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BackgroundNeonatal stroke is common and causes life-long motor and cognitive sequelae. Because neonates with stroke are not diagnosed until days-months after the injury, chronic targets for repair are needed. We evaluated oligodendrocyte maturity and myelination and assessed oligodendrocyte gene expression changes using single cell RNA sequencing (scRNA seq) at chronic timepoints in a mouse model of neonatal arterial ischemic stroke.MethodsMice underwent sixty minutes of transient right middle cerebral artery occlusion (MCAO) on postnatal day 10 (p10) and received 5-ethynyl-2’-deoxyuridine (EdU) on post-MCAO days 3-7 to label dividing cells. Animals were sacrificed 14 and 28-30 days post-MCAO for immunohistochemistry and electron microscopy. Oligodendrocytes were isolated from striatum 14 days post-MCAO for scRNA seq and differential gene expression analysis.ResultsThe density of Olig2+EdU+cells was significantly increased in ipsilateral striatum 14 days post-MCAO and the majority of oligodendrocytes were immature. Density of Olig2+EdU+cells declined significantly between 14 and 28 days post-MCAO without a concurrent increase in mature Olig2+EdU+cells. By 28 days post-MCAO there were significantly fewer myelinated axons in ipsilateral striatum. scRNA seq identified a cluster of “disease associated oligodendrocytes (DOLs)” specific to the ischemic striatum, with increased expression of MHC class I genes. Gene ontology analysis suggested decreased enrichment of pathways involved in myelin production in the reactive cluster.ConclusionsOligodendrocytes proliferate 3-7 days post-MCAO and persist at 14 days, but fail to mature by 28 days. MCAO induces a subset of oligodendrocytes with reactive phenotype, which may be a therapeutic target to promote white matter repair.

General anaesthesia in the neonatal period has detrimental effects on the developing mammalian brain. The impact of underlying inflammation on anaesthesia-induced developmental neurotoxicity remains largely unknown.Postnatal day 7 (PND7) rats were randomly assigned to receive sevoflurane (3 vol% for 3 h) or carrier gas 12 h after bacterial lipopolysaccharide (LPS; 1 μg gSevoflurane or LPS treatment increased activated caspase-3 and caspase-9 expression in the hippocampal subiculum and CA1, which was greater when sevoflurane was administered in the setting of LPS-induced inflammation. Neuronal injury induced by LPS+sevoflurane treatment resulted in sex-specific behavioural outcomes when rats were tested at 5-8 weeks of age, including learning and memory deficits in males and heightened anxiety-related behaviour in females. Hippocampal caspase-1 and NLRP1 (NLR family pyrin domain containing 1), but not NLRP3, were upregulated by LPS or LPS+sevoflurane treatment, along with related proinflammatory cytokines, interleukin (IL)-1β, and IL-18. Pretreatment with Vx-765, a selective caspase-1 inhibitor, led to reduced IL-1β in LPS and LPS+sevoflurane groups. Caspase-1 inhibition by Vx-765 significantly decreased activated caspase-3 and caspase-9 immunoreactivity in the subiculum.Systemic inflammation promotes developmental neurotoxicity by worsening anaesthesia-induced neuronal damage with sex-specific behavioural outcomes. This highlights the importance of studying anaesthesia-induced neurotoxicity in more clinically relevant settings.

Thalamocortical (TC) neurons within the ventrolateral thalamus (VL) receive projections from the cerebellum and the basal ganglia (BG) to facilitate motor and non-motor functions. Tonic and rebound firing patterns in response to excitatory cerebellar and inhibitory BG inputs, respectively, are a canonical feature of TC neurons and plays a key role in signal processing. The intrinsic excitability of TC neurons has a strong influence on how they respond to synaptic inputs, however, it is unknown whether their afferents influence their firing properties. Understanding the input-specific firing patterns could shed light into movement disorders with cerebellar or BG involvement. Here, we used whole-cell electrophysiology in brain slices from C57BL/6 mice to investigate the firing of TC neurons with optogenetic confirmation of cerebellar or BG afferents. TC neurons with cerebellar afferents exhibited higher tonic and rebound firing rates than those with BG afferents. This increased firing was associated with faster action potential depolarization kinetics and a smaller afterhyperpolarization potential. We also found differences in the passive membrane properties and sag currents during hyperpolarization. Despite higher rebound firing in TC neurons with cerebellar afferents, there were no differences in T-type calcium channel function compared to those with BG inputs. These data suggest input-specific differences in sodium and SK, but not T-type calcium channels, impact firing properties in TC populations. Altogether, we showed that the pronounced divergence observed in TC neuron firing properties correlate with its heterogeneous anatomical connectivity, which could signify a distinct signal integration and processing by these neurons. KEYPOINTS: Thalamocortical neurons in the VL with cerebellar afferents have higher intrinsic tonic and rebound firing properties than those with basal ganglia afferents. Membrane resistance and action potential depolarization slope were different based on the presence of cerebellar afferents. Despite elevated rebound burst firing, T-type mediated currents did not correlate with increased firing in neurons with cerebellar afferents.

We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.HighlightsMicrovesicles transfer mitochondria to endothelial cells and brain slice neuronsMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)Transfer of microvesicles increased mitochondrial function in hypoxic BECsTransfer of exosomes did not affect mitochondrial function in hypoxic BECsHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs

Summary Exposure to anaesthetic drugs during the fetal or neonatal period induces widespread neuronal apoptosis in the brains of rodents and non-human primates. Hundreds of published preclinical studies and nearly 20 clinical studies have documented cognitive and behavioural deficits many months or years later, raising the spectre that early life anaesthesia exposure is a long-term, perhaps permanent, insult that might affect the quality of life of millions of humans. Although the phenomenon of anaesthesia-induced developmental neurotoxicity is well characterised, there are important and lingering questions pertaining to sex differences and neurodevelopmental sequelae that might occur differentially in females and males. We review the relevant literature on sex differences in the field of anaesthesia-induced developmental neurotoxicity, and present an emerging pattern of potential sex-dependent neurodevelopmental abnormalities in rodent models of human infant anaesthesia exposure.

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.

Over the past three decades, we have been grappling with rapidly accumulating evidence that general anesthetics (GAs) may not be as innocuous for the young brain as we previously believed. The growing realization comes from hundreds of animal studies in numerous species, from nematodes to higher mammals. These studies argue that early exposure to commonly used GAs causes widespread apoptotic neurodegeneration in brain regions critical to cognition and socio-emotional development, kills a substantial number of neurons in the young brain, and, importantly, results in lasting disturbances in neuronal synaptic communication within the remaining neuronal networks. Notably, these outcomes are often associated with long-term impairments in multiple cognitive-affective domains. Not only do preclinical studies clearly demonstrate GA-induced neurotoxicity when the exposures occur in early life, but there is a growing body of clinical literature reporting similar cognitive-affective abnormalities in young children who require GAs. The need to consider alternative GAs led us to focus on synthetic neuroactive steroid analogues that have emerged as effective hypnotics, and analgesics that are apparently devoid of neurotoxic effects and long-term cognitive impairments. This would suggest that certain steroid analogues with different cellular targets and mechanisms of action may be safe alternatives to currently used GAs. Herein we summarize our current knowledge of neuroactive steroids as promising novel GAs.

Since its invention, general anesthesia has been an indispensable component of modern surgery. While traditionally considered safe and beneficial in many pathological settings, hundreds of preclinical studies in various animal species have raised concerns about the detrimental and long-lasting consequences that general anesthetics may cause to the developing brain. Clinical evidence of anesthetic neurotoxicity in humans continues to mount as we continue to contemplate how to move forward. Notwithstanding the alarming evidence, millions of children are being anesthetized each year, setting the stage for substantial healthcare burdens in the future. Hence, furthering our knowledge of the molecular underpinnings of anesthesia-induced developmental neurotoxicity is crucially important and should enable us to develop protective strategies so that currently available general anesthetics could be safely used during critical stages of brain development. In this mini-review, we provide a summary of select strategies with primary focus on the mechanisms of neuroprotection and potential for clinical applicability. First, we summarize a diverse group of chemicals with the emphasis on intracellular targets and signal-transduction pathways. We then discuss epigenetic and transgenerational effects of general anesthetics and potential remedies, and also anesthesia-sparing or anesthesia-delaying approaches. Finally, we present evidence of a novel class of anesthetics with a distinct mechanism of action and a promising safety profile.

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.

Large body of animal work and emerging clinical findings have suggested that early exposure to anesthetics may result in increased risk of learning disabilities and behavioral impairments [1–4]. Recent studies have begun to investigate anesthesia-induced epigenetic modifications to elucidate their role in behavioral and neurodevelopmental abnormalities. Here we examine sevoflurane-induced transgenerational modifications of subicular neuronal DNA methylation and expression of immediate early genes (lEGs), arc and junB, crucial to synaptic plasticity and normal neuronal development. We show that 6h sevoflurane exposure in postnatal day 7 rat pups resulted in a decrease in neuronal 5-methycytosine, indicating reduced DNA methylation. This effect is transgenerationally expressed in offspring born to exposed mothers which is of importance considering that decreased DNA methylation in the brain has been linked with functional decline in learning and memory [5]. We further show that sevoflurane exposure induces upregulation of Arc and JunB mRNA expression, 42.7% and 35.2%, respectively. Transgenerational changes in Arc and JunB mRNA were sexually dimorphic only occurring in males born to exposed females, expressed as upregulation of Arc and JunB mRNA, 71.6% and 74.0%, respectively. We further investigated correlation between altered arc promoter methylation and observed upregulation of Arc mRNA and observed that sevoflurane reduced methylation in the 5-upstream promoter region of females exposed to sevoflurane. Transgenerational hypomethylation and modifications to lEGs crucial to synaptic plasticity, observed following neonatal sevoflurane exposure could contribute to morphological and cognitive deficits known to occur with neonatal sevoflurane exposure.

Each year there are approximately 7000 out of hospital cardiac arrests in the pediatric population, with 30% resuscitation rate and a 6–10% rate of survival to hospital discharge. Survivors of cardiac arrest exhibit learning and memory deficits that are devastating during the school years. Delayed neuronal cell death occurs in the hippocampus following cardiac arrest and likely contributes to memory impairments. Circulating endogenous estrogen in young adult females has been shown to provide protection against ischemic cell death, as does chronic exogenous administration of 17β-estradiol (E2). Chronic estrogen benefit can have undesirable feminizing effects, particularly in pre-adolescents. Here, we tested if a single-dose of E2 is neuroprotective in our pediatric cardiac arrest mouse model performed in juvenile mice. We subjected P21 P25 C57Blk6 male and female mice to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). This developmental stage preceded the hormonal onset and serum estradiol and testosterone levels were not different in males and females. A single dose of E2 (100μg/kg) or vehicle was administered 30 min after resuscitation. Neuronal cell death measured 3 days after CA/CPR showed reduced hippocampal cell death in E2-treated females, but not males. Benefit of E2 in females was blocked by the P38 MAPK inhibitor, SB203580. Hippocampal-dependent memory function was equally impaired in E2-and vehicle-treated females measured in the contextual fear conditioning task at 7 days. Our findings demonstrate female-specific transient neuroprotection with E2 that does not provide sustained functional benefit.

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.

By 2050, 1 in 6 people will be 65 or older. As the aging population grows so too does the importance of understanding how injuries in this age group lead to a greater incidence of neurological complications and mortality. Neurological complications may be driven by elevated pro-inflammatory cytokines and astrocyte activation within the hippocampus (HPC), which reduces levels of phosphorylated cyclic-AMP response binding element (pCREB), leading to cognitive decline and memory impairment. To examine the effects of injury on neuroinflammation in advanced age, young and aged mice were anesthetized and subjected to a 15% total body surface area scald or sham injury. As an index of neuroinflammation, we observed an 85-fold (p Supported in part by NIH R01 AG018859 (EJK), R35 GM131831 (EJK), T32 AG000279 (KLM), and K08 134185 (JPI)

Background The most currently used general anaesthetics are potent potentiators of γ-aminobutyric acid A (GABAA) receptors and are invariably neurotoxic during the early stages of brain development in preclinical animal models. As causality between GABAA potentiation and anaesthetic-induced developmental neurotoxicity has not been established, the question remains whether GABAergic activity is crucial for promoting/enhancing neurotoxicity. Using the neurosteroid analogue, (3α,5α)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-24-ol (CDNC24), which potentiates recombinant GABAA receptors, we examined whether this potentiation is the driving force in inducing neurotoxicity during development. Methods The neurotoxic potential of CDNC24 was examined vis-a-vis propofol (2,6-diisopropylphenol) and alphaxalone (5α-pregnan-3α-ol-11,20-dione) at the peak of rat synaptogenesis. In addition to the morphological neurotoxicity studies of the subiculum and medial prefrontal cortex (mPFC), we assessed the extra-, pre-, and postsynaptic effects of these agents on GABAergic neurotransmission in acute subicular slices from rat pups. Results CDNC24, like alphaxalone and propofol, caused dose-dependent hypnosis in vivo, with a higher therapeutic index. CDNC24 and alphaxalone, unlike propofol, did not cause developmental neuroapoptosis in the subiculum and mPFC. Propofol potentiated post- and extrasynaptic GABAA currents as evidenced by increased spontaneous inhibitory postsynaptic current (sIPSC) decay time and prominent tonic currents, respectively. CDNC24 and alphaxalone had a similar postsynaptic effect, but also displayed a strong presynaptic effect as evidenced by decreased frequency of sIPSCs and induced moderate tonic currents. Conclusions The lack of neurotoxicity of CDNC24 and alphaxalone may be at least partly related to suppression of presynaptic GABA release in the developing brain.

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.

The role of biological sex in short-term and long-term outcome after traumatic brain injury (TBI) remains controversial. The observation that exogenous female sex steroids (progesterone and estrogen) reduce brain injury coupled with a small number of clinical studies showing smaller injury in women suggest that sex steroids may play a role in outcome from TBI. We used the controlled cortical impact (CCI) model of TBI in mice to test the hypothesis that after CCI, female mice would demonstrate less injury than male mice, related to the protective role of endogenous steroids. Indeed, adult females exhibit histological protection (3.7 ± 0.5 mm(3)) compared to adult male mice (6.8 ± 0.6 mm(3)), and females that lacked sex steroids (ovex) showed increased injury compared to intact females. Consistent with histology, sensorimotor deficits measured as reduced contralateral limb use were most pronounced in male mice (31.9 ± 6.9% reduced limb use) compared to a 12.7 ± 3.8% reduction in female mice. Ovex mice exhibited behavioral deficits similar to males (31.5 ± 3.9% reduced limb use). Ovex females demonstrated increased microglial activation relative to intact females in both the peri-injury cortex and the reticular thalamic nucleus. Ovex females also demonstrated increased astrogliosis in comparison to both females and males in the peri-injury cortex. These data indicate that female sex steroids reduce brain sensitivity to TBI and that reduced acute neuroinflammation may contribute to the relative protection observed in females.

GABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABA

SUMMARY The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling, but its role in pathological glutamate signaling (excitotoxicity) remains less clear, with indications for both neurotoxic and neuro-protective functions. Here, the role of CaMKII in ischemic injury is assessed utilizing our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). CaMKII inhibition (with tatCN21 or tatCN19o) at clinically relevant time points (30 min after resuscitation) greatly reduces neuronal injury. Importantly, CaMKII inhibition also works in combination with mild hypothermia, the current standard of care. The relevant drug target is specifically Ca2+-independent “autonomous” CaMKII activity generated by T286 autophosphorylation, as indicated by substantial reduction in injury in autonomy-incompetent T286A mutant mice. In addition to reducing cell death, tatCN19o also protects the surviving neurons from functional plasticity impairments and prevents behavioral learning deficits, even at extremely low doses (0.01 mg/kg), further highlighting the clinical potential of our findings., In Brief Deng et al. find that CaMKII and its phospho-T286-induced autonomous activity are a promising therapeutic drug target for global cerebral ischemia (induced by cardiac arrest followed by CPR in a mouse model). Pharmacological inhibition or T286A mutation leads to neuroprotection and improves synaptic and functional recovery following cardiac arrest.

Ischemic brain damage is triggered by glutamate excitotoxicity resulting in neuronal cell death. Previous research has demonstrated that N-methly-d-aspartate (NMDA) receptor activation triggers downstream calcium-dependent signaling pathways, specifically Ca2+/calmodulin-dependent protein kinase II (CaMKII). Inhibiting CaMKII is protective against hippocampal ischemic injury, but there is little known about its role in the cerebellum. To examine the neuroprotective potential of CaMKII inhibition in Purkinje cells, we subjected C57BL/6 or CaMKIIα KO male mice (8–12 weeks old) to cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). We performed a dose-response study for tat-CN19o and cerebellar injury was analyzed at 7 days after CA/CPR. Acute signaling was assessed at 6 h after CA/CPR using Western blot analysis. We observed increased phosphorylation of the T286 residue of CaMKII, suggesting increased autonomous activation. Analysis of Purkinje cell density revealed a decrease in cell density at 7 days after CA/CPR that was prevented with tat-CN19o at doses of 0.1 and 1 mg/kg. However, neuroprotection in the cerebellum required doses that were 10-fold higher than what was needed in the hippocampus. CaMKIIα KO mice subjected to sham surgery or CA/CPR had similar Purkinje cell densities, suggesting CaMKIIα is required for CA/CPR-induced injury in the cerebellum. We also observed a CA/CPR-induced activation of death-associated protein kinase (DAPK1) that tat-CN19o did not block. In summary, our findings indicate that inhibition of autonomous CaMKII activity is a promising therapeutic approach that is effective across multiple brain regions.

BACKGROUND: The cation-chloride cotransporters Na(+)–K(+)–2Cl(−)–1 (NKCC1) and K(+)–2Cl(−)–2 (KCC2) critically regulate neuronal responses to gamma-aminobutyric acid (GABA). NKCC1 renders GABA excitatory in immature neurons while expression of KCC2 signals GABA maturation to its inhibitory role. Imbalances in NKCC1/KCC2 alter GABA neurotransmission, which may contribute to hyperexcitability and blunted inhibition in neurocircuitry after neonatal exposure to anesthesia. Thus, we hypothesized that anesthetics may dysregulate NKCC1 and/or KCC2 in developing brain. METHODS: We exposed postnatal day (PND) 7 mice to sevoflurane or carrier gases and assessed NKCC1 and KCC2 expression across three brain regions 6 hours and 24 hours after initial exposure. To test differences in behavior, we challenged pups receiving sevoflurane or carrier gases on PND7 with propofol on PND8 and recorded parameters of anesthesia induction and maintenance. RESULTS: Sevoflurane exposure increased cortical NKCC1 at 6 hours (p = .03) and decreased cortical and hippocampal KCC2 at 24 hours (p = .009 and p = .007, respectively). NKCC1/KCC2 ratio was significantly increased at both 6 hours (p = .02) and 24 hours (p = .03) in cortex and at 24 hours (p = .02) in hippocampus. After propofol challenge on PND8, pups previously exposed to sevoflurane on PND7 regained righting reflex significantly faster than their non-exposed cohort (p < .001). CONCLUSIONS: Disturbing NKCC1/KCC2 balance may underlie circuit hyperexcitability and contribute to neurodevelopmental impairments we have observed in previous studies of neonatal anesthesia exposure. Human infants previously exposed to anesthesia may require higher concentrations of anesthetic drugs, potentially compounding their susceptibility for neurodevelopmental sequalae.

Sirtuins (Sirt) are a class of deacetylase enzymes that play an important role in cell proliferation. Sirt2 activation produces O-acetylated-ADPribose (OAADPr) which can act as a ligand for transient receptor potential cation channel, M2 (TRPM2). We tested the hypothesis that Sirt2 is activated following global cerebral ischemia and contributes to neuronal injury through activation of TRPM2.Adult male and female mice (8-12 weeks old) C57Bl/6 and TRPM2 knock-out mice were subjected to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). The Sirt2 inhibitor AGK-2 was administered intravenously 30 min after resuscitation. Hippocampal CA1 injury was analyzed at 3 days after CA/CPR. Acute Sirt2 activity was analyzed at 3 and 24 h after CA/CPR. Long-term hippocampal function was assessed using slice electrophysiology 7 days after CA/CPR.AGK-2 significantly reduced CA1 injury in WT but not TRPM2 knock-out males and had no effect on CA1 injury in females. Elevated Sirt2 activity was observed in hippocampal tissue from males at 24 h after cardiac arrest and was reduced by AGK-2. In contrast, Sirt2 activity in females was increased at 3 but not 24 h. Finally, we observed long-term benefit of AGK-2 on hippocampal function, with a protection of long-term potentiation at CA1 synapses at 7 and 30 days after ischemia.In summary, we observed a male specific activation of Sirt2 that contributes to neuronal injury and functional deficits after ischemia specifically in males. These results are consistent with a role of Sirt2 in activating TRPM2 following global ischemia in a sex specific manner. These results support the growing body of literature showing that oxidative stress mechanisms predominate in males and converge on TRPM2 activation as a mediator of cell death.

SUMMARY DAPK1 binding to GluN2B was prominently reported to mediate ischemic cell death in vivo. DAPK1 and CaMKII bind to the same GluN2B region, and their binding is mutually exclusive. Here, we show that mutating the binding region on GluN2B (L1298A/ R1300Q) protected against neuronal cell death induced by cardiac arrest followed by resuscitation. Importantly, the GluN2B mutation selectively abolished only CaMKII, but not DAPK1, binding. During ischemic or excitotoxic insults, CaMKII further accumulated at excitatory synapses, and this accumulation was mediated by GluN2B binding. Interestingly, extra-synaptic GluN2B decreased after ischemia, but its relative association with DAPK1 increased. Thus, ischemic neuronal death requires CaMKII binding to synaptic GluN2B, whereas any potential role for DAPK1 binding is restricted to a different, likely extra-synaptic population of GluN2B., In Brief Ischemic insults cause excitotoxic neuronal cell death via NMDA receptor overstimulation. Buonarati et al. find that excitotoxic insults cause DAPK1 movement to extra-synaptic NMDA receptors and CaMKII movement to synaptic NMDA receptors; importantly, preventing this CaMKII movement protects neurons from ischemic death., Graphical Abstract

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.

Ischemic long-term potentiation (iLTP) is a form of synaptic plasticity that occurs in acute brain slices following oxygen-glucose deprivation. In vitro, iLTP can occlude physiological LTP (pLTP) through saturation of plasticity mechanisms. We used our murine cardiac arrest and cardiopulmonary resuscitation (CA/CPR) model to produce global brain ischemia and assess whether iLTP is induced in vivo, contributing to the functionally relevant impairment of pLTP. Adult male mice were subjected to CA/CPR, and slice electrophysiology was performed in the hippocampal CA1 region 7 or 30 days later. We observed increased miniature excitatory postsynaptic current amplitudes, suggesting a potentiation of postsynaptic AMPA receptor function after CA/CPR. We also observed increased phosphorylated GluR1 in the postsynaptic density of hippocampi after CA/CPR. These data support the in vivo induction of ischemia-induced plasticity. Application of a low-frequency stimulus (LFS) to CA1 inputs reduced excitatory postsynaptic potentials in slices from mice subjected to CA/CPR, while having no effects in sham controls. These results are consistent with a reversal, or depotentiation, of iLTP. Further, depotentiation with LFS partially restored induction of pLTP with theta burst stimulation. These data provide evidence for iLTP following in vivo ischemia, which occludes pLTP and likely contributes to network disruptions that underlie memory impairments.

Extracorporeal membrane oxygenation (ECMO) has been a therapy of last resort for the treatment of severe cardiorespiratory failure since the 1970s [1]. In recent years, ECMO has seen a resurgence in its use in adults. Recent work examining rates of ECMO use in the US adult population, using Nationwide Inpatient Sample data, quotes an increase in use of 433% from 2006 to 2011 [2]. While much research has focused on neurologic injury after cardiac surgery and cardiopulmonary bypass (CPB), the effects of ECMO on neurocognitive function are less well described. This review aims to summarize recent findings as they pertain to pathophysiology, monitoring techniques, prevention, therapy, and emerging experimental concepts in the context of ECMO for adult patients. Given that neurocognitive outcomes after cardiac surgery have been recently reviewed [3,4], we will limit the discussion of findings from the cardiac surgery/CPB literature to those especially relevant for ECMO.

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.

Cerebral ischemia caused by loss of blood supply to the brain during cardiac arrest or stroke are major causes of death and disability. Biological sex is an important factor in predicting vulnerability of the brain to an ischemic insult, with males being at higher risk for cardio-cerebrovascular events than females of the same age. However, relative incidence of stroke between the genders appears to normalize at advanced ages. Therefore, many scientists have focused on the mechanisms of sex differences in outcome following brain ischemic injury, with a particular emphasis on the role of sex steroids. The majority of studies indicate that female sex steroids, such as estrogen and progesterone, play important roles in the relative neuroprotection following cerebral ischemia observed in females. However, less is known about male sex steroids and brain damage. This review describes the state of our knowledge of androgen-related contributions to neurological injury and recovery following cerebral ischemia that occurs following stroke. Experimental studies examining the effects of castration, androgenic agonists and antagonists and aging provide valuable insights into the role of androgens in clinical outcome following cerebrovascular events.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

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.

Every year in the United States, millions of individuals incur ischemic brain injury from stroke, cardiac arrest, or traumatic brain injury (TBI). These forms of acquired brain injury can lead to death, or in many cases long-term neurologic and neuropsychological impairments. The mechanisms of ischemic and traumatic brain injuries that lead to these deficiencies result from a complex interplay of multiple interdependent molecular pathways that include excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, inflammation, and apoptosis. This article briefly reviews several of the traditional, well-known mechanisms of brain injury and then discusses more recent developments and newer mechanisms. Although much is known concerning mechanisms of injury and the manipulation of these mechanisms to result in protection of neurons and increased behavioral performance in animal models of injury, it has been difficult to translate these effects to humans. Attention is given to why this is so and newer outcome measures of injury are discussed.

TRPM2 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.Fluorescent 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.TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) 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.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 clinically relevant therapeutic window.