Ischemic pre-conditioning preserves brain mitochondrial functions during the middle cerebral artery occlusion in rat

It is well known that a brief period of ischemia increases tolerance to a subsequent severe ischemic episode. In the present study, bilateral carotid arteries occlusion (BCAO) was applied as pre-conditioning to testify whether this kind of ischemia could preserve the function of mitochondria with the impairment induced by middle cerebral artery occlusion (MCAO) in brain. The activities of respiratory enzyme complex I to IV, mitochondria swelling, membrane potential, and membrane fluidity were investigated. The results showed that the percentage of infarct area decreased greatly due to the ischemic pre-conditioning (IP) revealing the preventive effect of IP on infarct size. The activities of respiratory enzyme complex III, IV were effectively preserved (p < 0.05, p < 0.05) compared with MCAO group through ischemic pre-conditioning. Mitochondrial swelling and membrane fluidity were protected by IP, and also an increased trend was found in membrane potential, which indicated that the integrity of mitochondrial membrane was maintained. It suggested that the function of mitochondrial energy metabolism in brain ischemia was effectively protected by this kind of pre-conditioning.     

Ischemic preconditioning increases antioxidants in the brain and peripheral organs after cerebral ischemia

BACKGROUND AND PURPOSE: Low molecular weight antioxidants (LMWA), which reflect tissue reducing power, are among the endogenous mechanisms for neutralizing reactive oxygen species (ROS). Ischemic preconditioning (IPC) was associated with decreased oxidative stress. We examined the effect of focal ischemia on LMWA and on prostaglandin E(2) (PGE(2), a product of arachidonic acid oxidation) in the brain, heart, liver, and lungs of rats subjected to 90 min of ischemia and in IPC rats subjected to similar insult. METHODS: Transient right middle cerebral artery occlusion (MCAO) was performed for 90 min and at 0, 5, 30, 60, or 240 min of reperfusion, LMWA and PGE(2) were evaluated by cyclic voltametry (CV) and radioimmunoassay, respectively. IPC was induced by 2 min of MCAO, 24 h prior to the major ischemic episode. RESULTS: LMWA decreased at 5 min of reperfusion in the brain, heart, liver, and lung and rose 4 h later only in the brain. PGE(2) levels increased three to fivefold in all tissues examined. Surprisingly, in IPC rats a dramatic increase of LMWA occurred at 5 min of reperfusion in the brain and in the peripheral organs. Uric acid, but not ascorbic, is the major LMWA increased. CONCLUSIONS: We propose that after ischemia, ROS rapidly consume the antioxidants reserves in the brain and also in peripheral organs, suggesting that the whole body is under oxidative stress. Moreover, part of the neuroprotection afforded by IPC is mediated by the brain's ability to mobilize antioxidants, especially uric acid, that attenuate the massive ROS-mediated oxidative stress.     

Calcium accumulation in synapses of the rat hippocampus after cerebral ischemia.

The ultrastructural localization of calcium deposits in the synapses of rat hippocampus after 10 min global cerebral ischemia was evaluated. Oxalate-pyroantimonate technique was applied. After 24 hours of postischemic recirculation enhancement of intracellular (pre- and postsynaptic parts) and extracellular (synaptic clefts) calcium deposits was found in great proportion of synapses in CA1 sector. Abundant Ca-precipitates appeared specially in synaptic clefts and in the postsynaptic parts near synaptic densities. Increased calcium deposits in some changed mitochondria were also observed. The results presented in this paper suggest synaptic modulation of Ca2+ homeostasis, disturbed after ischemic incident. Presence of Ca-precipitates in synaptic clefts and postsynaptic parts seems to be a sensitive indicator of increased calcium influx from the extracellular to the intracellular compartments.     

大鼠全脑缺血再灌注损伤后海马白噬相关蛋白LC3表达变化的研究

目的研究自噬微管相关蛋白轻链3（microtubule—associated protein 1 light chain3,LC3）在大鼠全脑缺血损伤后海马的表达情况,并探讨自噬在脑缺血损伤中的意义。方法使用四动脉结扎法制作大鼠全脑缺血模型再灌注损伤模型,实验动物随机分为：假手术组（sham组）、缺血再灌注纽。在全脑缺血15min后,分别再灌注0,30min,3,6,12,24h,1,3d,使用Westernblotting的方法检测各个时间点大鼠全脑缺血／复灌后海马LC3Ⅱ／LC3 Ⅰ蛋白的表达情况来研究脑缺血再灌注损伤后海马自噬现象的变化情况。结果与假手术组相比,大鼠全脑缺血再灌注损伤后3h,海马区LC3Ⅱ／LC3Ⅰ蛋白的表达开始上调,在全脑缺血再灌注损伤后1d表达最强（P〈0．05）。结论大鼠全脑缺血再灌注损伤后海马LC3Ⅱ／LC3Ⅰ蛋白表达上调,表明脑缺血再灌注损伤后海马区域的自噬活性上调。     

缺血预处理对沙土鼠脑缺血再灌注后线粒体功能的影响

目的 观察缺血预处理对沙土鼠脑缺血及再灌注后脑组织线粒体功能的影响。方法 用沙土鼠双侧颈总动脉结扎制成全脑缺血模型（缺血２０分钟,再灌注３０分钟）。动物随机分为预缺血组、模型组和假手术组。预缺血组给予两次（各２分钟）缺血预处理（间隔４８小时）。用氧电极法测定呼吸功能和呼吸链的氧化酶（ＮＡＤＨ氧化酶、琥珀酸氧化酶、细胞色素Ｃ氧化酶）活性。结果 缺血模型组动物的呼吸控制率、磷氧比及氧化磷酸化效率均较假     

Ischemic preconditioning upregulates expression of phospholipase D2 in the rat hippocampus

To investigate the possible involvement of phospholipase D2 (PLD2) in the induction of ischemic tolerance, we analyzed the distribution and time course of PLD2 expression in the rat hippocampus after a sublethal period of ischemia. Forebrain ischemia was induced by four-vessel occlusion for 3 min. Increased PLD2 immunoreactivity after this sublethal ischemia was observed in CA1 pyramidal neurons of the rat hippocampus. In tolerance-acquired CA1 neurons, PLD2 immunoreactivity was upregulated as early as 12 h post-ischemia and was most prominent at 1–3 days, with expression sustained for at least 7 days, as shown by a time course of immunoblotting and measurement of the enzymatic activity of PLD. PLD2 expression was also increased in ischemia-resistant CA3 neurons and dentate granule cells, although weaker staining intensity was noted. Further, we showed that, in cultured SK-N-BE(2)C human neuroblastoma cells, overexpression of PLD2 inhibited cell death by chemical hypoxia induced with potassium cyanide and deoxyglucose. These data suggest that upregulation of PLD2 might be involved in the neuroprotective mechanism of ischemic tolerance in the rat hippocampus. This research was supported by a grant (M103KV010010-06K2201-01010) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea.     

Pattern of neuronal vulnerability in the cat hippocampus after one hour of global cerebral ischemia

Summary The dorsal hippocampus of cat was investigated by light microscopy and immunohistochemistry following 1 h global cerebral ischemia and various recirculation times from 1 day to 1 year. Complete ischemia was produced by combining hypotension with intrathoracic occlusion of major arteries. Postischemic resuscitation was carried out using an intensive care regimen with continuous neurophysiological monitoring. Brains of controls (n=4) and postischemic animals (n=12) were fixed in formaldehyde and prepared for histology and immunohistochemistry of glial fibrillary acidic protein (GFAP). In all post-ischemic animals the hilus and the regio superior of dorsal hippocampus which encompasses the CA1 subfield were severely damaged. Neurons in these regions exhibited the typical sequela of neuronal death. GFAP staining revealed vivid astroglial proliferation in stratum lacunosum-moleculare and stratum oriens. Changes in the regio inferior of dorsal hippocampus, i.e., CA3 subfield, and in dentate gyrus granular layer, were variable. Although most animals exhibited moderate to severe neuronal and glial alterations, groups of surviving cells were observed in the stratum oriens and in the granular layer of dentate gyrus. In one animal the majority of CA3 pyramidal cells and granule cells was preserved. These findings demonstrate that after 1 h of complete cerebral ischemia dorsal hippocampus exhibits two different types of injury: a consistent pattern of selective vulnerability in the hilus and the regio superior, and a variable pattern of non-selective injury in the regio inferior and dentate gyrus. The two patterns can be best explained by intrinsic (pathoclitic) and extrinsic (hemodynamic/edema) factors, respectively and are likely to represent basically different mechanisms of ischemic injury.     

RNAi targeting µ-calpain increases neuron survival and preserves hippocampal function after global brain ischemia

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms has not been determined in in vivo models. Identification of the calpain isoform responsible for neuronal injury is particularly important given the differential role of calpain isoforms in normal physiology. This study evaluates the role of m-calpain and µ-calpain in an in vivo model of global brain ischemia. Adeno-associated viral vectors expressing short hairpin RNAs targeting the catalytic subunits of µ- or m-calpain were used to knockdown expression of the targeted isoforms in adult rat hippocampal CA1 pyramidal neurons. Knockdown of µ-calpain, but not m-calpain, prevented calpain activity 72 h after 6-min transient forebrain ischemia, increased long-term survival and protected hippocampal electrophysiological function. These findings represent the first in vivo evidence that reducing expression of an individual calpain isoform can decrease post-ischemic neuronal death and preserve hippocampal function.     

Co-localization of somatostatin mRNA and parvalbumin in the dorsal rat hippocampus after cerebral ischemia

Following transient global ischemia most of the neurons containing somatostatin in the fascia dentata of the dorsal hippocampal formation die, while somatostatinergic neurons in the CA1 region survive. These neurons react to ischemia with a transiently reduced expression of somatostatin mRNA and peptide. We have tested the hypothesis that this selective vulnerability is solely related to those somatostatinergic neurons which do not express the calcium-binding protein parvalbumin. Postischemic changes were studied in rat dorsal hippocampus at 2 and 16 days after 10 min of global cerebral ischemia using a four-vessel occlusion model. We performed a double-staining visualizing the mRNA coding for somatostatin by non-radioactive in situ hybridization and parvalbumin protein by immunocytochemistry. Only 5% of the somatostatinergic cells in the fascia dentata contained parvalbumin. The number of somatostatinergic cells was permanently reduced following ischemia. Among surviving neurons we found cells with and without parvalbumin expression. Thus, expression of parvalbumin is not predictive for survival of somatostatinergic cells in the fascia dentata. In contrast, in CA1, 37% of the somatostatinergic cells contained parvalbumin. These cells were unaffected by the transient ischemic period. The somatostatinergic cells lacking parvalbumin showed transiently reduced mRNA levels at day 2, but recovered to control values at the 16th postischemic day. Thus, expression of the calcium-buffering protein parvalbumin coincides with resistance of somatostatinergic neurons in CA1 to transient effects of ischemia. We conclude that the calcium-buffering capacity of parvalbumin may partially contribute to the protection of somatostatinergic neurons from ischemia in the dorsal hippocampus. However, the survival of somatostatinergic cells without parvalbumin indicates the importance of other factors as well. © 1995 Wiley-Liss, Inc.     

钾通道阻断剂TEA对大鼠全脑缺血后海马神经元损伤的保护作用

在大鼠四血管夹闭前脑缺血模型上,观察了侧脑室给予钾通道阻断剂四乙基铵（TEA）和4-氨基吡啶（4-AP）对脑缺血后海马CA1区锥体神经元迟发性死亡的保护作用。结果发现：再灌流30min后给予TEA组CA1区存活的锥体细胞数明显高于生理盐水对照组,而再灌流30min后给予4-AP组和缺血前30min给予TEA组的存活细胞数则与生理盐水对照组无明显差别。表明再灌流后给予TEA对脑缺血诱导的海马CA1区锥体神经元死亡具有明显的保护作用,提示钾通道可能在缺血后海马CA1区锥体细胞的迟发性死亡中发挥重要的作用。     

Seizure activity in the rat hippocampus,perirhinal and prefrontal cortex associated with transient global cerebral ischemia

Summary. Epileptiform EEG activity associated with ischemia can contribute to early damage of hippocampal neurons, and seizure activity may also lead to dysfunction in extrahippocampal regions. In this study, seizure activity associated with the four-vessel occlusion model of cerebral ischemia was monitored using chronically implanted electrodes in the CA1/subicular region, the perirhinal cortex, and the prefrontal cortex of the rat. Background EEG amplitude was reduced in all recording sites during occlusion, but spiking and bursting activity was also observed. Seizure activity occurred in most animals during the first several hours of reperfusion, but was not observed on subsequent days. Epileptiform spikes and bursts were often synchronous between two or three recording sites, and spikes in the CA1 region also often occurred just prior to spikes in other sites. These results demonstrate that the four-vessel occlusion model can lead to patterns of seizure activity in the hippocampus, prefrontal and perirhinal cortices. Correspondence: C. Andrew Chapman, Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 7141 Sherbrooke Street West, Rm. SP-244, Montreal, Quebec, H4B 1R6 Canada     

脑缺血后脑细胞线粒体LPO、SOD的变化

我们利用兔MCAO模型,分别测定脑缺血后4h(I_4)、24h(I_(24))脑组织的H_2o、Na~ 、Ca~(2 )、LPO,SOD以及线粒体的LPO,SOD,经与对照组(包括假手术和正常对照)对比,结果发现脑缺血后脑组织H_2o、Na~ 、C_a~(2 )LPO及线粒体LPO均明显增加,而SOD活性变化则相反地降低。结果提示脑缺血后脑细胞线粒体的LPO含量增加,而SOD活性则下降,说明脑缺血后线粒体产生的自由基参与了脑缺血脑损伤机制。     

Bradykinin antagonists reduce leukocyte-endothelium interactions after global cerebral ischemia.

The aim of the present study was to evaluate the influence of bradykinin on microcirculatory changes and outcome after global cerebral ischemia (15 minute) in Mongolian gerbils. The cerebral microcirculation was investigated by fluorescent intravital microscopy. Survival and functional outcome was evaluated up to 4 d after ischemia. Animals were treated with the selective B(1) and B(2) receptor antagonists B 9858 and CP 0597, respectively, and the nonselective B(1)/B(2) receptor antagonist B 9430. Leukocyte activation was significantly reduced by all antagonists as indicated by a significant decrease in the number of rolling (33 +/- 20, 6 +/- 8, 9 +/- 10, and 13 +/- 10) and adherent leukocytes (9 +/- 7, 3 +/- 4, 1 +/- 1, and 2 +/- 3. 100 microm(-1) x min(-1) in controls and in animals treated with B(1), B(2), and B(1)/B(2) antagonist, respectively). Arteriolar diameters were significantly reduced during reperfusion (35 +/- 11 before and 27 +/- 8 microm 40 minutes after ischemia) in animals treated with the B(2) antagonist. The postischemic hypoperfusion, however, was not affected. Mortality was significantly higher in animals treated with the B(1) and the B(1)/B(2) antagonist. The authors concluded that bradykinin is involved in postischemic disturbances of cerebral microcirculation. The therapeutic effect of specific bradykinin receptor antagonists on functional outcome, however, remains unclear.     

alpha-Melanocyte-stimulating hormone is neuroprotective in rat global cerebral ischemia

The aim of the study was to investigate the effects of alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from proopiomelanocortin (POMC), on the neurodegeneration following global cerebral ischemia and reperfusion in the rat. The biological activities of alpha-MSH include inhibition of inflammatory responses and anti-pyretic effects. Male Sprague-Dawley rats were subjected to four-vessel occlusion (4-VO) global cerebral ischemia followed by reperfusion, and treated with alpha-MSH (intraperitoneally, i.p.) at 30 min, and 24, 48, 72 and 96 h post-ischemia. Stereological quantification of the pyramidal cells in the CA1 area of the hippocampus showed that the number of viable neurons in ischemic rats was 96,945+/-18,610 (means+/-SD) as compared to 183,156+/-49,935 in sham-operated rats (P<0.05). The number of viable neurons after treatment of ischemic rats with alpha-MSH was 162,829+/-34,757, i.e. significantly different from the number of viable neurons in ischemic rats injected with saline (P<0.01). Astrocyte proliferation due to the ischemic insult was markedly reduced by the treatment with alpha-MSH, and the loss in body weight was reduced by alpha-MSH. In conclusion, post-ischemic administration of alpha-MSH was found to provide neuroprotection in the CA1 pyramidal cell layer in the hippocampus, concomitant with a reduction in glial activation, indicating that alpha-MSH or mimetics thereof may have a potential in the treatment of stroke or other neurodegenerative diseases. Further studies will be required to define the post-ischemic time window for administration of alpha-MSH.