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Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests pH homeostasis is a new cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption and reactive gliosis, which were both rescued by pharmacological or genetic inhibition of the NO-CCL2 pathwayin vivo.Together, our study identifies the astrocytic Slc4a4-NO-CCL2 axis as a pivotal mechanism controlling BBB integrity and repair, while providing insights for a novel therapeutic approach against BBB-related CNS disorders.

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.

Microglia, the resident innate immune cells of the brain, become more highly reactive with aging and diseased conditions. In collaboration with other cell types in brains, microglia can contribute both to worsened outcome following stroke or other neurodegenerative diseases and to the recovery process by changing their phenotype toward reparative microglia. Recently, IFITM3 (a member of the “interferon-inducible transmembrane” family) has been revealed as a molecular mediator between amyloid pathology and neuroinflammation. Expression of IFITM3 in glial cells, especially microglia following stroke, is not well described. Here, we present evidence that ischemic stroke causes an increase in IFITM3 expression along with increased microglial activation marker genes in aged brains. To further validate the induction of IFITM3 in post-stroke brains, primary microglia and microglial-like cells were exposed to a variety of inflammatory conditions, which significantly induced IFITM3 as well as other inflammatory markers. These findings suggest the critical role of IFITM3 in inducing inflammation. Our findings on the expression of IFITM3 in microglia and in aged brains following stroke could establish the basic foundations for the role of IFITM3 in a variety of neurodegenerative diseases, particularly those that are prevalent or enhanced in the aged brain.

Post-menopausal women become vulnerable to stroke and have poorer outcomes and higher mortality than age-matched men and previous studies suggested that sex chromosomes play a vital role in mediating stroke sensitivity in the aged. It is unknown if this is due to effects of the X or Y chromosome. The present study used the XY* mice model (with four genotypes: XX and XO gonadal females, and XY and XXY gonadal males) to compare the effect of the X vs. Y chromosome compliment in stroke. Aged (18-20 months) and gonadectomized young (8-12 weeks) mice were subjected to a 60-min middle cerebral artery occlusion. Infarct volume and behavioral deficits were quantified 3 days after stroke. Microglia activation and infiltration of peripheral leukocytes in the aged ischemic brain were assessed by flow cytometry. Plasma inflammatory cytokine levels by ELISA and brain expression of two X chromosome linked genes, KDM6A and KDM5C by immunochemistry, were also examined. Both aged and young XX and XXY mice had worse stroke outcomes compared to XO and XY mice respectively; however, difference between XX vs. XXY or XO vs. XY aged mice was minimal. Mice with two copies of the X chromosome showed more robust microglial activation, higher brain infiltrating leukocytes, elevated plasma cytokine levels, and enhanced colocalization of KDM6A and KDM5C with Iba-1+ cells after stroke than mice with one X. The number of X chromosomes mediates stroke sensitivity in aged mice, which might be processed through the X chromosome linked genes and the inflammatory responses.

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.

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. However, whether CCN1 contributes to senescence in CEC and how this is regulated requires further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. Stabilization of the G4 structures regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and these mice exhibited enhanced cognitive deficits. A sequence in the 3′-UTR of the human CCN1 mRNA has the ability to fold into G4s in vitro. We hypothesize that G4 stabilization regulates CCN1 in cultured primary CEC and induces endothelial senescence. We used cerebral microvessel fractions and cultured primary CEC from young (4-months old, m/o) and aged (18-m/o) mice to determine CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA-G4s for fluorescence spectra. We found that cultured CEC from aged mice exhibited enhanced levels of SA phenotypes, and higher levels of CCN1 and G4 stabilization. In cultured CEC, CCN1 induced SA phenotypes, such as SA β-galactosidase activity, and double-strand DNA damage. Furthermore, CCN1 levels were upregulated by a G4 ligand, and a G-rich motif in the 3′-UTR of the Ccn1 mRNA was folded into a G4. In conclusion, we demonstrate that CCN1 can induce senescence in cultured primary CEC, and we provide evidence that G4 stabilization is a novel mechanism regulating the SASP component CCN1.

In the past three decades, research on the gut microbiome and its metabolites, such as trimethylamines (TMA), trimethylamine N-oxide (TMAO), short-chain fatty acids (SCFAs), branched-chain amino acids (BCAAs), bile acids, tryptophan and indole derivatives, has attracted the attention of many scientists and industrialists. Among these metabolites, TMAO is produced from dietary choline, phosphatidylcholine, carnitine,andbetaine. TMAO and other gut metabolites, such as TMA and SCFAs, reach the brain by crossing the blood-brain barrier (BBB) and are involved in brain development, neurogenesis, and behavior. Gut-microbiota composition is influenced by diet, lifestyle, antibiotics, and age. Several studies have confirmed that altered TMAO levels contribute to metabolic, vascular, psychiatric, and neurodegenerative disorders. This review focuses on how altered TMAO levels impact oxidative stress, microglial activation, and the apoptosis of neurons, and may lead to neuroinflammation, which can subsequently result in the development of psychiatric, cognitive, and behavioral disorders.

Background CD146 is a tight junction-associated molecule involved in maintaining endothelial barrier, and balancing immune–inflammation response, in cardiovascular disease. Notably, peripheral CD146+ cells significantly upsurge under vessel dyshomeostasis such as acute myocardial injury (AMI), appearing to be a promising therapeutic target. In this study, with a new view of gene correlation, we aim at deciphering the complex underlying mechanism of CD146+ cells’ impact in the development of AMI. Methods Transcription dataset GSE 66,360 of CD146+ blood cells from clinical subjects was downloaded from NCBI. Pearson networks were constructed and the clustering coefficients were calculated to disclose the differential connectivity genes (DCGs). Analysis of gene connectivity and gene expression were performed to reveal the hub genes and hub gene clusters followed by gene enrichment analysis. Results and conclusions Among the total 23,520 genes, 27 genes out of 126 differential expression genes were identified as DCGs. These DCGs were found in the periphery of the networks under normal condition, but transferred to the functional center after AMI. Moreover, it was revealed that DCGs spontaneously crowded together into two functional models, CCL20 cluster and NR4A3 cluster, influencing the CD146-mediated signaling pathways during the pathology of AMI for the first time.

Secondary injury following cortical stroke includes delayed gliosis and eventual neuronal loss in the thalamus. However, the effects of aging and the potential to ameliorate this gliosis with NMDA receptor (NMDAR) antagonism are not established. We used the permanent distal middle cerebral artery stroke model (pdMCAO) to examine secondary thalamic injury in young and aged mice. At 3 days post-stroke (PSD3), slight microgliosis (IBA-1) and astrogliosis (GFAP) was evident in thalamus, but no infarct. Gliosis increased dramatically through PSD14, at which point degenerating neurons were detected. Flow cytometry demonstrated a significant increase in CD11b+/CD45int microglia (MG) in the ipsilateral thalamus at PSD14. CCR2-RFP reporter mouse further demonstrated that influx of peripheral monocytes contributed to the MG/Mϕ population. Aged mice demonstrated reduced microgliosis and astrogliosis compared with young mice. Interestingly, astrogliosis demonstrated glial scar-like characteristics at two years post-stroke, but not by 6 weeks. Lastly, treatment with memantine (NMDAR antagonist) at 4 and 24 h after stroke significantly reduced gliosis at PSD14. These findings expand our understanding of gliosis in the thalamus following cortical stroke and demonstrate age-dependency of this secondary injury. Additionally, these findings indicate that delayed treatment with memantine (an FDA approved drug) provides significant reduction in thalamic gliosis.

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.

Remote secondary injury in the thalamus has been observed following cortical infarct, however the mechanisms are not well understood. We used the distal MCAO stroke model (pdMCAO) to explore the cellular and temporal gliosis response in secondary thalamic injury in mice. At 3 days post-stroke (PSD3), primary infarct was limited to the cortex, with no infarct in the thalamus. However, at 2 weeks after stroke (PSD14), the ipsilateral thalamus demonstrated degenerating and severely damaged neurons. Staining for GFAP (astrogliosis) or IBA-1 (microgliosis) was first apparent in the ipsilateral thalamus by PSD3, and showed a progressive increase through PSD14. The number of activated microglia was increased within the thalamus at PSD14, reflecting proliferation of resident microglia as well as infiltration of peripheral monocytes. Interestingly, astrogliosis within the thalamus was enduring, as it was still evident at two years post-stroke. Furthermore, the astrogliosis at two years (but not at 6 weeks) demonstrated glial scar-like characteristics. Lastly, we demonstrated that post-stroke treatment with an NMDA receptor antagonist (memantine) reduces gliosis in the thalamus at PSD14. These findings highlight the development of lasting secondary injury in the thalamus following cortical stroke and support the value of memantine treatment in the mitigation of this injury.

Experimental guidelines have been proposed to improve the rigor and reproducibility of experimental stroke studies in rodents. As brain temperature is a strong determinant of ischemic injury, tight management of brain or body temperature (Tcore) during the experimental protocol is highly recommended. However, little guidance is provided regarding how or for how long temperature support should be provided. We compared a commonly used heat support method (cage on heating pad) with a low-cost custom built warm ambient air cage (WAAC) system. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra-ischemic period (60 min) and post-ischemic period (3 hrs). Neurologic deficit score showed significantly less variance at post-stroke day 1 (PSD1) in WAAC system mice. Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54% lower in the WAAC system group. In summary, we provide a simple low-cost heat support system that provides superior Tcore management in mice during the intra-ischemic and post-ischemic periods, which results in reduced variability of experimental outcomes.HighlightsWe describe the fabrication of a low-cost mouse cage warming system (warmed ambient air cage; WAAC system) that can be assembled and applied in any stroke laboratory.The WAAC system provides more precise control of post-stroke mouse body temperature compared with traditional heating pad warming system.The more precise control of post-stroke core temperature reduces variability in some experimental measures in more severely injured mice.

Background: CD146 is a tight junction associated molecule involved in maintaining endothelial barrier and balancing immune-inflammation response in cardiovascular disease. Notably, the peripheral CD146+ cells significantly upsurge under vessel dyshomeostasis like acute myocardial injury (AMI), appearing to be promising therapeutic targets. In this study, in a new view of gene correlation, we aim at deciphering the underlying complex mechanism of CD146+ cells in the development of AMI. Methods: Transcription dataset GSE 66360 of CD146+ blood cells from clinical subjects were downloaded from NCBI. Pearson networks were constructed and the clustering coefficients were calculated to disclose the differential connectivity genes (DCGs). Analysis of gene connectivity and gene expression was performed to reveal the hub genes and hub genes clusters followed by gene enrichment analysis. Results and Conclusions: Among the total 23520 genes, 27 genes out of 126 differential expression genes are identified as DCGs. Those DCGs normally stay in the peripheral of networks while transfer to the functional central position under AMI situation. Moreover, it is revealed that DCGs spontaneously crowd together into two functional models, CCL20 cluster and NR4A3 cluster, influencing the CD146-mediated signaling pathways during the pathology of AMI for the first time.

Background and Purpose: Delayed neurological deficits are a devastating consequence of subarachnoid hemorrhage (SAH), which affects about 30% of surviving patients. Although a very serious concern, delayed deficits are understudied in experimental SAH models; it is not known whether rodents recapitulate the delayed clinical decline seen in SAH patients. We hypothesized that mice with SAH develop delayed functional deficits and that microthrombi and infarction correlate with delayed decline. Methods: Adult C57BL/6J mice of both sexes were subjected to endovascular perforation to induce SAH. Mice were allowed to survive for up to 1 week post-ictus and behavioral performance was assessed daily. Postmortem microthrombi, large artery diameters (to assess vasospasm), and infarct volume were measured. These measures were analyzed for differences between SAH mice that developed delayed deficits and SAH mice that did not get delayed deficits. Correlation analyses were performed to identify which measures correlated with delayed neurological deficits, sex, and infarction. Results: Twenty-three percent of males and 47% of females developed delayed deficits 3 to 6 days post-SAH. Female mice subjected to SAH had a significantly higher incidence of delayed deficits than male mice with SAH. Mice that developed delayed deficits had significantly more microthrombi and larger infarct volumes than SAH mice that did not get delayed deficits. Microthrombi positively correlated with infarct volume, and both microthrombi and infarction correlated with delayed functional deficits. Vasospasm did not correlate with either infarction delayed functional deficits. Conclusions: We discovered that delayed functional deficits occur in mice following SAH. Sex differences were seen in the prevalence of delayed deficits. The mechanism by which microthrombi cause delayed deficits may be via formation of infarcts.

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.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Recent advances in three-dimensional (3D) fluorescence microscopy offer the ability to image the entire vascular network in entire organs, or even whole animals. However, these imaging modalities rely on either endogenous fluorescent reporters or involved immunohistochemistry protocols, as well as optical clearing of the tissue and refractive index matching. Conversely, X-ray-based 3D imaging modalities, such as micro CT, can image non-transparent samples, at high resolution, without requiring complicated or expensive immunolabeling and clearing protocols, or fluorescent reporters. Here, we compared two "homemade" barium-based contrast agents to the field standard, lead-containing Microfil, for micro-computed tomography (micro CT) imaging of the adult mouse cerebrovasculature. The perfusion pressure required for uniform vessel filling was significantly lower with the barium-based contrast agents compared to the polymer-based Microfil. Accordingly, the barium agents showed no evidence of vascular distension or rupture, common problems associated with Microfil. Compellingly, perfusion of an aqueous BaCl2 /gelatin mixture yielded equal or superior visualization of the cerebrovasculature by micro CT compared to Microfil. However, phosphate-containing buffers and fixatives were incompatible with BaCl2 due to the formation of unwanted precipitates. X-ray attenuation of the vessels also decreased overtime, as the BaCl2 appeared to gradually diffuse into surrounding tissues. A second, unique formulation composed of BaSO4 microparticles, generated in-house by mixing BaCl2 and MgSO4 , suffered none of these drawbacks. These microparticles, however, were unable to pass small diameter capillary vessels, conveniently labeling only the arterial cerebrovasculature. In summary, we present an affordable, robust, low pressure, non-toxic, and straightforward methodology for 3D visualization of the cerebrovasculature.

Neonatal hypoxia&ndash, ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase &beta, (CaMKK &beta, ) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK &beta, in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK &beta, knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK &beta, KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood&ndash, brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK &beta, inhibition. In conclusion, inhibition of CaMKK &beta, exacerbated neonatal hypoxia&ndash, ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic&ndash, ischemic brain injury.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Fil: Tomilin, Viktor N. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Mamenko, Mykola. Augusta University. Medical College of Georgia. Department of Physiology; Estados Unidos Fil: Zaika, Oleg. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Ren, Guohui. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Fil: Marrelli, Sean P. University of Texas. Health Science Center at Houston. Department of Neurology; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Pochynyuk, Oleh. University of Texas. Health Science Center at Houston. Department of Integrative Biology and Pharmacology; Estados Unidos Abstract: It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.

The conducted vasomotor response reflects electrical communication in the arterial wall and the distance signals spread is regulated by three factors including resident ion channels. This study defined the role of inward-rectifying K+channels (KIR) in governing electrical communication along hamster cerebral arteries. Focal KCl application induced a vasoconstriction that conducted robustly, indicative of electrical communication among cells. Inhibiting dominant K+conductances had no attenuating effect, the exception being Ba2+blockade of KIR. Electrophysiology and Q-PCR analysis of smooth muscle cells revealed a Ba2+-sensitive KIRcurrent comprised of KIR2.1/2.2 subunits. This current was surprisingly small and when incorporated into a model, failed to account for the observed changes in conduction. We theorized a second population of KIRchannels exist and consistent with this idea, a robust Ba2+-sensitive KIR2.1/2.2 current was observed in endothelial cells. When both KIRcurrents were incorporated into, and then inhibited in our model, conduction decay was substantive, aligning with experiments. Enhanced decay was ascribed to the rightward shift in membrane potential and the increased feedback arising from voltage-dependent-K+channels. In summary, this study shows that two KIRpopulations work collaboratively to govern electrical communication and the spread of vasomotor responses along cerebral arteries.

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

The prevalence of cardiovascular disease has increased among middle-aged women in the United States, yet has declined in middle-aged men. In experimental stroke, middle-aged females have larger strokes and greater inflammation than age-matched males or younger females. The mechanism underlying this shift from an “ischemia-protected” to an “ischemia-sensitive” phenotype in aging females is unknown. One potential factor is an age-related increase in systemic factors that induce inflammation. Increased abdominal fat deposition is seen in women during middle age. Adipose tissue plays a key role in obesity-induced systemic inflammation, including increased pro-inflammatory cytokines. We hypothesized that age and sex differences in adipose immune cells promote an augmented pro-inflammatory milieu in middle-aged females driven by a balance shift between pro-inflammatory and anti-inflammatory T cells. Abdominal adipose tissue immune cells from young (3–4 months) and middle-aged (15–16 months) male and female C57BL/6J mice were analyzed by flow cytometry. Plasma triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined with colorimetric assays. Middle-aged mice had higher adipose tissue mass compared to young mice. Lipid profiling showed no sex differences in TG and LDL, but middle-aged females had lower HDL (0.84 ± 0.07 μg/μl) than middle-aged males (1.35 ± 0.06 μg/μl). Flow cytometry data demonstrated an age-associated increase in adipose tissue CD8+ T cells that was augmented by female sex, with middle-aged females having a higher percentage of CD8+ cells (34.4 ± 3.2% of CD3+ T cells) than middle-aged males (24.4 ± 2.2%). This increase in CD8+ T-cell proportion was adipose tissue-specific, as this change was not observed in blood. Middle-aged females had higher numbers of activated (CD69+) CD8+ T cells than males. In addition, female CD8+ T cells produced higher levels of IFN-γ, TNF-α, and granzyme B ex vivo, and females had higher adipose levels of IFN-γ, RANTES and MIP-1β than middle-aged males. In parallel, females had lower levels of regulatory T cells (Tregs), an anti-inflammatory T-cell subtype, compared to age-matched males. In conclusion, middle-aged females have a detrimental combination of elevated pro-inflammatory T cells and decreased anti-inflammatory Tregs in adipose tissue, which may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.

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.

Hypothermia shows promise for stroke neuroprotection, but current cooling strategies cause undesirable side effects that limit their clinical applications. Increasing efforts have focused on pharmacological hypothermia as a treatment option for stroke. Previously, we showed that activation of a thermoregulatory ion channel, transient receptor potential vanilloid 1 (TRPV1), by dihydrocapsaicin (DHC) produces reliable hypothermia. In this study, we investigate the effects of TRPV1-mediated hypothermia by DHC on long-term ischemic stroke injury and functional outcome. Hypothermia initiated at 3.5 hours after stroke significantly reduced primary cortical injury. Interestingly, hypothermia by DHC also significantly reduced secondary thalamic injury, as DHC-treated stroke mice exhibited 53% smaller thalamic lesion size. DHC-treated stroke mice further demonstrated decreased neuronal loss and astrogliosis in the thalamus and less thalamic fiber loss by diffusion tensor imaging (DTI). Importantly, a single 8 hour treatment of hypothermia by DHC after stroke provided long-term improvement in functional outcome, as DHC-treated mice exhibited improved behavioral recovery at one month post-stroke. These findings indicate that TRPV1-mediated hypothermia is effective in reducing both primary cortical injury and remote secondary thalamic injury, and a single treatment can produce persistent effects on functional recovery. These data highlight the therapeutic potential for TRPV1 agonism for stroke treatment.

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.

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ∼30% in MCAs from rats undergoing apneas compared with those from a sham control group ( P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

Background/Aims: Canonical transient receptor potential (TRPC) channels modulate membrane potential and intracellular Ca2+. We examined the role of TRPC1 and TRPC3 channels in vasocontraction and relaxation in mouse aorta. Methods: Vasocontraction and relaxation of aorta from wild-type (WT), TRPC1 KO and TRPC3 knockout (KO) mice were measured for phenylephrine (Phe) and carbachol (CCh). Intracellular Ca2+ was measured in primary aorta endothelial cells (EC) and whole cell K+ current in freshly isolated smooth muscle cells (SMC). Results and Conclusion: TRPC1 KO aorta showed increased vasocontraction to Phe compared to WT and TRPC3 KO aorta due to diminished role of BKCa channels. BKCa mRNA (aorta) and whole cell current (SMC) were reduced versus WT. Contraction in WT aorta was increased to TRPC1 KO level by BKCa channel inhibition. Relaxation to CCh was reduced in TRPC1 KO and TRPC3 KO aortas with concomitant reduction in EC Ca2+ response. Pyr3 (TRPC3 blocker) reduced the Ca2+ response to CCh in EC from WT, but not TRPC3 KO mice. In summary, TRPC1 attenuates receptor-mediated contraction through activation and/or expression of SMC BKCa channels while TRPC3 does not contribute to receptor-mediated constriction. Both TRPC1 and TRPC3 participate in EC Ca2+ influx and vasorelaxation of aorta.