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Introduction: Advances in modern medicine have greatly increased chances of survival following traumatic events such as cardiac arrest, stroke, or heart attack. With more people able to recover from these ischemic insults, it is becoming apparent that survivors experience long-term effects as it relates to brain function. Despite clinical and animal model evidence of emotional dysfunctions following global ischemia, no study has attempted to identify amygdala dysfunction after Cardiac Arrest and subsequent Cardiopulmonary Resuscitation (CA/CPR). Utilizing a mouse model of CA/CPR-induced global ischemia, we have identified amygdala dysfunctions that are dependent on biological sex. We hypothesize that global ischemia results in deficits in amygdala-dependent learning tasks and circuit specific deficits of LTP in the basolateral amygdala (BLA). Methods: Seven days post CA-induced global ischemia, male and female adult C57BL/6 mice underwent amygdala dependent delay fear conditioning. In a separate cohort of animals acute-transverse slices were prepared seven days post global ischemia and extracellular field potential recordings were collected from the BLA. Results: We have revealed a sexually dimorphic deficit in amygdala dependent fear learning and memory. Both male and female mice display a previously reported contextual fear deficit (Male: 78.6 ± 2.6% sham freezing vs. 51.2±8.7% CA/CPR freezing, n=7; Female 70±5.1% sham freezing vs. 50.2±8.5% CA/CPR freezing, n=9), however, only male mice display a diminished freezing response to cued fear (56.4 ± 7.2% sham freezing vs. 31 ± 6.7% in CA/CPR, n=7). Similarly, plasticity involving cortical inputs to the basolateral amygdala is also disrupted in a sexually dimorphic manner, with only males displaying diminished LTP relative to sham controls (153.4 ± 8.1% of BL in controls, n=4 vs. 102.4 ± 3% of BL in CA/CPR, n=7). Conclusion: These results support the role of the amygdala in cognitive-affective impairments after CA-induced global ischemia and provide new insights into the role that biological sex plays in mediating brain dysfunction following CA. In the future, we will continue to unravel the mechanisms by which this sexually dimorphic impairment occurs and identify therapeutic targets.

Introduction: Emerging evidence has implicated post-stroke cognitive impairment (PSCI) as a major contributor to long-term disability following acute ischemic stroke. While the hippocampus is shown to contribute to PSCI, the exact mechanisms underlying PSCI have yet to be elucidated. Interestingly, a preclinical model of large artery stroke (middle cerebral artery occlusion; MCAO) causes hippocampal dysfunction, despite direct ischemic insult to brain regions distant from the hippocampus, suggesting the injury causes perturbations in neural circuits. Thus, we utilize electrophysiological recordings of hippocampal LTP as an indicator of network health following ischemia. We hypothesize activated astrocytes in the hippocampus following MCAO increase expression of the ectoenzyme, CD38, which signals to neurons to impair plasticity. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from MCAO in adult (8-12 week) mice. Immunohistochemistry was performed to assess hippocampal CD38 expression and astrogliosis. Acute slices were treated with CD38 inhibitors (78c & apigenin) to assess plasticity. Results: Recordings obtained in brain slices 30 days after MCAO exhibited a significant reduction in LTP; 161±9%, n=6 in sham compared to 115±4%, n=7 30 days after MCAO, in both ipsilateral and contralateral hippocampi. Immunohistochemical (IHC) staining indicates CD38 levels are increased and colocalize with activated astrocyte marker, GFAP, 30 days following MCAO. Bath application of CD38 inhibitors, 78c (vehicle n=6, 4.0±8.5% vs. 100nM 78c n=6, 78.3±15.8%; p Conclusion: These data indicate that MCAO provides a reproducible model of post-stroke memory dysfunction (PSCI) and that remote astrogliosis in the uninjured hippocampus may contribute to altered neuronal function (plasticity). Our data implicates increased levels of CD38 impair plasticity following stroke. Therefore, reducing CD38 activity at chronic timepoints following ischemia may represent a novel strategy to treat the symptoms of PSCI.

Background: Cognitive deficits are common long-term sequelae after neonatal stroke, occurring in to 70% of school aged children. Despite this, chronic cognitive deficits have not been evaluated in animal models of neonatal stroke. Objective: To determine whether neonatal mice have behavioral memory and hippocampal cellular and plasticity changes after transient middle cerebral artery occlusion (tMCAO). Methods: C57/BL6 mice underwent 60 minutes of right tMCAO using the intraluminal filament model, or sham surgery, followed by reperfusion on postnatal day 10 (p10). Mice underwent contextual fear conditioning (CFC) testing to evaluate spatial memory 14 days after tMCAO (p24, juvenile equivalent). A separate set of animals were sacrificed at the same timepoint for Crestly Violet staining and stereology of CA1 neurons, or for LTP recordings. Increase in field excitatory post-synaptic potential (fEPSP) slope 60 min after theta-burst stimulation (TBS) was analyzed as a measurement of synaptic plasticity (LTP). Results: Animals had a significant decrease in % time freezing in the CFC paradigm 14 days after p10 tMCAO compared to sham surgery animals, indicating behavioral spatial memory impairment (Figure 1A). Animals who underwent p10 tMCAO also had a deficit in LTP after TBS in ipsilateral compared to contralateral CA1 (figure 1B). Animals had a decrease density of CA1 hippocampal neurons in the ipsilateral hippocampus compared to contralateral hippocampus 14 days after p10 tMCAO (Figure 1C). Conclusion: Our results show that neonatal stroke causes CA1 hippocampal pyramidal cell death and synaptic plasticity deficits which contribute to chronic memory impairment.

Post-stroke cognitive impairment and dementia (PSCID) affects many survivors of large vessel cerebral ischemia. The molecular pathways underlying PSCID are poorly defined but may overlap with neurodegenerative pathophysiology. Specifically, synaptic dysfunction after stroke may be directly mediated by alterations in the levels of amyloid beta (Aβ), the peptide that accumulates in the brains of Alzheimer’s disease (AD) patients. In this study, we use the transient middle cerebral artery occlusion (MCAo) model in young adult mice to evaluate if a large vessel stroke increases brain soluble Aβ levels. We show that soluble Aβ40 and Aβ42 levels are increased in the ipsilateral hippocampus in MCAo mice 7 days after the injury. We also analyze the level and activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), an enzyme that generates Aβ in the brain, and observe that BACE1 activity is increased in the ipsilateral hippocampus of the MCAo mice. Finally, we highlight that treatment of MCAo mice with a BACE1 inhibitor during the recovery period rescues stroke-induced deficits in hippocampal synaptic plasticity. These findings support a molecular pathway linking ischemia to alterations in BACE1-mediated production of Aβ, and encourage future studies that evaluate whether targeting BACE1 activity improves the cognitive deficits seen with PSCID.

Cerebellar stroke-induced cognitive and affective symptoms (CCAS) have been observed in patients with infarcts localized to the posterior regions of the cerebellum. Our lab has developed a mouse model of cerebellar stroke in which mice with posterior infarcts display hippocampal-dependent memory deficits. Synaptic plasticity in the hippocampal area CA1 is essential for episodic memory consolidation. There is evidence of functional connectivity of cerebellum with contralateral hippocampus. Therefore, we hypothesize that changes in after cerebellar stroke are the result of synaptic plasticity impairments within the hippocampus. We used a photothrombotic model to induce stroke in the posterior cerebellar hemispheres. Seven days after stroke, we performed extracellular recordings of field excitatory postsynaptic potentials (fEPSP) within the CA1 region of acute brain slices. In shams, theta-burst stimulation (TBS) of Schaffer collateral inputs produced long-term potentiation (LTP). TBS failed to evoke an increase in fEPSP slope in contralateral hippocampus of mice at 7 days post-injury. These data provide strong evidence demonstrating that injury to the cerebellum can produce changes in synaptic function within the hippocampus. The lack of LTP impairment in ipsilateral hippocampus after cerebellar stroke suggests that the observed plasticity deficits are activity dependent. We also observed reduced NMDA receptor, Fos and Arc expression. Brain-derived neurotrophic factor (BDNF) signaling through TrkB receptors is required for LTP and memory formation. We tested whether enhancement of TrkB signaling with the agonist 7, 8 dihydroflavone (DHF) could improve hippocampal plasticity. We observed a full restoration of hippocampal LTP in slices from cerebellar stroke mice that were incubated with DHF (250 nM) prior to TBS. We provide strong evidence for hippocampal dysfunction following cerebellar stroke. Our data have implications for benefit of strategies to enhance neurotrophic signaling not only at the site of injury, but in non-injured regions as well.

Introduction: Post-stroke cognitive impairment (PSCI) is a major contributor to long-term disability following acute ischemic stroke. Learning and memory deficits are a common feature of PSCI and alterations in hippocampal function are a likely contributor. Interestingly, common experimental stroke models (middle cerebral artery occlusion; MCAO) cause hippocampal dysfunction, despite no direct ischemic insult to the hippocampus, suggesting perturbations in neural circuits. Thus, we utilize electrophysiological recordings of hippocampal plasticity in combination with neurobehavioral assessments of memory function. Hypothesis: Activated astrocytes in the hippocampus following MCAO increase expression of the surface enzyme CD38, which signals to neurons to impair plasticity. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from transient MCAO (60 min) in adult (6-8 week) mice. A behavioral fear conditioning paradigm (CFC) was used to evaluate contextual memory. Immunohistochemistry was performed to assess CD38 expression and slices were treated with CD38 inhibitors (78c) to assess plasticity. Results: Recordings obtained in brain slices 30 days after MCAO exhibited loss of hippocampal LTP; 134±6%, n=4 in sham and 107±12%, n=4 30 days after MCAO. Memory function, measured using CFC, was consistent with our LTP findings. MCAO decreased freezing behavior, indicating lack of memory (65±7% in sham mice (n=6) and 37±7% in MCAO mice, n=7). Immunohistochemical data indicates increased CD38 expression in activated astrocytes following MCAO in the hippocampus. Treatment of hippocampal slices with 78c, a potent CD38 inhibitor, after MCAO rescues LTP impairment. Finally, no additive increase in LTP when 78c is co-administered with a TRPM2 channel inhibitor was observed. Conclusion: These data indicate that MCAO is a reproducible model of post-stroke memory dysfunction (PSCI) and remote astrogliosis in the uninjured hippocampus may contribute to altered neuronal function (plasticity). Our data implicates increased levels of CD38 as an upstream activator of neuronal TRPM2 channel in the hippocampus following stroke, resulting in impaired synaptic plasticity.

Introduction: Cognitive impairments and memory loss are common after stroke, with an emerging awareness of a high risk of conversion to post-stroke dementia. It is increasingly clear that in addition to neuronal injury following cerebral ischemia, impaired functional networks contribute to long-term functional deficits. Synaptic plasticity (LTP) is the leading cellular model of learning and memory. Thus, we utilize electrophysiological recordings of hippocampal LTP as an indicator of network health following ischemia in combination with neurobehavioral assessments of memory function. Hypothesis: Focal ischemic stroke increases soluble amyloid beta (Aβ) in the hippocampus, causing impaired plasticity and memory function. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from transient MCAO (45 min) in adult (6-8 week) mice. A behavioral fear conditioning paradigm (CFC) was used to evaluate memory. ELISA assay was used to quantify soluble Aβ42 from the hippocampus. Slices were treated with Aβ42 oligomers with and without our newly developed peptide inhibitor of TRPM2, termed tatM2NX. Results: Recordings from brain slices 30 days after MCAO showed near complete loss of LTP; 161±9%, n=6 in sham compared to 115±4%, n=7 30 days after MCAO in the hippocampus. MCAO decreased freezing behavior, indicating lack of memory (65±7% in sham mice (n=6) and 37±7% in MCAO mice, n=7). We observed a 48% increase in Aβ42 in the hippocampus 30 days after MCAo. We observed that addition of Aβ42 oligomers (500 nM) impaired LTP. This impaired LTP was prevented with co-application of the TRPM2 channel inhibitor tatM2NX. Consistent with a role of TRPM2 channels in post-stroke cognitive impairment, MCAO mice treated with tatM2NX (20 mg/kg iv injection 24 hr before testing) on day 29 post MCA demonstrated increasing freezing to 72±5% (n=9). Conclusion: Our data implicates increased levels of soluble Aβ42 in the hippocampus following stroke, resulting in activation of TRPM2 channels and impaired synaptic plasticity. Therefore, reducing soluble Aβ42 and/or inhibition of TRPM2 channels at chronic time points following ischemia may represent a novel strategy to improve functional recovery following stroke.

Introduction: Cognitive impairments and memory loss are common after stroke, with an emerging awareness of a high risk of conversion to post-stroke dementia. It is increasingly clear that in addition to neuronal injury following cerebral ischemia, impaired functional networks contribute to long-term functional deficits. Synaptic plasticity (long term potentiation; LTP) is the leading cellular model of learning and memory. Thus, we utilize electrophysiological recordings of hippocampal LTP as an indicator of network health following ischemia in combination with neurobehavioral assessments of memory function. TRPM2 channels are oxidative stress sensitive ion channels that have been implicated in ischemic injury. Hypothesis: Inhibition of TRPM2 channels reverse stroke-induced cognitive deficits. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from transient MCAO (45 min) in adult (6-8 week) mice. A behavioral fear conditioning paradigm was used to evaluate contextual memory 30 days after MCAO. Slices or mice were treated with our newly developed peptide inhibitor of TRPM2, termed tatM2NX. Results: Recordings obtained in brain slices 30 days after MCAO exhibited near complete loss of LTP; 161±9%, n=6 in sham compared to 115±4%, n=7 30 days after MCAO in the ipsilateral hippocampus. Similar deficit in LTP observed in the contralateral hippocampus. Remarkably, iv injection of 20 mg/kg tatM2NX on day 29 after MCAO reversed MCAO-induced loss of LTP when recorded on day 30, recovering to 175±9% (n=3). Memory function, measured using contextual fear conditioning, was consistent with our LTP findings. MCAO decreased freezing behavior, indicating lack of memory (62±5% in sham mice (n=5) and 24±3% in MCAO mice, n=4). This was reversed in MCAO mice given tatM2NX (20 mg/kg iv injection 24 hr before testing) on day 29 post MCAO, increasing freezing to 73±12% (n=3). Conclusion: These data indicate that our new TRPM2 channel inhibitor, tatM2NX, restores synaptic plasticity and memory function after experimental stroke. Therefore, inhibition of TRPM2 channels at chronic timepoints following ischemia may represent a novel strategy to improve functional recovery following stroke.

Global ischemia in childhood often leads to poor neurologic outcomes, including learning and memory deficits. Using our novel model of childhood cardiac arrest/cardiopulmonary resuscitation (CA/CPR), we investigate the mechanism of ischemia-induced cognitive deficits and recovery. Memory is impaired seven days after juvenile CA/CPR and completely recovers by 30 days. Consistent with this remarkable recovery not observed in adults, hippocampal long-term potentiation (LTP) is impaired 7–14 days after CA/CPR, recovering by 30 days. This recovery is not due to the replacement of dead neurons (neurogenesis), but rather correlates with brain-derived neurotrophic factor (BDNF) expression, implicating BDNF as the molecular mechanism underlying impairment and recovery. Importantly, delayed activation of TrkB receptor signaling reverses CA/CPR-induced LTP deficits and memory impairments. These data provide two new insights (1) endogenous recovery of memory and LTP through development may contribute to improved neurological outcome in children compared to adults and (2) BDNF-enhancing drugs speed recovery from pediatric cardiac arrest during the critical school ages.

Objective: Each year in the U.S. there are an estimated 20,000 strokes resulting in cerebellar infarction. The neurological impairments observed in these patients include motor coordination and motor learning impairments. Surprisingly, cerebellar stroke patients also exhibit executive and emotional dysfunction. Establishing a model of cerebellar stroke that recapitulates aspects of human cerebellar infarction will allow for us to perform mechanistic studies of brain injury and identify therapeutic targets to improve recovery in stroke patients. The goal of this study was to develop and characterize a photo-thrombotic mouse model of focal cerebellar stroke. Methods: Adult male and female mice were head-fixed in a stereotaxic frame, were administered Rose Bengal (150 ug/g) and the superior cerebellar artery was illuminated for 15 minutes with a cold white LED light source. Behavioral testing of motor and memory function was performed in mice subjected to sham procedure or photo-thrombosis at 7 days following surgery. Stereological analysis of lesion volume was performed at 1 and 7 days after surgery. Extracellular field recordings were performed in the CA1 region of the hippocampus to assess long-term potentiation (LTP). Results: Cerebellar stroke volumes were larger at 1 day (2.4 mm 3 ) than 7 days (0.964 mm 3 ), likely due to edema at the earlier time point. Lesion volumes were similar between males and females at both time points. Cerebellar infarction resulted in gait abnormality, motor incoordination and ipsilateral limb impairments. Memory impairments were also observed at 7 days after cerebellar stroke. Memory deficits correlate with contralateral hippocampal long-term plasticity impairments at 7 days after stroke. Conclusions: We have developed a reproducible model of cerebellar stroke that results in cerebellar infarction and associated motor impairments. Importantly, we alsoobserve memory deficits and have identified a molecular mechanism that we can target to improve memory function after stroke. This novel model will be a valuable tool for understanding how alterations in the cerebellar network resulting from cerebellar infarction produce motor and non-motor deficits.

Introduction: Ischemic damage triggers glutamate excitotoxicity, resulting in neuronal cell death. Previous research has demonstrated that NMDA receptor activity triggers downstream calcium-dependent signaling pathways, specifically Ca2+/calmodulin-dependent protein kinase II (CaMKII). We hypothesize that Death Associated Protein Kinase I (DAPKI) and receptor interacting protein (RIP) are downstream of CaMKII, contributing to necroptosis. Focal and global ischemia studies have shown that inhibiting CaMKII is protective against hippocampal damage, but there is little known about cerebellar cell death mechanisms. The aim of this study is to examine the neuroprotective potential of inhibiting CaMKII in Purkinje cells using a global ischemia model. Methods: C57BL/6 male adult mice were subjected to 8 minutes of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Mice were randomized to tat-CN19o or control peptide (tat-SCR), administered 30 minutes after CA/CPR, and cerebellar injury analyzed 7 days after CA/CPR. Western blot analysis of cerebellar homogenates performed 6 hrs after CA/CPR. Phosphorylated and total CAMKII, DAPKI, and total RIP was quantified. Statistical analyses were performed using 1-WAY ANOVA, with P Results: Analysis of Purkinje cell death revealed injury at 7 days after CA/CPR that was prevented with tat-CN19o at doses of 0.1 and 1 mg/kg. We observed an increase in T286-P of CAMKII at 6 hours after CA/CPR, concurrent with increased DAPK1 activity (reduced phosphorylation) and increased RIP expression. Administration of tat-CN19o prevented CA/CPR-induced changes in DAPK1 and RIP, while having no effect on CAMKII. Conclusions: Autonomous activity of CAMKII is increased by ischemia in the cerebellum, which contributes to cell death. Neuroprotection with tat-CN19o occurred at doses 10-fold higher than that needed for protection in the hippocampus (Deng, Cell Reports, 2017). Importantly, we have identified signaling pathways downstream of CAMKII that may contribute to Purkinje cell death, DAPK1 and RIP. In summary, our findings indicate that inhibition of autonomous CaMKII activity is a promising therapeutic approach.

The current study focuses on the ability to improve cognitive function after stroke with interventions administered at delayed/chronic time points. In light of recent studies demonstrating delayed GABA antagonists improve motor function, we utilized electrophysiology, biochemistry and neurobehavioral methods to investigate the role of α5 GABAA receptors on hippocampal plasticity and functional recovery following ischemic stroke. Male C57Bl/6 mice were exposed to 45 min transient middle cerebral artery occlusion and analysis of synaptic and functional deficits performed 7 or 30 days after recovery. Our findings indicate that hippocampal long-term potentiation (LTP) is impaired 7 days after stroke and remain impaired for at least 30 days. We demonstrate that ex vivo administration of L655,708 reversed ischemia-induced plasticity deficits and importantly, in vivo administration at delayed time-points reversed stroke-induced memory deficits. Western blot analysis of hippocampal tissue reveals proteins responsible for GABA synthesis are upregulated (GAD65/67 and MAOB), increasing GABA in hippocampal interneurons 30 days after stroke. Thus, our data indicate that both synaptic plasticity and memory impairments observed after stroke are caused by excessive tonic GABA activity, making inhibition of specific GABA activity at delayed timepoints a potential therapeutic approach to improve functional recovery and reverse cognitive impairments after stroke.

ObjectiveDexmedetomidine, an α-2a adrenergic agonist, given pre- and postoperatively was previously shown to attenuate neuronal injury in a murine model of spinal cord ischemia-reperfusion. In the brain, α-2 agonists have been shown to induce the phosphorylation of cyclic AMP response-element binding protein (CREB), a transcription factor necessary for neuron survival. We hypothesized that the α-2a adrenergic agonist given preoperatively increases CREB-mediated neuroprotective proteins, attenuating neuronal injury and cytoarchitectural decay.MethodsMice (ie, C57BL/6 mice) underwent 5 minutes of aortic occlusion via median sternotomy. Mice received 25 μg/kg dexmedetomidine or equivalent normal saline at 24 hours, 12 hours, and 30 minutes preoperatively. Functional outcomes were recorded at 6 to 48 hours postoperatively when spinal cords were removed for histologic analysis. Spinal cords were examined for protein kinase B, CREB, B-cell lymphoma 2, and brain-derived neurotrophic factor following treatment alone or ischemia-reperfusion surgery.ResultsFollowing aortic occlusion, mice in the treatment group had preserved neurologic function at all time points (P

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

Cardiac arrest and cardiopulmonary resuscitation (CA/CPR) produce brain ischemia that results in cognitive and motor coordination impairments subsequent to injury of vulnerable populations of neurons, including cerebellar Purkinje neurons. To determine the effects of CA/CPR on plasticity in the cerebellum, we used whole cell recordings from Purkinje neurons to examine long-term depression (LTD) at parallel fiber (PF) synapses. Acute slices were prepared from adult male mice subjected to 8 min cardiac arrest at 1, 7, and 30 days after resuscitation. Concurrent stimulation of PF and climbing fibers (CFs) resulted in robust LTD of PF-evoked excitatory postsynaptic currents (EPSCs) in controls. LTD was absent in recordings obtained from mice subjected to CA/CPR, with no change in EPSC amplitude from baseline at any time point tested. AMPA and mGluR-mediated responses at the PF were not altered by CA/CPR. In contrast, CF-evoked NMDA currents were reduced following CA/CPR, which could account for the loss of LTD observed. A loss of GluN1 protein was observed following CA/CPR that was surprisingly not associated with changes in mRNA expression. These data demonstrate sustained impairments in synaptic plasticity in Purkinje neurons that survive the initial injury and which likely contribute to motor coordination impairments observed after CA/CPR.

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 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.

Female sex steroids, particularly estrogens, contribute to the sexually dimorphic response observed in cerebral ischemic outcome, with females being relatively protected compared to males. Using a mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR), we previously demonstrated that estrogen neuroprotection is mediated in part by the estrogen receptor β, with no involvement of estrogen receptor α. In this study we examined the neuroprotective effect of the novel estrogen receptor, G-protein coupled estrogen receptor 1 (GPER1/GPR30). Male mice administered the GPR30 agonist G1 exhibited significantly reduced neuronal injury in the hippocampal CA1 region and striatum. The magnitude of neuroprotection observed in G1 treated mice was indistinguishable from estrogen treated mice, implicating GPR30 in estrogen neuroprotection. Real-time quantitative RT-PCR indicates that G1 treatment increases expression of the neuroprotective ion channel, small conductance calcium-activated potassium channel 2. We conclude that GPR30 agonists show promise in reducing brain injury following global cerebral ischemia.

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel is activated following oxidative stress and has been implicated in ischemic damage; however, little experimental evidence exists linking TRPM2 channel activation to damage following cerebral ischemia. We directly assessed the involvement of TRPM2 channels in ischemic brain injury using pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of TRPM2 expression. Each of the four TRPM2 inhibitors tested provided significant protection to male neurons following in vitro ischemia (oxygen–glucose deprivation, OGD), while having no effect in female neurons. Similarly, TRPM2 knockdown by TRPM2 shRNA resulted in significantly reduced neuronal cell death following OGD only in male neurons. The TRPM2 inhibitor clotrimazole reduced infarct volume in male mice, while having no effect on female infarct volume. Finally, intrastriatal injection of lentivirus expressing shRNA against TRPM2 resulted in significantly smaller striatal infarcts only in male mice following middle cerebral artery occlusion, having no significant effect in female mice. Data presented in the current study demonstrate that TRPM2 inhibition and knockdown preferentially protects male neurons and brain against ischemia in vitro and in vivo, indicating that TRPM2 inhibitors may provide a new therapeutic approach to the treatment of stroke in men.

Objective: Experience-dependent memory acquisition, in vitro tetanic stimulation and simulated ischemia results in long-term potentiation (LTP) of hippocampal CA1 synapses. The goal of this study was to determine whether in vivo cerebral ischemia results in iLTP of CA1 synapses. We hypothesized that iLTP resulting from CA/CPR prevents physiological LTP. Methods: Cardiac arrest (8 minutes) followed by cardiopulmonary resuscitation (CA/CPR) or sham surgery was performed on adult (8-12 week) male mice. Electrophysiology was performed in hippocampal slices from sham and 7 and 30 days after CA/CPR. Field excitatory postsynaptic potentials (fEPSP) resulting from Schaffer collateral stimulation were recorded and rising slope was analyzed. Theta burst stimulation (TBS) was used for LTP induction and low frequency stimulation for reversal of LTP (depotentiation). Western blot analysis of synaptic isolations were performed from hippocampi collected from shams and 7 and 30 days after CA/CPR. Results: TBS resulted in an increased fEPSP to 164±9.8% (n=7, P Conclusions: Depotentiation in slices from CA/CPR mice suggests CA1 synapses are in a chronically potentiated state. Increased GluR1 phosphorylation and expression after CA/CPR also suggest in vivo ischemia causes iLTP. Our ability to induce physiological LTP after depotentiation in slices from CA/CPR suggests that reversing iLTP allows for normal synaptic plasticity. The results of this study are significant as they demonstrate in vivo iLTP and suggest a physiological stimulus has the potential to reverse synaptic impairments in the chronic phase after cerebral ischemia.

Objectives: The goal of this study was to assess neuronal injury and synaptic plasticity impairments in Purkinje cells in pediatric and adult mice following cardiac arrest and cardiopulmonary resuscitation. We hypothesized that synaptic impairments in pediatrics would be less severe than in adult mice. Methods: Pediatric ( 21-25 day old) and adult (8-12 week) male mice were subjected to 8 minutes cardiac arrest followed by cardiopulmonary resuscitation or sham surgery. Purkinje cells were labeled using anti-calbindin antibody and cell density was analyzed to examine neuronal injury. To examine synaptic function, whole-cell voltage clamp recording on acute cerebellar slices was performed at 1 and 7 days after CA/CPR or sham surgery. Excitatory postsynaptic currents (EPSCs) resulting from parallel (PF) or climbing fiber (CF) stimulation were recorded. Long-term depression (LTD) was induced by simultaneous parallel and climbing fiber stimulation (1 Hz, 5 min). Results: Purkinje cell loss was observed in pediatric (24% cell loss) and adult (32% cell loss) mice following CA/CPR. Long-term depression, a reduction of synaptic strength of PF EPSCs was observed in adult and pediatric sham controls following PF+CF stimulation (46.6 ± 10% of baseline n= 5 and 55.4 ± 6.1% n= 3, respectively). LTD was absent in pediatric and adult mice at 24 hours after CA/CPR (96.1 ± 5.2% n =6 and 89.4 ± 4.5% n=2, respectively). At 7 and 30 days after CA/CPR LTD was absent in adult mice (81.6 ± 10.8% n=6 and 86.5 ± 8.9% n=4) but had returned in pediatric mice at 7 days after CA/CPR (70 ± 5.4% n=3). Conclusions: The results of this study suggest that CA/CPR results in significant Purkinje cell loss in pediatric and adult mice. LTD was absent at 24 hours at both ages and was observed at 7 days after CA/CPR in pediatric but not adult mice. These results suggest enhanced recovery of synaptic plasticity at this developmental stage.

Background and Purpose— Pediatric stroke, birth to 18 years, is a significant cause of long-term disability in the United States; however, there is currently little experimental data on the pathophysiology of childhood stroke owing to lack of animal models. We developed a novel mouse model of experimental childhood-onset arterial ischemic stroke to characterize the sex-specific response of the adolescent brain to cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage. Methods— Postnatal day 20 to 25 mice were subjected to 90 minutes experimental stroke via the intraluminal filament middle cerebral artery occlusion model and ischemic damage assessed 22 hours after reperfusion. Real-time quantitative real-time polymerase chain reaction was performed 22 hours after middle cerebral artery occlusion to determine the effects of ischemia and estrogen treatment on the proapoptotic gene Bax . Results— Ischemic injury did not differ between male and female juvenile (postnatal day 20–25) mice after middle cerebral artery occlusion. However, estrogen reduced ischemic injury in female mice, whereas having no effect in juvenile males. No differences in estrogen receptor expression were observed on postnatal day between 20 males and females. In contrast, estrogen minimized the ischemia-induced increase in the proapoptotic gene Bax in female mice, whereas having no effect on Bax induction in the male brain. Conclusions— Focal ischemia has fundamentally different effects in the juvenile brain compared with the adult, as evidenced by the lack of sex difference in ischemic injury in the murine postnatal day 20 to 25 middle cerebral artery occlusion model and the sexually dimorphic response to estrogen neuroprotection.