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 O 2 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)
Stroke 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 Stroke Center, University Hospital 800,000 U.S. strokes/yr $73 billion annually (US) to provide long-term care
Ischemic stroke Coronal view Web MD 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
Stroke • Infarct Intact cortex Tissue necrosis, neuronal • death, possible loss of pre-lesion function Infarct • Peri-infarct CBF is 20-50% of normal • Peri-infarct values Cells are at risk of apoptosis • or necrosis
Middle cerebral territory Lateral view
Primary and secondary motor cortices in primates Primary motor cortex Premotor cortex Preuss et al., 1996; Dum and Strick, 2002; Dancause et al., 2005; owl monkey
Neurons that are interconnected to the infarct will undergo molecular responses immediately following infarct induction
medial Primary motor cortex rostral hand representation To sensory hand digit Premotor wrist cortex face hand proximal no response Previous work in the squirrel monkey model has highlighted neuronal changes following an infarct
medial rostral Infarct An infarct was induced in 30% of the M1 hand representation (Nudo and Milliken, 1996)
medial rostral Infarct Following spontaneous recovery, there is a further loss of hand representation (Nudo and Milliken, 1996)
medial rostral Infarct Following rehabilitative motor skill training, there was an actual increase in M1 hand representation (Nudo et al., 1996)
medial rostral To sensory Infarct Dancause et al, hand J Neurosci, 2005 PMv hand neurons undergo axonal sprouting to novel targets in primary somatosensory cortex (Dancause et al., 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 Luo et al., JCBFM (2008) 28, 973–983 Glasgow Experimental MRI Centre
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 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. University of Glasgow Glasgow Experimental MRI Centre
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 • High-resistance tight junctions • Capillaries are 40 m apart • No transcellular pathways Pardridge, 1997
Blood-Brain Barrier - Pericytes • Share basement membrane with EC • Antigen-presenting properties • May regulate blood vessel growth and EC proliferation in quiescent cortex Pardridge, 1997
Blood-Brain Barrier - Astrocytes • Foot processes cover more than 99% of brain capillary surface • Site of p-glycoprotein, product of the multi- drug resistance gene • Effective efflux system Pardridge, 1997
The BBB is physically uncoupled in areas of ischemic injury • 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
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 WT WT Contralateral 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
Tissue or Cellular Response necrosis apoptosis tolerance none * * Magnitude of Stress
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 stroke Flow cytometry on 3 or 5 week sedentary period 3 days brain and spleen SEDENTARY (SED) stroke 3 week exercise period 3 days Flow cytometry on EXERCISE brain and spleen (EX) stroke 3 week exercise period 3 days 2 week sedentary period DETRAINING Flow cytometry on (DET) brain and spleen
Detrained animals exhibit increased infarct volumes DET SED EX Unpublished data
Exercise intensity induces a non-linear, dose-dependent increase of immune cells in the ischemic brain that is lost after detraining All leukocytes in the brain All leukocytes EXERCISE R 2 = 0.8423 3000000 Average number of rotations/week 60000 EXERCISE *** # cells/hemisphere ** * 50000 * *** 2000000 (mean/SD) 40000 ** 30000 1000000 20000 10000 0 0 0 10000 20000 30000 40000 50000 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 Average Wheel Rotations Ex28 Ex34 Ex25 Ex26 Ex27 Ex29 Ex30 Ex31 Ex32 Ex33 All leukocytes DETRAINING Average number of rotations/week DETRAINING 2000000 * 60000 R 2 = 0.02860 * ** *** 50000 # cells/hemisphere ** 1500000 *** (mean/SD) ** 40000 * 1000000 30000 * *** 20000 500000 10000 0 0 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 W1 W2 W3 0 10000 20000 30000 40000 50000 Average Wheel Rotations Det12 Det14 Det15 Det16 Det17 Det19 Det20 Det11 Det13 Det18 Unpublished data
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