Anti-inflammatory Effect of Tanshinone I in Neuroprotection Against Cerebral Ischemia–Reperfusion Injury in the Gerbil Hippocampus

Tanshinone I (TsI) is an important lipophilic diterpene extracted from Danshen (Radix Salvia miltiorrhizae) and has been used in Asia for the treatment of cerebrovascular diseases such as ischemic stroke. In this study, we examined the neuroprotective effect of TsI against ischemic damage and its neuroprotective mechanism in the gerbil hippocampal CA1 region (CA1) induced by 5 min of transient global cerebral ischemia. Pre-treatment with TsI protected pyramidal neurons from ischemic damage in the stratum pyramidale (SP) of the CA1 after ischemia–reperfusion. The pre-treatment with TsI increased the immunoreactivities and protein levels of anti-inflammatory cytokines [interleukin (IL)-4 and IL-13] in the TsI-treated-sham-operated-groups compared with those in the vehicle-treated-sham-operated-groups; however, the treatment did not increase the immunoreactivities and protein levels of pro-inflammatory cytokines (IL-2 and tumor necrosis factor-α). On the other hand, in the TsI-treated-ischemia-operated-groups, the immunoreactivities and protein levels of all the cytokines were maintained in the SP of the CA1 after transient cerebral ischemia. In addition, we examined that IL-4 injection into the lateral ventricle did not protect pyramidal neurons from ischemic damage. In conclusion, these findings indicate that the pre-treatment with TsI can protect against ischemia-induced neuronal death in the CA1 via the increase or maintenance of endogenous inflammatory cytokines, and exogenous IL-4 does not protect against ischemic damage.     

Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.     

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.     

Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.     

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 of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic-ischemic conditions

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Mechanisms of Adenosine Release in the Developing and Adult Mouse Hippocampus

Adenosine is a neuromodulator known to inhibit the synaptic release of neurotransmitters, e.g., glutamate, and to hyperpolarize postsynaptic neurons. The release of adenosine is markedly enhanced under ischemic conditions. It may then act as an endogenous neuroprotectant against cerebral ischemia and excitotoxic neuronal damage. The mechanisms by which adenosine is released from nervous tissue are not fully known, particularly in the immature brain. We now characterized the release of [³H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. The properties of the release differed only partially in the immature and mature hippocampus. The K⁺-evoked release was Ca²⁺ and Na⁺ dependent. Anion channels were also involved. Ionotropic glutamate receptor agonists potentiated the release in a receptor-mediated manner. Activation of metabotropic glutamate receptors enhanced the release in developing mice, with group II receptors alone being effective. The evoked adenosine release apparently provides neuroprotective effects against excitotoxicity under cell-damaging conditions. Taurine had no effect on adenosine release in adult mice, but depressed the release concentration dependently in the immature hippocampus.     

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.     

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-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.     

Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK–Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats

Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)–Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague–Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p–c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK–Caspase3 signaling pathway.     

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.     

Chronic 17beta-estradiol pretreatment and ischemia-induced hippocampal degeneration and memory impairments: a 6-month survival study

Exogenous administration of estrogen has been shown to significantly reduce ischemia-induced neuronal degeneration. However, the long-term impact of such treatment on neuronal protection and functional recovery remain largely unknown. The present study assessed the effects of a 15-day pretreatment with 17beta-estradiol on memory deficits and neuronal damage up to 6 months following a 10-min global ischemia in rats. Four groups of ovariectomized female rats [sham-operated and ischemic rats receiving a 15-day pretreatment of either the vehicle or 17beta-estradiol (100 microg/kg)] were tested. The 8-arm radial maze and object recognition tests served to evaluate the impact of 17beta-estradiol treatment on ischemia-induced spatial and recognition memory impairments, respectively. Testing in the radial maze was initiated at two distinct time intervals following reperfusion (7 and 120 days) to evaluate changes in memory functions over time. Our findings revealed long-lasting neuroprotective effects of 17beta-estradiol treatment on hippocampal CA1 pyramidal cells in ovariectomized ischemic rats (43.5% greater neuronal survival than observed in vehicle-treated ischemic animals). Importantly, this neuronal protection translated into significant improvements of recognition and spatial memory functions in estradiol-treated ischemic rats.     

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.     

Neuroprotection by methanol extract of Uncaria rhynchophylla against global cerebral ischemia in rats

In traditional Oriental medicine, Uncaria rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Thus, in the current study, we evaluated neuroprotective effects of U. rhynchophylla after transient global ischemia using 4-vessel occlusion model in rats. Methanol extract of U. rhynchophylla administered intraperitoneally (100-1000 mg/kg at 0 and 90 min after reperfusion) significantly protected hippocampal CA1 neurons against 10 min transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxygenase-2 in hippocampus at 24 hr after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF-alpha and nitric oxide production in BV-2 mouse microglial cells in vitro. These anti-inflammatory actions of U. rhynchophylla extract may contribute to its neuroprotective effects.     

All-Trans-Retinoic Acid Rescues Neurons After Global Ischemia by Attenuating Neuroinflammatory Reactions

Retinoic acid (RA) plays an important role in the developing mammalian nervous system. Based on this concept, some studies have demonstrated the beneficial effects of RA administration on neurogenesis in neuropathological diseases. Some investigations have revealed the anti-inflammatory effects of RA treatment in multiple systems, in addition to its role in neurogenesis. To date, however, the neuroprotective efficacy of RA after cerebral ischemia, especially in the context of its anti-inflammatory effects, has been poorly demonstrated. Additionally, to the best of our knowledge, experiments of the therapeutic efficacy of RA treatment in a transient global ischemic model in the Mongolian gerbil have been lacking worldwide. Here, we studied the neuroprotective effects and neurobehavioral outcomes of intraperitoneally administered all-trans-RA (ATRA; a synthetic form of RA) on brains with transient global ischemia that was induced with the bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model in the gerbil. In order to identify whether these neuroprotective mechanisms were due to the anti-inflammatory effects of ATRA, in vivo hippocampal expression of proinflammatory cytokines including tissue necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) after ATRA injection and in vitro levels of release of nitric oxide, TNF-α and IL-6 from lipopolysaccharide (LPS)-stimulated BV2 microglial cells after ATRA treatment were evaluated. The results showed that ATRA can protect pyramidal neurons in the hippocampal CA1 region against BCCAO-induced neuronal apoptosis and significantly reduce the extent of astrocytosis and microglial activation. In addition, the ischemia-induced neurobehavioral changes were normalized by ATRA injection. Consistent with these phenotypic data, we observed the diminishing effects of ATRA treatment on the production of proinflammatory mediators (e.g., TNF-α and IL-6) in hippocampal homogenates and LPS-stimulated BV2 cells, and these effects were dose-dependent. These results suggest a beneficial role of ATRA in the attenuation of global cerebral ischemia due to its anti-inflammatory properties, resulting in, at least partly, the inhibition of microglial secretion of variable proinflammatory cytokines.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Comparison of the Immunoreactivity of Trx2/Prx3 Redox System in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we compared the immunoreactivities and levels of Trx/prx redox system, thioredoxin 2 (Trx2), thioredoxin reductase 2 (TrxR2) and peroxiredoxin 3 (Prx3), as well as neuronal death in the hippocampal CA1 region between the adult and young gerbil after 5 min of transient cerebral ischemia. At 4 days post-ischemia, pyramidal neurons (about 90%) in the adult stratum pyramidale of the CA1 region showed "delayed neuronal death (DND)"; however, at this time point, few pyramidal neurons showed DND in the young stratum pyramidale. At 7 days post-ischemia, about 56% of pyramidal neurons showed DND in the young stratum pyramidale. The immunoreactivities of all the antioxidants in the young sham-group were similar to those in the adult sham-group. At 4 days post-ischemia, the immunoreactivity of TrxR2, not Trx2 and Prx3 in the adult ischemia-group was dramatically decreased in CA1 pyramidal neurons. At this time point, the immunoreactivities of all the antioxidants in the young ischemia-group were apparently increased compared to the adult ischemia-group. From 7 days pots-ischemia, non-pyramidal cells showed the immunoreactivities of all the antioxidants in the ischemic CA1 region; however, in the young ischemia-groups, the immunoreactivities were much lower than those in the adult ischemia-groups. In brief, our results showed that the immunoreactivities of Trx2, TrxR2 and Prx3 were dramatically increased in CA1 pyramidal neurons of the young ischemia-groups at 4 days post-ischemia compared to those in the adult ischemia-groups induced by transient cerebral ischemia.     

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.