The Role of Taurine in Neuronal Protection Following Transient Global Forebrain Ischemia

Osmoregulation and post ischemic glutamate surge suppression (PIGSS) are important mechanisms in the neuroprotective properties of taurine. We studied the role of taurine in PIGSS following transient global forebrain ischemia (TGFI). A group of gerbils received a high dose of continuous intracerebral taurine during the peri-ischemic period. -alanine was given similarly to a negative control group. The control group consisted of animals undergoing only TGFI. On the fourth day following commencement of drug administration, TGFI was induced. Concurrently, half the animals from each group receiving an agent had intracerebral microdialysis. All animals underwent histological assessment at day 7. The microdialysis and histological data was analyzed. Our results showed that taurine treatment did not cause PIGSS. The histological difference between the three groups was statistically insignificant. We conclude that intracerebral taurine in the dosage administered during peri-ischemic period, does not result in PIGSS or histologically evident neuroprotection.     

Administration of thiazolidinediones for neuroprotection in ischemic stroke: a pre-clinical systematic review

Thiazolidinediones (TZDs) may prevent or attenuate CNS injury arising from an ischemic event. We performed meta-analysis of experimental studies in which a TZD (either rosiglitazone or pioglitazone) was administered in a rodent model of focal or global cerebral ischemia. Infarct volume was the primary endpoint for analysis of drug efficacy, and neurological outcome was also assessed. We identified 31 studies through the use of PubMed and Embase, 22 of which met our pre-specified inclusion criteria and were analyzed with the Cochrane Review Manager software. Treatment with TZDs decreased infarct volume and improved neurological outcome regardless of study quality, dose timing, or ischemia model (transient or permanent). Rosiglitazone and pioglitazone were similarly effective in reducing infarct volume and protecting neurologic function. Importantly, the collective data suggest that pre-treatment with a TZD is not required for neuroprotection, although additional studies are clearly needed to define the breadth of the therapeutic window. The data warrant further studies into the potential acute use of TZDs for ischemic stroke therapy in the general population.     

SM-31900, a novel NMDA receptor glycine-binding site antagonist,reduces infarct volume induced by permanent middle cerebral artery occlusion in spontaneously hypertensive rats

The purpose of this study was to investigate the effect of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900), an antagonist with high selectivity and affinity for the NMDA receptor glycine-binding site, on the cerebral infarct volume in a permanent middle cerebral artery occlusion (MCAo) model, which was constructed by electrocoagulation of a unilateral middle cerebral artery distal to the olfactory tract using spontaneously hypertensive rats (SHRs). To investigate the dose-response characteristics and the therapeutic time window of SM-31900 in this MCAo model, we conducted three experiments, in which the administration of SM-31900 was started 5min (experiment I), 30min (experiment II), or 60min (experiment III) after MCAo, respectively. In all the studies, SM-31900 was administered by intravenous bolus injection followed by continuous intravenous infusion to obtain a steady-state level of this compound in blood immediately after its administration. The treatment with SM-31900 was continued until 24h after MCAo, at which time the cerebral infarct volume was measured. In experiment I, SM-31900 significantly reduced the infarct volume by 37% at a dosage of 0.38mg/kg bolus followed by 1.5mg/kg/h continuous infusion (0.38mg/kg+1.5mg/kg/h). In experiment II, the neuroprotective effect of SM-31900 was also significant, with a 25% reduction in infarct volume at a dosage of 0.38mg/kg+1.5mg/kg/h, and a 40% reduction at 1.5mg/kg+6.0mg/kg/h. Furthermore, even in experiment III, SM-31900 exerted a significant neuroprotective effect, with a 20% reduction at 1.5mg/kg+6.0mg/kg/h. These studies revealed that SM-31900 can exert a neuroprotective effect when it is administered up to at least 60min after the onset of ischemia in the MCAo model, an animal model of stroke, indicating that SM-31900 is a good candidate for treating acute brain ischemia.     

A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice

Previous studies have demonstrated that a natural coumarin compound esculetin (Esc) possesses antioxidant, anti-tumor, and anti-inflammation activities and rescues cultured primary neurons from NMDA toxicity. In this study, we investigated the neuroprotective effects of Esc on cerebral ischemia/reperfusion (I/R) injury in a middle cerebral artery occlusion model in mice. Esc (20 μg) was administered intracerebroventricularly at 30 min before ischemia. We found that Esc significantly reduced infarct volume and decreased neurological deficit scores after 75 min of ischemia and 24 h of reperfusion. Post-treatment of Esc still provided neuroprotection even when Esc was administered after 4 h of reperfusion. Our data also indicated that intraperitoneal administration of Esc showed protective effects on cerebral I/R injury in a dose-dependent manner. We further explored the protective mechanisms of Esc on cerebral I/R injury and found that Esc decreased cleaved caspase 3 level, a marker of apoptosis. Finally, our data demonstrated that Esc exerted its anti-apoptotic activity by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, two apoptosis-related proteins. Because of its clinical use as an anticoagulant and its safety profile, Esc may have a therapeutic potential for the treatment of stroke in the future clinical trials.     

Topiramate Reduces Energy and Fat Gains in Lean (Fa/?) and Obese (fa/fa) Zucker Rats

Objective: This study examined the effects of topiramate (TPM), a novel neurotherapeutic agent reported to reduce body weight in humans, on the components of energy balance in female Zucker rats. Research Methods and Procedures: A 2 × 3 factorial experiment was performed in which two cohorts of Zucker rats differing in their phenotype (phenotype: lean, Fa/?; obese, fa/fa) were each divided into three groups defined by the dose of TPM administered (dose: TPM 0, vehicle; TPM 15, 15 mg/kg; TPM 60, 60 mg/kg). Results: The reduction in body weight gain induced by TPM in both lean and obese rats reflected a decrease in total body energy gain, which was more evident in obese than in lean rats. Whereas TPM administration did not influence the intake of digestible energy in lean rats, it induced a reduction in food intake in obese animals. In lean, but not in obese rats, apparent energy expenditure (as calculated by the difference between energy intake and energy gain) was higher in rats treated with TPM than in animals administered the vehicle. The low dose of TPM decreased fat gain (with emphasis on subcutaneous fat) without affecting protein gain, whereas the high dose of the drug induced a reduction in both fat and protein gains. The effects of TPM on muscle and fat depot weights were representative of the global effects of TPM on whole body fat and protein gains. The calculated energetic efficiency (energy gain/energy intake) was decreased in both lean and obese rats after TPM treatment. TPM dose independently reduced hyperinsulinemia of obese rats, but it did not alter insulinemia of lean animals. Discussion: The present results provide sound evidence for the ability of TPM to reduce fat and energy gains through reducing energetic efficiency in both lean and obese Zucker rats.     

NG-硝基-L-精氨酸对大鼠局灶性缺血脑区线粒体损伤的影响

目的:观察非选择性一氧化氮合酶抑制剂NG-硝基-L-精氨酸(NG-nitro-L-arginine,L-NA)对局灶性脑缺血大鼠脑线粒体的损伤作用,以探讨其改善缺血性脑损伤的作用机制。方法:将大鼠随机分为假手术组、缺血对照组、L-NA治疗组,采用线栓法阻断大鼠大脑中动脉(MCAO)复制局灶性脑缺血模型,分别于缺血后2h、6h、12h给药治疗3d,迅速断头取脑,差速离心法提取缺血侧脑组织线粒休,迅速测定线粒体膜肿胀度及线粒体活力,测定线粒体总ATP酶、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性,以及线粒休一氧化氮(NO)、丙二醛(MDA)含量:电镜观察缺血后皮层神经元超微结构的改变及L-NA对其影响。结果:在大鼠MCAO后线粒体膜肿胀度增加,线粒体活力下降,线粒体NO、MDA含量明显增加,线粒体总ATP酶、SOD、GSH-Px活性均明显下降:缺血后2h、6h、12h给予L-NA治疗3d与缺血对照组相比NO含量明显下降,缺血后12h治疗组线粒体膜肿胀度、线粒体活力、总ATP酶、SOD、GSH-Px活性均显著升高、MDA含量下降。电镜结果显示脑缺血后皮层神经元水肿,线粒体肿胀、嵴断裂、溶解、消失,且随缺血时间延长损伤加重;缺血后12h给予L-NA治疗能明显改善脑缺血引起的神经元水肿、线粒体肿胀和空泡化。结论:L-NA能明显抑制脑缺血后线粒体NO生成,在缺血早期给予L-NA对缺血性脑损伤无改善作用:缺血后期给予L-NA,能明显降低线粒体膜肿胀程度,改善线粒体能量供应,增强线粒体抗氧化作用及其活力,从而减轻脑缺血损伤。     

Neuroprotective Effects of Poly(ADP-ribose)polymerase Inhibitor Olaparib in Transient Cerebral Ischemia

Olaparib was the first poly(ADP-ribose)polymerase inhibitor approved by Food and Drug Administration for oncology treatment. However, its neuroprotective effects have not been elucidated. This study aimed to evaluate the effects of olaparib in transient cerebral ischemia. A mouse model of transient middle cerebral artery occlusion was used. Reperfusion was performed at 2 h after ischemia. Different doses of olaparib (1, 3, 5, 10 and 25 mg/kg) were administered intraperitoneally immediately after reperfusion. Twenty-four hours after ischemia, the neurological score was assessed, and grip and string tests were performed to evaluate the behavioral deficits in the mice. Cresyl violet staining was used to assess cerebral edema and the lesion volume. Immunohistochemistry was performed to evaluate the expression of blood–brain barrier proteins collagen IV and claudin-5, as well as extravasation of IgG. Ischemia induced a neurological deficit, which was significantly ameliorated by olaparib at 3 and 5 mg/kg. However, this neuroprotective effect was not observed in mice treated with either low-dose or high-dose olaparib. Both 3 and 5 mg/kg olaparib markedly reduced cerebral infarction volume, but not cerebral edema. The expression of collagen IV decreased after cerebral ischemia, which was improved by olaparib at 3 and 5 mg/kg. These results were confirmed by the reduction of IgG extravasation with olaparib. Olaparib showed clear neuroprotective effects in transient ischemic mice mainly through the reduction of cerebral infarction and blood–brain barrier damage.     

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。     

The influence of proglumide, a putative CCK antagonist, on cerebral ischemia in gerbil

Studies were conducted to clarify the possible role of CCK in cerebral ischemia and to evaluate the effects of proglumide, a competitive and reversible CCK antagonist, as a potential therapeutic or prophylactic tool in the treatment of cerebral ischemia. Proglumide at the doses of 10, 50 and 150 mg/kg was administered to gerbils before unilateral carotid ligation, and its effect on stroke index score, incidence and mortality rate was observed. Our results show that proglumide injected prior to carotid ligation at the dose of 150 mg/kg significantly reduces both incidence and mortality rate and changes the distribution of the stroke index score in gerbils. There was a significant inverse relationship between the dose of proglumide and both incidence and mortality: the greater the injection dose of proglumide, the lower the incidence and mortality. These results suggest that CCK may be involved in the pathological processes of cerebral ischemia and proglumide or related compounds prove to be effective in the pharmacological prophylaxis of ischemic brain damage.     

Automated label‐free detection of injured neuron with deep learning by two‐photon microscopy

Stroke is a significant cause of morbidity and long‐term disability globally. Detection of injured neuron is a prerequisite for defining the degree of focal ischemic brain injury, which can be used to guide further therapy. Here, we demonstrate the capability of two‐photon microscopy (TPM) to label‐freely identify injured neurons on unstained thin section and fresh tissue of rat cerebral ischemia‐reperfusion model, revealing definite diagnostic features compared with conventional staining images. Moreover, a deep learning model based on convolutional neural network is developed to automatically detect the location of injured neurons on TPM images. We then apply deep learning‐assisted TPM to evaluate the ischemic regions based on tissue edema, two‐photon excited fluorescence signal intensity, as well as neuronal injury, presenting a novel manner for identifying the infarct core, peri‐infarct area, and remote area. These results propose an automated and label‐free method that could provide supplementary information to augment the diagnostic accuracy, as well as hold the potential to be used as an intravital diagnostic tool for evaluating the effectiveness of drug interventions and predicting potential therapeutics.     

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。     

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.     

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.     

Bone morphogenetic protein-7 (BMP-7) mediates ischemic preconditioning-induced ischemic tolerance via attenuating apoptosis in rat brain

A mild cerebral ischemic insult, also known as ischemic preconditioning (IPC), confers transient tolerance to a subsequent ischemic challenge in the brain. This study was conducted to investigate whether bone morphogenetic protein-7 (BMP-7) is involved in neuroprotection elicited by IPC in a rat model of ischemia. Ischemic tolerance was induced in rats by IPC (15 min middle cerebral artery occlusion, MCAO) at 48 h before lethal ischemia (2 h MCAO). The present data showed that IPC increased BMP-7 mRNA and protein expression after 24 h reperfusion following ischemia in the brain. In rats of ischemia, IPC-induced reduction of cerebral infarct volume and improvement of neuronal morphology were attenuated when BMP-7 was inhibited either by antagonist noggin or short interfering RNA (siRNA) pre-treatment. Besides, cerebral IPC-induced up-regulation of B-cell lymphoma 2 (Bcl-2) and down-regulation of cleaved caspase-3 at 24 h after ischemia/reperfusion (I/R) injury were reversed via inhibition of BMP-7. These findings indicate that BMP-7 mediates IPC-induced tolerance to cerebral I/R, probably through inhibition of apoptosis.     

Prevention and repair of cerebral ischemia-reperfusion injury by Chinese herbal medicine, Shengmai San, in rats

The protective activity of Shengmai San, a traditional Chinese herbal medicine, was studied in cerebral ischemia-reperfusion injury in rats. Shengmai San consists of three herbal components, Panax Ginseng, Ophiopogon Japonicus and Schisandra Chinensis and is routinely being used for treating coronary heart disease.

When Shengmai San was injected directly into rat duodenum 2 h before cerebral ischemia by bilateral carotid artery occlusion, thiobarbituric acid reactive substance (TBARS) formation during reperfusion following ischemia was almost completely suppressed in the brain. The loss of glutathione perioxidase activity after the ischemia-reperfusion was also effectively prevented by the Shengmai San pre-administration whereas the activity was considerably decreased in the damaged brain.

It was found that Shengmai San also effectively suppressed the TBARS formation even when it was administered after 45 min reperfusion following ischemia, indicating that Shengmai San improves the oxidative damage already established in the brain. Likewise, the decrease of glutathione peroxidase activity was minimized in the damaged brain by the post-administration of Shengmai San.

On the other hand, none of the Shengmai San components were active in protecting the ischemia-reperfusion brain damage when they were independently administered.

These experiments suggest the potential of Shengmai San in both preventive and therapeutic usages for cerebral ischemia-reperfusion injury.     

miR-126调控脑缺血大鼠细胞凋亡机制的研究

摘要 目的：探讨脑缺血大鼠microRNA-126（MiR-126）在脑组织和血浆中表达的动态变化及与细胞凋亡的关系。方法：健康雄性SD大鼠45只随机分为3组,其中3只大鼠不作任何处理,用于检测正常大鼠中MiR-126在脑组织和血浆的表达,剩余大鼠随机分为假手术组和模型组,模型组用线栓法造脑缺血大鼠模型。TTC法检测模型组大鼠脑组织梗死病变用以确定造模是否成功;RT-qPCR检测大鼠造模后1、3、6、9、12、24和48小时脑组织和血浆中MiR-126的动态表达变化;采用苏木精-伊红染色法检测脑组织形态学变化;采用Western blot 方法分析相关凋亡蛋白表达的变化。结果：TTC法和H-E染色结果表明,大鼠脑缺血造模成功,模型组大鼠出现脑组织大面积梗死病变;MiR-126和Caspase-3表达在模型组大鼠脑组织和血浆中随着脑缺血时间的延长其表达水平逐渐提高,24 h到达高峰后,在48 h Mi-R126表达强度又降低,但仍然高于假手术组大鼠水平;Caspase-3在正常对照组和假手术组表达量都较少（P>0.05）,其他凋亡蛋白Bax和 STAT3相对表达水平趋势同 Caspase-3相似;而Bcl-2表达水平明显降低,趋势与其他凋亡蛋白相反。结论：MiR-126在脑缺血大鼠脑组织和血浆中表达水平明显升高,而且细胞凋亡明显增加,因此MiR-126可能通过促进细胞凋亡对脑缺血大鼠有保护作用,此研究为脑缺血发病机制和诊断提供更多依据。     

7,8-dihydroxyflavone,a small-molecule tropomyosin-related kinase B (TrkB) agonist,attenuates cerebral ischemia and reperfusion injury in rats

7,8-dihydroxyflavone (7,8-DHF) is a recently identified potent agonist of tropomyosin-related kinase B that can cross the blood–brain barrier after oral or intraperitoneal administration. The aim of the present study was to determine whether 7,8-DHF has neuroprotective effects against cerebral ischemia and reperfusion (I/R) injury and, if so, to investigate the possible underlying mechanisms. Cerebral I/R injury rats were induced by middle cerebral artery occlusion for 90 min followed by reperfusion for 24 h. 7,8-DHF was administered intraperitoneally at a dose of 5 mg/kg immediately after ischemia. Our results showed that 7,8-DHF significantly reduced neurological deficit scores, infarct volumes, and neuronal apoptosis in brains of I/R rats. Meanwhile, 7,8-DHF also increased Bcl-2 expression, decreased expression of cleaved caspase-3, Bax and inducible nitric oxide synthase, and inhibited nuclear factor-κB activation in ischemic cortex. Finally, malondialdehyde and nitric oxide contents were reduced, but activities of glutathione, glutathione peroxidase and superoxide dismutase were restored in ischemic cortex treated with 7,8-DHF. Taken together, our findings demonstrated that 7,8-DHF is able to protect against cerebral I/R injury, which may be, at least in part, attributable to its anti-apoptotic, anti-oxidative and anti-inflammatory actions.     

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.