Frank C. Barone

Mouse Strain Differences in Susceptibility to Cerebral Ischemia are Related to Cerebral Vascular Anatomy

The consequences of cerebral ischemia were studied in three different strains (BDF, CFW, and BALB/C) of mice. The different strains exhibited significant differences in susceptibility to 24-h focal ischemia. Following middle cerebral artery occlusion (MCAO), infarct volumes (mm3) were 5 ± 3 in BDF, 15 ± 5 in CFW, and 23 ± 3 in BALB/C mice (p < 0.05). MCAO plus ipsilateral common carotid artery occlusion (CCAO) resulted in infarct volumes of 15 ± 9 in BDF, 38 ± 10 in CFW, and 72 ± 12 in BALB/C mice (p < 0.05). In addition, MCAO plus CCAO produced death by 24 h in 42% of CFW and 67% of BALB/C mice, but not in any BDF mice (p < 0.05). CCAO alone produced multifocal hemispheric infarctions in 36% of BALB/C mice but not in the other two strains. Brains of all mouse strains subjected to sham surgery were free of any ischemic injury. Arterial blood pressures, blood gases, and blood cell profiles were relatively similar for the three mouse strains. However, carbon black studies of the cerebrovascular anatomy revealed an incomplete circle of Willis (i.e., a significant decrease in the frequency of patent posterior communicating arteries) for BALB/C compared with BDF mice (p < 0.05), with CFW mice being intermediary. Based on these anatomical data, BALB/C mice also were evaluated following transient global brain ischemia produced by bilateral CCAO. BALB/C mice exhibited a >85% reduction in cortical microvascular perfusion and EEG power within 1 min of bilateral CCAO. Also, hippocampal neuronal CA1 damage and mortality over 7 days were related to the duration of global brain ischemia (p < 0.05). These data demonstrate a significant difference between mouse strains in their sensitivity to cerebral ischemia that appears to be related, at least in part, to the functional vascular anatomy at the level of the posterior communicating arteries. In particular, we point out the potential usefulness of BALB/C mice as a sensitive and reproducible model of focal and global ischemia.

Thrombopoietin Protects the Brain and Improves Sensorimotor Functions: Reduction of Stroke-Induced MMP-9 Upregulation and Blood—Brain Barrier Injury

This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 μg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood—brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 μg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPOs efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.

Long-Term Post-Stroke Changes Include Myelin Loss, Specific Deficits in Sensory and Motor Behaviors and Complex Cognitive Impairment Detected Using Active Place Avoidance

Persistent neurobehavioral deficits and brain changes need validation for brain restoration. Two hours middle cerebral artery occlusion (tMCAO) or sham surgery was performed in male Sprague-Dawley rats. Neurobehavioral and cognitive deficits were measured over 10 weeks included: (1) sensory, motor, beam balance, reflex/abnormal responses, hindlimb placement, forepaw foot fault and cylinder placement tests, and (2) complex active place avoidance learning (APA) and simple passive avoidance retention (PA). Electroretinogram (ERG), hemispheric loss (infarction), hippocampus CA1 neuronal loss and myelin (Luxol Fast Blue) staining in several fiber tracts were also measured. In comparison to Sham surgery, tMCAO surgery produced significant deficits in all behavioral tests except reflex/abnormal responses. Acute, short lived deficits following tMCAO were observed for forelimb foot fault and forelimb cylinder placement. Persistent, sustained deficits for the whole 10 weeks were exhibited for motor (p<0.001), sensory (p<0.001), beam balance performance (p<0.01) and hindlimb placement behavior (p<0.01). tMCAO produced much greater and prolonged cognitive deficits in APA learning (maximum on last trial of 604±83% change, p<0.05) but only a small, comparative effect on PA retention. Hemispheric loss/atrophy was measured 10 weeks after tMCAO and cross-validated by two methods (e.g., almost identical % ischemic hemispheric loss of 33.4±3.5% for H&E and of 34.2±3.5% for TTC staining). No visual dysfunction by ERG and no hippocampus neuronal loss were detected after tMCAO. Fiber tract damage measured by Luxol Fast Blue myelin staining intensity was significant (p<0.01) in the external capsule and striatum but not in corpus callosum and anterior commissure. In summary, persistent neurobehavioral deficits were validated as important endpoints for stroke restorative research in the future. Fiber myelin loss appears to contribute to these long term behavioral dysfunctions and can be important for cognitive behavioral control necessary for complex APA learning.

A novel calpain inhibitor for treatment of transient retinal ischemia in the rat.

After an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the blood–retinal barrier would provide for novel systemic intervention that protects the retina from acute injury and loss of function. Herein, we study a novel calpain peptide inhibitor, cysteic–leucyl–argininal (CYLA), in an in-vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA prodrug was administered intraperitoneally before and/or after ischemia–reperfusion at concentrations of 20–40 mg/kg. We found that administering 20 mg/kg of CYLA only after ischemia provides significant preservation of retinal function.

Post-stroke pharmacological intervention: Promoting brain recovery from injury in the future☆

Many vascular approaches to ischemic stroke intervention and prevention include as their goalmaintenance of normal circulationor the reversal of vascular occlusion. Thrombolytic agents/mechanical instruments (e.g., tissue plasminogen activator; tPA, urokinase and mechanical devices) and anti-platelet and anti-thrombotic agents can protect the brain primarily by hemodynamic mechanisms. The majority of clinical trials have evaluated these (Ginsberg, 2008). Intravenous administration of tPA (i.e., to achieve thrombolysis of the occluded cerebral vessel) within 3 h is the only approved pharmacological treatment for ischemic stroke (Elijovich and Chong, 2010; National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). Beyond 3 h after stroke onset, tPA administration exhibits increased risk of hemorrhagic conversion in the infarcted brain, generally results in negative outcome, and is contraindicated for safety reasons. Even though efforts are beingmade to extend tPA use to 4.5 h post-stroke for certain eligible patients (Del Zoppo et al., 2009), only a limited number (<5%) of patients qualify for its safe use. Therefore, in addition to preventative therapies, it is imperative to develop additional treatment interventions for ischemic stroke. Additional treatments of interest include agents that can: A) extend the time for safe tPA thrombolysis, B) directly protect brain cells if administered soon after stroke (i.e., neuroprotective interventions-Neuroprotection) and/or C) can improve brain recovery from injury even if administered after brain injury has occurred (i.e., neurorestorative interventions that can improve lost neurological function-Neurorestoration). Approaches beyond stroke prevention (e.g., extending the use of tPA, protecting the brain once a stroke

Cytokines in Brain Ischemia—The Role of TNFα

Abstract1. The role of cytokines and other inflammatory mediators in the progression of ischemic brain injury is a new and exciting era of research. Evidence in support for a role for TNFα in this respect is emerging as evidence on de novo upregulation of TNFα following ischemia is now well established.2. TNFα administered directly to the brain parenchyma elicits local microvascular injury in the form of pericapillary edema and leukocyte adhesion to cerebral capillaries.3. TNFα administered into the cerebroventricular space prior to ischemia augment the extent of tissue damage and neurological deficits.4. Specific and potent inhibitors of TNFα synthesis or TNFα receptors must be developed and tried to prove firmly a role for TNFα in ischemic brain injury.

Calcium Channel Blockers and Neuroprotection

Stroke is most commonly the outcome of an obstruction of blood flow in a major cerebral vessel (usually the middle cerebral artery), which, if not resolved within a short period of time (minutes), will lead to a core of severely ischemic tissue that may not be salvaged. However, the ultimate size of the brain infarct also depends on the “penumbra,” a zone of tissue around the core of the infarct where blood flow is still maintained above a neuronal disabling level or the critical 20–25% of normal blood flow. If blood flow in the penumbral zone further decreases below a critical level of flow, the infarct zone will inevitably expand. The blood flow, therefore, is the driving force in determining the size of the core infarct and penumbral zones by providing the conditions essential to maintenance of cellular energy hemostasis (Siesjo, 1988a). The decrease in blood flow leads to reduction in phosphocreatinine (PCr) and ATP (especially the latter), and, if ischemia is prolonged, the energy source depletion will be sufficient to lead to severe impairment of cellular function by disruption of ATP-dependent processes (e.g., N+ /K+ ATPase). However, anoxia (lack of oxygen per se) may allow prolonged cellular survival (1 hr), which upon restoration of energy is not necessarily associated with the neuronal death seen in shorter episodes of brain ischemia. This discrepancy has forced researchers to examine the rapid consequences of ATP depletion which may not be readily reversed upon restoration of energy levels such as loss of ion homeostasis.

First translational 'Think Tank' on cerebrovascular disease, cognitive impairment and dementia.

Abstract and introduction to the workshopAs the human population continues to age, an increasing number of people will exhibit significant deficits in cognitive function and dementia. It is now recognized that cerebrovascular, metabolic and neurodegenerative diseases all play major roles in the evolution of cognitive impairment and dementia. Thus with our more recent recognition of these relationships and our need to understand and more positively impact on this world health problem, “The Leo and Anne Albert Charitable Trust” (Gene Pranzo, Trustee with significant support from Susan Brogan, Meeting Planner) provided generous support for this inaugural international workshop that was held from April 13–16, 2015 at the beautiful Ritz Carlton Golf Resort in North Naples, Florida. Researchers from SUNY Downstate Medical Center, Brooklyn, NY organized the event by selecting the present group of translationally inclined preclinical, clinical and population scientists focused on cerebrovascular disease (CVD) risk and its progression to vascular cognitive impairment (VCI) and dementia. Participants at the workshop addressed important issues related to aging, cognition and dementia by: (1) sharing new data, information and perspectives that intersect vascular, metabolic and neurodegenerative diseases, (2) discussing gaps in translating population risk, clinical and preclinical information to the progression of cognitive loss, and (3) debating new approaches and methods to fill these gaps that can translate into future therapeutic interventions. Participants agreed on topics for group discussion prior to the meeting and focused on specific translational goals that included promoting better understanding of dementia mechanisms, the identification of potential therapeutic targets for intervention, and discussed/debated the potential utility of diagnostic/prognostic markers. Below summarizes the new data-presentations, concepts, novel directions and specific discussion topics addressed by this international translational team at our “First Leo and Anne Albert Charitable Trust ‘Think Tank’ VCI workshop”.

Comparison of passive leg raising and hyperemia on macrovascular and microvascular responses

Passive leg raising is a simple diagnostic maneuver that has been proposed as a measure of arterial vasodilator reserve and possibly endothelial function. While passive leg raising has previously been shown to lower blood pressure, increase flow velocity and cause brachial artery dilation, its effects on microvascular flow has not been well studied. Also, passive leg raising has been directly compared previously to upper arm but never to lower arm occlusion of blood flow induced hyperemia responses. We compared changes in macrovascular indices measured by brachial artery ultrasound and microvascular perfusion measured by Laser Doppler Flowmetry induced by passive leg raising to those provoked by upper arm and lower arm induced hyperemia in healthy subjects. Upper arm induced hyperemia increased mean flow velocity by 398%, induced brachial artery dilatation by 16.3%, and increased microvascular perfusion by 246% (p<.05 for all). Lower arm induced hyperemia increased flow velocity by 227%, induced brachial artery dilatation by 10.8%, and increased microvascular perfusion by 281%. Passive leg raising increased flow velocity by 29% and brachial artery dilatation by 5.6% (p<.05 for all), but did not change microvascular perfusion (-5%, p=ns). In conclusion, passive leg raising increases flow velocity orders of magnitude less than does upper arm or lower arm induced hyperemia. Passive leg raising-induced brachial artery dilatation is less robust than either of these hyperemic techniques. Finally, although upper arm and lower arm hyperemia elicits macrovascular and microvascular responses, passive leg raising elicits only macrovascular responses.

The March of Thrombolytic Therapy for Acute Ischemic Stroke to Clinical Trials: Pre-clinical Thrombolysis and Adjuncts to Thrombolysis Research

Data from experimental and clinical cerebral ischemia studies demonstrate that acute stroke must be treated within a few hours or less to effectively reduce stroke morbidity and mortality. Time from symptom onset (presumed time of acute cerebral arterial occlusion) to treatment initiation is considered the single most important factor in the effective treatment of acute focal cerebral ischemia/infarction. Recently the time window for use of tissue plasminogin activator (rt-PA), the only approved thrombolytic pharmacotherapy for acute stroke intervention, has been extended from 3 to 4.5 h. Hemorrhagic conversion can occur and limits it use beyond this time and the efficacy drops off considerably as well.