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Introduction: VWF is an endothelial glycoprotein with known roles in vascular hemostasis and thrombosis. We previously showed increased expression of VWF in leptomeningeal collateral arterioles (LMCs) that underwent outward remodeling in the post-stroke brain. Interestingly, recent studies indicate that VWF may act as a novel smooth muscle mitogen and contribute to smooth muscle proliferation. The present study tested the hypothesis that VWF is required for LMC outward remodeling in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos, males) or VWF KO and WT mice (cohort 2; 10 mos, males). At 3 days after pdMCAO, the intact leptomeningeal vasculature was “planed off” for VWF immunofluorescence and morphometric analyses of LMCs and distalmost arterioles (DMAs) from the ipsilateral (ischemic) and contralateral hemispheres. VWF density in LMCs was normalized to the connected DMAs and presented as a fold difference. In a subset of samples, VWF location in LMCs was imaged by super-resolution microscopy (STED imaging, Nikon). Results: At 3 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF expression in ipsilateral LMCs (5.34 ipsi LMC vs 1.89 contra LMCs; p Conclusions: Our study revealed novel evidence for the essential role of VWF in the outward remodeling of LMCs in the post-stroke brain. Our findings also provide direct evidence for VWF within the smooth muscle layer, supporting the potential role of VWF as a critical smooth muscle mitogen in LMC remodeling.

Introduction: Piezo1 is a calcium-permeable mechanosensitive ion channel that is opened by membrane stretch or increased shear force. In peripheral vasculature, activation of endothelial Piezo1 promotes vasodilation. Whether endothelial Piezo1 regulates cerebral blood flow and the consequences of endothelial cell (EC) Piezo1 loss-of-function in the brain are unknown. The present study was designed to test the overall hypothesis that loss of EC Piezo1 results in reduced cerebral blood flow and subsequent brain pathology consistent with chronic hypoperfusion. Methods: EC specific Piezo1 knockout mice were generated by crossing Piezo1 fl/fl mice with inducible EC-specific Cre mice (Cdh5(Pac)CreERT2). Tamoxifen (75 mg/kg) was administrated to generate EC Piezo1 KO or as control in Piezo1 “floxed only” mice. At 1 month after tamoxifen injection, three cohorts were evaluated: (cohort 1) measure CBF by MRI ASL, (cohort 2) gait analysis (Digigait) and subsequent immunofluorescence (IF), (cohort 3) scRNAseq analysis. Gait analysis was chosen as a behavioral measure sensitive to white matter injury. Results: EC Piezo1 KO resulted in >8% reduction in resting CBF in striatum (n=5; pCtss and C1q family genes. In choroid plexus cluster, EC Piezo1 KO showed increased expression of Hif1a target genes. Conclusions: We conclude that EC Piezo1 loss-of-function promotes hypoperfusion sufficient to induce induction of inflammatory signaling and hypoxia-dependent transcription. White matter injury was evident as gliosis in the corpus callosum and by alterations in gait.

Intro: Mild hypothermia has been shown to improve outcomes in cardiac arrest and experimental stroke models. Unfortunately, hypothermia protocols in awake subjects are hampered by inconsistent temperature management due to incomplete suppression of cold defense mechanisms (e.g. shiver response). To address a need for better strategies for cooling conscious subjects, we sought to promote mild hypothermia by pharmacological activation of heat-sensing nerve fibers in the thermoregulatory system. We hypothesize that capsinoids - non-pungent agonists of the transient receptor potential vanilloid 1 (TRPV1) channel - activate vagal afferents, triggering thermoregulatory regions of the brain to induce mechanisms of whole-body cooling, protecting the brain and preventing injury progression after a stroke. Methods: C57BL6 male mice of 11wks underwent permanent distal middle cerebral artery occlusion (pdMCAO) or sham. Mice recovered for 2hrs in warming cages to maintain normal body temperatures during post-stroke recovery before being administered capsinoids or vehicle (4 IP injections, 90 min interval) to produce 6 hours of mild hypothermia (or normothermic control). After 1 month, mice underwent functional tests and brains were collected for histological analysis. Results: Capsinoids provided a significant and consistent hypothermia whereas vehicle maintained a baseline temperature (two-way ANOVA, n=7, p=0.0036). Capsinoid-treated mice showed ~50% infarct volume reduction 1 month after stroke, measured by cresyl-violet staining (0.7% ± 0.1% SEM vs 1.5% ± 0.2% SEM, n=7, two-tailed unpaired t-test: p=0.0046). Additionally, a stroke-induced breathing dysfunction (decrease in tidal volume by plethysmography) was significantly restored toward normal in the capsinoid-treated group (7.7e-3 ± 5.6e-4 SEM vs 5.8e-3 ± 2.4e-4 SEM, n=8, one-way ANOVA: p=0.033). Conclusion: Capsinoids delivered to TRPV1-containing visceral afferents promotes reversible mild hypothermia in freely moving conscious mice. Application of this form of cooling in the pdMCAO model provides significant reduction of stroke injury volume and long-term improvement in functional outcome.

Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. We compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq) and identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG following ischemic stroke. However, the functional role of Ifi27l2a in ischemic stroke is not known. Methods: Single cell suspensions were obtained from the brains subjected to permanent distal middle cerebral artery occlusion (pdMCAO) in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke. qRT-PCR and RNAscope were used to validate our scRNAseq findings for Ifi27l2a expression and compare regional transcriptional changes. To determine the role of Ifi27l2a, we used WT and Ifi27l2a +/- (Het) mice. Infarction was measured at 3-day post stroke by TTC staining. Thalamic gliosis were detected by IBA1 and GFAP staining at 2 week post-stroke. Results: Using unbiased scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes and senescence-related genes. Our qRT-PCR results validated the scRNAseq findings and further showed that Ifi27l2a was significantly up regulated in thalamus of aged brains (p = 0.04) and trending in cortex (p=0.23, n=4-6) as well as other proinflammatory genes. Using knockout mice, we found that the thalamic gliosis was significantly reduced in the brains of Het, compared to WT (n=6, p Conclusions: We identified Ifi272la as a highly up-regulated gene in MG following stroke and demonstrated that Ifi27l2a partial deletion results in reduced brain injury acutely and chronically. Our findings suggest that Ifi27l2a is a novel mediator of neuroinflammation and neurodegeneration, and thus provide the basis for a new therapeutic target to reduce brain injury in aging and stroke.

Introduction: The leptomeningeal collaterals (LMCs) are specialized brain surface arterioles that interconnect the distalmost arterioles (DMAs) of the major cerebral arteries. In normal conditions, flow in LMCs is negligible. However, during ischemic stroke, the LMCs provide a vital route for rescue blood flow from neighboring vessel territories, thereby lessening ischemic injury and improving outcome. With sustained flow, the LMCs rapidly remodel to further increase perfusion capacity. The LMCs differ in function and molecular composition compared to DMAs, however, a comprehensive transcriptional signature of these two vessel types is not available. Therefore, we developed methods to identify and isolate LMCs and DMAs from pig brain for subsequent unbiased RNAseq analysis. Methods: Duroc pigs were used to due to their larger vascular anatomy and gyrencephalic brains. We first mapped the cerebral vasculature and location of LMCs using blue dye infusion (surface vessel tracing) and BaSO4 microparticle infusion (3D microCT imaging). Having established location of LMCs, we infused an RNAlater/blue ink solution to facilitate vessel visualization and RNA stabilization during dissection. Harvested LMCs and DMAs were pooled by type and processed for RNAseq and analysis by R. Results: RNAseq analysis of LMCs and DMAs revealed 349 differentially expressed genes (DEGs) (adjusted p value < 0.05, fold change > 0.5), of which 241 were upregulated and 108 were down regulated in LMCs. Gene ontology (GO) analysis showed upregulation of biological processes including “vasculature development”, “sprouting angiogenesis”, “angiogenesis”, and “regulation of smooth muscle cell proliferation” in the LMC group. Angiogenesis related genes ( Cldn5 , Kdr , Vegfc , and Vwf ) were markedly upregulated in LMCs compared to DMAs (1.75 to 2.75 fold). In contrast, “extracellular matrix organization” and “positive regulation of smooth muscle cell migration” were among the most downregulated biological processes in LMCs. Conclusions: Our unbiased RNAseq approach revealed numerous DEGs between LMCs and DMAs. Interestingly, GO analysis revealed a proangiogenic signature in LMCs, which may shed new light on how they are able to rapidly remodel following ischemic stroke.

Microglia (MG) play critical roles in modulating inflammation and interferon signaling is a driver of neuroinflammation in various neurodegenerative diseases. Recently, Ifitm3 (a member of the “interferon-inducible transmembrane” family) has been revealed as a molecular mediator between neuroinflammation and neurodegeneration. However, expression of Ifitm3 in MG under inflammatory conditions, is not well described. Methods: To define the expression of Ifitm3 following stroke, permanent distal middle cerebral artery occlusion was performed with aged mice (18-22 mos). Brains were isolated at post-stroke day (PSD) 14 for single-cell RNAseq (scRNAseq), and at PSD 3, 7, and 14 for immunostaining and qRT-PCR. Primary microglial culture was established to evaluate mRNA levels of Ifitm3 and other pro-inflammatory genes to cytokine treatment. Results: Our scRNAseq revealed that Ifitm3 is among the top 10 genes upregulated in MG following stroke in aged brains. To validate these findings, we utilized a stroke model in aged mice and primary cell culture. We found that ischemic injury causes an induction of Ifitm3 in the striatum and peri-infarcted area following stroke, with peak induction PSD 7 (n=3-5, pIfitm3 was markedly induced in the damaged cortex of PSD 3 brains (n=4-6). We found that Ifitm3 expression is induced in primary MG following treatment with proinflammatory mediators (TNFα/IFNγ) for 6 hours (n=5-6, pIl1b and Casp1 . There was a close correlation between Ifitm3 and Casp1 expression, suggesting a potential of Ifitm3 in inflammasome activation. Conclusions: We demonstrated the profound induction of Ifitm3 in activated MG within the stroke brain in aged mice and in response to inflammatory factors in primary microglia culture. These studies identify Ifitm3 as a novel player involved in microglial modulation in response to a variety of inflammatory conditions. Future studies are warranted to determine if Ifitm3 deletion or inhibition can reduce neuroinflammation in the stroke brain.

Introduction: The leptomeningeal collaterals (LMCs) are specialized brain surface arterioles that interconnect the major cerebral artery territories. Good outcome following ischemic stroke is highly correlated with high collateral number and function. Unfortunately, with aging and disease, the LMCs demonstrate impaired dilatory capacity, reduced vessel diameter, and even complete vessel loss (rarefaction). The present study examined the chronic remodeling capacity of LMCs following permanent stroke in young and aged mice. Methods: Young and aged C57BL6 mice (5 and 20 months) were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). At 2 weeks or 3 months, LMCs were evaluated for inner diameter and VWF expression (% LMC coverage). We compared the LMCs feeding the occluded MCA territory (LMC ipsi) to the contralateral equivalent vessels (LMC contra). Diameter change is reported as fold change relative to the distal-most arteriole (DMA) associated with each LMC. Results: Young male mice demonstrated increased diameter of LMC ipsi relative to DMA at both 2 weeks (1.36 ± 0.075; n=24 vessels/4 mice, P Conclusions: Our data show lasting LMC remodeling in young stroke brain, consisting of increased LMC diameter and expression of VWF. With aging, LMC remodeling is transient and evidence of LMC rarefaction is apparent by 3 months.

Interferon signaling drives neuroinflammation in AD and stroke. Microglia (MG) play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. We compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq) to identify novel key players in inflammation. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice (3 & 20 months). scRNAseq was performed to define the transcriptional signature in brains at 14-days after stroke versus sham surgery. Primary MG culture was used to compare the mRNA levels of Ifi27l2a and Ifitm3 after cytokine treatment. We used qRT-PCR to validate scRNAseq findings and compare specific MG genes. Results: Using comprehensive scRNAseq analysis, our unbiased approach identified Ifi27l2a and Ifitm3 as the most highly up-regulated genes in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and Ifitm3 in MG ( R =0.4, pp =0.01), and trending at PSD 14 ( p =0.08) (n=5-6 stroke vs. sham). In contrast, Ifitm1 and Ifitm2 were not changed. Interestingly, Ifitm3 was recently proposed as an activator to γ-secretase. In vitro exposure of MG to inflammatory cytokines (TNFα and IFNγ, 6 hrs) caused significant increase in Ifi27l2a ( p =0.03, n=5-6) and Ifitm3 mRNA (p=0.02, n=5-6) compared to untreated MG. The in vitro co-expression of these genes was also well correlated ( R =0.5). Conclusions: We identified Ifi27l2a and Ifitm3 as highly up-regulated and correlated genes in MG in aging and following stroke. Our findings suggest that co-expression of Ifi27l2a and Ifitm3 may contribute to neuroinflammation. Further studies will be required to identify the specific functions of these genes and to determine the therapeutic potential of targeting these proteins to regulate neuroinflammation in aging and stroke.

Vascular contributions to cognitive impairment and dementia (VCID) includes multiple disorders that are identified by cognitive deficits secondary to cerebrovascular pathology. The risk of VCID is higher in people after the age of 70, and, currently, there is no effective treatment. Vascular endothelial cells (VEC) are critical components of the brain vasculature and neurovascular unit and their health is vital to the capacity of the brain vasculature to respond to stressors. However, aged VEC may enter an irreversible replicative-arrest state (senescence), which has been associated with dementia. E2F transcription factor 1 (E2F1) regulates cell cycle progression and DNA damage repair. Importantly, E2F1 deficiency is associated with cell senescence. We hypothesized that E2F1 downregulation contributes to senescence in the cerebral endothelium during aging. We used cultured primary VEC from young (4-months old, mo) and aged (18-mo) male and female mice for RNA sequencing, plasmid-based gene delivery, high-resolution microscopy, and (4-, 12-, and 18-mo) mice of the bilateral carotid artery stenosis (BCAS) model, which produces chronic cerebral hypoperfusion and recapitulates some of the features seen in patients with VCID. We found that overexpression of E2F1 reduced the levels of senescence-associated phenotypes in cultured VEC from young mice that were exposed to oxygen and glucose deprivation (pE2f1 was reduced (~40%) in cultured primary VEC from aged mouse brains compared with young cells (pIn conclusion , our study identifies E2F1 as a potential regulator of endothelial senescence in mice and highlights the contribution of aging as an important factor in losing endothelial resilience.

Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. However, how the transcriptional signature of these cells is altered in aging and stroke remains poorly defined. Here, we compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq). Our unbiased approach identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG with aging or ischemic stroke. Further analyses suggested that this gene may be a novel regulator of MG inflammation in aging and stroke. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke and sham controls. We used qRT-PCR to validate our scRNAseq findings and compare regional transcriptional changes. Human microglial cells (HMC3; subjected to OGD) and human autopsy brain sections (stroke versus non-stroke) were used to examine IFI27l2 protein expression. Results: Using comprehensive scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes. In contrast, a negative correlation was shown with genes involved in promoting the phagocytic MG phenotype (e.g. Spp1, Cst7, Lpl, and Itgax). Our qRT-PCR results validated the central scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in thalamus of aged brains (p = 0.0412) and trending in cortex (p = 0.23) (n=4 young/aged). Following stroke, Ifi27l2a and other markers of MG activation were significantly increased in both brain regions (n= 4-6). In human tissues, we observed an increase in IFI27l2 protein in HMC3 cells with OGD and in brain of stroke patients. Conclusions: We identified Ifi272la as a highly up-regulated gene in MG in aging and following stroke. Our findings suggest that Ifi27l2a may be a novel regulator of neuroinflammation in MG, and thus provide the basis for a new therapeutic target to reduce inflammation in aging and stroke.

Introduction: The leptomeningeal collaterals (LMCs) are arterial anastomoses that interconnect the distal branches of the major cerebral arteries. They distribute blood flow from adequately to poorly perfused vascular territories when needed. In normal conditions, the LMCs experience little net flow. However, during ischemic stroke, the LMCs are recruited to provide rescue flow to the ischemic territory. During prolonged or permanent stroke, the LMCs can rapidly remodel to provide increased perfusion capacity. Von Willebrand factor (VWF) is a secreted protein expressed in cerebral endothelium and has recently been implicated in angiogenesis. We sought to determine if VWF expression was increased in remodeling LMCs in the post-stroke brain. Methods: C57BL6 mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). At 3 days after pdMCAO, mice were perfused with fluorescent tomato lectin to label vascular endothelium. The cortical surface was “planed off” by vibratome to provide largely contiguous cortical surface vessel preparations containing distalmost arterioles (DMAs) and the connected LMCs. VWF immunofluorescence intensity was measured in LMCs and the connecting DMAs of hemispheres ipsilateral and contralateral to the stroke. Inner diameter of the LMCs and DMAs were measured with Image J. Results: At 3 days post-stroke, LMCs of the stroke hemisphere demonstrated significant outward remodeling. Mean inner diameter of the ipsilateral versus contralateral LMCs were 44.5 ± 5.9 um versus 28.2 ± 1.2 um, respectively (p Conclusions: We demonstrate that VWF is highly expressed in the remodeling LMCs (but not connecting DMAs) of the post-stroke brain. These findings suggest a novel and selective role for endothelial VWF in the LMC response to prolonged recruitment during ischemic stroke.

Secondary injury in the thalamus has been observed following cortical stroke in rodents and humans and is associated with worsened recovery. Interruption of this progressive injury reflects an important therapeutic goal. However, the mechanisms whereby primary cortical infarction leads to remote injury in distant regions of brain are not well defined. We used a mouse model of cortical stroke (which demonstrates delayed thalamic injury) to define the time course of thalamic gliosis and neuronal injury and then test the potential of delayed memantine treatment (an NMDA receptor antagonist) to attenuate this secondary injury. Methods: Cortical infarction was induced by permanent occlusion of the distal middle cerebral artery (pdMCAO) in male C57BL/6J mice (young and aged) and CCR2-RFP mice. Brain infarct, cell-specific injury, and gliosis were measured by cresyl violet, Fluoro-jade C (FJC), TTC, FACS, and immunofluorescence. In young mice, memantine was injected at 4 and 24 hours post-stroke (100 and 50 mg/kg, ip). Brains were evaluated at post-stroke day 3 and 14 (PSD3 and PSD14). Results: At PSD3, the primary infarct was restricted to the cortex of the MCA territory, with no infarct detected in the thalamus of young mice. However, by PSD 14, neurons in the ipsilateral thalamus exhibited significant injury (FJC positive, condensed pyknotic nuclei). Gliosis was first detectable in the ipsilateral thalamus at PSD3 and progressively increased to PSD14 (anti-GFAP and Iba1). Infiltration of peripheral-derived monocytes was determined to be one source of the activated microglia in the thalamus (CCR2-RFP reporter mice, n=3). Interestingly, pdMCAO mice allowed to recover for two years demonstrated persistent astrogliosis (cortex and thalamus), though microgliosis was no longer evident (n=2). Aged mice subjected to pdMCAO also demonstrated gliosis in thalamus at PSD14, albeit to a lesser extent than young mice (n=5 each age). Finally, delayed treatment with memantine resulted in significantly attenuated gliosis and neuronal loss in the thalamus at PSD14 (young mice, n=9 each). Conclusions: These results further define gliosis in the mechanism of secondary injury and importantly demonstrate attenuation of secondary injury by delayed NMDA receptor antagonism.

Delta-like 4 (Dll4) is a Notch receptor ligand classically thought to be expressed exclusively in endothelial cells. However, little is known about the cellular expression or functional role of Dll4 following ischemic stroke. Here, we present novel findings that Dll4 expression is shifted from endothelial cells to glial cells in the sub-acute stroke phase. Method: The permanent distal middle cerebral artery occlusion (dMCAO) model was performed to induce stroke. To investigate expression patterns of Dll4 protein and mRNA, immunostaining as well as single-cell RNA sequencing and single-cell fluorescent in situ hybridization were performed. To verify the role of endothelial Dll4 , tamoxifen-inducible, endothelial-specific Dll4 KO mice (Dll4iecKO) and Dll4 fl/fl (“floxed-only”) female mice (3 mos) were injected with tamoxifen (n=4) at 24 and 48 hrs post-stroke. To determine the effect of Notch inhibition at different time points, DAPT (100 mg/kg) or vehicle was administered at 24 or 72 hours post-stroke (n=4-5). Behavioral tests (foot fault) were conducted at Post Stroke Day (PSD) 1, 3, 7, and 14 and functional angiogenesis was assessed at PSD 14. Results: Elevated expression of Dll4 protein was observed in the microvasculature of the peri-infarct cortex (piCtx) as early as PSD 1. By PSD 3, however, we discovered Dll4 expression in microglia within the piCtx, with robust expression by PSD 14. Post-stroke deletion of endothelial Dll4 resulted in significant improvement in neurological function and increased capillary density in piCtx[l1] . DAPT given at 24 hours post-stroke improved outcome and capillary density; however, DAPT given at 72 hours (when microglia Dll4 expression is evident) appeared to worsen outcome. Conclusions: After stroke, expression of Dll4 shifts from exclusively endothelial (PSD 1) to mixed endothelial/microglial (PSD 3) to primarily microglial (PSD 14). Notch inhibition beginning at 24 hours post-stroke or endothelial-only deletion of Dll4 leads to increased angiogenesis and improved functional recovery. In contrast, Notch inhibition initiated at 72 hours post-stroke leads to worse outcome[l2] . These findings suggest a potential novel role of microglia in supporting reparative angiogenesis through Dll4/Notch signaling.

Background and Purpose— VWF (von Willebrand factor) strings mediate spontaneous platelet adhesion in the vascular lumen, which may lead to microthrombi formation and contribute to stroke pathology. However, the mechanism of VWF string attachment at the endothelial surface is unknown. We tested the novel hypothesis that VWF strings are tethered to the endothelial surface through an interaction between extracellular vimentin and the A2 domain of VWF. We further explored the translational value of blocking this interaction in a model of ischemic stroke. Methods— Human endothelial cells and pressurized cerebral arteries were stimulated with histamine to elicit VWF string formation. Recombinant proteins and antibodies were used to block VWF string formation. Mice underwent transient middle cerebral artery occlusion with reperfusion. Just before recanalization, mice were given either vehicle or A2 protein (recombinant VWF A2 domain) to disrupt the vimentin/VWF interaction. Laser speckle contrast imaging was used to monitor cortical perfusion. Results— Pressurized cerebral arteries produced VWF strings following histamine stimulation, which were reduced in arteries from Vim KO (vimentin knockout) mice. VWF string formation was significantly reduced in endothelial cells incubated with A2 protein or antivimentin antibodies. Lastly, A2 protein treatment significantly improved cortical reperfusion after middle cerebral artery occlusion. Conclusions— We provide the first direct evidence of cerebral VWF strings and demonstrate that extracellular vimentin significantly contributes to VWF string formation via A2 domain binding. Lastly, we show that pharmacologically targeting the vimentin/VWF interaction through the A2 domain can promote improved reperfusion after ischemic stroke. Together, these studies demonstrate the critical role of VWF strings in stroke pathology and offer new therapeutic targets for treatment of ischemic stroke.

Remote secondary injury in the thalamus has been observed following rodent and human stroke and is associated with poorer recovery. However the mechanisms and resultant molecular changes whereby cortical infarction leads to remote injury in distant regions of brain are not well defined. The present studies utilize a mouse model of permanent distal MCAO (pdMCAO) with delayed secondary thalamic injury. We have measured time dependent molecular and cellular events, focusing on glial activation and neuronal death in the thalamus. These studies provide an important foundation for future studies aimed at reducing secondary brain injury. Methods: Cortical infarction by pdMCAO was induced by microcauterization of the distal MCA in male C57BL/6J mice (11-13 wks). Cresyl violet (CV), fluoro-jade C (FJC) and immunochemical staining were performed at 24 hours, 3 days, 1 week, and 2 weeks after stroke (n=5, each time point) and in shams (n=3). Results: First, we confirmed with TTC staining that the primary infarction induced by pdMCAO was restricted to the cortex of the MCA territory at 72 hours, and that no infarct was seen in the thalamus. Microglia and astrocyte activation (IBA-1 and GFAP, respectively) was first detectable in the ipsilateral ventral posteromedial nucleus (VPN) of the thalamus at 3 days after stroke, and progressively increased at 1 and 2 weeks. However, thalamic neuronal injury (by FJC staining) was not evident until 1 week after stroke. At 2 weeks following stroke, the number of FJC positive neurons further increased and significant morphological changes were evident by CV staining, including shrunken cytoplasm and condensed pyknotic nuclei. Conclusions: We present a mouse stroke model for studying the mechanisms of secondary thalamic injury. In this model, primary cortical injury results in delayed cellular events in the ipsilateral thalamus, including an initial activation of microglia and astrocytes with subsequent neuronal injury and death. These studies provide necessary foundation for designing future interventional strategies aimed at reducing secondary injury following stroke.

Introduction: The TRPV1 channel is a non-selective ion channel that is activated by capsaicinoids, temperature (>43 °C), and multiple factors produced during inflammation or injury. At this time, there is very little reported regarding a role of this channel in regulation of cerebral blood flow. Here we provide new evidence for TRPV1 in promoting microvascular reperfusion following stroke. Hypothesis: Endothelial TRPV1 channels contribute to cerebral reperfusion following ischemic stroke. Methods: Experiments were performed in male WT and TRPV1 knockout mice of C57BL/6 background. Immunofluorescence was performed in brain sections with TRPV1 and Pecam-1 antibodies. Pressurized artery preparations were performed with isolated superior cerebellar arteries. Relative cerebral blood flow measurements were performed by laser Doppler and laser speckle flowmetry in mice before, during, and after MCAO (20 or 30 min). Reperfusion was monitored continuously for 2-3 hours and again at 24 hours. Results: TRPV1 expression was positively detected by immunofluorescence in the cerebral microvasculature, as assessed by Pecam-1 co-localization. TRPV1 KO mice were used as a negative control. Pressurized cerebral arteries demonstrated modest vasodilation to TRPV1 agonist (capsaicin) that was significantly potentiated with H 2 O 2 pre-treatment. The potentiated component of vasodilation was lost with removal of the endothelium, demonstrating the endothelial source of the response. Following brief MCA occlusion (20 or 30 min), WT mice demonstrated an initial return of cerebral perfusion followed by a transient drop in perfusion (~25% drop). This drop in perfusion started at 20-30 minutes into the reperfusion phase and lasted approximately one hour, at which point perfusion was restored to pre-stroke baseline value. In TRPV1 KO mice, the drop in perfusion was more profound (~65% drop) and persisted through 3 hours. By 24 hours, flow was restored to baseline in both groups. Conclusions: These data demonstrate a functional role for TRPV1 channels during reperfusion. We speculate that endothelial TRPV1 channels are functionally potentiated during injury and contribute to the vasodilatory capacity of the cerebral microvasculature following stroke.

Introduction: VWF strings can form within the vascular lumen following endothelial activation and have been implicated in platelet adhesion and worsened outcome following stroke. However, the molecular interactions facilitating VWF string anchorage to the endothelial surface are currently unknown. Here we examined the novel role of endothelial vimentin in mediating the anchorage of VWF strings within the cerebrovasculature. We hypothesize that VWF released from activated endothelium remains anchored at the luminal surface (i.e. VWF strings) through direct interaction with extracellular vimentin. Methods: Cultured endothelial cell (EC) experiments were performed with human umbilical vein EC (HUVECs) and human brain microvascular EC (HBMVECs). EC were stimulated with histamine (10 uM) under flow conditions. Specific protein interactions were probed with recombinant vimentin and A2 domain of VWF. Mouse middle cerebral and superior cerebellar arteries from WT and vimentin KO mice were set up in a pressurized artery chamber, stimulated with histamine, and then processed for VWF immunofluorescence to quantify VWF strings. VWF string formation was further evaluated by cranial window preparation using labeled platelets to detect real-time in vivo string formation. Results: In cultured EC, histamine stimulation promoted the production of long VWF strings that were significantly attenuated in the presence of recombinant vimentin or A2 domain of VWF. In pressurized cerebral arteries, histamine stimulation promoted VWF strings that aligned along the luminal endothelial surface. VWF string formation was reduced 2.9 fold in arteries from vimentin knockout mice (P=0.02). Histamine stimulation in the cranial window produced platelet-adherent strings in the pial microvasculature. Conclusions: These studies provide in vitro and in vivo evidence for a novel interaction between vimentin and the A2 domain of VWF at the endothelial surface which contributes to the anchorage of VWF strings in the microvasculature. This vimentin/VWF interaction critically regulates VWF-mediated platelet adhesion at the surface of activated endothelium and could thus provide a novel therapeutic target in the treatment of acute ischemic stroke.

Mild decrease in core temperature (therapeutic hypothermia) provides lasting neuroprotection following cardiac arrest or cerebral ischemia. However, current methods for producing therapeutic hypothermia trigger a cold-defense response that must be countered by sedatives, muscle paralytics, and mechanical ventilation. We aimed to determine methods for producing hypothermia in the conscious mouse by targeting two transient receptor potential channels involved in thermoregulation, two transient receptor potential (TRP) channels involved in thermoregulation, TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8).Controlled prospective animal study.Research laboratory at academic medical center.Conscious unrestrained young and aged male mice.Mice were treated with the TRPV1 agonist dihydrocapsaicin, a TRPM8 inhibitor ("compound 5"), or their combination and the effects on core temperature (Tcore) were measured by implanted thermocouples and wireless transponders.TRPV1 agonist dihydrocapsaicin produced a dose-dependent (2-4 mg/kg s.c.) drop in Tcore. A loading dose followed by continuous infusion of dihydrocapsaicin produced a rapid and prolonged (6 hr) drop of Tcore within the therapeutic range (32-34°C). The hypothermic effect of dihydrocapsaicin was augmented in aged mice and was not desensitized with repeated administration. TRPM8 inhibitor "compound 5" (20 mg/kg s.c.) augmented the drop in core temperature during cold exposure (8°C). When "compound 5" (30 mg/kg) was combined with dihydrocapsaicin (1.25-2.5 mg/kg), the drop in Tcore was amplified and prolonged.Activating warm receptors (TRPV1) produced rapid and lasting hypothermia in young and old mice. Furthermore, hypothermia induced by TRPV1 agonists was potentiated and prolonged by simultaneous inhibition of TRPM8.

Background: There is a lack of widely applicable treatments of stroke. One of the potential new treatment strategies is the induction of heat shock proteins (HSPs), such as Hsp27 and Hsp70, in the brain, which was shown to confer neuroprotection from ischemia. In epithelial cells, HSPs were shown to be induced by agonists of the Transient Receptor Potential Vanilloid Member 1 ion channel (TRPV1), also known as the heat and capsaicin receptor. Therefore, our overall hypothesis is that systemic administration of TRPV1 agonist dihydrocapsaicin (DHC) induces HSPs in the brain cortical neurons and thus provides neuroprotection from stroke. In the present study we focus on the initial portion of the hypothesis, that TRPV1 agonism promotes HSP induction in the brain. Methods: We determined relative expression of candidate HSP genes by quantitative real-time polymerase chain reaction after DHC treatment in cultured rat primary cortical neurons as well as in cortex tissue samples of in vivo DHC-treated wild-type and TRPV1 knock-out mice. Results: First, we established that DHC injection (4 mg/kg i.p.) 4 hours before tissue harvest led to 3- and 5-fold upregulation of mRNA of the Hsp27 and Hsp70 respectively in the cortex of mice (compared to vehicle injection, n=3 mice per group). This effect was specific to TRPV1 channels, as it was absent in TRPV1 knock-out mice. Second, we determined that 3-hour DHC treatment led to upregulation of Hsp70 mRNA in cultured rat primary cortical neurons, which was dependent on the dose of DHC (1-1000 nM). Conclusions: We present the first demonstration that systemic delivery of TRPV1 agonists promotes an upregulation of neuroprotective heat shock proteins in the brain. We have further demonstrated that cortical neurons have the capacity to respond to TRPV1 agonism directly rather than through other TRPV1-mediated physiologic responses. We propose that systemic delivery of TRPV1 agonists may offer a novel treatment option in the protection or recovery from ischemic stroke.

Background: Therapeutic hypothermia is a promising strategy for neuroprotection following stroke. However, current widely available methods are not compatible with conscious patients and produce unwanted physiological stress. We have recently demonstrated that pharmacological hypothermia (PH) through transient receptor potential vanilloid 1 (TRPV1) channel agonism is effective and safe in the conscious mouse and provides significant neuroprotection following ischemic stroke at 24 hours reperfusion. We now evaluate whether this method of PH provides sustained neuroprotection through one month reperfusion. Methods: Three experimental groups were evaluated: sham operated (Sham), stroke with normothermia (Stroke/NT), and stroke with PH (Stroke/PH) (n=8, 10, 7). Stroke was induced by transient occlusion of left distal middle cerebral artery (MCA) plus left common carotid artery for two hours. For the Stroke/PH group, hypothermia (32-34°C) was induced by infusion of TRPV1 agonist (dihydrocapsaicin) for 8 hours; beginning 90 minutes after reperfusion. We evaluated several behavioral tests from pre-stroke up to 28 days reperfusion. Total cerebral infarct was then evaluated in Nissl-stained brain sections at 30 days reperfusion. Results: The foot fault test was the most sensitive in demonstrating extended functional deficit after stroke. With this test, Stroke/NT demonstrated prolonged deficiency (through 21 days reperfusion) compared to Sham. PH treatment (Stroke/PH) demonstrated significant improvement in function compared to Stroke/NT. Other tests demonstrated non-statistically significant performance reduction of Stroke/NT in swimming time, grip side preference, turning and climbing time. Histological analysis demonstrated significant ipsilateral necrosis and hemisphere shrinkage in Stroke/NT brains compared to Stroke/PH brains. Conclusions: In summary, we demonstrate long-term neuroprotection with TRPV1-mediated PH following stroke. These studies support the therapeutic potential of TRPV1 agonism for promoting hypothermia in the conscious stroke subject.

There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.

Background and Purpose— The contribution of endothelium-derived hyperpolarizing factor (EDHF) to ATP-mediated dilations is significantly attenuated in the rat middle cerebral artery of intact and estrogen-treated ovariectomized (OVX) females compared with males and vehicle-treated OVX females. Since an increase in endothelial calcium appears to be a critical prerequisite in the EDHF response, we tested the hypothesis that endothelial cell intracellular calcium ([Ca 2+ ] i ) fails to reach sufficient levels to elicit robust EDHF-mediated dilations in females and that this effect is mediated by estrogen. Methods— Vascular diameter and [Ca 2+ ] i were measured concomitantly in perfused middle cerebral artery segments with the use of videomicroscopy and fura 2 fluorescence, respectively. Results— In the presence of N G -nitro- l -arginine methyl ester and indomethacin, the dilation to 10 −5 mol/L ATP was significantly reduced ( P 2+ ] i increased to comparable levels in intact females (461±116 nmol/L), estrogen-treated OVX females (417±50 nmol/L), intact males (421±77 nmol/L), and vehicle-treated OVX females (530±92 nmol/L). In response to luminal ATP (10 −5 mol/L), smooth muscle cell [Ca 2+ ] i decreased to a greater degree in males (37±4%; n=8) compared with females (21±5%; n=7) and in vehicle-treated OVX females (18±7%; n=7) compared with estrogen-treated OVX females (3±5%; n=9). Conclusions— Our data suggest that loss of a factor coupling EDHF to reduction of ionized smooth muscle cell [Ca 2+ ] i accounts for the attenuated EDHF-mediated dilations in the female middle cerebral artery.

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF)–mediated dilations are potentiated after several pathologies, including ischemia/reperfusion (I/R). However, no study to date has addressed the mechanism by which this potentiation occurs. This study tested the hypothesis that potentiated EDHF-mediated dilations are due to altered endothelial Ca 2+ handling after I/R. Methods— Rat middle cerebral arteries (MCAs) were isolated after 2 hours of MCA occlusion and 24 hours of reperfusion (or sham surgery). This model has been previously demonstrated to produce potentiated EDHF-mediated dilations. MCAs were studied in a pressurized/perfused vessel chamber equipped for the simultaneous measurement of endothelial Ca 2+ (with fura 2) and artery diameter. Measures were made after luminal administration of UTP (P2Y 2 purinoceptor agonist), 2 MeS-ATP (P2Y 1 purinoceptor agonist), and Br-A23187 (receptor-independent Ca 2+ ionophore) for sham and I/R MCAs. Results— I/R resulted in significantly potentiated UTP-mediated dilations (through a P2Y 2 purinoceptor) and endothelial Ca 2+ responses in the presence of N G -nitro- l -arginine methyl ester (L-NAME) and indomethacin. Endothelial Ca 2+ and diameter responses were also significantly potentiated with 2 MeS-ATP (through a P2Y 1 purinoceptor) when L-NAME and indomethacin were absent. Br-A23187, a receptor-independent Ca 2+ ionophore, produced significantly potentiated endothelial Ca 2+ responses after I/R in the presence of L-NAME/indomethacin. Evaluation of artery diameter as a function of endothelial Ca 2+ demonstrated no differences between sham and I/R groups. Conclusions— These findings demonstrate that I/R results in augmented endothelial Ca 2+ responses that appear to be downstream of the receptor level. Moreover, these data suggest that this augmented Ca 2+ response contributes to the potentiated EDHF-mediated dilations after I/R.

Background Transient receptor potential C3 ( TRPC 3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell ( EC ) hyperpolarization and endothelium‐dependent hyperpolarization–mediated vasodilation. However, the mechanism by which TRPC 3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC 3 trafficking to the plasma membrane, where it provides the source of Ca 2+ influx for small conductance calcium‐activated K + (SK Ca ) channel activation and sustained EC hyperpolarization. Methods and Results Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC 3 blocker (Pyr3) produced significant attenuation of endothelium‐dependent hyperpolarization–mediated vasodilation and endothelial Ca 2+ response ( EC ‐specific Ca 2+ biosensor) to intraluminal ATP . Pyr3 treatment also resulted in a reduced ATP ‐stimulated global Ca 2+ and Ca 2+ influx in primary cultures of cerebral endothelial cells. Patch‐clamp studies with freshly isolated cerebral EC s demonstrated 2 components of EC hyperpolarization and K + current activation in response to ATP . The early phase was dependent on intermediate conductance calcium‐activated K + channel activation, whereas the later sustained phase relied on SK C a channel activation. The SK C a channel–dependent phase was completely blocked with TRPC 3 channel inhibition or in ECs of TRPC 3 knockout mice and correlated with increased trafficking of TRPC 3 (but not SK C a channel) to the plasma membrane. Conclusions We propose that TRPC 3 dynamically regulates SK C a channel activation through receptor‐dependent trafficking to the plasma membrane, where it provides the source of Ca 2+ influx for sustained SK C a channel activation, EC hyperpolarization, and endothelium‐dependent hyperpolarization–mediated vasodilation.

It is well established that stimulation of receptors on the endothelium can elicit dilation of arteries and arterioles by initiating the synthesis and release of nitric oxide (NO) and/or metabolites of the cyclooxygenase pathway (most often prostacyclin). Recent evidence now suggests that there is at least one other endothelium-dependent dilator mechanism that does not involve NO or a cyclooxygenase metabolite. This mechanism has been termed “endothelium-derived hyperpolarizing factor” (EDHF). (Note that EDHF is different from endothelium-derived relaxing factor [EDRF], which is often associated with NO. It should also be noted that recent studies suggest that EDHF may not be a “factor” but rather a process or mechanism. To be more accurate, the term “EDHF” should be referred to as a non-NO, noncyclooxygenase endothelium-dependent hyperpolarization. However, in order to maintain consistency in the literature, we will refer to it as “EDHF.”) We believe that EDHF is a major regulator of cerebral blood flow during physiological states and may become even more important following pathological insults such as ischemia or traumatic brain injury. The purpose of this editorial is to familiarize the reader with EDHF and to highlight the potential importance of EDHF in the cerebral circulation. While the defining criteria for EDHF or the mechanism of endothelium-dependent hyperpolarizations can vary, we will characterize the process as (1) requiring endothelium, (2) being distinct from NO and a cyclooxygenase metabolite, (3) hyperpolarizing the vascular smooth muscle (VSM), and (4) involving calcium-activated potassium (KCa) channels. The existence of a third pathway was alluded to in the late 1980s, when some endothelium-dependent relaxations were shown to be resistant to inhibitors of NO and cyclooxygenase.1,2⇓ Although it has been suggested that EDHF-dependent dilations reflect incomplete inhibition of nitric oxide synthase (NOS),3 there is now convincing evidence of the existence of a non-NO, …