蛋白激酶C抑制剂对缺血/再灌注海马CAl区迟发性神经元死亡的作用研究

目的:蛋白激酶C与脑组织缺血性损害有密切关系,且证明可调节一氧化氮合成酶的活性。作为PKC抑制剂,灯盏花素可抑制蛋白激酶C的活性,但其对大鼠海马CAl区缺血/再灌注损害的作用和机制需深入研究。方法:四血管闭塞复制大鼠前脑缺血／再灌注模型,观察PKC抑制剂灯盏花素对海马CAl区NO浓度、局部脑血流量及CAl区锥体细胞密度变化的影响。结果:PKC抑制剂灯盏花素对大鼠海马CAl区缺血／再灌注脑组织的作用为降低CAl区局部NO的产生、明显改善脑组织的rCBF和显著降低该区锥体细胞的脱失。结论:PKC抑制剂对大鼠前脑缺血／再灌注所致海马CAl区迟发性神经元死亡的保护作用与其降低局部NO的产生及增加局部脑血流量有密切关系。     

蛋白激酶C抑制剂对缺血／再灌注海马CA1区迟发性神经元死亡的作用研究

目的:蛋白激酶C与脑组织缺血性损害有密切关系,且证明可调节一氧化氮合成酶的活性。作为PKC抑制剂,灯盏花素可抑制蛋白激酶C的活性,但其对大鼠海马CAl区缺血/再灌注损害的作用和机制需深入研究。方法:四血管闭塞复制大鼠前脑缺血／再灌注模型,观察PKC抑制剂灯盏花素对海马CAl区NO浓度、局部脑血流量及CAl区锥体细胞密度变化的影响。结果:PKC抑制剂灯盏花素对大鼠海马CAl区缺血／再灌注脑组织的作用为降低CAl区局部NO的产生、明显改善脑组织的rCBF和显著降低该区锥体细胞的脱失。结论:PKC抑制剂对大鼠前脑缺血／再灌注所致海马CAl区迟发性神经元死亡的保护作用与其降低局部NO的产生及增加局部脑血流量有密切关系。     

Cholinergic deafferentation prevents delayed neuronal death of the hippocampal CA1 pyramidal neurons after transient forebrain ischaemia

Abstract

The relation between CA1 neurons, fimbria-fornix and cholinergic neurons of the basal forebrain was examined with the aid of Acetylcholine esterase (AChE) staining, Woelcke's staining and immunohistochemistry of Choline-acetyl transferase (ChAT). The transected side of the hippocampus was poorly stained by AChE two weeks after the transection, when the ipsilateral medial septum ChAT-positive neurons were reduced, but showed good recovery with AChE six weeks later.’ Nerve growth factor (NGF) was added at a dose of 10 jig/100 \i\ immediately after the aspiration>, and after that once per week with cisternal puncture. As a result, ipsilateral medial septum ChAT-positive neurons were preserved, but cross innervation with relation to hypertrophy of the cholinergic neurons was not detectable even six weeks after the transection. Furthermore, delayed CA1 neuronal death on the transected side of the hippocampus following occlusion of four vessels for 30 minutes was not detectable two weeks after the operation, although neuronal density was reduced after six weeks. The density of neurons on the transected side of the hippocampus in the CA1 subfield with treated NGF had not decreased significantly six weeks later. Therefore, we suspect that the input from cholinergic fibres must be transported to the hippocampal pyramidal neurons responding to NCF, and it was confirmed that cholinergic deafferentation prevents the delayed neuronal death of CA1 pyramidal neurons during transient ischaemia. [Neurol Res 1992; 14: 340-344]     

Ischemic delayed neuronal death: Role of the cysteine proteases calpain and cathepsins

A few days after a transient brain ischemia, the pyramidal neurons in the cornu Ammonis (CA) 1 sector of the hippocampus undergo selective death, a process named delayed neuronal death (DND). Cell death may occur as necrosis and/or apoptosis, and both have been reported to take place in DND. The cell's decision between apoptosis and necrosis may depend on the strength of the insult, the balance of downstream signal transduction systems, and the expression level of pro- and anti-apoptotic or necrotic factors. Cytosolic calcium (Ca²⁺) overload specifically occurs in the CA1 neurons after ischemia and thus is considered a common triggering event of the death cascade. As Ca²⁺ activates a wide array of intracellular enzymes, many Ca²⁺-targeted enzymes have been implicated in DND. Among these, the present review will focus on the cysteine proteases calpain and cathepsins (B and L). In addition, their possible interactions with another family of cysteine proteases, caspases, will be discussed in relation to the cellular fate toward apoptosis or necrosis.     

Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates

Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of micro-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of micro-calpain. The immunoreactivity of activated micro-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2-3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates.     

Gerbil hippocampal extracellular glutamate and neuronal activity after transient ischemia

In order to elucidate the role of glutamate in the pathogenesis of delayed neuronal death, we analyzed changes in extracellular levels of glutamate induced by transient ischemia in the Mongolian gerbil hippocampus by a new brain microdialysis method combined with an enzymatic cycling technique. We also studied the effect of this change in glutamate on CA1 spontaneous neuronal discharges. The level of glutamate significantly increased during the 5 min of ischemia and during the first 5 min of recirculation. However, neuronal hyperactivity anticipated as a result of the increased extracellular glutamate was not observed. Spike discharges disappeared during and shortly after 5 min of ischemia; CA1 spontaneous spike discharges reappeared about 15 min after the recirculation. The frequency and amplitude of the discharges of CA1 neurons returned to normal by 30 min of the recirculation. However, the pattern of discharges was different from that recorded before the ischemia. CA1 neurons were found dead 4 days after the ischemia. Brief exposure to toxic concentrations of glutamate may cause the delayed neuronal death.     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.     

Effect of hyperthermia on calbindin-D 28k immunoreactivity in the hippocampal formation following transient global cerebral ischemia in gerbils

Calbindin D-28K(CB), a Ca2+-binding protein, maintains Ca2+ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We investigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei(Neu N) and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia(39.5 ± 0.2°C) was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neuronal damage/death in the pyramidal layer of CA1–3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreactivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immunoreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.     

Immunohistochemical investigation of caspase-1 and effect of caspase-1 inhibitor in delayed neuronal death after transient cerebral ischemia

The localization of caspase-1 protein, interleukin-1beta (IL-1beta)-converting enzyme, was immunohistochemically examined in the hippocampal CA-1 subfield by a transient occlusion of bilateral common carotid arteries in Mongolian gerbils. Immunoreactivities for caspase-1 were found in microglias, astrocytes, endothelial cells of capillaries and some non-pyramidal neurons. Immunopositive microglias increased in number from 3 days until 7 days from the transient ischemia, and astrocytes also increased in number from 3 days until 28 days. At the electron microscopic level, caspase-1 immunoreaction endproducts were associated with Golgi apparatus in glial cells, endothelial cells of blood vessels and non-pyramidal neurons. The delayed neuronal death of CA-1 pyramidal cells was significantly protected by the treatment of specific caspase-1 inhibitor (Ac-WEHD-CHO) or broad caspase family inhibitor (z-VAD-FMK). Cell death was protected in a dose dependent manner by the former by 43-57%, and by the latter by 66-91% when injected at 1 and 10 microg, respectively. On the other hand, the protective effect of specific caspase-3 inhibitor (Ac-DMQD-CHO) was less significant at higher dose (10 microg) by 33% (P<0.05), and not detectable at lower dose (1 microg) by 13% (P=0.27). Furthermore, a significant decrease of microglias and astrocytes was found in the CA-1 as well as the reduction of IL-1beta and caspase-1 immunoreactivities by the treatment of Ac-WEHD-CHO. Extravasation of serum albumin was also extremely reduced by this treatment. These findings suggest that the inhibition of caspase-1 activity ameliorates the ischemic injury by inhibiting the activity of IL-1beta.     

银杏叶制剂对沙土鼠短暂性脑缺血后海马DND的影响

实验在沙土鼠短暂性脑缺血模型上应用含有PAF受体拮抗剂的FGP,以观察其对沙土鼠短暂性脑缺血后海马CA1区DND的影响。结果表明：沙土鼠双侧颈总动脉结扎10分钟再灌4天模型,与对照组相比,银杏叶制剂明显增加海马CA1区存活神经元数,分别为38．50±16．31／mm和133．13±20．99／mm（P＜0．01）,与Nimodipine组（182±67／mm）结呆相近（P＞0．05）。提示：银杏叶制剂能明显对沙土鼠短暂性脑缺血后海马CA1区DND有保护作用。     

Regional distribution of selective neuronal loss and microglial activation across the MCA territory after transient focal ischemia: quantitative versus semiquantitative systematic immunohistochemical assessment

Histopathologic assessment in transient middle cerebral artery occlusion (MCAo) rodent models generally lacks comprehensiveness and exposes to interobserver bias. Here we compared a novel quantitative assessment of regional infarction, selective neuronal loss (SNL) and microglial activation (MA) across the MCA territory to a previously published semiquantitative visual protocol. NeuN and OX42 immunohistochemistry was applied after either 15 or 45 minutes distal MCAo to maximize SNL and infarction, respectively. Survival times varied from 28 to 60 days to cover potential biases such as delayed tissue shrinkage. Damage was assessed using a template of 44 cytoarchitectonic regions of interest (ROIs) mapped onto a subset of digitized coronal sections spanning the MCA territory. For each ROI were obtained a semiquantitative visually determined index of histopathologic changes (method 1), and lpsilateral/contralesional ratios of remaining neurons and activated microglia cell counts (method 2). There was excellent agreement between the two methods for 28-day survival for both MCAo durations, whereas method 2 more sensitively detected subtle SNL and MA at 45 days and 60 days after 15-minute MCAo. Thus the visual method is accurate for usual degrees of ischemic damage, but absolute cell quantification is superior to detect subtle changes and should therefore be preferred in brief MCAo models, although requires optimal staining quality.     

Proteasome alteration and delayed neuronal death in hippocampal CA1 and dentate gyrus regions following transient cerebral ischemia

BACKGROUND： Proteasome dysfunction has been reported to induce abnormal protein aggregation and cell death. OBJECTIVE： To investigate the effect of proteasome changes on delayed neuronal death in CA1 and dentate gyrus （DG） regions of the rat hippocampus following transient cerebral ischemia. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment. The study was performed at the Department of Biochemistry and Molecular Biology, Norman Bethune Medical College of Jilin University, from September 2006 to May 2008. MATERIALS： Rabbit anti-19S S10B polyclonal antibody was purchased from Bioreagents, USA; propidium iodide and fluorescently-labeled goat anti-rabbit IgG were purchased from Jackson Immunoresearch, USA; hematoxylin and eosin staining solution was purchased from Sigma, USA; LSM 510 confocal microscope was purchased from Zeiss, Germany. METHODS： A total of 40 healthy Wistar rats, male, 4 months old, were randomly divided into sham surgery group （n = 8） and model group （n = 32）. Ischemic models were established in the model group by transient clamping of the bilateral carotid arteries and decreased blood pressure. After 20 minutes of global ischemia, the clamp was removed to allow blood flow for 30 minutes, 4, 24 and 72 hours, respectively, with 8 rats at each time point. The bilateral carotid arteries were not ligated in the sham surgery group. MAIN OUTCOME MEASURES： Neuronal death in the CA1 and DG regions was observed by hematoxylin-eosin staining. Proteasome expression in CA1 and DG region neurons was detected by immunohistochemistry. RESULTS： Hematoxylin-eosin staining showed neuronal death in the CA1 region alone at 72 hours of reperfusion following ischemia. In comparison to the sham surgery group, a significant decrease in proteasome expression was observed, by immunohistochemistry, in the CA1 and DG regions in the model group, following 30 minutes, 4, 24, and 72 hours of reperfusion （P 〈 0.01）. After 72 hours of reperfusion following ischemia, proteasome expression had almost completely disappeared in the CA1 region. In contrast, neurons of the DG region showed minimized proteasome expression at 24 hours, with a slight increase at 72 hours （P 〈 0.01）. CONCLUSION： The alteration of proteasome following ischemia/reperfusion in the neurons of hippocampal CA1 and DG regions reduces the ability of cells to degrade abnormal protein, which may be an important factor resulting in delayed neuronal death following transient cerebral ischemia.     

Dynamic expression of p38beta MAPK in neurons and astrocytes after transient focal ischemia

Here we report the dynamically regulated expression of p38beta MAPK isoform in specific subsets of cells in postischemic brain. The activity of p38beta MAPK in the postischemic brain revealed biphasic induction at 30 min and 4 days after 1 h MCAO. During the early surge period, p38beta MAPK was preferentially localized in the nucleus and dendrites of neurons in the future infarction area, while during the delayed surge p38beta MAPK was heavily induced in reactive astrocytes in penumbra. The temporally and spatially regulated pattern of p38beta MAPK expression in the postischemic brain suggests distinct roles of p38beta MAPK in neuronal death and in the astrocyte activation.     

Hypothyroid state does not protect but delays neuronal death in the hippocampal CA1 region following transient cerebral ischemia: Focus on oxidative stress and gliosis

We investigated protective effects of hypothyroidism on delayed neuronal death, gliosis, lipid peroxidation and Cu,Zn‐superoxide dismutase (SOD1) in the gerbil hippocampal CA1 region (CA1) after 5 min of transient cerebral ischemia. The hypothyroidism was induced by 0.025% methimazole treatment. Free triiodothyronine and thyroxine levels were markedly decreased in the hypothyroid group. Four days after ischemia/reperfusion, only a few NeuN‐immunoreactive (+) neurons were detected in the CA1 of euthyroid‐ischemia (eu‐ischemia) group; however, at this time point, the number of NeuN⁺ neurons was significantly higher in the hypothyroid‐ischemia (hypo‐ischemia) group than in the eu‐ischemia group. At 5 days postischemia, NeuN⁺ neurons were significantly decreased in the hypo‐ischemia group: The number of NeuN⁺ neurons in this group was similar to that in the eu‐ischemia group. Activations of GFAP⁺ astrocytes and Iba‐1⁺ microglia in the CA1 were higher in the eu‐ischemia group 3 and 4 days after ischemia/reperfusion. At 5 days postischemia, the activations of both the glial cells in the CA1 were similar between the two groups. 4‐Hydroxy‐2‐nonenal (HNE), a marker for lipid peroxidation, immunoreactivity in the eu‐ischemia group was higher than in the hypo‐ischemia group; at 5 days postischemia, the immunoreactivity was similar between the two groups. In contrast, SOD1 level was lower in the CA1 of the eu‐ischemia group. These results suggest that hypothyroid state does not protect against delayed neuronal death but only delays the neuronal death in the hippocampal CA1 region after transient cerebral ischemia by reducing lipid peroxidation and increasing SOD1. © 2010 Wiley‐Liss, Inc.     

Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat

The immunohistochemical localization of calcium-binding protein (CaBP) in the cerebellum, hippocampal formation and olfactory bulb of the rat was examined using rabbit anti-human or sheep anti-chick antisera purified by affinity chromatography. CaBP-like immunoreactivity was observed within the somata and dendrites of: (1) cerebellar Purkinje cells; (2) dentate granule cells, CA1 pyramidal cells and scattered interneurons in the stratum radiatum of the hippocampus; (3) periglomerular cells in the olfactory bulb. Staining was conspicuously absent in certain major cell types in each of these structures including cerebellar granule cells, hippocampal pyramidal cells in the CA3 region and both mitral and granule cells in the olfactory bulb. Immunoreactive fibers in the cerebellum presumably corresponding to climbing fiber inputs from the inferior olive and efferent projections to the deep cerebellar nuclei, were also observed. In the hippocampus intense staining was present in the mossy fiber projection to the CA3 region and in the terminal regions of the perforant path projection from entorhinal cortex.     

Postischemic alterations of complex spike cell discharges and evoked potentials in rat hippocampal CA1 region

Postischemic alterations of spontaneous discharges of complex spike cells (CS cells) and evoked potential in the rat hippocampal CA1 region were studied. Following 5 min of ischemia, CS cell discharge reappeared approximately 5 min after reperfusion and the frequency remained low, reaching a final value of 66.1 +/- 16.0% (n = 11) of preischemic frequency 2 h later. However, only one of 7 CS cells subjected to 20 min of ischemia exhibited discharges 2 h later. In the group with 5 min of ischemia, we obtained CS cell discharges from all rats at both 1 and 2 days after ischemia, with cluster frequencies indistinguishable from preischemic levels. In the group with 20 min of ischemia, discharges were noted in 7 neurons of 11 rats after 1 day, and in only 2 neurons of 8 rats after 2 days: their mean frequencies were lower than preischemic levels. In experiments of evoked potentials, the mean percentages of amplitudes of the post-synaptic potential (psp) 2 h after 3, 5 and 20 min of ischemia were 98.0 +/- 10.7 (n = 8), 70.7 +/- 8.22 (n = 9) and 45.1 +/- 6.34% (n = 7) of preischemic amplitudes, respectively. These results suggest that the functional deterioration of spike generation, as well as synaptic transmission, starts during transient ischemia and/or at the early stage of reperfusion.     

Hypothermic prevention of nuclear DNA fragmentation in gerbil hippocampus following transient forebrain ischemia

The protective effect of hypothermia on DNA fragmentation following transient forebrain ischemia in mongolian gerbils was investigated. The DNA fragmentation demonstrated in situ in gerbil hippocampal CA1 was compared between intra- and post-ischemic hypothermia. Intra- ischemic hypothermia prevented the DNA fragmentation in hippocampal CA1 completely while severe DNA damage was observed in post-ischemic hypothermia group. The degree of DNA fragmentation of hippocampal CA1 in the post-ischemic hypothermia group was equal to that in the ischemic control group. The results suggest that hypothermia during a transient forebrain ischemia exerts a protective effect on the post-ischemic hippocampal damage by preventing the DNA fragmentation in CA1 neurons. [Neurol Res 1995; 17; 461-464]     

Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course

An important feature of ischemic brain damage is the selective vulnerability of specific neuronal populations. We studied the distribution and time course of neuronal damage following transient cerebral ischemia in the gerbil, using light microscopy and 45Ca autoradiography. Following 5 min of ischemia, selective neuronal damage determined by abnormal 45Ca accumulation was recognized only in the hippocampal CA1 subfield and part of the inferior colliculus. Ischemia for 10 to 15 min caused extensive neuronal injury in the 3rd and 5th layers of neocortex, the striatum, the septum, the whole hippocampus, the thalamus, the medial geniculate body, the substantia nigra, and the inferior colliculus. Progression of the damage was rapid in the medial geniculate body and the inferior colliculus, moderate in the neocortex, striatum, septum, thalamus, and the substantia nigra, and was delayed in the hippocampal CA1 sector. However, the delayed damage of the hippocampus occurred earlier when the ischemia period was prolonged. Histological observation revealed neuronal loss in the identical sites of the 45Ca accumulation. This study revealed that the distribution and time course of selective neuronal damage by ischemia proceeded with different order of susceptibility and different speed of progression.     

Nitrotyrosine immunoreactivity in gerbil hippocampal CA1 region after transient forebrain ischemia

We examined whether or not nitration of tyrosine residues takes place in the gerbil hippocampal CA1 region after transient forebrain ischemia. The nitration of tyrosine residues to produce nitrotyrosine is a footprint of peroxynitrite, a reaction product of nitric oxide (NO) with superoxide. Nitrotyrosine immunoreactivity had been detected in the CA1 region from the early stage in a reperfused brain at 30 min after transient ischemia until DNA fragmentation and neuronal death appeared at 4 days after transient ischemia. In electron microscopy, we detected, prominently, nitrotyrosine immunoreactivity after transient ischemia in the cytoplasm of the CA1 neurons. Therefore, it is considered that the nitration of tyrosine residues by peroxynitrite may be closely related to apoptosis after transient ischemia.