Stroke In Male Mice Research Articles

Differences in Acute Expression of Matrix Metalloproteinases-9, 3, and 2 Related to the Duration of Brain Ischemia and Tissue Plasminogen Activator Treatment in Experimental Stroke.

Matrix metalloproteinases (MMPs) such as MMP-9, 3, and 2 degrade the cellular matrix and are believed to play a crucial role in ischemic stroke. We examined how the duration of ischemia (up to 4 h) and treatment with recombinant tissue plasminogen activator altered the comparative expression of these MMPs in experimental ischemic stroke with reperfusion. Both prolonged ischemia and r-tPA treatment markedly increased MMP-9 expression in the ischemic hemisphere (all p < 0.0001). The duration of ischemia and r-tPA treatment also significantly increased MMP-2 expression (p < 0.01-0.001) in the ischemic hemisphere (p < 0.01) but to a lesser degree than MMP-9. In contrast, MMP-3 expression significantly decreased in the ischemic hemisphere (p < 0.001) with increasing duration of ischemia and r-tPA treatment (p < 0.05-0001). MMP-9 expression was prominent in the vascular compartment and leukocytes. MMP-2 expression was evident in the vascular compartment and MMP-3 in NeuN+ neurons. Prolonging the duration of ischemia (up to 4 h) before reperfusion increased brain hemorrhage, infarction, swelling, and neurologic disability in both saline-treated (control) and r-tPA-treated mice. MMP-9 and MMP-2 expression were significantly positively correlated with, and MMP-3 was significantly negatively correlated with, infarct volume, swelling, and brain hemorrhage. We conclude that in experimental ischemic stroke with reperfusion, the duration of ischemia and r-tPA treatment significantly altered MMP-9, 3, and 2 expression, ischemic brain injury, and neurological disability. Each MMP showed unique patterns of expression that are strongly correlated with the severity of brain infarction, swelling, and hemorrhage. In summary, in experimental ischemic stroke in male mice with reperfusion, the duration of ischemia, and r-tPA treatment significantly altered the immunofluorescent expression of MMP-9, 3, and 2, ischemic brain injury, and neurological disability. In this model, each MMP showed unique patterns of expression that were strongly correlated with the severity of brain infarction, swelling, and hemorrhage.

Integrating single-cell and spatially resolved transcriptomic strategies to survey the astrocyte response to stroke in male mice

Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO (tissue-digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5. Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.

Amino acid solution mitigates hypothermia response and intestinal damage following exertional heat stroke in male mice.

Increased gut permeability is implicated in the initiation and extent of the cytokine inflammatory response associated with exertional heat stroke (EHS). The primary objective of this study was to determine if a five amino acid oral rehydration solution (5AAS), specifically designed for the protection of the gastrointestinal lining, would prolong time to EHS, maintain gut function and dampen the systemic inflammatory response (SIR) measured during EHS recovery. Male C57/BL6J mice instrumented with radiotelemetry were gavaged with 150 μL of 5AAS or H2 O, and ≈12 h later were either exposed to an EHS protocol where mice exercised in a 37.5°C environmental chamber to a self-limiting maximum core temperature (Tc,max) or performed the exercise control (EXC) protocol (25°C). 5AAS pretreatment attenuated hypothermia depth and length (p < 0.005), which are indicators of EHS severity during recovery, without any effect on physical performance or thermoregulatory responses in the heat as determined by percent body weight lost (≈9%), max speed (≈6 m/min), distance (≈700 m), time to Tc,max (≈160 min), thermal area (≈550°C∙min), and Tc,max (42.2°C). EHS groups treated with 5AAS showed a significant decrease in gut transepithelial conductance, decreased paracellular permeability, increased villus height, increased electrolyte absorption and changes in tight junction protein expression pattern suggestive of improved barrier integrity (p < 0.05). No differences were witnessed between EHS groups in acute phase response markers of liver, circulating SIR markers, or indicators of organ damage during recovery. These results suggest that a 5AAS improves Tc regulation during EHS recovery through maintaining mucosal function and integrity.

Vascular perfusion differs in two distinct PDGFRβ-positive zones within the ischemic core of male mice 2 weeks following photothrombotic stroke.

Stroke therapy has largely focused on preventing damage and encouraging repair outside the ischemic core, as the core is considered irreparable. Recently, several studies have suggested endogenous responses within the core are important for limiting the spread of damage and enhancing recovery, but the role of blood flow and capillary pericytes in this process is unknown. Using the Rose Bengal photothrombotic model of stroke, we illustrate blood vessels are present in the ischemic core and peri-lesional regions 2 weeks post stroke in male mice. A FITC-albumin gel cast of the vasculature revealed perfusion of these vessels, suggesting cerebral blood flow (CBF) may be partially present, without vascular leakage. The length of these vessels is significantly reduced compared to uninjured regions, but the average width is greater, suggesting they are either larger vessels that survived the initial injury, smaller vessels that have expanded in size (i.e., arteriogenesis), or that neovascularization begins with larger vessels. Concurrently, we observed an increase in platelet-derived growth factor receptor beta (PDGFRβ, a marker of pericytes) expression within the ischemic core in two distinct patterns, one which resembles pericyte-derived fibrotic scarring at the edge of the core, and one which is vessel associated and may represent blood vessel recovery. We find little evidence for dividing cells on these intralesional blood vessels 2 weeks post stroke. Our study provides evidence flow is present in PDGFRβ-positive vessels in the ischemic core 2 weeks post stroke. We hypothesize intralesional CBF is important for limiting injury and for encouraging endogenous repair following cerebral ischemia.

A New Model of Exertional Heat Stroke in Male Mice Reveals Sustained Metabolic Dysfunction in the Myocardium

Exertional heat stroke (EHS) is a life threating condition that has grown in prevalence with rising global temperatures. Development and use of new preclinical models are necessary to understand the pathophysiology. In our previous model of EHS, a metabolic disorder occurred in the ventricular myocardium (VM) after 14 days of recovery only in female mice. However, females were able to achieve much higher levels of exercise than males. In this study we developed a new model, specific for male mice, that prolonged exercise time and intensity but at a lower environmental temperature (Te). We then tested whether this approach would induce similar metabolic disorders of the VM in male hearts that was seen previously in females.MethodsFourteen C57BL\6 male mice were exposed to EHS by running on a forced running wheel in an environmental chamber until symptom limitation (loss of consciousness). The new model reduced Te to 34.5°C compared to 37.5°C in the original EHS model. Another group of mice was exercise‐matched to the EHS mice, but at a Te of 22.5°C. After symptom limitation was achieved, Te was kept at 37.5°C for 2 hours, post EHS. After 2 weeks of recovery from EHS, the mice were sacrificed and ventricles rapidly removed and stored at ‐80°C. The tissues were later submitted for untargeted metabolomic analysis (Metabolome, Inc. Raleigh, NC).ResultsWe found that the average max temp. achieved was 41.4±0.4°C, which is ~1°C cooler than the original EHS model. The mice lost an average of 6.7±2.1%BW during EHS. The ascending thermal load they achieved was only 54.7±41.2 °C•min, approximately ~1/3 of the original model. Within 804 metabolites identified from the VM, 113 were significantly (P &lt; 0.05) elevated; whereas, 15 were significantly reduced. Pathway categories of prominently and significantly changed metabolites included the: 1) pentose phosphate:­ribose 1‐P, ­ribonate; 2) glycolysis:­glucose, ­pyruvate, ­fructose, ­fructose‐6‐phosphate, decreased 3‐phosphoglycerate; 3) TCA cycle: ­isocitrate, ­itaconate, ­succylcarnitine, ­succinate; 4) methionine metabolism:­S‐methylmethionine, ­N‐formylmethionine, ­S‐adenosylmethionine, ­4 MeTHF; 5) antioxidant defense: ­glutathione, ­cysteine, ­cystathionine, ­ascorbate;6) fat metabolism: ­variety of long and med chain fatty acids, ­carnitine, ­deoxycarnitine, decreases in a variety of acylcarnitines, and 6) injury, inflammation and stress:­corticosterone, ­kynurenine, ­triethylamine N‐oxide, ­N‐methylproline, and ­AICA.ConclusionsBy increasing the time and intensity of exercise in the heat, even at a much lower Te, male mice exhibited strikingly similar metabolic disorders in the myocardium, after 2 weeks of recovery, that were previously reported in female mice only. These results suggest that it is not the actual thermal load or the max temperature that is critical, but rather the “exertional” load or time of exercise exposure in a warm environment that is the trigger for inducing profound metabolic disorders in the VM.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice

ObjectiveStroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery. The aim of this study was to determine if the delayed secondary diapedesis of CD8 T cells into the ischemic brain negatively impacts functional recovery after transient ischemic stroke in male mice. ResultsMice exhibited an increased number of leukocytes in the ipsilesional hemispheres at 14 days (3-fold; p < 0.001) and 30 days (2.2-fold; p = 0.02) after transient middle cerebral artery occlusion (tMCAo) compared to 8 days post-tMCAo, at which time acute neuroinflammation predominantly resolves. Moreover, mice with higher ipsilesional CD8 T cells at 30 days (R2 = 0.52, p < 0.01) exhibited worse functional recovery. To confirm a detrimental role of chronic CD8 T cell diapedesis on recovery, peripheral CD8 T cells were depleted beginning 10 days post-tMCAo. Delayed CD8 T cell depletion improved motor recovery on the rotarod (F(1,28) = 4.264; p = 0.048) compared to isotype control-treated mice. CD8 T cell-depleted mice also exhibited 2-fold (p < 0.001) reduced leukocyte infiltration at 30 days post-tMCAo. Specifically, macrophage, neutrophil, and CD4 T cell numbers were reduced in the ipsilesional hemisphere of the CD8 T cell-depleted mice independent of inflammatory status of the post-stroke CNS (e.g. microglial phenotype and cytokine production). RNAseq identified a unique profile for brain infiltrating CD8 T cells at 30 days post-tMCAo, with 46 genes differentially expressed relative to CD8 T cells at 3 days post-tMCAo. ConclusionOur data reveal a role for CD8 T cells in the chronic phase post-stroke that can be therapeutically targeted. We demonstrate long-term CD8 T cell recruitment into the ipsilesional hemisphere that affects both immune cell numbers present in the injured brain and functional recovery through one month after stroke onset.

The Effect of Histone Deacetylase Inhibitors Panobinostat or Entinostat on Motor Recovery in Mice After Ischemic Stroke.

Using rigorous and clinically relevant experimental design and analysis standards, in this study, we investigated the potential of histone deacetylase (HDAC) inhibitors panobinostat and entinostat to enhance recovery of motor function after photothrombotic stroke in male mice. Panobinostat, a pan-HDAC inhibitor, is a FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor in late stage of clinical investigation. The drugs were administered every other day (panobinostat-3 or 10mg/kg; entinostat-1.7 or 5mg/kg) starting from day 5 to 15 after stroke. To imitate the current standard of care in stroke survivors, i.e., physical rehabilitation, the animals run on wheels (2h daily) from post-stroke day 9 to 41. The predetermined primary end point was motor recovery measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. In addition, we evaluated the running distance and speed throughout the study, and the number of parvalbumin-positive neurons in medial agranular cortex (AGm) and infarct volumes at the end of the study. Both sensorimotor tests revealed that combination of physical exercise with either drug did not substantially affect motor recovery in mice after stroke. This was accompanied by negligible changes of parvalbumin-positive neurons recorded in AGm and comparable infarct volumes among experimental groups, while dose-dependent increase in acetylated histone 3 was observed in peri-infarct cortex of drug-treated animals. Our observations suggest that add-on panobinostat or entinostat therapy coupled with limited physical rehabilitation is unlikely to offer therapeutic modality for stroke survivors who have motor dysfunction.

Increased brain plasmin levels following experimental ischemic stroke in male mice.

Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24hr following stroke in comparison to healthy mice (F(3, 72)=39.5, p<0.0001, repeated measures ANOVA) after the induction of permanent middle cerebral artery occlusion (PMCAo). Plasmin activity was higher in the ischemic hemisphere (F(1,36)=9.1, p=0.005) and there was a significant interaction between time and ischemic hemisphere (F(3,36)=4.4, p=0.009). Plasmin activity was correlated with infarct volume (R2 =0.5289, p=0.0009 by Spearman). The specificity of the assay was verified utilizing tissue-type plasminogen activator (tPA)-deficient mice which, as expected, had significantly lower levels of plasmin 24hr following ischemia compared to wild-type mice (ischemic (0.6±0.23 and 1.94±0.5, respectively), p=0.049 and contralateral hemispheres (0.13±0.14 and 0.75±0.10, respectively), p=0.018 by t test). There is a time-dependent increase in plasmin levels and an association of higher levels of plasmin with larger infarct volumes in an experimental stroke model. This suggests caution in the use of recombinant tPA (rtPA) and that plasmin inhibition in the brain may be a therapeutic target in acute ischemic stroke.

Methylenetetrahydrofolate reductase deficiency alters cellular response after ischemic stroke in male mice

ABSTRACT Objective: Elevated homocysteine concentrations are a risk factor for stroke. A common genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR 677 C→T) results in elevated levels of homocysteine. MTHFR plays a critical role in the synthesis of S-adenosylmethionine (SAM), a global methyl donor. Our previous work has demonstrated that Mthfr +/- mice, which model the MTHFR polymorphism in humans, are more vulnerable to ischemic damage. The aim of this study was to investigate the cellular mechanisms by which the MTHFR-deficiency changes the brain in the context of ischemic stroke injury. Methods: In the present study, three-month-old male Mthfr +/- and wild-type littermate mice were subjected to photothrombosis (PT) damage. Four weeks after PT damage, animals were tested on behavioral tasks, in vivo imaging was performed using T2-weighted MRI, and brain tissue was collected for histological analysis. Results: Mthfr +/- animals used their non-impaired forepaw more to explore the cylinder and had a larger damage volume compared to wild-type littermates. In brain tissue of Mthfr +/- mice methionine adenosyltransferase II alpha (MAT2A) protein levels were decreased within the damage hemisphere and increased levels in hypoxia-induced factor 1 alpha (HIF-1α) in non-damage hemisphere. There was an increased antioxidant response in the damage site as indicated by higher levels of nuclear factor erythroid 2-related factor 2 (Nrf2) in neurons and astrocytes and neuronal superoxide dismutase 2 (SOD2) levels. Conclusions: Our results suggest that Mthfr +/- mice are more vulnerable to PT-induced stroke damage through the regulation of the cellular response. The increased antioxidant response we observed may be compensatory to the damage amount.

Dose-dependent and long-term cerebroprotective effects of intranasal delivery of progesterone after ischemic stroke in male mice

Intranasal administration is emerging as a very promising route to deliver therapeutics to the brain. We have recently shown that the intranasal delivery of progesterone at 8 mg/kg is neuroprotective after stroke in male mice. To explore the translational potential of intranasal progesterone treatment, we performed a dose-response study and analyzed outcomes at 48 h after middle cerebral artery occlusion (MCAO). The effects on functional outcomes at long-term were examined by using the optimal dose. In the first experiment, male C57BL/6JRj mice were treated with progesterone at 8, 16 or 24 mg/kg, or with placebo at 1, 6 and 24 h post-MCAO. Our results show that the dose of 8 mg/kg was optimal in counteracting the early histopathological impairments as well as in improving functional recovery. Steroid profiling in plasma showed that the dose of 8 mg/kg is the one that leads to sustained high levels of progesterone and its neuroactive metabolites. In the second experiment, the dose of 8 mg/kg was used and analyzes were performed at 2, 7 and 21 days post-MCAO. Progesterone increased survival, glycemia and body weight. Furthermore, progesterone decreased neurological deficits and improved performances of mice on the rotarod and pole as early as 2 days and up to 21 days post-MCAO. These findings show that intranasal administration of progesterone has a significant translational potential as a cerebroprotective treatment after stroke that can be effective to reduce mortality, to limit tissue and cell damage at the acute phase; and to confer a long-term functional recovery.

Parenchymal pericytes are not the major contributor of extracellular matrix in the fibrotic scar after stroke in male mice.

Scar formation after injury of the brain or spinal cord is a common event. While glial scar formation by astrocytes has been extensively studied, much less is known about the fibrotic scar, in particular after stroke. Platelet‐derived growth factor receptor ß‐expressing (PDGFRß+) pericytes have been suggested as a source of the fibrotic scar depositing fibrous extracellular matrix (ECM) proteins after detaching from the vessel wall. However, to what extent these parenchymal PDGFRß+ cells contribute to the fibrotic scar and whether targeting these cells affects fibrotic scar formation in stroke is still unclear. Here, we utilize male transgenic mice that after a permanent middle cerebral artery occlusion stroke model have a shift from a parenchymal to a perivascular location of PDGFRß+ cells due to the loss of regulator of G‐protein signaling 5 in pericytes. We find that only a small fraction of parenchymal PDGFRß+ cells co‐label with type I collagen and fibronectin. Consequently, a reduction in parenchymal PDGFRß+ cells by ca. 50% did not affect the overall type I collagen or fibronectin deposition after stroke. The redistribution of PDGFRß+ cells to a perivascular location, however, resulted in a reduced thickening of the vascular basement membrane and changed the temporal dynamics of glial scar maturation after stroke. We demonstrate that parenchymal PDGFRß+ cells are not the main contributor to the fibrotic ECM, and therefore targeting these cells might not impact on fibrotic scar formation after stroke.

Peptidase neurolysin functions to preserve the brain after ischemic stroke in male mice.

Previous studies documented up-regulation of peptidase neurolysin (Nln) after brain ischemia, however, the significance of Nln function in the post-stroke brain remained unknown. The aim of this study was to assess the functional role of Nln in the brain after ischemic stroke. Administration of a specific Nln inhibitor Agaricoglyceride A (AgaA) to mice after stroke in a middle cerebral artery occlusion model, dose-dependently aggravated injury measured by increased infarct and edema volumes, blood-brain barrier disruption, increased levels of interleukin 6 and monocyte chemoattractant protein-1, neurological and motor deficit 24h after stroke. In this setting, AgaA resulted in inhibition of Nln in the ischemic hemisphere leading to increased levels of Nln substrates bradykinin, neurotensin, and substance P. AgaA lacked effects on several physiological parameters and appeared non-toxic to mice. In a reverse approach, we developed an adeno-associated viral vector (AAV2/5-CAG-Nln) to overexpress Nln in the mouse brain. Applicability of AAV2/5-CAG-Nln to transduce catalytically active Nln was confirmed in primary neurons and in vivo. Over-expression of Nln in the mouse brain was also accompanied by decreased levels of its substrates. Two weeks after in vivo transduction of Nln using the AAV vector, mice were subjected to middle cerebral artery occlusion and the same outcome measures were evaluated 72h later. These experiments revealed that abundance of Nln in the brain protects animals from stroke. This study is the first to document functional significance of Nln in pathophysiology of stroke and provide evidence that Nln is an endogenous mechanism functioning to preserve the brain from ischemic injury.

Abstract WMP83: cGMP-Dependent Protein Kinase I in Smooth Muscle Cells Protects Against Stroke in Female Mice

Introduction: The role of cGMP-dependent protein kinase I (cGKI) in regulating vascular smooth muscle cells (SMC) during stroke has not been completely clarified. We showed that SMC cGKI has protective effect against stroke in male mice. To elucidate whether cGKI in SMC renders its protective effect on stroke outcome by sex-dependent mechanism, we conducted study on female mice. Animals: We used a novel tamoxifen-inducible SMC-specific gene knockout mice, that expresses CreER T2 recombinase under the control of smooth muscle alpha-actin promoter. SMA-CreER T2 _cGKIfloxflox adult 12-14 weeks-old female mice after tamoxifen (TAM) administration (100 μl TAM solution (1 mg/ml) i.p. per day for 5 consecutive days at the age 5-6 weeks) were used as experimental animals (cGKI SMKO mice). Control animals consisted of three types of female mice: (1) C57BL/6 (WT) mice without TAM treatment; (2) SMA-CreER T2 _cGKIfloxflox mice without TAM treatment (cGKI control); (3) cGKIfloxflox mice (without SMA-CreER T2 transgene) treated with TAM (cGKI control TAM). Methods: Mice (n=12/group) were anesthetized (1.5% of isoflurane in 30% of O 2 and 70% of N 2 O), body temperature was maintained (36.5-37.0 0 C) during 30-min filament-mediated middle cerebral artery occlusion (MCAO) and 30 min of reperfusion. Cerebral laser-Doppler flowmetry was utilized during the surgery to evaluate the regional cerebral blood flow (CBF). Results: The cGKI SMKO mice revealed greater cerebral infarct volume (93±12 mm 3 , Mean±SD) than WT (64±14), cGKI control (62±14), and cGKI control TAM (68±10) mice (TTC staining, indirect calculation, p&lt;0.001, one-way analysis of variance with Bonferroni correction). Neurological deficit by 4-point scale was worse in the cGKI SMKO mice (2.3 points) as compared with WT (1.5), cGKI control (1.2), and cGKI control TAM (1.3) mice (p&lt;0.05). The CBF during reperfusion was less in cGKI SMKO mice (44±19%) than in control groups (70±16 in WT; 81±33 in cGKI control; 70±19 in cGKI control TAM (15-th minute of reperfusion), p&lt; 0.05). In conclusion, cGKI in SMC protects against stroke injury in female mice in a similar way that we previously detected in male mice. These results suggest that cGKI in SMC should be targeted in studies designed to protect brain tissue against stroke.

Claudin-1-Dependent Destabilization of the Blood-Brain Barrier in Chronic Stroke.

Recent evidence suggests that blood-brain barrier (BBB) recovery and reestablishment of BBB impermeability after stroke is incomplete. This could influence stroke recovery, increase the risk of repeat stroke, and be a solid substrate for developing vascular dementia. Although accumulating evidence has defined morphological alterations and underlying mechanisms of tight junction (TJ) changes during BBB breakdown in acute stroke, very little is known about the type of alterations and mechanisms in BBB "leakage" found subacutely or chronically. The current study examined BBB structural alterations during the "BBB leakage" associated with the chronic phase of stroke in male mice and both genders of humans. We found significant upregulation of claudin-1 mRNA and protein, a nonspecific claudin for blood vessels, and downregulation in claudin-5 expression. Morphological and biochemical as well as fluorescence resonance energy transfer and fluorescence recovery after photobleaching analysis of postischemic brain endothelial cells and cells overexpressing claudin-1 indicated that newly synthesized claudin-1 was present on the cell membrane (∼45%), was incorporated into the TJ complex with established interaction with zonula occludens-1 (ZO-1), and was building homophilic cis- and trans-interactions. The appearance of claudin-1 in the TJ complex reduced claudin-5 strands (homophilic claudin-5 cis- and trans-interactions) and claudin-5/ZO-1 interaction affecting claudin-5 incorporation into the TJ complex. Moreover, claudin-1 induction was associated with an endothelial proinflammatory phenotype. Targeting claudin-1 with a specific C1C2 peptide improved brain endothelial barrier permeability and functional recovery in chronic stroke condition. This study highlights a potential "defect" in postischemic barrier formation that may underlie prolonged vessel leakiness.SIGNIFICANCE STATEMENT Although rarely expressed at the normal blood-brain barrier (BBB), claudin-1 is expressed in pathological conditions. Analyzing poststroke human and mouse blood microvessels we have identified that claudin-1 is highly expressed in leaky brain microvessels. Our results reveal that claudin-1 is incorporated in BBB tight junction complex, impeding BBB recovery and causing BBB leakiness during poststroke recovery. Targeting claudin-1 with a claudin-1 peptide improves brain endothelial barrier permeability and consequently functional neurological recovery after stroke.

Abstract WP254: Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase (CaMKK) Exacerbates Endothelial Cell Impairment After Cerebral Ischemia

CaMKK (α and β) is a major kinase activated by elevated intracellular calcium. We have previously demonstrated CaMKK is neuroprotective in stroke. Pharmacological inhibition of CaMKK exacerbated outcome while overexpression reduced brain injury in mice. Brain microvascular endothelial cells play an essential role in maintaining BBB integrity, disruption of which aggravates ischemic injury. Interestingly, CaMKK activates a key endothelial protectant Sirtuin1 (SIRT1), although this interaction has never been studied in stroke. We hypothesized that CaMKK protects brain endothelial cells (EC) through activation of SIRT1 after ischemia. Oxygen-glucose deprivation (OGD, 18 hours) with reoxygenation (24 hours) was performed in human brain microvascular endothelial cells (HBEC-5i). Cell viability was assessed using a CCK-8 assay. Middle cerebral artery occlusion (MCAO, 90 mins) was used to induce stroke in male mice (20 to 25 g). The CaMKK inhibitor STO-609, SIRT 1 inhibitor EX-527, or CaMKK β siRNA was added to EC before OGD. Data are presented as mean±SEM. STO-609 treatment reduced cell viability (%) after OGD (vehicle 71.45 ± 5.60 vs. STO-609 55.06 ± 5.02, n=4, p&lt;0.05). Knockdown of CaMKK β using siRNA increased cell death following OGD. Mechanistically, inhibition of CaMKK by STO-609 led to significant down-regulation of phosphorated SIRT1 (Ser-27) after OGD. Levels of total SIRT1 were also reduced by STO-609, consistent with the notion that phosphorylation prevents the degradation of this protein. Changes in downstream targets (e.g., eNOS) of SIRT1 were accordingly observed with STO-609 treatment. Further, the effect of STO-609 on cell viability after OGD was absent when SIRT1 was concurrently inhibited by EX-527 (STO-609+EX-527 45.19 ± 2.09 vs. EX-527 44.42 ± 2.89, n=4, p&gt;0.05). Finally, in a MCAO model, intracerebroventricular injection of STO-609 (pre-stroke) significantly worsened BBB integrity (Evans blue assay) after 24-hour reperfusion (MCAO+DMSO 17.83 ± 6.34 versus MCAO+STO-609 55.74 ± 8.88, n=5/6; P &lt;0.05). In conclusion, we demonstrate that CaMKK inhibition reduces EC viability after ischemia via SIRT 1. Our data suggest CaMKK attenuates ischemic brain injury by protecting the microvascular system and preserving BBB integrity.

Macro-to-micro cortical vascular imaging underlies regional differences in ischemic brain.

The ability to non-invasively monitor and quantify hemodynamic responses down to the capillary level is important for improved diagnosis, treatment and management of neurovascular disorders, including stroke. We developed an integrated multi-functional imaging system, in which synchronized dual wavelength laser speckle contrast imaging (DWLS) was used as a guiding tool for optical microangiography (OMAG) to test whether detailed vascular responses to experimental stroke in male mice can be evaluated with wide range sensitivity from arteries and veins down to the capillary level. DWLS enabled rapid identification of cerebral blood flow (CBF), prediction of infarct area and hemoglobin oxygenation over the whole mouse brain and was used to guide the OMAG system to hone in on depth information regarding blood volume, blood flow velocity and direction, vascular architecture, vessel diameter and capillary density pertaining to defined regions of CBF in response to ischemia. OMAG-DWLS is a novel imaging platform technology to simultaneously evaluate multiple vascular responses to ischemic injury, which can be useful in improving our understanding of vascular responses under pathologic and physiological conditions, and ultimately facilitating clinical diagnosis, monitoring and therapeutic interventions of neurovascular diseases.

Regulatory CD8(+)CD122 (+) T-cells predominate in CNS after treatment of experimental stroke in male mice with IL-10-secreting B-cells.

Clinical stroke induces inflammatory processes leading to cerebral and splenic injury and profound peripheral immunosuppression. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that transfer of IL-10(+) B-cells reduced infarct volume in male C57BL/6J (wild-type, WT) recipient mice when given 24h prior to or 4h after middle cerebral artery occlusion (MCAO). The purpose of this study was to determine if passively transferred IL-10(+) B-cells can exert therapeutic and immunoregulatory effects when injected 24h after MCAO induction in B-cell-sufficient male WT mice. The results demonstrated that IL-10(+) B-cell treated mice had significantly reduced infarct volumes in the ipsilateral cortex and hemisphere and improved neurological deficits vs. Vehicle-treated control mice after 60min occlusion and 96h of reperfusion. The MCAO-protected B-cell recipient mice had less splenic atrophy and reduced numbers of activated, inflammatory T-cells, decreased infiltration of T-cells and a less inflammatory milieu in the ischemic hemispheres compared with Vehicle-treated control mice. These immunoregulatory changes occurred in concert with the predominant appearance of IL-10-secreting CD8(+)CD122(+) Treg cells in both the spleen and the MCAO-affected brain hemisphere. This study for the first time demonstrates a major neuroprotective role for IL-10(+) B-cells in treating MCAO in male WT mice at a time point well beyond the ~4h tPA treatment window, leading to the generation of a dominant IL-10(+)CD8(+)CD122(+) Treg population associated with spleen preservation and reduced CNS inflammation.