Mitochondrial porin required for ischemia-induced mitochondrial dysfunction and neuronal damage

The precise molecular events of mitochondrial dysfunction, one of the last steps that irreversibly determines cellular degeneration and death, remain unknown. We introduce a novel strategy to isolate and assess the molecular mechanisms underlying mitochondrial dysfunction. Using an in vitro ischemia model, we obtained evidence for prolonged mitochondrial depolarization in rat organotypic hippocampal brain slices during reperfusion. Then, mitochondria were isolated from brain slices and mitochondrial proteins were purified on a cyclosporin-A affinity column. Cyclosporin-A is the most potent inhibitor of mitochondrial dysfunction, in particular the mitochondrial permeability transition, and therefore we hypothesized that it may interact with proteins involved in the permeability transition after mitochondria were subjected to manipulations that promote this event. Mitochondrial porin was reproducibly eluted from the affinity column using proteins from ischemic brain mitochondria, or from mitochondria exposed to oxidative stress that were used as a positive control. Anti-porin antibodies prevented mitochondrial depolarization and electrophysiological deterioration of hippocampal neurons during hypoxia-reperfusion, as measured by simultaneous fluorescence imaging and whole-cell recordings.These observations provide biochemical and functional evidence that porin is directly involved in mitochondrial dysfunction and neuronal impairment during ischemia-reperfusion, and indicate that porin could be a novel therapeutic target to prevent cellular degeneration.     

Combination effects of normobaric hyperoxia and edaravone on focal cerebral ischemia-induced neuronal damage in mice

We evaluated the potential neuroprotective effects of combination treatment with normobaric hyperoxia (NBO) and edaravone, a potent scavenger of hydroxyl radicals, on acute brain injuries after stroke. Mice subjected to 2-h filamental middle cerebral artery occlusion were treated with NBO (95% O2, during the ischemia) alone, with edaravone (1.5 mg/kg, intravenously after the ischemia) alone, with both of these treatments (combination), or with vehicle. The histological and neurological score were assessed at 22-h after reperfusion. Infarct volume was significantly reduced in the combination group [36.3+/-6.7 mm3 (n=10) vs. vehicle: 65.5+/-5.9 mm3 (n=14) P<0.05], but not in the two monotherapy-groups [NBO: 50.5+/-5.8 mm3 (n=14) and edaravone: 56.7+/-5.8 mm3 (n=10)]. The combination therapy reduced TUNEL-positive cells in the ischemic boundary zone both in cortex [6.0+/-1.4 x 10(2)/mm2 (n=5) vs. vehicle: 18.9+/-2.4 x 10(2)/mm2 (n=5), P<0.01] and subcortex [11.6+/-1.5 x 10(2)/mm2 (n=5) vs. vehicle: 22.5+/-2.1 x 10(2)/mm2 (n=5), P<0.01]. NBO and combination groups exhibited significantly reduced neurological deficit scores at 22-h after reperfusion (vs. vehicle, P<0.05). Combination therapy with NBO plus edaravone prevented the neuronal damage after focal cerebral ischemia and reperfusion in mice, compared with monotherapy of NBO or edaravone.     

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, whether these events have a causal role in ischemia-induced neuronal death is unclear. We found that the Bcl-2 and Bcl-x(L) inhibitor ABT-737, which enhances death of tumor cells, protected rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-x(L) is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment, ΔN-Bcl-x(L). We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-x(L)-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-x(L), we generated knock-in mice expressing a caspase-resistant form of Bcl-x(L). The knock-in mice exhibited markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings suggest that truncated Bcl-x(L) could be a potentially important therapeutic target in ischemic brain injury.     

内源性组胺减轻小鼠前脑缺血再灌注后期脑损伤*

 目的： 研究内源性组胺在前脑缺血再灌注后期的神经保护作用。方法: 将野生型（WT）小鼠和组氨酸脱羧酶基因敲除（HDC-KO）小鼠各随机分为对照组和缺血组,缺血组小鼠双侧颈总动脉夹闭30 min以建立前脑缺血模型,比较再灌后WT和HDC-KO小鼠的体重变化和死亡率,并在再灌14 d时对各组存活小鼠进行条件性恐惧学习记忆测试,再灌3 d及15 d时,对各组小鼠取脑,制作冰冻切片,进行甲苯胺蓝染色,观察海马CA1区神经元损伤情况。结果: 再灌1 d后,WT和HDC-KO小鼠均出现体重下降,再灌4 d、5 d、6 d及7 d后,HDC-KO小鼠的体重恢复程度均显著低于WT小鼠。前脑缺血再灌8~14 d,HDC-KO小鼠的死亡率显著高于WT小鼠（P<0.05）。再灌14 d后,HDC-KO小鼠的背景及线索记忆能力均显著低于WT小鼠（P<0.05）。再灌3 d后,HDC-KO和WT小鼠的海马CA1区神经元密度无显著差异,而再灌15 d后,HDC-KO小鼠海马CA1区神经元密度显著低于WT小鼠（P<0.05）。结论: 内源性组胺可减轻脑缺血再灌注后期的学习记忆能力下降及神经元缺失,但其作用机制有待进一步研究。     

Long-term treadmill exercise overcomes ischemia-induced apoptotic neuronal cell death in gerbils

It has been suggested that exercise may ameliorate neurologic impairment by impeding neuronal loss following various brain insults. In the present study, the effect of long-term treadmill exercise on short-term memory and apoptotic neuronal cell death in the hippocampus following transient global ischemia in gerbils was investigated. A step-down inhibitory avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were used for this study. Ischemia was induced by occlusion of both the common carotid arteries of gerbils for 5 min. Gerbils in the exercise groups were forced to run on a treadmill for 30 min once a day for 4 consecutive weeks. The present results reveal that treadmill exercise for 4 weeks improved short-term memory by suppressing the ischemia-induced apoptotic neuronal cell death in the hippocampus. Here in this study, we show that long-term treadmill exercise for 4 weeks overcomes the ischemia-induced apoptotic neuronal cell death and thus facilitates the recovery of short-term memory impairment induced by ischemic cerebral injury.     

Ibuprofen protects ischemia-induced neuronal injury via up-regulating interleukin-1 receptor antagonist expression

The inflammatory response accompanies and exacerbates the developing injury after cerebral ischemia. Ibuprofen, a non-steroidal anti-inflammatory drug, has been shown to attenuate injuries in animal models of various neurological diseases. In the present study, we investigated ibuprofen's neuroprotective effects in rats exposed to transient forebrain ischemia and in cultures exposed to oxygen glucose deprivation (OGD). Rats treated with ibuprofen after transient forebrain ischemia displayed long-lasting protection of CA1 hippocampal neurons. There were selective increases in interleukin-1 receptor antagonist gene and protein expression in ibuprofen-treated OGD microglia. Furthermore, treatment with ibuprofen in neuron/microglia co-cultures increased the number of surviving HC2S2 neurons against OGD whereas IL-1ra neutralizing antibody reversed the ibuprofen-induced neuroprotection. The data indicate that ibuprofen-induced IL-1ra secretion is involved in neuroprotection against ischemic conditions.     

Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus

Injury to the brain often results in loss of synapses or cell death in the damaged area. Subsequent to the injury, the areas that are not directly affected often exhibit enhanced neuronal plasticity. Although there are many reports of morphological changes resulting from such plasticity, their functional consequences are poorly understood. In this study we examined electrophysiological changes associated with ischemia-induced neurogenesis in the hippocampus, a brain region that is particularly vulnerable but also exceptionally plastic. Transient global ischemia was induced in Sprague-Dawley rats by occlusion of both carotid arteries and a reduction in blood pressure for 12 min. The procedure resulted in delayed cell death in the CA1 field of the hippocampus while the dentate gyrus (DG) was spared. To assess neurogenesis and synaptic changes in parallel we used both hemispheres from each animal. One side was used for immunohistochemistry and the other for in vitro electrophysiological experiments in brain slices. Synaptic field responses and synaptic plasticity (LTP) in perforant path within the DG were reduced by 50% at 10 days after the ischemic injury but recovered at 35 days. Synaptic responses in non-neurogenic CA1 were abolished in parallel with cell death and did not recover. Gamma irradiation applied focally to the head selectively prevented neurogenesis and the synaptic recovery in the DG. These experiments reveal electrophysiological changes associated with reactive neural plasticity in the hippocampus.     

Treadmill exercise improves short-term memory by suppressing ischemia-induced apoptosis of neuronal cells in gerbils

In the present study, the effect of treadmill exercise on short-term memory, apoptotic neuronal cell death, and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils was investigated. Step-down inhibitory avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 and 5-bromo-2'-deoxyuridine (BrdU) were used. Ischemia was induced by the occlusion of both common carotid arteries (CCA) of gerbils for 5 min. Gerbils in exercise groups were forced to run on a treadmill for 30 min once a day for 10 consecutive days. Such treadmill exercise improved short-term memory by suppressing the ischemia-induced apoptotic neuronal cell death in the dentate gyrus. In addition, treadmill running suppressed the ischemia-induced cell proliferation in the dentate gyrus. The present results suggest that treadmill exercise overcomes the ischemia-induced apoptotic neuronal cell death and thus facilitates the recovery following ischemic cerebral injury.     

Pharmacologic amelioration of severe hypoglycemia-induced neuronal damage

Hypoglycemia is a common complication for insulin treated people with diabetes. Severe hypoglycemia, which occurs in the setting of excess or ill-timed insulin administration, has been shown to cause brain damage. Previous pre-clinical studies have shown that memantine (an N-methyl-d-aspartate receptor antagonist) and erythropoietin can be neuroprotective in other models of brain injury. We hypothesized that these agents might also be neuroprotective in response to severe hypoglycemia-induced brain damage. To test this hypothesis, 9-week old, awake, male Sprague-Dawley rats underwent hyperinsulinemic (0.2 U kg(-1)min(-1)) hypoglycemic clamps to induce severe hypoglycemia (blood glucose 10-15 mg/dl for 90 min). Animals were randomized into control (vehicle) or pharmacological treatments (memantine or erythropoietin). One week after severe hypoglycemia, neuronal damage was assessed by Fluoro-Jade B and hematoxylin and eosin staining of brain sections. Treatment with both memantine and erythropoietin significantly decreased severe hypoglycemia-induced neuronal damage in the cortex by 35% and 39%, respectively (both p<0.05 vs. controls). These findings demonstrate that memantine and erythropoietin provide a protective effect against severe hypoglycemia-induced neuronal damage.     

迟发性神经元损伤发生中与自由基的作用机制及防治

本文用沙土鼠制作迟发性神经元损伤动物模型。从自由基代谢角度观察了动物脑6个分区5种生化指标在缺血前后7个时相的变化情况。结果表明,海马CA_1区有其独特变化规律,突出地表现为:在短时脑缺血后1—96小时的重灌流过程中,自由基含量呈持续性升高,线粒体中的锰超氧化物歧化酶则呈进行性降低,铜锌超氧化物歧化酶和脂质过氧化物含量的变化则都具有波动性。在除CA_2区外的其余部分脑组织中则没有上述这些变化。本实验的结果表明:自由基代谢紊乱是迟发性神元经损伤发生机理中的关键环节。经进一步研究发现,Aniacetam、豆腐果甙和尼卡地平对这种自由基代谢紊乱都有不同程度的改善作用。     

Cilostazol protects against hemorrhagic transformation in mice transient focal cerebral ischemia-induced brain damage

Cilostazol, an antiplatelet drug used to treat intermittent claudication, has been reported to offer neuroprotection and endothelial protection in animals with ischemic brain injury. Here, we evaluated the protection afforded by cilostazol against ischemic brain injury and hemorrhagic transformation. Mice subjected to a 2-h filamental middle cerebral artery (MCA) occlusion were treated with cilostazol (10 mg/kg, intraperitoneally just after the occlusion) or with vehicle. Histological outcomes (infarct volume and hemorrhagic transformation) and Evans blue extravasation were assessed after reperfusion. Mean infarct volume, hemorrhagic transformation, and Evans blue extravasation were all significantly reduced in the cilostazol-treated group. Thus, cilostazol protected against ischemic brain injury and hemorrhagic transformation in mice subjected to transient focal cerebral ischemia.     

Protective effect of high glucose against ischemia-induced synaptic transmission damage in rat hippocampal slices

Cerebral ischemic damage is an important cause of morbidity and mortality. However, there is conflicting evidence regarding the effect of the extracellular glucose concentration in focal and global ischemic injury. This study was designed to investigate this effect in ischemia-induced synaptic transmission damage in rat hippocampal slices. Slices were superfused with artificial cerebrospinal fluid (ACSF) containing various concentrations of glucose before and after ischemia. The evoked somatic postsynaptic population spike (PS) and dendritic field excitatory postsynaptic potential (fEPSP) were extracellularly recorded in the CA1 stratum pyramidal cell layer and s. radiatum after stimulation of the Schaeffer collaterals, respectively. The glucose concentration in ACSF before and after ischemia determined the duration of ischemia tolerated by synaptic transmission as demonstrated by complete recovery of the somatic PS and dendritic fEPSP. Specifically, the somatic PS and dendritic fEPSP completely recovered following 3, 4, and 5 min of ischemia only when slices were superfused with ACSF containing 4, 10, and 20 mM glucose before and after ischemia, respectively. The latencies of the somatic and dendritic ischemic depolarization (ID) occurrence in the CA1 s. pyramidal cell layer and s. radiatum were significantly longer with 10 than 4 mM glucose in ACSF before ischemia and significantly longer with 20 than 10 mM glucose in ACSF before ischemia. Regardless of the glucose concentration in ACSF before and after ischemia, the somatic PS and dendritic fEPSP only partially recovered when ischemia was terminated at the occurrence of ID. These results indicate that high glucose in ACSF during the period before and after ischemia significantly protects CA1 synaptic transmission against in vitro ischemia-induced damage through postponing the occurrence of ID.     

Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia

Reactive oxygen species have been implicated in brain injury after cerebral ischemia. These oxidants can damage proteins, lipids, and DNA, and lead to cell injury and necrosis. Oxidants are also initiators in intracellular cell death signaling pathways that may lead to apoptosis. The possible targets of this redox signaling include mitochondria, death membrane receptors, and DNA repair enzymes. Genetic manipulation of intrinsic antioxidants and the factors in the signaling pathways has provided substantial progress in understanding the mechanisms in cell death signaling pathways and involvement of oxygen radicals in ischemic brain injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.     

Cell wall-mediated neuronal damage in early sepsis

Neuronal dysfunction can occur in the course of sepsis without meningitis. Sepsis-associated neuronal damage (SAND) was observed in the hippocampus within hours in experimental pneumococcal bacteremia. Intravascular challenge with purified bacterial cell wall recapitulated SAND. SAND persisted in PAFr(-/-) mice but was partially mitigated in mice lacking cell wall recognition proteins TLR2 and Nod2 and in mice overexpressing interleukin-10 (IL-10) in macrophages. Thus, cell wall drives SAND through IL-10-repressible inflammatory events. Treatment with CDP-choline ameliorated SAND, suggesting that it may be an effective adjunctive therapy to increase survival and reduce organ damage in sepsis.     

Beta-hydroxybutyric acid attenuates neuronal damage in epileptic mice

β-Hydroxybutyric acid (BHBA) reportedly has neuroprotective and anti-oxidation properties. The present study aimed to investigate the protective effects of BHBA against epilepsy. C57BL/6 J mice were exposed to lithium chloride and pilocarpine to induce epilepsy and then were administrated with 300 mg/kg/day BHBA for 30 days. The learning impairment was evaluated via Morris Water Maze. Neuron loss and cell apoptosis were detected through Nissl staining and TUNEL staining. The levels of oxidative stress-related factors were determined by commercial kits. The protein expression levels of AMP-activated protein kinase (AMPK), p-AMPK, peroxisome proliferator-activated receptor alpha (PPARα), anti-apoptotic Bcl-2, and pro-apoptotic Bax were measured through Western blots. It was found BHBA improved epilepsy- caused learning deficiency and attenuated epilepsy-mediated neuron loss and cell apoptosis in the hippocampus. BHBA ameliorated oxidative stress via decreasing the levels of reactive oxygen species and malondialdehyde plus strengthening the activities of glutathione peroxidase and superoxide dismutase. BHBA also promoted the phosphorylation of AMPK and upregulated PPARα in the epileptic hippocampus. In conclusion, BHBA attenuates neuronal damage in epileptic mice, which is associated with its anti-apoptotic and anti-oxidative effects as well as the activation of AMPK and PPARα.     

Pneumolysin causes neuronal cell death through mitochondrial damage

Bacterial toxins such as pneumolysin are key mediators of cytotoxicity in infections. Pneumolysin is a pore-forming toxin released by Streptococcus pneumoniae, the major cause of bacterial meningitis. We found that pneumolysin is the pneumococcal factor that accounts for the cell death pathways induced by live bacteria in primary neurons. The pore-forming activity of pneumolysin is essential for the induction of mitochondrial damage and apoptosis. Pneumolysin colocalized with mitochondrial membranes, altered the mitochondrial membrane potential, and caused the release of apoptosis-inducing factor and cell death. Pneumolysin induced neuronal apoptosis without activating caspase-1, -3, or -8. Wild-type pneumococci also induced apoptosis without activation of caspase-3, whereas pneumolysin-negative pneumococci activated caspase-3 through the release of bacterial hydrogen peroxide. Pneumolysin caused upregulation of X-chromosome-linked inhibitor of apoptosis protein and inhibited staurosporine-induced caspase activation, suggesting the presence of actively suppressive mechanisms on caspases. In conclusion, our results indicate additional functions of pneumolysin as a mitochondrial toxin and as a determinant of caspase-independent apoptosis. Considering this, blocking of pneumolysin may be a promising cytoprotective strategy in pneumococcal meningitis and other infections.     

Motor impairment and neuronal damage following hypothermia in tropical amphibians

Although the induction of mild to moderate cerebral hypothermia in mammals can have neuroprotective activity, some deleterious effects have been described when inducing deep hypothermia during cooling of the brain. In the spinal cord, rapid deep cooling can induce seizure activity accompanied by release of the excitatory neurotransmitters, glutamate and aspartate. We used cold-sensitive tropical amphibians as a model to determine (a) the critical temperature inside the central nervous system necessary to induce seizures during rapid cooling; (b) the survival rate during slow deep cooling of the whole animal; and (c) whether deep cooling can cause neuronal cell damage. Seizures induced by deep rapid (or=30 min) deep cooling of the whole animal (12 h at 2-3 degrees C), around 70% of animals died. Spinal reflexes were enhanced when temperatures within the spinal cord reached between 9.0 degrees C and 11.6 degrees C. A fivefold increase in blood glucose level was observed during slow deep cooling. Recovery after slow deep cooling was accompanied by motor impairment and the main histological findings were condensation of the cytoplasm and nuclear pyknosis. Severe neuronal cell damage was characterized by swelling, vacuolated cytoplasm with distended neuronal bodies. These results indicate that deep cooling can easily induce neuronal cell damage in the central nervous system of cold-sensitive animals. They also warn us to the potential sequels associated with the use of deep brain cooling as a neuroprotective strategy.