Ren-Zhi Zhan

Both Caspase-Dependent and Caspase-Independent Pathways May Be Involved in Hippocampal CA1 Neuronal Death Because of Loss of Cytochrome c From Mitochondria in a Rat Forebrain Ischemia Model

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

Intravenous anesthetics differentially reduce neurotransmission damage caused by oxygen-glucose deprivation in rat hippocampal slices in correlation with N-methyl-D-aspartate receptor inhibition.

ObjectiveTo examine the relation between the effect of intravenous anesthetics on ischemic neurotransmission damage and their actions on N-methyl-d-aspartate (NMDA) receptors in an in vitro cerebral ischemic model. DesignProspective, randomized study in freshly prepared rat hippocampal slices. SettingUniversity research laboratory. SubjectsHippocampal slices were prepared from male Wistar rats (4–5 wks old). Interventions and Measurements In vitro ischemia was induced by exposing slices to glucose-free Krebs solution gassed with 95% N2 /5% CO2 at 37.1–37.3°C. Ischemic neurotransmission damage was indicated by the amplitudes of population spikes (PS) recorded from the CA1 pyramidal layer after stimulation of the Schaffer collaterals. The effect of anesthetics on NMDA receptors was determined by measuring the NMDA-mediated changes in intracellular calcium in the CA1 pyramidal layer with a calcium indicator, fura-2. ResultsFollowing 4, 6, and 7.5 mins ischemia in vitro, the recoveries of PS (% control) were 100%, 17.5 ± 21.8%, and 5.4 ± 2.1%, respectively. 3-(R)-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 5 &mgr;M), an NMDA receptor antagonist, increased the recovery of PS to 88.3 ± 24.5% after 6 mins ischemia, and to 42.1 ± 18.7% after 7.5 mins ischemia. Thiopental (400 &mgr;M), thiamylal (400 &mgr;M), and ketamine (100 &mgr;M), but not propofol (100 &mgr;M) and etomidate (10 &mgr;M), improved the recovery of PS after 6 and 7.5 mins ischemia; the degrees of their protection were comparable to that of 5 &mgr;M CPP. The NMDA-mediated increases in intracellular calcium were almost completely inhibited by thiamylal, reduced to half by ketamine and thiopental, augmented by propofol, and not affected by etomidate. ConclusionsThe results indicate that the efficacy of intravenous anesthetics in attenuating ischemic neuronal damage varies among agents, relating to their effects on NMDA receptors.

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice.

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca2+ and glycolysis

To elucidate the mechanism of pHi changes induced by membrane depolarization, the variations in pHi and [Ca2+]i induced by a number of depolarizing agents, including high K+, veratridine, N-methyl-D-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pHi and an elevation of intracellular calcium ([Ca2+]i) in the CA1 pyramidal cell layer. The increases in [Ca2+]i caused by the depolarizing agents almost completely disappeared in the absence of Ca2+ (0 mM Ca2+ with 1 mM EGTA). In Ca2+ free media, pHi acid shifts produced by high K+, veratridine or NMDA were attenuated by 10-25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pHi acid shifts induced by both high K+ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K+, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na+-K+ ATPase activity. A Ca2+-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H+ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.

Ischemic preconditioning is capable of inducing mitochondrial tolerance in the rat brain.

Background Preconditioning to ischemia is a phenomenon whereby a brief episode of sublethal ischemia and other nonlethal stressors produce protection against a subsequent detrimental ischemic insult. As mitochondrial dysfunction is related to necrotic and apoptotic neuronal death after cerebral ischemia, the authors examined if ischemic preconditioning is capable of inducing mitochondrial tolerance. Methods Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 8 min in Wistar rats (275–300 g). A 3-min ischemic episode performed 48 h before the 8-min ischemia was used for preconditioning. The extents of hippocampal CA1 neuronal damage were evaluated 7 days after reperfusion by neuro-specific nuclear protein immunostaining. Brain mitochondria were isolated 48 h after animals were subjected to the sham operation or the 3-min conditioning ischemia. Loss of cytochrome c from mitochondria after cerebral ischemia in vivo and after exposure of brain mitochondria to calcium in vitro was used as an indication of mitochondrial dysfunction. Results Results showed that ischemic preconditioning induced by a 3-min ischemic episode dramatically reduced the loss of hippocampal CA1 neurons resulting from a subsequent 8-min ischemia 7 days after reperfusion, and this protection was associated with a preservation of mitochondrial cytochrome c as examined after early reperfusion. Exposure of isolated brain mitochondria to calcium produced a dose-dependent increase in cytochrome c release either at 30°C or at 37°C. Compared with those animals receiving only sham operation, cytochrome c release caused by 100 &mgr;m calcium was significantly reduced in conditioned animals. Conclusion Regarding the importance of mitochondrial dysfunction in mediating ischemic neuronal death, the above results indicate that mitochondria may serve as end-effecting organelles to ischemic preconditioning.

Sublethal cerebral ischemia inhibits caspase-3 activation induced by subsequent prolonged ischemia in the C57Black/Crj6 strain mouse

Caspase-3 activation has been implicated in ischemic neuronal death. In the present study, we examined if cerebral ischemic tolerance induced by sublethal ischemia is associated with an attenuation of caspase-3 activation in a mouse forebrain ischemia model. Forebrain ischemia in C57Black/Crj6 strain mice was induced by bilateral common carotid artery occlusion (BCCAO) for 18 min. Two episodes of 6-min ischemia were carried out as preconditioning 48 and 72 h before the 18-min BCCAO. Caspase-3-like activity was determined by fluorescently monitoring the release of amino-4-methylcoumarin from N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin in the striatal protein extracts at 4, 24, and 72 h after reperfusion. The results showed that the ischemic preconditioning significantly attenuated caspase-3 activation at 4, 24, and 72 h after reperfusion, and reduced neuronal loss caused by the 18-min ischemia as examined on the 7th day after reperfusion. The present results suggest that the neuroprotection achieved by ischemic preconditioning is related to an attenuation of caspase-3 activation.

The effects of thiopental and propofol on cell swelling induced by oxygen/glucose deprivation in the CA1 pyramidal cell layer of rat hippocampal slices.

Cellular swelling has been implicated as an early process after cerebral ischemia. We compared the effects of two commonly used IV anesthetics, thiopental and propofol, on hippocampal CA1 pyramidal cell swelling induced by oxygen/glucose deprivation (OGD) in vitro. Experiments were performed in rat hippocampal slices. Cell swelling in the CA1 pyramidal cell layer was evaluated by determining light transmittance (LT) change through the slices and by histopathological examination. For LT experiments, OGD was induced for 10 min by superfusing slices with glucose-free artificial cerebrospinal fluid equilibrated with 95% nitrogen and 5% CO2. Thiopental and propofol were present 10 min before and during the period of OGD. The results showed that thiopental (100 and 400 &mgr;M), but not propofol (40 and 160 &mgr;M), significantly prolonged latency to the peak of LT increase after the onset of OGD. Consistent with the LT experiments, histopathological examination revealed that thiopental, but not propofol, attenuated CA1 pyramidal cell expansion and the gap diminution between CA1 pyramidal cells induced by OGD. These results suggest that thiopental, but not propofol, reduces the neuronal cell swelling caused by OGD. Whether the reduction of cell swelling is related to reduction in cell injury caused by OGD remains to be investigated.

Regionally Different Elevation Of Intracellular Free Calcium In Hippocampus Of Septic Rat Brain

ABSTRACT The effect of sepsis on cellular calcium homeostasis in the central nervous system (CNS) was investigated using hippocampal slices of rats in which sepsis was induced by cecal ligation and puncture (CLP). Hippocampal slices were prepared from septic or sham-operated rats at 24 h after abdominal surgery. The basal intracellular calcium ([Ca2+]i) and its response to oxygen-glucose deprivation in hippocampal slices were measured for assessing cellular calcium homeostasis using fura-2 fluorescent imaging technique. The levels of [Ca2+]i- were estimated by the fluorescence ratio (R340/380). Twenty-four hours after CLP, spontaneous movement was reduced and plasma lactate was increased in the septic rats in comparison with the sham-operated rats in which laparotomy was performed without CLP. Basal level of R34O/380 in the CA4 area (.72 ± .07) was significantly higher (p < .001) in the septic group than that in the sham-operated group (.55 ± .06). The fluorescence ratio of septic vs. sham-operated in other hippocampal regions were .55 ± .09 vs. .48 ± .06 in CA1 (not significant) and .65 ± .10 vs. .59 ± .08 (not significant) in CA3, respectively. Increase in [Ca2+]i- due to oxygen-glucose deprivation was significant in CA1 and CA3 of the septic group and in all hippocampal regions of sham-operated group. However, it was not significantly increased in CA4 of the septic group. These results suggest that regional deregulation of cellular calcium occurs in the CNS following CLP. Cellular calcium deregulation may be one of the pathogeneses occurred in clinically observed septic encephalopathy.

NMDA receptor antagonists improve recovery of synaptic transmission from ischemic insult in rat cortical slices: A comparison between competitive and noncompetitive agents

1Dcpt. of Pharmacol, Yokohama City Univ Sch Mcd, Yokohama 236-0001. 7-Dept. for Ncurosci, Tokyo Mctrop Inst of Ncurosci, Tok~~o 183-8526 DOPA itself is a ncuromodulator in striata (hog A’eurohiol 19, 415451, 1996). Esogcr~ousl~~ applied DOPA stcrcosclccti\.cl!. c\,okcd ncuronal glutamate (Glu) with competitive inhibition by a DOPA antagonist in slices and caused delayed neuronal death via Glu rclcase in cultured fetal neurons, suggesting involvcmcnt of DOPA in an upstream process of mechanisms for Glu rcleasc and ncuronal death. WC tried to clarify whether DOPA is rclcascd. being a factor for Glu rclcasc and ncuronal death b! transient ischemia. Four vessels vxrc occluded for 10 min during striatal microdialysis of conscious rats. DOPA, DA and Glu wcrc mcasurcd by HPLC-ECD and spcctrophotomctcr. Ncuronal death H’as c\.aIuatcd 96 hr after ischcmia. lschcmia indeed cwkcd DOPA with DA and Glu. rcspcctivc peak being 5.6. __ 770and 8.3.fold of a basal Ic\,cl. Moderate striatal and SC! crc hippocampal ncuronal death occurred. Intrastriatal perfusion of NSD-1015 30 uM. a DOPA dccarbosylase inhibitor, markcdl!, incrcascd DOPA and Glu rclcasc by ischcmia with slight inhibition of DA rclcasc and csaggcratcd striatal ncuronal death. Furthcrmorc, DOPA cyclohcsyl cstcr (CHE) 10-100 nM, H no\ cl. poicnt and stable compctiti\ c DOPA antagonist, inhibit4 dose-dcpcndcntly incrcascs in Glu rclcasc without change of DA rclcasc. CHE 100 nM protcctcd striata from dclaycd ncuronal death. Hippocampal ncuronal death ~‘as ncithcr affected by KSD-1015 nor CHE. DOPA woked seems to bc a causal f’actor b!, itself \,ia its recognition site for incrcasc in Glu rclcasc and resultant dclayxl ncuronal death b\, tnrnsicnt ischcmia in rats.

120 Intracellular acidification produced by depolarizing agents in rat hippocampal slices

Naoshi FUJIWARA, Ren-Zhi ZHAN, Koki Shimoji To elucidate the mechanism of pHi changes induced by membrane depolarization, changes in pHi and [Ca2+]i induced by high K+, veratridine, NMDA were investigated in rat hippocampal slices using BCECF or fura-2. All these agents elicited a decrease in pHi and an elevation of [Ca2+]i in the CA1 pyramidal cell layer. Although the [Ca2+]i increase was completely suppressed in Ca 2+ free media, a major part of each pHi acid shift remained unchanged. Glucose-deprivation reduced pHi acid shifts induced by high K+ or NMDA by twothird. Anthermore, lactate contents significantly increased in slices exposed to the depolarizing agents. The results suggest that pHi acid shifts produced by the depolarizing agents are mainly due to lactate accumulation. A Ca2+-dependent process may partially cause pHi acid shifts. Since an increase in [H+] enables to attenuate neuronal activity, glycolytic acid production promoted by depolarization may contribute to prevent excessive neuronal excitation.