Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia

The beneficial effect of hemodilution on cerebral blood flow (CBF) during focal cerebral ischemia is mitigated by reduced arterial oxygen content (CaO2). In anesthetized cats subjected to permanent middle cerebral artery occlusion, the time course of regional CBF was evaluated after isovolemic exchange transfusion with either albumin or a tetrameric hemoglobin-based oxygen carrier. The transfusion started 30 min after arterial occlusion. We tested the hypothesis that bulk oxygen transport (CBF x CaO2) to ischemic tissue is increased by hemoglobin transfusion at a hematocrit of 18% compared with albumin-transfused cats at a hematocrit of 18% or control cats at a hematocrit of 30% and equivalent arterial pressure. In the nonischemic hemisphere, CBF increased selectively after albumin transfusion, and oxygen transport was similar among groups. In the ischemic cortex, albumin transfusion increased CBF, but oxygen transport was not increased above that of the control group. Hemoglobin transfusion increased both CBF and oxygen transport in the ischemic cortex above values in the control group, but the increase was delayed until 4 h of ischemia. Consequently, acute injury volume measured at 6 h of ischemia was not significantly attenuated. In contrast to the cortex, CBF in the ischemic caudate nucleus was not substantially increased by either albumin or hemoglobin transfusion. Therefore, in a large animal model of permanent focal ischemia in which transfusion starts 30 min after ischemia, tetrameric cross-linked hemoglobin transfusion can augment oxygen transport to the ischemic cortex, but the increase can be delayed and not necessarily provide protection. Moreover, an end-artery region such as the caudate nucleus is less likely to benefit from hemodilution.     

Influence of the corticotropin releasing hormone (CRH) on the brain-blood barrier permeability in cerebral ischemia in rats.

The increase in the blood-brain barrier (BBB) permeability and a developing cerebral oedema due to the ischemic infarction appear a few hours, and intensify during a few days, after closing the carotid arteries. It fails to be clear, however, what causes the increase in the microvessels damage, and whether the damage is a secondary result of the vasoactive substances released by the neurones and glia cells damaged by the ischemia. CRH, which plays an essential role in integrative the nervous, endocrine, and immunological systems, has a positive effect on the decrease in the permeability of the BBB damaged by various physical and chemical factors. Therefore, the examination of the CRH role in the cerebral ischemia may prove useful for explaining the processes taking place in the foci of the cerebral infarction and their environment. The experiment was carried out on rats which, 20 minutes before closing of both internal carotid arteries, was administered 10 microg CRH to cerebrospinal fluid via cisterna magna of the brain. The BBB permeability was measured 30 minutes, 3 hours, 3 days, and 7 days after closing the arteries. The experiment has shown the CRH protective effect on the BBB and its consequent effect on the decrease in the BBB permeability which appears in the 3 hours after closing the arteries (p<0.05), and is high significant during the chronic phase of the cerebral ischemia (p<0.03). It can be thus concluded that CRH, by affecting directly the endothelium of the cerebral vessels, decreases the endothelial damage in the acute phase of the ischemia. The decrease is noted to be more significant in the chronic phase of the ischemia; such an effect can be attributed to CRH stimulating the hypothalamic-adrenal axis, and to the secondary activation of the mechanisms decreasing the BBB permeability.     

MDA, oxypurines, and nucleosides relate to reperfusion in short-term incomplete cerebral ischemia in the rat.

Short-term incomplete cerebral ischemia (5 min) was induced in the rat by the bilateral clamping of the common carotid arteries. Reperfusion was obtained by removing carotid clamping and was carried out for the following 10 min. Animals were sacrificed either at the end of ischemia or reperfusion. Controls were represented by a group of sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. Neutralized perchloric acid extracts of brain tissue were analyzed by a highly sensitive high-performance liquid chromatography (HPLC) method for the direct determination of malondialdehyde, oxypurines, nucleosides, nicotinic coenzymes, and high-energy phosphates. In addition, plasma concentrations of malondialdehyde, hypoxanthine, xanthine, inosine, uric acid, and adenosine were determined by the same HPLC technique. Incomplete cerebral ischemia induced the appearance of a significant amount (8.05 nmol/g w.w.; SD = 2.82) of cerebral malondialdehyde (which was undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde (53.30 nmol/g w.w.; SD = 17.77) and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia (0.050 mumol/L; SD = 0.015), significantly increased after 5 min of ischemia (0.277 mumol/L; SD = 0.056) and was strikingly augmented after 10 min of reperfusion (0.682 mumol/L; SD = 0.094). A similar trend was observed for xanthine, uric acid, inosine, and adenosine, while hypoxanthine reached its maximal concentration after 5 min of incomplete ischemia, being significantly decreased after reperfusion. From the data obtained, it can be concluded that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat. In particular, these results suggest the possibility of using the variation of malondialdehyde, oxypurines, and nucleosides in peripheral blood as a potential biochemical indicator of reperfusion damage occurring to postischemic tissues.     

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

目的：探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法：健康雄性SD大鼠60只,随机分为：假手术（Sham）组（n=10）,脑缺血和缺血/再灌注模型组（n=50）.模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果：脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组（P<0.01）;缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组（P<0.05）;缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组（P<0.01）。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异（P<0.01）。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组（P<0.05）。HE染色显示：仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论：局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。     

HBO suppresses Nogo-A,Ng-R,or RhoA expression in the cerebral cortex after global ischemia

Nogo-A, a myelin-associated neurite outgrowth inhibitory protein, binds with the Ng-R receptor to activate RhoA intracellular signals and inhibit the plasticity after CNS injury. We evaluated the effect of hyperbaric oxygen (HBO) on the expression of Nogo-A, Ng-R, and RhoA after transient global ischemia in a rat 2 vessel occlusion global ischemic model. Male SD rats (n=78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia, and 6 groups of HBO treatment after global ischemia. HBO (3ATA) was applied for 2 hr at 1 hr after global ischemia. Rats were sacrificed at 6, 12, 24, 48, and 96 hr and 7 days. Global ischemia (10 min) produced a marked increase of Nogo-A/B, Nogo-A, Ng-R, and RhoA expression. Immunohistochemistry showed increased Nogo-A/B and Nogo-A located in the myelin sheath of ischemic brain cortex. Ng-R expressed on the surface of neurons and their processes, and RhoA expressed inside the cytoplasm of neurons in ischemic brain. HBO significantly reduced neurological injury, decreased the levels of Nogo-A, Ng-R, and RhoA in ischemic injured cortex (p<0.05).     

扩布性阻抑与脑缺血

研究表明,扩布性阻抑（ＳＤ）不仅与脑缺血,偏头痛,癫痫,颅脑创伤等疾患有关,而且可影响睡眠类型,觉醒等生理过程。本文重点对ＳＤ与脑缺血的关系进行了综述,订 叙述了以下几个问题：（１）ＳＤ的发现及研究简史;（２）完整脑内的ＳＤ;（３）局灶性脑梗塞周围的ＳＤ。     

Effects of transient, global, cerebral ischemia on striatal extracellular dopamine, serotonin and their metabolites

Rat striatal extracellular fluid levels of dopamine, serotonin, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured before, during and after transient, global cerebral ischemia in awake rats using in vivo brain microdialysis. Before ischemia, extracellular levels of dopamine, DOPAC, HVA and 5-HIAA were detectable and consistent from sample to sample. During cerebral ischemia, there was a large increase in extracellular dopamine levels and a decrease in the extracellular levels of DOPAC, HVA, and 5-HIAA. During reperfusion, dopamine levels returned to normal as did those of DOPAC, HVA and 5-HIAA. Dialysate serotonin and 3-methoxytyramine concentrations were below detection limits except for samples collected during ischemia and early reperfusion.     

The angiotensin I converting enzyme inhibitors, captopril and Wy-44,655 attenuate the consequences of cerebral ischemia in renovascular hypertensive rats

Global cerebral ischemia (four vessel model) was induced in renovascular hypertensive rats (two kidney, one clip model) chronically treated with intraperitoneal administration of angiotensin I converting enzyme inhibitors, either captopril (100 mg/kg per day) or Wy-44,655 (10 mg/kg per day). Mortality following cerebral ischemia was higher in renovascular hypertensive rats than in normotensive controls. Reduction of blood pressure with captopril or Wy-44,655, lowered mortality. In surviving renovascular hypertensive and normotensive rats cerebral ischemia induced hyperactivity and lesions of the CA1 area of the hippocampus. Prolonged treatment with captopril--but not with Wy-44,655--reduced hyperactivity and the extent of the CA1 lesions. In conclusion, hypertension increases mortality following cerebral ischemia but does not affect the extent of brain injury in survivors. Prior treatment with converting enzyme inhibitors lowers mortality. Treatment with captopril attenuates brain injury in survivors.     

Changes in brain energy metabolism,neurotransmitters, and choline during and after incomplete cerebral ischemia in spontaneously hypertensive rats

In order to investigate changes in energy metabolism, neurotransmitters, and membrane disorder accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats was utilized. We measured concentrations of ATP, phosphocreatine (PCr), lactate (Lac), glucose (Glu), acetylcholine (ACh), choline (Ch), and -aminobutyric acid (GABA) in both the cerebral cortex and the subcortical regions after 1 h ischemia, 2 h ischemia, and 2 h reflow following 2 h ischemia, and then examined changes in concentrations of these substances during and after incomplete cerebral ischemia. Also examined were interrelations of changes in these substance levels during ischemia. In the cerebral cortex, levels of ATP, PCr, Glu, and ACh decreased, and levels of Lac, Ch, and GABA increased during ischemia. After recirculation, levels of ATP, PCr, Ch, and GABA tended to return to the normal range. On the other hand, the Lac level remained in the ischemic range and the Glu level rose and greatly exceeded the normal range. With regard to ACh, most animals showed normal levels but some exceeded the normal range. Changes in the subcortical regions were qualitatively the same as those in the cerebral cortex during and after ischemia (except with Glu), but only smaller in degrees. Glu levels remained unchanged during ischemia. Correlation of the levels of these substances in the cerebral cortex was examined using normal and ischemic values. A high correlation was generally observed between ATP and other substance levels. The relations between ATP and either PCr or Glu levels were linear. The relation between ATP and ACh levels was logarithmic. The relations between ATP and either Lac, Ch, or GABA levels were exponential. Namely, ACh, Lac, Ch, and GABA levels stayed constant until ATP fell to some fixed low level, suggesting the existence of a threshold. High correlations were also observed among Lac, Ch, and GABA levels.     

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.     

Expression of prostaglandin E2 receptor subtypes, EP2 and EP4, in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the distribution and time course of expression of two subtypes of prostaglandin E₂ (PGE₂) receptors, EP2 and EP4, in a rat model of cerebral ischemia and ischemic tolerance. Adult male Sprague-Dawley rats were subjected to either lethal global ischemia (10 min) with or without sublethal ischemic preconditioning (3 min), or ischemia only (3 min). A short 3-min cerebral ischemia and a 3-min ischemia followed by a second lethal ischemia enhanced the expression of EP2 and EP4 receptors in CA1 pyramidal neurons of the hippocampus. In tolerance-acquired CA1 neurons, the immunoreactivities of EP2 and EP4 were upregulated after 4 h and 12 h, respectively. The immunoreactivities were most prominent at 3 days and were sustained for at least 14 days, consistent with results of immunoblotting experiments. However, immunoreactivities for these PGE₂ receptors increased in reactive glial cells in the vulnerable CA1 and hilar regions of rats subjected to lethal ischemia without ischemic preconditioning. Most of the EP2 immunoreactivity occurred in microglial cells and some astrocytes, whereas increased immunoreactivity for EP4 was found only in astrocytes. These data suggest that ischemia and the induction of ischemia tolerance have different regulatory effects on the expression of EP2 and EP4 receptors. Moreover, PGE₂ may exert its unique pathophysiological functions in relation to delayed neuronal death and ischemic tolerance induction in the rat hippocampus via specific PGE₂ receptors.This research was supported by a grant (M103KV010019 04K2201 01930) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.     

Changes in cerebral free fatty acids and triacylglycerols in focal cerebral ischemia

1. Focal cerebral ischemia was induced in anesthetized rats by occluding the stem of the proximal middle cerebral artery. 2. The levels of free fatty acids, such as stearic and arachidonic acids, in the ischemic cerebral cortex increased progressively until 60 min after occlusion, but thereafter they decreased rapidly. 3. In contrast to the time-dependent changes in free fatty acids, the levels of triacylglycerol (TAG) in the ischemic cerebral cortex continued to increase for 120 min after occlusion. Increases in TAG-palmitate, -stearate and -arachidonate accounted for the increase in the triacylglycerol level. 4. The pattern of the lipid changes in focal cerebral ischemia differs from those reported in bilateral diffuse cerebral ischemia induced by arterial occlusion or in decapitation ischemia.     

Ginkgo biloba extract (EGb 761) protects Na,K-ATPase isoenzymes during cerebral ischemia.

Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.     

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.     

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2-7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.     

Protective effect of resveratrol against neuronal damage following transient global cerebral ischemia in mice

Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol which is rich in grape seeds and skin. Several studies have revealed that resveratrol possesses neuroprotective effects. In the case of global brain ischemia, there are few reports regarding the protective effect of resveratrol. Therefore, the influence of resveratrol on neuronal damage after transient global brain ischemia remains to be clarified. In the current study, C57BL/6 black mice were subjected to 20 min of transient global brain ischemia and followed by 72 h of reperfusion. Resveratrol (20 or 40 mg/kg, once daily, dissolved in 0.5% carboxymethylcellulose) was administered orally for 7 days before ischemia and daily until the mice were euthanized. The effect of lower or higher dose of resveratrol on neuronal damage, matrix metalloproteinase (MMP) activity and in situ DNA fragmentation (TUNEL) assay in the hippocampus after global ischemia was examined. Neuronal damages were remarkable in CA1 and CA2 pyramidal cell layers after global ischemia. In resveratrol-treated mice (40 mg/kg), neuronal damage was significantly reduced compared with vehicle-treated mice. Mice treated with resveratrol showed reduced MMP-9 activity. Resveratrol also inhibited TUNEL staining. These data suggest that resveratrol, a natural polyphenol, reduces hippocampal neuronal cell damage following transient global ischemia by reducing MMP-9 activity.     

Neuroendocrine mechanism for tolerance to cerebral ischemia‐reperfusion injury in male rats

Testosterone has been shown to exacerbate cerebral ischemia‐reperfusion injury, which suggests that the well‐known stress‐induced testosterone reduction could be a protective response. We hypothesized that stress‐induced testosterone reduction contributes to ischemia tolerance in cerebral ischemia‐reperfusion injury in male rats. In intact male rats, stress was induced by brief anesthesia at 6 h before transient middle cerebral artery occlusion (MCAO). Testosterone levels were significantly decreased 6 h after stress. Testosterone reduction was associated with a 50% reduction in cerebral lesion volume in the stressed animals. Further, the stress‐induced cerebral ischemia tolerance was eliminated by testosterone replacement in castrated males. Immunohistochemical staining showed that androgen receptors were up‐regulated after cerebral ischemia‐reperfusion injury and partially colocalized with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in the parietal cortex and extensively colocalized in the caudate putamen. Heat shock protein 70 (Hsp70) and 90 (Hsp90) are involved in ischemia tolerance, and were not colocalized with TUNEL in the immunohistochemical staining, suggesting an antiapoptotic role of Hsp's. To determine the effect of testosterone on MCAO‐induced Hsp70 and ‐90 expression, a testosterone replacement or withdrawal paradigm was used. Testosterone‐replaced animals exhibited a decrease in Hsp expression, whereas testosterone withdrawal (mimicking the stress‐induced testosterone suppression) normalized this deficit. In summary, stress‐induced testosterone reduction contributes to ischemia tolerance in cerebral ischemia‐reperfusion injury in males, which could be related to the loss of inhibition by testosterone of Hsp70 and ‐90 expression. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Carvacrol, a food-additive, provides neuroprotection on focal cerebral ischemia/reperfusion injury in mice

Carvacrol (CAR), a naturally occurring monoterpenic phenol and food additive, has been shown to have antimicrobials, antitumor, and antidepressant-like activities. A previous study demonstrated that CAR has the ability to protect liver against ischemia/reperfusion injury in rats. In this study, we investigated the protective effects of CAR on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that CAR (50 mg/kg) significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. This neuroprotection was in a dose-dependent manner. Post-treatment with CAR still provided protection on infarct volume when it was administered intraperitoneally at 2 h after reperfusion; however, intracerebroventricular post-treatment reduced infarct volume even when the mice were treated with CAR at 6 h after reperfusion. These findings indicated that CAR has an extended therapeutic window, but delivery strategies may affect the protective effects of CAR. Further, we found that CAR significantly decreased the level of cleaved caspase-3, a marker of apoptosis, suggesting the anti-apoptotic activity of CAR. Finally, our data indicated that CAR treatment increased the level of phosphorylated Akt and the neuroprotection of CAR was reversed by a PI3K inhibitor LY-294002, demonstrating the involvement of the PI3K/Akt pathway in the anti-apoptotic mechanisms of CAR. Due to its safety and wide use in the food industry, CAR is a promising agent to be translated into clinical trials.     

脑缺血大鼠海马信号转导与转录激活子-3的激活及其调控

以往的研究表明,在脑缺血/再灌注的皮层和纹状体组织中信号转导与转录激活子-3(STAT3)被激活。本实验旨在研究SD大鼠四动脉结扎诱导的全脑缺血是否引起海马组织STAT3的快速激活及其调控机制。结果表明,脑缺血导致STAT3快速磷酸化激活及DNA结合活性增加。胞浆STAT3的磷酸化水平从缺血5min起就显著增高,10min达高峰(增加约1．7倍),然后开始下降。核内STAT3的磷酸化水平则逐渐增加,缺血30min时达高峰(增加约2．3倍)。电泳迁移率改变分析法显示,STAT3的DNA结合活性从缺血5min起就显著增加,30min达高峰(增加约3．2倍)。进一步的研究表明,缺血前20min腹腔注射给药,然后缺血30min,发现蛋白酪氨酸激酶抑制剂染料木黄酮和抗氧化剂N-乙酞半胱氨酸能显著地抑制核内STAT3的磷酸化水平及DNA结合活性的增加(磷酸化水平从2．3和2．5倍分别降为1．2和1．4倍,DNA结合活性则从2．8和3．7倍分别降为1．1和1．5倍),而蛋白酪氨酸磷酸酶抑制剂矾酸钠则能明显地促进他们的增高(磷酸化水平从2．0倍增到3．4倍,DNA结合活性从3．1倍增为5．1倍)。这些结果提示,蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶可能共同参与了缺血诱导STAT3的激活调控,STAT3的激活可能有助于海马神经元适应氧化应激。     

Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.