PGE2-receptor subtype EP4-dependent adherence of mastocytoma P-815 cells to matrix components in subcutaneous tissues overlaying inside surface of air pouch cavity in CDF1 mouse.

OBJECTIVES: It remains to be fully clarified how adhesion of mast cells is regulated in vivo. We previously reported that PGE2-receptor EP4 stimulated the adhesion of mouse mastocytoma P-815 cells to plate-bound fibronectin. Our purpose in this study is to evaluate the adhesion using a system, which can mimic the in vivo adhesion. METHODS: P-815 cells were transplanted in an air pouch produced in the transplantable mice, CDF1. The number of cells that adhere to the subcutaneous tissues overlaying the inside cavity surface was determined. RESULTS: The number of adhered cells was decreased in mice administered with ibuprofen or an EP4 antagonist, ONO AE3-208. A local administration of PGE(2) or a phorbol ester, PMA, increased the number of adhered cells, which was also suppressed in the mice treated with ONO AE3-208. CONCLUSION: Our results suggest that PGE(2)-mediated adhesion of P-815 cells in the subcutaneous tissues of the air pouch is mediated by the EP4 subtype.

Hematopoietic stem cells prevent hair cell death after transient cochlear ischemia through paracrine effects.

Transplantation of hematopoietic stem cells (HSCs) is regarded to be a potential approach for promoting repair of damaged organs. Here, we investigated the influence of hematopoietic stem cells on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Intrascalar injection of HSCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. We also showed that the protein level of glial cell line-derived neurotrophic factor (GDNF) in the organ of Corti was upregulated after cochlear ischemia and that treatment with HSCs augmented this ischemia-induced upregulation of GDNF. A tracking study revealed that HSCs injected into the cochlea were retained in the perilymphatic space of the cochlea, although they neither transdifferentiated into cochlear cell types nor fused with the injured hair cells after ischemia, suggesting that HSCs had therapeutic potential possibly through paracrine effects. Thus, we propose HSCs as a potential new therapeutic strategy for hearing loss.

Protective effect of vitamin E against focal brain ischemia and neuronal death through induction of target genes of hypoxia-inducible factor-1.

Vitamin E has been shown to have protective effects against cerebral ischemia, possibly due to its anti-oxidant effects. However, its non-anti-oxidant, intracellular molecular mechanism remains elusive. For in vivo experiments in rats, orally administered vitamin E significantly reduced not only the brain infarct volume but also space navigation disability after permanent middle cerebral artery (MCA) occlusion. The level of anti-oxidant after MCA occlusion was significantly increased specifically in the ipsilateral brain tissues of vitamin E-treated rats. For in vitro experiments, posttreatment with vitamin E protected primary cultured neurons from nitric oxide-induced insult. Vitamin E induced the expression of the alpha subunit of hypoxia-inducible factor-1 (HIF-1) and its target genes, including vascular endothelial growth factor (VEGF) and heme oxygenase-1. The hypoxia response element on the VEGF promoter was responsible for this vitamin E-induced transcriptional activation of VEGF gene. Taken together, these results suggest that cerebral infarction increased the permeability of vitamin E across the blood-brain barrier, and this increased vitamin E in brain tissue elicited neuroprotective effects not only through scavenging oxidants, as are previously well reported, but also by transactivating HIF-1-dependent genes, which results in protection of brains from ischemic insults.

High expression of interleukin-1beta in the corneal epithelium of MRL/lpr mice is under the control of their genetic background.

MRL/Mp mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), spontaneously develop polyarthritis, sialoadenitis and dacryoadenitis, resembling rheumatoid arthritis (RA), and also corneal involvement such as keratopathy and scleritis, which is a major complication in RA patients. In this study, we found that the expression levels of IL-1beta and MMP-1 mRNAs in cornea were high in both MRL/lpr and MRL/Mp-+/+ strains of mice at an age younger than when they develop any inflammatory lesions. This was not true of other inbred strains, even those bearing the lpr gene, and also not of (NZB x NZW) F1 lupus mice. There was no significant difference in the expression of IL-1alpha and TGFbeta in cornea in these strains. Using crosses between MRL/lpr and C3H/HeJ-lpr/lpr (C3H/lpr) mice, at least the expression of IL-1beta was found to be under the control of the MRL genetic background, likely with a recessive mode of inheritance. Considering that IL-1beta in cornea was detected particularly in the epithelial layer, the high expression of IL-1beta in cornea is most likely involved in the genetic predisposition for corneal involvement and possibly also for arthritis in an MRL strain of mice.

Protective effect of a prosaposin-derived, 18-mer peptide on slowly progressive neuronal degeneration after brief ischemia.

SUMMARY: Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide-induced neuronal damage as expected and significantly up-regulated the expressions of bcl-x(L) mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-x(L).

Expressions of neuropilin-1, neuropilin-2 and semaphorin 3A mRNA in the rat brain after middle cerebral artery occlusion.

This study investigated the spatial and temporal expressions of mRNA encoding neuropilin (Npn)-1, Npn-2 and semaphorin3A (Sema3A) in the rat brain after occlusion of the middle cerebral artery (MAC) distal to the striate branches. The expression of Npn-1 mRNA was transiently upregulated in layers V and VI of the parietal cortex not entering infarction on the lesion side from 3 to 6 h after MCA occlusion. The transient up-regulation of Npn-1 mRNA expression was presumably accompanied by an increase in Npn-1 protein as shown by immunohistochemistry in combination with in situ hybridization histochemistry. Intense Npn-2 mRNA expression was noted temporarily in layer II of the parietal cortex on the lesion side from 1 to 6 h after MCA occlusion. The expression of Sema3A mRNA was upregulated in layer VI of the non-infarcted parietal cortex on the lesion side at 6 h after MCA occlusion. The above increases in mRNA expression were no longer observed at 12 h after MCA occlusion. The expressions of Npn-1, -2 and Sema3A mRNA were not detected in the ventroposterior thalamic nucleus undergoing secondary degeneration after MCA occlusion. In the infarct lesion or ischemic core, neuronal expressions of Npn-1, -2 and Sema3A disappeared by 3 days after MCA occlusion as the neurons in situ entered apoptosis or necrosis. In contrast, ED-1-positive microglia/macrophages with Npn-1 and Npn-2 mRNA were observed in the infarct lesion at 1 week after MCA occlusion. These findings suggest that the temporal up-regulation of Npn-1 and Sema 3A mRNA expressions in the non-infarcted parietal cortex on the lesion side is insufficient to induce neuronal cell death possibly because the up-regulated mRNA molecules are not fully translated and that the overexpression of Npn-1 and/or Npn-2 in the ischemic core with degenerating neurons enables activated microglial cells to contact the damaged neurons in situ for phagocytosis.

Up-regulation of calcineurin Abeta mRNA in the Alzheimer's disease brain: assessment by cDNA microarray.

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.

L-Serine regulates the activities of microglial cells that express very low level of 3-phosphoglycerate dehydrogenase, an enzyme for L-Serine biosynthesis.

Microglia are well known to become activated during various kinds of neuropathological events. The factors that are responsible for the activation, however, are not fully determined. In the present study, L-Ser was shown to enhance production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) by lipopolysaccharide (LPS)-stimulated cultured rat microglial cells. L-Ser, however, did not enhance the expression of mRNAs encoding inducible NO synthase, IL-6 and TNF alpha. On the other hand, astrocytes did not depend on L-Ser for release of IL-6 and TNF alpha. The expression of an enzyme 3-phosphoglycerate dehydrogenase (3PGDH), which is essential for L-Ser biosynthesis from a glycolytic intermediate 3-phosphoglycerate, was investigated. As revealed by Western blotting and immunocytochemical staining, 3PGDH-protein expression in vitro was the highest in astrocytes, intermediate in neurons and the lowest in microglial cells. Semiquantitative RT-PCR showed that microglial cells expressed 3PGDH-mRNA at a lower level than astrocytes. In frozen sections from rat forebrain, only astrocytes were immunoreactive for 3PGDH. The present study suggested that L-Ser is able to modulate microglial function mainly at the translation level because microglial cells cannot synthesize sufficient amount of L-Ser due to the scarce expression of 3PGDH.

The expression of basic fibroblast growth factor and vascular endothelial growth factor in prostatic adenocarcinoma: correlation with neovascularization.

BACKGROUND: Neovascularization associated with tumor invasion and metastasis may be stimulated by factors which are released from tumor cells, tumor-associated inflammatory cells or extracellular matrix. Although basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been characterized as promoters of angiogenesis, their precise localization in prostatic adenocarcinoma remains unclear. MATERIALS AND METHODS: In this study, the immunohistochemical expression of the growth factors and their receptors were studied using paraffin-embedded archival tissues before and after neoadjuvant hormonal therapy. The mRNA expression of the growth factors was also examined using an in situ hybridization (ISH) technique. RESULTS: The ISH study demonstrated that bFGF mRNA was present only in the stromal cells whilst that VEGF mRNA was present only in the adenocarcinoma cells. In contrast, the immunohistochemical study showed that bFGF, FGF receptor, VEGF and VEGF receptor proteins were expressed in adenocarcinoma cells and in endothelial cells. We also observed that microvessel density in prostatic adenocarcinoma was correlated with the degree of the expression of growth factors in the cases without neoadjuvant hormonal therapy. Additionally, the expression of those receptor proteins were much frequently identified in the cases with neoadjuvant hormonal therapy than those without it, while virtually no change in the expression of ligands was observed between the cases without and with neoadjuvant therapies. CONCLUSIONS: In prostatic adenocarcinoma, bFGF and VEGF may correlate with neovascularization through each characteristic pathway. In addition, we assumed that neoadjuvant hormonal therapy may have minimal inhibitory effects on bFGF, VEGF and their receptors.

Induction of phosphorylated-Stat3 following focal cerebral ischemia in mice.

It has been shown that Stat3 is induced following transient cerebral ischemia in rat. However there is no evidence that cerebral ischemia stimulates the expression of phosphorylated-Stat3 (p-Stat3), which can activate cytokine-mediated signal transduction from the membrane to the nucleus. In the present study, we investigated the changes in p-Stat3 expression following middle cerebral artery occlusion in mice. Western blot analysis revealed a significant increase in the p-Stat3 protein in the peripheral part of the ischemic area, starting from 6 h after ischemia. p-Stat3 immunoreactivity was detected only in neurons, but not in astrocytes or microglia, and p-Stat3-positive neurons were increased in number in the peripheral part of the ischemic area at 24 h after ischemia. Double staining with aTdT-mediated biotinylated UTP nick end labeling (TUNEL) kit and the p-Stat3 antibody indicated that p-Stat3-positive neurons were also TUNEL-positive. Subsequent immuno-electron microscopic observations showed that p-Stat3-positive neurons were at different stages of degeneration. The present findings suggest that the increased expression of p-Stat3 after cerebral ischemia could play a crucial role in ischemia-induced neuron death.

Improvement of the viability of cultured rat neurons by the non-essential amino acids L-serine and glycine that upregulates expression of the anti-apoptotic gene product Bcl-w.

The non-essential amino acids L-serine (Ser) and glycine (Gly) have recently been shown to exhibit specific actions in the nervous system. In the present study, L-Ser and Gly promoted the survival of cultured rat cerebrocortical neurons in a concentration-dependent manner as revealed by Alamar blue assay and microtubule-associated protein-2 (MAP2) immunoblotting. The maximum effects of the amino acids were detected at the concentrations of 30-100 microM. L-Ser was more effective than Gly. D-Ser failed to promote neuronal survival. L-Ser and Gly upregulated expression of the anti-apoptotic gene product Bcl-w, while they did not affect the expression of Bcl-xL. The promotion of neuronal survival by L-Ser and Gly may be, at least in part, attributable to the upregulated Bcl-w.

Morphological changes of capillaries in the rat soleus muscle following experimental tenotomy.

We examined the structural changes of capillaries in the rat soleus muscle 4, 7, 14, and 35 days after experimental limb tenotomy. In the soleus muscles after tenotomy, muscle fibres degenerated and some of them were destroyed; the muscle did not seem to recover until the 35th day. On the 14th day, some small muscle fibres, probably regenerating muscle fibres, started forming within the basal-lamina tube and remained after necrosis of a pre-existing muscle fibre. Most capillaries at each stage were of the continuous type. However, about 10% of the capillaries around degenerated muscle fibres at days 4, 7 and 14 consisted of endothelial cells with a small number of fenestrae bridged by a single-layered diaphragm. On the 14th day, capillaries around small regenerating muscle fibres also often had a small number of fenestrations. Even on the 35th day, capillaries occasionally had fenestrations. Additionally, some of the fenestrated capillaries formed small pores at the fenestrated portion of the endothelial cells. The untreated muscles contained only continuous capillaries. These findings suggest that fenestrations in the endothelial cells may occur in intramuscular capillaries not only around degenerated muscle fibres but also around regenerating muscle fibres after tenotomy.

Histochemical cytochrome c oxidase activity and caspase-3 in gerbil hippocampal CA1 neurons after transient forebrain ischemia.

We examined the cytochrome c oxidase (COX) activity in gerbil hippocampal CA1 neurons after 5-min ischemia by a histochemical method in the presence or absence of exogenous cytochrome c. In the CA1 neurons, COX activity without exogenous cytochrome c decreased from 1 h after ischemia, but was restored by the addition of exogenous cytochrome c in the following 6 h after ischemia. These results suggest that it is not COX activity but endogenous cytochrome c that is changed in the early phase after ischemia, and that COX activity begins to decrease 9 h after ischemia. We examined caspase-3 in the CA1 region by immunoblotting, as caspase-3 is known to take part in the cell-death cascade downstream from cytochrome c. Although pro-caspase-3 was strongly detected, active caspase-3 was not detected before and until 84 h after 5-min ischemia. Our data suggested that delayed neuronal death is likely to progress via cytochrome c-release but not via caspase-3 activation.

Cytochrome c release from mitochondria to the cytosol was suppressed in the ischemia-tolerance-induced hippocampal CA1 region after 5-min forebrain ischemia in gerbils.

Cytochrome c was detected by immunoblotting in the cytosolic fraction 3 h after 5-min ischemia in the non-ischemia-tolerant CA1 region in which about 96% of neurons had developed delayed neuronal death, while less cytosolic cytochrome c was detected in the ischemia-tolerance-induced CA1 region where many more neurons survived. In the immunohistochemical study using anti-non-native cytochrome c monoclonal antibody, immunoreactivity was observed throughout the cytoplasm in the non-ischemia-tolerant CA1 neurons, but not in the normal and ischemia-tolerant CA1 neurons. Then we determined whether Bcl-2, Bax, Bcl-xL and Bcl-xS, which regulate the release of cytochrome c from mitochondria, were altered in the ischemia-tolerant CA1 region. Bcl-2 and Bax were up-regulated in the ischemia-tolerant group, but Bcl-xL and Bcl-xS showed no apparent difference in their expression. These results suggest that cytochrome c release is prevented in CA1 neurons in gerbils in which ischemia-tolerance had been induced and that the altered ratio of Bcl-2 to Bax may play a part in this mechanism.

Release of cytochrome c from mitochondria to cytosol in gerbil hippocampal CA1 neurons after transient forebrain ischemia.

We examined cytosolic cytochrome c in gerbil hippocampal CA1 and CA3 regions after induction of 5-min ischemia by immunoblotting. In the CA1 region, cytochrome c was detected in the cytosolic fraction from 1 to 6 h after ischemia by Western blotting, while it was not detected in the CA3 region. Following intraventricular administration of cyclosporin A (CsA), detectable cytosolic cytochrome c was dramatically decreased, and about 80% of CA1 neurons survived after ischemia. The present studies demonstrate that cytochrome c is translocated from mitochondria to the cytosol in the early stage of delayed neuronal cell death, and suggest the involvement of the mitochondrial permeability transition.

Changes in guanylate cyclase activity in arteriolar smooth muscle cells and hemodynamics after ischemia-reperfusion in rats.

Participation of nitric oxide (NO) and hydroxyl radicals in the pathogenesis of hemodynamic alterations after postischemic recirculation were examined by measuring cerebral blood flow (CBF) and estimating guanylate cyclase activities in arteriolar smooth muscle cells using a reversible 2-h thread occlusion model in rats and an electron microhistochemical technique. In the reversible 2-h ischemia model, guanylate cyclase activity in the arteriolar smooth muscle cells increased at the peak of hyperemia and decreased during postischemic hypoperfusion. Administration of N(omega)-nitro-l-arginine (L-NNA), a NO synthase inhibitor, in this model decreased infarct volume and completely inhibited both hyperemia and guanylate cyclase activation at hyperemia. Administration of 1,2-bis(nicotinamido)-propane (AVS), a free radical scavenger, affected neither CBF nor guanylate cyclase activity during hyperemia despite a significant reduction in infarct volume. Administration of L-NNA and AVS significantly suppressed the decrease in CBF during postischemic hypoperfusion and the effect of AVS was greater than that of L-NNA. Although continuous infusion of sodium nitroprusside (SNP) following postischemic hypoperfusion in the reversible 2-h ischemia rats without treatment with L-NNA and AVS did not alter either CBF or guanylate cyclase activity, it significantly elevated both CBF and guanylate cyclase activities in rats administered L-NNA and AVS. The responses of CBF and guanylate cyclase to SNP were also greater in AVS- than L-NNA-treated rats. These results suggest that a physiological vasodilative mechanism is involved in the induction of postischemic hyperemia through the NO-guanylate cyclase pathway in arteriolar smooth muscle cells. Both NO-related and non-related radicals are involved in the pathogenesis of postischemic delayed hypoperfusion through the loss of arteriolar smooth muscle relaxation capability.

Morphological differentiation of microglial cells in culture: involvement of insoluble factors derived from astrocytes.

It is believed that ramified resting microglial cells in the brain are differentiated from macrophage-like ameboid cells, although the mechanism for the differentiation is not fully understood. In the present study, we investigated whether the differentiation of microglial cells is observable in mixed brain cell culture prepared from newborn rat forebrains. In confluent mixed brain cell culture, both ramified and ameboid microglial cells were simultaneously present. The ramified cells were located in or under the astrocyte monolayer, while the ameboid cells were over the layer as revealed by confocal laser scan microscopy. The majority of ramified cells appeared after the astrocyte layer was completely formed and they downregulated the expression of the major histocompatibility complex antigen. Fibronectin was detected around ramified microglial cells, and laminin was also present in the astrocyte monolayer in mixed brain cell culture, while both proteins were not distributed near ameboid cells over the monolayer. When purified microglial cells were cultured on astrocyte-derived extracellular matrix in serum-free medium, they ramified. These results show that the differentiation of microglial cells is observable in culture and that astrocytes may play pivotal roles in the differentiation mainly by secreting insoluble factors.

Astrocytes prevent neuronal death induced by reactive oxygen and nitrogen species.

Reactive oxygen and nitrogen species (RO/NS) such as nitric oxide (NO), hydroxyl radical (OH.), and superoxide anion (O(2)(-)) are generated in a variety of neuropathological processes and damage neurons. In the present study, we investigated the neuroprotective effects of rat astrocytes against RO/NS-induced damage using neuron-glia cocultures, and the effects were compared to those of microglial cells. Sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and FeSO(4) were used to generate NO, O(2)(-) and NO, and OH., respectively. Solely cultured neurons, which were transiently exposed to these agents, degenerated, possibly through apoptotic mechanisms as revealed by in situ detection of DNA fragmentation, whereas neurons cocultured with either astrocytes or microglial cells were viable even after exposure to RO/NS. In contrast, most neurons cocultured with meningeal fibroblasts degenerated. Astrocyte-conditioned medium partially attenuated RO/NS-induced neuronal damage. When neurons were cultured on astrocyte-derived extracellular matrix (AsECM), neuronal death induced by SNP and FeSO(4) was almost completely inhibited. AsECM contained significant amounts of laminin and fibronectin, and pure fibronectin and laminin also protected neurons against RO/NS-induced damage in the same manner as AsECM. These results suggest that astrocytes can protect neurons against RO/NS-induced damage by secreting soluble and insoluble factors.

Increase in p53 protein expression following cortical infarction in the spontaneously hypertensive rat.

Using stroke-prone spontaneously hypertensive (SH-SP) rats with permanent occlusion of the middle cerebral artery (MCA), we investigated the expression of wild type p53 (wt-p53) protein and the occurrence of DNA fragmentation in cerebral neurons after ischemia. Three days following MCA occlusion, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL staining) revealed a distinct pattern of nuclear staining in many neurons around the ischemic core. On the lesioned side of the cerebral cortex one day after MCA occlusion, wt-p53 immunoreactivity was observed specifically in the cortical neurons, in the same regions as the TUNEL staining. Mutant type p53 (mt-p53) immunoreactivity was not observed at any time following MCA occlusion. These findings suggest that wt-p53 dependent cell death of cortical neurons occurred in the ischemic periphery following cerebral ischemia and that this pathway for the induction of cell death may play an important role in the exaggeration of cerebral ischemic injury.

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.