[14C]Leucine Incorporation into Brain Proteins in Gerbils After Transient Ischemia: Relationship to Selective Vulnerability of Hippocampus

Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to approximately 20-40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to less than 50%. After 1 day, protein synthesis in the CA1 sector returned to approximately 70% of control values, followed by a secondary decline to less than 50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.     

Altered Network Timing in the CA3-CA1 Circuit of Hippocampal Slices from Aged Mice

Network patterns are believed to provide unique temporal contexts for coordinating neuronal activity within and across different regions of the brain. Some of the characteristics of network patterns modeled in vitro are altered in the CA3 or CA1 subregions of hippocampal slices from aged mice. CA3–CA1 network interactions have not been examined previously. We used slices from aged and adult mice to model spontaneous sharp wave ripples and carbachol-induced gamma oscillations, and compared measures of CA3–CA1 network timing between age groups. Coherent sharp wave ripples and gamma oscillations were evident in the CA3–CA1 circuit in both age groups, but the relative timing of activity in CA1 stratum pyramidale was delayed in the aged. In another sample of aged slices, evoked Schaffer collateral responses were attenuated in CA3 (antidromic spike amplitude) and CA1 (orthodromic field EPSP slope). However, the amplitude and timing of spontaneous sharp waves recorded in CA1 stratum radiatum were similar to adults. In both age groups unit activity recorded juxtacellularly from unidentified neurons in CA1 stratum pyramidale and stratum oriens was temporally modulated by CA3 ripples. However, aged neurons exhibited reduced spike probability during the early cycles of the CA3 ripple oscillation. These findings suggest that aging disrupts the coordination of patterned activity in the CA3–CA1 circuit.     

Effects of Transient Cerebral Ischemia on the Expression of DNA Methyltransferase 1 in the Gerbil Hippocampal CA1 Region

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia–reperfusion (I–R), NeuN-positive (⁺) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)⁺ cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.     

Effects of a New Calcium Channel Blocker, KB-2796, on Protein Synthesis of the CA1 Pyramidal Cell and Delayed Neuronal Death Following Transient Forebrain Ischemia

The effects of a new calcium channel blocker, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (KB-2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1, 2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB-2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recirculation. KB-2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB-2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the mammalian hippocampal formation

The distributional pattern of serotonin-immunoreactive nerve fibers in the hippocampal formation of six different mammalian species (rat, chipmunk, hamster, cat, dog and monkey) was studied in detail by means of a modified peroxidase-antiperoxidase immunohistochemical method, using a specific serotonin antiserum. Furthermore, the density of varicosities distributed in each layer of the hippocampus was semiquantitatively analyzed. In all species investigated, the routes of serotonin fibers entering the hippocampal formation were found to be almost the same. These fibers were extensively distributed throughout the hippocampal formation, and had a characteristic arrangement corresponding to the laminar structure of this region. A dense innervation by varicose serotonin fibers was found in the stratum lacunosum-moleculare, but a few serotonin fibers were also distributed in the stratum lucidum of the CA2 and CA3 fields. The stratum pyramidale and the granule cell layer of the dentate gyrus contained a small number of serotonin fibers. The concentration and the direction of serotonin fibers were different in each area of each animal. Two peculiar observations should be stressed: (1) in the rat, the strata oriens and radiatum of CA2 and CA3 as well as the stratum lacunosum-moleculare displayed abundant serotonin fibers; (2) in the dog, abundant serotonin fibers were diffusely distributed in the CA1 field except for the stratum pyramidale and the most dense concentration of serotonin fibers was seen in the stratum oriens of CA3. The present study provides a morphological basis for further study of the functional significance of serotonin in the limbic system.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Iron Deposition after Transient Forebrain Ischemia in Rat Brain

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perl's reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.     

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.     

Time Course of Changes in Immunoreactivities of GABA Degradation Enzymes in the Hippocampal CA1 Region after Adrenalectomy in Gerbils

Adrenalectomy (ADX) has been useful for a good in vivo model for apoptosis in the hippocampus by the absence of corticosteroids following ADX. In some neurodegenerative diseases, GABAergic neurons are more resistant to neuronal damage as compared with glutamatergic neurons. In the present study, we observed chronological changes in three GABA degradation enzymes, e.g., GABA transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivity and protein levels in the gerbil hippocampal CA1 region after ADX. Changes in their immunoreactivities were distinct in the stratum pyramidale of the CA1 region. GABA-T immunoreactivity and protein level were significantly increased in the CA1 region 3 h after ADX, in contrast, SSAR and SSADH immunoreactivity and protein level were increased 12 h and 3–12 h, respectively, after ADX. These results suggest that the increases of GABA-T, SSADH and SSAR immunoreactivity and protein levels in the hippocampal CA1 region in ADX gerbils may be associated with the control of GABA levels in this region.     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Insulin Stimulates Phosphatidylinositol 3-Kinase Activity in Rat Neuronal Primary Cultures

Abstract: The effect of cortical spreading depression (CSD) on cerebral protein synthesis (CPS) was examined. CSD was evoked in normal rats with KCI, and CPS was measured autoradiographically with [1-¹⁴C]leucine. Average rates (mean ± SD) of CPS in layers I-IV of cortex decreased significantly from 10.7 ± 0.6 (sham-operated controls; n = 4) to 6.7 ± 0.7 nmol/g/min (n = 4; p < 0.01) and in layers V-VI from 10.9 ± 0.5 to 9.4 ± 0.4 nmol/g/min (p < 0.05) during 60 min of repetitive CSD. Spreading depression did not affect CPS rates in other subcortical brain regions. These results indicate that KCl-evoked CSD induces inhibition but not suppression of cortical protein synthesis.     

Alpha-methyl-para-tyrosine pretreatment protects from striatal neuronal death induced by four-vessel occlusion in the rat

Rats were treated with alpha-methyl-para-tyrosine (AMT, 250 mg/kg, i.p), an hydroxylase inhibitor, in order to decrease brain levels of catecholamines. Six hours later, when cerebral dopamine (DA) and norepinephrine were reduced by about 80%, a transient forebrain ischemia of 30 min duration was induced by four-vessel occlusion technique. Evaluation of brain damage 72 hours after ischemia showed that AMT treatment significantly decreased neuronal necrosis in the striatum but had no cytoprotective effect in the CA1 sector of the hippocampus and in the neocortex. AMT treatment reduced mortality within the ischemic period but did not affect either the mortality within the recirculation period or the postischemic neurologic deficit. These results suggest that the striatal cytoprotective effect of AMT is linked to cerebral DA depletion and that excessive release of DA during ischemia or dopaminergic hyperactivity during recirculation play a detrimental role in the development of ischemic cell damage in the striatum.     

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.     

外侧隔—海马CA1通路电生理分析

电刺激外侧隔区可在海马 CA1区锥体细胞层记录到群锋电位,在 CA1辐射层顶树突记录到兴奋性突触后电位(EPSP)。侧脑室注射微量海人酸损毁海马 CA3-CA4区锥体细胞后,电刺激外侧隔区在 CA1顶树突不再诱发 EPSP,由此认为外侧隔-CA1顶树突的神经联系是通过同侧海马 CA3锥体细胞侧支实现的。但 CA3-CA4损毁后,电刺激外侧隔区在海马 CA1起层锥体细胞底树突仍可记录到 EPSP。这一在正常情况下被掩盖的外侧隔-CA1底树突神经联系及其来源尚有待探讨。     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英文)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠 15min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠15 min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高.     

Differential response to ischemia in adjacent hippocampalsectors: neuronal death in CA1 versus neurogenesis in dentate gyrus

Two hippocampal sectors show distinct responses to transient ischemia: the cornu Ammonis (CA)1 sector undergoes a delayed neuronal death followed by a lack of neuronal generation, while the dentate gyrus (DG) shows slight postischemic damage followed by an increased neurogenesis. Using the monkey experimental paradigm of transient whole brain global ischemia, the 'calpain-cathepsin hypothesis' was formulated in 1998. This hypothesis proposes that following ischemia calpain compromises the integrity of lysosomal membrane, causing a leakage of degrading hydrolytic enzymes--cathepsins--into the cytoplasm. Ischemia induces Ca(2+) mobilization, calpain activation, lysosomal membrane disruption, and cathepsin release, which all occur specifically in the CA1 sector and cause neuronal death. In the postischemic DG, a vascular niche has been implicated in adult neurogenesis, in that adventitial cells of the DG microvascular environment provoke postischemic up-reguation of neurogenesis with the aid of brain-derived neurotrophic factor and polysialylated form of the neural cell adhesion molecule. In parallel, Down's syndrome cell adhesion molecule has recently been shown to be expressed specifically in the neural progenitor cells of DG. In this review, we focus on the monkey experimental paradigm to reveal the remarkable contrasts between CA1 and DG in response to the ischemic insult.