Minutes Of Middle Cerebral Artery Occlusion Research Articles

Abstract TP287: Estrous Cycle Stages and Cerebral Blood Flow: Implications for Ischemic Tolerance in Stroke

Introduction: Previous stroke studies in female rodents have demonstrated that the severity of ischemic damage is differentially impacted by the estrous cycle. Estrogen is considered to be neuroprotective, but it is unclear whether this protection is dependent on the vasodilator properties of estrogen. In this study, we investigated the impact of estrus cycle stage on stroke lesion volume and whether that impact was dependent on cerebral blood flow (CBF) changes during stroke. Methods: Ischemia was induced in adult ( 10 weeks) male and female Sprague-Dawley rats by 30 minutes middle cerebral artery occlusion (MCAO) using the suture model. Prior to the stroke procedure, the phases of the estrous cycle (proestrus(P), estrus(E), metestrus(M) and diestrus (D)) were determined by vaginal smear tests in female rats. Cerebral blood flow (CBF) was measured by laser doppler and infarct volume were measured by TTC staining at d2 post-stroke. Results: Females in proestrus and diestrus (P/D) stages of the estrous cycle, which have reportedly high levels of estrogen, had significantly (p<0.05) smaller CBF decreases during ischemia and a trend for smaller infarcts than those in estrous and metestrus stages, which have lower estrogen levels. This suggests that the protective effects of estrogen may be related to effects on blood flow. However, when compared to male rats, females in P/D stages had significantly (p<0.05) smaller brain infarcts despite having similar CBF decreases. This implies that additional mechanisms are responsible for the neuroprotection observed in females when compared to males. Conclusion: Estrous cycle stage affects CBF decrease during ischemia, but this does not account for the difference in infarct size observed between male and female rats. Our study indicates that multiple mechanisms are responsible for ischemic tolerance in specific stages of the estrous cycle. Keywords: stroke, estrous cycle, CBF, estrogen

Abstract WP233: Histone Lactylation in Neuronal Mitochondria Fission: Implications for Stress-Exacerbated Ischemic Stroke Outcomes

Introduction: Mental stress was linked with adverse outcomes following ischemic stroke. Accumulation of lactate due to mental stress is well-documented, especially in skeletal muscles. Importantly, lactate serves as a key substrate for lactylation, a novel post-translational protein modification, which triggers mitochondrial fission. This study investigates the pivotal role of histone lactylation in driving neuronal mitochondrial fission, which may amplify the detrimental outcomes of stroke under mental stress. Methods: A total of 120 C57BL/6J mice were divided into four groups: control, chronic restraint stress (CRS), lactylationantagonist (C646, 10mg/kg, i.p.) and C646+CRS. CRS for mice was induced in a 50mL tube for 6 hours per day for consecutive 28 days. On the day 29 th , Western blot was applied to detect the expression of fission related proteins (DRP1, MFF and FIS1). The effects of lactylation on mitochondria function was determined through JC-1 and mitoSOX™ staining by adding exogenous lactate (10mM) and C646 (20mM) in neuronal cells. The stressed mice then were subjected to 45 minute-middle-cerebral -artery-occlusion (MCAO). At 6 and 24 hours of reperfusion, apoptotic cell death and apoptotic proteins (Bcl-2, BAX and cleaved caspase-3) were measured by TUNEL and Western blot. Infarct volume and neurological deficits were determined by TTC staining and scoring systems at 48 hours of reperfusion. Results: CRS aggravated ischemic injury and apoptosis after stroke. C646, a lactylation antagonist, significantly decreased protein expressions of DRP1, MFF and FIS1 and reversed CRS-associated stroke injury. Furthermore, mitochondria membrane potential (MMP) was decreased and ROS was increased upon exogenous lactate in neuronal cells, accompanied by increased mitochondria fission. Again, C646 reverses mitochondria dysfunction and fission. Conclusions: Our results shed light on how CRS intensifies the ischemic damage post-stroke. Central mechanism is histone lactylation which triggers neuronal mitochondria fission, thereby impacting the outcomes related to stress-associated stroke. Lactylation antagonists offer promising avenues for therapeutic interventions, mitigating the adverse effects of stress on stroke outcomes.

Abstract TP252: Intranasal Insulin Treatment Improves Stroke Outcome In Both Male And Female Mice

Insulin resistance has been linked to increased risk of stroke recurrence, death, and poor outcome following stroke. Insulin resistance, a common phenomenon following stroke, is associated with elevated levels of insulin circulating in the periphery, which leads to brain insulin deficiency. Brain insulin promotes neuroplasticity and synaptogenesis, has anti-inflammatory, anti-thrombotic, vasodilatory, and anti-apoptotic properties. The impact of brain insulin deficiency and the therapeutic potential of increasing brain insulin levels has yet to be fully understood. Hence, purpose of this study is to assess the potential therapeutic benefits of intranasal insulin treatment on stroke outcomes in a mouse model of brain insulin deficiency. Ischemic stroke was induced using a 30 minute middle cerebral artery occlusion (MCAO) in 16 week old (12 week diet) male and female mice on either a standard diet (control) or a high-fat diet induced at 4 weeks of age, a model of brain insulin deficiency. Recovery assessments begin on day 0 following stroke and continue until day 14 post stroke. Recovery assessments entail observing survival, neurological, motor, and cognitive function after receiving either intranasal saline (0.9%) or intranasal insulin (1.75 U). These assessments were evaluated by survival, and completion of the neurological severity score, ladder, and problem solving tasks on days 0-14 post stroke. High-fat diet-induced brain insulin deficiency was associated with worse survival and neurological deficits following ischemic stroke, intranasal insulin improved stroke mortality and neurological assessments in high-fat diet mice. Thus, intranasal insulin may be a useful therapy for improving stroke outcomes.

Neuroprotective Effects of Normobaric Hyperoxia and Transplantation of Encapsulated Choroid Plexus Epithelial Cells on The Focal Brain Ischemia.

Objective Choroid plexus epithelial cells (CPECs) have the epithelial characteristic, produce cerebrospinal fluid, contribute to the detoxification process in the central nervous system (CNS), and are responsible for the synthesis and release of many nerve growth factors. On the other hand, studies suggest that normobaric hyperoxia (HO) by induction of ischemic tolerance (IT) can protect against brain damage and neurological diseases. We examined the effect of combination therapy of encapsulated CPECs and HO to protect against ischemic brain injury. Materials and MethodsIn this experimental study, six groups of adult male Wistar rats were randomly organized: sham, room air (RA)+middle cerebral artery occlusion (MCAO), HO+MCAO, RA+MCAO+encapsulated CPECs, HO+MCAO+encapsulated CPECs, RA+MCAO+empty capsules. RA/HO were pretreatment. The CPECs were isolated from the brain of neonatal Wistar rats, cultured, and encapsulated. Then microencapsulated CPECs were transplanted in the neck of the animal immediately after the onset of reperfusion in adult rats that had been exposed to 60 minutes MCAO. After 23 hours of reperfusion, the neurologic deficit score (NDS) was assessed. Next, rats were killed, and brains were isolated for measuring brain infarction volume, blood-brain barrier (BBB) permeability, edema, the activity of superoxide dismutase (SOD), and catalase (CAT) and also, the level of malondialdehyde (MDA).Results Our results showed that NDS decreased equally in HO+MCAO, RA+MCAO+encapsulated CPECs, and HO+MCAO+encapsulated CPECs groups. Brain infarction volume decreased up 79%, BBB stability increased, edema decreased, SOD and CAT activities increased, and MDA decreased in the combination group of HO and transplantation of encapsulated CPECs in the ischemic brain as compared with when HO or transplantation of encapsulated CPECs was applied alone. ConclusionThe combination of HO and transplantation of encapsulated CPECs for stroke in rats was more effective than the other treatments, and it can be taken into account as a promising treatment for ischemic stroke.

Aflibercept, a VEGF (Vascular Endothelial Growth Factor)-Trap, Reduces Vascular Permeability and Stroke-Induced Brain Swelling in Obese Mice.

Brain edema is an important underlying pathology in acute stroke, especially when comorbidities are present. VEGF (Vascular endothelial growth factor) signaling is implicated in edema. This study investigated whether obesity impacts VEGF signaling and brain edema, as well as whether VEGF inhibition alters stroke outcome in obese subjects. High-fat diet-induced obese mice were subjected to a transient middle cerebral artery occlusion. VEGF-A and VEGFR2 (receptor) expression, infarct volume, and swelling were measured 3 days post-middle cerebral artery occlusion. To validate the effect of an anti-VEGF strategy, we used aflibercept, a fusion protein that has a VEGF-binding domain and acts as a decoy receptor, in human umbilical vein endothelial cells stimulated with rVEGF (recombinant VEGF; 50 ng/mL) for permeability and tube formation. In vivo, aflibercept (10 mg/kg) or IgG control was administered in obese mice 3 hours after transient 30 minutes middle cerebral artery occlusion. Blood-brain barrier integrity was assessed by IgG staining and dextran extravasation in the postischemic brain. A separate cohort of nonobese (lean) mice was subjected to 40 minutes middle cerebral artery occlusion to test the effect of aflibercept on malignant infarction. Compared with lean mice, obese mice had increased mortality, infarct volume, swelling, and blood-brain barrier disruption. These outcomes were also associated with increased VEGF-A and VEGFR2 expression. Aflibercept reduced VEGF-A-stimulated permeability and tube formation in human umbilical vein endothelial cells. Compared with the IgG-treated controls, mice treated with aflibercept had reduced mortality rates (40% versus 17%), hemorrhagic transformation (43% versus 27%), and brain swelling (28% versus 18%), although the infarct size was similar. In nonobese mice with large stroke, aflibercept neither improved nor exacerbated stroke outcomes. The study demonstrates that aflibercept selectively attenuates stroke-induced brain edema and vascular permeability in obese mice. These findings suggest the repurposing of aflibercept to reduce obesity-enhanced brain edema in acute stroke.

Molecular Mechanisms of Skeletal Muscle Atrophy in Cerebral Ischemic Stroke

Stroke is the leading cause of death and the top cause of adult disability in patients world‐wide. Skeletal muscle is the main effector organ accounting for disability in stroke patients due to severe muscle atrophy and related weakness. However, the molecular signaling mechanisms that cause post‐stroke muscle atrophy remains elusive. To determine the mechanisms, we used a preclinical model of stroke in which we induced 60 minutes middle cerebral artery occlusion (MCAO) followed by 3 days reperfusion. We found that stroke caused a significant brain lesion and motor deficit. Stroke induced severe skeletal muscle atrophy in the paretic tibialis anterior muscle compared to the corresponding tibialis anterior muscle of sham mice, as evidenced by reduced muscle mass and FCA (fiber cross‐sectional area). Our transcriptome analysis showed that stroke altered mRNA expression profile in skeletal muscle. Some of the genes associated with catabolic pathways are highly dysregulated in post‐stroke muscle. We also found that stroke dramatically repressed the protein deacetylase Sirtuin1 (SirT1) gene expression and its function in skeletal muscle. Interestingly, restoring SirT1 function in post‐stroke muscle notably reversed muscle atrophy, in part, through regulating the key players of autophagy and ubiquitin proteasomal pathway. Collectively, our data uncovered a potential mechanism that causes cerebral ischemic stroke‐induced muscle atrophy and identified SirT1 as a critical regulator of post‐stroke muscle mass.

Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model

After ischemic stroke, in the lesion core as well as in the ischemic penumbra, evolution of tissue damage and repair is strongly affected by neuroinflammatory events that involve activation of local specialized glial cells, release of inflammatory mediators, recruiting of systemic cells and vascular remodelling. To take advantage of this intricate response in the quest to devise new protective therapeutic strategies we need a better understanding of the territorial and temporal interplay between stroke-triggered inflammatory and cell death-inducing processes in both parenchymal and vascular brain cells. Our goal is to describe structural rearrangements and functional modifications occurring in glial and vascular cells early after an acute ischemic stroke. Low and high scale mapping of the glial activation on brain sections of mice subjected to 30 minutes middle cerebral artery occlusion (MCAO) was correlated with that of the neuronal cell death, with markers for microvascular changes and with markers for pro-inflammatory (IL-1β) and reparative (TGFβ1) cytokines. Our results illustrate a time-course of the neuroinflammatory response starting at early time-points (1 h) and up to one week after MCAO injury in mice, with an accurate spatial distribution of the observed phenomena.

Pre-stroke surgery is not beneficial to normotensive rats undergoing sixty minutes of transient focal cerebral ischemia.

Experimental stroke in rodents, via middle cerebral artery occlusion (MCAO), can be associated with a negative impact on wellbeing and mortality. In hypertensive rodents, pre-stroke craniotomy increased survival and decreased body weight loss post-MCAO. Here we determined the effect, in normotensive Sprague-Dawley rats following 60 minutes MCAO, with or without pre-surgical craniotomy, on post-stroke outcomes in terms of weight loss, neurological deficit, lesion volume and functional outcomes. There was no effect of pre-stroke craniotomy on indicators of wellbeing including survival rate (P = 0.32), body weight loss (P = 0.42) and neurological deficit (P = 0.75). We also assessed common outcome measures following experimental stroke and found no effect of pre-stroke craniotomy on lesion volume as measured by T2-weighted MRI (P = 0.846), or functional performance up to 28 days post-MCAO (staircase test, P = 0.32; adhesive sticker test, P = 0.49; cylinder test, P = 0.38). Thus, pre-stroke craniotomy did not improve animal welfare in terms of body weight loss and neurological deficit. However, it is important, given that a number of drug delivery studies utilise the craniotomy procedure, to note that there was no effect on lesion volume or functional outcome following experimental stroke.

Abstract WMP74: Therapeutic Effect of Human Dental Pulp Stem Cells Overexpressing Hepatocyte Growth Factor in Experimental Ischemic Stroke

Objective: Human dental pulp stem cell (DPSC) is an attractive cell source because they can be easily obtained as medical waste without ethical and logistical problems. Transplantation of DPSC decreases tissue injury in the rat brain and enhances motor function recovery after the middle cerebral artery occlusion (MCAO). We aimed increasing therapeutic effects combining DPSC and ex vivo human hepatocyte growth factor (HGF) gene transfer with adeno-associated virus (AAV) vector. Methods: Male Sprague-Dawley rats were subjected to transient 90 minute MCAO, followed by intravenous administration of 1 x 10 6 cells of DPSC, AAV-1/HGF transferred DPSC (DPSC/HGF) or vehicle immediately after reperfusion (each, n=8). Infarct volumes, neurological deficits and immunochemistry were assessed at 24 and 72 h after reperfusion. We also detected concentrations of TNF-α and IL-1β in the brain tissue extracts from the ischemic hemisphere by ELISA. Results: Infarct volumes at 24 h after reperfusion were diminished in both DPSC (155.7 ± 35.6 mm 3 , p < 0.01) and DPSC/HGF (110.2 ± 34.7 mm 3 , p < 0.01) transplantation compared with vehicle (213.1 ± 29.1 mm 3 ). DPSC/HGF transplantation showed more reduction of infarct volume compared with DPSC (110.2 ± 34.7 mm 3 vs 155.7 ± 35.6 mm 3 , p < 0.05). Motor function recovery was also observed at 24 and 72 h by both DPSC and DPSC/HGF transplantation. Furthermore, we determined that DPSC/HGF transplantation significantly suppressed expression of Iba-1 and TNF-α compared with DPSC in the cortical ischemic boundary zone (p < 0.05, p < 0.01). Also, DPSC/HGF markedly decreased brain tissue TNF-α (11.5 ± 4.2 pg/ml) and IL-1β (22.2 ± 4.2 pg/ml) concentrations compared with vehicle (TNF-α, 54.6 ± 9.8 pg/ml, p < 0.01; IL-1β, 95.2 ± 9.8 pg/ml, p < 0.01) and DPSC (TNF-α, 26.8 ± 11.2 pg/ml, p < 0.05; IL-1β, 40.6 ± 11.2 pg/ml, p < 0.05) in the ischemic hemisphere. Both DPSC and DPSC/HGF group improved neuronal degeneration compared with vehicle (p < 0.05, p < 0.01). Conclusions: These findings show that overexpression of HGF by AAV vector would enhance the neuroprotective effects of DPSC transplantation through the modulation of inflammation after acute ischemic stroke.

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1, accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders.

Abstract WMP41: Neuronal Sirt1 is Required for Resveratrol Preconditioning Induced Ischemic Tolerance

Introduction: Our previous work demonstrates that resveratrol, a naturally occurring polyphenol, protects against cerebral ischemia when administered 2 or 14 days prior to injury. Resveratrol activates Sirt1, an NAD + -dependent deacylase that regulates cellular metabolism. It has been postulated that neuronal Sirt1 directly mediates this neuroprotection but it remains to be empirically tested. Objective: The objective of this study was to generate an inducible, neuronal-specific Sirt1 knockout mouse and determine whether neuronal Sirt1 is necessary for resveratrol-induced ischemic tolerance. Methods: Twenty to twenty-five gram neuronal-specific Sirt1 knockout mice (Sirt1neu-/-) and WTs were induced with tamoxifen. Mice were randomized for 1) western blot; 2) resveratrol preconditioning (RPC; 10 mg/kg resveratrol i.p.) or vehicle (1.5% DMSO; 0.9% saline) treatment 2 days prior to 60 minute middle cerebral artery occlusion (MCAo); 3) untargeted primary metabolomics by GC-TOF-MS; or 4) transcription factor activation profiling. Twenty-four hours following MCAo, neurological score was used to assess functional outcome and infarct volume was quantified by TTC staining. Results: Tamoxifen treatment removed WT Sirt1 protein from major brain regions but not from heart (Figure 1A, n=3). In WT, RPC reduced infarct volume by 43.7% and improved neurological score by nearly 3 points, however these effects were lost in Sirt1neu-/- (Figure 1B, n=5-9). Compared to WT, metabolic profiles from Sirt1neu-/- displayed significantly altered glycolysis metabolites (Figure 1C, n=8). Activation of hypoxia inducible factor (HIF) was reduced by 48% in Sirt1neu-/- (Figure 1D, n=3). Conclusions: We generated and utilized an inducible, neuronal-specific knockout mouse to demonstrate that neuronal Sirt1 specifically is required for RPC-induced ischemic tolerance. Additionally, Sirt1 regulates glycolysis in the brain, possibly through its interaction with HIF.

Abstract 161: Age and Sex Influence the Neutrophil Response to Ischemic Stroke in Mice and Humans

Background and Purpose: Age and sex have important effects on stroke-induced inflammation. We conducted CBC analysis and RNA-sequencing of ischemic stroke patient blood, with follow-up studies in an experimental mouse model, to test the hypothesis that there are sex differences in neutrophil function and neutrophil-mediated damage after ischemic stroke. Methods: Blood from male and female stroke patients was collected 24 hours after stroke, with transient ischemic attack patients serving as controls. Absolute neutrophil count was quantified and RNA-sequencing was performed on an age and severity matched subset of patients. Results were analyzed by multivariate regression and differential expression analysis, respectively. For animal experiments, aged (18-20 month) and young (8-10 week) male and female mice were subjected to 60 minute middle cerebral artery occlusion and sacrificed at 24 hours. Neutrophils from blood, bone marrow, lung, brain and spleen were quantified and phenotyped by flow cytometry. Results were analyzed by student T-test and two way ANOVA. Results: Absolute neutrophil counts increased significantly after stroke in both sexes (p=.01). Interestingly, females had more differentially expressed genes after stroke than males, including matrix metalloproteinase 9 and TNFa. In animal experiments, aged animals were found to have higher percentages of neutrophils in the blood, spleen, lungs and bone marrow than young animals. In addition, aged female animals were found to have more activated neutrophils 24 hours after stroke than aged males (p=<.05). Conclusion: These results suggest that the immune response to ischemic stroke differs in aged males and females. Female neutrophils appear to exhibit enhanced activation, which may contribute to the poorer stroke outcomes seen in aged female patients. Understanding sex and age differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.

Abstract 221: The P2x4 Receptor is Required for Neuroprotection via Ischemic Preconditioning

Background: Ischemic preconditioning (IPC), a procedure consisting of transient ischemia and subsequent reperfusion, provides ischemic tolerance against prolonged ischemia in the brain. Although the blood flow changes mediated by IPC are primarily perceived by vascular endothelial cells, the role of these cells in ischemic tolerance has not fully clarified. In this research, we focused on the role of P2X4 receptor, which sense blood flow changes and is expressed on vascular endothelial cells. Methods: We administrated P2X4 receptor inhibitor into lateral ventricle of C57BL/6J male mice (8-10 weeks) and then conducted middle cerebral artery occlusion (MCAO). Fifteen minutes MCAO was done as IPC 48 hours before 60 minutes MCAO. To examine the necessity of P2X4 receptor expression in vascular endothelial cells, we generated a conditional knockout (CKO) mouse in which the P2X4 receptor was knocked down in VE-cadherin-positive vascular endothelial cells. To investigate molecular change by IPC, we obtained cerebrovascular endothelial cells of mice 48 hours after IPC, and real time PCR and ELISA were evaluated. To examine the molecular expression change on vascular endothelial cells by blood flow, we used in vitro culture system which generates fluid flow and real time PCR was evaluated. Inhibition of P2X4 receptor expression was conducted by P2X4 receptor siRNA transfection. Results: P2X4 receptor antagonist abolished neuroprotection via IPC. Moreover, the effect of IPC to P2X4 receptor CKO mice was smaller than control mice, the infarct volume of P2X4 receptor CKO was larger than control mice after 60 minutes MCAO (p<0.05, Control, n=4; CKO, n=6). IPC induced expression of osteopontin mRNA (p<0.05, n=5). Osteopontin administration attenuates the increase of infarct formation induced by P2X4 receptor inhibition (p<0.05, Control, n=5; Osteopontin, n=6). In vitro, shear stress upregulated expression of osteopontin mRNA (p<0.05, n=3). This upregulation was inhibited by P2X4 receptor siRNA (p<0.05, Control siRNA, n=6; P2X4 receptor siRNA, n=7). Conclusion: These results demonstrate a novel mechanism whereby vascular endothelial cells are involved in ischemic tolerance by way of the pathway about P2X4 receptor and osteopontin.

Effect of umbilical cord blood stem cells transplantation on bladder dysfunction induced by cerebral ischemia in rats

ObjectiveTo demonstrate the effect of human umbilical cord blood-derived CD34+ cells on bladder dysfunction induced by cerebral ischemia in rats. Materials and MethodsFemale rats were subjected to either 60 minute middle cerebral artery occlusion (MCAO) or a sham operation. Rats were divided into four groups: sham operation, MCAO without treatment, infusion with 1×106 CD34+ cells 30 minutes before MCAO, and infusion with 1×106 CD34+ cells 3 hours after MCAO. Bladder function was analyzed by cystometry at 1 day, 3 days, and 7 days after MCAO. Expressions of nerve growth factor (NGF), M2 and M3 muscarinic receptors were measured by immunohistochemistry and real time polymerase chain reaction. ResultsCystometric results showed that, following MCAO, rats have a significant increase in peak voiding pressure and residual volume. Conversely, there is a significant decrease in voided volumes and intercontraction intervals. Cystometric variables after pre- and postischemic CD34+ treatment nearly returned to levels found in sham-operated rats. The expression of bladder NGF and M3 was decreased after MCAO, but significantly increased following preischemic CD34+ treatment. There was decreased expression of bladder M2 mRNA despite an increased level of M2 immunoreactivity at 3 days and 7 days after MCAO. Expression of bladder M2 immunoreactivity and mRNA nearly returned to sham group levels after preischemic CD34+ treatment. ConclusionBladder dysfunction in a rat model caused by MCAO may be restored to normal micturition by treatment with human umbilical cord blood-derived CD34+ cells and may be related to the expressions of NGF, M2, and M3 in the bladder.

Depletion of Cultivatable Gut Microbiota by Broad-Spectrum Antibiotic Pretreatment Worsens Outcome After Murine Stroke.

Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain-gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.

Infarct Volume Prediction by Early Magnetic Resonance Imaging in a Murine Stroke Model Depends on Ischemia Duration and Time of Imaging.

Despite standardization of experimental stroke models, final infarct sizes after middle cerebral artery occlusion (MCAO) vary considerably. This introduces uncertainties in the evaluation of drug effects on stroke. Magnetic resonance imaging may detect variability of surgically induced ischemia before treatment and thus improve treatment effect evaluation. MCAO of 45 and 90 minutes induced brain infarcts in 83 mice. During, and 3 and 6 hours after MCAO, we performed multiparametric magnetic resonance imaging. We evaluated time courses of cerebral blood flow, apparent diffusion coefficient (ADC), T1, T2, accuracy of infarct prediction strategies, and impact on statistical evaluation of experimental stroke studies. ADC decreased during MCAO but recovered completely on reperfusion after 45 and partially after 90-minute MCAO, followed by a secondary decline. ADC lesion volumes during MCAO or at 6 hours after MCAO largely determined final infarct volumes for 90 but not for 45 minutes MCAO. The majority of chance findings of final infarct volume differences in random group allocations of animals were associated with significant differences in early ADC lesion volumes for 90, but not for 45-minute MCAO. The prediction accuracy of early magnetic resonance imaging for infarct volumes depends on timing of magnetic resonance imaging and MCAO duration. Variability of the posterior communicating artery in C57Bl6 mice contributes to differences in prediction accuracy between short and long MCAO. Early ADC imaging may be used to reduce errors in the interpretation of post MCAO treatment effects on stroke volumes.

Delayed reperfusion deficits after experimental stroke account for increased pathophysiology.

Cerebral blood flow and oxygenation in the first few hours after reperfusion following ischemic stroke are critical for therapeutic interventions but are not well understood. We investigate changes in oxyhemoglobin (HbO2) concentration in the cortex during and after ischemic stroke, using multispectral optical imaging in anesthetized mice, a remote filament to induce either 30 minute middle cerebral artery occlusion (MCAo), sham surgery or anesthesia alone. Immunohistochemistry establishes cortical injury and correlates the severity of damage with the change of oxygen perfusion. All groups were imaged for 6 hours after MCAo or sham surgery. Oxygenation maps were calculated using a pathlength scaling algorithm. The MCAo group shows a significant drop in HbO2 during occlusion and an initial increase after reperfusion. Over the subsequent 6 hours HbO2 concentrations decline to levels below those observed during stroke. Platelets, activated microglia, interleukin-1α, evidence of BBB breakdown and neuronal stress increase within the stroked hemisphere and correlate with the severity of the delayed reperfusion deficit but not with the ΔHbO2 during stroke. Despite initial restoration of HbO2 after 30 min MCAo there is a delayed compromise that coincides with inflammation and could be a target for improved stroke outcome after thrombolysis.

Essential Role of Program Death 1-Ligand 1 in Regulatory T-Cell–Afforded Protection Against Blood–Brain Barrier Damage After Stroke

Our recent research revealed that adoptively transferred regulatory T cells (Tregs) reduced acute ischemic brain injury by inhibiting neutrophil-derived matrix metalloproteinase-9 (MMP-9) and protecting against blood-brain barrier damage. The mechanisms underlying Treg interactions with neutrophils remain elusive. This study evaluates the contribution of program death 1-ligand 1 (PD-L1) to Treg-mediated neutrophil inhibition and neuroprotection after cerebral ischemia. In vitro experiments were performed using a transwell system or a coculture system allowing cell-to-cell contact. Focal cerebral ischemia was induced in mice for 60 minutes. Tregs (2×10(6)) isolated from donor animals (wild-type or PD-L1-/-) were intravenously injected into ischemic recipients 2 hours after middle cerebral artery occlusion (MCAO). MMP-9 production, blood-brain barrier permeability, and brain infarct were assessed at 1 or 3 days after MCAO. In vitro experiments reveal that Treg-mediated inhibition of neutrophil MMP-9 required direct cell-to-cell contact. The suppression of MMP-9 was abolished when Tregs were pretreated with PD-L1 neutralizing antibodies or when neutrophils were pretreated with PD-1 antibodies. In vivo studies confirmed that intravenous administration of Tregs pretreated with PD-L1 antibodies or Tregs isolated from PD-L1-deficient mice failed to inhibit MMP-9 production by blood neutrophils 1 day after 60 minutes MCAO. Furthermore, the blood-brain barrier damage after MCAO was greatly ameliorated in PD-L1-competent Treg-treated mice but not in PD-L1-compromised Treg-treated mice. Consequently, PD-L1 dysfunction abolished Treg-mediated brain protection and neurological improvements 3 days after MCAO. PD-L1 plays an essential role in the neuroprotection afforded by Tregs against cerebral ischemia by mediating the suppressive effect of Tregs on neutrophil-derived MMP-9.

Abstract T MP111: Gpr124 Regulates Post-Stroke Cerebrovascular Integrity

Introduction: GPR124/TEM5 is an orphan G-protein coupled receptor (GPCR) with a large extracellular domain. We and others have previously demonstrated that GPR124 exerts CNS-specific angiogenesis regulation with knockout mice exhibiting embryonic lethality from hemorrhagic glomeruloid vascular malformations in forebrain and neural tube (c.f. Kuhnert et al., Science , Nov 12;330(6006):985-9 . (2010)). Hypothesis: GPR124 regulates adult angiogenesis and blood-brain barrier (BBB) integrity during homeostasis or after stroke. Methods: To bypass GPR124 embryonic lethality, we generated GPR124 conditional knockout (cko) mice allowing temporally-regulated deletion. Tamoxifen treatment of GPR124 flox/- ; ROSA-CreER mice versus GPR124 flox/+; ROSA-CreER littermate controls allowed GPR124 cko versus heterozygosity, respectively, in adult mice. GPR124 deletion was followed by analyses of microvascular structure and patterning and blood-brain barrier (BBB) integrity). GPR124 cko mice versus controls were also subjected to 60 minute middle cerebral artery occlusion (MCAO) and effects on stroke volume, survival and microvascular structure assessed. Results: GPR124 deletion in neonatal or adult mice was well-tolerated without impairment of postnatal vascular patterning, BBB maturation or BBB integrity. However, GPR124 cko mice subjected to the middle cerebral artery occlusion (MCAO) stroke model exhibited impaired survival and a profound microvascular hemorrhagic transformation that was confined to the infarct region relative to wild-type controls. GPR124 cko stroke vasculature also exhibited numerous cellular and architectural defects relative to controls that will be discussed. Conclusions: GPR124 deletion is well tolerated in adult mice but results in marked hemorrhagic transformation in the MCAO stroke model. GPR124 represents a novel receptor whose function is essential for cerebrovascular integrity in the post-stroke setting, with attendant therapeutic implications.

Abstract 10: Human Placenta-Derived Adherent Cell Treatment of Stroke Induces Angiogenesis, Increases Synaptic Plasticity and Improves Functional Outcome in Older-Adult Rats

Background and purpose: Human Placenta-Derived Adherent cells (PDAC TM ) are a novel adherent mesenchymal-like cell population derived from normal, full-term human placental tissue. PDA-001 (cenplacel-L) is a clinical formulation of PDAC TM developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in older-adult rats. Methods: Older-adult male Wistar rats (600-750g, 10-12 months) were subjected to 90 minutes of MCAo. To test the efficacy of PDA-001 treatment in older-adult rats, at 1 day after MCAo, randomly selected animals (n=12 per group) were injected, via a tail vein, with vehicle control (cell media), or with PDA-001 (4x10 6 or 8x10 6 cells). Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. All rats were sacrificed 29 days after MCAo, and lesion volumes were measured using hematoxylin and eosin (H&E). To assess cerebral tissue for angiogenesis and synaptogenesis, immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed, respectively. Results: PDA-001 treatment at the 4X10 6 and 8X10 6 doses significantly improved functional outcome after stroke in older-adult rats (p<0.05). The neurological benefit of PDA-001 treatment was associated with significant increases in the number of BrdU immunoreactive endothelial cells, increases in vascular density and perimeter in the ischemic brain (p<0.05), as well as significantly increased synaptic plasticity as measured by increased synaptophysin expression in the ischemic border zone (IBZ) (p<0.05). Conclusion: PDA-001 treatment significantly improved functional outcome after stroke in older-adult rats. The neurorestorative effects induced by PDA-001 treatment may be related to the increase in angiogenesis and synaptic plasticity.