Although inflammatory responses increase stroke severity, the role of immune cells specific for central nervous system (CNS) antigens remains controversial. Disruption of the blood-brain barrier (BBB) during stroke allows CNS antigens to leak into the peripheral circulation and enhances access of circulating leukocytes to the brain, including those specific for CNS antigens such as myelin oligodendrocyte glycoprotein (MOG) that can induce experimental autoimmune encephalomyelitis (EAE). We here demonstrate for the first time that myelin reactive splenocytes specific for MOG transferred into severe combined immunodeficient (SCID) mice can migrate into the infarct hemisphere of recipients subjected to 60 min middle cerebral artery occlusion (MCAO) and 96 h reperfusion; moreover these cells exacerbate infarct volume and worsen neurological deficits compared to animals transferred with naïve splenocytes. These findings indicate that autoimmunity in the CNS can exert detrimental injury on brain cells and worsen the damage from ischemic stroke.

A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a therapeutically relevant timing after middle cerebral artery occlusion (MCAO) and verify functional benefit to complement histological outcome measures. We observed that the administration of mouse-specific RTL551 reduced infarct size and improved sensorimotor outcome when administered within a 3 h post-ischemic therapeutic window. RTL551 treatment reduced cortical, caudate putamen, and total infarct volume as compared to vehicle-treated mice. Using a standard behavioral testing repertoire, we observed that RTL551 reduced sensorimotor impairment 3 days after MCAO. Humanized RTL1000 (HLA-DR2 moiety linked to hMOG-35-55 peptide) also reduced infarct size in HLA-DR2 transgenic mice. These data indicate that this neuroantigen-specific immunomodulatory agent reduces damage when administered in a therapeutically relevant reperfusion timeframe.

BACKGROUND Peripheral nerve blocks with local anesthetics (LAs) are commonly performed to provide surgical anesthesia or postoperative analgesia. Nerve injury resulting in persistent numbness or weakness is a potentially serious complication. Local anesthetics have previously been shown to damage neuronal and Schwann cells via several mechanisms. We sought to test the hypothesis that LAs are toxic to Schwann cells and that the degree of toxicity is directly related to the concentration of LA and duration of exposure. Intraneural injection of LAs has been shown to produce nerve injury. We sought to test the hypothesis that a prolonged extraneural infusion of LA can also produce injury. METHODS Schwann cells cultured from neonatal rat sciatic nerves were incubated with LAs at different concentrations (10, 100, 500, and 1000 μM), and each concentration was assessed for toxicity after 4, 24, 48 and 72 hours of exposure. Local anesthetics tested were lidocaine, mepivacaine, chloroprocaine, ropivacaine, and bupivacaine. Cell death was assessed by lactate dehydrogenase release measured by optical density.In a separate experiment, a microcatheter was placed along the sciatic nerves of Sprague-Dawley rats. Rats were randomly assigned to receive either 0.9% saline (n = 8) or bupivacaine (0.5%, n = 4; 0.75%, n = 4) via the perineural catheters for 72 hours. The rats were then killed, and their nerves sectioned and stained for analysis. Sections were stained for myelin and with an antimacrophage (CD68) antibody. RESULTS None of the LAs tested produced significant Schwann cell death at very low concentrations (10 μM, or 0.0003%) even after prolonged exposure. With prolonged exposure (48 or 72 hrs) to high concentrations (1000 μM, or 0.03%), all of the LAs tested produced significant Schwann cell death (increased lactate dehydrogenase release relative to control as measured by optical density, 0.384-0.974; all P values < 0.001). Only bupivacaine produced significant cell death (0.482, P < 0.001) after prolonged exposure to low concentrations (100 μM, or 0.003%). At intermediate concentrations (500 μM, or 0.015%), cell death was more widespread with bupivacaine (0.768, P < 0.001) and ropivacaine (0.675, P < 0.001) than the other agents (0.204-0.368; all P values < 0.001). Prolonged extraneural exposure of rat sciatic nerves to bupivacaine caused significant demyelination and infiltration of nerves with inflammatory cells. CONCLUSIONS Local anesthetics induce Schwann cell death in a time- and concentration-dependent manner. Bupivacaine and ropivacaine have greater toxicity at intermediate concentrations, and prolonged exposure to bupivacaine produces significant toxicity even at low concentrations. Brief exposure to high concentrations of bupivacaine damages Schwann cells. Prolonged extraneural infusion of bupivacaine results in nerve injury.

BACKGROUND AND PURPOSE Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion strongly implicates a mixture of both pathogenic and regulatory immune cell subsets that affect stroke outcome. Our goal was to evaluate the contribution of the well-described coinhibitory pathway, programmed death (PD)-1, to the development of middle cerebral artery occlusion. METHODS Infarct volumes, functional outcomes, and effects on infiltrating immune cell populations were compared in wild-type C57BL/6 versus PD-1-deficient mice after 60 minutes middle cerebral artery occlusion and 96 hours reperfusion. RESULTS The results clearly demonstrate a previously unrecognized activity of the PD-1 pathway to limit infarct volume, recruitment of inflammatory cells from the periphery, activation of macrophages and central nervous system microglia, and functional neurological deficits. These regulatory functions were associated with increased percentages of circulating PD-ligand-1 and PD-ligand-2 expressing CD19(+) B-cells in blood, the spleen, and central nervous system with the capacity to inhibit activation of inflammatory T-cells and central nervous system macrophages and microglial cells through upregulated PD-1. CONCLUSIONS Our novel observations are the first to implicate PD-1 signaling as a major protective pathway for limiting central nervous system inflammation in middle cerebral artery occlusion. This inhibitory circuit would likely be pivotal in reducing stroke-associated Toll-like receptor-2- and Toll like receptor-4-mediated release of neurotoxic factors by activated central nervous system microglia.

Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B cells to the development of MCAO by comparing infarct volumes and functional outcomes in wild-type (WT) versus B-cell-deficient μMT(-/-) mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits, and increased numbers of activated T cells, macrophages, microglial cells, and neutrophils in the affected brain hemisphere of MCAO-treated μMT(-/-) versus WT mice. These MCAO-induced changes were completely prevented in B-cell-restored μMT(-/-) mice after transfer of highly purified WT GFP(+) B cells that were detected in the periphery, but not the CNS. In contrast, transfer of B cells from IL-10(-/-) mice had no effect on infarct volume when transferred into μMT(-/-) mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B cells to limit infarct volume, mortality rate, recruitment of inflammatory cells, and functional neurological deficits 48 h after MCAO. Our novel observations are the first to implicate IL-10-secreting B cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B cells might have application as a novel therapy for this devastating neurologic condition.

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.

BACKGROUND AND PURPOSE Treatment of ischemic stroke by activation of endogenous plasminogen using tissue plasminogen activator is limited by bleeding side effects. In mice, treatment of experimental ischemic stroke with activated protein C improves outcomes; however, activated protein C also has bleeding side effects. In contrast, activation of endogenous protein C using thrombin mutant W215A/E217A (WE) is antithrombotic without hemostasis impairment in primates. Therefore, we investigated the outcome of WE-treated experimental ischemic stroke in mice. METHODS The middle cerebral artery was occluded with a filament for 60 minutes to induce ischemic stroke. Vehicle, recombinant WE, or tissue plasminogen activator was administered during middle cerebral artery occlusion or 2 hours after middle cerebral artery occlusion. Neurological performance was scored daily. Intracranial bleeding and cerebral infarct size, defined by 2,3,5-triphenyltetrazolium chloride exclusion, were determined on autopsy. Hemostasis was evaluated using tail bleeding tests. RESULTS WE improved neurological performance scores, increased laser Doppler flowmetry-monitored post-middle cerebral artery occlusion reperfusion of the parietal cortex, and reduced 2,3,5-triphenyltetrazolium chloride-defined cerebral infarct size versus vehicle controls. However, unlike tissue plasminogen activator, WE did not increase tail bleeding or intracranial hemorrhage. CONCLUSIONS WE treatment is neuroprotective without hemostasis impairment in experimental acute ischemic stroke in mice and thus may provide an alternative to tissue plasminogen activator for stroke treatment.

Stroke induces a biphasic effect on the peripheral immune response that involves early activation of peripheral leukocytes followed by severe immunosuppression and atrophy of the spleen. Peripheral immune cells, including T lymphocytes, migrate to the brain and exacerbate the developing infarct. Recombinant T-cell receptor (TCR) Ligand (RTL)551 is designed as a partial TCR agonist for myelin oligodendrocyte glycoprotein (MOG)-reactive T cells and has demonstrated the capacity to limit infarct volume and inflammation in brain when administered to mice undergoing middle cerebral artery occlusion (MCAO). The goal of this study was to determine if RTL551 could retain protection when given within the therapeutically relevant 4 h time window currently in clinical practice for stroke patients. RTL551 was administered subcutaneously 4 h after MCAO, with repeated doses every 24 h until the time of euthanasia. Cell numbers were assessed in the brain, blood, spleen and lymph nodes and infarct size was measured after 24 and 96 h reperfusion. RTL551 reduced infarct size in both cortex and striatum at 24 h and in cortex at 96 h after MCAO and inhibited the accumulation of inflammatory cells in brain at both time points. At 24 h post-MCAO, RTL551 reduced the frequency of the activation marker, CD44, on T-cells in blood and in the ischemic hemisphere. Moreover, RTL551 reduced expression of the chemokine receptors, CCR5 in lymph nodes and spleen, and CCR7 in the blood and lymph nodes. These data demonstrate effective treatment of experimental stroke with RTL551 within a therapeutically relevant 4 h time window through immune regulation of myelin-reactive inflammatory T-cells.

Male animals exhibit greater neuronal damage following focal cerebral ischemic injury in many experimental injury models, however the mechanism of this is unknown. This study used cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in male mice exposed to physiological vs. pharmacological doses of testosterone and tested the hypothesis that testosterone increases damage following global cerebral ischemia. Analysis of histological damage 72h after resuscitation revealed a complex dose-response curve for testosterone, such that low and high doses of testosterone exacerbated ischemic neuronal damage, while intermediate doses had no effect on neuronal survival. In agreement with these histological observations of neuronal damage, both low and high doses of testosterone increased sensorimotor deficit following CA/CPR compared to vehicle treated animals. Finally, the androgen receptor antagonist flutamide inhibited the increase in neuronal damage and sensorimotor impairment observed in testosterone treated mice. Our data showed that low and supra-physiological levels of testosterone increase neuronal damage following global cerebral ischemia and that blockade of androgen receptors limits this injury. Therefore, this study indicated that testosterone may have a role in determining sex-linked differences in cerebrovascular disease as well as having important health implications in clinical conditions of elevated testosterone.

Sex steroids are essential for reproduction and development in animals and humans, and sex steroids also play an important role in neuroprotection following brain injury. New data indicate that sex-specific responses to brain injury occur at the cellular and molecular levels. This review summarizes the current understanding of neuroprotection by sex steroids, particularly estrogen, androgen, and progesterone, based on both in vitro and in vivo studies. Better understanding of the role of sex steroids under physiological and pathological conditions will help us to develop novel effective therapeutic strategies for brain injury.