Min Of Middle Cerebral Artery Occlusion Research Articles

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke.

ObjectiveInhibitors of the voltage‐gated K+ channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up‐regulated on “M1‐like” microglia, we here tested the therapeutic hypothesis that the brain‐penetrant small‐molecule Kv1.3‐inhibitor PAP‐1 reduces secondary inflammatory damage after ischemia/reperfusion.MethodsWe studied microglial Kv1.3 expression using electrophysiology and immunohistochemistry, and evaluated PAP‐1 in hypoxia‐exposed organotypic hippocampal slices and in middle cerebral artery occlusion (MCAO) with 8 days of reperfusion in both adult male C57BL/6J mice (60 min MCAO) and adult male Wistar rats (90 min MCAO). In both models, PAP‐1 administration was started 12 h after reperfusion.ResultsWe observed Kv1.3 staining on activated microglia in ischemic infarcts in mice, rats, and humans and found higher Kv1.3 current densities in acutely isolated microglia from the infarcted hemisphere than in microglia isolated from the contralateral hemisphere of MCAO mice. PAP‐1 reduced microglia activation and increased neuronal survival in hypoxia‐exposed hippocampal slices as effectively as minocycline. In mouse MCAO, PAP‐1 dose‐dependently reduced infarct area, improved neurological deficit score, and reduced brain levels of IL‐1β and IFN‐γ without affecting IL‐10 and brain‐derived nerve growth factor (BDNF) levels or inhibiting ongoing phagocytosis. The beneficial effects on infarct area and neurological deficit score were reproduced in rats providing confirmation in a second species.InterpretationOur findings suggest that Kv1.3 constitutes a promising therapeutic target for preferentially inhibiting “M1‐like” inflammatory microglia/macrophage functions in ischemic stroke.

Analysis of long non-coding RNA expression profiles following focal cerebral ischemia in mice.

Long noncoding RNAs (lncRNAs) have a variety of biological functions and play key roles in many diseases. However, the knowledge of lncRNA function during a stroke is limited. We analyzed the expression profiles of lncRNAs in the brain ischemic region of mice after a 45min middle cerebral artery occlusion (MCAO) with a 48h reperfusion. Gene ontology and pathway analysis were used to elucidate the potential functions of the differentially expressed mRNAs. A total of 255 lncRNAs (217 up-regulated and 38 down-regulated) and 894 mRNAs (870 up-regulated and 24 down-regulated) showed significantly altered expression in the ischemic brain compared to the sham controls (fold change ≫>2, P≪0.05). The gene ontology terms were mainly associated with neutrophil chemotaxis, positive regulation of inflammatory response, cell cycle, positive regulation of apoptotic process, and apoptotic process. The pathway analysis indicated that the mRNAs were mainly associated with inflammatory pathways. Additionally, the interactions between the differentially expressed lncRNAs and mRNAs are revealed by a dynamic lncRNA-mRNA network. Our findings provide an overview of aberrantly expressed lncRNAs in stroke and further broaden the understanding of stroke pathogenesis.

Stage 1 Registered Report: Effect of deficient phagocytosis on neuronal survival and neurological outcome after temporary middle cerebral artery occlusion (tMCAo).

Stroke is a major cause of death and disability worldwide. In addition to neuronal death resulting directly from energy depletion due to lack of blood supply, inflammation and microglial activation following ischemic brain injury has been increasingly recognized to be a key contributor to the pathophysiology of cerebrovascular disease. However, our understanding of the cross talk between the ischemic brain and the immune system is limited. Recently, we demonstrated that following focal ischemia, death of mature viable neurons can be executed through phagocytosis by microglial cells or recruited macrophages, i.e. through phagoptosis. It was shown that inhibition of phagocytic signaling pathways following endothelin-1 induced focal cerebral ischemia leads to increased neuronal survival and neurological recovery. This suggests that inhibition of specific phagocytic pathways may prevent neuronal death during cerebral ischemia. To further explore this potential therapeutic target, we propose to assess the role of phagocytosis in an established model of temporary (45min) middle cerebral artery occlusion (tMCAo), and to evaluate neuronal survival and neurological recovery in mice with deficient phagocytosis. The primary outcome of this study will be forelimb function assessed with the staircase test. Secondary outcomes constitute Rotarod performance, stroke volume (quantified on MR imaging or brain sections, respectively), diffusion tensor imaging (DTI) connectome mapping, and histological analyses to measure neuronal and microglial densities, and phagocytic activity. Male mice aged 10-12 weeks will be used for experiments.

Adipose-derived perivascular stem cells promote sensorimotor recovery after ischemic stroke in rats

Neuronal damage after transient cerebral ischemia is exacerbated by signaling pathways involving activated platelet-activating factor (PAF) and ameliorated by PAF-acetylhydrolase (PAF-AH); but whether cerebral neurons can be rescued by human recombinant PAF-AH (rPAF-AH) remains unknown. Adult male mice underwent a 60 min middle cerebral artery occlusion (MCAO) and reperfusion for 24 h. Then, the mice received intravenous tail injections with different drugs. Neurological behavioral function was evaluated by Bederson's test, and cerebral infarction volume was assessed with tetrazolium chloride (TTC) staining. mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2, collagenase-1), MMP-9 (gelatinase-B), and vascular endothelial growth factor (VEGF) were determined by quantitative real-time PCR (RT-PCR) and western blot analysis, respectively. Compared with the vehicle group, rPAF-AH significantly improved sensorimotor function (42%, P = 0.0001). The volume of non-infarcted brain tissue was increased by the rPAF-AH treatment (16.3 ± 4.6% vs. 46.0 ± 10.3%, respectively). rPAF-AH significantly reduced mRNA and protein levels of MMP-2 and MMP-9, but increased the mRNA (P < 0.001) and protein levels (P < 0.01) of VEGF. These results demonstrate that rPAF-AH provides neuroprotection against ischemic injury. Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression.

Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke

Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90 min MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hr after ischemia reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hr transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hr transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hr and 7 days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.

YO-05 - Long-term contribution of Nrf2 to behavioral recovery following focal cerebral ischemia-reperfusion injury

Ischemic stroke is a leading cause of death and adult disability worldwide. The redox sensitive transcription factor Nrf2 is a key target for protection in rodent stroke models. We investigated protection afforded by 5 mg/kg dietary sulforaphane in C57BL6/J WT and Nrf2-/- adult male mice subjected to 60min middle cerebral artery occlusion and 72 h or 21 days reperfusion. Behavioural outcomes were measured and brains were sectioned for high throughput imaging and infarct quantification. SFN pretreatment improved functional rotating rod and grip strength performance, significantly reduced cresyl violet infarct volumes and upregulated HO-1 and PPARγ expression in cerebrovascular endothelium. We subsequently conducted the first long-term (>14 day) follow up studies of stroke in Nrf2-deficient and in wild-type mice subjected to long-term pretreatment with SFN (5 mg/kg/day). Basal and inducible contributions of Nrf2 to alternative activation of resident microglia and recruited macrophages, long-term cytokine expression (IL-1β, IL-6, IL-10), post-infarct neurogenesis and angiogenesis and functional recovery were investigated. Our findings highlight the translational potential of Nrf2 inducers in stroke research and establish that dietary levels of sulforaphane afford protection in rodent models of focal cerebral ischaemia.

Sex differences in regulatory cells in experimental stroke

Stroke is the leading cause of disability in the United States. Sex differences, including smaller infarcts in females and greater involvement of immune-mediated inflammation in males may affect the efficacy of immune-modulating interventions. To address these differences, we sought to identify distinct stroke-modifying mechanisms in female vs. male mice. The current study demonstrated smaller infarcts and increased levels of regulatory CD19+CD5+CD1dhi B10 cells as well as anti-inflammatory CD11b+CD206+ microglia/macrophages in the ipsilateral vs. contralateral hemisphere of female but not male mice undergoing 60min middle cerebral artery occlusion followed by 96h of reperfusion. Moreover, female mice with MCAO had increased total spleen cell numbers but lower B10 levels in spleens. These results elucidate differing sex-dependent regulatory mechanisms that account for diminished stroke severity in females and underscore the need to test immune-modulating therapies for stroke in both males and females.

Abstract WMP74: Sex Differences in T Cell Immune Responses and Outcome After Stroke in Aged Mice

Introduction: Sex differences are increasingly recognized in stroke. Stroke incidence is higher in men than in women until an advanced age and women have higher mortality and morbidity. Inflammatory responses are key determinants of outcome but have not been well studies in aged animals. Hypothesis: We hypothesize that there are important sex differences in inflammation in aged male and female mice. Results: We quantified T cells and T regulatory cells, as well as functional outcome after stroke in aged mice. Male and female C57BL/6 mice (20-21 months) were subjected to 60 min middle cerebral artery occlusion (n=16) by the filament model, or sham surgery (n=7). Immune responses in brain, blood and spleen were investigated 15 days post-stroke by flow cytometry. Functional outcomes were assessed at day 3, 7 and 14. Mortality was 25% in females and occurred at day 1, while in males 50% mortality was seen between day 3 and 9. Hemorrhagic transformation was seen in 53% of the males that died (5/8) and in none of the females (0/4). Flow cytometry analysis at day 15 showed significantly higher levels of CD8 + T cells in the blood after stroke in males compared to females (73±5% of CD3 + T cells vs 52±3%, P&lt;0.001). CD8 + T cells were found in brains from males and females after stroke but levels were not different from sham (Male Stroke: 64±7%, Female Stroke: 58±5%). Independent of the type of surgery (stroke or sham), we observed higher levels of splenic CD8 + T cells in males (P&lt;0.01). T regulatory cells were significantly increased in the brain after stroke in males compared to sham (P&lt;0.01), but not in females. Open-field testing showed a decrease in center visits after stroke in both sexes at 3 days, however males spent more time immobile (P&lt;0.01) indicating decreased locomotor activity and/or anxiety. This difference equalized at 7 and 14 days post-stroke. Conclusion: Higher mortality and hemorrhagic transformation rates were seen in aged males than in females after stroke. This may be associated with the elevated levels of CD8 + T cells in blood and possible unfavorable effects on blood brain barrier permeability.

Adipose tissue and metabolic and inflammatory responses to stroke are altered in obese mice.

ABSTRACTObesity is an independent risk factor for stroke, although several clinical studies have reported that obesity improves stroke outcome. Obesity is hypothesised to aid recovery by protecting against post-stroke catabolism. We therefore assessed whether obese mice had an altered metabolic and inflammatory response to stroke. Obese ob/ob mice underwent a 20-min middle cerebral artery occlusion and 24-h reperfusion. Lipid metabolism and expression of inflammatory cytokines were assessed in the plasma, liver and adipose tissue. The obese-specific metabolic response to stroke was assessed in plasma using non-targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics coupled with univariate and multivariate analysis. Obesity had no effect on the extent of weight loss 24 h after stroke but affected the metabolic and inflammatory responses to stroke, predominantly affecting lipid metabolism. Specifically, obese mice had increases in plasma free fatty acids and expression of adipose lipolytic enzymes. Metabolomics identified several classes of metabolites affected by stroke in obese mice, including fatty acids and membrane lipids (glycerophospholipids, lysophospholipids and sphingolipids). Obesity also featured increases in inflammatory cytokines in the plasma and adipose tissue. Overall, these results demonstrate that obesity affected the acute metabolic and inflammatory response to stroke and suggest a potential role for adipose tissue in this effect. These findings could have implications for longer-term recovery and also further highlight the importance of considering comorbidities in preclinical stroke research, especially when identifying biomarkers for stroke. However, further work is required to assess whether these changes translate into long-term effects on recovery.

Water-soluble fullerene derivatives (C60(OH)18-22) decrease infarct volume and edema after middle cerebral artery occlusion in rat

Overproduction of oxygen free radicals (ROS) and oxidative stress during reperfusion period plays a critical role in pathophysiology of cerebral ischemia-reperfusion injury. Since the antioxidant properties of fullerene nanoparticles has been extensively demonstrated, we assessed the possible protective effects of water-soluble fullerene derivatives (C60(OH)18-22) on infarct volume and edema after middle cerebral artery occlusion in rat.Twenty four rats were randomly divided into three groups (each group; n=8): sham, control ischemia (IR) and ischemic treatment groups. Cerebral ischemia was induced by 90 min middle cerebral artery occlusion (MCAO) followed by 24 hours reperfusion. Fullerene nanoparticles were given orally at dose of 1mg/kg immediately after termination of MCAO and the beginning of reperfusion period. The brains were processed for histopathological assessment and quantitation of the infarct volume (TTC staining method). Finally, the brain hemispheres were weighed as an index of brain edema.There was no infarction and edema in right hemispheres of sham rats. MCAO induced brain infarction both in cortex (261±23mm3) and subcortex (138±23mm3) and also right hemispheres weights (23%) of control ischemic rats. Administration of fullerene reduced the infarction both in cortex (49±26mm3) and subcortex (43±21mm3) and also the value of right hemisphere weight (20%) of ischemic treatment rats. Additionally in histopathological assessment, the perivascular areas decreased in ischemic treatment rats.The findings of present study are revealing the protective effects of water-soluble fullerene derivatives against reperfusion-induced injury such as infarction and edema after middle cerebral artery occlusion in rat.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice.

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 μg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.

Tetramethylpyrazine-2'-O-sodium ferulate attenuates blood-brain barrier disruption and brain oedema after cerebral ischemia/reperfusion.

Disruption of blood-brain barrier (BBB) and subsequent oedema are major causes of the pathogenesis in ischaemic stroke with which the current clinical therapy remains unsatisfied. In this study, we examined the therapeutic effect of tetramethylpyrazine-2'-O-sodium ferulate (TSF)-a novel analogue of tetramethylpyrazine in alleviating BBB breakdown and brain oedema after cerebral ischaemia/reperfusion (I/R). Then, we explored the potential mechanism of the protection on BBB disruption in cerebral I/R rat models. Male Sprague-Dawley rats (250-300 g) were subjected to 120 min middle cerebral artery occlusion (MCAO), followed by 48 h reperfusion. TSF (10.8, 18 and 30 mg kg-1) and ozagrel (18 mg kg-1) were administrated by intravenous injection immediately for the first time and then received the same dose every 24 h for 2 days. We found that TSF treatment significantly attenuated the cerebral water content, infarction volume and improved neurological outcomes in MCAO rats compared to I/R models. Moreover, we investigated the effect of TSF on the BBB for that cerebral oedema is closely related to the permeability of the BBB. We found that the permeability of BBB was improved significantly in TSF groups compared to I/R model group by Evans blue leakage testing. Furthermore, the expressions of tight junction (TJ) proteins junction adhesion molecule-1 and occludin significantly decreased, but the protein expression of matrix metalloproteinase-9 (MMP-9) and aquaporin 4 (AQP4) increased after cerebral I/R, all of which were alleviated by TSF treatment. In conclusion, TSF significantly reduced BBB permeability and brain oedema, which were correlated with regulating the expression of TJ proteins, MMP-9 and AQP4. These findings provide a novel approach to the treatment of ischaemic stroke.

Polyhydroxylated fullerene nanoparticles attenuate brain infarction and oxidative stress in rat model of ischemic stroke.

Oxidative stress is the common underlying mechanism of damage in ischemic stroke. Therefore, we aimed to evaluate the possible protective effects of polyhydroxylated fullerene derivatives on brain infarction and oxidative/nitrosative stress in a rat model of ischemic stroke. The experiment was performed by four groups of rats (each; n=12); Sham, Control ischemia, and ischemic treatment groups (Pretreatment and Posttreatment). Brain ischemia was induced by 90 min middle cerebral artery occlusion (MCAO) followed by 24 hours reperfusion. Rats received fullerene nanoparticles at dose of 1 mg/kg 30 min before MCAO and immediately after beginning of reperfusion. Infarct volume, contents of malondialdehyde (MDA), glutathione (GSH) and nitrate as well as superoxide dismutase (SOD) activity were assessed 24 hours after termination of MCAO. Brain infarct volume was 310 ± 21 mm3 in control group. Administration of fullerene nanoparticles before and after MCAO significantly decreased the infarct volume by 53 % (145 ± 45 mm3) and 81 % (59 ± 13 mm3), respectively. Ischemia also enhanced MDA and nitrate contents of ischemic hemispheres by 45 % and 25 % , respectively. Fullerene nanoparticles considerably reduced the MDA and nitrate contents of ischemic hemispheres before MCAO by 58 % and 17 % , respectively, and after MCAO by 38 % and 21 % , respectively. Induction of MCAO significantly decreased GSH content (19 % ) and SOD activity (52 % ) of ischemic hemispheres, whereas fullerene nanoparticles increased the GSH content and SOD activity of ischemic hemispheres by 19 % and 52 % before MCAO, respectively, and 21 % and 55 % after MCAO, respectively. Our findings indicate that fullerene nanoparticles, as a potent scavenger of free radicals, protect the brain cells against ischemia/reperfusion injury and inhibit brain oxidative/nitrosative damage.

Extended therapeutic window of a novel peptide inhibitor of TRPM2 channels following focal cerebral ischemia

IntroductionTRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor. MethodsFluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2–3months) and aged (18–20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining. ResultsTRPM2 inhibition by tat-M2NX was observed by decreased Ca2+ influx following H2O2 exposure human TRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2−/− mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX 3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. ConclusionsThese data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.

Methylene blue and normobaric hyperoxia combination therapy in experimental ischemic stroke.

IntroductionIschemic stroke is a global burden that contributes to the disability and mortality of millions of patients. This study aimed to evaluate the efficacy of combined MB (methylene blue) and NBO (normobaric hyperoxia) therapy in experimental ischemic stroke.MethodsRats with transient (60 min) MCAO (middle cerebral artery occlusion) were treated with: (1) air + vehicle (N = 8), (2) air + MB (N = 8), (3) NBO + vehicle (N = 7), and (4) NBO + MB (N = 9). MB (1 mg/kg) was administered at 30 min, again on days 2, 7, and 14 after stroke. NBO was given during MRI (30–150 min) on day 0, and again 1 h each during MRI on subsequent days. Serial diffusion, perfusion and T2 MRI were performed to evaluate lesion volumes. Foot‐fault and cylinder tests were performed to evaluate sensorimotor function.ResultsThe major findings were: (1) NBO + MB therapy showed a greater decrease in infarct volume compared to NBO alone, but similar infarct volume compared to MB alone, (2) NBO + MB therapy accelerated sensorimotor functional recovery compared to NBO or MB alone, (3) Infarct volumes on day 2 did not change significantly from those on day 28 for all four groups, but behavioral function continued to show improved recovery in the NBO + MB group.ConclusionsThese findings support the hypothesis that combined NBO + MB further improves functional outcome and reduces infarct volume compared to either treatment alone and these improvements extended up to 28 days.

Abstract 142: Extended Therapeutic Window of a Novel Peptide Inhibitor of TRPM2 Channels Following Focal Cerebral Ischemia

Introduction: Recent data demonstrates that inhibition of TRPM2 channels protect the brain against ischemia, specifically in males. A major hindrance to our research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor developed in our laboratory. Methods: Fluorescent calcium imaging was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. tat-peptides were detected by immunohistochemistry using an anti-tat antibody. Adult (2-3 mo) and aged (18-20 mo) were subjected to 60 min middle cerebral artery occlusion (MCAo) and injected iv with tat-M2NX (20 mg/kg), control tat-SCR (20 mg/kg) or clotrimazole (CTZ) (30 mg/kg) either 20min prior or 3hr after reperfusion. Infarct size was assessed using TTC staining. Results: Successful TRPM2 inhibition in HEK293 cells was observed by decreased Ca2+ influx following H2O2 exposure. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR; 29.2±4.6% (n=8) vs. 43.4±3.2% (n=8; p&lt;0.01), respectively. No effect of tat-M2NX was observed in female mice. Immunostaining demonstrates tat-M2NX accumulation in striatal and cortical neurons 4-6 hours of IV injection. Importantly, male TRPM2-/- mice were not further protected by tat-M2NX, demonstrating selectivity of our new inhibitor. Administration of tat-M2NX at a delayed time point (3hr) provided significant protection to males when analyzed at 24 hr (28.33 ± 5.1% (n=7) vs. 46.45 ± 3.0% (n=6; p&lt;0.01) or 4 days after MCAo (23.84 ± 4.416, (n=8) vs. 41.41 ± 2.831; (n=8; p&lt;0.01). In contrast, delayed administration of CTZ failed to reduce infarct volume. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. Conclusions: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a relatively wide therapeutic window. Furthermore, we took advantage of TRPM2-/- mice to confirm specificity of tat-M2NX protection.

Abstract WP273: Focal Knockdown of Matrix Metalloprotease 3 Reduces Hemorrhagic Transformation and Improves Neurobehavioral Outcomes in Hyperglycemic Stroke

Matrix metalloprotease 3 (MMP3) is an endopeptidase with broad substrate specificity that can target and degrade the components of the neurovascular unit. MMP3 was shown to be a critical mediator of the tPA-induced hemorrhagic transformation (HT) after stroke. The role of MMP3 in mediating neurovascular injury in hyperglycemic stroke was not previously reported. Accordingly, this study tested the hypothesis that MMP3 focal knockdown in the brain reduces HT and improves functional outcomes in hyperglycemic stroke. Methods: MMP3 shRNA lentiviral particles or an empty vector (EV) was injected into both hemispheres to the lateral ventricles of male Wistar rats. Two weeks later, Control and mildly HG rats (140-200 mg/dl, achieved by 30% glucose injection (IP) 15 min prior to surgery, n=7-9/group) were subjected to 90 min middle cerebral artery occlusion and 24 h reperfusion. At 24 h, neurological deficit, infarct size, edema, Hb content in ischemic hemispheres and HT occurrence rate (HT index) were measured. In an additional group of animals, the spatial expression of MMP3 within the neurovascular unit was determined by immunohistochemistry. Results: MMP3 knockdown significantly reduced HT index, Hb content and edema with no effect on infarct size and this was associated with improved functional outcomes (Table). MMP3 expression was more evident in HG animals and co-localized with the neurons, blood vessels and pericytes but not astrocytes. Conclusion: MMP3 knockdown reduced the vascular injury and improved the functional outcomes. Our findings point out MMP3 as a potential therapeutic target in hyperglycemic stroke.

Abstract 65: Restoration of Growth Differentiation Factor 11 Protects Against Stroke in Mice

Introduction: GDF 11 is a member of the transforming growth factor β superfamily. Loss of GDF11 occurs with aging and declining levels correlate with several detrimental age-associated phenotypes in both peripheral tissues and brain. Restoration of GDF11 enhances neurogenesis and cognitive function in aged mice. Brain expression of GDF11 has not been investigated after stroke. Stroke differentially affects the elderly. In this work we examined the role of GDF11 in aging, stroke and its potential utility as a neuroprotective agent. Methods: Male C57/BL6NCrl young (2-3 months) and aged (19-21) mice were used. Brain GDF11 expression was evaluated in young and aged mice by western blot. Focal ischemia was induced with a transient middle cerebral artery occlusion (MCAO). Mice were randomly assigned into two groups and were subjected to 90 min MCAO. Group 1 received vehicle (phosphate buffered saline) and group 2 was administered rGDF11 (100 ug/kg., ip) at the onset of ischemia. In additional experiments, the efficacy of delayed treatment (3 h after ischemia) with rGDF11 was tested. These mice were subjected to a 60 min MCAO. Mice were euthanized after 24 hours and 7 days respectively and brains were harvested to estimate infarct area. Results: A significant decrease in brain GDF11 levels was observed in aged mice as compared to young (p&lt;0.05). Additionally, a significant decline in brain GDF11 expression was observed after stroke at 24 hours vs. sham groups (p&lt;0.05). A significant decrease in cortical and hemispheric infarct area was observed in the rGDF11 group (cortical 48.73±1.05; hemisphere 49.68±3.58) as compared to vehicle group (60.54±4.88; 61.35±6.03), when GDF was administered at the time of ischemia. Delayed treatment with rGDF11 also reduced infarct at 7 days. Conclusions: Brain GDF11 levels decline with age and after stroke. Supplementation with rGDF11 ameliorates stroke induced injury in young mice at 24h and 7 days. These finding suggest potential role of GDF11 in age and stroke. Restoration of age-related loss of GDF may be a viable therapy for stroke.

Post-treatment with cocaine- and amphetamine-regulated transcript enhances infarct resolution, reinnervation, and angiogenesis in stroke rats - an MRI study.

Recent studies have shown that post-treatment with cocaine- and amphetamine-regulated transcript (CART) has neuroregenerative effects in animal models of stroke. The purpose of this study was to characterize CART-mediated neuronal and vascular repairments using non-invasive MRI techniques. Adult male rats were subjected to a 90 min middle cerebral artery occlusion (MCAo). Animals were separated into two groups with similar infarction sizes, measured by T2 -weighted MRI on Day 2 after MCAo, and were treated with CART or vehicle intranasally from Day 3 to Day 12. Diffusion tensor imaging was used to examine changes in plasticity of white matter elements. Susceptibility-weighted imaging (SWI) was used to measure angiogenesis. Post-treatment with CART significantly increased fractional anisotropy (FA) in lesioned cortex on Days 10 and 25 post stroke. A significant correlation between the behavioral recovery in body asymmetry and the change in FA was shown, suggesting that behavioral recovery was associated with reinnervation to the lesioned hemisphere. CART also increased the intensity of SWI and the immunoreactivity of the vascular marker alpha-smooth muscle actin in lesioned cortex. Together, our data support a non-invasive treatment strategy for stroke through angiogenesis and reinnervation by CART. Copyright © 2016 John Wiley & Sons, Ltd.

Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo.

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a ‘stringent’ one [60min MCAO/48h reperfusion] and a ‘mild’ one [30min MCAO/72h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the ‘mild’ model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.