Domoic acid-induced neurotoxicity in the hippocampus of adult rats

Domoic acid (DA), an agonist of non-N-methyl-D-aspartate (non-NMDA) receptor subtype including kainate receptor, was identified as a potent neurotoxin showing involvement in neuropathological processes like neuronal degeneration and atrophy. In the past decade evidence indicating a role for excitatory amino acids in association with neurological disorders has been accumulating. Although the mechanisms underlying the neuronal damage induced by DA are not yet fully understood, many intracellular processes are thought to contribute towards DA-induced excitotoxic injury, acting in combination leading to cell death. In this review article, we report the leading hypotheses in the understanding of DA-induced neurotoxicity, which focus on the role of DA in neuropathological manifestations, the formation of the retrograde messenger molecule nitric oxide (NO) for the production of free radicals in the development of neuronal damage, the activation of glial cells (microglia and astrocytes) in response to DA-induced neuronal damage and the neuroprotective role of melatonin as a free radical scavenger or antioxidant in DA-induced neurotoxicity. The possible implications of molecular mechanism underlying the neurotoxicity in association with necrosis, apoptosis, nitric oxide synthases (nNOS and iNOS) and glutamate receptors (NMDAR1 and GluR2) related genes and their expression in DA-induced neuronal damage in the hippocampus have been discussed.     

Enhanced neurogenesis during trimethyltin-induced neurodegeneration in the hippocampus of the adult rat

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.     

大鼠轻型颅脑损伤后星形胶质细胞与神经元的病理改变

目的 探讨轻型颅脑损伤（TBI）后神经元及星形胶质细胞改变的病理生理过程。方法 将24只成年SD大鼠随机分为轻型TBI组（n=18）和假手术组（n=6）,轻型TBI组又分为伤后3 h（n=6）、伤后24 h（n=6）、伤后72 h（n=6）三亚组。采用液压冲击法制作轻型TBI模型。采用胶质纤维酸性蛋白（GFAP）染色检测星形胶质细胞,采用Fluoro-Jade B（FJ-B）荧光染色检测变性神经元。结果 与假手术组相比,轻型TBI后3 h、24 h、72 h邻近顶叶皮质、海马CA2/3区GFAP阳性细胞数量均明显减少（P<0.05）;缺失区周围星形胶质细胞肿胀增生明显。FJ-B阳性神经元在损伤后3 h无明显增加（P>0.05）,伤后24 h皮层区FJ-B阳性神经元显著增加（P<0.05）,伤后72 h海马区FJ-B阳性神经元显著增加（P<0.05）。伤后72 h伤侧皮层区与海马区GFAP阳性细胞数和FJ-B阳性细胞数呈显著负相关（r=-0.8285,P<0.05）。结论 轻型TBI后星形胶质细胞超急性期（3 h）即出现损害和胶质反应,神经元则在急性期（24 h）至亚急性期（72 h）出现明显损害,星形胶质细胞缺失程度可以反应神经元损伤程度。     

Early loss of astrocytes after experimental traumatic brain injury

Neuronal-glial interactions are important for normal brain function and contribute to the maintenance of the brain's extracellular environment. Damage to glial cells following traumatic brain injury (TBI) could therefore be an important contributing factor to brain dysfunction and neuronal injury. We examined the early fate of astrocytes and neurons after TBI in rats. A total of 27 rats were euthanized at 0.5, 1, 2, 4, or 24 h after moderate lateral fluid percussion TBI or after sham TBI. Ipsilateral and contralateral hippocampi were examined in coronal sections from -2.12 to -4.80 mm relative to bregma. Adjacent sections were processed with markers for either astrocytes or degenerating neurons. Astrocytes were visualized using glial fibrillary acidic protein (GFAP) or glutamine synthetase immunohistochemistry. Neuronal degeneration was visualized using Fluoro-Jade (FJ) histofluorescence. At 30 min, there was a significant loss of GFAP immunoreactivity in ipsilateral hippocampal CA3 with some loss of normal astrocyte morphology in the remaining cells. The number of normal staining astrocytes decreased progressively over time with extensive astrocyte loss at 24 h. At 4 h, lightly stained FJ-positive neurons were scattered in the ipsilateral CA3. The intensity and number of FJ-positive neurons progressively increased over time with moderate numbers of degenerating neurons in the ipsilateral hippocampal CA3 evident at 24 h. We conclude that astrocyte loss occurs in the hippocampus early after TBI. The data suggest that loss of supporting glial cell may contribute to subsequent neuronal degeneration.     

Nestin expression in hippocampal astrocytes after injury depends on the age of the hippocampus

Analysis of the expression of nestin in reactive astrocytes facilitates quantification of the extent of activation of astrocytes after injury in the mature CNS. We hypothesize that the capability of astrocytes for re-expressing nestin in response to CNS injury diminishes as a function of age. We quantified astrocytes positive for S-100beta protein, glial fibrillary acidic protein (GFAP) and nestin in the hippocampus of young adult, middle-aged, and aged Fischer 344 rats after an intracerebroventricular kainic acid (KA) administration. In all age groups, KA administration induced degeneration of CA3 pyramidal neurons, which led to a significant deafferentation in the CA1 region. The KA-induced neurodegeneration and deafferentation resulted in an increased population of astrocytes positive for S-100beta and glial fibrillary acidic protein (GFAP) in all age groups. Interestingly, these increases were highly comparable across the three age groups. However, in areas of both neurodegeneration and deafferentation, the overall numerical density of nestin-positive reactive astrocytes varied depending on the age at the time of injury with noticeably decreased numerical density in the injured middle-aged and aged hippocampus. In contrast, nestin-immunoreactive radial glia framework after lesion is not impaired with aging in the ependymal lining of the CA3 region.     

Neuronal degeneration and glial cell-responses following trimethyltin intoxication in the rat

Trimethyltin (TMT) preferentially induces neuronal changes in the hippocampus and pyriform cortex. In the present study we investigated the time course of microglial and astroglial responses associated with neurodegeneration after the administration of TMT (i.p. 9 mg/kg or 12 mg/kg body weight) in the rat. At a dosage of 9 mg/kg TMT, neurodegeneration was clearly demonstrated in the CA1 and CA3 regions of the hippocampus as argyrophilic (dark) neurons by day 4 using the Gallyas-Braak (G-B) impregnation method that has been shown to be sensitive and specific for neurodegeneration. Early microglial response was immunohistochemically shown with anti-microglial response factor-1 (MRF-1) antibody in the CA3 by day 1, preceding neurodegeneration morphologically detected by the G-B method. Activation of astrocytes was revealed by immunohistochemical staining for glial fibrillary acidic protein (GFAP) by day 2. In parallel with the maximal neurodegeneration, large numbers of hypertrophied microglia and astrocytes were observed in the CA1 and CA3 by day 7. Numbers of degenerative neurons appeared to be closely associated with adjacent microglia by the double staining of G-B impregnation and MRF-1 immunohistochemistry. The number of reactive microglia considerably decreased to the resting state by day 14, while hypertrophied astrocytes were still prominent in the CA3 up to day 21. With the high dose of TMT, granule cells in the dentate gyrus and CA1 and CA3 pyramidal cells were significantly impregnated. After TMT treatment, accompaning neurodegeneration we observed early response of microglia and prolonged activation of astrocytes, suggesting an individual role of glial cells in maintenance and repair of damaged neurons following brain injury.     

Degeneration and proliferation of astrocytes in the mouse dentate gyrus after pilocarpine-induced status epilepticus

Astrocytes are relatively resistant to injury compared to neurons and oligodendrocytes. Here, we report transient region-specific loss of astrocytes in mice early after pilocarpine-induced status epilepticus (SE). In the dentate hilus, immunoreactivity for glial acidic fibrillary protein (GFAP) was decreased, and the number of healthy appearing GFAP- or S100beta-positive cells was significantly reduced (> or =65%) 1 and 3 days after pilocarpine-induced SE. Many remaining GFAP-positive cells were shrunken, and 1 day after SE electron microscopy revealed numerous electron-dense degenerating astrocyte processes and degenerating glial somata in the hilus. Degeneration of GFAP-expressing cells may be linked to hilar neuronal death, because we did not observe loss of astrocytes after kainate-induced SE, after which hilar neurons remained intact. Ten days after SE, hilar GFAP immunoreactivity had returned, partially from GFAP-positive cells in the hilus. Unlike control mice, many GFAP-positive hilar processes originated from cell bodies located in the subgranular zone (SGZ). To investigate whether proliferation contributes to hilar repopulation, we injected 5-bromo-2'-deoxyuridine (BrdU) 3 days after SE. Five hours later and up to 31 days after SE, many BrdU/GFAP colabeled cells were found in the hilus and the SGZ, some with hilar processes, indicating that proliferation in both areas contributes to generation of hilar astrocytes and astrocyte processes. In contrast to pilocarpine-induced SE in mice, astrocyte degeneration was not found after pilocarpine-induced SE in rats. These findings demonstrate astrocyte degeneration in the mouse dentate hilus specifically in the mouse pilocarpine epilepsy model, followed by astrogenesis leading to hilar repopulation.     

Disturbance of hippocampal long-term potentiation after transient ischemia in GFAP deficient mice.

GFAP (glial fibrillary acidic protein) is an intermediate filament protein found exclusively in the astrocytes of the central nervous system. We studied the role of GFAP in the neuronal degeneration in the hippocampus after transient ischemia using knockout mice. Wild-type C57 Black/6 (GFAP(+/+)) mice and mutant (GFAP(-/-)) mice were subjected to occlusion of both carotid arteries for 5-15 min. Hippocampal slices were prepared 3 days after reperfusion and the field excitatory postsynaptic potentials (fEPSP) in the CA1 were recorded. High frequency stimulation induced robust long-term potentiation (LTP) in GFAP(-/-), as in GFAP(+/+) mice. After ischemia, however, the LTP in GFAP(-/-) was significantly depressed. Similarly, paired pulse facilitation (PPF) displayed little difference between GFAP(+/+) and GFAP(-/-), but after ischemia, the PPF in GFAP(-/-) showed a depression. Histological study revealed that loss of CA1 and CA3 pyramidal neurons after ischemia was marked in GFAP(-/-). MAP2 (dendritic) immunostaining in the post-ischemic hippocampus showed little difference but NF200 (axonal) immunoreactivity was reduced in GFAP(-/-). S100beta (glial) immunoreactivity was similar in the post-ischemic hippocampus of the GFAP(+/+) and GFAP(-/-), indicating that reactive astrocytosis did not require GFAP. Our results suggest that GFAP has an important role in astrocyte-neural interactions and that ischemic insult impairs LTP and accelerates neuronal death.     

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.     

Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia

We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration.     

Transient forebrain ischemia-induced neuronal degeneration in fascia dentata transplants

Fascia dentata tissue blocks from newborn rats were grafted into one-week-old, ibotenic acid-induced lesions of the fascia dentata, or the normal fascia dentata of adult rats. After at least 2 months survival the recipient rats were subjected to 10 min of forebrain ischemia (4-vessel occlusion), and examined 2 or 4 days later for neuronal degeneration in the host hippocampi and the transplants, by silver staining and immunohistochemistry. Transplants survived well in both normal and lesioned host brains, with easily recognizable subfields and layers and presence of normal types of principal and non-principal neurons. As expected, argyrophilic, degenerating neurons were present in the pyramidal cell layer of CAl and CA3c of the non-grafted contralateral host hippocampus and in the contralateral dentate hilus (CA4). In the hilus the degeneration corresponded to the loss of somatostatin-immunoreactive neurons, while parvalbumin-immunoreactive neurons were spared. In the dentate transplants degenerating neurons were observed in the granule cell layer, the hilus and the adjacent CA3 pyramidal cell layer. There was no obvious loss of either somatostatin- or parvalbumin-immunoreactive neurons. The degeneration varied considerably between transplants, from a few to large groups of silver stained neurons, but this difference did not display any obvious relation to grafting into normal or lesioned hosts, the exact location of the grafts or the general organization and distribution of intrinsic or extrinsic host afferents in the grafts. The results demonstrate that both ischemia-susceptible and -resistant types of neurons grafted to normal and lesioned adult rat brains are susceptible to transient forebrain ischemia after transplantation. In spite of an extensive reorganization of transplant nerve connections, the physiologicalbiochemical mechanisms necessary for the induction of ischemic cell death were accordingly present in the transplants.     

Reaction of astrocytes in the gerbil hippocampus following transient ischemia: immunohistochemical observations with antibodies against glial fibrillary acidic protein, glutamine synthetase, and S-100 protein.

An immunohistochemical method was used to study the distribution and changes with time of the astrocytic reaction in the gerbil hippocampus following transient ischemia. Three markers were investigated with specific antibodies to glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and S-100 protein. On Day 2 after ischemia, and more prominently on Day 3, reactive astrocytes were intensely stained for GFAP in the hippocampal formation, especially in the CA1 region and dentate gyrus. This response by astrocytes preceded CA1 pyramidal cell degeneration, which became apparent on Day 5. On Day 5, immunoreactive cells were not stained as intensely as on Day 3, but cells in the CA1 region and dentate gyrus were still more intensely stained than those in normal animals. GS and S-100 showed similar changes in distribution after ischemia, although the change in GS was less prominent: the hilus of the dentate gyrus was most intensely stained. Both immunoreactivities seemed to increase rather transiently on Day 2 or 3 and to decrease to the initial level on Day 5. The fact that reactive astrocytes appeared in CA1 before the onset of visible neural degeneration indicates that signals from indisposed neurons may be transmitted to astrocytes for their quick functioning. It is also suggested that degenerative changes occur in the dentate gyrus and may be involved in the delayed neural death of CA1 pyramidal cells. These observations indicate that astrocytes play a role in the neural degeneration induced by ischemia and that several types of astrocytes seem to react differently.     

Neurotoxicant-induced elevation of adrenomedullin expression in hippocampus and glia cultures.

Adrenomedullin (AM), a vasoactive peptide first isolated from pheochromocytoma, has been reported to be present in neurons in the central nervous system and in tumors of neural and glial origin. In this study, we investigated AM expression both in the hippocampus and in glial cell cultures using a chemical-induced model of injury. An acute intraperitoneal injection of the organometal trimethyltin (TMT) results in neurodegeneration of the hippocampal CA3-4 pyramidal cell layer. Within 4 days of injection, sparse, punctate staining for AM and lectin was evident in the CA3-4 region; by 10 days, a minimal level of CA3-4 neuronal degeneration was evident, with an increase in glial fibrillary acidic protein (GFAP)-positive astrocytes throughout the hippocampus. Degeneration progressed in severity until 30 days post-TMT, with distinct positive immunoreactivity for AM in the CA4 region. mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, GFAP, and AM in the hippocampus were increased over control levels within 4 days following TMT. In cultured glial cells, a 6 hr exposure to TMT (10 microM) produced a morphological response of the cells and increased immunoreactivity for vimentin, GFAP, and AM. mRNA levels for TNFalpha, IL-1alpha, GFAP, vimentin, and AM were elevated within 3-6 hr of exposure. In culture, neutralizing antibodies to IL-1alpha and TNFalpha were effective in inhibiting the TMT-induced elevation of AM mRNA. These data suggest an interaction between the proinflammatory cytokines and glia response in the regulation of AM in response to injury.     

Abnormalities of the hippocampus are similar in deoxycorticosterone acetate-salt hypertensive rats and spontaneously hypertensive rats

Hippocampal neuropathology is a recognised feature of the brain in spontaneously hypertensive rats (SHR), but similar studies are lacking in another model of hypertension, the mineralocorticoid-salt-treated rat. The present study aimed to compare changes in hippocampal parameters in 16-week-old male SHR (blood pressure approximately 190 mmHg) and their normotensive Wistar-Kyoto controls, with those of male Sprague-Dawley rats receiving (i) 10 mg deoxycorticosterone acetate (DOCA) every other day during 3 weeks and drinking 1% NaCl solution (blood pressure approximately 160 mmHg) and normotensive controls treated with (ii) DOCA and drinking water, (iii) drinking water only or (iv) 1% NaCl only. In these experimental groups, we determined: (i) cell proliferation in the dentate gyrus (DG) using the 5-bromo-2'-deoxyuridine-labelling technique; (ii) the number of glial fibrillary acidic protein (GFAP) positive astrocytes under the CA1, CA3 and DG; (iii) the number of apolipoprotein E (ApoE) positive astrocytes as a marker of potential neuronal damage; and (iv) the number of neurones in the hilus of the DG, taken as representative of neuronal density in other hippocampal subfields. Changes were remarkably similar in both models, indicating a decreased cell proliferation in DG, an increased number of astrocytes immunopositive for GFAP and ApoE and a reduced number of hilar neurones. Although hypertension may be a leading factor for these abnormalities, endocrine mechanisms may be involved, because hypothalamic-pituitary function, mineralocorticoid receptors and sensitivity to mineralocorticoid treatment are stimulated in SHR, whereas high exogenous mineralocorticoid levels circulate in DOCA-treated rats. Thus, in addition to the deleterious effects of hypertension, endocrine factors may contribute to the abnormalities of hippocampus in SHR and DOCA-treated rats.     

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down‐regulated proportionally to neuronal loss in the human epileptic hippocampus

Human mesial temporal lobe epilepsy (MTLE) features subregion‐specific hippocampal neurodegeneration and reactive astrogliosis, including up‐regulation of the glial fibrillary acidic protein (GFAP) and down‐regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE‐associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high‐resolution image analysis for digital pathology and whole‐slide imaging. In the cortex, GS‐positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP‐positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1–5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up‐regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS‐pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down‐regulated proportionally to the degree of neuronal loss.     

Lactation Reduces Glial Activation Induced by Excitotoxicity in the Rat Hippocampus

Motherhood induces a series of adaptations in the physiology of the female, including an increase of maternal brain plasticity and a reduction of cell damage in the hippocampus caused by kainic acid (KA) excitotoxicity. We analysed the role of lactation in glial activation in the hippocampal fields of virgin and lactating rats after i.c.v. application of 100 ng of KA. Immunohistochemical analysis for glial fibrillary acidic protein (GFAP) and ionised calcium binding adaptor molecule 1 (Iba-1), which are markers for astrocytes and microglial cell-surface proteins, respectively, revealed differential cellular responses to KA in lactating and virgin rats. A significant astrocyte and microglial response in hippocampal areas of virgin rats was observed 24 h and 72 h after KA. By contrast, no increase in either GFAP- or Iba-1-positive cells was observed in response to KA in the hippocampus of lactating rats. Western blot analysis of GFAP showed an initial decrease at 24 h after KA treatment, with an increase at 72 h in the whole hippocampus of virgin but not of lactating rats. The number of GFAP-positive cells was increased by lactation in the dentate gyrus of the hippocampus but not in CA1 and CA3 areas. The present results indicate that lactating rats exhibit diminished responses of astrocyte and microglial cells in the hippocampus to damage induced by KA, supporting the notion that the maternal hippocampus is resistant to excitotoxic insults.     

Nitric oxide synthase immunoreactivity in the rat hippocampus after status epilepticus induced by perforant pathway stimulation

Nitric oxide has recently been implicated in mediation of neuronal excitotoxicity and damage. This study aimed at elucidating the changes in the expression of neuronal isoform of nitric oxide synthase (nNOS) in the hippocampus after status epilepticus induced by perforant pathway stimulation. nNOS-immunoreactivity (nNOS-ir) and neuronal damage, assessed by silver staining, were evaluated separately in different hippocampal subfields 2 weeks after induction of status epilepticus. Perforant pathway stimulation resulted in an increase in the number of nNOS-immunoreactive neurons in the stratum radiatum of the CA1 and CA3 subfields of the hippocampus proper, and the hilus of the dentate gyrus. The morphology and distribution of the nNOS-ir neurons resembled that of interneurons. No correlation of the number of nNOS-ir neurons to the neuronal damage score was observed. Our results suggest that status epilepticus provokes a de novo expression of nNOS protein, and the nNOS expressing neurons may be selectively resistant to epileptic brain injury.     

An immunohistochemical study of APG-2 protein in the rat hippocampus after transient forebrain ischemia

The cellular localization and spatiotemporal expression pattern of APG-2 protein, a member of the heat shock protein 110 family, were investigated in the rat hippocampus after transient forebrain ischemia. The spatiotemporal patterns of immunoreactivity of both APG-2 and glial fibrillary acidic protein were very similar, indicating that reactive astrocytes express APG-2, which was confirmed by double immunofluorescence histochemistry. Colocalization of APG-2 and a neuronal marker NeuN in the neurons of the CA2 and CA3 subfields was also confirmed.