How we use animal studies to understand

recovery from brain injury

Ann M. Stowe, PhD Assistant Professor

Neurology & Neurotherapeutics

Overview

Introduction to clinical stroke

Post-stroke plasticity in non-human primates

Stroke models in rodents

Methods to promote recovery after stroke

Educational resources for the use of animals in

biomedical research

General considerations

The brain is highly aerobic tissue Dependent upon a steady supply of well-oxygenated

blood 650 – 750 ml of arterial blood /minute

15% of total cardiac output 20% of body’s total O2 consumption

Global interruption in blood flow results in loss of consciousness within 10 seconds!

Irreversible CNS injury occurs of blood flow drops to less than 15 ml / 100 gm tissue / minute

Blood flow to the brain is maintained at a constant rate over a wide range of blood pressure (autoregulation)

Global mortality, all causes: 65 million people

Global mortality, stroke: 6.5 million people – 10%

In the US, stroke #1 cause of long-term adult disability

800,000 U.S. strokes/yr

$73 billion annually (US) to provide long-term care

Stroke

Stroke Center, University Hospital

Ischemic stroke

88% of all strokes are ischemic Thrombotic – blood clots formed in the artery (50%) Embolic – blood clots dislodged from the body and

trapped in arteries in brain

Coronal view

WebMD

• Infarct • Tissue necrosis, neuronal

death, possible loss of pre-lesion function

• Peri-infarct • CBF is 20-50% of normal

values

• Cells are at risk of apoptosis or necrosis

Infarct

Peri-infarct

Intact cortex

Stroke

Middle cerebral territory

Lateral view

Primary and secondary motor cortices in primates

Preuss et al., 1996; Dum and Strick, 2002; Dancause et al., 2005; owl monkey

Primary motor cortex

Premotor cortex

Neurons that are interconnected to the infarct will undergo molecular responses immediately following infarct induction

To sensory hand

Primary motor cortex hand representation

Premotor cortex hand

digit

wrist

face

proximal

no response

medial

rostral

Previous work in the squirrel monkey model has highlighted neuronal changes following an infarct

medial

rostral

An infarct was induced in 30% of the M1 hand representation (Nudo and Milliken, 1996)

Infarct

medial

rostral

Following spontaneous recovery, there is a further loss of hand representation (Nudo and Milliken, 1996)

Infarct

medial

rostral

Following rehabilitative motor skill training, there was an actual increase in M1 hand representation (Nudo et al., 1996)

Infarct

medial

rostral

Infarct

PMv hand neurons undergo axonal sprouting to novel targets in primary somatosensory cortex (Dancause et al., 2005)

To sensory hand Dancause et al,

J Neurosci, 2005

Findings:

Disuse of the hand can decrease the number of neurons that directly control hand movement after brain injury

Rehabilitation after stroke directly affects neuronal plasticity during recovery

Recovery after brain injury takes months to complete in larger brains, especially during new connections

Stroke models in rodents – why?

Much more readily available Behavioral recovery can be monitored in rats

and mice We know their genetics… …which allows for genetic manipulation Develop various models to ask different

questions Myriad ways we can quantify injury and repair –

genetic, molecular, behavioral, imaging

Variety of rodent models of stroke:

University of Glasgow Glasgow Experimental MRI Centre

Luo et al., JCBFM (2008) 28, 973–983

Middle Cerebral Artery Occlusion (tMCAo)

Procedure Intraluminal filament is threaded to the origin of the MCA, with retraction comes brain reperfusion. Infarct Volume 2,3,5-triphenyltetrazolium chloride (TTC) Transient: Excellent for looking at post-stroke inflammation Permanent: Much larger infarct volumes

Imaging techniques to measure infarct volumes

University of Glasgow Glasgow Experimental MRI Centre

T2 weighted MRI 24h after permanent MCAo to measure edema

University of Glasgow Glasgow Experimental MRI Centre

Diffusion tensor imaging after permanent MCAo to measure

white matter tracks

Water moves along the axons of neurons faster than in cerebral cortex. This allows for quantification of direction based on the rate of diffusion.

This means we can track the in vivo

progression of the infarct, along with

behavioral recovery, to assess the efficacy of

drug or behavioral interventions

60-min tMCAo PBS (n=14), WT B cell (n=12), RHP B cell (n=11)

Unpublished data

Motor recovery can be measured as a

secondary outcome

Human CD20 transgenic mice B cells depleted with Rituximab 8-12 week males 60-min tMCAo (n=11 WT, n=13 B cell-depleted) Unpublished data

Blood-Brain Barrier - Endothelial Cells

Pardridge, 1997

• High-resistance tight junctions

• Capillaries are 40m apart

• No transcellular pathways

Pardridge, 1997

• Share basement membrane with EC

• Antigen-presenting properties

• May regulate blood vessel growth and EC proliferation in quiescent cortex

Blood-Brain Barrier - Pericytes

Pardridge, 1997

•Foot processes cover more than 99% of brain capillary surface

•Site of p-glycoprotein, product of the multi-drug resistance gene

•Effective efflux system

Blood-Brain Barrier - Astrocytes

• Angiogenic factors facilitate endothelial/pericyte dissociation and disruption of tight junctions

• Astrocyte end-feet withdraw from the vasculature

• Increase in vascular permeability into peri-infarct tissue

• Cerebral edema

The BBB is physically uncoupled in areas of ischemic injury

Post-Ischemic Inflammation: Leukocyte diapedesis occurs in the post-

capillary venules

modified from Eltzschig and Collard, 2004

• Selectins mediate rolling along the vessel wall

• Integrins mediate firm adherence to the vessel wall

Flow cytometry can be used to quantify leukocyte populations within the injured (i.e. ischemic) hemisphere, spleen, or blood

Abcam.com

B cells support post-stroke neurogenesis Ipsilateral Contralateral WT WT

B cell-depleted B cell-depleted

Scale bar = 20µm hCD20Tg mice, WT littermate controls All receive Rituximab Bottom border- subgranular zone Dendrites extending into the molecular layer

Unpublished data

Recap:

Several models for inducing stroke, can ask different questions

Concurrent quantification of outcomes Use of genetic manipulation to generate new mouse

strains Look at important mechanisms that can not be studied

in the clinical population

Help to understand the contribution of genetic, environmental, and physiological factors to stroke outcome in the individual

“Preconditioning” The presentation of a non-injurious stimulus that

promotes adaptive responses at the level of the cell, tissue, organ, and/or whole animal to afford protection against an injurious or lethal intervention.

“Tolerance” The state of relative resistance to a normally injurious or

lethal intervention.

(Dirnagl et al., Trends Neurosci., 2003)

In Vivo Preconditioning Stimuli

Local • brief ischemia • mild trauma Systemic • hypoxia and hypoxia-mimetic drugs • hyperoxia, hypoglycemia, caloric restriction • heat shock • cytokines, LPS, anesthetics, metabolic inhibitors, antibiotics • distant tissue ischemia (“remote” PC) • exercise

Magnitude of Stress

Tiss

ue o

r C

ellu

lar

Res

pons

e

none

tolerance

apoptosis

necrosis

* *

Sustained exercise – but not the magnitude of exercise – creates a unique B cell phenotype in the blood

Unpublished data

Hypothesis: Exercise-mediated changes in adaptive immunity are lost with detraining

3 week exercise period 2 week sedentary period Flow cytometry on brain and spleen

Flow cytometry on brain and spleen

3 week exercise period

3 or 5 week sedentary period Flow cytometry on brain and spleen

stroke

stroke

stroke

3 days

3 days

3 days

SEDENTARY (SED)

EXERCISE (EX)

DETRAINING (DET)

Detrained animals exhibit increased infarct volumes

SED EX DET

Unpublished data

Exercise intensity induces a non-linear, dose-dependent increase of immune cells in the ischemic brain that is lost

after detraining

EXER

CIS

E D

ETR

AIN

ING

All leukocytes in the brain

W1W

2W

3W

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Ex27 Ex28 Ex29 Ex30 Ex31 Ex32 Ex33 Ex34Ex25 Ex26

**

******

*** *

EXERCISE

W1W

2W

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Det13 Det14 Det15 Det16 Det17 Det18 Det19 Det20Det11 Det12

**

*****

****

*** **

***

DETRAINING

0 10000 20000 30000 40000 500000

1000000

2000000

3000000

Average Wheel Rotations

# c

ells

/he

mis

ph

ere

(me

an

/SD

)

All leukocytes

R2 = 0.8423

0 10000 20000 30000 40000 500000

500000

1000000

1500000

2000000

Average Wheel Rotations

# c

ells

/he

mis

ph

ere

(me

an

/SD

)

All leukocytes

R2 = 0.02860

Unpublished data

Recap:

Spontaneously hypertensive rodents Obese and aged rodents Other environmental factors- exercise vs. sedentary

lifestyle

We can use these interventions to determine the mechanisms by which lifestyle and genetics can contribute to injury and recovery after stroke.

http://www.pewinternet.org/2015/07/01/chapter-7-opinion-about-the-use-of-animals-in-research/

Bringing up the concept of animals in biomedical research

Pew research into the public opinion of animal research

American Physiological Society Advocacy and

Outreach Presentations available online http://www.the-aps.org/mm/SciencePolicy/Advocacy/Research-Benefits

NIH RePORT website- search by any disease http://report.nih.gov/NIHfactsheets/Default.aspx?key=S#S

AN D T H AN K S !

Questions?