Background and Purpose Remote ischemic conditioning is cardioprotective in myocardial infarction and neuroprotective in mechanical occlusion models of stroke. However, there is no report on its therapeutic potential in a physiologically relevant embolic stroke model (eMCAO) in combination with intravenous (IV) tissue plasminogen activator (tPA). Methods We tested remote ischemic perconditioning therapy (RIPerC) at 2 hours after eMCAO in the mouse with and without IV tPA at 4 hours. We assessed cerebral blood flow (CBF) up to 6 hours, neurologic deficits, injury size and phosphorylation of Akt (Serine473; p-Akt) as a pro-survival signal in the ischemic hemisphere at 48 hours post stroke. Results RIPerC therapy alone improved the CBF and neurologic outcomes. tPA alone at 4 hours did not significantly improve the neurologic outcome even after successful thrombolysis. Individual treatments with RIPerC and IV tPA reduced the infarct size (25.7% and 23.8%, respectively). Combination therapy of RIPerC and tPA resulted in additive effects in further improving the neurologic outcome, and reducing the infarct size (50%). All the therapeutic treatments upregulated p-Akt in the ischemic hemisphere. Conclusions RIPerC is effective alone after eMCAO and has additive effects in combination with IV tPA. RIPerC may be a simple, safe and inexpensive combination therapy with IV tPA.

Remote ischemic conditioning is neuroprotective in young male rodents after experimental stroke. However, it has never been tested in females whom remain at higher risk of stroke injury after menopause. We tested remote ischemic perconditioning therapy (RIPerC) at 2 h after embolic stroke in ovariectomized (OVX) female mice with and without intravenous tissue plasminogen activator (IV-tPA) treatment. We assessed cerebral blood flow (CBF), neurobehavioral outcomes, infarction, hemorrhage, edema, and survival. RIPerC therapy with and without IV-tPA improved the CBF and neurobehavioral outcomes and reduced the infarction, hemorrhage, and edema significantly. Late IV-tPA alone at 4 h post-stroke neither improved the neurobehavior nor reduced the infarction but aggravated hemorrhage and mortality in OVX mice. RIPerC therapy prevented the increased mortality during late IV-tPA. Our study demonstrates for the first time that RIPerC therapy is effective in OVX females.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues—such as incomplete mechanistic knowledge and faulty clinical trial design—a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Background: Stroke is a leading cause of disability and death worldwide. There is evidence that there is a circadian rhythm in stroke with peak occurrence in the morning (6 to 10 am). However it is not clear if the size of infarcts and the outcomes of stroke also varies during the 24 hour period Hypothesis: We hypothesized that the size of cerebral infarct and outcome from stroke would show circadian variation in a mouse suture occlusion model. Methods: Seven to eight-month-old C57BL/6J (Wild Type, n=10-15 mice/group) mice were randomly assigned to do stroke at the different time points of the day following zeitgeber time at ZT0, ZT6, ZT12, and Z18. Cerebral Ischemia was induced by occlusion of the middle cerebral artery (MCAO) for 60 min. Blood flow was monitored by Laser Speckle before, after occlusion, and at 24h. Neurological deficit was observed by using Bederson score at 24h and 48h. The corner test was used to detect unilateral abnormalities of sensory and motor functions in the stroke mice at 48h. TTC staining was done, 48 hours after stroke, to estimate brain infarction, and the infarct area was measured by using NIH-Image J software. Results: We did not find a significant difference in CBF at any time points. There was a significant increased ( p <0.05) neurological deficit (Bederson score) at 48h during deep sleep period (ZT6, noon) stroke (1.55±0.17) in comparison to fully awake period stroke (1.1±0.1). In the corner test, we found right turn preference significantly higher ( p <0.005) at noon/ZT6 (9.5±0.34) compared to the fully awake (5.5±0.34) (midnight, ZT18) period. Similarly, the infarction volume was significantly higher ( p <0.05) during the sleep (ZT6, noon) period (29.32±5.03) in comparison to a fully awake midnight/ZT18 period (15.68±2.38). Conclusion: This is the first report demonstrating that mice have larger infarcts and worse short term outcomes during their sleep period (noon/ZT6) than during their awake period (midnight/ZT18).

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Background: Stroke leads to disability and death worldwide. There is evidence that stroke affects the immune system function, and the clock gene controls the immune system. Stroke elevates the inflammatory cascade. Immune response controls the stroke pathology. However, it is unclear if the circadian rhythm influences the immune system in ischemic stroke mice and affects stroke outcomes. Hypothesis: We hypothesized that immune response might be affected by a circadian rhythm that aggravates stroke pathology in a mouse suture occlusion model Methods: Seven to eight-month-old C57BL/6J (Wild Type, n=8-10 mice/group) mice were randomly assigned to do stroke at the different time points of the day following zeitgeber time at ZT0, ZT6, ZT12, and Z18. Cerebral Ischemia was induced by occlusion of the middle cerebral artery (MCAO) for 60 min. Whole blood was analyzed using flow cytometry, and we observed macrophages (M1, M2), neutrophils (N1, N2), Anti-inflammatory IL10, and pro-inflammatory TNF-α cytokines at 24h and 48h. Forty-eight hours after stroke, TTC staining was done to estimate brain infarction, and the infarct area was measured using NIH-Image J software. Results: There was a significant increase ( p <0.005) in TNF-α (9±2.50) and significant low IL-10 (5.12±1.35) ( p <0001) at (ZT6, noon) stroke at 48h during a deep sleep period (ZT6, noon) stroke in comparison to fully awake period stroke. We found a significant increase ( p <001) in M1 (54.12±5.16) macrophage and a significant decrease ( p <001) in M2 (45.87±5.16) macrophage at ZT6 (noon) compare to other zeitgeber time points. We also found a significantly higher M1:M2 ratio (1.17) at ZT6. Additionally, we found that neutrophil N1 (66.88±4.39) level was significantly ( p <0001) elevated while neutrophil N2 (33.88±4.43) was significantly reduced at ZT6 (noon) sleep period in comparison to ZT0, ZT12, and Z18 time points. We also found a considerably higher N1:N2 ratio (1.97) at ZT6. Conclusion: This study demonstrates that mice brain infarcts are influenced by immune responses that aggravate stroke pathology during their sleep period (noon/ZT6) than during their awake period (midnight/ZT18).

Background and Purpose: Chronic remote ischemic conditioning (C-RIC) is effective at improving cerebral blood flow (CBF) inducing vascular remodeling, and improving cognition in a bilateral carotid artery stenosis (BCAS) mouse model, a model for Vascular Cognitive Impairment and Dementia (VCID). This improvement is associated with elevations of plasma nitrite. Our aim was to determine if the beneficial effect of C-RIC was eNOS dependent. Methods: Microcoil (01.8 mm) induced BCAS model was used to induce chronic hypoperfusion. Adult eNOS-KO male mice (5-6 months) were randomly assigned to 2-groups (N=5): (1) BCAS and (2) BCAS+RIC. RIC was started 7d post-surgery daily for 3 weeks. Behavioral test and CBF was performed before termination. Functional outcomes were assessed using novel object recognition (NOR) test for non-spatial working memory, and hanging wire and beam walk test for motor/muscular impairment. Mice were followed for 4 weeks. Results: C-RIC-therapy for 3 weeks did not improve CBF in the BCAS+RIC groups at either a 2 nd weeks or 4 th weeks compared to BCAS-Sham RIC groups. There was no significant change between the BCAS and BCAS+RIC groups in the discrimination index as determined by the NOR test or poor motor function as determined by hanging wire and beam walk test. Conclusions: The beneficial effect of C-RIC in the BCAS model is abrogated in eNOS KO mice indicating that the effect of C-RIC is eNOS dependent.