Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

Mineralocorticoid and glucocorticoid receptor expressions in astrocytes and microglia in the gerbil hippocampal CA1 region after ischemic insult

In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia. In blood, corticosterone levels were increased biphasically at 30 min and 12 h after ischemia/reperfusion, and thereafter its levels were decreased. In the sham-operated group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region: at 12 h after ischemia, GR was expressed in a few neurons in the CA1 region, whereas MR was not expressed in any neurons after ischemic insult. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia, MR and GR immunoreactivities peaked in the hippocampal CA1 region. At this time, 55% of astrocytes and 30% of microglia showed MR immunoreactivity, and 20% of astrocytes and 40% of microglia showed GR immunoreactivity. Western blot analyses showed that the pattern of changes in MR and GR protein levels was similar to the immunohistochemical changes observed after transient forebrain ischemia. From 4 days after ischemia, MR and GR protein levels were increased time-dependently after ischemia. In conclusion, enhanced MR and GR expressions in astrocytes and microglia were detected in the hippocampal CA1 region 4-7 days after ischemia/reperfusion. At this time, GR immunoreactivity was abundant in microglia, whereas MR immunoreactivity was prominent in astrocytes. The specific distribution of corticosteroid receptors in the astrocytes and microglia may be associated with the differences of MR and GR functions against ischemic damage.     

孕酮对沙土鼠脑缺血再灌海马小白蛋白免疫反应的影响

目的:观察孕酮对沙土鼠脑缺血再灌海马小白蛋白免疫反应的影响。方法:以双侧颈总动脉夹闭法制作脑缺血再灌注模型,免疫细胞化学方法显示海马小白蛋白免疫反应的变化。结果:脑缺血再灌后1～7 d,海马小白蛋白阳性神经元增多;第7天背侧海马CA1区锥体细胞层呈现小白蛋白阳性致密条带。脑缺血再灌孕酮处理后小白蛋白阳性神经元进一步增加,可见许多串珠状小白蛋白阳性突起,未见海马CA1区锥体细胞层小白蛋白阳性致密条带结构。结论:孕酮可能通过对小白蛋白的表达调控发挥对脑缺血再灌损伤的部分神经保护作用。     

Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus

Neuronal degeneration followed by glial activation (microglia and astrocytes) and nitric oxide synthase (NOS) expression in the hippocampus was investigated at 3 months after domoic acid (DA) administration and compared with DA treated rats at 5 days time interval which was reported earlier. Massive degeneration with complete absence of neurons in the hippocampal CA1 and CA3 regions and hypertrophied microglial cells showing intense immunoreaction with the antibody OX-42 was observed at 3 months after DA administration. Sparsely distributed OX-42 positive microglial cells were observed in the hippocampus of control rats at 3 months after saline treatment No apparent changes could be observed in the immunoreactivity of GFAP at 3 months after saline and DA administration. Neuronal nitric oxide synthase immunoreactive neurons were completely absent in the hippocampus at 3 months after DA administration. In contrast, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical analysis revealed absence of NADPH-d reactivity in the neurons, but positive reactivity in the microglial cells of CA1-CA3 regions in the hippocampus after DA treatment. Double immunofluorescense revealed co-expression of inducible nitric oxide synthase with immunoreactive OX-42 positive microglial cells in the hippocampal subfields at 3 months after DA administration. The microglia-produced NO appears to be a secondary phenomenon in the prolonged inflammatory process following DA-induced neuronal degeneration.     

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Ontogeny of enkephalin-like immunoreactivity in the rat hippocampus

The postnatal development of leucine5-enkephalin-like immunoreactivity within the hippocampal formation of the rat has been analyzed using immunocytochemical techniques. From the day of birth to postnatal day three, no intrinsic hippocampal elements exhibit immunoreactivity although labeled axons are found within the fimbria, within the alveus, and in the vicinity of the angular bundle. On postnatal day 4, a few immunoreactive hippocampal neurons can be seen in stratum radiatum of the region CA3 and by postnatal day 8, within the hilus, strata pyramidale and oriens of regio superior, and the subiculum. There is a dramatic increase in the incidence of immunoreactive perikarya between postnatal days 8 and 10 in all fields as well as the appearance of labeled neurons in CA1 stratum pyramidale and stratum granulosum of the dentate gyrus. Two days after the first appearance of immunoreactive perikarya, intensely immunoreactive neurons, labeled much more extensively than is ever seen in the adult, are encountered in each subfield of the hippocampus. The spatio-temporal order in both the emergence of perikaryal immunoreactivity and the transient appearance of intensely immunoreactive neurons follows that of neurogenesis, with immunoreactivity developing 12-14 days after the peak period of last cell division for a given hippocampal region. The incidence of immunoreactive perikarya in the dentate gyrus was quantified in rat pups ranging from postnatal days 8 to 19. The appearance of labeled neurons followed the spatio-temporal gradients that have been described for neurogenesis in this region as well. Immunoreactive perikarya emerged in the suprapyramidal stratum granulosum prior to their emergence in the infrapyramidal zone and in the temporal pole of the dentate earlier than in the mid-dorsoventral dentate. The lateral perforant path and mossy fiber axons, seen to exhibit enkephalin-like immunoreactivity in the adult hippocampal formation, differ in their relative maturity at the age immunoreactivity first appears. Immunoreactivity appears as early as postnatal day 4 in the lateral perforant path, an age at which these axons are just growing into their target field while it is not found within the mossy fibers until after postnatal day 10, an age at which mossy fiber bouton elaboration is well advanced and physiologically competent mossy fiber synapses with the regio inferior pyramidal cells have been established. The latter observation indicates that enkephalin is not necessary for synaptic transmission at the mossy fiber synapse.     

GABAergic neurons containing somatostatin-like immunoreactivity in the rat hippocampus and dentate gyrus

Summary The distribution of somatostatin-like immunoreactive (SS-LI) material and its colocalization with glutamic acid decarboxylase (GAD)-like immunoreactivity were studied in the rat hippocampus and dentate gyrus neurons using immunohistochemistry. In the dentate gyrus and CA1 region, SS-LI perikarya were concentrated in the hilus and in the stratum oriens, respectively, whereas immunoreactive cell bodies were rarely seen in other layers. Approximately half of the SS-LI neurons of the CA3 region were situated in the stratum oriens, the other half being scattered in strata pyramidale, lucidum and radiatum. About 90% of SS-LI neurons were also GAD-like immunoreactive, whereas about 14% of GAD-like immunoreactive (GAD-LI) neurons were SS-like immunoreactive. The percentage of GAD-LI neurons which were also immunoreactive for SS varied from one layer to the other. This percentage was about 30% in the hilus of the dentate gyrus and in the stratum oriens of the CA1 and CA3 regions; it was 5–10% in the strata pyramidale, lucidum and radiatum of the CA3 region and reached only 2% in the granule cell layer and molecular layer of the dentate gyrus and in the stratum pyramidale and stratum radiatum in the CA1 region. These observations indicate that the majority of SS-LI neurons in the rat hippocampal formation are a subpopulation of GABAergic neurons.     

在戊四氮致痫大鼠早期前脑中0X-42和Fos蛋白的表达变化

研究大鼠在戊四氮导致癫痫发作早期前脑内小胶质细胞的变化及其与神经元的关系，本研究应用免疫组织化学法分别显示前脑内OX-42和Fos蛋白表达的时程变化，并用双重标记显示OX-42和Fos阳性细胞的相互关系。结果发现：在戊四氮导致大鼠癫痫发作早期（从15min到360min），前脑的小胶质细胞0X42表达阳性，随着存活时间的变化，OX42的阳性反应经历逐渐升高又降低的过程；Fos蛋白在神经元和小胶质细胞中有表达，也呈现逐渐升高又降低的变化；Fos在小胶质细胞表达高峰的时间早于在神经元的表达；另外0X-42阳性小胶质细胞和Fos阳性神经元在前脑分布基本相同，主要分布在大脑皮层．海马．杏仁核等部位。以上结果表明，前脑的小胶质细胞和神经元一样在戊四氮所致癫痫发作的早期表现明显的反应，但小胶质细胞反应的意义有待进一步研究。     

在戊四氮致痫大鼠早期前脑中OX-42和Fos蛋白的表达变化

研究大鼠在戊四氮导致癫痫发作早期前脑内小胶质细胞的变化及其与神经元的关系,本研究应用免疫组织化学法分别显示前脑内OX-42和Fos蛋白表达的时程变化,并用双重标记显示OX-42和Fos阳性细胞的相互关系。结果发现:在戊四氮导致大鼠癫痫发作早期(从15min到360min),前脑的小胶质细胞OX-42表达阳性,随着存活时间的变化,OX-42的阳性反应经历逐渐升高又降低的过程;Fos蛋白在神经元和小胶质细胞中有表达,也呈现逐渐升高又降低的变化;Fos在小胶质细胞表达高峰的时间早于在神经元的表达;另外OX-42阳性小胶质细胞和Fos阳性神经元在前脑分布基本相同,主要分布在大脑皮层、海马、杏仁核等部位。以上结果表明,前脑的小胶质细胞和神经元一样在戊四氮所致癫痫发作的早期表现明显的反应,但小胶质细胞反应的意义有待进一步研究。     

Long-term changes in calbindin D(28K) immunoreactivity in the rat hippocampus after cardiac arrest

Calbindin D(28K) (CB) expression was analyzed in the rat hippocampus following 10-min-cardiac arrest-induced ischemia within a year after reperfusion. In rats examined 3 days after ischemia, CB immunoreactivity disappeared completely from CA1 pyramidal neurons and from most CA2 pyramids. In the stratum granulosum of the dentate gyrus, mossy fibers, and hippocampal interneurons, CB immunoreactivity was preserved, although staining was somewhat paler than that in control rats. A similar pattern of CB immunoreactivity was found in rats sacrificed 14 days and 1 month after cardiac arrest. From the 14th postischemic day, neuronal loss in the stratum pyramidale of CA1 but not in that of CA2 became apparent. The reappearance of CB immunoreactivity in CA1 and CA2 pyramidal neurons was noticed 6 months after ischemia, and the pattern was identical to that observed in animals sacrificed 12 months after the ictus. The prolonged loss and delayed reappearance of CB immunoreactivity in the hippocampus demonstrate that ischemia may induce long-term disturbances of protein expression, which may in turn result in impairment of hippocampal functioning.     

Inhibition of the canonical Wnt pathway by Dickkopf-1 contributes to the neurodegeneration in 6-OHDA-lesioned rats

We investigated the cellular localization and progressive changes of corticotropin releasing factor (CRF) in the mouse hippocampus, during and after pilocarpine induced status epilepticus (PISE) and subsequent epileptogenesis. We found that CRF gene expression was up-regulated significantly at 2h during and 1d after PISE in comparison to control mice. Immunohistochemical analysis showed that the number of CRF and Fos immunoreactive cells was increased significantly in the strata oriens and pyramidale of CA1 area and in the stratum pyramidale of CA3 area at 2h during and 1d after PISE. CRF was induced in calbindin (CB) or calretinin (CR) immunoreactive interneurons in stratum oriens at 2h during PISE. It suggests that induced CRF may be related to the over excitation of hippocampal neurons and occurrence of status epilepticus. It may also cause excitoneurotoxicity and delayed loss of CA3 and CA1 pyramidal neurons, leading to the onset of epilepsy.     

人胎海马结构小白蛋白免疫反应性神经元的分布

目的 观察人胎海马结构小白蛋白（PV）免疫反应性神经元的分布。方法 取孕龄为30周的人胎尸体,用ABC免疫细胞化学方法显示PV免疫反应性神经元。结果 海马结构的各区域内均有丰富的PV免疫反应性神经元分布,以锥体细胞怪最为密集。CA1、CA2、CA3始层PV免疫反应性神经元呈散在分布,胞体形态多样,细胞的突起伸向浅怪的始层和深层的分子层;分子层PV免疫反应性神经元较稀少。门区PV免疫反应性神经元分布密集,但细胞分层不明显,可见部分细胞的突起伸向齿状回;齿状回PV免疫反应性神经元集中分布于颗粒细胞层,其余各层在有少量散在PV免疫反应性神经元,细胞染色浅谈,无明显突起,下托复合体PV免疫反应性神经元主要分布于锥体细胞层,始层和分子层较稀少,细胞淡染,突起不明显。结论 海马结构的各区域均有丰富的PV免疫反应性神经元分布,主要分布于锥体细胞层和齿状回的颗粒层。各区域PV免疫反应性神经元发育成熟的时间可能并不同步,CA1－3和门区PV免疫反应性神经元发育成熟早于齿状回和下托复合体。     

Alzheimer's disease: is the decrease of the cholinergic innervation of the hippocampus related to intrinsic hippocampal pathology?

Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerative changes in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32-33%). In contrast, a significant loss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.     

Activation of Src tyrosine kinase in microglia in the rat hippocampus following transient forebrain ischemia

To better understand the pathophysiological role of Src protein, a non-receptor protein tyrosine kinase of 60kDa, in the ischemic brain, we investigated the time course and regional distribution of active Src expression by using a specific antibody against Tyr416 phosphorylated Src (phospho-Src) in the rat hippocampus after transient forebrain ischemia. In the hippocampus of the control animals, active Src expression was too low to be detected by immunolabeling. Beginning 4h after reperfusion, active Src expression became evident and, after 1 day, had increased preferentially in the CA field of the hippocampus proper and the dentate gyrus. By day 3, active Src expression markedly increased in the pyramidal cell layer of CA1 and the dentate hilar region in temporal correlation with neuronal cell death occurring in these areas, where cells typical of phagocytic microglia showed phospho-Src immunoreactivity. Double-labeling experiments revealed that cells expressing active Src were microglia that stained for biotinylated lectin derived from Griffonia simplicifolia (GSI-B4). Active Src expression began to decline at day 7 and returned to the basal level by day 14 after reperfusion. These results demonstrate increased phosphorylation of Src in activated microglia of the post-ischemic hippocampus, indicating that Src signaling may be involved in the microglial reaction to an ischemic insult.     

Increase in choline acetyltransferase activity in septum of rats after transient forebrain ischemia: a possible role of factors released in the hippocampus

Choline acetyltransferase (ChAT) activity increased in rat septum 2 weeks after a transient forebrain ischemia. Extracts were prepared from hippocampus in which CA1 pyramidal neurons had been selectively destroyed by the ischemic insult. ChAT activity in septal neuronal cultures treated with these extracts for 6 days was significantly higher than that in control cultures.     

Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil

Delayed neuronal death was produced in the CA1 area of the hippocampus following 5 min of forebrain ischemia in adult gerbils. Immunohistochemistry and Western blotting to Bcl-2, Bax, and Bcl-x was examined in control (age-matched, non-operated and sham-operated) and ischemic gerbils. Bcl-2 immunoreactivity was low in CA1 neurons, but Bax was highly expressed in CA1 neurons of control gerbils. Moderate Bcl-x immunoreactivity was observed in control CA1 neurons. Strong Bcl-2 and Bcl-x immunoreactivity was found in CA1 neurons following ischemia. Bcl-2, Bax, and Bcl-x were localized in dying cells, thus suggesting that expression of Bcl-2 was not sufficient to prevent nerve cells from dying. Although the Bcl-x antibody does not discriminate between Bcl-xL and Bcl-xS content in tissue sections, Western blots disclosed a marked increase in the intensity of the band corresponding to Bcl-xS, but not of the band corresponding to Bcl-xL in ischemic hippocampi, thus indicating that the increase in Bcl-xS is associated with delayed cell death following transient forebrain ischemia in the adult gerbil. Received: 24 June 1997 / Accepted: 29 January 1998     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, reduces delayed neuronal death following transient forebrain ischemia in the adult rat hippocampus

Recent evidence indicates that statins have beneficial effects on the brain in the ischemic condition. However, there is a lack of studies related to the effect of statins on delayed neuronal death. We investigated the effect of prophylactic therapy with pravastatin on delayed neuronal death in the rat hippocampus. The rats were given a daily dose of 20 mg/kg of pravastatin orally for 14 days. Transient forebrain ischemia was induced by the four-vessel occlusion method. Three days after ischemia, surviving neurons of the hippocampal CA1 subfield were counted. Our results demonstrated that prophylactic statin treatment significantly reduced delayed neuronal death after transient forebrain ischemia. Our findings suggest that prophylactic statin treatment may be useful in preventing functional neurological disorders after transient cerebral ischemic insult.