Blood, Brains, and Lasers: Imaging the Development of Brain Disease

Chris B. Schafferhttp://www.bme.cornell.edu/schafferlab

Our labs efforts center on in vivo experiments investigating the cellular dynamics that underlie neurological disease, supported by the development of optical techniques.

Alzheimers disease has been with us for 100 years, but it is the pandemic of the 21st century First described by Aloys Alzheimer in 1907Patients:1 in 8 over 65 have AD1 in 2 over 85 have AD$200 billion 2012 in direct medical expenses$1.1 trillion by 2050Caregivers:15 million Americans provide 17 billion hours of unpaid care for AD patientsValue of $210 billionDepression, health problems

Preclinical stageminimal cognitive effectscan take 20 years to progresspathological changes occurring in brainMild cognitive impairementNoticeable cognitive dysfunction to patient and familyHalf will progress to ADAlzheimers diseaseImpaired ability to function in daily lifeAlzheimers disease leads to progressive loss of cognitive functionAuguste Deter, first AD patient at 51

In AD, the brain is filled with amyloid plaquesand shows severe atrophy

Amyloid plaques

Amyloid plaques are composed of a small protein called amyloid-beta (A) A is a cleavage product of amyloid precursor protein (APP) produced in neurons

Alzheimers is caused by neuronal loss due to neurotoxic effects of aggregated A peptideK. Blennow, et al., Lancet (2006)

K. Blennow, et al., Lancet (2006)Alzheimers is caused by neuronal loss due to neurotoxic effects of aggregated A peptide

A is produced by neurons and cleared primarily through the vasculature

Familial Alzheimers is caused by mutations that increase A production, leading to aggregation

Familial Alzheimers disease only accountsfor a tiny fraction of AD patientsAbout 0.5% of AD cases are caused by specific genetic mutations in APP or the enzymes that cleave itCauses rapid accumulation of ALeads to early-onset of the disease

Most AD patients have sporadic form of diseaseLater onsetWhile there are genetic risk factors, they do not guarantee getting ADModifiable risk-factors: high blood pressure, high cholesterol, brain traumaProtective factors: education, exercise

Our understanding of familial AD is critical for experimental research on this disease The mutations found in the familial form of AD have been added to the genome of mice

These mice develop pathology and cognitive deficits like AD patients

Drugs that emerged from such animal studies treat some symptoms, but there remains no cureacetylcholinesterase inhibitorsDonepezil (Aricept)Galantamine (Razadyne)Rivastigmine (Exelon)

NMDA blockerMemantine (Namenda)

Humans:Dozens of studiesCerebral blood flow reductions of 10-50% Mouse models of AD:~30% reduction in cerebral blood flowOccurs before any amyloid depositionFlow decrease likely cognitively significant, but origin remains unclearBaseline cortical blood flow is dramatically reduced in Alzheimers diseaseASL MRI reveals as much as 40%reduction in CBF in AD patientsControlADDifferenceI. Asllani, et al., JCBFM (2008)K. Niwa, et al., Neurobio of Disease (2002)mL/100 g min

A major aim of my lab is to try to understand whatcauses this decreased brain blood flow in AD

Hard to see what is wrong in vasculature because blood vessels implicated are so smallEdvinsson (1993)

Two-photon excitation of fluorescent dyes leads to emission that originates only from the focal volumeZ. Huang, et al., belfield.cos.ucf.edu/one vs two-photon excitation.html

Image is formed by scanning laser focus through the sample and recording fluorescence intensity

Image is formed by scanning laser focus through the sample and recording fluorescence intensity

Image fluorescently-labeled features in brain of anesthetized rodent with glass-covered craniotomy

Imaging fluorescent blood plasma yields a 3-D cortical micro-angiography

In vivo imaging of vasculature and amyloid plaques in AD mouse modelsplaques labeled by methoxy-X04vasculature with intravenous dye injectionAged APPswe/PS1 mice with craniotomies

Dye labels blood plasma, but not blood cells, allowing identification of flowing vs. stalled vessels

Fraction of capillaries with stalled blood flow increased in mouse models of AD~1,000 capillaries imaged/mouse; 25000 AD capillaries; 10000 WT capillaries

In AD mice, about half the stalls are transient, but many last for hours and vessels tend to re-stall

Stalls reappear in the same capillaries, suggesting special locations for stall occurrence

Majority of capillary stalls in AD mouse models are caused by leukocytes that plug a capillary segment

How much do these capillary stalls affect blood flow?

We can track RBC motion to monitor the changes in blood flow caused by a single capillary clot

mm/sN. Nishimura, et al., Nature Methods 3, 99 (2006) baseline flowpost-clot flow

A single stalled capillary causes reduced blood flow in multiple downstream vessel branchesN. Nishimura, et al., Nature Methods 3, 99 (2006)

Post-clot blood flow speed(fraction of baseline)

Map of blood vessels and amyloidplaques from AD mouse

Location of stalled capillaries

Simulate blood flow changes in capillaries downstream from plugged vessels

Simulate blood flow changes in capillaries downstream from plugged vessels

Simulate blood flow changes in capillaries downstream from plugged vessels

Increases in number of stalled capillaries causes decreases in average cerebral blood flow2% of capillaries stalled predicts a 30% decrease in flow compared to controls

Blocking leukocyte adhesion reduces capillary stalls

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Blocking leukocyte adhesion improves blood flow

BaselineAfter administration of anti-Ly6G

Flow improves by ~30% when capillary stalls are reduced

Take away message: Blood flow alterations in AD2% of capillaries are spontaneous stalled in AD mouse modelsStalls are caused by leukocytes plugging capillary segmentsThis rate of capillary stalling likely produces a ~30% decrease in cerebral blood flow

Suggests that A aggregates cause vascular inflammation that leads to firm adhesion of leukocytes to the endothelium

Increasing A concentration in the brain drives Alzheimers by promoting A aggregation

Familial AD: increased A productionSporadic AD: decreased A clearance

Normally, A produced by neuronsand cleared through vasculature

Brain hypoperfusion likely contributes to the progression of Alzheimers disease

Novel therapeutic target?

Preliminary evidence of immediate cognitive improvement after blocking capillary stalls

Team members

Oliver Bracko*Jean Carlos Cruz-Hernandez*Mohammad Javaherian*Calvin KersbergenIryna IvasykSydney GehrkingVictorine Muse*Elizabeth SlackGabriel OtteJeffery BeverlyJoan ZhouPuifai SantisakultarmYiming KangRaymond Xu

Sylvie LorthoisNozomi NishimuraCostantino IadecolaChris B. Schaffer

Thank you for your attention

http://www.bme.cornell.edu/schafferlab