Immunoreactivity of astroglia in the hippocampus of the Mongolian gerbil during short survival following brief ischemia.

Mongolian gerbils subjected to 5-min cerebral ischemia by common carotid artery ligation were decapitated after 24, 48, 72 and 96 h of survival to investigate the immunoreactivity of astroglia in the hippocampus. The sections from formalin-fixed, paraffin-embedded brains were stained histologically and with ABC method (Hsu et al. 1981). Control animals (normal and shame-operated) presented positive GFAP immunostaining in corpus callosum, in subventricular regions, in temporal subcortical white matter, in fimbria hipocampi and perivascularly in stratum lacunosum-moleculare. Experimental animals, independently of postischemic survival time showed various individual GFAP reactivity. Differences concerning the number and localization of immunoreactive astrocytes in both cerebral hemispheres of the same animal stressed the asymmetry of the reaction. The authors did not observe any accumulation of reactive astrocytes in the area of synaptic terminals of glutaminergic fibers (mossy fibers, Schaffer's collaterals) or in the neighbourhood of CA1 and CA3 sectors. In particular, there was complete lack or only sporadic reactive astrocytes among pyramidal neurons of CA1 and among granular cells of dentate gyrus in all examined animals.     

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.     

Expression and changes of Ca2+-ATPase in neurons and astrocytes in the gerbil hippocampus after transient forebrain ischemia

Ca2+-ATPase is one of the most powerful modulators of intracellular calcium levels. In this study, we focused on chronological changes in the immunoreactivity and protein levels of Ca2+-ATPase in the hippocampus after 5 min of transient forebrain ischemia. Ca2+-ATPase immunoreactivity was significantly altered in the hippocampal CA1 region and in the dentate gyrus, but not in the CA2/3 region after ischemic insult. In the sham-operated group, Ca2+-ATPase immunoreactivity was detected in the hippocampus. Ca2+-ATPase immunoreactivity in the CA1 region and in the dentate gyrus, and its protein levels peaked 3 h after ischemic insult. At this time, CA1 pyramidal cells and dentate polymorphic cells showed strong Ca2+-ATPase immunoreactivity. Thereafter, Ca2+-ATPase immunoreactivity reduced in the CA1 region and in the dentate gyrus. One day after ischemic insult, Ca2+-ATPase immunoreactivity was observed in some CA1 non-pyramidal cells, and 4 days after ischemic insult, Ca2+-ATPase immunoreactivity was detected in astrocytes throughout the CA1 region, but Ca2+-ATPase immunoreactivity in the dentate gyrus had nearly disappeared. Our results suggest that Ca2+-ATPase changes may be associated with a response to ischemic damage in hippocampal CA1 pyramidal cells, and that increased Ca2+-ATPase immunoreactivity in the reactive astrocytes may be associated with the maintenance of intracellular calcium levels.     

Glucose metabolism and neurogenesis in the gerbil hippocampus after transient forebrain ischemia

Recent evidence exists that glucose transporter 3 (GLUT3) plays an important role in the energy metabo-lism in the brain. Most previous studies have been conducted using focal or hypoxic ischemia models and have focused on changes in GLUT3 expression based on protein and mRNA levels rather than tissue levels. In the present study, we observed change in GLUT3 immunoreactivity in the adult gerbil hippocampus at various time points after 5 minutes of transient forebrain ischemia. In the sham-operated group, GLUT3 immunoreactivity in the hippocampal CA1 region was weak, in the pyramidal cells of the CA1 region in-creased in a time-dependent fashion 24 hours after ischemia, and in the hippocampal CA1 region decreased signiifcantly between 2 and 5 days after ischemia, with high level of GLUT3 immunoreactivity observed in the CA1 region 10 days after ischemia. In a double immunolfuorescence study using GLUT3 and gli-al-ifbrillary acidic protein (GFAP), we observed strong GLUT3 immunoreactivity in the astrocytes. GLUT3 immunoreactivity increased after ischemia and peaked 7 days in the dentate gyrus after ischemia/reperfu-sion. In a double immunolfuorescence study using GLUT3 and doublecortin (DCX), we observed low level of GLUT3 immunoreactivity in the differentiated neuroblasts of the subgranular zone of the dentate gyrus after ischemia. GLUT3 immunoreactivity in the sham-operated group was mainly detected in the subgran-ular zone of the dentate gyrus. These results suggest that the increase in GLUT3 immunoreactivity may be a compensatory mechanism to modulate glucose level in the hippocampal CA1 region and to promote adult neurogenesis in the dentate gyrus.     

Differential expression of S100beta and glial fibrillary acidic protein in the hippocampus after kainic acid-induced lesions and mossy fiber sprouting in adult rat

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.     

Morphological transformations of cells immunopositive for GFAP, TrkA or p75 in the CA1 hippocampal area following transient global ischemia in the rat. A quantitative study

Transient global ischemia induces intensive neuronal degeneration in the hippocampal CA1 pyramidal layer, accompanied by reactive transformation of glial cells. Previously, we have shown using the double immunostaining method that the NGF receptors (NGFR) p75 and TrkA are expressed mainly on subpopulations of GFAP+ astrocytes, and this expression increases progressively after ischemia. In the presented study, we analyzed quantitatively the morphological transformations of cells immunopositive for GFAP or NGF receptors in the stratum radiatum of the CA1 hippocampal area in different survival periods after ischemia, evoked by 10-min cardiac arrest in adult rats. In control brains, NGF receptors were expressed only on small cells with poorly ramified processes. After ischemia, the NGFR+ cells increased in size and morphological complexity (measured using fractal analysis). However, even 2 weeks after ischemia these cells did not reach the size and value of the fractal dimension typical of the largest GFAP+ astrocytes. Moreover, the reaction of NGFR+ cells was significantly delayed in comparison with the total astrocyte population. The obtained results suggest that NGF receptors are expressed mainly by immature astrocytes and ischemia induces the maturation of these cells.     

Upregulation of phospholipase D in astrocytes in response to transient forebrain ischemia

Previous in vitro studies using cell cultures or brain slices have demonstrated that phospholipase D (PLD) in the nervous system is involved in the signaling mechanism in response to a variety of agonists. However, little is known about the pathophysiological role of PLD-mediated signaling in the adult brain. We examined the changes in the expression of a PLD isozyme, PLD1, in the adult rat hippocampus, using immunological approaches and an assay for PLD activity after transient forebrain ischemia (four-vessel occlusion model) that results in the selective delayed death of CA1 pyramidal cells and induces reactive astrocytes in the CA1 subfield. In the control hippocampus, PLD1 the level of immunoreactivity was very low. After ischemia, in parallel with the results of Western blot analysis and the PLD activity assay, immunohistochemical analysis of PLD1 demonstrated that the immunoreactive proteins peaked at 7-14 days and were most prominent in the CA1 and the dentate hilar region. The temporal and spatial patterns of immunoreactivity of both PLD1 and glial fibrillary acidic protein (GFAP) were very similar, indicating that reactive astrocytes express PLD1, confirmed by double staining for PLD1 and GFAP. These results demonstrate that reactive astrocytes upregulate PLD in vivo after injury in the adult rat hippocampus.     

Changes of neuronal transmission in the hippocampus after transient ischemia in spontaneously hypertensive rats and the protective effects of MK-801.

BACKGROUND AND PURPOSE: I studied the mechanism of postischemic neuronal degeneration in the hippocampus by an electrophysiological method. METHODS: Sequential changes of field potentials evoked by perforant path stimulation in the dentate gyrus and the CA1 region of the hippocampus were evaluated in spontaneously hypertensive rats up to 7 days after transient global ischemia induced by bilateral occlusion of the carotid arteries for 20 minutes after electrocauterization of the vertebral arteries. Animals were treated with vehicle or the excitotoxin antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10 amine (MK-801, 2 mg/kg or 5 mg/kg) intraperitoneally 30 minutes before ischemia. RESULTS: Complete recovery of the population spike was observed in the dentate gyrus within 24 hours after recirculation, followed by a gradual reduction of population spike amplitude. In contrast, population spike in the CA1 region showed partial recovery 24 hours after recirculation, and an abrupt reduction of population spike amplitude occurred on day 2. There was no significant enhancement of population spike amplitude in either region throughout the experiment. Interneuronal recurrent inhibition in the dentate gyrus was enhanced on day 4, and ischemic changes were apparent in the CA1 pyramidal cells on day 7. Pretreatment with 5 mg/kg MK-801 prevented field potential and pathological changes completely in the dentate gyrus and partially in the CA1 region. CONCLUSIONS: My results indicate that pathological changes of the CA1 pyramidal neurons after transient ischemia may not be the result of postischemic overstimulation. However, neuronal transmission in the CA1 region may be persistently impaired during or after transient ischemia.     

Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.

The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field.     

Differential expression of Musashi1 and nestin in the adult rat hippocampus after ischemia

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin.     

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.     

Post-ischemic and kainic acid-induced c-fos protein expression in the rat hippocampus

The c-fos protein is a gene regulatory third messenger involved in long-term responses of cells to various stimuli. It can be used as a marker of neuronal activity. In the present immunohistochemical study the presence of c-fos protein (FP) in the rat brain from 1 h to 14 days after 10 min of cerebral ischemia was compared with that 3 h after an intraventricular injection of kainic acid. The kainic acid injection resulted in staining of dentate hilar cells, granule cells and hippocampal interneurones. The postischemic changes at Day 1 were sporadic CA1 pyramidal cells expressing the FP. At Day 2 FP was expressed with variable intensity in many pyramidal cells in the CA1. At Day 3 many necrotic CA1 pyramidal cells were seen. They did not express the FP, and the expression was less intense and found in fewer cells than at Day 2. At Days 3, 7 and 14 there was increasing gliosis without c-fos expression in the CA1. The study demonstrates a delayed postischemic synthesis of the gene regulatory protein c-fos preceding the necrosis in the selectively vulnerable CA1 region.     

Reactive astrocytes express bis,a bcl-2-binding protein,after transient forebrain ischemia

Bis (also called Bag-3), identified as a novel Bcl-2-interacting protein, has been shown to enhance anti-cell death activity of Bcl-2. Because ischemia/reperfusion induces expression of Bcl-2, we examined the changes in the pattern of Bis expression in the adult rat hippocampus after transient forebrain ischemia. Western blot analysis with protein extracts from the hippocampus showed that, compared with controls, levels of Bis were markedly increased seven days after ischemia. An immunohistochemical study showed that the expression of Bis increased preferentially in the CA1 and the dentate hilar regions, and peaked at 3-7 days after reperfusion. The temporal and spatial patterns of expression for both Bis and glial fibrillary acidic protein (GFAP) were very similar, and double immunofluorescence histochemistry showed that Bis was expressed in reactive astrocytes, which express GFAP. Immunolabeling of adjacent sections with anti-Bcl-2 and anti-Hsp70 antibodies revealed that the pattern of Bis expression closely correlates with that of Bcl-2, but clearly differs from that of Hsp70. Coexpression of Bis and Bcl-2 in reactive astrocytes was confirmed by double immunofluorescence histochemistry. Our results demonstrate that reactive astrocytes transiently up-regulate Bis after ischemia/reperfusion in the adult rat hippocampus. However, the precise role of Bis in the astrocytic response to ischemia/reperfusion in relation to Bcl-2 remains to be determined.     

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.     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

Differential expression of phospholipase D isozymes in the hippocampus following kainic acid-induced seizures

To investigate the pathophysiological role of phospholipase D (PLD)-mediated signaling, changes in the expression of the PLD isozymes PLD1 and PLD2 were investigated in the rat kainic acid (KA) model of human temporal lobe epilepsy. Western blot analysis showed a significant increase in the expression of PLD1 and PLD2 in the postictal hippocampus. PLD1 immunoreactivity increased preferentially in the CA3 and CA1 regions, where pyramidal neurons are susceptible to temporal lobe epilepsy. Experiments employing double immunofluorescence revealed that the cells expressing PLD1 were GFAP-expressing reactive astrocytes. By contrast, PLD2 immunoreactivity increased strikingly in infrapyramidal, but not in suprapyramidal granule cells of the postictal dentate gyrus, fitting well with results of the PLD activity assay. Considering that PLD belongs to a key signaling pathway, this result suggests that changes in granule cell activity in the dentate gyrus after seizures occurs specifically between the supra- and infrapyramidal blades. In addition, enhanced immunoreactivity of PLD2 was observed in the reactive astrocytes of the CA1, CA3, and hilar subregions, but its temporal pattern is different from that of PLD1. Taken together, our results suggest that PLD1 and PLD2 exercise their unique pathophysiological functions in the rat hippocampus after KA-induced seizures.     

Immunohistochemical study on the distribution of insulin-like growth factor I (IGF-I) receptor in the central nervous system of SOD1(G93A) mutant transgenic mice

In the present study, we used the SOD1(G93A) mutant transgenic mice as an in vivo model of ALS and performed immunohistochemical studies to investigate the changes of insulin-like growth factor I (IGF-I) receptor in the central nervous system. IGF-I receptor-immunoreactive astrocytes were detected in the spinal cord, brainstem, central gray and cerebellar nuclei of SOD1(G93A) transgenic mice. In contrast to transgenic mice, no IGF-I receptor-immunoreactive astrocytes were observed in any brain region of wtSOD1 transgenic mice although a few moderately stained neurons were observed. In the hippocampal formation of SOD1(G93A) transgenic mice, IGF-I receptor immunoreactivity was increased in the pyramidal cells of the CA1-3 regions and granule cells of the dentate gyrus. The present study provides the first evidence that IGF-I receptor immunoreactivity was increased in reactive astrocytes in the central nervous system of SOD(G93A) transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for IGF-I receptor, and the functional implications of these increases, require elucidation.     

Hippocampal pathology in refractory temporal lobe epilepsy: T2-weighted signal change reflects dentate gliosis.

BACKGROUND: The MR and pathologic features of hippocampal sclerosis (HS) are well described and include volume decrease and T2-weighted signal increase for MRI, and neuron cell loss and gliosis for pathology. OBJECTIVE: To confirm the established correlation between hippocampal volumes and neuron cell counts, and to study the still controversial association between signal change and gliosis. METHODS: The authors studied 44 patients (22 men and 22 women; mean age at surgery, 37 years) with refractory temporal lobe epilepsy. Quantitative assessment of hippocampal volumes and T2 relaxometry, and neuron and glial cell count in the region CA1 and molecular layer of the dentate gyrus was performed. The proportion of glial fibrillary acidic protein (GFAP)-positive glial cells (reactive astrocytes) was indicated. RESULTS: In a stepwise regression, the ipsilateral hippocampal volume was predicted best by the neuron cell count in the dentate gyrus (p = 0.005, r = 0.4). Hippocampal T2 time, however, was predicted best by the glial cell count in the dentate gyrus (p = 0.01, r = 0.4). None of the other cell counts contributed to either model. In the dentate, 31% of the glial cells were reactive astrocytes, whereas in CA1, 5% were reactive. CONCLUSION: The results confirmed the correlation between hippocampal volumes and neuron cell counts. T2-weighted signal increase in the hippocampus was mainly influenced by gliosis in the dentate gyrus, where a high proportion of glial cells show abnormal activity. This activity may reflect changes important in the development of hippocampal epileptogenicity.     

Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.     

Microglial and Astrocytic Cell Responses in the Rat Hippocampus after an Intracerebroventricular Kainic Acid Injection

After central nervous system injury activated microglial cells and reactive astrocytes secrete neurotrophic factors which may provide an environment conducive to axonal sprouting. The present study has used a unilateral intracerebroventricular (ICV) injection of kainic acid (KA) to produce a lesion of the CA3 pyramidal neurons in the rat hippocampus. The time course of the microglial and astrocytic response was studied throughout a 3-month period using an antibody to proliferating cell nuclear antigen to identify proliferating cells as well as OX-42 and GFAP antibodies to identify the activated microglia and reactive astrocytes, respectively. There was no proliferation of reactive astrocytes whereas activated microglial cells continued to proliferate throughout the duration of the study. During the first month there were some activated microglial cells in the CA1 field and in the fascia dentata but this was short-lived in comparison to the persistance of activated microglia and reactive astrocytes in the CA3 field which were still present 3 months after the initial injection. The discussion attempts to correlate this ipsilateral microglial and astrocytic response with the bilateral mossy fiber axonal sprouting, which occurs in the dentate gyrus after a unilateral ICV injection of KA. The discussion concludes that the two events, the microglial and astrocytic response and the mossy fiber sprouting, are not directly related since the contralateral sprouting occurs in the absence of any astrocytic or microglial response.