Copper chaperone for Cu,Zn-SOD supplement potentiates the Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus

In the present study, we investigated the chronological alterations in SOD1 and its copper chaperone (chaperone for superoxide dismutase, CCS) immunoreactivities and their neuroprotective effects against neuronal damage in the gerbil hippocampus after 5 min of transient forebrain ischemia. SOD1 and CCS immunoreactivities were significantly increased in the stratum pyramidale of the CA1 region at 24 and 12 h after ischemic insult, respectively. At 24 h after ischemic insult, the SOD1 and CCS immunoreactivities were colocalized in the CA1 pyramidal cells of the stratum pyramidale. Thereafter, their immunoreactivities were significantly decreased in the CA1 region. To elucidate the effects of CCS or CCS/SOD1, we constructed the expression vectors PEP-1-SOD and PEP-1-CCS. In the CCS-treated group and the CCS/SOD1-treated group, 43.9 and 78.9% pyramidal cells, respectively, compared to the sham-operated group, were stained with cresyl violet 5 or 7 days after ischemic insult. The distribution pattern of active astrocytes and microglia in the PEP-CCS/SOD1-treated group 5 days after ischemic insult was similar to that of the sham-operated group. In addition, the SOD activity in the PEP-CCS- or PEP-CCS/SOD1-treated group was maintained by 10 days after ischemic insult. The SOD activity was higher in the PEP-CCS/SOD1-treated group vs the CCS-treated group. These results suggest that the enhanced expression of SOD1 and CCS may be related to compensatory mechanisms against ischemic damage and that cotreatment with CCS and SOD1 has a greater neuroprotective effect than treatment with CCS or SOD1 in isolation.     

Ischemia-related change of ceruloplasmin immunoreactivity in neurons and astrocytes in the gerbil hippocampus and dentate gyrus

In the present study, we investigated the temporal and spatial alterations of ceruloplasmin immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. In sham-operated animals, ceruloplasmin immunoreactivity in the hippocampal CA2/3 areas was higher than that of other areas. Ceruloplasmin immunoreactivity and its protein content significantly increased and were highest in the CA1 area 1 day after ischemia-reperfusion. At this time point, the immunoreactivity was shown in pyramidal cells of the CA1 area. Four days after ischemia-reperfusion, ceruloplasmin immunoreactivity was shown in astrocytes in the hippocamapal CA1 area. These results suggest that reactive oxygen species (ROS) do not immediately damage neuronal cytosol, unlike DNA. An interval of time is required for the full expression of the cytoplasmic protein injury by ROS. This delayed neuronal injury 1 day after ischemic insult might provide a window of opportunity for therapeutic interventions using antioxidants.     

Ischemia-Induced Changes of PRAS40 and p-PRAS40 Immunoreactivities in the Gerbil Hippocampal CA1 Region After Transient Cerebral Ischemia

Proline-rich Akt substrate of 40-kDa (PRAS40) is one of the important interactive linkers between Akt and mTOR signaling pathways. The increase of PRAS40 is related with the reduction of brain damage induced by cerebral ischemia. In the present study, we investigated time-dependent changes in PRAS40 and phospho-PRAS40 (p-PRAS40) immunoreactivities in the hippocampal CA1 region of the gerbil after 5 min of transient cerebral ischemia. PRAS40 immunoreactivity in the CA1 region was decreased in pyramidal neurons from 12 h after ischemic insult in a time-dependent manner, and, at 5 days post-ischemia, PRAS40 immunoreactivity was newly expressed in astrocytes. p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was hardly found 12 h and apparently detected again 1 and 2 days after ischemic insult. At 5 days post-ischemia, p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was not found. These results indicate that ischemia-induced changes in PRAS40 and p-PRAS40 immunoreactivities in CA1 pyramidal neurons and astrocytes may be closely associated with delayed neuronal death in the hippocampal CA1 region following transient cerebral ischemia.     

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.     

Na+/HCO3- cotransporter immunoreactivity changes in neurons and expresses in astrocytes in the gerbil hippocampal CA1 region after ischemia/reperfusion

The maintenance of intracellular pH is important in neuronal function. Na⁺/HCO₃ ⁻ cotransporter (NBC), a bicarbonate-dependent acid–base transport protein, may contribute to cellular acid–base homeostasis in pathophysiological processes. We examined the alterations of NBC immunoreactivity and its protein levels in the hippocampal CA1 region after transient cerebral ischemia in gerbils. In the sham-operated group, moderate NBC immunoreactivity was detected in CA1 pyramidal neurons, and, 12 h after I/R, the immunoreactivity in the pyramidal neurons was markedly increased over controls. Three days after I/R, NBC immunoreactivity nearly disappeared in the CA1 pyramidal neurons. However, NBC immunoreactivity was detected in the non-pyramidal neurons of the ischemic CA1 region at 3 days after I/R. From double immunofluorescence study with glial markers, NBC immunoreactivity was detected in astrocytes, not in microglia, at 4 days after I/R. NBC protein level in the CA1 region was significantly increased at 12 h post-ischemia and significantly decreased at 2 days post-ischemia. Thereafter, NBC protein level was again increased and returned to the level of the sham-operated group at 4 days post-ischemia. On the other hand, treatment with 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an inorganic anion exchanger blocker including Cl-bicarbonate exchanger, protected CA1 pyramidal neurons from I/R injury at 4 days post-ischemia. These results indicate that changes in NBC expressions may play an important role in neuronal damage and astrocytosis induced by transient cerebral 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.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Comparison of Phosphorylated Extracellular Signal-Regulated Kinase 1/2 Immunoreactivity in the Hippocampal Ca1 Region Induced by Transient Cerebral Ischemia Between Adult and Aged Gerbils

In this study, the authors examined the difference of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the hippocampal CA1 region (CA1) between adult and aged gerbils after 5 min of transient cerebral ischemia. Delayed neuronal death in the CA1 of the aged group was much slower than that in the adult group after ischemia/reperfusion (I/R). pERK1/2 immunoreaction was observed in the CA1 region of the sham-operated adult gerbil. pERK1/2 immunoreactivity and protein levels in the ischemic CA1 region of the adult group were markedly increased 4 days after I/R, and then reduced up to 10 days after I/R. In contrast, pERK1/2 immunoreaction was hardly detected in the CA1 region of sham-operated aged gerbils, and the immunoreactivity increased from 1 day after the ischemic insult, and still observed until 10 days post-ischemia. In addition, pERK1/2-immunoreaction was expressed in astrocytes in the ischemic CA1 region: The expression in the ischemia-operated aged gerbils was later than that in the ischemia-operated adult gerbils. These results indicate that different patterns of ERK1/2 immunoreactivity may be associated with different processes of delayed neuronal death in adult and aged animals.     

Increases of Catalase and Glutathione Peroxidase Expressions by Lacosamide Pretreatment Contributes to Neuroprotection Against Experimentally Induced Transient Cerebral Ischemia

Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin–eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment.     

Thyroxine attenuates hippocampal neuronal damage caused by ischemia in the rat.

We investigated the effect of thyroxine against neuronal damage caused by ischemia in the rat. Neuronal damage was evaluated in the hippocampal CA1 subfield 7 days after a 10 min forebrain ischemia. Thyroxine was administered to animals divided in three groups: 15 min prior to ischemia (group 1), immediately after ischemia (group 2), and both before and after ischemia (group 3). The treatment of rats with a single dose of thyroxine given pre- or postischemia failed to prevent the loss of CA1 pyramidal cells. In contrast, repetitive administration of thyroxine before and after ischemia reduced the damage of the CA1 pyramidal cells. The mechanisms possibly underlying this neuroprotective effect are discussed.     

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult. Ki-Yeon Yoo and In Koo Hwang equally contributed to this article.     

Immediate and Delayed Treatments with Curcumin Prevents Forebrain Ischemia-Induced Neuronal Damage and Oxidative Insult in the Rat Hippocampus

Oxidative stress is believed to contribute to neurodegeneration following ischemic injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of curcumin (Cur) on neuronal death of hippocampal CA1 neurons following transient forebrain ischemia in rat. Treatment of Cur (200 mg/kg/day, i.p.) at three different times (immediately, 3 h and 24 h after ischemia) significantly (P<0.01) reduced neuronal damage 7 days after ischemia. Also, treatment of ischemic rats with Cur decreased the elevated levels of MDA and increased GSH contents, catalase and SOD activities to normal levels. In the in vitro, Cur was as potent as antioxidant (IC₅₀ = 1 μM) as butylated hydroxytoluene. The present study demonstrates that curcumin treatment attenuates forebrain ischemia-induced neuronal injury and oxidative stress in hippocampal tissue. Thus treatment with curcumin immediately or even delayed until 24 h may have the potential to be used as a protective agent in forebrain ischemic insult in human.     

Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

Extract from Terminalia chebula Seeds Protect Against Experimental Ischemic Neuronal Damage Via Maintaining SODs and BDNF Levels

The fruit of Terminalia chebula Retz has been used as a traditional medicine in Asia and contains tannic acid, chebulagic acid, chebulinic acid and corilagin. Extract from T. chebula seeds (TCE) has various biological functions. We observed the neuroprotective effects of TCE against ischemic damage in the hippocampal C1 region (CA1) of the gerbil that had received oral administrations of TCE (100?mg/kg) once a day for 7?days before the induction of transient cerebral ischemia. In the TCE-treated ischemia group, neuronal neuclei (a marker for neurons)-positive neurons were distinctively abundant (62% of the sham group) in the CA1 4?days after ischemia-reperfusion (I-R) compared to those (12.2% of the sham group) in the vehicle-treated ischemia group. Four days after I-R TCE treatment markedly decreased the activation of astrocytes and microglia in the ischemic CA1 compared with the vehicle-treated ischemia group. In addition, immunoreactivities of Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2) and brain-derived neurotrophic factor (BDNF) in the CA1 of the TCE-treated ischemia group were much higher than those in the vehicle-ischemia group 4?days after I-R. Protein levels of SOD1, SOD2 and BDNF in the TCE-treated ischemia group were also much higher than those in the vehicle-ischemia group 4?days after I-R. These results indicate that the repeated supplement of TCE protected neurons from ischemic damage induced by transient cerebral ischemia by maintaining SODs and BDNF levels as well as decreasing glial activation.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of 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.     

Change in platelet endothelial cell adhesion molecule-1 immunoreactivity in the dentate gyrus in gerbils fed a folate-deficient diet

Folate deficiency increases stroke risk. We examined whether folate deficiency affects platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-associated cell adhesion molecule and mediates the final common pathway of neutrophil transendothelial migration, in blood vessels in the gerbil dentate gyrus after transient forebrain ischemia. Gerbils were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to common carotid artery occlusion for 5 min. In the control diet (CD)- and FAD-treated sham-operated groups, weak PECAM-1 immunoreactivity was detected in the blood vessels located in the dentate gyrus. PECAM-1 immunoreactivity in both groups was increased by 4 days after ischemic insult. PECAM-1 immunoreactivity in the FAD-treated group was twice as high that in the CD-treated-sham-operated group 4 days after ischemic insult. Western blot analyses showed that the change patterns in PECAM-1 protein levels in the dentate gyrus in both groups after ischemic insult were similar to changes in PECAM-1 immunohistochemistry in the ischemic dentate gyrus. Our results suggest that folate deficiency enhances PECAM-1 in the dentate gyrus induced by transient ischemia.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult

In the present study, we focused upon expression and changes of endogenous insulin-like growth factor-1 (IGF-1) in the hippocampus of the Mongolian gerbil after ischemic insult. In sham-operated animals, IGF-1 immunoreactivity was absent from the hippocampus. IGF-1-immunoreactive (IR) neurons were detected at 12 h and 1 day after ischemic insult. In the hippocampal CA1 area, the IGF-IR neurons were non-pyramidal cells (GABAergic neurons). In the hippocampal CA2/3 areas, the IGF-1-IR neurons were pyramidal and non-pyramidal cells, and in the dentate gyrus the IGF-1-IR neurons were hilar neurons. Four days after ischemia-reperfusion, IGF-1 immunoreactivity disappeared from neurons, and significantly increased in astrocytes and microglia. These results suggest that the induction of IGF-1 in the CA1 area during the early stage (12-24 h after ischemic insult) is associated with the relative vulnerabilities of pyramidal glutamatergic neurons and non-pyramidal GABAergic neurons. The later increase (4 days after ischemic insult) of IGF-1 expression and protein content was found to promote the activities of astrocytes and microglia. These increases of IGF-1 in astrocytes and in microglia are associated with mechanisms that compensate for the effects of delayed neuronal death.