Search

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 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 p p p p 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).

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

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 p p 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).

The circadian system is widely involved in the various pathological outcomes affected by time dimension changes. In the brain, the master circadian clock, also known as the "pacemaker," is present in the hypothalamus's suprachiasmatic nucleus (SCN). The SCN consists of molecular circadian clocks that operate in each neuron and other brain cells. These circadian mechanisms are controlled by the transcription and translation of specific genes such as the clock circadian regulator (Clock) and brain and muscle ARNT-Like 1 (Bmal1). Period (Per1-3) and cryptochrome (Cry1 and 2) negatively feedback and regulate the clock genes. Variations in the circadian cycle and these clock genes can affect stroke outcomes. Studies suggest that the peak stroke occurs in the morning after patients awaken from sleep, while stroke severity and poor outcomes worsen at midnight. The main risk factor associated with stroke is high blood pressure (hypertension). Blood pressure usually dips by 15-20% during sleep, but many hypertensives do not display this normal dipping pattern and are non-dippers. A sleep blood pressure is the primary determinant of stroke risk. This article discusses the possible mechanism associated with circadian rhythm and stroke outcomes.

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 effect is lost in NOS3-KO mice. We wanted to determine if the beneficial effect of C-RIC can be restored by bone marrow cell-derived NOS3. Methods: We prepared BM chimera from adult WT (B6.SJL-Ptprc a ; CD45.1) to adult NOS3-KO (B6.129; CD45.2) & vice versa in male mice (5-6 months) following confirmation for BM engrafting with flow cytometry at 2 weeks. Microcoil (0.18 mm) induced BCAS model was used to induce chronic hypoperfusion. Mice were randomly assigned to 3-groups: (1) Sham (2) BCAS and (3) BCAS+RIC. RIC was started 7d post-surgery daily for 3-4 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. We measured whole blood P-NOS3, P-AMPK and VEGFR2/CD31 by flow cytometry. Results: C-RIC-therapy for 3-4 weeks improves CBF in engrafted BM of WT into NOS3-KO in the BCAS+RIC groups at both 2 weeks and 4 weeks compared to BCAS (Sham RIC groups).No effect of C-RIC was shown in engrafted BM of NOS3 into WT. There was significant change between the BCAS and BCAS+RIC groups in the functional outcomes and histopathological evidences also reflects major changing in the groups in the BM of WT into NOS3-KO mice. Whole blood P-NOS3, P-AMPK and VEGFR2/CD31 was increased by C-RIC. However, no effect was shown in the NOS3-KO into WT chimera. Conclusions: The Beneficial effect of C-RIC is dependent upon bone marrow derived NOS3.Further studies are needed to determine if the red blood cell is the key cell carrying NOS3

Background and Purpose: There is presently no specific therapy for the treatment of VCID. We reported that daily C-RIC (4mo) is effective in young mice by improving cerebral blood flow (CBF), minimizing white matter (WM) damage and improving functional outcomes in Bilateral Carotid Artery Stenosis model (BCAS.) The purpose of this study was to evaluate whether 4 months (4 MO-C-RIC) is effective in male and female aged mouse and if the treatment effects are durable out to 6MOS. Methods: Microcoil induced BCAS model was used to induce chronic hypoperfusion. Adult C57BL/6 both male & female mice (14-months ) were randomly assigned to 3-different groups (N=10), and subjected to Sham, BCAS+sham RIC, BCAS+RIC. C-RIC was started 7d post-surgery daily for 4MOs. Behavioral test and CBF was performed at 1, 4 and 6MO after BCAS and Sham surgery. 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. Histopathology as well as immunohistochemistry for CD31/α-SMA; GFAP, myelin basic protein (MBP) and beta-amyloid were also performed on the brain tissue collected after the neurobehavioral tests. Results: C-RIC-therapy for 4MO significantly improved CBF in the male and female BCAS+C-RIC groups at all time points compared to BCAS-Sham RIC groups. Mice from the BCAS group +Sham C-RIC group showed significant loss in the discrimination index as determined by the NOR test, and poor motor function in hanging wire and beam walk test. C-RIC- significantly improved these functional outcomes. Histopathology showed prevention of WM degeneration and myelin basic protein by C-RIC. Immunohistochemical analysis at 6 MOs showed increased CD31 (angiogenesis) and a-SMA staining (arteriogenesis) indicating collateral remodeling in the C-RIC group compared to BCAS-sham RIC. Conclusions: Daily 4MO C-RIC-therapy improves long term CBF and vascular architecture at 6 MO and reduces WM damage and improves functional outcomes in aged males and females C-RIC induces durable long term collateral remodeling. C-RIC is a promising therapy for VCID and is ready for future clinical trials in VCID.

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.

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 (embolic middle cerebral artery occlusion) in combination with intravenous tissue-type plasminogen activator (tPA). Methods— We tested remote ischemic perconditioning therapy (RIPerC) at 2 hours after embolic middle cerebral artery occlusion in the mouse with and without intravenous tPA at 4 hours. We assessed cerebral blood flow up to 6 hours, neurological deficits, injury size, and phosphorylation of Akt (Serine 473 ) as a prosurvival signal in the ischemic hemisphere at 48 hours poststroke. Results— RIPerC therapy alone improved the cerebral blood flow and neurological outcomes. tPA alone at 4 hours did not significantly improve the neurological outcome even after successful thrombolysis. Individual treatments with RIPerC and intravenous 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 neurological outcome and reducing the infarct size (50%). All the therapeutic treatments upregulated phosphorylation of Akt in the ischemic hemisphere. Conclusions— RIPerC is effective alone after embolic middle cerebral artery occlusion and has additive effects in combination with intravenous tPA. RIPerC may be a simple, safe, and inexpensive combination therapy with intravenous 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.

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 effect is lost in NOS3-KO mice. We wanted to determine if the beneficial effect of C-RIC can be restored by bone marrow cell-derived NOS3. Methods: We prepared BM chimera from adult WT (B6.SJL-Ptprca; CD45.1) to adult NOS3-KO (B6.129; CD45.2) & vice versa in male mice (5-6 months) following confirmation for BM engrafting with flow cytometry at 2 weeks. Microcoil (0.18 mm) induced BCAS model was used to induce chronic hypoperfusion. Mice were randomly assigned to 3-groups: (1) Sham (2) BCAS and (3) BCAS+RIC. RIC was started 7d post-surgery daily for 3-4 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. We measured whole blood P-NOS3, P-AMPK and VEGFR2/CD31 by flow cytometry. Results: C-RIC-therapy for 3-4 weeks improves CBF in engrafted BM of WT into NOS3-KO in the BCAS+RIC groups at both 2 weeks and 4 weeks compared to BCAS (Sham RIC groups).No effect of C-RIC was shown in engrafted BM of NOS3 into WT. There was significant change between the BCAS and BCAS+RIC groups in the functional outcomes and histopathological evidences also reflects major changing in the groups in the BM of WT into NOS3-KO mice. Whole blood P-NOS3, P-AMPK and VEGFR2/CD31 was increased by C-RIC. However, no effect was shown in the NOS3-KO into WT chimera. Conclusions: The Beneficial effect of C-RIC is dependent upon bone marrow derived NOS3.Further studies are needed to determine if the red blood cell is the key cell carrying NOS3