Human umbilical cord blood‐derived mesenchymal stem cells improve functional recovery through thrombospondin1, pantraxin3, and vascular endothelial growth factor in the ischemic rat brain

Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)‐derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB‐MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB‐MSC group was significantly improved compared with that of the vehicle‐injected control group. The infarct was also remarkably smaller in the hUCB‐MSC group. Additionally, hUCB‐MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB‐MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB‐MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB‐MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti‐inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia. © 2015 Wiley Periodicals, Inc.     

Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia‐induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 × 10⁶ immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotorphic factors and cytokines was evaluated by quantitative real‐time RT‐PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC‐transplanted brain, among many neurotrofic factors, only human insulin‐like growth factor 1 (IGF‐1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF‐1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain. © 2009 Wiley‐Liss, Inc.     

Long‐term fate of grafted hippocampal neural progenitor cells following ischemic injury

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long‐term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB‐Cg‐Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP‐positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long‐term fate of grafted GFP‐positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP‐positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury. © 2014 Wiley Periodicals, Inc.     

Mesenchymal stem cells attenuate MRI‐identifiable injury,protect white matter,and improve long‐term functional outcomes after neonatal focal stroke in rats

Cell therapy has emerged as a potential treatment for many neurodegenerative diseases including stroke and neonatal ischemic brain injury. Delayed intranasal administration of mesenchymal stem cells (MSCs) after experimental hypoxia‐ischemia and after a transient middle cerebral artery occlusion (tMCAO) in neonatal rats has shown improvement in long‐term functional outcomes, but the effects of MSCs on white matter injury (WMI) are insufficiently understood. In this study we used longitudinal T2‐weighted (T2W) and diffusion tensor magnetic resonance imaging (MRI) to characterize chronic injury after tMCAO induced in postnatal day 10 (P10) rats and examined the effects of delayed MSC administration on WMI, axonal coverage, and long‐term somatosensory function. We show unilateral injury‐ and region‐dependent changes in diffusion fraction anisotropy 1 and 2 weeks after tMCAO that correspond to accumulation of degraded myelin basic protein, astrocytosis, and decreased axonal coverage. With the use of stringent T2W‐based injury criteria at 72 hr after tMCAO to randomize neonatal rats to receive intranasal MSCs or vehicle, we show that a single MSC administration attenuates WMI and enhances somatosensory function 28 days after stroke. A positive correlation was found between MSC‐enhanced white matter integrity and functional performance in injured neonatal rats. Collectively, these data indicate that the damage induced by tMCAO progresses over time and is halted by administration of MSCs. © 2016 Wiley Periodicals, Inc.     

GluN2B N‐methyl‐D‐aspartic acid receptor subunit mediates atorvastatin‐Induced neuroprotection after focal cerebral ischemia

Statins are potent cholesterol biosynthesis inhibitors that exert protective effects in humans and in experimental models of stroke. The mechanisms involved in these protective actions are not completely understood. This study evaluates whether atorvastatin (ATV) treatment affects the GluN1 and GluN2B subunits of the N‐methyl‐D‐aspartic acid receptor in the somatosensory cerebral cortex at short and long periods following ischemia. Sham and ischemic male Wistar rats received 10 mg/kg of ATV or placebo by gavage every 24 hr for 3 consecutive days. The first dose was administered 6 hr after ischemia–reperfusion or the sham operation. ATV treatment resulted in faster recovery of neurological scores than placebo, prevented the appearance of pyknotic neurons, and restored microtubule‐associated protein 2 and neuronal nuclei staining to control values in the somatosensory cerebral cortex and the hippocampus at 72 hr and 15 days postischemia. Furthermore, ATV prevented spatial learning and memory deficits caused by cerebral ischemia. Cerebral ischemia reduced the number of GluN1/PSD‐95 and GluN2B/PSD‐95 colocalization clusters in cortical pyramidal neurons and reduced the levels of brain‐derived neurotrophic factor (BDNF) in the cerebral cortex. These effects of the ischemic insult were prevented by ATV, which also induced GluN2B/PSD‐95 colocalization in neuronal processes and an association of GluN2B with TrkB. The GluN2B pharmacological inhibitor ifenprodil prevented the increase in BDNF levels and the motor and cognitive function recovery caused by ATV in ischemic rats. These findings indicate that GluN2B is involved in the neuroprotective mechanism elicited by ATV to promote motor and cognitive recovery after focal cerebral ischemia. © 2014 Wiley Periodicals, Inc.     

Rat model of perinatal hypoxic‐ischemic brain damage

To gain insights into the pathogenesis and management of perinatal hypoxic‐ischemic brain damage, the authors have used an immature rat model which they developed many years ago. The model entails ligation of one common carotid artery followed thereafter by systemic hypoxia. The insult produces permanent hypoxic‐ischemic brain damage limited to the cerebral hemisphere ipsilateral to the carotid artery occlusion. The mini‐review describes recently accomplished research pertaining to the use of the immature rat model, specifically, investigations involving energy metabolism, glucose transporter proteins, free radical injury, and seizures superimposed upon cerebral hypoxia‐ischemia. Future research will focus on molecular mechanisms of neuronal injury with a continuing focus on therapeutic strategies to prevent or minimize hypoxic‐ischemic brain damage. J. Neurosci. Res. 55:158–163, 1999. © 1999 Wiley‐Liss, Inc.     

Neuronal death/survival signaling pathways in cerebral ischemia

Cumulative evidence suggests that apoptosis plays a pivotal role in cell deathin vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury inin vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.     

Therapeutic hypothermia in experimental models of focal and global cerebral ischemia and intracerebral hemorrhage

Experimental evidence shows that therapeutic hypothermia (TH) protects the brain from cerebral injury in multiple ways. In different models of focal and global cerebral ischemia, mild-to-moderate hypothermia reduces mortality and neuronal injury and improves neurological outcome. In models of experimental intracerebral hemorrhage (ICH), TH reduces edema formation but does not show consistent benefi cial effects on functional outcome parameters. However, the number of studies of hypothermia on ICH is still limited. TH is most effective when applied before or during the ischemic event, and its neuroprotective properties vary according to species, strains and the model of ischemia used. Intrinsic changes in body and brain temperature frequently occur in experimental models of focal and global cerebral ischemia, and may have infl uenced studies on other neuroprotectants. This might be one explanation for the failure of a large amount of translational clinical neuroprotective trials. Hypothermia is the only neuroprotective therapeutic agent for cerebral ischemia that has successfully managed the transfer from bench to bedside, and it is an approved therapy for patients after cardiac arrest and children with hypoxic-ischemic encephalopathy. However, the implementation of hypothermia in the treatment of stroke patients is still far from routine clinical practice. In this article, the authors describe the development of TH in different models of focal and global cerebral ischemia, point out why hypothermia is so efficient in experimental cerebral ischemia, explain why temperature regulation is essential for further neuroprotective studies and discuss why TH for acute ischemic stroke still remains a promising but controversial therapeutic option.     

12 hours after cerebral ischemia is the optimal time for bone marrow mesenchymal stem cell transplantation

Cell therapy using stem cell transplantation against cerebral ischemia has been reported. However, it remains controversial regarding the optimal time for cell transplantation and the transplantation route. Rat models of cerebral ischemia were established by occlusion of the middle cerebral artery. At 1, 12 hours, 1, 3, 5 and 7 days after cerebral ischemia, bone marrow mesenchymal stem cells were injected via the tail vein. At 28 days after cerebral ischemia, rat neurological function was evaluated using a 6-point grading scale and the pathological change of ischemic cerebral tissue was observed by hematoxylin-eosin staining. Under the fluorescence microscope, the migration of bone marrow mesenchymal stem cells was examined by PKH labeling. Caspase-3 activity was measured using spectrophotometry. The optimal neurological function recovery, lowest degree of ischemic cerebral damage, greatest number of bone marrow mesenchymal stem cells migrating to peri-ischemic area, and lowest caspase-3 activity in the ischemic cerebral tissue were observed in rats that underwent bone marrow mesenchymal stem cell transplantation at 12 hours after cerebral ischemia. These findings suggest that 12 hours after cerebral ischemia is the optimal time for tail vein injection of bone marrow mesenchymal stem cell transplantation against cerebral ischemia, and the strongest neuroprotective effect of this cell therapy appears at this time.     

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia–ischemia

Perinatal hypoxia–ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia–ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia–ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24 h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia–ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia–ischemia and a high-dose of MSCs improved motor function.     

骨髓基质细胞立体定向移植治疗大鼠脑缺血损伤的实验研究

目的:观察骨髓基质细胞立体定向移植对大鼠脑缺血损伤后神经功能恢复的作用并探讨其作用机制。方法:制作SD大鼠大脑中动脉缺血模型(MCAO);体外培养骨髓基质细胞,观察其生物学特性以及立体定向移植后对脑缺血损伤后神经功能改善情况。结果:骨髓基质细胞体外可以长期传代、扩增,分泌NGF、VEGF等多种神经保护性因子;立体定向移植后,骨髓基质细胞在脑内存活、迁徙,小部分分化成具有神经元表面标志的细胞,与对照组相比,骨髓基质细胞移植组神经功能改善情况好于对照组。结论:骨髓基质细胞具有多向分化潜能,表达并分泌多种神经保护性营养因子。立体定向移植MSCs,对改善脑缺血损伤后的神经功能状况具有积极作用。     

No barbiturate protection in a dog model of complete cerebral ischemia.

Complete global ischemia was produced in 39 dogs by temporary ligation of the aorta. Prior to the ischemic episode, pentobarbital (30 to 45 mg per kilogram of body weight) was administered to 19 of these dogs. The neurological effects of cerebral ischemia episodes lasting 8, 9, or 10 minutes were compared in dogs treated with pentobarbital and those not treated. At 48 hours following the ischemic episode most of the dogs made ischemic for 8 minutes were normal, whereas most animals made ischemic for 10 minutes were dead or comatose. The 9-minute ischemic period resulted in a relatively even distribution of normal and damaged dogs. There were no differences between treated and untreated dogs. Cerebral blood flow, cerebral metabolic rate for oxygen, and various cerebral metabolites were measured in dogs surviving 48 hours. Again, there were no differences between treated and undertreated dogs. We conclude that barbiturates provide no protection in this model of complete global ischemia. This conclusion supports the hypothesis that the likely mechanism of barbiturate protection in models of incomplete ischemia or hypoxia is based on cerebral metabolic depression; such a mechanism would not be expected to be effective in complete global ischemia.     

Validation of a four-vessel occlusion model for transient global cerebral ischemia in dogs

A new model of transient global cerebral ischemia in dogs with minimal measures of intervention is described together with a simple scale for evaluation of functional outcome. During pentobarbital anesthesia, a global cerebral ischemia lasting seven minutes was induced by a four-vessel occlusion and a controlled systemic hypotension. The reperfusion phase begun after removal of arterial clamps, and the animals were sacrificed by perfusion fixation 24 hours latter. The efficiency of controlled systemic hypotension in diminishing collateral blood flow was validated in two experimental groups with different cerebral filling pressure (CFP). Severe ischemia group (CFP 1.0-1.5 kPa) underwent near-complete ischemia as indicated by rCBF, electroencephalography, and histologically documented ischemic neuronal changes. Mild ischemia group (CFP 2.5-3 kPa) animals experienced reduction in cerebral blood flow well above the ischemic threshold, had better functional outcome as well as no ischemic neuronal changes on light microscopy. This model consistently produces global cerebral ischemia in dogs with minimal surgical intervention and pharmacological support, and without intracranial hypertension, cardiac arrest or asphyxia. We recommend this model for outcome-oriented studies of complete forebrain ischemia in dogs.     

转染肝细胞生长因子的骨髓间质细胞治疗大鼠缺血性脑卒中

目的探讨转染肝细胞生长因子（HGF）的骨髓间质细胞（MSCs）治疗大鼠脑缺血的效果。方法制备大鼠一过性大脑中动脉缺血（MCAO）模型。MCAO后24h分别将磷酸盐溶液（PBS）、MSCs和转染HGF的MSCs（MSC—HGF）通过立体定向技术植人缺血脑组织。通过改良神经功能严重性评分（mNSS）评价神经功能,应用免疫组织化学染色分析脑组织HGF表达、缺血脑组织边缘带细胞凋亡及存活神经元。结果治疗后2周,MSC—HGF组mNSS明显低于PBS组和EMCs组（P〈0.05）。治疗后第1天MSC—HGF组病变脑组织中HGF蛋白表达明显高于PBS组和EMCs组（P〈0．05）。治疗后1周,与PBS组和EMCs组相比,MSC—HGF组在梗死边缘带凋亡细胞的百分比明显减少（P〈0．05）,存活神经元的百分比明显增加（P〈0.05）。结论转染HGF的MSCs治疗缺血性脑卒中具有抗细胞凋亡、神经保护作用。     

Suppression of hypoxia‐inducible factor‐1α and its downstream genes reduces acute hyperglycemia‐enhanced hemorrhagic transformation in a rat model of cerebral ischemia

We evaluated a role of hypoxia‐inducible factor‐1α (HIF‐1α) and its downstream genes in acute hyperglycemia‐induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague‐Dawley rats weighing 280–300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF‐1α inhibitors, 2‐methoxyestradiol (2ME2) or 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole (YC‐1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF‐1α inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF‐1α was increased in ischemic brain tissues after MCAO and reduced by HIF‐1α inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase‐2 and ?9 (MMP‐2/MMP‐9) in the ipsilateral hemisphere. Both 2ME2 and YC‐1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF‐1α and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia. © 2010 Wiley‐Liss, Inc.     

骨髓基质细胞移植治疗大鼠实验性脑梗死的疗效分析及组织病理学观察

目的 观察大脑中动脉闭塞(MCAO)模型大鼠脑内移植骨髓基质细胞fBMSCs)的治疗作用,分析植入梗死灶不同区域的BMSCs的存活、迁移情况以及植入细胞的行为与脑内微环境中GFAP阳性细胞的形态关系.方法 75只成年SD大鼠采用随机数字表法分为MCAO组(n=50)和BMSCs移植组(n=25),所有动物均采用线栓法制作MCAO 1 h模型,24 h后BMSCs移植组脑内注射BrdU标记的同种异体BMSCs(2x106个),MCAO组注射等量PBS.MCAO前及MCAO后第1(移植前)、3、5、7、10、14天应用加速转轮试验和贴纸去除试验检测神经功能缺损情况;第14天处死动物,取脑组织切片应用HE染色观察两组的缺血病灶范围,行BrdU和GFAP免疫组化染色观察BMSCs在不同区域和不同胶质细胞环境下的存活和迁移情况.结果 BMSCs移植组MCAO后7 d加速转轮试验结果优于MCAO组,差异有统计学意义(P＜0.05);组织学观察发现植入缺血半暗带区的细胞存活数量最多,且向病变方向放射状迁移,植入缺血病灶核心的细胞甚少存活,且无迁移现象.结论 BMSCs脑内移植可改善MCAO后大鼠神经运动功能;活化的星形胶质细胞构成适合植入细胞存活、迁移的环境,而胶质瘢痕阻碍了细胞的迁移.     

Neuroprotection against permanent focal cerebral ischemia by ginkgolides A and B is associated with obstruction of the mitochondrial apoptotic pathway via inhibition of c‐Jun N‐terminal kinase in rats

We have previously reported that ginkgolides containing ginkgolides A and B (GKAB) reduce infarct size in a rat model of focal ischemia. c‐Jun N‐terminal kinase (JNK), also known as stress‐activated kinase (SAPK), is a critical stress‐responsive kinase activated by various brain insults. Previous studies have demonstrated a brief increase in p‐SAPK/JNK levels after focal ischemic brain injuries. In this study, we sought to investigate whether the neuroprotective effects of GKAB in rat models of permanent focal cerebral ischemia are associated with the JNK signaling pathway. Sprague‐Dawley rats were subjected to permanent middle cerebral artery occlusion by intraluminal suture blockade. GKAB was injected intravenously immediately after ischemia onset. Here we demonstrate in rats that GKAB reduces neuronal apoptosis and blocks the increase of p‐SAPK/JNK levels and nuclear translocation after cerebral ischemia in a dose‐dependent manner. Furthermore, we report that cerebral ischemia increases ischemia‐induced induction of reactive oxygen species, and this effect was blocked by GKAB. In addition, we show that BimL is induced and attenuated by GKAB. GKAB also repressed the ischemia‐induced increase in the expression of Bax and reversed the decline in expression of Bcl‐2. Likewise, there was a reduction in the release or activation of several mitochondrial proapoptotic molecules, including cytochrome c, caspases 3 and 9, and PARP. Taken together, our findings strongly suggest that GKAB‐mediated neuroprotective effects against focal ischemia act through the inhibition of p‐SAPK/JNK activation, in which the obstruction of the mitochondrial apoptotic pathway via the JNK signaling pathway is a key downstream mechanism of GKAB. © 2013 Wiley Periodicals, Inc.     

Sexually Dimorphic Response of TRPM2 Inhibition Following Cardiac Arrest-Induced Global Cerebral Ischemia in Mice

Transient global cerebral ischemia due to cardiac arrest followed by resuscitation (CA/CPR) causes significant neurological damage in vulnerable neuron populations within the brain, such as hippocampal CA1 neurons. In recent years, we have implicated the transient receptor potential M2 (TRPM2) channel as a mediator of ischemic injury to neurons. We previously demonstrated that genetic and pharmacological strategies that reduce TRPM2 function preferentially protect male neurons in vitro and reduce infarct volume following experimental stroke. Due to the narrow therapeutic window for intervention following ischemic stroke, it is important to assess the role of TRPM2 in other models of cerebral ischemia. Therefore, this study utilized a modified mouse model of CA/CPR to mimic more accurately the clinical condition by maintaining body and head temperatures near the physiological range throughout. Here, we report that inhibition of TRPM2 activity with clotrimazole reduces hippocampal CA1 neuronal injury when administered 30 min after resuscitation from cardiac arrest. Consistent with our previous observations, neuroprotection was observed in male mice and no effect on injury was observed in the female. These findings provide further evidence for TRPM2 as a target for protection against cerebral ischemia in the male brain.