Antagonists of group I metabotropic glutamate receptors do not inhibit induction of ischemic tolerance in gerbil hippocampus

In this study we tested the effect of antagonists of two subtypes of the group I metabotropic glutamate receptors (mGluRs GI) on the induction of ischemic tolerance in relation to brain temperature. These experiments were prompted by indications that glutamate receptors may participate in the mechanisms of ischemic preconditioning. The role of NMDA receptors in the induction of ischemic tolerance has been debated while there is lack of information concerning the involvement of mGluRs GI in this phenomenon. The tolerance to injurious 3 min forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Brain temperature was measured using telemetry equipment. EMQMCM and MTEP, antagonists of mGluR1 and mGluR5, respectively, were injected i.p. at a dose of 5 mg/kg. They were administered either before preconditioning ischemia in a single dose or after 2 min ischemia three times every 2 h. Both antagonists did not inhibit the induction of ischemic tolerance. Thus, our data indicate that group I metabotropic glutamate receptors do not play an essential role in the induction of ischemic tolerance.     

The Free Radical Scavenger, α-Lipoic Acid, Protects Against Cerebral Ischemia-Reperfusion Injury in Gerbils

-Lipoic acid (thioctic acid) was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion. Adult gerbils were treated for 7 days with two intraperitoneal injections per day of -lipoic acid (20 mg/kg), vehicle or saline and on the 7th day the animals were subjected to 5 min of forebrain ischemia. Ischemic injury was assessed by monitoring the increases in locomotor activity and from the extent of damage to the CA1 hippocampal pyramidal cell layer after 5 days of recovery. By both criteria, -lipoic acid was neuroprotective against ischemia/reperfusion evoked cerebral injury.     

Parallel changes in brain tissue blood flow and mitochondrial function during and after 30 minutes of bilateral forebrain ischemia in the gerbil

Forebrain ischemia was induced in Mongolian gerbils by bilateral occlusion of the common carotid arteries for 30 minutes. These animals do not have a complete circulus arteriosus Willisii. Mitochondria were prepared from the forebrain tissue at the end of the 30 minutes occlusion period as well as at different time points after the release of the occlusion. Tissue blood flow in the forebrain was also determined by measuring the brain tissue accumulation of 14C-iodoantipyrine. Tissue blood flow in the forebrain decreased from a control level of 1.43 +/- 0.03 ml/min/gr to 0.13 +/- 0.03 ml/min/gr by the 30th minute of ischemia, increased to 1.12 +/- 0.25 ml/min/gr after 5 minutes of reflow, but decreased again to 0.41 +/- 0.07 ml/min/gr after 1 1/2 hours of reflow. Oxygen consumption rate of mitochondria prepared from the forebrain (glutamate + malate as substrates in the presence of ADP) was 98 +/- 13 nmoles O2/min/mg protein in control animals, decreased to 61 +/- 9 nmoles O2/min/mg protein after 30 minutes of occlusion, recovered to 106 +/- 9 nmoles O2/min/mg protein during the first 30 minutes of reperfusion. During extended reperfusion, mitochondrial respiratory activity declined reaching 20 +/- 5 nmoles O2/min/mg protein after 5 1/2 hours of reperfusion. Respiratory control ratio of the mitochondria (relative increase of respiration upon addition of ADP) was 9.2 +/- 1.3 in control animals, 7.0 +/- 1.5 after 30 minutes of carotid occlusion, 9.0 +/- 1.2 after 30 minutes of reperfusion, and 5.8 +/- 0.8 after 5 1/2 hours of reperfusion. Superoxide dismutase activity of the forebrain mitochondria was 5.10 +/- 0.7 I.U./mg protein in control animals, decreased to 3.3 +/- 1.6 I.U./mg protein after 30 minutes of occlusion and remained at this level throughout the reperfusion period. These data confirm earlier reports that deterioration of mitochondrial function may contribute to the development of ischemic and post-ischemic brain tissue damage. It also appears possible that postischemic damage of mitochondrial function develops secondary to postischemic deterioration of tissue blood flow.     

Chronic daily administration of ethyl docosahexaenoate protects against gerbil brain ischemic damage through reduction of arachidonic acid liberation and accumulation

Recently, we reported that dietary ethyl docosahexaenoate (Et-DHA) intake decreases the level of membrane arachidonic acid (AA), which reduces the generation of AA metabolites in ischemic gerbil brain. As an extended study, we further investigated the influence of the chronic administration of Et-DHA on free AA levels after ischemia. In addition, Na,K-ATPase activity, cation content, cerebral edema and brain damage were also evaluated. Weanling male gerbils were orally pretreated with either Et-DHA (200 mg/kg) or vehicle, once a day for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. Time-course analyses revealed that pretreatment with Et-DHA, compared with pretreatment with the vehicle, significantly decreased the brain's free AA levels during ischemia (5, 15 and 30 min) and after reperfusion (5, 10, 15 and 30 min), and attenuated the decline of Na,K-ATPase activity at examined time points. Pretreatment with Et-DHA significantly prevented an increase in Na(+) concentration and a decrease in K(+) concentration after 24 h of reperfusion, which resulted in lower cerebral water content. Reduced brain infarct volume and low animal mortality were also observed in Et-DHA-treated animals. These data suggest that the reduction of ischemia-induced AA liberation and accumulation by Et-DHA pretreatment may be attributable to (a) protection against the decline of Na,K-ATPase activity, (b) postischemic cerebral edema and brain damage and (c) animal mortality.     

Chronic administration of ethyl docosahexaenoate reduces gerbil brain eicosanoid productions following ischemia and reperfusion

Arachidonic acid (AA) and its vasoactive metabolites have been implicated in the pathogenesis of brain damage induced by cerebral ischemia. The membrane AA concentrations can be reduced by changes in dietary fatty acid intake. The purpose of the present study was to investigate the effects of chronic ethyl docosahexaenoate (E-DHA) administration on the generation of eicosanoids of AA metabolism during the period of reperfusion after ischemia in gerbils. Weanling male gerbils were orally pretreated with either E-DHA (100, 200 mg/kg) or vehicle, once a day, for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 10 min. E-DHA (200 mg/kg) pretreatment significantly decreased the content of brain lipid AA at the termination of treatment, prevented postischemic impaired regional cerebral blood flow (rCBF) and reduced the levels of brain prostaglandin (PG) PGF(2alpha) and 6-keto-PGF(1alpha), and thromboxane B(2) (TXB(2)), as well as leukotriene (LT) LTB(4) and LTC(4) at 30 and 60 min of reperfusion compared with the vehicle, which was well associated with the attenuated cerebral edema in the E-DHA-treated brain after 48 h of reperfusion. These data suggest that the E-DHA (200 mg/kg) pretreatment reduces the postischemic eicosanoid productions, which may be due to its reduction of the brain lipid AA content.     

Ex vivo and in vivo neuroprotection induced by argon when given after an excitotoxic or ischemic insult

In vitro studies have well established the neuroprotective action of the noble gas argon. However, only limited data from in vivo models are available, and particularly whether postexcitotoxic or postischemic argon can provide neuroprotection in vivo still remains to be demonstrated. Here, we investigated the possible neuroprotective effect of postexcitotoxic-postischemic argon both ex vivo in acute brain slices subjected to ischemia in the form of oxygen and glucose deprivation (OGD), and in vivo in rats subjected to an intrastriatal injection of N-methyl-D-aspartate (NMDA) or to the occlusion of middle-cerebral artery (MCAO). We show that postexcitotoxic-postischemic argon reduces OGD-induced cell injury in brain slices, and further reduces NMDA-induced brain damage and MCAO-induced cortical brain damage in rats. Contrasting with its beneficial effect at the cortical level, we show that postischemic argon increases MCAO-induced subcortical brain damage and provides no improvement of neurologic outcome as compared to control animals. These results extend previous data on the neuroprotective action of argon. Particularly, taken together with previous in vivo data that have shown that intraischemic argon has neuroprotective action at both the cortical and subcortical level, our findings on postischemic argon suggest that this noble gas could be administered during but not after ischemia, i.e. before but not after reperfusion has occurred, in order to provide cortical neuroprotection and to avoid increasing subcortical brain damage. Also, the effects of argon are discussed as regards to the oxygen-like chemical, pharmacological, and physical properties of argon.     

Ischemia-Induced Inhibition of Active Calcium Transport into Gerbil Brain Microsomes: Effect of Anesthetics and Models of Ischemia

The excessive increase in intracellular Ca²⁺ concentration is associated with events linking cerebral blood flow reduction to neuronal cell damage. We have investigated the possible effect of ischemia and ischemia-reperfusion injury on endoplasmic reticulum (ER) Ca²⁺ transport. Two different models of ischemia as well as two different anesthetics were used. 5 min and 15 min of global forebrain ischemia caused significant depression of the rate of microsomal Ca²⁺ accumulation in pentobarbital anesthetised gerbils. The Ca²⁺ uptake activity recovered partially after 1 hour of reperfusion. Unlike pentobarbital anesthetised gerbils, no significant changes were detected in the active microsomal Ca²⁺-transport after 10 min of global forebrain ischemia in gerbil forebrain and hippocampus under halothane anesthesia. In addition, using the model of decapitation ischemia, we observed significant changes of the Ca²⁺ uptake in both halothane and pentobarbital anesthetised gerbils. These findings indicate that ischemic insult alters the brain microsomal Ca²⁺ transport which is not due to inhibition of the Ca²⁺-ATPase activity. However, the effect of ischemia on this transport system is dependent on the model of ischemia and on the type of anesthetics.     

Neuroprotective efficiency of NMDA receptor blockade in the striatum and CA3 hippocampus after various durations of cerebral ischemia in gerbils

The purpose of this study was to investigate neuroprotective efficiency of N-methyl D-aspartate (NMDA) receptor (NMDAR) blockade on the neuronal damage in the less studied and allegedly less affected CA3 hippocampus and striatum in the Mongolian gerbil model of global cerebral ischemia. The common carotid arteries of gerbils were occluded for 5, 10 or 15 minutes. Gerbils were given a low dose of non-competitive NMDA antagonist (MK-801, 3 mg/kg i.p.) or saline immediately after the occlusion in normothermic conditions. Neuronal damage was examined on 4th, 14th and 28th day after reperfusion. The effect of NMDAR blockade was followed in vivo by monitoring the neurological status of whole animals or at the cellular level by standard light- and confocal microscopy on brain slices. Increased duration of cerebral ischemia resulted in a progressive loss of striatal and CA3 hippocampal neurons. The most beneficial NMDAR blockade effect was observed when the neuronal damage was most severe - on the 28th day after 15-min ischemia. As judged by morphological and neurological data, the effect of ischemia is also apparent in the presumed less vulnerable regions (CA3 and striatum) which are functionally important in stroke plasticity. So, NMDAR blockade in normothermic conditions showed neuroprotective efficiency.     

脑源性促红细胞生成素在短暂性前脑缺血沙土鼠海马的表达变化

目的 探讨短暂性前脑缺血鼠海马脑源性促红细胞生成素蛋白的表达变化,揭示脑缺血时中枢神经系统发生内源性脑保护的机制。方法 阻断沙土鼠双侧颈总动脉3．5min造成前脑缺血模型,再灌注1h,6h,12h,1d,3d,7d,应用免疫组织化学和免疫印迹法观察海马脑源性促红细胞生成素蛋白的表达变化。结果 脑缺血再灌注6h,可检测到脑源性促红细胞生成素的表达,再灌注12h,脑源性促红细胞生成素表达达到较高水平,以后随时间延长逐渐下调。结论 脑源性促红细胞生成素在脑缺血再灌注后的表达,可能是机体发生内源性脑保护的机制之一。     

Accurate Evaluation of 1,2-Diacylglycerol in Gerbil Forebrain Using HPLC and In Situ Freezing Technique

Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.     

缺血引起沙土鼠纹状体DARPP—32磷酸化状态的变化

采用蒙古沙土双侧颈总动脉阻断前脑缺血模型,以放射自显影（反向磷酸化,back-phosphorylation)及免疫印迹（Western blotting)法体外测定缺血时纹状体DARPP－32磷酸化水平和蛋白含量的变化,结果表明,短暂性缺血纹状体DARPP－32的免疫学活性和蛋白含量地明显改变。在缺血10min内,随缺血时间的延长,体外DARPP－32的[^32P]的掺入量在缺血5min时升高,在缺血2,7,10min时均降低,而反向磷酸化的测定结果表明体内DARPP－32磷酸化水平增高,说明缺血可诱导DARPP－32磷酸化水平变化。     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

Effect of Brain Ischemia on Protein Kinase C

We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6-min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane-bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane-bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca(2+)-dependent, antiprotease-insensitive down-regulation of PKC during incubation. This down-regulation was significantly enhanced by prior phorbol (PDBu) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vinpocetine prevents ischemic cell damage in rat hippocampus

The effects of vinpocetine on hippocampal cell damage and local cerebral blood flow (LCBF) were measured in a rat model of forebrain ischemia (2-vessel occlusion and hypotension). Duration of ischemia was 10 min. LCBF was determined after 2 min of recirculation using the 14C-iodoantipyrine technique. Hippocampal cell loss was quantified histologically 7 days post-ischemia. Intraperitoneal application of vinpocetine (10 mg/kg) 15 min prior to ischemia significantly reduced neuronal cell loss in hippocampal CA 1 sector from 60% to 28%. The drug led to a marked increase in blood flow in cortical areas, whereas LCBF remained unchanged in hippocampus and all other structures measured. It is suggested that the protective effect of vinpocetine does not depend on increased postischemic blood flow.     

Comparison of In Vitro Ischemia-Induced Disturbances in Energy Metabolism and Protein Synthesis in the Hippocampus of Rats and Gerbils

Abstract: To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue-specific or hemodynamic factors, ischemia-induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37°C, 34°C, or 31°C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia-induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.     

Naloxone or TRH fails to improve neurologic deficits in gerbil models of “stroke”

The effects of naloxone or thyrotropin releasing hormone (TRH) upon neurologic outcome were evaluated in gerbil models of cerebral ischemia. Following temporary bilateral carotid occlusion, hypotension was transiently reversed by these endorphin antagonists. However, neither drug altered time to awaken, time to death, or the severity of neurologic signs (ptosis, movement, retracted paws, circling, righting reflexes, seizures, or opisthotonus) when evaluated by a blinded rater. Hot plate escape and roto-rod performance were also unaltered by naloxone or TRH; TRH, but not naloxone, increased respiratory rates. Thus, the transient improvement of cardiorespiratory function produced by these drugs is unrelated to the morbidity and mortality associated with temporary cerebral ischemia in the gerbil. Additional studies evaluating the effects of naloxone or TRH upon neurologic outcome following permanent unilateral carotid occlusion also failed to show any therapeutic effects of these drugs. Both morphine and TRH exacerbated the effects of ischemia. Of gerbils which developed neurologic impairment, the deficit was usually ipsilateral to the occluded carotid. Collectively, these results indicate that neither naloxone nor TRH prevents ischemic deficits in the gerbil. Further studies with different cerebral ischemia models in other species are required to clarify the possible therapeutic effects of these drugs in experimental stroke.     

A Dual-Probe Microdialysis Study in Simultaneously Monitoring Extracellular Pyruvate, Lactate, and Biogenic Amines in Gerbil Striata during Unilateral Cerebral Ischemia

A dual-probe microdialysis technique coupled with liquid chromatographic assays was developed for the simultaneous monitoring of neurochemicals in gerbil striata during cerebral ischemia. Isocratic separation of lactate and pyruvate was achieved within 5 min whereas the separation of biogenic amines was completed within 30 min. An unilateral ligation was produced by occlusion of the right common carotid artery for 30 mins in anethetized gerbils to perform a typical focal cerebral ischemia. Microdialysis probes were inserted in both sides of the striata to simultaneously monitor biogenic amines, lactate and pyruvate during cerebral ischemia. Dynamic and comparative changes of these analytes in ipsilateral and contralateral sides of the brain can be simultaneously measured by the assay. The present assay can be used as a research tool to explore neurochemical substances and their relationships during cerebral ischemia.     

Carotid ligation alters cholecystokinin-8 (CCK-8) immunoreactivity in gerbil brains

The unilateral or bilateral carotid arteries were ligated in gerbils used as a model of cerebral ischemia. The effect of different times of bilateral ischemia on the content of CCK-8 in fore regions of gerbil brain and the effect of 30 min of unilateral ischemia on the content of CCK-8 of the same regions in gerbils with or without neurological signs were observed. Our results show that the content of CCK-8 of cortex, basal ganglia, thalamus and hypothalamus decreased significantly. But, in brain stem it remained basically unchanged no matter whether the ischemia was unilateral or bilateral. This suggests that there is a close relationship between CCK-8 and cerebral ischemia, and raises the possibility that CCK-8 may be involved in cerebral ischemia through a yet unclear mechanism.     

Evolution of the regional blood deficit and energy metabolism after induction of transient cerebral ischemia by occlusion of the vertebral and carotid arteries in the rat

A transient brain ischemia of 10 min duration was produced in rats by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. Ischemia reduced blood flow to 10-18% of the control values in forebrain structures (cortex, striatum, thalamus) and to 25-50% in the mesencephalon, cerebellum and brain stem. In these last structures, after 30 min of recirculation, the flow rates returned to normal values but a 20-35% reduction of blood flow was present in the forebrain structures, indicating that the development of the postischemic hypoperfusion was related to the severity of the preceding ischemia. After 30 min of recirculation, there was a near complete recovery of the high energy compounds but a residual metabolic dysfunction was evidenced by an increase in lactate/pyruvate ratio and an elevation of the glucose content, suggesting a depression of cerebral metabolism which may account for the brain hypoperfusion.     

Cerebokrast as a corrector of postischemic phenomena in acute transient cerebral ischemia]

Acute cerebral ischemia in cats (both carotid arteries occlusion during 30 min after permanent occlusion of both vertebral arteries) was accompanied by postischemic hypoperfusion and hypo-oxygenation of the cerebral tissue. Intravenous infusion of cerebrocrast (1 micrograms.kg-1.min-1 during 60 min) prevented manifestation of the postischemic phenomena. Antihypoxic effect of cerebrocrast involved the cerebral blood flow increase, brain oxygen consumption lowering and Hb-O2-affinity decrease.