Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia

We investigated changes of immunoreactivities of N-methyl-D-aspartate receptor (NR) and of excitatory amino acid carrier 1 (EAAC-1), the neuronal glutamate transporter, in the vulnerable CA1 area and the less vulnerable subiculum of the gerbil hippocampus at various times following transient forebrain ischemia. At 30 min after ischemia-reperfusion, the intensity of NR immunoreactivity increased markedly in neurons of CA1 and subiculum, particularly NR2A/B, while EAAC-1 immunoreactivity was reduced in CA1. At 3 hr after reperfusion, the density of NR1 immunoreactivity markedly decreased in CA1. In contrast EAAC-1 immunoreactivity increased in CA1 and in the subiculum. At 12 hr after reperfusion, the decrease of NR1 immunoreactivity was not detected whereas EAAC-1 immunoreactivities in the CA1 area were intensified. In the subiculum, both NR subunits immunoreactivities decreased significantly, in contrast to the maintenance of EAAC-1 immunoreactivity. At 24 hr after reperfusion, both NR2A/B and EAAC-1 immunoreactivities decreased markedly in CA1 and subiculum. We tentatively suggest that the increase of NR immunoreactivity in CA1 at early times after ischemia-reperfusion may increase the delayed neuronal death, and that the increase or maintenance of EAAC-1 immunoreactivity at early times after ischemia-reperfusion may be an important factor in survival of neurons.     

Post-ischemic regional changes in acetylcholine synthesis following transient forebrain ischemia in gerbils

The synthesis rate of brain acetylcholine (ACh) was estimated 30 min and 5 days following transient forebrain ischemia performed by 10 min bilateral carotid occlusion in gerbils. ACh synthesis was evaluated from the conversion of radiolabeled choline (Ch) into ACh after an i.v. administration of [methyl-³H]Ch. Endogenous and labeled Ch and ACh were quantified by HPLC. The synthesis rate of ACh was significantly decreased following 30 min of recirculation. The reductions reached 55.4% in the hippocampus, 51.2% in the cerebral cortex and 44.4% in the striatum. Five days after ischemia, the values returned to normal in the cerebral cortex and in the striatum, while ACh synthesis remained selectively lowered (–30.4%, p<0.01) in the hippocampus. These cholinergic alterations may account for both early and delayed post-ischemic behavioral and mnesic deficits.     

Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils.

Although galanin (GAL) protects hippocampal neurons from ischemic damage, no study has examined ischemia-related changes in endogenous GAL in the hippocampal dentate gyrus. We investigated the chronological changes of GAL, well-known as the potassium channel opener, expression in the dentate gyrus at various times after 5 min of transient forebrain ischemia in gerbils. A few GAL-immunoreactive (IR) neurons were found in the polymorphic layer of the sham-operated group. Three hours after ischemia-reperfusion, the pattern of GAL immunoreactivity was similar to that of the sham-operated group and the number of GAL-IR neurons and immunoreactivity were highest 12 h after ischemic insult. At this time, GAL-IR neurons in the polymorphic layer showed strong GAL immunoreactivity. Thereafter, GAL-IR neurons and immunoreactivity significantly decreased in the dentate hilar region. Four days after ischemic insult, GAL-IR neurons were not detectable. In addition, the results of a Western blot study showed a pattern of GAL expression similar to the immunohistochemical changes. GAL protein content also was highest 12 h after ischemia. In conclusion, the increased expression of endogenous GAL in the dentate gyrus after ischemia is related to response to the ischemic damage.     

Censoring the editor in transient forebrain ischemia

A molecular explanation for why some neurons are more vulnerable than others to ischemic injury has long remained elusive. In this issue of Neuron, Peng et al. propose that CREB-dependent downregulation of the RNA editing enzyme ADAR2, resulting in defective Q/R editing of AMPA receptor GluR2 subunits and increased availability of calcium and zinc-permeable death-promoting AMPA receptors, underlies the vulnerability of some neuronal populations to ischemia.     

Neurophysiological changes associated with selective neuronal damage in hippocampus following transient forebrain ischemia.

Neurophysiological changes of hippocampal neurons were compared before and after transient forebrain ischemia using intracellular recording and staining techniques in vivo. Ischemic depolarization (ID) was used as an indication of severe ischemia. Under halothane anesthesia, approximately 13 min of ID consistently produced severe neuronal damage in the CA1 region of rat hippocampus, while CA3 pyramidal neurons and dentate granule cells remained intact. After such severe ischemia, approximately 60% of the CA1 neurons exhibited a synaptic potentiation. The excitability of these neurons progressively decreased following reperfusion. Approximately 30% of the CA1 neurons showed a synaptic depression following ischemia. The excitability of these neurons transiently decreased following reperfusion. After ischemia of the same severity, both synaptic transmission and excitability of CA3 and granule cells transiently depressed. These data suggest that ischemia-induced synaptic potentiation may be associated with the pathogenesis of neuronal damage following ischemia, and that the synaptic depression may have protective effects on hippocampal neurons after ischemic insult.     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.     

Changes in parvalbumin immunoreactivity in the parietofrontal cortex after transient forebrain ischemia in the Mongolian gerbil

We investigated the changes in parvalbumin (PV)-immunoreactive (IR) neurons in the parietofrontal cortex after transient forebrain ischemia. In the sham-operated group, PV-IR neurons were present in all layers of the parietofrontal cortex except layer I. Shortly after ischemia the number of PV-IR neurons in layer II/III first increased, and then declined dramatically 12 h after ischemic insult, followed by a second increase after 2 days. At this time the PV immunoreactivity was very weak and only present in the peripheral neuronal cytoplasm. The reversible increase in the number of PV-IR neurons and in the level of their immunoreactivity could result from a transient ischemia-induced increase in intracellular calcium. This pattern of expression was particularly pronounced in layer II/III of the parietofrontal cortex, suggesting that these neurons are especially\susceptible to ischemic insult.     

Regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia

Neurogenesis in the dentate gyrus occurs throughout life. We observed regional differences in neurogenesis in the dentate gyrus of adult rats following transient forebrain ischemia. Nine days after ischemic-reperfusion or sham manipulation, rats were given 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU), a marker for dividing cells. They were killed 1 or 28 days later to distinguish between cell proliferation and survival. Neurogenesis was evaluated by BrdU incorporation as well by identifying neuronal and glial markers in six regions of the dentate gyrus: rostral, middle and caudal along the rostrocaudal axis, each further divided into suprapyramidal and infrapyramidal blade subregions. In control rats BrdU-positive cells in the rostral subregions were significantly lower in the suprapyramidal than in the infrapyramidal blades at both 1 and 28 days after BrdU injection. One day after injection, BrdU-positive cells had increased more in five of the subregions in the ischemic rats than in the controls, the exception being the suprapyramidal blade of the rostral subregion. At 28 days after BrdU injection, numbers of BrdU-positive cells were higher in four subregions in the ischemic group, the exceptions being the rostral suprapyramidal and middle infrapyramidal blades. At 28 days after BrdU injection, the percentages of BrdU positive cells that expressed a neuronal marker (NeuN) were the same in the dentate granule cell layers of ischemic and control rats. Our data thus demonstrate regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia.     

Immunohistochemical and in situ hybridization studies on glycine transporter 1 after transient ischemia in the rat forebrain

Glycine is a critical factor in ischemia as reduced astrocytic and increased extracellular glycine levels aggravate the neurotoxic effect of glutamate and consequently, increase the extent of brain damage. Extracellular levels of glycine are primarily regulated by the plasma membrane glycine transporter 1. In the present study, we examined the effects of transient ischemia (1 h occlusion of the middle cerebral artery; followed by 0 h, 0.5 h, 1 h, 2 h, 4 h, 24 h or 48 h reperfusion) on immunoreactivity and mRNA expression of glycine transporter 1 in the rat forebrain. In control animals, glycine transporter 1-immunoreactivity was strong in diencephalic and certain telencephalic structures, moderate in the globus pallidus, and rather low in the cortex and striatum. In situ hybridization studies revealed a similar distribution pattern of glycine transporter 1 mRNA expression. One hour occlusion of the middle cerebral artery resulted in a significant decrease in ipsilateral glycine transporter 1-immunoreactivity and mRNA expression in a circumscribed region of the preoptic/hypothalamic area; both the immunoreactivity and mRNA exhibited further reductions with increasing reperfusion time. In contrast, the cerebral cortex and the globus pallidus showed an increase of glycine transporter 1-immunoreactivity after 0.5 h reperfusion; the elevation proved to be transient in the somatosensory cortex and remained sustained in the globus pallidus after longer reperfusion times. Western blot analysis of globus pallidus samples from the ipsilateral side confirmed higher glycine transporter 1 protein levels. These results suggest an elevated expression of the transporter protein facilitating the glial uptake of glycine from the extracellular space. However, glycine transporter 1 mRNA expression was not significantly different in the penumbra regions from the corresponding contralateral sites of the injury. Together, these findings indicate that post-translational mechanisms are of primary importance in elevating glycine transporter 1 protein levels following transient ischemia.     

Hyperphosphorylation at serine 199/202 of tau factor in the gerbil hippocampus after transient forebrain ischemia

We examined the phosphorylation state of tau factor in hippocampal delayed neuronal death (DND) after transient forebrain ischemia. A transient phosphorylation increase at serine 199/202 but not serine 396 of tau factor after transient ischemia was clearly observed. Intraventricular injections of olomoucine and U-0126 (CDK5 and MAP kinase inhibitors, respectively) inhibited hyperphosphorylation. In contrast, wortmannin (PI3 kinase inhibitor) increased phosphorylation at serine 199/202 and corresponded with an increase in GSK3 phosphorylation. Our findings suggest that CDK5, MAP kinase, and GSK3 phosphorylate these sites after ischemia. We prepared recombinant normal human tau (N-Tau40) with TAT-HA protein and dephosphorylated-form human Tau-40 (D-tau40) in which 199/202 serines were changed to alanine by site-directed mutagenesis. Intraventricularly injected D-tau40 protected somewhat against DND while N-Tau40 did not. These data suggest that hyperphosphorylation at serine 199/202 of tau factor is induced by MAP kinase, CDK5, and GSK3, and contributes to ischemic neuronal injury.     

Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus.2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism.3. Between 12 and 24 hr postischemia, there was marked restoration of the Na⁺, K⁺, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to fill in gaps along the basal lamina caused by necrotic epithelial cell disintegration.4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system.5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms.6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance.7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus–CSF–brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.     

Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats

Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.     

Sustained expression of osteopontin is closely associated with calcium deposits in the rat hippocampus after transient forebrain ischemia

The present study was designed to evaluate the extent and topography of osteopontin (OPN) protein expression in the rat hippocampus 4 to 12 weeks following transient forebrain ischemia, and to compare OPN expression patterns with those of calcium deposits and astroglial and microglial reactions. Two patterns of OPN staining were recognized by light microscopy: 1) a diffuse pattern of tiny granular deposits throughout the CA1 region at 4 weeks after ischemia and 2) non-diffuse ovoid to round deposits, which formed conglomerates in the CA1 pyramidal cell layer over the chronic interval of 8 to 12 weeks. Immunogold-silver electron microscopy and electron probe microanalysis demonstrated that OPN deposits were indeed diverse types of calcium deposits, which were clearly delineated by profuse silver grains indicative of OPN expression. Intracellular OPN deposits were frequently observed within reactive astrocytes and neurons 4 weeks after ischemia but rarely at later times. By contrast, extracellular OPN deposits progressively increased in size and appeared to be gradually phagocytized by microglia or brain macrophages and some astrocytes over 8 to 12 weeks. These data indicate an interaction between OPN and calcium in the hippocampus in the chronic period after ischemia, suggesting that OPN binding to calcium deposits may be involved in scavenging mechanisms.     

Lack of evidence in neurite growth in the gerbil hippocampal CA1 region 15 days after transient forebrain ischemia

Transient forebrain ischemia promotes a robust increase in neuroblast differentiation in the hippocampal dentate gyrus that peaks 7–15 days after the surgery. In this study, we compared the glucose transporter 3 (GLUT3)-dependent glucose utilization and the dynamin-1 (DNM1)-dependent neurite growth in the hippocampus of Mongolian gerbils 15 days after the induction of transient forebrain ischemia. The animals were subjected to a 5 min transient ischemia protocol and sacrificed 15 days after the surgery. Both doublecortin (DCX) immunoreactive neuroblasts and DCX total protein levels were abundantly increased in the ischemic group compared to the levels observed in the control group. In addition, animals in the ischemic group showed elevated GLUT3 immunoreactivity in the subgranular zone of the dentate gyrus compared to animals in the control group. Based on the double immunofluorescent study, increased DCX-immunoreactive neuroblasts were co-localized with GLUT3-immunoreactive components in the dentate gyrus. However, both the immunoreactivity and the total protein levels of DNM1 were significantly decreased in the dentate gyrus and hippocampal CA1 regions of the ischemic group. These results suggest that the regeneration process such as neurite growth is lacking in the hippocampus 15 days after ischemia/reperfusion although neuroblasts production and glucose utilization increased in the hippocampus.     

Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection

We have previously demonstrated that cytidine 5'-diphosphocholine (CDP-choline or citicoline) attenuated arachidonic acid (ArAc) release and provided significant protection for the vulnerable hippocampal CA(1) neurons of the cornu ammonis after transient forebrain ischemia of gerbil. ArAc is released by the activation of phospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis. Released ArAc is metabolized by cyclooxygenases/lipoxygenases to form eicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicity through generation of lipid peroxides and the cytotoxic byproducts 4-hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase to produce ceramide, a potent pro-apoptotic agent. In the present study, we examined the changes and effect of CDP-choline on ceramide and phospholipids including PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (an exclusive inner mitochondrial membrane lipid essential for electron transport) following ischemia/1-day reperfusion. Our studies indicated significant decreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin and loss of ArAc from PtdEtn in gerbil hippocampus after 10-min forebrain ischemia/1-day reperfusion. CDP-choline (500 mg/kg i.p. immediately after ischemia and at 3-h reperfusion) significantly restored the PtdCho, sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdCho and PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiple beneficial effects of CDP-choline: (1) stabilizing the cell membrane by restoring PtdCho and sphingomyelin (prominent components of outer cell membrane), (2) attenuating the release of ArAc and limiting its oxidative metabolism, and (3) restoring cardiolipin levels.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

Regulation of XIAP and Smac/DIABLO in the rat hippocampus following transient forebrain ischemia

We investigated the expression of XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac/DIABLO, a newly identified mitochondrial apoptogenig molecule in the hippocampus following transient global ischemia. Transient global ischemia produced by two-vessel occlusion triggers the delayed neuronal death of CA1 neurons in the hippocampus. We demonstrate that CA1 neuronal loss induced by ischemia (10 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. XIAP (X chromosome-linked inhibitor of apoptosis protein) is a member of the inhibitor of apoptosis (IAP) gene family that, in addition to suppressing cell death by inhibition of caspases, is involved in an increasing number of signalling cascades. The present study shows alterations in the levels of XIAP and of Smac/DIABLO (second mitochondrial activator of caspase) after cerebral ischemia. The protein levels of XIAP and the number of XIAP-positive cells were regulated by cerebral ischemia in a strictly time and region dependent manner. The largest change in XIAP-IR was observed in the CA1 sub field, which is the most vulnerable area of hippocampus. The mitochondrial expression level of Smac/DIABLO increased during reperfusion. Smac/DIABLO expression was associated with alteration of the XIAP levels and the appearance of activated form of caspase-3 within the hippocampus during reperfusion in spatial and temporal manners.     

Phosphorylation of PTEN and Akt in astrocytes of the rat hippocampus following transient forebrain ischemia

To ascertain whether the PTEN (phosphatase and tensin homolog deleted on chromosome 10)/Akt signaling pathway is activated during ischemic brain injury, we investigated the expression and phosphorylation of PTEN and Akt by immunohistochemistry in the rat hippocampus after transient forebrain ischemia. Weak immunoreactivity for PTEN and its phosphorylated form (p-PTEN) was constitutively expressed in hippocampal neurons and astrocytes of the control rats, but their upregulation was detected mainly in reactive astrocytes in the ischemic hippocampus. Increased immunoreactivity for PTEN and p-PTEN occurred specifically in astrocytes by day 1 and was sustained for more than 2 weeks. The spatiotemporal activation of Akt in the ischemic hippocampus mirrored that of p-PTEN expression. Post-ischemic activation of Akt, revealed by phosphorylated Akt (p-Akt) immunoreactivity, was first detected at day 1 and was maintained for at least 2 weeks. Double-labeling experiments revealed that the cells expressing PTEN, p-PTEN, or p-Akt were reactive astrocytes expressing glial fibrillary acidic protein. These results demonstrate the increased phosphorylation of PTEN and Akt in reactive astrocytes of the post-ischemic hippocampus, suggesting that the PTEN/Akt pathway is involved in the astroglial reaction in the rat hippocampus after transient forebrain ischemia.This research was supported by Korea Science and Engineering Foundation (R01-2002-000-00334-0(2002)).