Mice were administered IP injections of cadaverine conjugated to Alexa

Fluor 555 (250μg/25g mouse, dissolved in 100μL sterile PBS, A30677, Life

Technologies Inc, Waltham, MA) 2h prior to transcardial perfusion. Cadaverine is the

decarboxylation product of the amino acid lysine, typically present in decaying tissue but

also present in small amounts in live animals [Stepita-Klauco 1974. The conjugated dye

is a water-soluble tracer with molecular weight of approximately 950Da. Transcardial

perfusion was performed after induction of general anesthesia with IP ketamine

hydrochloride (200mg/kg) and xylazine hydrochloride (20mg/kg). The descending aorta

was clamped prior to left ventricular puncture with a 23G needle. Subsequently, the

right atrium was punctured. Perfusion was performed with a pressurized tubular system

with the sphygmomanometer set to maintain 100mmHg. Brains were extracted and

placed in ice-cold PBS. The right kidney was also extracted to assess systemic

absorbance of the cadaverine dye. Successful absorbance of the cadaverine dye was

also confirmed by the urine demonstrating a bright pink colour. Whole brains and

kidneys were imaged with a Leica DFC365FX camera (Leica Microsystems Inc.)

mounted to a Leica M205FA upright fluorescent microscope (Leica Microsystems Inc.)

with GFP and mCherry filter cubes. Brains were imaged in the ventral, dorsal, left

lateral, and right lateral positions under the same exposure time, gain and intensity

settings. Brains were then fixed in 4% PFA at 4oC for 6h followed by coronal sectioning

at 50µm thickness using the Leica VT1200S vibratome (Leica Microsystems Inc.).

These coronal slices were placed on microscope slides and the nuclei were stained with

DAPI for 15min followed by several washes. Aqueous-based CC/mount (Sigma-Aldrich,

St. Louis, MO) was added to the specimen slides and then cover slipped. Confocal

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images of left and right sides of coronal sections were taken using the Zeiss LSM700

confocal microscope (Carl Zeiss Canada LTD.) in the ventral medial, ventral lateral,

lateral, and dorsal regions under the same exposure time, gain and power settings. For

quantification, whole brain ventral images were analyzed with Image J (NIH, Bethesda,

MD). Coronal slices were quantified using the triangle algorithm for auto-thresholding

and “analyze particle” function from Image J for identifying positive cells.

4.2.5 Transmission electron microscopy

Whole brain specimens were fixed in a phosphate buffer solution containing 4% PFA

and 1% glutaraldehyde. The samples were then post-fixed in 1% osmium tetroxide.

Using a graded series of acetone solution, the samples were dehydrated and then

infiltrated and subsequently embedded in Epon-Araldite epoxy resin, carried out in a

microwave oven, Pelco BioWave 34770 (Pelco International, Clovis, CA). A diamond

knife on the Reichert Ultracut E ultramicrotome (Leica Microsystems, Buffalo Grove, IL)

was used to cut ultra-thin coronal sections 4mm from the anterior pole. These sections

were then stained with uranyl acetate and lead citrate before being visualized on the

transmission electron microscope, JEM-1011 (JEOL USA Corp., Peabody, MA). Digital

electron micrographs were acquired with a 2336 x 2048 pixels CCD camera (AMT

Corp., Danvers, MA, USA).

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4.2.6 Brain endothelial cell isolation protocol #1: AutoMACS Method

(Figure 13)

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Figure 13. Overview of brain endothelial cell isolation protocols. Total brain cell

suspension was created with a combination of mechanical and enzymatic dissociation

followed by magnetic-based myelin depletion. Subsequently, brain endothelial cells

were isolated either using an AutoMACS protocol, a magnetic-based sorting method for

CD45-CD31+ cells, or by a FACS protocol, a sorting method for Tie2+PDGFRβ- cells

derived from transgenic Tie2-GFP mice.

Twelve-week-old male FVB mice (n=4 per group for microarray studies, n=3 per group

for celecoxib studies) were anesthetized with IP ketamine hydrochloride (200mg/kg) and

xylazine hydrochloride (20mg/kg) and subsequently transcardially perfused with ice-cold

sterile PBS (14190-144, Gibco by Life Technologies, Waltham, MA) for 2min, similar to

the perfusions for the cadaverine-based studies described above. Brains were extracted

and placed in a petri dish with ice-cold sterile PBS. The cerebellum and

brainstem/diencephalon were removed by sharp dissection. The cerebral hemispheres

were sharply separated. Because the majority of cadaverine leakage occurred in the left

ventral region, only the left cerebral hemisphere was used for the brain endothelial cell

isolation protocol to maximize the signal magnitude. The meninges of the left cerebral

hemisphere were dissected off and the brain tissue was gently rolled on sterile filter

paper to remove pial vessels from the dorsal brain surface, and circle of Willis arteries

and subarachnoid blood clot from the basal surface. The purpose of these maneuvers

was to minimize inclusion of endothelial cells from the arteries and larger arterioles and

to minimize ex vivo activation of isolated endothelial cells by the subarachnoid blood

clot. The left cerebral hemisphere was reproducibly dissociated by a combination of

mechanical means with the brain dissociation protocol from the gentleMACS system

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using C-tubes (130-093-237, Miltenyi Biotec, Auburn, CA) and by enzymatic means with

the papain-based Neural Tissue Dissociation Kit (130-092-628, Miltenyi Biotec, Auburn,

CA). Because our cell surface marker of interest, PECAM1/CD31, is somewhat

sensitive to papain enzymatic digestion, we used 10% of the usual volume of stock

papain enzyme (5μL instead of 50μL). The left cerebral hemisphere was dissociated in

2mL volume of buffer containing 0.067mM of β-mercaptoethanol (M6250, Sigma-

Aldrich, St. Louis, MO) and enzyme mix as per the instruction manual. After a series of

incubation and mechanical dissociation steps, the suspension was then passed through

a 70μm nylon cell strainer (352350, Falcon), washed with sterile Hank’s Balanced Salt

Solution without calcium or magnesium (HBSS-/-, 14175-095, Gibco by Life

Technologies, Waltham, MA), and centrifuged at 300g for 10min. The supernatant fluid

was discarded and pellet was resuspended with 1,800μL of buffer containing HBSS-/-

and 0.5% BSA (30% stock solution, A9576, Sigma-Aldrich, St. Louis, MO). All

subsequent steps used the HBSS-/- with 0.5% BSA as buffer. The brain suspension

was incubated with 200μL of myelin removal beads II for 15min (IgM mouse anti-mouse

[Immunoglobulin M], 130-096-733, Miltenyi Biotec, Auburn, CA). After another wash and

centrifugation at 300g for 10min, the pellet was resuspended with 2mL of buffer and

transferred to 5mL polypropylene round-bottom tubes (352063, Falcon). Using the

“deplete” protocol from the AutoMACS Pro Separator (130-092-545, Miltenyi Biotec,

Auburn, CA) and fresh pack of AutoMACS separation columns (130-021-101, Miltenyi

Biotec, Auburn, CA), the brain suspension was depleted of myelin, creating a 4mL

output of “Total Cell Suspension.” For all AutoMACS magnetic sorting, we used the

AutoMACS Running Buffer which contains BSA (130-091-221, Miltenyi Biotec, Auburn,

CA). This suspension was centrifuged at 300g for 10min. After removal of the

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supernatant fluid, the cell pellet was resuspended with 85μL of buffer and 5μL of Fc

receptor blocker (CD16/32, IgG rat anti-mouse [Immunoglobulin G], 553141, BD

Pharmingen) for 5min, followed by incubation with 10μL of CD45 microbeads (IgG2b rat

anti-mouse, 130-052-301, Miltenyi Biotec, Auburn, CA) for 15min. After a wash and

centrifugation at 300g for 5min, the cell pellet was resuspended with 500μL of buffer.

Using the “depleteS” protocol from the AutoMACS Pro Separator, the total cell

suspension was depleted of CD45, creating 2.5mL output of CD45- cell suspension. A

CD45 depletion step was essential as leukocytes also express CD31 which could

confound results. This suspension was centrifuged at 300g for 10min and the

supernatant fluid was removed. The cell pellet was resuspended with 80μL of buffer and

incubated with 20μL of CD31/PECAM1 (endothelial marker) microbeads (IgG2a rat anti-

mouse, 130-097-418, Miltenyi Biotec, Auburn, CA) for 15min. After a wash and

centrifugation at 300g for 5min, the cell pellet was resuspended with 500μL of buffer.

Using the “posseld” protocol from the AutoMACS Pro Separator which utilizes 2

magnetic columns and creates a small elution volume ideal for antigen expression of

less than 5% of cells, the CD45- cell suspension was enriched for CD31, creating 500μL

output of CD45-CD31+ cells or “Endothelial Cell Suspension.” The negative fraction

CD45-CD31- cell suspension was also collected and designated as the “Parenchymal

Cell Suspension.” Quality control included assessment of cell viability by uptake of

intracellular dye propidium iodide (PI, 1:1,000, P4864, Sigma-Aldrich, St. Louis, MO) on

flow cytometry (MACSQuant, Miltenyi Biotec, Auburn, CA). Endothelial cell suspension

(CD45-CD31+) purity was assessed by flow cytometry using conjugated fluorescent

antibodies (Rat anti-mouse CD31-FITC [Fluorescein isothiocyanate], 553372, BD

Pharmingen; Rat anti-mouse CD45-APC [Allophycocyanin], 17-0451-82, eBioscience)

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and their respective rat isotype controls (IgG2a-FITC, 553929, BD Pharmingen; IgG2a-

APC, 553932, BD Pharmingen). Typical viability was > 80% and CD45-CD31+ purity >

90%. All buffers (HBSS-/-, HBSS-/- with 0.5% BSA) were kept on ice, equilibrated with

oxygen probes (95% O2, 5% CO2) and pH adjusted to 7.4. All steps after enzymatic

digestion were performed at 4oC. Two independent biological samples were processed

simultaneously per day (typically one SAH left hemisphere and one Sham left

hemisphere), and the order of processing alternated by day (e.g. SAH followed by

Sham one day, Sham followed by SAH the next day). Time from transcardial perfusion

to placement in RLT extraction buffer (Qiagen Inc, Venlo, Netherlands) was typically

3.5h.

RNA Extraction. Endothelial cell suspension derived from the AutoMACS isolation

protocol (CD45-CD31+ cells), as well as the total cell and parenchymal cell

suspensions, was centrifuged for 5min at 300g at 4oC. The supernatant fluid was

carefully aspirated followed by addition of 75μL of RLT extraction buffer (Qiagen Inc,

Venlo, Netherlands) containing 1% β-mercaptoethanol. Addition of 0.025ng of luciferase

mRNA plasmid (pSP-luc+NF cloning vector, U47123.2) in a 10μL volume was

performed for determining first strand synthesis and amplification efficiencies of each

sample. Samples were then transferred to a QIAshredder spin column (79654, Qiagen

Inc, Venlo, Netherlands) and centrifuged at 25,000g for 2min to ensure complete cell

lysis. Total RNA was extracted using the RNeasy micro kit (74004, Qiagen Inc, Venlo,

Netherlands) as per the provided instructions manual. On-column DNase I was used

(10μL reconstituted stock DNase I, 70μL RDD buffer, 79254, Qiagen Inc, Venlo,

Netherlands). For RNA elution, 15μL volume of RNase-free water was used. RNA

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quantity and quality were assessed using Nanodrop 2000 (Thermo Scientific, Waltham,

MA) and Agilent 2100 Bioanalyzer with the RNA 6000 Pico Reagents Part I Series II

(Agilent Technologies, Santa Clara, CA). Remaining RNA samples were stored at -80oC

until further downstream gene expression assays. Typical RNA concentrations were

0.5-2ng/μL with RNA integrity number (RIN) > 7.0.

4.2.7 Brain endothelial cell isolation protocol #2: FACS Method (Figure 13)

An alternative strategy of FACS was used in 12-15-month-old male Tg(Tie2-

GFP)287Sato/J mice (n=4 per group) to isolate brain endothelial cells with significant

modifications to a previously-reported isolation strategy [Daneman 2010]. Brain

extraction and dissociation steps used the papain-based Neural Tissue Dissociation Kit

and gentleMACS systems, like the AutoMACS method described above. However, in

this protocol, the full 50µL volume of papain was used. Magnetic-based myelin depletion

using the AutoMACS Pro Separator was used like above, creating 4mL output of “Total

Cell Suspension.” We incubated this cell suspension with PDGFRβantibodies

conjugated to the fluorescent dye APC (1:1,000, A18383, molecular probes by Life

Technologies, Waltham, MA) for 10min at 4oC. PI (1:1,000) was added and cells were

sorted by FACS with BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA) at

4oC. Non-fluorescent FVB mouse brain homogenates were used to gate for GFP+ cells.

Total cell suspension derived from Tg(Tie2-GFP)287Sato/J mice but not stained with

PDGFRβ-APC was used to gate for APC+ cells. Gating parameters were selected for

live single cells that were GFP+ and APC- (Tie2+PDGFRβ-), which were sorted into

200µL of HBSS-/- with 0.5% BSA and 10% 0.5M ethylenediaminetetraacetic acid

(EDTA). For brain endothelial cell purity assessment, a small aliquot of cell suspension

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underwent a second confirmatory sort. FACS time was typically 1h. Time from

transcardial perfusion to placement in RNA extraction buffer was typically 3h. Only a

single biological replicate was processed per day. FACS analysis was performed with

BD FACSDiva Software Version 8.0.1 (BD Biosciences, San Jose, CA).

RNA Extraction. Total RNA from brain endothelial cells derived from the FACS method

(Tie2+PDGFRβ-) was extracted using the Arcturus PicoPure RNA isolation kit

(KIT0204, Applied Biosystems, Foster City, CA). After obtaining FACS-derived

Tie2+PDGFRβ- cells, the cell suspension was centrifuged at 1,000g for 10min at 4oC.

The supernatant fluid was carefully removed followed by addition of 100µL of RNA

extraction buffer containing 1% β-mercaptoethanol. This mixture was incubated at 42oC

for 30min with gentle agitation. RNA extraction was performed according to the

instruction manual of the kit. On-column DNase was added (10µL of DNase stock, 30μL

of RDD buffer per sample). For RNA elution, 11.5μL of elution buffer was used. RNA

quantity and quality were assessed using Nanodrop 2000 and Agilent 2100 Bioanalyzer

like above. Remaining RNA samples were stored at -80oC until further downstream

gene expression assays were performed. Typical RNA concentrations were 0.5-1ng/μL.

However, the RINs from FACS-derived brain endothelial cells were generally poor

(range 2.0-7.0) compared to RNA derived from AutoMACS-derived brain endothelial

cells. Hence, only the RNA from AutoMACS-derived brain endothelial cells (CD45-

CD31+ cells) were used for amplification and microarray analysis.

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4.2.8 Microarray

Equal quantities of extracted RNA derived from CD45-CD31+ brain endothelial cells

(n=4 per group, with each sample being a single unique biological replicate without

pooling) were amplified using the Ovation Pico WTA System V2 (NuGEN, San Carlos,

CA), which utilizes unique RNA-DNA hybrid oligo-dT and random hexamer primers for

linear amplification. Subsequently, 5.0μg of cDNA (Complementary deoxyribonucleic

acid) was fragmented and labeled using the Encore Biotin Module (NuGEN, San Carlos,

CA) and hybridized to Affymetrix Mouse Gene 2.0 ST Arrays (Affymetrix, Hercules, CA)

for 18h at 45oC at 60 revolutions per minute. This microarray platform utilizes 21

perfect-match oligo-probes for each of 41,345 probe-sets representing 35,240 RefSeq

transcripts including 26,191 well-established protein-coding genes and 3,391 well-

established non-coding genes, which includes approximately 2000 lncRNAs. Arrays

were then washed using GeneChip Fluidics Station P450 and scanned with Affymetrix

GeneChip Scanner 7G (Affymetrix, Hercules, CA). Hybridization controls were similar

across all arrays. Microarray hybridizations were performed with the assistance of the

Princess Margaret Genomics Centre (PMGC, Toronto, Canada, www.pmgenomics.ca).

Microarray raw intensity values were normalized using the robust multi-array average

(RMA) algorithm with R statistical software (R Foundation for Statistical Computing, R

version 3.2.2), with the “oligo” package derived from Bioconductor version 3.2 [Irizarry

2003]. Clustering of sample groups were determined using unsupervised hierarchical

clustering, Pearson’s correlation and principal component analysis (PCA). Differentially-

expressed genes (SAH vs. Sham) were identified using independent 2-tailed t-tests with

multiple testing correction via the Benjamini-Hochberg false discovery rate (FDR)

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method (significance level of 0.05) [Benjamini 2001]. Heatmaps were generated using

the “gplots” package. Volcano plot was generated using the “plot” function and

“calibrate” packages. PCA plot was generated using the GeneSpring v13.1 software.

Gene Set Enrichment Analysis (GSEA) was used to identify pathways relevant to the

patterns of gene expression observed [Subramaniam 2005]. Mouse-specific gene

pathways were interrogated using the “gskb” (Gene Set Knowledgebase) package,

which contains 33,261 gene sets from 40 sources involving gene ontology (GO),

chromosome location, metabolic pathways, curated pathways, and target genes of

transcription factors.

4.2.9 RT-PCR Validations

Microarray results were validated by RT-PCR using amplified cDNA samples derived

from CD45-CD31+ cells. As well, unamplified total RNA derived from CD45-CD31+ and

Tie2+PDGFRβ- was converted to cDNA using Superscript III First-Strand Synthesis

SuperMix for quantitative RT-PCR (11762050, Invitrogen by Life Technologies,

Waltham, MA). Primers were designed using Oligo Primer Analysis Software version 7

(Molecular Biology Insights, Inc., Cascade, CO) (Table 5). Primers were obtained from

IDT (Integrated DNA Technologies, Coralville, IA) for the following genes: luciferase

(pSP-luc+NF cloning vector), Actb (β-actin), Ppia (Peptidylprolyl isomerase A,

Cyclophilin A), Ptgs1 (Prostaglandin-endoperoxide synthase 1, also called

Cyclooxygenase-1 or COX-1), Ptgs2 (COX-2), Angpt1 (Angiopoietin-1), Angpt2, Tie2,

Vegfr2, Mfsd2a, Pecam1 (CD31), Cdh5 (VE-Cad), Vcam1, and Klf2 (Krüppel-like factor

2). Equal amounts of cDNA underwent RT-PCR with technical triplicates with SYBR

Green PCR master mix (4309155, Applied Biosystems, Foster City, CA) and ABI Prism

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7900 Real-Time PCR System (Applied Biosystems, Foster City, CA), undergoing 40

cycles of 15s at 95oC followed by 1min at 60oC. Relative mRNA expression was

determined using ΔΔCt method with the sham sample as the experimental control and

β-actin as the house-keeping gene [Schmittgen 2008]. First-strand synthesis efficiency

was determined by dividing the measured absolute copy numbers of luciferase plasmid

using a standard curve from RT-PCR with the known quantity of luciferase plasmid

added at the time of RNA extraction. Similarly, amplification efficiency was determined

by dividing the measured absolute copy numbers of luciferase plasmid from amplified

CD45-CD31+ cDNA with the absolute copy numbers of luciferase plasmid from

unamplified CD45-CD31+ cDNA.

Table 5: Forward and reverse primer sequences used for RT-PCR.

Gene name

Accession

Number Forward Primer (5' - 3') Reverse Primer (5' - 3')

Actb (β-actin) NM_007393.5 AAC CGT GAA AAG ATG ACC CAG AT CAC AGC CTG GAT GGC TAC GTA

Angpt1 NM_009640.4 GGC CAC CAT GCT TGA GAT AG CAA GTC GGG ATG TTT GAT TTA

Angpt2 NM_007426.4 GCA GCT TCT CCA ACA TTC TA CCT CCA TGT CCA GAA CTT TC

Cdh5 (VE-cadherin) NM_009868.4 TGC CCA CCA TCG CCA AAA GAG AGA C CTG GCG GTT CAC GTT GGA CTT G

Hif1a NM_001313919.1 TCA GTT GCC ACT TCC CCA CAA AGA CCA CCG GCA TCC AGA AGT T

Kdr/Vegfr2 NM_010612.2 ACC GGG ACG TCG ACA TAG CAC TGA CAG AGG CGA TGA A

Klf2 NM_008452.2 CTG CGG CAA GAC CTA CAC CAA CGC ATC CTT CCC AGT TGC AAT GAT A

Luciferase U47123.2 ACT CCT CTG GAT CTA CTG GTC GTA ATC CTG AAG GCT CCT CA

Mfsd2a NM_029662.2 GGC TGC GCA CTG GGA TTC T CCA GGC TCG GCC CAC AAA

Pecam1 (CD31) NM_008816.3 TCA GAA CCC ATC AGG AGT GAA TAC G TGC TTG GAG GTG GCT ACA ATC

Ptgs1 (COX-1) NM_008969.4 GAC CAC TCG CCT CAT CCT TAT A TCA AAC TTG AGC TGC AGG AAA TA

Ptgs2 (COX-2) NM_011198.4 CCT TCC TGC GAA GTT TAA T GGT GGA CTG TCA ATC AAA TAT

Rela (p65) NM_009045.4 TGCG GTG GGG ATG AGA TCT T CAG CCT GGT CCC GTG AAA TA

Tie2 NM_013690.3 GTT CGA GGA CAG GCT ATA A TGT CCA CGG TCA TAG TTA AA

Vcam1 NM_011693.3 GGG AAG CTG GAA CGA AGT AT GGG GCC ACT GAA TTG AAT CTC

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4.2.10 Immunofluorescence

Mice were transcardially perfused with PBS for 2min followed by 4% PFA for 5min at a

constant pressure of 100mmHg. Brains were extracts and fixed in 4% PFA at 4oC for

24h. A coronal cut was made 4mm from the anterior pole. Brain tissue were then

sequentially dehydrated using the Leica TP 1020 Automatic Tissue Processor (Leica,

Wetzlar, Germany) followed by paraffin embedding. Coronal slices 5µm thick were

created using a microtome. For immunofluorescence histology, slides with coronal brain

slices were gradually rehydrated, followed by heat-mediated antigen retrieval.

Specimens were then blocked with 10% goat serum. Primary antibodies were incubated

for 1h at room temperature or overnight at 4oC. After washes, secondary antibodies

were incubated for 1h at room temperature. After more washes, specimens were

incubated with DAPI for 15min, followed by more washes, and mounting with aqueous

media and glass coverslip. Images were taken using the Olympus inverted fluorescent

microscope (Olympus, Center Valley, PA) and Zeiss confocal microscope (Zeiss,

Oberkochen, Germany). Primary antibodies included rabbit anti-mouse COX-2 (Abcam,

Cambridge, UK, 1:200), mouse anti-mouse NeuN (Millipore, Darmstadt, Germany,

1:400, neuronal marker) and rabbit anti-mouse Caspase3 (BD Pharmingen, San Jose,

CA, 1:300). Secondary antibodies included goat anti-rabbit Alexa Fluor 568 (Life

Technologies, Waltham, MA) and goat anti-mouse Alexa Fluor 488 (Life Technologies,

Waltham, MA). Image processing included using the triangle algorithm for auto

thresholding and the “analyze particles” function on Image J (NIH, Bethesda, MD).

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4.2.11 Fluoro-jade b staining

Paraffin-embedded coronal slices were gradually deparaffinized and rehydrated.

Microscope slides were placed in 0.06% KMnO4 (Sigma-Aldrich, St. Louis, MO) for 8min

and then rinsed multiple times with deionized water. Microscope slides were then

placed in 0.001% FJB solution (Histo-Chem Inc., Jefferson, Arkansas) for 30min. Slides

were then washed, dried, and placed in xylene prior to being cover slipped with non-

aqueous DPX mounting medium (Sigma-Aldrich, St. Louis, MO). Images were taken

using the Olympus inverted fluorescent microscope (Olympus, Center Valley, PA).

Image processing included using the triangle algorithm for auto thresholding and the

“analyze particles” function on Image J (NIH, Bethesda, MD).

4.2.12 Enzyme-linked immunosorbent assay (ELISA)

Whole blood samples were obtained from mice via cardiac puncture at the time of

transcardial perfusion. Serum samples were obtained by allowing whole blood samples

to clot at room temperature for 2h, followed by centrifugation at 2,000g x 20min, and

storage of the supernatant component at -20oC until the time of usage. Mouse brain

tissue was extracted at the time of transcardial perfusion, snap frozen in liquid nitrogen

and stored at -80oC until the time of homogenization. Brain tissue was homogenized in

1mL of sterile PBS and centrifuged at 5,000g x 5min, with the supernatant component

subsequently transferred to a separate tube and stored at -20oC until the time of usage.

Brain tissue homogenates and serum samples were assessed using commercially-

available ELISA (Enzyme-linked immunosorbent assay) kits for TIE2 (Mouse TIE-2

Quantikine ELISA kit, MTE200, R&D Systems, Minneapolis, MN), ANGPT1 (Mouse

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Angiopoietin-1 ELISA kit, LS-F13065, LSBio Inc, Seattle, WA), and ANGPT2 (Mouse/rat

Angiopoietin-2 Quantikine ELISA kit, MANG20, R&D Systems, Minneapolis, MN).

Serum samples were diluted 2-fold, 40-fold, and 40-fold for the TIE2, ANGPT1, and

ANGPT2 ELISA experiments, respectively. Brain homogenates were diluted 2-fold, 1-

fold, and 10-fold for the TIE2, ANGPT1, and ANGPT2 ELISA experiments, respectively.

Absorbance values were detected by spectrophotometer (SpectraMax M5e, Molecular

Devices, Sunnyvale, CA) and analyzed with SoftMax Pro 5 (Molecular Devices,

Sunnyvale, CA).

4.3 Results

4.3.1 Experimental SAH caused neurobehavioural deficits and histological

brain injury

In our SAH model, CBF decreased consistently after SAH induction with a gradual rise

over 10min, but not quite reaching base-line CBF (Figure 14). CBF was consistently

lower in SAH mice compared to sham mice at all time points after SAH induction. SAH

resulted in 27% (3/11) mortality, with mortality typically occurring within 30min after

blood injection, whereas sham procedure resulted in no mortality (0/8) (Figure 15A).

Weight did not change significantly from base-line after SAH although there was a trend

toward decreasing weight at 48h after SAH (p=0.06, Figure 14). Mice displayed

significant neurobehavioural impairment after SAH at both measured time-points as

determined by the MGS (24h and 48h, Figure 15B). SAH mice also demonstrated

decreased spontaneous activity after SAH (Figure 14). Twenty-four hours after SAH,

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there was an increase in neuronal apoptosis (Caspase3+NeuN+ cells) and neuronal

degeneration (FJB+ cells), particularly in the ventral cortical region (Figure 15C-F).

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Figure 14. SAH model characteristics. (a) Intraoperative cerebral blood flow (CBF)

measurements after SAH induction. Data points represent means ± SEM. (b) Weight

change from base-line, 24h and 48h after SAH. (c) Spontaneous activity assessment, a

component of the Modified Garcia Score (MGS), determined by the amount of time

required for mice to touch 3 out of 4 walls with 2 paws simultaneously. n=4 per group.

Data presented as means ± SEM. Two-way ANOVA with Holm-Sidak post hoc

correction (CBF). Kruskal-Wallis test (Weight change, spontaneous activity). *p<0.05,

**p<0.01, ***p<0.001 (relative to Sham 24h or Sham 48h).

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Figure 15. SAH model neurobehavioural and histological outcomes. (a) Mortality after

SAH. (b) Modified Garcia Score (MGS) at 24h and 48h after SAH. (c) Representative

confocal double-immunofluorescence images of NeuN (red), a neuronal marker, and

activated Caspase3 (green), an apoptosis marker, from coronal brain slices. DAPI stain

for nuclei (blue). Arrows indicate cells that are positive for both NeuN and activated

Caspase3. (d) Quantitative assessment of apoptotic neurons in the left ventral region.

(e) Representative fluorescent microscopy images of FJB positive cells (arrows) from

coronal brain slices, indicative of neuronal degeneration. (f) Quantitative assessment of

FJB positive cells in the left ventral region. n=4-5 per group. Data presented as means ±

SEM. Kruskal-Wallis test (MGS). t-test (Caspase3, FJB). *p<0.05, **p<0.01.

4.3.2 Experimental SAH caused blood-brain barrier disruption

BBB disruption, as evidenced by cadaverine dye extravasation, was most evident 24h

after SAH compared with 48h (Figure 16A, C). The BBB disruption was predominantly

ipsilateral to the needle insertion (left cerebral hemisphere) with a ventral cortex bias.

Confocal images demonstrated that the cadaverine dye was clearly extravascular with

parenchymal cell dye uptake at 24h (Figure 16B, D). Consistent reabsorption of

cadaverine dye from the IP space into the systemic circulation was observed, based on

fluorescent imaging of the kidneys. Because BBB disruption was most prominent at 24h

compared with 48h after SAH, further studies involving brain endothelial cells used the

24h time-point. At 24h after SAH, the tight junctions did not qualitatively change based

on transmission electron microscopy (TEM) (Figure 16E).

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Figure 16. Blood-brain barrier (BBB) disruption after SAH. (a) Representative whole

brain fluorescent imaging of the ventral surface after IP cadaverine dye injections (red).

Intensity measurements taken from the left ventral region of interest (yellow marking).

(b) Representative merged confocal microscopy images of coronal brain slices after

cadaverine dye injections (red) with endothelial cells marked endogenously by GFP

driven by the Tie2 promoter (grey) and nuclei marked by DAPI (blue). (c) Quantification

of cadaverine dye seen in the left ventral region after SAH, normalized to Sham 24h

group. (d) Quantification of extravascular parenchymal cells with cadaverine dye uptake

from the left cerebral hemisphere after SAH. n=4-5 per group. Data presented as means

± SEM. One-way ANOVA with Holm-Sidak post-hoc correction, *p<0.05 (relative to

Sham 24h or Sham 48h). (e) Transmission electron microscopy (TEM) of left ventral

region showing intraparenchymal blood vessel and tight junctions marked by the black

circles. n=3 per group.

4.3.3 Mouse brain endothelial cell isolation by magnetic-based and

fluorescence-activated cell sorting

Using a single freshly-extracted left cerebral hemisphere, which represented the region

of highest BBB disruption, brain endothelial cells were successfully isolated by both

magnetic-based (CD45-CD31+) and FACS-based (Tie2+PDGFRβ-) methods with >

90% purity on flow cytometry (Figure 17A-B). These brain endothelial cells also had >

80% viability (Figure 18). On average, approximately 50,000 and 25,000 brain

endothelial cells were isolated from the magnetic-based and FACS-based protocols

respectively. Prototypical endothelial genes Pecam1, Tie2, and Vegfr2 were significantly

enriched in CD45-CD31+ cells compared to the corresponding parenchymal cell fraction

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(CD45-CD31- cells) and total cell suspension, as determined by RT-PCR (Figure 17C).

Using microarray raw intensity values, amplified RNA derived from CD45-CD31+ brain

endothelial cells showed significant enrichment of prototypical brain endothelial cell

genes and relatively low levels of prototypical genes of other cell types (Figure 19).

There was also significant enrichment of pan-endothelial (Tie2, Cdh5/VE-Cad, Pecam1)

and BBB endothelial genes (Slc2a1/Glut1, Abcb1a/Mdr1a) compared with low levels of

genes more specifically expressed by arterial, venous, or lymphatic endothelial cells

(Figure 17D). Hence, the majority of isolated CD45-CD31+ endothelial cells were

derived from the BBB.

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Figure 17. Brain endothelial cell isolation. (a) Representative flow cytometry

assessment of magnetically-sorted CD45-CD31+ brain endothelial cells demonstrating

viability > 80% and purity > 90%. (b) Representative FACS of single, viable

Tie2+PDGFRβ- brain endothelial cells demonstrating both viability and purity > 95%.

Tie2+PDGFRβ+ cells are designated as perivascular cells (pericytes and perivascular

macrophages). (c) Enrichment of prototypical endothelial genes Pecam1 (CD31), Tie2,

and Vegfr2 in CD45-CD31+ brain endothelial cells compared to parenchymal cells

(CD45-CD31-) and total cell suspension, using RT-PCR. n=4 per group. Data presented

as means ± SEM. One-way ANOVA with Holm-Sidak post-hoc correction, *p < 0.05

compared to CD45-CD31- cells or total cell suspension. (d) Microarray intensities of

endothelial genes typically expressed from different regions of the cerebrovascular tree

and lymphatic vessels. n=4 per group. Data presented as means ± SEM. One-way

ANOVA with Holm-Sidak post-hoc correction. *p<0.05 vs. “All EC” genes or “BBB EC”

genes. Abbreviations: Abcb1a/Pgp/Mdr1a: ATP-binding cassette, sub-family B

(MDR/TAP), member 1A / P-glycoprotein / Multidrug resistance protein 1a; APC:

Allophycocyanin; Cdh5/VE-Cad: Cadherin 5; EC: Endothelial cell; Efnb2: Ephrin-B2;

Ephb4: Ephrin type-B receptor 4; FITC: Fluorescein isothiocyanate; Flt4/Vegfr3: Fms-

related tyrosine kinase 4 / Vascular endothelial growth factor receptor 3; FSC-A:

Forward scatter area; FSC-H: Forward scatter height; GFP: Green fluorescent protein;

Lefty2: Left-right determination factor 2; Lyve1: Lymphatic vessel endothelial hyaluronan

receptor 1; Nos3/eNOS: Nitric oxide synthase 3 / endothelial nitric oxide synthase;

PDGFRβ: Platelet-derived growth factor receptor beta; Pecam1: Platelet endothelial cell

adhesion molecule 1; PI: Propidium iodide; PVC: Perivascular cell; SSC-A: Side scatter

area; Slc2a1/Glut1: Solute carrier family 2, member 1 / Glucose transporter 1; SSC-W:

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Side scatter width; Tie2: Tyrosine kinase with immunoglobulin and epidermal growth

factor homology domains 2; Vegfr2: Vascular endothelial growth factor receptor 2.

Figure 18. Brain endothelial cell viability. Average viability of isolated CD45-CD31+

cells (a) and Tie2+PDGFRβ- cells (b). Data presented as means ± SEM

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Figure 19. Expression levels of prototypical genes of various cell types in isolated

CD45-CD31+ cells. Microarray intensities of prototypical RNA transcripts of various cell

types including (a) neurons, (b) microglia, (c) oligodendrocytes, (d) red blood cells, (e)

vascular smooth muscle cells, (f) astrocytes, (g) pericytes, and (h) blood-brain barrier

endothelial cells. n=4 per group. Data presented as means ± SEM. Abbreviations: Abo:

Transferase A, alpha 1-3-N-acetylgalactosaminyltransferase, transferase B, alpha 1-3-

galactosyltransferase; Aldh1l1: Aldehyde dehydrogenase 1 family, member L1; Anpep:

Alanyl (membrane) aminopeptidase; Aqp4: Aquaporin 4; Cspg4/NG-2: Chondroitin

sulfate proteoglycan 4; Gad1: Glutamate decarboxylase 1; Gfap: Glial fibrillary acidic

protein; Hba-a2/Hba-a1: Hemoglobin alpha, adult chain 1 / hemoglobin alpha, adult

chain 2; Itgam/Iba1: Integrin alpha M; Lgals3/Galectin3: Lectin, galactose binding,

soluble 3; Mbp: Myelin basic protein; Mog: Myelin oligodendrocyte glycoprotein; Myh2:

Myosin, heavy polypeptide 2; Myocd: Myocardin; Olig2: Oligodendrocyte transcription

factor 2; PDGFRβ: Platelet-derived growth factor receptor beta; Pecam1/CD31:

Platelet/endothelial cell adhesion molecule 1; Ptprc/CD45: Protein tyrosine

phosphatase, receptor type, C; Spta1: Spectrin alpha, erythrocytic 1; Syp:

Synaptophysin; Tagln3/SM22: Transgelin 3; Tie2: Tyrosine kinase with immunoglobulin

and epidermal growth factor homology domains 2; Th: Tyrosine hydroxylase; Tjp1/ZO-1:

Tight junction protein 1 / Zonula occludens 1.

Because the amount of RNA extracted from brain endothelial cells was relatively low, an

amplification step was critical to be able to perform whole genome expression profiling.

Absolute copy numbers of exogenously-added luciferase mRNA plasmid from pre-

amplified and post-amplified samples reveal similar amplification efficiencies between

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brain endothelial cells derived from Sham and SAH mice (Figure 20). A priori, we

identified 3 genes that we predicted would remain relatively unchanged (Pecam1/CD31,

Cdh5/VE-Cad, Icam2 [Intercellular adhesion molecule 2]), 1 gene that would increase

after SAH (Vcam1), and 1 gene that would decrease after SAH (Klf2). We found similar

expression patterns among these genes among pre-amplified and post-amplified

samples by RT-PCR, suggesting that the amplification process likely did not significantly

distort the global gene expression patterns (Figure 20).

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Figure 20. Effect of RNA amplification on gene expression. (a) Amplification efficiency,

as determined by fold change in absolute copy number of exogenously-added luciferase

mRNA plasmid after RNA linear amplification with Ovation Pico WTA System V2

(NuGEN, San Carlos, CA). (b, c) RT-PCR of several endothelial genes, selected a

priori, in unamplified and amplified samples derived from brain endothelial cells (CD45-

CD31+). n=4 per group. Data presented as means ± SEM. t-test, *p<0.05, **p<0.01.

Abbreviations: Cdh5/VE-Cad: Cadherin 5 / Vascular-endothelial cadherin; Icam2:

Intercellular adhesion molecule 2; Klf2: Kruppel-like factor 2; Pecam1/CD31: Platelet-

endothelial cell adhesion molecule 1; Vcam1: Vascular cell adhesion molecule 1.

4.3.4 Gene expression patterns distinguish SAH from sham brain

endothelial cells

Pearson’s correlation, PCA, and unsupervised hierarchical clustering reveal distinct

SAH and Sham brain endothelial cell sample groupings (Figure 21A-C). There were

707 (2.8%) significantly differentially-expressed genes in brain endothelial cells after

SAH out of 24,865 interrogated probe sets which passed quality control measures. The

Affymetrix Mouse Gene 2.0 ST Array contains 35,240 total transcripts including 26,191

coding transcripts with well-established annotation and approximately 2000 lncRNA

transcripts. There were 403 significantly upregulated genes (236 gene upregulated at

least 1.5-fold) and 304 significantly downregulated genes (200 genes downregulated to

75% of base-line or less) (Figure 21D). The top 50 upregulated and downregulated

genes are displayed in Figure 22A-B.

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Figure 21. Clustering of SAH and sham samples based on brain endothelial cells

expression patterns. (a) Pearson’s correlation, (b) principal component analysis (PCA),

and (c) unsupervised hierarchical clustering demonstrating clustering of SAH and sham

samples. (d) Volcano plot showing 707 (2.8%) significant differentially-expressed genes

(403 upregulated [236 upregulated > 1.5-fold – red dots], 304 downregulated [200

downregulated to < 75% of baseline – blue dots]) out of 24,865 interrogated gene probe

sets. Select genes of interest are labeled with green dots. n=4 per group. Abbreviations:

Angpt2: Angiopoietin-2; Mfsd2a: Major facilitator superfamily domain containing 2a;

Ptgs2/COX-2: Prostaglandin-endoperoxide synthase 2; Tie2: Tyrosine kinase with

immunoglobulin and epidermal growth factor homology domains 2; Vegfr2: Vascular

endothelial growth factor receptor 2.

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Figure 22. Top differentially-expressed genes. Top 50 significantly (a) upregulated and

(b) downregulated genes in brain endothelial cells after SAH, ranked by fold change.

n=4 per group.

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4.3.5 Gene expression changes demonstrate enrichment in inflammatory

response genes

Examining expression of genes known to be enriched in BBB endothelial cells, there

was surprisingly very little change in endothelial cell barrier genes and an overall

modest downregulation of BBB transporter genes after SAH (Figure 23, 24). GSEA

revealed that the term “Inflammatory Response” to be the top GO biological process hit

(Table 6) and “Prostaglandin Synthesis and Regulation” to be one of the top pathways

involved (Table 7, Figure 25).

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Figure 23. Expression of BBB intercellular junction genes in brain endothelial cells after

SAH. *corrected p<0.05.

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Figure 24. Expression of BBB transporter genes in brain endothelial cells after SAH.

*corrected p<0.05 (upregulated), **corrected p<0.05 (downregulated).

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Table 6: Gene Set Enrichment Analysis (GSEA) with Gene Ontology (GO) data sets.

GENE ONTOLOGY (GO) DATA SETS NES FDR q-value

GO - BIOLOGICAL PROCESS

INFLAMMATORY RESPONSE 1.76 0.091

HETEROPHILIC CELL-CELL ADHESION 1.73 0.095

OOGENESIS 1.73 0.087

REGULATION OF APOPTOTIC PROCESS 1.72 0.081

RESPONSE TO HYDROGEN PEROXIDE 1.71 0.086

POSITIVE REGULATION OF CAMP

BIOSYNTHETIC PROCESS 1.71 0.084

CELL-CELL ADHESION 1.70 0.082

PEPTIDE CROSS-LINKING 1.70 0.080

CYTOKINE PRODUCTION 1.69 0.084

RESPONSE TO LIPOPOLYSACCHARIDE 1.68 0.087

ACTIVATION OF MAPK ACTIVITY 1.68 0.086

PLACENTA DEVELOPMENT 1.67 0.092

REGULATION OF BLOOD PRESSURE 1.67 0.089

SUPEROXIDE METABOLIC PROCESS 1.67 0.086

RESPONSE TO OXIDATIVE STRESS 1.66 0.095

ADULT LOCOMOTORY BEHAVIOUR -2.14 0.01

SYNAPTIC TRANSMISSION -1.95 0.024

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GO - CELLULAR COMPONENT

SECRETORY GRANULE 1.73 0.081

HIGH-DENSITY LIPOPROTEIN PARTICLE 1.71 0.081

GO - MOLECULAR FUNCTION

CALCIUM-DEPENDENT PROTEIN BINDING 1.79 0.093

CYTOKINE ACTIVITY 1.75 0.097

PROTEASE BINDING 1.70 0.083

METALLOPEPTIDASE ACTIVITY 1.70 0.083

TRANSPORTER ACTIVITY 1.69 0.077

Abbreviations: FDR: False-discovery rate; NES: Normalized enrichment score.

Table 7: Gene Set Enrichment Analysis (GSEA) with Pathway Analysis data sets.

PATHWAY ANALYSIS DATA SETS NES FDR q-value

TH1 TH2 DIFFERENTIATION 1.81 0.042

PROSTAGLANDIN SYNTHESIS AND REGULATION 1.75 0.067

FOLATE METABOLISM 1.70 0.071

REGULATION OF ACTIN CYTOSKELETON 1.70 0.056

SELENIUM PATHWAY 1.68 0.054

MONOAMINE TRANSPORT 1.67 0.050

INFLAMMATORY RESPONSE PATHWAY 1.66 0.051

EBV LMP1 SIGNALING 1.65 0.056

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SELECTIVE EXPRESSION OF CHEMOKINE

RECEPTORS DURING T-CELL POLARIZATION 1.64 0.072

TYPE II DIABETES MELLITUS 1.64 0.076

APOPTOSIS 1.62 0.091

COMPLEMENT AND COAGULATION CASCADES 1.62 0.086

IL4 SIGNALING PATHWAY 1.61 0.093

FOLLICLE STIMULATING HORMONE SIGNALING

PATHWAY 1.61 0.088

MATRIX METALLOPROTEINASES 1.60 0.089

IL-4 SIGNALING PATHWAY 1.60 0.086

CYTOSKELETAL REGULATION BY RHO GTPASE 1.60 0.082

ROLE OF MITOCHONDRIA IN APOPTOTIC SIGNALING 1.58 0.097

CASPASE CASCADE IN APOPTOSIS 1.57 0.102

HIV-I NEF NEGATIVE EFFECTOR OF FAS AND TNF 1.56 0.098

CHAPERONES MODULATE INTERFERON SIGNALING

PATHWAY 1.55 0.099

KIT RECEPTOR SIGNALING PATHWAY 1.55 0.100

IL-2 RECEPTOR BETA CHAIN IN T CELL ACTIVATION 1.54 0.099

Abbreviations: FDR: False-discovery rate; NES: Normalized enrichment score.

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Figure 25. Gene set enrichment analysis (GSEA) demonstrating enrichment of genes

relevant to prostaglandin synthesis and regulation in SAH brain endothelial cell samples

(leading edge peak on upper left).

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4.3.6 Brain endothelial cells show increased Ptgs2 (COX-2) and Angpt2

expression after SAH

Validation studies confirmed increased mRNA expression of Ptgs2/COX-2

and Angpt2 in CD45-CD31+ brain endothelial cells after SAH (Figure 26A). COX-2, an

inducible enzyme associated with inflammation, was chosen for further study, given that

prostaglandin synthesis and regulation appeared to be a significantly upregulated pathway

in brain endothelial cells after SAH [FitzGerald 2003]. Angpt2 was chosen given its role in

the destabilization of the endothelial lining [Augustin 2009]. Congruent with the microarray

results, RT-PCR studies reveal no changes in expression of related genes Ptgs1 (COX-1)

and Angpt1. There was significantly decreased expression of Tie2 , Vegfr2 and Mfsd2a

after SAH (Figure 26B), based on both microarray intensities and RT-PCR.

Tie2+PDGFRβ- brain endothelial cells demonstrate similar gene expression findings

compared with CD45-CD31+ brain endothelial cells, demonstrating the robustness of our

results (Figure 26C-D). On visual inspection, there appeared to be increased COX-2

protein expression in the wall of parenchymal blood vessels on immunofluorescence

microscopy (Figure 26E). The protein expression of ANGPT2 was assessed by ELISA of

brain homogenates, revealing increased ANGPT2 expression and increased

ANGPT2/ANGPT1 ratio in brain tissue but not in serum after SAH (Figure 26F-G, Figure

27).

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Figure 26. Microarray validation studies. (a) RT-PCR of magnetic-sorted CD45-CD31+

brain endothelial cells validated increased expression of Ptgs2 (COX-2) and Angpt2,

confirming no change in Ptgs1 (COX-1) and Angpt1, and (b) validated decreased

expression of Tie2, Vegfr2, and Mfsd2a after SAH. Similar gene expression patterns

were seen with FACS-derived Tie2+PDGFRβ- brain endothelial cells (c, d). Relative

mRNA expression was normalized to Actb (β-actin). n=4 per group. t-test with Holm-

Sidak post-hoc correction *p<0.05, **p<0.01, ***p<0.001. Data presented as means ±

SEM. (e) Representative double-immunofluorescence confocal images of coronal brain

slices for NeuN (green), a neuronal marker, COX-2 (red), and DAPI (blue),

demonstrating increased COX-2 protein expression in the wall of intraparenchymal

vessels. n=5 per group. (f, g) Brain protein expression of ANGPT1, ANGPT2, and TIE2

based on ELISA measurements. n=5. t-test with Holm-Sidak post-hoc correction

*p<0.05. Abbreviations: Angpt1/2: Angiopoietin-1/2; COX-1/2: Cyclooxygenase-1/2;

Mfsd2a: Major facilitator superfamily domain containing 2a; ND: Not detected; Ptgs1/2:

Prostaglandin-endoperoxide synthase 1/2; Vegfr2: Vascular endothelial growth factor

receptor 2.

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Figure 27. Serum protein expression of ANGPT1, ANGPT2, and TIE2 (a, b). n=5. t-test

with Holm-Sidak post-hoc correction *p<0.05. Abbreviations: ANGPT1/2: Angiopoietin-

1/2; ND: Not detected; sTIE2: Soluble tyrosine kinase with immunoglobulin and

epidermal growth factor homology domains 2; TIE2:

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2.

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4.3.7 Treatment with selective COX-2 inhibitor celecoxib blunted brain

endothelial Ptgs2 upregulation after SAH

Celecoxib treatment significantly increased overall activity level. There is some suggestion

that celecoxib treatment may have limited neurobehavioural deficits after SAH, although

not reaching statistical significance (Figure 28). After celecoxib treatment, there was a

trend toward decreased BBB disruption in the left ventral cortex (p=0.15) as assessed by

extravasation of cadaverine dye (Figure 29A-B). There is some suggestion that celecoxib

treatment may have decreased neuronal apoptosis (Figure 29C-D). However, celecoxib

treatment had no significant effect on neuronal degeneration (Figure 28). After celecoxib

treatment, there did not appear to be any obvious qualitative difference in COX-2

expression in microglia-like cells and the walls of intraparenchymal vessels (Figure 29E).

Finally, celecoxib treatment blunted the induction of endothelial Ptgs2 gene expression

after SAH, but not Angpt2 gene expression (Figure 29F).

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Figure 28. Effect of selective COX-2 inhibitor celecoxib in SAH. (a) Modified Garcia

Score (MGS) 24h after SAH. Kruskal-Wallis test, **p<0.01. (b) Assessment of

spontaneous activity after SAH. One-way ANOVA with Holm-Sidak post-hoc correction,

**p<0.01. (c, d) Representative coronal brain slice images and quantification of

neuronal degeneration in left ventral region with fluoro-jade b (FJB) staining in SAH

mice treated with vehicle vs. celecoxib. n=6-7 per group. Data presented as means ±

SEM.

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Figure 29. Effect of selective COX-2 inhibitor celecoxib in SAH. (a) Representative

whole brain ventral imaging with the region of interest demarcated by yellow dash

marks. (b) Mean intensity measurements of the left ventral cortex. (c) Representative

coronal brain slice imaging staining for apoptotic neurons (Caspase3+NeuN+) and (d)

quantification of apoptotic neurons in the left ventral region. (e) Representative coronal

brain slice imaging showing COX-2 expression. (f) RT-PCR gene expression of isolated

brain endothelial cells (CD45-CD31+) after celecoxib treatment, normalized to Actb (β-

actin). n=3-4 per group. Data presented as means ± SEM. One-way ANOVA with Holm-

Sidak post-hoc correction. *p<0.05.

4.4 Discussion

In this study, we accomplished the goal of isolating viable, relatively pure brain

endothelial cells using 2 distinct methods, and then characterizing the gene expression

of these cells in a mouse model of SAH. In doing so, we identify Ptgs2/COX-2 as a

potential therapeutic target in SAH. This target is druggable with the selective COX-2

inhibitor, celecoxib, which is already available clinically. Our study is the first to

investigate genome-wide expression changes in brain endothelial cells after SAH.

Indeed, research scientists have reported an urgent need for more brain endothelial

gene expression data sets, especially in the setting of neurological diseases [Huntley

2014].

When surveying the top differentially-expressed genes in brain endothelial cells after

SAH, it became apparent that many of these genes have sizable expression changes

and are relevant to endothelial cell pathophysiology (Figure 22). Prior published gene

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expression data sets in experimental SAH may have been limited by inclusion of

multiple brain cell types (neurons, astrocytes, oligodendrocytes, microglia, vascular

smooth muscle cells, etc.) [Peng 2017, Zheng 2015, Kikkawa 2014, Kurogi 2015, Lee

2012, Sasahara 2008, Vikman 2006]. In doing so, the signals for potentially important

changes in gene expression in specific cell types, may be blunted by the presence of

other cell types. Future experimental studies in SAH will need to investigate gene

expression changes in these other brain cell types. However, it is critical that the

isolation process be gentle and efficient to limit the ex vivo effects of the isolation

process itself on gene expression. Key endothelial transcription factors important for

BBB maturation (Foxf2, Foxl2, Foxq1, Lef1, Pparδ, Zfp551, and Zic3) identified in a

study by Hupe and colleagues, were not significantly changed 24h after SAH in our

study (data not shown) [Hupe 2017]. Genes important for BBB maturation may be

distinct from genes essential for BBB maintenance.

In addition, genes identified from human GWAS studies associated with aneurysm

formation (ANRIL, HDAC9, THSD1), angiographic vasospasm (HP, haptoglobin), and

DCI (NOS3), were not significantly changed. From our results, SOX17, associated with

aneurysm formation from human GWAS studies, was significantly downregulated in

endothelial cells after experimental SAH.

We also identified a general trend of decreased BBB transporter mRNA expression after

SAH. Downregulation of BBB transporters has been implicated in neurodegenerative

disorders such as Alzheimer’s Disease and Parkinson’s Disease, and developmental

neurological disorders [Zlokovic 2011, Kersseboom 2013].

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In our prechiasmatic blood injection model of SAH, we found increased leakage of

cadaverine dye 24h after SAH induction, especially in the left ventral cortical region

(Figure 16A-B). This left ventral cortical region is where the spinal needle traverses and

typically most of the hematoma is situated in this region. The cadaverine dye

(approximately 1kDa in size) may have transported itself into the brain interstitium either

by a paracellular route due to disrupted interendothelial junctions (e.g. tight junctions,

adherens junctions), or by a transcellular route (e.g. transcytosis), or both.

Ultrastructurally, the tight junctions looked unchanged on TEM after SAH (Figure 16E).

It should be emphasized that not all BBB disruption involves intercellular junctions but

could also be attributed to an increase rate of transcytosis [De Bock 2016]. It is unclear

if increased transcytosis across the BBB is harmful or protective.

4.4.1 COX-2 as a therapeutic target

We chose to investigate further Ptgs2/COX-2, an inducible cyclooxygenase, due to the

prominence of prostaglandin synthesis pathways from the GSEA results. COX-2 is an

attractive target as its pathways have been well-studied and it is targeted by the

clinically-available celecoxib [FitzGerald 2003]. Celecoxib is known to easily cross the

BBB [Dembo 2005]. COX-2 inhibitors have been used clinically in SAH patients without

any clear adverse effects [Nassiri 2016].

A comprehensive review of prostaglandin synthesis has been published [FitzGerald

2003]. Prostaglandins are important signaling molecules [Ricciotti 2011]. Phospholipids

derived from cell membranes can be mobilized to release the hairpin-shaped

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arachidonic acid by the enzyme phospholipase A2 [FitzGerald 2003]. Arachidonic acid

is then metabolized to the transitional prostaglandin H2 by COX-1 or COX-2 [FitzGerald

2003]. Subsequently, tissue-specific isomerases and synthases convert prostaglandin

H2 to various prostanoids including prostacyclin (PGI2), thromboxane A2 (TXA2),

prostaglandin D2, PGE2, and prostaglandin F2a, which then act on specific G-protein

coupled receptors [Simmons 2004]. COX-1 activity is generally thought to have

constitutive but low-level expression in many cell lineages whereas COX-2 activity is

inducible, especially with inflammatory stimuli [Simmons 2004]. COX-1 derived

prostaglandins have house-keeping functions, provide gastric cytoprotection and

maintain hemostasis, whereas COX-2 derived prostaglandins are regulated by

cytokines and mitogens and have increased expression in inflammation and cancer, but

are constitutively expressed in brain and kidney [Simmons 2004]. COX-2 is generated

from the Ptgs2 gene, which has several transcription factor binding sites in its promoter

including C/EBP (CCAAT-enhancer-binding protein), NF-κB, Sp1 (Specificity protein 1),

AP1 (Activator protein 1), and a TATA box [Thorn 2011].

The COX-2 inhibitor NS398 was studied previously in a mouse endovascular perforation

SAH model and was found to improve neurological outcomes at the 30mg/kg dose

[Ayer 2011]. COX-2 has been implicated in development of large artery vasospasm

after SAH secondary to upregulation of endothelin-1 and activation of the JAK-STAT

pathway (Janus kinase-Signal transducer and activator of transcription), with celecoxib

treatment decreasing this vasospasm [Munakata 2016, Osuka 1998, Osuka 2006, Tran

Dinh 2001]. COX-2 expression has been demonstrated to be increased after ischemic

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stroke in humans, particularly among infiltrating neutrophils, peri-infarct neurons, and

vascular cells [Iadecola 1999]. COX-2 inhibitors have been found to be beneficial in

hemorrhagic and ischemic stroke animal models. Celecoxib was found to decrease

brain injury and improve outcomes in a rat model of ICH using collagenase [Chu 2004].

Akram and colleagues used a mouse MCAO model and found NS-398 to limit stroke

volume [Akram 2013]. However, they also found decreased stroke volume with EP4

receptor agonist treatment, suggesting that the PGE2 receptors EP2 and EP4 have

neuroprotective functions while PGE2 receptors EP1 and EP3 exacerbate stroke

[Akram 2013]. In a rat endovascular perforation SAH model, Xu and colleagues

administered a selective EP4 agonist treatment and at 24h found improved neurologic

scores, decreased inflammation, decreased BBB disruption, decreased neuronal cell

death, while the administration of a selective EP4 antagonist exacerbated the

neurologic injury and BBB disruption [Xu 2017]. Frankowski and colleagues found that

EP1 receptor pathway to be particularly responsible for BBB breakdown in ischemic

stroke, in which EP1 antagonist and EP1 genetic deletion attenuated BBB disruption

and hemorrhagic transformation in transient MCAO [Frankowski 2015]. Similar results

were identified by Kawano and colleagues [Kawano 2006]. In contrast, Jiang and

colleagues found EP2 antagonism to reduce neuroinflammation in a mouse model of

epilepsy [Jiang 2013]. Also, Leclerc and colleagues found EP1 antagonism to increase

brain injury in a mouse ICH model, whereas EP1 agonist decreased brain injury [Leclerc

2015]. The complete picture regarding the role of the EP receptors is still being

elucidated, with each receptor likely to have a mixed pro-inflammatory and anti-

inflammatory signal depending on the context.

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However, COX-2 inhibitors have been shown to significantly increase pro-thrombotic

events such as ischemic stroke and myocardial infarction, typically in the setting of

prolonged use in patients with cardiovascular risk factors [Pirlamarla 2016]. Concern

with COX-2 inhibitors was clearly demonstrated in the VIGOR (Vioxx Gastrointestinal

Outcomes Research) study comparing the COX-2 inhibitor rofecoxib versus non-

selective NSAID (non-steroidal anti-inflammatory drug, naproxen) treatment in patients

with rheumatoid arthritis, which revealed less serious GI adverse events but five times

the cardiovascular risk with rofecoxib [Bombardier 2000]. The etiology of these events is

controversial but may be related to suppression of vascular-protective PGI2 in addition

to the suppression of the pro-inflammatory PGE2 [FitzGerald 2003]. However, in the

PRECISION (Prospective Randomized Evaluation of Celecoxib Integrated Safety

versus Ibuprofen Or Naproxen) study of patients with osteoarthritis and rheumatoid

arthritis treated for a mean of 20 months, the COX-2 inhibitor celecoxib was found to be

non-inferior to the non-selective NSAIDs ibuprofen or naproxen regarding occurrence of

myocardial infarction and/or stroke (risk of 2.3-2.7%) [Nissen 2016]. This study included

patients with cardiovascular diseases, which emphasizes the safety of celecoxib

[Nissen 2016]. The safety of celecoxib regarding cardiovascular risk was also confirmed

in large retrospective series [Hirayama 2014, Andersohn 2006].

A potential benefit of COX-2 inhibitors in SAH is the sodium retention and increased

blood pressure, which may be beneficial for improving overall blood flow and preventing

a negative fluid balance and alterations in sodium homeostasis such as cerebral salt

wasting. Also, celecoxib unlike other COX-2 inhibitors, is known to activate KCNQ (Kv7)

potassium channels and antagonize L-type calcium channels, causing vascular smooth

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muscle relaxation and decrease vascular tone [Brueggemann 2010]. This may explain

why celecoxib is associated with less thrombosis than rofecoxib [Brueggemann 2010].

Using clinical trial data from the CONSCIOUS-1 trial, comparing clazosentan to placebo

in patients with aneurysmal SAH, the subgroup of patients who were treated with

NSAIDs were compared to a propensity-score matched group of patients not treated

with NSAIDs in hospital [Nassiri 2016]. The authors found that NSAID treatment was

associated with lower mortality rate and decreased ICU and hospital length of stay with

a trend toward improved clinical outcome at 12 weeks [Nassiri 2016]. However, there

were not enough patients taking COX-2 inhibitors to make any meaningful comparisons.

COX-2 expression has also been implicated in aneurysm formation in both animal

models and clinically. Aneurysm fundus samples from patients demonstrated increased

expression of COX-2 and EP2, one of the pro-inflammatory receptors of PGE2 [Aoki

2011]. The authors confirmed this expression profile in a mouse model of cerebral

aneurysms as well as an in vitro model of increased shear stress over cultured primary

human carotid endothelial cells [Aoki 2011]. Chu and colleagues also identified

increased COX-2 expression in cerebral arteries in a mouse model of cerebral

aneurysm, which was dependent on the expression of myeloperoxidase [Chu 2015]. In

mice, COX-2 inhibitors did not decrease the formation of aneurysms but decreased the

risk of rupture [Chalouhi 2016].

The timing of celecoxib administration is important. Surprisingly, in a rat kainic acid-

induced brain injury model, post-injury celecoxib helped decrease brain injury and

improved cognitive outcomes although there was no benefit observed with pre-injury

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celecoxib [Gobbo 2004]. COX-2 inhibition in freshly isolated human brain capillaries

prevented glutamate-mediated induction of PGP transport activity, which may

theoretically improve the CNS penetration of concurrently administered drugs [Avemary

2013]. The cellular origin of COX-2 expression may also matter. In our study we only

addressed the fact that COX-2 expression was increased in brain endothelial cells. Aid

and colleagues have shown that prostaglandins derived from neuronal COX-2 to have

little role in neuroinflammatory response to LPS, although An and colleagues found

neuronal COX-2 to increase brain injury after administration of a neurotoxin [Aid 2010,

An 2014].

4.4.2 Angiopoietin-2 as a therapeutic target

In this study we found an upregulation of Angpt2 mRNA in endothelial cells and

increased ANGPT2 protein expression in brain tissue after SAH. ANGPT2 is a secreted

angiocrine factor approximately 70kDa in size that is known to destabilize vascular

integrity by antagonizing the trophic effects of its counterpart ANGPT1 on the TIE2

receptor on endothelial cells, resulting in increased vascular permeability and loss of

pericytes on the vascular wall [Augustin 2009]. However, ANGPT2 may also act as an

agonist for TIE2 receptor under certain circumstances including the presence of VEGF,

and can promote endothelial survival, proliferation, migration, angiogenesis, and

remodeling of blood vessels [Beck 2000]. ANGPT2 expression is upregulated in sepsis

and in tumours [Tanaka 1999, Stratmann 1998, Ziegler 2013]. Hypoxia, VEGF, basic

fibroblast growth factor (bFGF), and TNFα also can increase endothelial expression of

ANGPT2 [Pichiule 2004, Kim 2000].

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The endothelial TIE2 receptor can undergo proteolytic cleavage of the extracellular

domain and released into the systemic circulation in soluble form [Augustin 2009]. TIE2

signaling is essential for maintenance of vascular integrity, mediating communication

between endothelial cells and vascular smooth muscle cells and inhibiting endothelial

apoptosis [Augustin 2009]. TIE2 is also essential for embryonic development of the

endocardium and is embryonic lethal in knock-out mice [Augustin 2009].

We observed a decrease in GFP expression after SAH. The Tie2 promoter that drives

the GFP expression in the Tie2-GFP mice has sequences specific for GATA and Ets

transcription factors, although neither of these family of transcription factors significantly

changed in brain endothelial cells after SAH [Minami 2003, Song 2009].

Gu and colleagues used a rat endovascular perforation SAH model and identified

increased ANGPT2 expression and decreased ANGPT1 expression in brain tissue

starting at 6h and maximal at 24h, with measurements made by immunohistochemistry

and western blots [Gu 2016]. These results confirm our ELISA-based results regarding

increased ANGPT2 and decreased ANGPT1 expression in brain tissue 24h after SAH.

Fernandez-Lopez and colleagues also found an increase in ANGPT2 expression in

brain endothelial cells 24h after 3h transient MCAO in rats, but data regarding COX-2

was not available [Fernandez-Lopez 2012]. Gurnik and colleagues found that mice

undergoing MCAO with gain of function mutation for Angpt2 resulted in an increase in

BBB permeability and increase in stroke size, suggesting that ANGPT2 leads to

cerebrovascular damage and edema [Gurnik 2016]. Beck and colleagues found

170

upregulation of VEGF and ANGPT2 in the peri-infarct area beginning at 6h after rat

MCAO, resulting in endothelial proliferation [Beck 2000].

There have only been a few studies looking at angiopoietins in clinical SAH. Fischer and

colleagues found that decreased serum ANGPT1 and not ANGPT2, was associated

with development of aVSP and DCI in patients with SAH [Fischer 2011]. However,

serum ANGPT2 was significantly elevated in patients with Fisher grade IV SAH versus

grade II or III [Fischer 2011]. Although Fischer and colleagues observed a significant

decrease in serum ANGPT1 over the first 3 days after aneurysm rupture, Wang and

colleagues observed an increase serum ANGPT1 over the first 3 days [Wang 2015].

Both studies had small numbers of patients, so at this point the prognostic significance

of serum angiopoietin levels is unclear. Chittiboina and colleagues have suggested that

the ratio of serum and CSF ANGPT1 to ANGPT2 is more important than the actual

individual levels [Chittiboina 2013]. More studies are needed to investigate ANGPT1,

ANGPT2, and soluble TIE2 as prognostic biomarkers.

4.4.3 Limitations

This study had several limitations. The brain endothelial cell isolation procedure is

several hours long, which may significantly affect the gene expression profile from what

is occurring in vivo. It is possible that some of the gene expression changes identified in

brain endothelial cells may be due to ex vivo activation by thrombin in the residual blot

clot, as thrombin itself may upregulate endothelial COX-2 expression [Minami 2004].

Our studies also do not consider the heterogeneity of gene expression of endothelial

cells derived from different brain compartments. By using systemic administration of

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celecoxib, we have not excluded COX-2 derived from other brain cell types contributing

to BBB pathophysiology. Future studies will investigate downstream COX-2 pathways

including mPGES-1, PGE2 and its multiple receptors EP1-4 expression and activity.

4.5 Conclusions

Using whole genome-profiling of freshly isolated brain endothelial cells derived from an

SAH mouse model, we identified COX-2 as a potential therapeutic target. Our brain

endothelial-specific gene expression data set may open new translational and clinical

research avenues aimed at improving clinical outcomes in SAH and may be applicable

to other neurological diseases characterized by BBB disruption including ischemic

stroke, TBI, epilepsy, multiple sclerosis, brain tumours and neurodegenerative

disorders.

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Chapter 5

Future Directions

173

Future Directions

5.1 Brain endothelial cell gene expression studies

In this study, we provide one of the first whole genome expression data sets in an

animal model of a neurological disease. The brain endothelial cell isolation methods

developed can continue to be optimized in terms of improving viability, purity, and

efficiency. Although RNA amplification and microarray techniques assisted in achieving

the goals of this study, more detailed molecular studies may be performed using next-

generation RNAseq to have a better understanding of more rare mRNA transcripts,

alternatively-spliced transcripts, and lncRNAs [Shin 2014]. Single cell RNAseq would

allow the ability to characterize gene expression of individual cells [Zeisel 2015, Lovatt

2014]. It is known that even among the same cell type, there can be significant

heterogeneity in gene expression [Darmanis 2015]. Although our study focused on brain

endothelial gene expression changes, it would be informative to understand the gene

expression changes of other cell types in the brain (neurons, astrocytes,

oligodendrocytes, microglia, etc.) and create a disease-specific cell expression data

sets, like what has already been performed for naïve mouse brains [Zhang 2014].

Finally, the brain endothelial cell isolation and gene expression techniques can be

applied to other animal models of brain injury and ischemia, to identify common

pathways and to identify therapeutic targets and agents that may apply to a broader

range of neurological diseases. Also, finding commonalities among various animal

models of diseases would allow a greater understanding of the brain’s response to

ischemia and injury. Currently, it is not possible to ascertain the gene expression

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changes in the BBB within the brains of patients with SAH. Thus, it is essential to have

these preclinical studies in the field of BBB research. There has been significant interest

in non-invasive methods to determine the status of the BBB clinically. One method is to

interrogate endothelial microparticles and exosomes derived from the BBB but

circulating in the blood as potential biomarkers of disease presence or progression, but

much more study is needed in this arena [Tenreiro 2016].

5.2 COX-2 studies in experimental SAH

Ptgs2 knockout mice can be congenitally lethal, with a neonatal mortality of 35%, due to

the failure of the ductus arteriosus to close after birth [Loftin 2001]. Knockout of Ptgs2

can result in congenital kidney abnormalities, elevated blood pressure, increased

vasoconstriction in response to angiotensin II, cardiomyopathy, and reproductive

defects in females [Thorn 2011]. These confounding factors may affect the ability to

properly assess brain injury after SAH in Ptgs2 knockout mice. It is possible to consider

COX-2 inducible knockouts in brain endothelial cells, in which the Cre recombinase is

driven by brain endothelial-specific promoter such as a specialized transporter (e.g.

Abcb1a). Also, further downstream effects of COX-2 upregulation can be explored after

SAH such as looking at PGE synthase expression and activity, looking at expression of

various prostanoids such as PGE2 and PGI2, and investigating the signaling through

PGE2 receptors EP1-EP4 as well as their downstream signaling.

We performed our experiments in male mice to avoid the potential issue of cycling

estrogen/progesterone to affect the degree of brain injury. However, as majority of

patients who have SAH are female, more studies are needed in females. Friederich and

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colleagues found differential brain expression levels of eNOS and thrombomodulin in

male and female rats undergoing the endovascular perforation SAH model [Friederich

2015]. This suggests that preclinical studies in male animals might not necessarily be

validly extrapolated to female animals.

Much of the research in BBB has focused on tight junction integrity and paracellular

leakage. Within the last several years there has been increasing appreciation of

elevated endothelial transcytosis as a major component of BBB pathophysiology [De

Bock 2016, Ben-Zvi 2014, Knowland 2014]. Whether this increased transcytosis is

protective or harmful, and over which time periods, is still not clarified and needs further

attention.

5.3 Clinical trial design in SAH

Many in the SAH research community have suggested targeting EBI after SAH as

opposed to DCI and aVSP [Conzen 2016]. It has been shown that acute cerebral

infarcts from the initial hemorrhage and aneurysm-securing procedure to be a greater

determinant of poor outcome than delayed infarcts [Ayling 2016].

5.3.1 Consideration of a clinical trial with VPA in patients with SAH

VPA in the context of clinical SAH has special considerations. Because of the potential

for coagulopathy secondary to decreased and/or dysfunctional platelets, it may not be

possible to initiate VPA prior to the aneurysm-securing procedure due to the theoretical

risk of increased rebleeding. In the context of a clinical trial, VPA can be started post-

176

procedure, but that limits how quickly the drug can be initiated from SAH onset. One

reason why recent SAH clinical trials have failed may be related to the timing of initiation

of drug treatment. Most recently, clazosentan (within 56h) in CONSCIOUS-1,

magnesium (within 72h) in MASH-2, and simvastatin (within 96h) in STASH have all

had relatively delayed drug initiation times [Macdonald 2011, Dorhout Mees 2012,

Kirkpatrick 2014]. Drug initiation within 24h after ictus may be the most practical timing

in a VPA clinical trial. Pregnancy screening is important as prenatal VPA exposure is

associated with neural tube defects and autism spectrum disorder [Chateauvieux 2010].

VPA levels must be monitored closely as other drugs can affect plasma concentrations

of VPA including the commonly administered antibiotic meropenem [Mink 2011].

Duration of drug treatment probably should be 2 weeks or less and not months, as there

were adverse effects with VPA treatment in a randomized trial of TBI patients [Temkin

1999].

Given the wealth of pre-clinical data showing VPA to be beneficial effect in various

models of neurological diseases including several different models of SAH, it is

reasonable to study VPA treatment in SAH in a phase IIa trial to demonstrate safety,

identify the optimal dose, and show the feasibility for a larger clinical trial. Systemic

parameters can also be investigated as VPA treatment has been shown to be

associated with decreased acute respiratory failure after SAH [Liao 2017]. However, the

lack of patentability of this drug may limit financial interest from pharmaceutical

companies to fund this clinical trial.

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5.3.2 Consideration of a clinical trial with celecoxib in patients with SAH

COX-2 inhibitors in the context of clinical SAH have special considerations too. They

can potentially be administered early, even prior to the aneurysm-securing procedure,

as there are typically no bleeding issues with COX-2 inhibitors. For patients undergoing

endovascular coiling, the theoretical risk for increased thromboembolic complications

exists although unclear if this would be the case after only one or two doses of the

COX-2 inhibitor. Also, after deployment of the first framing coil, the patient is typically

heparinized to prevent thromboembolic events.

A potential relative exclusion for COX-2 inhibitor treatment would be patients with

known coronary artery disease due to the potential thrombotic risks [Grosser 2010,

Bavry 2011]. Also, COX-2 inhibitor treatment may need to be stopped at day 3 prior to

the onset of aVSP due to its inhibition of prostacyclin/PGI2 production, a potent

vasodilator, as well as its pro-thrombotic effects which may increase microthrombi

burden in the brain, a potential pathophysiological process contributing to DCI [Pober

2007].

Non-selective NSAIDs, in particular indomethacin, have been associated with

decreased ICP, particularly in patients with TBI [Sader 2015]. This would potentially be

beneficial for patients with SAH who frequently have high ICP. However, because of

concerns of NSAID decreasing platelet aggregation, the use of traditional NSAID in

SAH might only be considered after securing the aneurysm, which would delay the

initiation of the drug. NSAIDs are known to slow the rate of platelet plug formation in

178

SAH patients [Parkhutik 2012]. ASA, which has predominantly COX-1 inhibition without

affecting COX-2 activity, was found to be safe in a small pilot randomized trial, with ASA

started within 5 days of aneurysm rupture, and immediately after surgical clipping [Hop

2000]. In a larger randomized controlled trial of 161 patients with SAH treated with

100mg ASA or placebo within 12h after aneurysm-securing procedure and within 4 days

of aneurysm rupture, there was no significant difference in DCI or clinical outcome and

no change in bleeding outcomes [van den Bergh 2006].

COX-2 inhibitors used during craniotomy procedures administered at the time of dural

closure intraoperatively, were found to be safe without increased risk of postoperative

complications [Williams 2011]. In a small pilot randomized controlled trial of celecoxib

versus placebo after non-surgical spontaneous ICH, the authors found slightly

decreased expansion of perihematomal edema in patients receiving the COX-2 inhibitor

(400mg bid x 14 days, initiated within 24h) [Lee 2013B]. This trial was not powered to

assess an effect on clinical outcomes.

Although we did not observe any serum changes in ANGPT1 or ANGPT2 in our SAH

model, others have observed changes in ANGPT1 and ANGPT2 in clinical SAH

[Fischer 2011, Wang 2015]. In a study of patients with SAH or ICH undergoing

endovascular cooling, all patients were observed to have a decrease in serum ANGPT1

and increase in serum ANGPT2 at day 4 [Fischer 2012]. NSAID treatment resulted in an

increase in serum ANGPT1 and decrease in serum ANGPT2 by day 7 [Fischer 2012].

Perhaps, when designing a clinical trial with a COX-2 inhibitor, the serum and CSF

levels of ANGPT1 and ANGPT2 can be measured as an indicator of COX-2 efficacy.

179

Chapter 6

Concluding Remarks

180

Concluding Remarks

6.1 Summary of results

In a mouse model of SAH, VPA treatment resulted in improved neurobehavioural

outcomes. VPA also limited brain injury, decreased the microthrombi burden, decreased

BBB disruption, but did not affect large artery vasospasm after SAH. VPA, with its

HDAC inhibitor properties, increased the acetylation of histone H3 in brain tissue. VPA

limited brain injury, independent of AKT phosphorylation. Using propensity-score

matched groups of patients with SAH, VPA treatment did not significantly affect clinical

outcome, although most patients had short duration and delayed initiation of treatment.

VPA is a potential therapeutic agent for SAH treatment and needs further clinical study.

Using the same SAH mouse model, two protocols were developed and optimized to

obtain relatively pure and viable brain endothelial cells in an efficient manner. These

purified brain endothelial cells were interrogated for whole genome expression changes

after SAH. A list of potential therapeutic targets was generated, with several targets

validated, and relevant pathways determined. One specific pathway, COX-2

upregulation, was investigated further with celecoxib treatments, a selective COX-2

inhibitor. Celecoxib treatment resulted in improved neurobehavioural outcomes,

decreased BBB disruption, and decreased endothelial COX-2 mRNA expression.

Celecoxib is also a potential therapeutic agent for SAH treatment and needs further

clinical study.

181

6.2 Final comments

Aneurysmal SAH continues to be a disease causing devastating morbidity and mortality.

With the absence of successful phase III drug clinical trials in SAH since 1989, there is

a need to identify and investigate new therapeutics in SAH. Neither VPA or celecoxib is

a ‘perfect’ drug. Each has its specific drawbacks. VPA may cause more bleeding and

celecoxib may cause more thrombosis. Although the prostaglandin synthesis pathway

was one of the top pathways in our analysis, there are many other important pathways

relevant to BBB pathophysiology that have yet to be explored, which stresses the

importance of making published data sets available to the scientific community.

The study of the BBB continues to be a worthwhile endeavour. There is still much

mystery to it and is still incompletely understood. BBB and its dysfunction appears to be

relevant to many neurological diseases and may contribute or cause neurological injury.

At the same time, BBB can serve as an indicator of CNS health, with possible release of

biomarkers into the systemic circulation in times of disease.

182

References

Aid S, Parikh N, Palumbo S, Bosetti F. Neuronal overexpression of cyclooxygenase-2

does not alter the neuroinflammatory response during brain innate immune activation.

Neurosci Lett 2010 Jul 12;478(3):113-8.

Aird[A] WC. Phenotypic heterogeneity of the endothelium: I. Structure, function, and

mechanisms. Circ Res 2007 Feb 2;100(2):158-73.

Aird[B] WC. Phenotypic heterogeneity of the endothelium: II. Representative vascular

beds. Circ Res 2007 Feb 2;100(2):174-90.

Akhigbe T, Zolnourian A, Bulters D. Cholesterol-Reducing Agents for Treatment of

Aneurysmal Subarachnoid Hemorrhage: Systematic Review and Meta-Analysis of

Randomized Controlled Trials. World Neurosurg 2017 May;101:476-85.

Akhondzadeh S. The Importance of Clinical Trials in Drug Development. Avicenna J

Med Biotechnol 2016 Oct;8(4):151.

Akram A, Gibson CL, Grubb BD. Neuroprotection mediated by the EP(4) receptor

avoids the detrimental side effects of COX-2 inhibitors following ischaemic injury.

Neuropharmacology 2013 Feb;65:165-72.

Al-Mufti F, Amuluru K, Smith B, Damodara N, El-Ghanem M, Singh IP, et al. Emerging

Markers of Early Brain Injury and Delayed Cerebral Ischemia in Aneurysmal

Subarachnoid Hemorrhage. World Neurosurg 2017 Nov;107:148-59.

183

Alvarez JI, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, Terouz S, et al. The

Hedgehog pathway promotes blood-brain barrier integrity and CNS immune

quiescence. Science 2011 Dec 23;334(6063):1727-31.

An Y, Belevych N, Wang Y, Zhang H, Herschman H, Chen Q, et al. Neuronal and

nonneuronal COX-2 expression confers neurotoxic and neuroprotective phenotypes in

response to excitotoxin challenge. J Neurosci Res 2014 Apr;92(4):486-95.

Andersohn F, Schade R, Suissa S, Garbe E. Cyclooxygenase-2 selective nonsteroidal

anti-inflammatory drugs and the risk of ischemic stroke: a nested case-control study.

Stroke 2006 Jul;37(7):1725-30.

Anderson GD, Temkin NR, Chandler WL, Winn HR. Effect of valproate on hemostatic

function in patients with traumatic brain injury. Epilepsy Res 2003 Dec;57(2-3):111-9.

Ansar S, Edvinsson L. Subtype activation and interaction of protein kinase C and

mitogen-activated protein kinase controlling receptor expression in cerebral arteries and

microvessels after subarachnoid hemorrhage. Stroke 2008 Jan;39(1):185-90.

Aoki T, Nishimura M, Matsuoka T, Yamamoto K, Furuyashiki T, Kataoka H, et al.

PGE(2) -EP(2) signalling in endothelium is activated by haemodynamic stress and

induces cerebral aneurysm through an amplifying loop via NF-kappaB. Br J Pharmacol

2011 Jul;163(6):1237-49.

Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, et al. Pericytes

regulate the blood-brain barrier. Nature 2010 Nov 25;468(7323):557-61.

184

Asano T, Sano K. Pathogenetic role of no-reflow phenomenon in experimental

subarachnoid hemorrhage in dogs. J Neurosurg 1977 Apr;46(4):454-66.

Ashburn TT, Thor KB. Drug repositioning: identifying and developing new uses for

existing drugs. Nat Rev Drug Discov 2004 Aug;3(8):673-83.

Atangana E, Schneider UC, Blecharz K, Magrini S, Wagner J, Nieminen-Kelha M, et al.

Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to

Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH). Transl

Stroke Res 2017 Apr;8(2):144-56.

Augustin HG, Koh GY, Thurston G, Alitalo K. Control of vascular morphogenesis and

homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol 2009

Mar;10(3):165-77.

Avemary J, Salvamoser JD, Peraud A, Remi J, Noachtar S, Fricker G, et al. Dynamic

regulation of P-glycoprotein in human brain capillaries. Mol Pharm 2013 Sep

3;10(9):3333-41.

Ayer R, Jadhav V, Sugawara T, Zhang JH. The neuroprotective effects of

cyclooxygenase-2 inhibition in a mouse model of aneurysmal subarachnoid

hemorrhage. Acta Neurochir Suppl 2011;111:145-9.

Ayling OG, Ibrahim GM, Alotaibi NM, Gooderham PA, Macdonald RL. Dissociation of

Early and Delayed Cerebral Infarction After Aneurysmal Subarachnoid Hemorrhage.

Stroke 2016 Dec;47(12):2945-51.

185

Bache S, Rasmussen R, Rossing M, Laigaard FP, Nielsen FC, Moller K. MicroRNA

Changes in Cerebrospinal Fluid After Subarachnoid Hemorrhage. Stroke 2017

Sep;48(9):2391-8.

Backes D, Vergouwen MD, Tiel Groenestege AT, Bor AS, Velthuis BK, Greving JP, et

al. PHASES Score for Prediction of Intracranial Aneurysm Growth. Stroke 2015

May;46(5):1221-6.

Bambakidis T, Dekker SE, Sillesen M, Liu B, Johnson CN, Jin G, et al. Resuscitation

with Valproic Acid Alters Inflammatory Genes in a Porcine Model of Combined

Traumatic Brain Injury and Hemorrhagic Shock. J Neurotrauma 2016 Aug

15;33(16):1514-21.

Bavry AA, Khaliq A, Gong Y, Handberg EM, Cooper-Dehoff RM, Pepine CJ. Harmful

effects of NSAIDs among patients with hypertension and coronary artery disease. Am J

Med 2011 Jul;124(7):614-20.

Beck H, Acker T, Wiessner C, Allegrini PR, Plate KH. Expression of angiopoietin-1,

angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat. Am J

Pathol 2000 Nov;157(5):1473-83.

Bederson JB, Germano IM, Guarino L. Cortical blood flow and cerebral perfusion

pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat. Stroke

1995 Jun;26(6):1086-91.

186

Ben-Zvi A, Lacoste B, Kur E, Andreone BJ, Mayshar Y, Yan H, et al. Mfsd2a is critical

for the formation and function of the blood-brain barrier. Nature 2014 May

22;509(7501):507-11.

Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery rate in

behavior genetics research. Behav Brain Res 2001 Nov 1;125(1-2):279-84.

Berthiaume AA, Grant RI, McDowell KP, Underly RG, Hartmann DA, Levy M, et al.

Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult

Mouse Brain. Cell Rep 2018 Jan 2;22(1):8-16.

Blanchette M, Daneman R. Formation and maintenance of the BBB. Mech Dev 2015

Nov;138 Pt 1:8-16.

Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, et al.

Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with

rheumatoid arthritis. VIGOR Study Group. N Engl J Med 2000 Nov 23;343(21):1520-8,

2.

Brandt C, Gastens AM, Sun M, Hausknecht M, Loscher W. Treatment with valproate

after status epilepticus: effect on neuronal damage, epileptogenesis, and behavioral

alterations in rats. Neuropharmacology 2006 Sep;51(4):789-804.

Britz GW, Meno JR, Park IS, Abel TJ, Chowdhary A, Nguyen TS, et al. Time-dependent

alterations in functional and pharmacological arteriolar reactivity after subarachnoid

hemorrhage. Stroke 2007 Apr;38(4):1329-35.

187

Brueggemann LI, Mani BK, Mackie AR, Cribbs LL, Byron KL. Novel Actions of

Nonsteroidal Anti-Inflammatory Drugs on Vascular Ion Channels: Accounting for

Cardiovascular Side Effects and Identifying New Therapeutic Applications. Mol Cell

Pharmacol 2010;2(1):15-9.

Bundgaard M, Abbott NJ. All vertebrates started out with a glial blood-brain barrier 4-

500 million years ago. Glia 2008 May;56(7):699-708.

Buoli M, Serati M, Botturi A, Altamura AC. The Risk of Thrombocytopenia During

Valproic Acid Therapy: A Critical Summary of Available Clinical Data. Drugs R D 2017

Dec 19.

Cach R, Smock T, Popejoy S. Blood-borne factors regulating microvascular constriction

in the rat hippocampal slice. Brain Res 1987 Jun 23;414(1):1-7.

Calabria AR, Shusta EV. A genomic comparison of in vivo and in vitro brain

microvascular endothelial cells. J Cereb Blood Flow Metab 2008 Jan;28(1):135-48.

Chalouhi N, Starke RM, Correa T, Jabbour PM, Zanaty M, Brown RD, Jr., et al.

Differential Sex Response to Aspirin in Decreasing Aneurysm Rupture in Humans and

Mice. Hypertension 2016 Aug;68(2):411-7.

Chang CZ, Wu SC, Lin CL, Kwan AL. Valproic acid attenuates intercellular adhesion

molecule-1 and E-selectin through a chemokine ligand 5 dependent mechanism and

subarachnoid hemorrhage induced vasospasm in a rat model. J Inflamm (Lond)

2015;12:27.

188

Chateauvieux S, Morceau F, Dicato M, Diederich M. Molecular and therapeutic potential

and toxicity of valproic acid. J Biomed Biotechnol 2010;2010.

Chen S, Wu H, Klebe D, Hong Y, Zhang J. Valproic acid: a new candidate of

therapeutic application for the acute central nervous system injuries. Neurochem Res

2014 Sep;39(9):1621-33.

Chi JT, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, et al.

Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci U S

A 2003 Sep 16;100(19):10623-8.

Chittiboina P, Ganta V, Monceaux CP, Scott LK, Nanda A, Alexander JS. Angiopoietins

as promising biomarkers and potential therapeutic targets in brain injury.

Pathophysiology 2013 Feb;20(1):15-21.

Chow BW, Gu C. The molecular constituents of the blood-brain barrier. Trends Neurosci

2015 Oct;38(10):598-608.

Chu K, Jeong SW, Jung KH, Han SY, Lee ST, Kim M, et al. Celecoxib induces

functional recovery after intracerebral hemorrhage with reduction of brain edema and

perihematomal cell death. J Cereb Blood Flow Metab 2004 Aug;24(8):926-33.

Chu Y, Wilson K, Gu H, Wegman-Points L, Dooley SA, Pierce GL, et al.

Myeloperoxidase is increased in human cerebral aneurysms and increases formation

and rupture of cerebral aneurysms in mice. Stroke 2015 Jun;46(6):1651-6.

Chuang DM. The antiapoptotic actions of mood stabilizers: molecular mechanisms and

therapeutic potentials. Ann N Y Acad Sci 2005 Aug;1053:195-204.

189

Chun HB, Scott M, Niessen S, Hoover H, Baird A, Yates J, III, et al. The proteome of

mouse brain microvessel membranes and basal lamina. J Cereb Blood Flow Metab

2011 Dec;31(12):2267-81.

Connolly ES, Jr., Rabinstein AA, Carhuapoma JR, Derdeyn CP, Dion J, Higashida RT,

et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage: a

guideline for healthcare professionals from the American Heart Association/American

Stroke Association. Stroke 2012 Jun;43(6):1711-37.

Conzen C, Schubert GA. About the importance of the acute phase of subarachnoid

hemorrhage and the chances for successful translation. J Neurol Sci 2016 Nov

15;370:310-1.

Cooke DL, McCoy DB, Halbach VV, Hetts SW, Amans MR, Dowd CF, et al.

Endovascular Biopsy: In Vivo Cerebral Aneurysm Endothelial Cell Sampling and Gene

Expression Analysis. Transl Stroke Res 2018 Feb;9(1):20-33.

Costa C, Martella G, Picconi B, Prosperetti C, Pisani A, Di FM, et al. Multiple

mechanisms underlying the neuroprotective effects of antiepileptic drugs against in vitro

ischemia. Stroke 2006 May;37(5):1319-26.

Cremers CH, van der Schaaf IC, Wensink E, Greving JP, Rinkel GJ, Velthuis BK, et al.

CT perfusion and delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage:

a systematic review and meta-analysis. J Cereb Blood Flow Metab 2014 Feb;34(2):200-

7.

190

D'Abbondanza JA, Ai J, Lass E, Wan H, Brathwaite S, Tso MK, et al. Robust effects of

genetic background on responses to subarachnoid hemorrhage in mice. J Cereb Blood

Flow Metab 2016 Nov;36(11):1942-54.

Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. The mouse blood-

brain barrier transcriptome: a new resource for understanding the development and

function of brain endothelial cells. PLoS One 2010 Oct 29;5(10):e13741.

Daneman R. The blood-brain barrier in health and disease. Ann Neurol 2012

Nov;72(5):648-72.

Darmanis S, Sloan SA, Zhang Y, Enge M, Caneda C, Shuer LM, et al. A survey of

human brain transcriptome diversity at the single cell level. Proc Natl Acad Sci U S A

2015 Jun 9;112(23):7285-90.

Dash PK, Orsi SA, Zhang M, Grill RJ, Pati S, Zhao J, et al. Valproate administered after

traumatic brain injury provides neuroprotection and improves cognitive function in rats.

PLoS One 2010 Jun 30;5(6):e11383.

de Rooij NK, Linn FH, van der Plas JA, Algra A, Rinkel GJ. Incidence of subarachnoid

haemorrhage: a systematic review with emphasis on region, age, gender and time

trends. J Neurol Neurosurg Psychiatry 2007 Dec;78(12):1365-72.

De BM, Van H, V, Vandenbroucke RE, Decrock E, Wang N, Leybaert L. Into rather

unexplored terrain-transcellular transport across the blood-brain barrier. Glia 2016

Jul;64(7):1097-123.

191

Dekker SE, Bambakidis T, Sillesen M, Liu B, Johnson CN, Jin G, et al. Effect of

pharmacologic resuscitation on the brain gene expression profiles in a swine model of

traumatic brain injury and hemorrhage. J Trauma Acute Care Surg 2014 Dec;77(6):906-

12.

Dekker SE, Nikolian VC, Sillesen M, Bambakidis T, Schober P, Alam HB. Different

resuscitation strategies and novel pharmacologic treatment with valproic acid in

traumatic brain injury. J Neurosci Res 2017 Jul 25.

Delgado TJ, Brismar J, Svendgaard NA. Subarachnoid haemorrhage in the rat:

angiography and fluorescence microscopy of the major cerebral arteries. Stroke 1985

Jul;16(4):595-602.

Dembo G, Park SB, Kharasch ED. Central nervous system concentrations of

cyclooxygenase-2 inhibitors in humans. Anesthesiology 2005 Feb;102(2):409-15.

Doczi[A] T, Joo F, Adam G, Bozoky B, Szerdahelyi P. Blood-brain barrier damage

during the acute stage of subarachnoid hemorrhage, as exemplified by a new animal

model. Neurosurgery 1986 Jun;18(6):733-9.

Doczi[B] T, Joo F, Sonkodi S, Adam G. Increased vulnerability of the blood-brain barrier

to experimental subarachnoid hemorrhage in spontaneously hypertensive rats. Stroke

1986 May;17(3):498-501.

Dorhout Mees SM, Rinkel GJ, Feigin VL, Algra A, van den Bergh WM, Vermeulen M, et

al. Calcium antagonists for aneurysmal subarachnoid haemorrhage. Cochrane

Database Syst Rev 2007 Jul 18;(3):CD000277.

192

Dorhout Mees SM, Algra A, Vandertop WP, van KF, Kuijsten HA, Boiten J, et al.

Magnesium for aneurysmal subarachnoid haemorrhage (MASH-2): a randomised

placebo-controlled trial. Lancet 2012 Jul 7;380(9836):44-9.

Dreier JP. The role of spreading depression, spreading depolarization and spreading

ischemia in neurological disease. Nat Med 2011 Apr;17(4):439-47.

Ecker A. The discovery of human cerebral arterial spasm in angiograms: an

autobiographical note (Arthur Ecker). Neurosurgery 1982 Jan;10(1):90.

Endo H, Nito C, Kamada H, Yu F, Chan PH. Akt/GSK3beta survival signaling is involved

in acute brain injury after subarachnoid hemorrhage in rats. Stroke 2006

Aug;37(8):2140-6.

Enerson BE, Drewes LR. The rat blood-brain barrier transcriptome. J Cereb Blood Flow

Metab 2006 Jul;26(7):959-73.

Erdo F, Erdo SL. Bimoclomol protects against vascular consequences of experimental

subarachnoid hemorrhage in rats. Brain Res Bull 1998;45(2):163-6.

Fernandez-Lopez D, Faustino J, Daneman R, Zhou L, Lee SY, Derugin N, et al. Blood-

brain barrier permeability is increased after acute adult stroke but not neonatal stroke in

the rat. J Neurosci 2012 Jul 11;32(28):9588-600.

Fiedler U, Scharpfenecker M, Koidl S, Hegen A, Grunow V, Schmidt JM, et al. The Tie-2

ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial

cell Weibel-Palade bodies. Blood 2004 Jun 1;103(11):4150-6.

193

Findlay JM, Nisar J, Darsaut T. Cerebral Vasospasm: A Review. Can J Neurol Sci 2016

Jan;43(1):15-32.

Fischer M, Broessner G, Dietmann A, Beer R, Helbok R, Pfausler B, et al. Angiopoietin-

1 is associated with cerebral vasospasm and delayed cerebral ischemia in

subarachnoid hemorrhage. BMC Neurol 2011 May 26;11:59.

Fischer M, Dietmann A, Lackner P, Beer R, Helbok R, Pfausler B, et al. Endovascular

cooling and endothelial activation in hemorrhagic stroke patients. Neurocrit Care 2012

Oct;17(2):224-30.

Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid

hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980

Jan;6(1):1-9.

Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, et al. Update of the

stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009

Jun;40(6):2244-50.

FitzGerald GA. COX-2 and beyond: Approaches to prostaglandin inhibition in human

disease. Nat Rev Drug Discov 2003 Nov;2(11):879-90.

Foroud T, Koller DL, Lai D, Sauerbeck L, Anderson C, Ko N, et al. Genome-wide

association study of intracranial aneurysms confirms role of Anril and SOX17 in disease

risk. Stroke 2012 Nov;43(11):2846-52.

194

Foroud T, Lai D, Koller D, Van't Hof F, Kurki MI, Anderson CS, et al. Genome-wide

association study of intracranial aneurysm identifies a new association on chromosome

7. Stroke 2014 Nov;45(11):3194-9.

Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C. PI3K/Akt and apoptosis: size

matters. Oncogene 2003 Dec 8;22(56):8983-98.

Frankowski JC, DeMars KM, Ahmad AS, Hawkins KE, Yang C, Leclerc JL, et al.

Detrimental role of the EP1 prostanoid receptor in blood-brain barrier damage following

experimental ischemic stroke. Sci Rep 2015 Dec 9;5:17956.

Friedrich B, Muller F, Feiler S, Scholler K, Plesnila N. Experimental subarachnoid

hemorrhage causes early and long-lasting microarterial constriction and

microthrombosis: an in-vivo microscopy study. J Cereb Blood Flow Metab 2012

Mar;32(3):447-55.

Friedrich[A] V, Flores R, Muller A, Sehba FA. Luminal platelet aggregates in functional

deficits in parenchymal vessels after subarachnoid hemorrhage. Brain Res 2010 Oct

1;1354:179-87.

Friedrich[B] V, Flores R, Muller A, Sehba FA. Escape of intraluminal platelets into brain

parenchyma after subarachnoid hemorrhage. Neuroscience 2010 Feb 3;165(3):968-75.

Friedrich V, Flores R, Muller A, Bi W, Peerschke EI, Sehba FA. Reduction of neutrophil

activity decreases early microvascular injury after subarachnoid haemorrhage. J

Neuroinflammation 2011 Aug 19;8:103.

195

Friedrich V, Bi W, Sehba FA. Sexual dimorphism in gene expression after aneurysmal

subarachnoid hemorrhage. Neurol Res 2015;37(12):1054-9.

Frontera JA, Claassen J, Schmidt JM, Wartenberg KE, Temes R, Connolly ES, Jr., et al.

Prediction of symptomatic vasospasm after subarachnoid hemorrhage: the modified

fisher scale. Neurosurgery 2006 Jul;59(1):21-7.

Fujii M, Yan J, Rolland WB, Soejima Y, Caner B, Zhang JH. Early brain injury, an

evolving frontier in subarachnoid hemorrhage research. Transl Stroke Res 2013

Aug;4(4):432-46.

Gabathuler R. Approaches to transport therapeutic drugs across the blood-brain barrier

to treat brain diseases. Neurobiol Dis 2010 Jan;37(1):48-57.

Galeffi F, Degan S, Britz G, Turner DA. Dysregulation of oxygen hemodynamic

responses to synaptic train stimulation in a rat hippocampal model of subarachnoid

hemorrhage. J Cereb Blood Flow Metab 2016 Apr;36(4):696-701.

Geier EG, Chen EC, Webb A, Papp AC, Yee SW, Sadee W, et al. Profiling solute

carrier transporters in the human blood-brain barrier. Clin Pharmacol Ther 2013

Dec;94(6):636-9.

Geraghty JR, Testai FD. Delayed Cerebral Ischemia after Subarachnoid Hemorrhage:

Beyond Vasospasm and Towards a Multifactorial Pathophysiology. Curr Atheroscler

Rep 2017 Oct 23;19(12):50.

196

Germano A, d'Avella D, Imperatore C, Caruso G, Tomasello F. Time-course of blood-

brain barrier permeability changes after experimental subarachnoid haemorrhage. Acta

Neurochir (Wien ) 2000;142(5):575-80.

Germano A, Costa C, DeFord SM, Angileri FF, Arcadi F, Pike BR, et al. Systemic

administration of a calpain inhibitor reduces behavioral deficits and blood-brain barrier

permeability changes after experimental subarachnoid hemorrhage in the rat. J

Neurotrauma 2002 Jul;19(7):887-96.

Gilad R, Boaz M, Dabby R, Sadeh M, Lampl Y. Are post intracerebral hemorrhage

seizures prevented by anti-epileptic treatment? Epilepsy Res 2011 Aug;95(3):227-31.

Gobbo OL, O'Mara SM. Post-treatment, but not pre-treatment, with the selective

cyclooxygenase-2 inhibitor celecoxib markedly enhances functional recovery from kainic

acid-induced neurodegeneration. Neuroscience 2004;125(2):317-27.

Goldstein GW, Wolinsky JS, Csejtey J, Diamond I. Isolation of metabolically active

capillaries from rat brain. J Neurochem 1975 Nov;25(5):715-7.

Gram L, Bentsen KD. Valproate: an updated review. Acta Neurol Scand 1985

Aug;72(2):129-39.

Grosser T, Yu Y, FitzGerald GA. Emotion recollected in tranquility: lessons learned from

the COX-2 saga. Annu Rev Med 2010;61:17-33.

Gu H, Fei ZH, Wang YQ, Yang JG, Zhao CH, Cai Y, et al. Angiopoietin-1 and

Angiopoietin-2 Expression Imbalance Influence in Early Period After Subarachnoid

Hemorrhage. Int Neurourol J 2016 Dec;20(4):288-95.

197

Guez-Barber D, Fanous S, Harvey BK, Zhang Y, Lehrmann E, Becker KG, et al. FACS

purification of immunolabeled cell types from adult rat brain. J Neurosci Methods 2012

Jan 15;203(1):10-8.

Gules I, Satoh M, Nanda A, Zhang JH. Apoptosis, blood-brain barrier, and subarachnoid


Guo S, Zhou Y, Xing C, Lok J, Som AT, Ning M, et al. The vasculome of the mouse

brain. PLoS One 2012;7(12):e52665.

Gurnik S, Devraj K, Macas J, Yamaji M, Starke J, Scholz A, et al. Angiopoietin-2-

induced blood-brain barrier compromise and increased stroke size are rescued by VE-

PTP-dependent restoration of Tie2 signaling. Acta Neuropathol 2016 May;131(5):753-

73.

Hall CN, Reynell C, Gesslein B, Hamilton NB, Mishra A, Sutherland BA, et al. Capillary

pericytes regulate cerebral blood flow in health and disease. Nature 2014 Apr

3;508(7494):55-60.

Hamming AM, van der Toorn A, Rudrapatna US, Ma L, van Os HJ, Ferrari MD, et al.

Valproate Reduces Delayed Brain Injury in a Rat Model of Subarachnoid Hemorrhage.

Stroke 2017 Feb;48(2):452-8.

Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and

disease. Pharmacol Rev 2005 Jun;57(2):173-85.

He Y, Yao Y, Tsirka SE, Cao Y. Cell-culture models of the blood-brain barrier. Stroke

2014 Aug;45(8):2514-26.

198

Herz DA, Baez S, Shulman K. Pial microcirculation in subarachnoid hemorrhage. Stroke

1975 Jul;6(4):417-24.

Hirayama A, Tanahashi N, Daida H, Ishiguro N, Chachin M, Sugioka T, et al. Assessing

the cardiovascular risk between celecoxib and nonselective nonsteroidal

antiinflammatory drugs in patients with rheumatoid arthritis and osteoarthritis. Circ J

2014;78(1):194-205.

Hop JW, Rinkel GJ, Algra A, Berkelbach van der Sprenkel JW, van GJ. Randomized

pilot trial of postoperative aspirin in subarachnoid hemorrhage. Neurology 2000 Feb

22;54(4):872-8.

Huntley MA, Bien-Ly N, Daneman R, Watts RJ. Dissecting gene expression at the

blood-brain barrier. Front Neurosci 2014;8:355.

Hupe M, Li MX, Kneitz S, Davydova D, Yokota C, Kele-Olovsson J, Hot B, Stenman JM,

Gessler M. Gene expression profiles of brain endothelial cells during embryonic

development at bulk and single-cell levels. Sci Signal 2017 Jul;10(487):1-12.

Iadecola C, Forster C, Nogawa S, Clark HB, Ross ME. Cyclooxygenase-2

immunoreactivity in the human brain following cerebral ischemia. Acta Neuropathol

1999 Jul;98(1):9-14.

Iadecola C, Nedergaard M. Glial regulation of the cerebral microvasculature. Nat

Neurosci 2007 Nov;10(11):1369-76.

199

Ibrahim GM, Fallah A, Macdonald RL. Clinical, laboratory, and radiographic predictors

of the occurrence of seizures following aneurysmal subarachnoid hemorrhage. J

Neurosurg 2013 Aug;119(2):347-52.

Imperatore C, Germano A, d'Avella D, Tomasello F, Costa G. Effects of the radical

scavenger AVS on behavioral and BBB changes after experimental subarachnoid

hemorrhage. Life Sci 2000 Jan 21;66(9):779-90.

Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of

Affymetrix GeneChip probe level data. Nucleic Acids Res 2003 Feb 15;31(4):e15.

Ishikawa M, Kusaka G, Yamaguchi N, Sekizuka E, Nakadate H, Minamitani H, et al.

Platelet and leukocyte adhesion in the microvasculature at the cerebral surface

immediately after subarachnoid hemorrhage. Neurosurgery 2009 Mar;64(3):546-53.

Ishikawa M, Kajimura M, Morikawa T, Tsukada K, Tsuji T, Kusaka G, et al. Leukocyte

plugging and cortical capillary flow after subarachnoid hemorrhage. Acta Neurochir

(Wien ) 2016 Jun;158(6):1057-67.

Ivanidze J, Kesavabhotla K, Kallas ON, Mir D, Baradaran H, Gupta A, et al. Evaluating

blood-brain barrier permeability in delayed cerebral infarction after aneurysmal

subarachnoid hemorrhage. AJNR Am J Neuroradiol 2015 May;36(5):850-4.

Jaja BNR, Schweizer TA, Claassen J, Le RP, Mayer SA, Macdonald RL. The SAFARI

Score to Assess the Risk of Convulsive Seizure during Admission for Aneurysmal

Subarachnoid Hemorrhage. Neurosurgery 2017 Jun 27.

200

James RF, Kramer DR, Aljuboori ZS, Parikh G, Adams SW, Eaton JC, et al. Novel

Treatments in Neuroprotection for Aneurysmal Subarachnoid Hemorrhage. Curr Treat

Options Neurol 2016 Aug;18(8):38.

Jiang J, Quan Y, Ganesh T, Pouliot WA, Dudek FE, Dingledine R. Inhibition of the

prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and

brain inflammation. Proc Natl Acad Sci U S A 2013 Feb 26;110(9):3591-6.

Johnston SC, Selvin S, Gress DR. The burden, trends, and demographics of mortality

from subarachnoid hemorrhage. Neurology 1998 May;50(5):1413-8.

Johshita H, Kassell NF, Sasaki T, Ogawa H. Impaired capillary perfusion and brain

edema following experimental subarachnoid hemorrhage: a morphometric study. J

Neurosurg 1990 Sep;73(3):410-7.

Kabakus N, Ay I, Aysun S, Soylemezoglu F, Ozcan A, Celasun B. Protective effects of

valproic acid against hypoxic-ischemic brain injury in neonatal rats. J Child Neurol 2005

Jul;20(7):582-7.

Kajita Y, Dietrich HH, Dacey RG, Jr. Effects of oxyhemoglobin on local and propagated

vasodilatory responses induced by adenosine, adenosine diphosphate, and adenosine

triphosphate in rat cerebral arterioles. J Neurosurg 1996 Nov;85(5):908-16.

Kamii H, Kato I, Kinouchi H, Chan PH, Epstein CJ, Akabane A, et al. Amelioration of

vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing CuZn-

superoxide dismutase. Stroke 1999 Apr;30(4):867-71.

201

Kang YS, Terasaki T, Tsuji A. Acidic drug transport in vivo through the blood-brain

barrier. A role of the transport carrier for monocarboxylic acids. J Pharmacobiodyn 1990

Feb;13(2):158-63.

Kassner A, Merali Z. Assessment of Blood-Brain Barrier Disruption in Stroke. Stroke

2015 Nov;46(11):3310-5.

Katusic ZS, Milde JH, Cosentino F, Mitrovic BS. Subarachnoid hemorrhage and

endothelial L-arginine pathway in small brain stem arteries in dogs. Stroke 1993

Mar;24(3):392-9.

Kawano T, Anrather J, Zhou P, Park L, Wang G, Frys KA, et al. Prostaglandin E2 EP1

receptors: downstream effectors of COX-2 neurotoxicity. Nat Med 2006 Feb;12(2):225-

9.

Kerro A, Woods T, Chang JJ. Neurogenic stunned myocardium in subarachnoid

hemorrhage. J Crit Care 2017 Apr;38:27-34.

Kersseboom S, Kremers GJ, Friesema EC, Visser WE, Klootwijk W, Peeters RP, Visser

TJ. Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its

cellular distribution. Mol Endocrinol 2013 May;27(5):801-813.

Kikkawa Y, Matsuo S, Kurogi R, Nakamizo A, Mizoguchi M, Sasaki T. Upregulation of

relaxin after experimental subarachnoid hemorrhage in rabbits. Biomed Res Int

2014;2014:836397.

202

Kikkawa Y, Ogura T, Nakajima H, Ikeda T, Takeda R, Neki H, et al. Altered Expression

of MicroRNA-15a and Kruppel-Like Factor 4 in Cerebrospinal Fluid and Plasma After

Aneurysmal Subarachnoid Hemorrhage. World Neurosurg 2017 Dec;108:909-16.

Kim HJ, Rowe M, Ren M, Hong JS, Chen PS, Chuang DM. Histone deacetylase

inhibitors exhibit anti-inflammatory and neuroprotective effects in a rat permanent

ischemic model of stroke: multiple mechanisms of action. J Pharmacol Exp Ther 2007

Jun;321(3):892-901.

Kim I, Kim JH, Ryu YS, Liu M, Koh GY. Tumor necrosis factor-alpha upregulates

angiopoietin-2 in human umbilical vein endothelial cells. Biochem Biophys Res

Commun 2000 Mar 16;269(2):361-5.

Kinnecom K, Pachter JS. Selective capture of endothelial and perivascular cells from

brain microvessels using laser capture microdissection. Brain Res Brain Res Protoc

2005 Dec;16(1-3):1-9.

Kirkpatrick PJ, Turner CL, Smith C, Hutchinson PJ, Murray GD. Simvastatin in

aneurysmal subarachnoid haemorrhage (STASH): a multicentre randomised phase 3

trial. Lancet Neurol 2014 Jul;13(7):666-75.

Kishore S, Ko N, Soares BP, Higashida RT, Tong E, Bhogal S, et al. Perfusion-CT

assessment of blood-brain barrier permeability in patients with aneurysmal

subarachnoid hemorrhage. J Neuroradiol 2012 Dec;39(5):317-25.

203

Kleinloog R, Verweij BH, van d, V, Deelen P, Swertz MA, de ML, et al. RNA Sequencing

Analysis of Intracranial Aneurysm Walls Reveals Involvement of Lysosomes and

Immunoglobulins in Rupture. Stroke 2016 May;47(5):1286-93.

Kniesel U, Wolburg H. Tight junctions of the blood-brain barrier. Cell Mol Neurobiol

2000 Feb;20(1):57-76.

Knight RA, Nagaraja TN, Ewing JR, Nagesh V, Whitton PA, Bershad E, et al.

Quantitation and localization of blood-to-brain influx by magnetic resonance imaging

and quantitative autoradiography in a model of transient focal ischemia. Magn Reson

Med 2005 Oct;54(4):813-21.

Knowland D, Arac A, Sekiguchi KJ, Hsu M, Lutz SE, Perrino J, et al. Stepwise

recruitment of transcellular and paracellular pathways underlies blood-brain barrier

breakdown in stroke. Neuron 2014 May 7;82(3):603-17.

Koch S, Gidal BE. Phenytoin and cognitive decline. Stroke 2005 Oct;36(10):2070-1.

Koide M, Bonev AD, Nelson MT, Wellman GC. Inversion of neurovascular coupling by

subarachnoid blood depends on large-conductance Ca2+-activated K+ (BK) channels.

Proc Natl Acad Sci U S A 2012 May 22;109(21):E1387-E1395.

Koide M, Wellman GC. SAH-induced suppression of voltage-gated K(+) (K (V)) channel

currents in parenchymal arteriolar myocytes involves activation of the HB-EGF/EGFR

pathway. Acta Neurochir Suppl 2013;115:179-84.

Kumar TP, Vasudevan A. Isolation and culture of endothelial cells from the embryonic

forebrain. J Vis Exp 2014 Jan 23;(83):e51021.

204

Kurki MI, Hakkinen SK, Frosen J, Tulamo R, von Und Zu FM, Wong G, et al.

Upregulated signaling pathways in ruptured human saccular intracranial aneurysm wall:

an emerging regulative role of Toll-like receptor signaling and nuclear factor-kappaB,

hypoxia-inducible factor-1A, and ETS transcription factors. Neurosurgery 2011

Jun;68(6):1667-75.

Kurogi R, Kikkawa Y, Matsuo S, Nakamizo A, Mizoguchi M, Sasaki T. Upregulation of

tissue inhibitor of metalloproteinase-1 contributes to restoration of the extracellular

matrix in the rabbit basilar artery during cerebral vasospasm after subarachnoid

hemorrhage. Brain Res 2015 Aug 7;1616:26-36.

Kusaka G, Ishikawa M, Nanda A, Granger DN, Zhang JH. Signaling pathways for early

brain injury after subarachnoid hemorrhage. J Cereb Blood Flow Metab 2004

Aug;24(8):916-25.

Kwon KJ, Kim JN, Kim MK, Kim SY, Cho KS, Jeon SJ, et al. Neuroprotective effects of

valproic acid against hemin toxicity: possible involvement of the down-regulation of

heme oxygenase-1 by regulating ubiquitin-proteasomal pathway. Neurochem Int 2013

Feb;62(3):240-50.

Lai NS, Zhang JQ, Qin FY, Sheng B, Fang XG, Li ZB. Serum microRNAs are non-

invasive biomarkers for the presence and progression of subarachnoid haemorrhage.

Biosci Rep 2017 Feb 28;37(1).

Lampugnani MG. Endothelial adherens junctions and the actin cytoskeleton: an 'infinity

net'? J Biol 2010;9(3):16.

205

Lanzino G, D'Urso PI, Suarez J. Seizures and anticonvulsants after aneurysmal

subarachnoid hemorrhage. Neurocrit Care 2011 Sep;15(2):247-56.

Larsson P, Ulfhammer E, Magnusson M, Bergh N, Lunke S, El-Osta A, et al. Role of

histone acetylation in the stimulatory effect of valproic acid on vascular endothelial

tissue-type plasminogen activator expression. PLoS One 2012;7(2):e31573.

Larsson P, Alwis I, Niego B, Sashindranath M, Fogelstrand P, Wu MC, et al. Valproic

acid selectively increases vascular endothelial tissue-type plasminogen activator

production and reduces thrombus formation in the mouse. J Thromb Haemost 2016

Dec;14(12):2496-508.

Leclerc JL, Ahmad AS, Singh N, Soshnik-Schierling L, Greene E, Dang A, et al.

Intracerebral hemorrhage outcomes following selective blockade or stimulation of the

PGE2 EP1 receptor. BMC Neurosci 2015 Aug 1;16:48.

Lee CI, Chou AK, Lin CC, Chou CH, Loh JK, Lieu AS, et al. Immune and inflammatory

gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

Mol Med Rep 2012 Jan;5(1):118-25.

Lee[A] JH, Kim K, Jo YH, Lee SH, Kang C, Kim J, et al. Effect of valproic acid on

survival and neurologic outcomes in an asphyxial cardiac arrest model of rats.

Resuscitation 2013 Oct;84(10):1443-9.

Lee[B] SH, Park HK, Ryu WS, Lee JS, Bae HJ, Han MK, et al. Effects of celecoxib on

hematoma and edema volumes in primary intracerebral hemorrhage: a multicenter

randomized controlled trial. Eur J Neurol 2013 Aug;20(8):1161-9.

206

Li JY, Boado RJ, Pardridge WM. Blood-brain barrier genomics. J Cereb Blood Flow

Metab 2001 Jan;21(1):61-8.

Li JY, Boado RJ, Pardridge WM. Rat blood-brain barrier genomics. II. J Cereb Blood

Flow Metab 2002 Nov;22(11):1319-26.

Li Q, Chen Y, Li B, Luo C, Zuo S, Liu X, et al. Hemoglobin induced NO/cGMP

suppression Deteriorate Microcirculation via Pericyte Phenotype Transformation after

Subarachnoid Hemorrhage in Rats. Sci Rep 2016 Feb 25;6:22070.

Liebner S, Corada M, Bangsow T, Babbage J, Taddei A, Czupalla CJ, et al. Wnt/beta-

catenin signaling controls development of the blood-brain barrier. J Cell Biol 2008 Nov

3;183(3):409-17.

Lin CL, Calisaneller T, Ukita N, Dumont AS, Kassell NF, Lee KS. A murine model of

subarachnoid hemorrhage-induced cerebral vasospasm. J Neurosci Methods 2003 Feb

15;123(1):89-97.

Liu D, Han L, Wu X, Yang X, Zhang Q, Jiang F. Genome-wide microRNA changes in

human intracranial aneurysms. BMC Neurol 2014 Oct 10;14:188.

Liu XS, Chopp M, Kassis H, Jia LF, Hozeska-Solgot A, Zhang RL, et al. Valproic acid

increases white matter repair and neurogenesis after stroke. Neuroscience 2012 Sep

18;220:313-21.

Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, Epstein JA, et al. Failure of ductus

arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and

cyclooxygenase-2. Proc Natl Acad Sci U S A 2001 Jan 30;98(3):1059-64.

207

Lovatt D, Ruble BK, Lee J, Dueck H, Kim TK, Fisher S, et al. Transcriptome in vivo

analysis (TIVA) of spatially defined single cells in live tissue. Nat Methods 2014

Feb;11(2):190-6.

Macdonald JA, Murugesan N, Pachter JS. Endothelial cell heterogeneity of blood-brain

barrier gene expression along the cerebral microvasculature. J Neurosci Res 2010 May

15;88(7):1457-74.

Macdonald RL, Zhang ZD, Ono S, Komuro T. Up-regulation of parathyroid hormone

receptor in cerebral arteries after subarachnoid hemorrhage in monkeys. Neurosurgery

2002 May;50(5):1083-91.

Macdonald RL, Kassell NF, Mayer S, Ruefenacht D, Schmiedek P, Weidauer S, et al.

Clazosentan to overcome neurological ischemia and infarction occurring after

subarachnoid hemorrhage (CONSCIOUS-1): randomized, double-blind, placebo-

controlled phase 2 dose-finding trial. Stroke 2008 Nov;39(11):3015-21.

Macdonald RL, Higashida RT, Keller E, Mayer SA, Molyneux A, Raabe A, et al.

Clazosentan, an endothelin receptor antagonist, in patients with aneurysmal

subarachnoid haemorrhage undergoing surgical clipping: a randomised, double-blind,

placebo-controlled phase 3 trial (CONSCIOUS-2). Lancet Neurol 2011 Jul;10(7):618-25.

Macdonald RL, Higashida RT, Keller E, Mayer SA, Molyneux A, Raabe A, et al.

Randomized trial of clazosentan in patients with aneurysmal subarachnoid hemorrhage

undergoing endovascular coiling. Stroke 2012 Jun;43(6):1463-9.

208

Macdonald RL. Delayed neurological deterioration after subarachnoid haemorrhage.

Nat Rev Neurol 2014 Jan;10(1):44-58.

Macdonald RL, Schweizer TA. Spontaneous subarachnoid haemorrhage. Lancet 2017

Feb 11;389(10069):655-66.

Macdonald RL. Origins of the Concept of Vasospasm. Stroke 2016 Jan;47(1):e11-e15.

Maddaluno L, Rudini N, Cuttano R, Bravi L, Giampietro C, Corada M, et al. EndMT

contributes to the onset and progression of cerebral cavernous malformations. Nature

2013 Jun 27;498(7455):492-6.

Manaenko A, Chen H, Kammer J, Zhang JH, Tang J. Comparison Evans Blue injection

routes: Intravenous versus intraperitoneal, for measurement of blood-brain barrier in a

mice hemorrhage model. J Neurosci Methods 2011 Feb 15;195(2):206-10.

Mayberg MR, Okada T, Bark DH. Morphologic changes in cerebral arteries after

subarachnoid hemorrhage. Neurosurg Clin N Am 1990 Apr;1(2):417-432.

McNamee LM, Walsh MJ, Ledley FD. Timelines of translational science: From

technology initiation to FDA approval. PLoS One 2017;12(5):e0177371.

Michaux G, Cutler DF. How to roll an endothelial cigar: the biogenesis of Weibel-Palade

bodies. Traffic 2004 Feb;5(2):69-78.

Minami T, Kuivenhoven JA, Evans V, Kodama T, Rosenberg RD, Aird WC. Ets motifs

are necessary for endothelial cell-specific expression of a 723-bp Tie-2

promoter/enhancer in Hprt targeted transgenic mice. Arterioscler Thromb Vasc Biol

2003 Nov 1;23(11):2041-7.

209

Minami T, Sugiyama A, Wu SQ, Abid R, Kodama T, Aird WC. Thrombin and phenotypic

modulation of the endothelium. Arterioscler Thromb Vasc Biol 2004 Jan;24(1):41-53.

Minami T, Aird WC. Endothelial cell gene regulation. Trends Cardiovasc Med 2005

Jul;15(5):174-84.

Mink S, Muroi C, Seule M, Bjeljac M, Keller E. Levetiracetam compared to valproic acid:

plasma concentration levels, adverse effects and interactions in aneurysmal

subarachnoid hemorrhage. Clin Neurol Neurosurg 2011 Oct;113(8):644-8.

Molyneux A, Kerr R, Stratton I, Sandercock P, Clarke M, Shrimpton J, et al.

International Subarachnoid Aneurysm Trial (ISAT) of neurosurgical clipping versus

endovascular coiling in 2143 patients with ruptured intracranial aneurysms: a

randomised trial. Lancet 2002 Oct 26;360(9342):1267-74.

Montagne A, Toga AW, Zlokovic BV. Blood-Brain Barrier Permeability and Gadolinium:

Benefits and Potential Pitfalls in Research. JAMA Neurol 2016 Jan;73(1):13-4.

Morgan S, Grootendorst P, Lexchin J, Cunningham C, Greyson D. The cost of drug

development: a systematic review. Health Policy 2011 Apr;100(1):4-17.

Motoike T, Loughna S, Perens E, Roman BL, Liao W, Chau TC, et al. Universal GFP

reporter for the study of vascular development. Genesis 2000 Oct;28(2):75-81.

Munakata A, Naraoka M, Katagai T, Shimamura N, Ohkuma H. Role of

Cyclooxygenase-2 in Relation to Nitric Oxide and Endothelin-1 on Pathogenesis of

Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbit. Transl Stroke Res

2016 Jun;7(3):220-7.

210

Murphy A, Manoel AL, Burgers K, Kouzmina E, Lee T, Macdonald RL, et al. Early CT

perfusion changes and blood-brain barrier permeability after aneurysmal subarachnoid

hemorrhage. Neuroradiology 2015 Aug;57(8):767-73.

Naidech AM, Kreiter KT, Janjua N, Ostapkovich N, Parra A, Commichau C, et al.

Phenytoin exposure is associated with functional and cognitive disability after

subarachnoid hemorrhage. Stroke 2005 Mar;36(3):583-7.

Nakaoka H, Tajima A, Yoneyama T, Hosomichi K, Kasuya H, Mizutani T, et al. Gene

expression profiling reveals distinct molecular signatures associated with the rupture of

intracranial aneurysm. Stroke 2014 Aug;45(8):2239-45.

Nassiri F, Ibrahim GM, Badhiwala JH, Witiw CD, Mansouri A, Alotaibi NM, et al. A

Propensity Score-Matched Study of the Use of Non-steroidal Anti-inflammatory Agents

Following Aneurysmal Subarachnoid Hemorrhage. Neurocrit Care 2016 Dec;25(3):351-

8.

Neuwelt EA, Bauer B, Fahlke C, Fricker G, Iadecola C, Janigro D, et al. Engaging

neuroscience to advance translational research in brain barrier biology. Nat Rev

Neurosci 2011 Mar;12(3):169-82.

Nguyen LN, Ma D, Shui G, Wong P, Cazenave-Gassiot A, Zhang X, et al. Mfsd2a is a

transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 2014

May 22;509(7501):503-6.

211

Nissen SE, Yeomans ND, Solomon DH, Luscher TF, Libby P, Husni ME, et al.

Cardiovascular Safety of Celecoxib, Naproxen, or Ibuprofen for Arthritis. N Engl J Med

2016 Dec 29;375(26):2519-29.

Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, et al. Size-selective loosening

of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 2003 May

12;161(3):653-60.

Nolan DJ, Ginsberg M, Israely E, Palikuqi B, Poulos MG, James D, et al. Molecular

signatures of tissue-specific microvascular endothelial cell heterogeneity in organ

maintenance and regeneration. Dev Cell 2013 Jul 29;26(2):204-19.

Nystoriak MA, O'Connor KP, Sonkusare SK, Brayden JE, Nelson MT, Wellman GC.

Fundamental increase in pressure-dependent constriction of brain parenchymal

arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.

Am J Physiol Heart Circ Physiol 2011 Mar;300(3):H803-H812.

Obermeier B, Daneman R, Ransohoff RM. Development, maintenance and disruption of

the blood-brain barrier. Nat Med 2013 Dec;19(12):1584-96.

Ohkuma H, Itoh K, Shibata S, Suzuki S. Morphological changes of intraparenchymal

arterioles after experimental subarachnoid hemorrhage in dogs. Neurosurgery 1997

Jul;41(1):230-5.

Ohkuma H, Suzuki S. Histological dissociation between intra- and extraparenchymal

portion of perforating small arteries after experimental subarachnoid hemorrhage in

dogs. Acta Neuropathol 1999 Oct;98(4):374-82.

212

Ohkuma H, Suzuki S, Ogane K. Phenotypic modulation of smooth muscle cells and

vascular remodeling in intraparenchymal small cerebral arteries after canine

experimental subarachnoid hemorrhage. Neurosci Lett 2003 Jul 3;344(3):193-6.

Ohtsuki S, Yamaguchi H, Asashima T, Terasaki T. Establishing a method to isolate rat

brain capillary endothelial cells by magnetic cell sorting and dominant mRNA expression

of multidrug resistance-associated protein 1 and 4 in highly purified rat brain capillary

endothelial cells. Pharm Res 2007 Apr;24(4):688-94.

Ohtsuki S, Yamaguchi H, Katsukura Y, Asashima T, Terasaki T. mRNA expression

levels of tight junction protein genes in mouse brain capillary endothelial cells highly

purified by magnetic cell sorting. J Neurochem 2008 Jan;104(1):147-54.

Onda H, Kasuya H, Takakura K, Hori T, Imaizumi T, Takeuchi T, et al. Identification of

genes differentially expressed in canine vasospastic cerebral arteries after

subarachnoid hemorrhage. J Cereb Blood Flow Metab 1999 Nov;19(11):1279-88.

Ostergaard L, Aamand R, Karabegovic S, Tietze A, Blicher JU, Mikkelsen IK, et al. The

role of the microcirculation in delayed cerebral ischemia and chronic degenerative

changes after subarachnoid hemorrhage. J Cereb Blood Flow Metab 2013

Dec;33(12):1825-37.

Osuka K, Suzuki Y, Watanabe Y, Takayasu M, Yoshida J. Inducible cyclooxygenase

expression in canine basilar artery after experimental subarachnoid hemorrhage. Stroke

1998 Jun;29(6):1219-22.

213

Osuka K, Watanabe Y, Yamauchi K, Nakazawa A, Usuda N, Tokuda M, et al. Activation

of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid

hemorrhage. Brain Res 2006 Feb 9;1072(1):1-7.

Pardridge WM, Eisenberg J, Yamada T. Rapid sequestration and degradation of

somatostatin analogues by isolated brain microvessels. J Neurochem 1985

Apr;44(4):1178-84.

Pardridge WM. Blood-brain barrier genomics. Stroke 2007 Feb;38(2 Suppl):686-90.

Pardridge WM. Delivery of Biologics Across the Blood-Brain Barrier with Molecular

Trojan Horse Technology. BioDrugs 2017 Dec;31(6):503-19.

Park IS, Meno JR, Witt CE, Chowdhary A, Nguyen TS, Winn HR, et al. Impairment of

intracerebral arteriole dilation responses after subarachnoid hemorrhage. Laboratory

investigation. J Neurosurg 2009 Nov;111(5):1008-13.

Park KW, Metais C, Dai HB, Comunale ME, Sellke FW. Microvascular endothelial

dysfunction and its mechanism in a rat model of subarachnoid hemorrhage. Anesth

Analg 2001 Apr;92(4):990-6.

Park KW, Dai HB, Metais C, Comunale ME, Sellke FW. Isoflurane does not further

impair microvascular vasomotion in a rat model of subarachnoid hemorrhage. Can J

Anaesth 2002 Apr;49(4):427-33.

Parkhutik V, Lago A, Tembl JI, Rubio C, Fuset MP, Valles J, et al. Influence of COX-

inhibiting analgesics on the platelet function of patients with subarachnoid hemorrhage.

J Stroke Cerebrovasc Dis 2012 Nov;21(8):755-9.

214

Peng J, Wu Y, Tian X, Pang J, Kuai L, Cao F, et al. High-Throughput Sequencing and

Co-Expression Network Analysis of lncRNAs and mRNAs in Early Brain Injury Following

Experimental Subarachnoid Haemorrhage. Sci Rep 2017 Apr 18;7:46577.

Pera J, Korostynski M, Krzyszkowski T, Czopek J, Slowik A, Dziedzic T, et al. Gene

expression profiles in human ruptured and unruptured intracranial aneurysms: what is

the role of inflammation? Stroke 2010 Feb;41(2):224-31.

Perrone RD, Malek AM, Watnick T. Vascular complications in autosomal dominant

polycystic kidney disease. Nat Rev Nephrol 2015 Oct;11(10):589-98.

Pichiule P, Chavez JC, LaManna JC. Hypoxic regulation of angiopoietin-2 expression in

endothelial cells. J Biol Chem 2004 Mar 26;279(13):12171-80.

Pickard JD, Murray GD, Illingworth R, Shaw MD, Teasdale GM, Foy PM, et al. Effect of

oral nimodipine on cerebral infarction and outcome after subarachnoid haemorrhage:

British aneurysm nimodipine trial. BMJ 1989 Mar 11;298(6674):636-42.

Pirlamarla P, Bond RM. FDA labeling of NSAIDs: Review of nonsteroidal anti-

inflammatory drugs in cardiovascular disease. Trends Cardiovasc Med 2016

Nov;26(8):675-80.

Pisapia JM, Xu X, Kelly J, Yeung J, Carrion G, Tong H, et al. Microthrombosis after

experimental subarachnoid hemorrhage: time course and effect of red blood cell-bound

thrombin-activated pro-urokinase and clazosentan. Exp Neurol 2012 Jan;233(1):357-63.

Pober JS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev

Immunol 2007 Oct;7(10):803-15.

215

Porte B, Hardouin J, Zerdoumi Y, Derambure C, Hauchecorne M, Dupre N, et al. Major

remodeling of brain microvessels during neonatal period in the mouse: A proteomic and

transcriptomic study. J Cereb Blood Flow Metab 2017 Feb;37(2):495-513.

Prunell GF, Mathiesen T, Svendgaard NA. A new experimental model in rats for study

of the pathophysiology of subarachnoid hemorrhage. Neuroreport 2002 Dec

20;13(18):2553-6.

Prunell GF, Svendgaard NA, Alkass K, Mathiesen T. Delayed cell death related to acute

cerebral blood flow changes following subarachnoid hemorrhage in the rat brain. J

Neurosurg 2005 Jun;102(6):1046-54.

Radu M, Chernoff J. An in vivo assay to test blood vessel permeability. J Vis Exp 2013

Mar 16;(73):e50062.

Ramakrishna R, Sekhar LN, Ramanathan D, Temkin N, Hallam D, Ghodke BV, et al.

Intraventricular tissue plasminogen activator for the prevention of vasospasm and

hydrocephalus after aneurysmal subarachnoid hemorrhage. Neurosurgery 2010

Jul;67(1):110-7.

Raslan F, Albert-Weissenberger C, Westermaier T, Saker S, Kleinschnitz C, Lee JY. A

modified double injection model of cisterna magna for the study of delayed cerebral

vasospasm following subarachnoid hemorrhage in rats. Exp Transl Stroke Med 2012

Nov 29;4(1):23.

Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies

revisited. FASEB J 2008 Mar;22(3):659-61.

216

Reglero-Real N, Colom B, Bodkin JV, Nourshargh S. Endothelial Cell Junctional

Adhesion Molecules: Role and Regulation of Expression in Inflammation. Arterioscler

Thromb Vasc Biol 2016 Oct;36(10):2048-57.

Ren M, Leng Y, Jeong M, Leeds PR, Chuang DM. Valproic acid reduces brain damage

induced by transient focal cerebral ischemia in rats: potential roles of histone

deacetylase inhibition and heat shock protein induction. J Neurochem 2004

Jun;89(6):1358-67.

Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc

Biol 2011 May;31(5):986-1000.

Rosalind Lai PM, Du R. Role of Genetic Polymorphisms in Predicting Delayed Cerebral

Ischemia and Radiographic Vasospasm After Aneurysmal Subarachnoid Hemorrhage:

A Meta-Analysis. World Neurosurg 2015 Oct;84(4):933-41.

Rosengart AJ, Huo JD, Tolentino J, Novakovic RL, Frank JI, Goldenberg FD, et al.

Outcome in patients with subarachnoid hemorrhage treated with antiepileptic drugs. J

Neurosurg 2007 Aug;107(2):253-60.

Rubin DB. Estimating causal effects from large data sets using propensity scores. Ann

Intern Med 1997 Oct 15;127(8 Pt 2):757-63.

Rush B, Wiskar K, Fruhstorfer C, Hertz P. Association between seizures and mortality in

patients with aneurysmal subarachnoid hemorrhage: A nationwide retrospective cohort

analysis. Seizure 2016 Oct;41:66-9.

217

Sabri M, Jeon H, Ai J, Tariq A, Shang X, Chen G, et al. Anterior circulation mouse

model of subarachnoid hemorrhage. Brain Res 2009 Oct 27;1295:179-85.

Sabri M, Ai J, Knight B, Tariq A, Jeon H, Shang X, et al. Uncoupling of endothelial nitric

oxide synthase after experimental subarachnoid hemorrhage. J Cereb Blood Flow

Metab 2011 Jan;31(1):190-9.

Sabri M, Ai J, Lakovic K, D'abbondanza J, Ilodigwe D, Macdonald RL. Mechanisms of

microthrombi formation after experimental subarachnoid hemorrhage. Neuroscience

2012 Nov 8;224:26-37.

Sader N, Zeiler FA, Gillman LM, West M, Kazina CJ. Indomethacin for control of ICP.

Neurocrit Care 2015 Jun;22(3):437-49.

Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, et al. Complex

phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell

2000 Dec;11(12):4131-42.

Santiago-Sim T, Fang X, Hennessy ML, Nalbach SV, DePalma SR, Lee MS, et al.

THSD1 (Thrombospondin Type 1 Domain Containing Protein 1) Mutation in the

Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage. Stroke 2016

Dec;47(12):3005-13.

Sasahara A, Kasuya H, Krischek B, Tajima A, Onda H, Sasaki T, et al. Gene expression

in a canine basilar artery vasospasm model: a genome-wide network-based analysis.

Neurosurg Rev 2008 Jul;31(3):283-90.

218

Saunders NR, Dreifuss JJ, Dziegielewska KM, Johansson PA, Habgood MD, Mollgard

K, et al. The rights and wrongs of blood-brain barrier permeability studies: a walk

through 100 years of history. Front Neurosci 2014;8:404.

Schallner N, Pandit R, LeBlanc R, III, Thomas AJ, Ogilvy CS, Zuckerbraun BS, et al.

Microglia regulate blood clearance in subarachnoid hemorrhage by heme oxygenase-1.

J Clin Invest 2015 Jul 1;125(7):2609-25.

Schmidt-Kastner R, Meller D, Bellander BM, Stromberg I, Olson L, Ingvar M. A one-step

immunohistochemical method for detection of blood-brain barrier disturbances for

immunoglobulins in lesioned rat brain with special reference to false-positive labelling in

immunohistochemistry. J Neurosci Methods 1993 Feb;46(2):121-32.

Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)

method. Nat Protoc 2008;3(6):1101-8.

Schoknecht K, Prager O, Vazana U, Kamintsky L, Harhausen D, Zille M, et al.

Monitoring stroke progression: in vivo imaging of cortical perfusion, blood-brain barrier

permeability and cellular damage in the rat photothrombosis model. J Cereb Blood Flow

Metab 2014 Nov;34(11):1791-801.

Scholler K, Trinkl A, Klopotowski M, Thal SC, Plesnila N, Trabold R, et al.

Characterization of microvascular basal lamina damage and blood-brain barrier

dysfunction following subarachnoid hemorrhage in rats. Brain Res 2007 Apr

20;1142:237-46.

219

Sehba FA, Mostafa G, Knopman J, Friedrich V, Jr., Bederson JB. Acute alterations in

microvascular basal lamina after subarachnoid hemorrhage. J Neurosurg 2004

Oct;101(4):633-40.

Sehba FA, Friedrich V, Jr., Makonnen G, Bederson JB. Acute cerebral vascular injury

after subarachnoid hemorrhage and its prevention by administration of a nitric oxide

donor. J Neurosurg 2007 Feb;106(2):321-9.

Sehba FA, Flores R, Muller A, Friedrich V, Chen JF, Britz GW, et al. Adenosine A(2A)

receptors in early ischemic vascular injury after subarachnoid hemorrhage. Laboratory

investigation. J Neurosurg 2010 Oct;113(4):826-34.

Sehba FA, Friedrich V. Early micro vascular changes after subarachnoid hemorrhage.

Acta Neurochir Suppl 2011;110(Pt 1):49-55.

Sehba FA, Friedrich V. Cerebral microvasculature is an early target of subarachnoid


Seibenhener ML, Wooten MC. Use of the Open Field Maze to measure locomotor and

anxiety-like behavior in mice. J Vis Exp 2015 Feb 6;(96):e52434.

Sherchan P, Lekic T, Suzuki H, Hasegawa Y, Rolland W, Duris K, et al. Minocycline

improves functional outcomes, memory deficits, and histopathology after endovascular

perforation-induced subarachnoid hemorrhage in rats. J Neurotrauma 2011

Dec;28(12):2503-12.

Shin J, Ming GL, Song H. Decoding neural transcriptomes and epigenomes via high-

throughput sequencing. Nat Neurosci 2014 Nov;17(11):1463-75.

220

Shusta EV, Boado RJ, Mathern GW, Pardridge WM. Vascular genomics of the human

brain. J Cereb Blood Flow Metab 2002 Mar;22(3):245-52.

Siakotos AN, Rouser G. Isolation of highly purified human and bovine brain endothelial

cells and nuclei and their phospholipid composition. Lipids 1969 May;4(3):234-9.

Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of

prostaglandin synthesis and inhibition. Pharmacol Rev 2004 Sep;56(3):387-437.

Sinn DI, Kim SJ, Chu K, Jung KH, Lee ST, Song EC, et al. Valproic acid-mediated

neuroprotection in intracerebral hemorrhage via histone deacetylase inhibition and

transcriptional activation. Neurobiol Dis 2007 May;26(2):464-72.

Smith RR, Clower BR, Grotendorst GM, Yabuno N, Cruse JM. Arterial wall changes in

early human vasospasm. Neurosurgery 1985 Feb;16(2):171-176.

Smith SL, Scherch HM, Hall ED. Protective effects of tirilazad mesylate and metabolite

U-89678 against blood-brain barrier damage after subarachnoid hemorrhage and lipid

peroxidative neuronal injury. J Neurosurg 1996 Feb;84(2):229-33.

Solomon RA, Antunes JL, Chen RY, Bland L, Chien S. Decrease in cerebral blood flow

in rats after experimental subarachnoid hemorrhage: a new animal model. Stroke 1985

Jan;16(1):58-64.

Song H, Suehiro J, Kanki Y, Kawai Y, Inoue K, Daida H, et al. Critical role for GATA3 in

mediating Tie2 expression and function in large vessel endothelial cells. J Biol Chem

2009 Oct 16;284(42):29109-24.

221

Spadoni I, Zagato E, Bertocchi A, Paolinelli R, Hot E, Di SA, et al. A gut-vascular barrier

controls the systemic dissemination of bacteria. Science 2015 Nov 13;350(6262):830-4.

Stein SC, Browne KD, Chen XH, Smith DH, Graham DI. Thromboembolism and

delayed cerebral ischemia after subarachnoid hemorrhage: an autopsy study.

Neurosurgery 2006 Oct;59(4):781-7.

Stepita-Klauco M, Dolezalova H. Cadaverine in the brain of axenic mice. Nature 1974

Nov 8;252(5479):158-9.

Storck SE, Meister S, Nahrath J, Meissner JN, Schubert N, Di SA, et al. Endothelial

LRP1 transports amyloid-beta(1-42) across the blood-brain barrier. J Clin Invest 2016

Jan;126(1):123-36.

Stratmann A, Risau W, Plate KH. Cell type-specific expression of angiopoietin-1 and

angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am J Pathol 1998

Nov;153(5):1459-66.

Straus I, Globus JH, Ginsburg SW. Spontaneous subarachnoid hemorrhage. Archives

of Neurology and Psychiatry 1932;1080-132.

Stylli SS, Adamides AA, Koldej RM, Luwor RB, Ritchie DS, Ziogas J, et al. miRNA

expression profiling of cerebrospinal fluid in patients with aneurysmal subarachnoid

hemorrhage. J Neurosurg 2017 Apr;126(4):1131-9.

Su XW, Chan AH, Lu G, Lin M, Sze J, Zhou JY, et al. Circulating microRNA 132-3p and

324-3p Profiles in Patients after Acute Aneurysmal Subarachnoid Hemorrhage. PLoS

One 2015;10(12):e0144724.

222

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al.

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-

wide expression profiles. Proc Natl Acad Sci U S A 2005 Oct 25;102(43):15545-50.

Sun BL, Zheng CB, Yang MF, Yuan H, Zhang SM, Wang LX. Dynamic alterations of

cerebral pial microcirculation during experimental subarachnoid hemorrhage. Cell Mol

Neurobiol 2009 Mar;29(2):235-41.

Suwatcharangkoon S, Meyers E, Falo C, Schmidt JM, Agarwal S, Claassen J, et al.

Loss of Consciousness at Onset of Subarachnoid Hemorrhage as an Important Marker

of Early Brain Injury. JAMA Neurol 2016 Jan;73(1):28-35.

Suzuki H, Kanamaru K, Tsunoda H, Inada H, Kuroki M, Sun H, et al. Heme oxygenase-

1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats. J

Clin Invest 1999 Jul;104(1):59-66.

Suzuki H, Ayer R, Sugawara T, Chen W, Sozen T, Hasegawa Y, et al. Protective effects

of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats.

Crit Care Med 2010 Feb;38(2):612-8.

Sweeney MD, Ayyadurai S, Zlokovic BV. Pericytes of the neurovascular unit: key

functions and signaling pathways. Nat Neurosci 2016 May 26;19(6):771-83.

Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, et al. Astrocyte-mediated

control of cerebral blood flow. Nat Neurosci 2006 Feb;9(2):260-7.

223

Tam SJ, Richmond DL, Kaminker JS, Modrusan Z, Martin-McNulty B, Cao TC, et al.

Death receptors DR6 and TROY regulate brain vascular development. Dev Cell 2012

Feb 14;22(2):403-17.

Tanaka S, Mori M, Sakamoto Y, Makuuchi M, Sugimachi K, Wands JR. Biologic

significance of angiopoietin-2 expression in human hepatocellular carcinoma. J Clin

Invest 1999 Feb;103(3):341-5.

Tartara A, Galimberti CA, Manni R, Parietti L, Zucca C, Baasch H, et al. Differential

effects of valproic acid and enzyme-inducing anticonvulsants on nimodipine

pharmacokinetics in epileptic patients. Br J Clin Pharmacol 1991 Sep;32(3):335-40.

Temkin NR, Dikmen SS, Anderson GD, Wilensky AJ, Holmes MD, Cohen W, et al.

Valproate therapy for prevention of posttraumatic seizures: a randomized trial. J

Neurosurg 1999 Oct;91(4):593-600.

Tenreiro MM, Ferreira R, Bernardino L, Brito MA. Cellular response of the blood-brain

barrier to injury: Potential biomarkers and therapeutic targets for brain regeneration.

Neurobiol Dis 2016 Jul;91:262-73.

Terpolilli NA, Brem C, Buhler D, Plesnila N. Are We Barking Up the Wrong Vessels?

Cerebral Microcirculation After Subarachnoid Hemorrhage. Stroke 2015

Oct;46(10):3014-9.

Thorn CF, Grosser T, Klein TE, Altman RB. PharmGKB summary: very important

pharmacogene information for PTGS2. Pharmacogenet Genomics 2011 Sep;21(9):607-

13.

224

Titova E, Ostrowski RP, Zhang JH, Tang J. Experimental models of subarachnoid

hemorrhage for studies of cerebral vasospasm. Neurol Res 2009 Jul;31(6):568-81.

Tran Dinh YR, Jomaa A, Callebert J, Reynier-Rebuffel AM, Tedgui A, Savarit A, et al.

Overexpression of cyclooxygenase-2 in rabbit basilar artery endothelial cells after

subarachnoid hemorrhage. Neurosurgery 2001 Mar;48(3):626-33.

Tran KA, Zhang X, Predescu D, Huang X, Machado RF, Gothert JR, et al. Endothelial

beta-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity

and Central Nervous System Homeostasis. Circulation 2016 Jan 12;133(2):177-86.

Tso MK, Macdonald RL. Acute microvascular changes after subarachnoid hemorrhage

and transient global cerebral ischemia. Stroke Res Treat 2013;2013:425281.

Tso MK, Macdonald RL. Subarachnoid hemorrhage: a review of experimental studies

on the microcirculation and the neurovascular unit. Transl Stroke Res 2014

Apr;5(2):174-89.

Tso MK, Lass E, Ai J, Loch MR. Valproic Acid treatment after experimental

subarachnoid hemorrhage. Acta Neurochir Suppl 2015;120:81-5.

Tso MK, Ibrahim GM, Macdonald RL. Predictors of Shunt-Dependent Hydrocephalus

Following Aneurysmal Subarachnoid Hemorrhage. World Neurosurg 2016 Feb;86:226-

32.

Tuma P, Hubbard AL. Transcytosis: crossing cellular barriers. Physiol Rev 2003

Jul;83(3):871-932.

225

Tyagi S, Gupta P, Saini AS, Kaushal C, Sharma S. The peroxisome proliferator-

activated receptor: A family of nuclear receptors role in various diseases. J Adv Pharm

Technol Res 2011 Oct;2(4):236-40.

van den Bergh WM, Algra A, Dorhout Mees SM, van KF, Dirven CM, van GJ, et al.

Randomized controlled trial of acetylsalicylic acid in aneurysmal subarachnoid

hemorrhage: the MASH Study. Stroke 2006 Sep;37(9):2326-30.

van der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S, O'Collins V, et al. Can

animal models of disease reliably inform human studies? PLoS Med 2010 Mar

30;7(3):e1000245.

Vasilache AM, Qian H, Blomqvist A. Immune challenge by intraperitoneal administration

of lipopolysaccharide directs gene expression in distinct blood-brain barrier cells toward

enhanced prostaglandin E(2) signaling. Brain Behav Immun 2015 Aug;48:31-41.

Vasudevan A, Long JE, Crandall JE, Rubenstein JL, Bhide PG. Compartment-specific

transcription factors orchestrate angiogenesis gradients in the embryonic brain. Nat

Neurosci 2008 Apr;11(4):429-39.

Veelken JA, Laing RJ, Jakubowski J. The Sheffield model of subarachnoid hemorrhage

in rats. Stroke 1995 Jul;26(7):1279-83.

Vergouwen MD, Knaup VL, Roelofs JJ, de Boer OJ, Meijers JC. Effect of recombinant

ADAMTS-13 on microthrombosis and brain injury after experimental subarachnoid

hemorrhage. J Thromb Haemost 2014 Jun;12(6):943-7.

226

Vikman P, Beg S, Khurana TS, Hansen-Schwartz J, Edvinsson L. Gene expression and

molecular changes in cerebral arteries following subarachnoid hemorrhage in the rat. J

Neurosurg 2006 Sep;105(3):438-44.

Vollmer DG, Takayasu M, Dacey RG, Jr. An in vitro comparative study of conducting

vessels and penetrating arterioles after experimental subarachnoid hemorrhage in the

rabbit. J Neurosurg 1992 Jul;77(1):113-9.

Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and

related forms of learning and memory. Nat Protoc 2006;1(2):848-58.

Wallgard E, Larsson E, He L, Hellstrom M, Armulik A, Nisancioglu MH, et al.

Identification of a core set of 58 gene transcripts with broad and specific expression in

the microvasculature. Arterioscler Thromb Vasc Biol 2008 Aug;28(8):1469-76.

Wang W, Li H, Yu L, Zhao Z, Wang H, Zhang D, et al. Aberrant expression of lncRNAs

and mRNAs in patients with intracranial aneurysm. Oncotarget 2017 Jan 10;8(2):2477-

84.

Wang Y, Tian Y, Wang D, Wei H, Zhao Z, Jiang R, et al. High Angiopoietin-1 levels

predict a good functional outcome within 72 h of an aneurysmal subarachnoid

hemorrhage: A prospective study from a single center. J Neurol Sci 2015 Sep 15;356(1-

2):72-6.

Wang Z, Zuo G, Shi XY, Zhang J, Fang Q, Chen G. Progesterone administration

modulates cortical TLR4/NF-kappaB signaling pathway after subarachnoid hemorrhage

in male rats. Mediators Inflamm 2011;2011:848309.

227

Weir B, Grace M, Hansen J, Rothberg C. Time course of vasospasm in man. J

Neurosurg 1978 Feb;48(2):173-8.

Wiernsperger N, Schulz U, Gygax P. Physiological and morphometric analysis of the

microcirculation of the cerebral cortex under acute vasospasm. Stroke 1981

Sep;12(5):624-7.

Williams DL, Pemberton E, Leslie K. Effect of intravenous parecoxib on post-craniotomy

pain. Br J Anaesth 2011 Sep;107(3):398-403.

Winkler MK, Chassidim Y, Lublinsky S, Revankar GS, Major S, Kang EJ, et al. Impaired

neurovascular coupling to ictal epileptic activity and spreading depolarization in a

patient with subarachnoid hemorrhage: possible link to blood-brain barrier dysfunction.

Epilepsia 2012 Nov;53 Suppl 6:22-30.

Won C, Lin Z, Kumar TP, Li S, Ding L, Elkhal A, et al. Autonomous vascular networks

synchronize GABA neuron migration in the embryonic forebrain. Nat Commun

2013;4:2149.

Wong GK, Poon WS. Use of phenytoin and other anticonvulsant prophylaxis in patients

with aneurysmal subarachnoid hemorrhage. Stroke 2005 Dec;36(12):2532.

Wong GK, Chan DY, Siu DY, Zee BC, Poon WS, Chan MT, et al. High-dose simvastatin

for aneurysmal subarachnoid hemorrhage: multicenter randomized controlled double-

blinded clinical trial. Stroke 2015 Feb;46(2):382-8.

228

Wu Z, Hofman FM, Zlokovic BV. A simple method for isolation and characterization of

mouse brain microvascular endothelial cells. J Neurosci Methods 2003 Nov

30;130(1):53-63.

Wylot B, Konarzewska K, Bugajski L, Piwocka K, Zawadzka M. Isolation of vascular

endothelial cells from intact and injured murine brain cortex-technical issues and pitfalls

in FACS analysis of the nervous tissue. Cytometry A 2015 Oct;87(10):908-20.

Xu J, Xu Z, Yan A. Prostaglandin E2 EP4 Receptor Activation Attenuates

Neuroinflammation and Early Brain Injury Induced by Subarachnoid Hemorrhage in

Rats. Neurochem Res 2017 Apr;42(4):1267-78.

Yagi K, Lidington D, Wan H, Fares JC, Meissner A, Sumiyoshi M, et al. Therapeutically

Targeting Tumor Necrosis Factor-alpha/Sphingosine-1-Phosphate Signaling Corrects

Myogenic Reactivity in Subarachnoid Hemorrhage. Stroke 2015 Aug;46(8):2260-70.

Yan J, Chen C, Hu Q, Yang X, Lei J, Yang L, et al. The role of p53 in brain edema after

24 h of experimental subarachnoid hemorrhage in a rat model. Exp Neurol 2008

Nov;214(1):37-46.

Yan J, Li L, Khatibi NH, Yang L, Wang K, Zhang W, et al. Blood-brain barrier disruption

following subarchnoid hemorrhage may be faciliated through PUMA induction of

endothelial cell apoptosis from the endoplasmic reticulum. Exp Neurol 2011

Aug;230(2):240-7.

229

Yan J, Manaenko A, Chen S, Klebe D, Ma Q, Caner B, et al. Role of SCH79797 in

maintaining vascular integrity in rat model of subarachnoid hemorrhage. Stroke 2013

May;44(5):1410-7.

Yatsushige H, Calvert JW, Cahill J, Zhang JH. Limited role of inducible nitric oxide

synthase in blood-brain barrier function after experimental subarachnoid hemorrhage. J

Neurotrauma 2006 Dec;23(12):1874-82.

Yatsushige H, Ostrowski RP, Tsubokawa T, Colohan A, Zhang JH. Role of c-Jun N-

terminal kinase in early brain injury after subarachnoid hemorrhage. J Neurosci Res

2007 May 15;85(7):1436-48.

Ying GY, Jing CH, Li JR, Wu C, Yan F, Chen JY, et al. Neuroprotective Effects of

Valproic Acid on Blood-Brain Barrier Disruption and Apoptosis-Related Early Brain

Injury in Rats Subjected to Subarachnoid Hemorrhage Are Modulated by Heat Shock

Protein 70/Matrix Metalloproteinases and Heat Shock Protein 70/AKT Pathways.

Neurosurgery 2016 Aug;79(2):286-95.

Yousif S, Marie-Claire C, Roux F, Scherrmann JM, Decleves X. Expression of drug

transporters at the blood-brain barrier using an optimized isolated rat brain microvessel

strategy. Brain Res 2007 Feb 23;1134(1):1-11.

Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La MG, Jureus A, et al.

Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell

RNA-seq. Science 2015 Mar 6;347(6226):1138-42.

230

Zhang S, Wang L, Liu M, Wu B. Tirilazad for aneurysmal subarachnoid haemorrhage.

Cochrane Database Syst Rev 2010 Feb 17;(2):CD006778.

Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, et al. An RNA-

sequencing transcriptome and splicing database of glia, neurons, and vascular cells of

the cerebral cortex. J Neurosci 2014 Sep 3;34(36):11929-47.

Zhao Z, Zlokovic BV. Blood-brain barrier: a dual life of MFSD2A? Neuron 2014 May

21;82(4):728-30.

Zheng B, Liu H, Wang R, Xu S, Liu Y, Wang K, et al. Expression signatures of long non-

coding RNAs in early brain injury following experimental subarachnoid hemorrhage. Mol

Med Rep 2015 Jul;12(1):967-73.

Zheng VZ, Wong GKC. Neuroinflammation responses after subarachnoid hemorrhage:

A review. J Clin Neurosci 2017 Aug;42:7-11.

Zhou N, Xu T, Bai Y, Prativa S, Xu JZ, Li K, et al. Protective effects of urinary trypsin

inhibitor on vascular permeability following subarachnoid hemorrhage in a rat model.

CNS Neurosci Ther 2013 Sep;19(9):659-66.

Ziegler T, Horstkotte J, Schwab C, Pfetsch V, Weinmann K, Dietzel S, et al.

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis. J Clin

Invest 2013 Jul 1.

Zlokovic BV. Neurovascular pathways to neurodegeneration in Alzheimer’s disease and

other disorders. Nat Rev Neurosci 2011 Nov;12(12):723-738.

231

Zubkov AY, Tibbs RE, Aoki K, Zhang JH. Prevention of vasospasm in penetrating

arteries with MAPK inhibitors in dog double-hemorrhage model. Surg Neurol 2000

Sep;54(3):221-7.

Zuchero YJ, Chen X, Bien-Ly N, Bumbaca D, Tong RK, Gao X, et al. Discovery of Novel

Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies.

Neuron 2016 Jan 6;89(1):70-82.

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