Antithrombotic effect of SP-8008, a benzoic acid derivative, through the selective inhibition of shear stress-induced platelet aggregation.

BACKGROUND AND PURPOSE: Bleeding is one of the most critical adverse effects of antithrombotic drugs, and many efforts have been made to discover novel antiplatelet agents without bleeding complications. Shear stress-induced platelet aggregation (SIPA), where the interaction of von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib constitutes the initial step, is a promising target to overcome bleeding problems, as SIPA occurs only in pathological conditions. Here, we describe SP-8008, a novel modulator of vWF-GP Ib interactions and evaluated its antiplatelet/antithrombotic effects. EXPERIMENTAL APPROACH: Newly synthesized compounds were screened for antiplatelet effects in vitro, using human platelets exposed to high shear stress. Aggregation, intracellular calcium level, granule secretion, and integrin activation were assessed. Molecular modelling using virtual docking and flow cytometry were used to evaluate effects on vWF-GP Ib interactions. Antithrombotic effects in vivo were determined in rats, using arterial thrombosis and shear stress-specific thrombosis. Transection tail bleeding time was used to evaluate adverse effects. KEY RESULTS: SP-8008 was a potent inhibitor of SIPA, with IC50 of 1.44 ± 0.09 µM. SP-8008 effectively and broadly blocked shear stress-induced platelet activation events, without any significant toxicity. Importantly, SP-8008 was highly selective against SIPA, effectively interfering with vWF-GP Ib engagement. Most importantly, SP-8008 exerted significant antithrombotic effects in vivo in both shear stress-specific and arterial thrombosis, without prolonging bleeding time. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated that SP-8008 can be a novel selective antiplatelet agent with improved safety profile.

Effects of chronic scopolamine treatment on cognitive impairment and neurofilament expression in the mouse hippocampus.

Neurofilaments (NFs) including neurofilament­200 kDa (NF­H), neurofilament­165 kDa (NF­M) and neurofilament­68 kDa (NF­L) are major protein constituents of the brain, and serve important roles in the regulation of axonal transport. NF alteration is a key feature in the pathogenesis of neurological disorders involving cognitive dysfunction. In the present study, cognitive impairments were investigated, via assessments using the Morris water maze and passive avoidance tests, in mice following chronic systemic treatment with 1 mg/kg scopolamine (SCO) for 4 weeks. SCO­induced cognitive impairments were significantly observed 1 week following the SCO treatment, and these cognitive deficits were maintained for 4 weeks. However, the NF immunoreactivities and levels were altered differently according to the hippocampal subregion following SCO treatment. NF­H immunoreactivity and levels were markedly altered in all hippocampal subregions, and were significantly increased 1 week following the SCO treatment; thereafter, the immunoreactivity and levels significantly decreased with time. NF­M immunoreactivity and levels gradually decreased in the hippocampus and were significantly decreased 4 weeks following SCO treatment. NF­L immunoreactivity and levels gradually decreased in the hippocampus, and were significantly decreased 2 and 4 weeks following SCO treatment. In conclusion, the results of the present study demonstrated that chronic systemic treatment with SCO induced cognitive impairment from 1 week following SCO treatment, and NF expression was diversely altered according to the hippocampal subregion from 1 week following SCO treatment. These results suggest that SCO­induced changes in NF expression may be associated with cognitive impairment.

Effect of hyperthermia on calbindin-D 28k immunoreactivity in the hippocampal formation following transient global cerebral ischemia in gerbils.

Calbindin D-28K (CB), a Ca2+-binding protein, maintains Ca2+ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We investigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei (NeuN) and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia (39.5 ± 0.2°C) was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neuronal damage/death in the pyramidal layer of CA1-3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreactivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immunoreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.

Effects of ischemic preconditioning on PDGF-BB expression in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Ischemic preconditioning (IPC) is induced by exposure to brief durations of transient ischemia, which results in ischemic tolerance to a subsequent longer or lethal period of ischemia. In the present study, the effects of IPC (2 min of transient cerebral ischemia) were examined on immunoreactivity of platelet­derived growth factor (PDGF)­BB and on neuroprotection in the gerbil hippocampal CA1 region following lethal transient cerebral ischemia (LTCI; 5 min of transient cerebral ischemia). IPC was subjected to a 2­min sublethal ischemia and a LTCI was given 5­min transient ischemia. The animals in all of the groups were given recovery times of 1, 2 and 5 days and change in PDGF­BB immunoreactivity was examined as was the neuronal damage/death in the hippocampus induced by LTCI. LTCI induced a significant loss of pyramidal neurons in the hippocampal CA1 region 5 days after LTCI, and significantly decreased PDGF­BB immunoreactivity in the CA1 pyramidal neurons from day 1 after LTCI. Conversely, IPC effectively protected the CA1 pyramidal neurons from LTCI and increased PDGF­BB immunoreactivity in the CA1 pyramidal neurons post­LTCI. In conclusion, the results demonstrated that LTCI significantly altered PDGF­BB immunoreactivity in pyramidal neurons in the hippocampal CA1 region, whereas IPC increased the immunoreactivity. These findings indicated that PDGF­BB may be associated with IPC­mediated neuroprotection.

Neuroprotection of ischemic preconditioning is mediated by thioredoxin 2 in the hippocampal CA1 region following a subsequent transient cerebral ischemia.

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox-regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)-mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham-operated group, ischemia-operated group, IPC plus (+) sham-operated group, IPC + ischemia-operated group, IPC + auranofin (a TrxR2 inhibitor) + sham-operated group and IPC + auranofin + ischemia-operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-group 5 days after ischemia-reperfusion; in the IPC + ischemia-operated-group, pyramidal neurons in the SP were well protected. In the IPC + ischemia-operated-group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham-operated-group after ischemia-reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( O2-) production, denatured cytochrome c expression and TUNEL-positive cells in the IPC + ischemia-operated-group were similar to those in the sham-operated-group. Conversely, the treatment of auranofin to the IPC + ischemia-operated-group significantly increased cell damage/death and abolished the IPC-induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, O2- production, denatured cytochrome c expression and TUNEL-positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.

Roles of HIF-1α, VEGF, and NF-κB in Ischemic Preconditioning-Mediated Neuroprotection of Hippocampal CA1 Pyramidal Neurons Against a Subsequent Transient Cerebral Ischemia.

Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic insults by specific mechanisms. We tested the hypothesis that IPC attenuates post-ischemic neuronal death in the gerbil hippocampal CA1 region (CA1) throughout hypoxia inducible factor-1α (HIF-1α) and its associated factors such as vascular endothelial growth factor (VEGF) and nuclear factor-kappa B (NF-κB). Lethal ischemia (LI) without IPC increased expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) in CA1 pyramidal neurons at 12 h and/or 1-day post-LI; thereafter, their expressions were decreased in the CA1 pyramidal neurons with time and newly expressed in non-pyramidal cells (pericytes), and the CA1 pyramidal neurons were dead at 5-day post-LI, and, at this point in time, their immunoreactivities were newly expressed in pericytes. In animals with IPC subjected to LI (IPC/LI)-group), CA1 pyramidal neurons were well protected, and expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) were significantly increased compared to the sham-group and maintained after LI. Whereas, treatment with 2ME2 (a HIF-1α inhibitor) into the IPC/LI-group did not preserve the IPC-mediated increases of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) expressions and did not show IPC-mediated neuroprotection. In brief, IPC protected CA1 pyramidal neurons from LI by upregulation of HIF-1α, VEGF, and p-IκB-α expressions. This study suggests that IPC increases HIF-1α expression in CA1 pyramidal neurons, which enhances VEGF expression and NF-κB activation and that IPC may be a strategy for a therapeutic intervention of cerebral ischemic injury.

Effect of ischemic preconditioning on the expression of c-myb in the CA1 region of the gerbil hippocampus after ischemia/reperfusion injury.

OBJECTIVES: In the present study, we investigated the effect of ischemic preconditioning (IPC) on c-myb immunoreactivity as well as neuronal damage/death after a subsequent lethal transient ischemia in gerbils. MATERIALS AND METHODS: IPC was subjected to a 2 min sublethal ischemia and a lethal transient ischemia was given 5 min transient ischemia. The animals in all of the groups were given recovery times of 1 day, 2 days and 5 days and we examined change in c-myb immunoreactivity as well as neuronal damage/death in the hippocampus induced by a lethal transient ischemia. RESULTS: A lethal transient ischemia induced a significant loss of cells in the stratum pyramidale (SP) of the hippocampal CA1 region at 5 days post-ischemia, and this insult showed that c-myb immunoreactivity in cells of the SP of the CA1 region was significantly decreased at 2 days post-ischemia and disappeared at 5 days post-ischemia. However, IPC effectively prevented the neuronal loss in the SP and showed that c-myb immunoreactivity was constitutively maintained in the SP after a lethal transient ischemia. CONCLUSION: Our results show that a lethal transient ischemia significantly decreased c-myb immunoreactivity in the SP of the CA1 region and that IPC well preserved c-myb immunoreactivity in the SP of the CA1 region. We suggest that the maintenance of c-myb might be related with IPC-mediated neuroprotection after a lethal ischemic insult.

Post-ischemic treatment of WIB801C, standardized Cordyceps extract, reduces cerebral ischemic injury via inhibition of inflammatory cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Anti-inflammatory therapy has been intensively investigated as a potential strategy for treatment of cerebral stroke. However, despite many positive outcomes reported in animal studies, anti-inflammatory treatments have not proven successful in humans as yet. Although immunomodulatory activity and safety of Cordyceps species (Chinese caterpillar fungi) have been proven in clinical trials and traditional Asian prescriptions for inflammatory diseases, its anti-ischemic effect remains elusive. AIM OF THE STUDY: In the present study, therefore, we investigated the potential therapeutic efficacy of WIB801C, the standardized extract of Cordyceps militaris, for treatment of cerebral ischemic stroke. MATERIALS AND METHODS: The anti-chemotactic activity of WIB801C was assayed in cultured rat microglia/macrophages. Sprague-Dawley rats were subjected to ischemic stroke via either transient (1.5-h tMCAO and subsequent 24-h reperfusion) or permanent middle cerebral artery occlusion (pMCAO for 24-h without reperfusion). WIB801C was orally administered twice at 3- and 8-h (50mg/kg each) after the onset of MCAO. Infarct volume, edema, blood brain barrier and white matter damages, neurological deficits, and long-term survival rates were investigated. The infiltration of inflammatory cells into ischemic lesions was assayed by immunostaining. RESULTS: WIB801C significantly decreased migration of cultured microglia/macrophages. This anti-chemotactic activity of WIB-801C was not mediated via adenosine A3 receptors, although cordycepin, the major ingredient of WIB801C, is known as an adenosine receptor agonist. Post-ischemic treatment with WIB801C significantly reduced the infiltration of ED-1-and MPO-positive inflammatory cells into ischemic lesions in tMCAO rats. WIB801C-treated rats exhibited significantly decreased infarct volume and cerebral edema, less white matter and blood-brain barrier damages, and improved neurological deficits. WIB801C also improved survival rates over 34 days after ischemia onset. A significant reduction in infarct volume and neurobehavioral deficits by WIB801C was also observed in rats subjected to pMCAO. CONCLUSIONS: In summary, post-ischemic treatment of WIB801C reduced infiltration of inflammatory cells into ischemic lesions via inhibition of chemotaxis, which confers long-lasting histological and neurological protection in ischemic brain. WIB801C may be a promising anti-ischemic drug candidate with clinically relevant therapeutic time window and safety.

Monocarboxylate transporter 4 plays a significant role in the neuroprotective mechanism of ischemic preconditioning in transient cerebral ischemia.

Monocarboxylate transporters (MCTs), which carry monocarboxylates such as lactate across biological membranes, have been associated with cerebral ischemia/reperfusion process. In this study, we studied the effect of ischemic preconditioning (IPC) on MCT4 immunoreactivity after 5 minutes of transient cerebral ischemia in the gerbil. Animals were randomly designated to four groups (sham-operated group, ischemia only group, IPC + sham-operated group and IPC + ischemia group). A serious loss of neuron was found in the stratum pyramidale of the hippocampal CA1 region (CA1), not CA2/3, of the ischemia-only group at 5 days post-ischemia; however, in the IPC + ischemia groups, neurons in the stratum pyramidale of the CA1 were well protected. Weak MCT4 immunoreactivity was found in the stratum pyramidale of the CA1 in the sham-operated group. MCT4 immunoreactivity in the stratum pyramidale began to decrease at 2 days post-ischemia and was hardly detected at 5 days post-ischemia; at this time point, MCT4 immunoreactivity was newly expressed in astrocytes. In the IPC + sham-operated group, MCT4 immunoreactivity in the stratum pyramidale of the CA1 was increased compared with the sham-operated group, and, in the IPC + ischemia group, MCT4 immunoreactivity was also increased in the stratum pyramidale compared with the ischemia only group. Briefly, present findings show that IPC apparently protected CA1 pyramidal neurons and increased or maintained MCT4 expression in the stratum pyramidale of the CA1 after transient cerebral ischemia. Our findings suggest that MCT4 appears to play a significant role in the neuroprotective mechanism of IPC in the gerbil with transient cerebral ischemia.

Simvastatin Reduces Lipopolysaccharides-Accelerated Cerebral Ischemic Injury via Inhibition of Nuclear Factor-kappa B Activity.

Preceding infection or inflammation such as bacterial meningitis has been associated with poor outcomes after stroke. Previously, we reported that intracorpus callosum microinjection of lipopolysaccharides (LPS) strongly accelerated the ischemia/reperfusion-evoked brain tissue damage via recruiting inflammatory cells into the ischemic lesion. Simvastatin, 3-hydroxy-3-methylgultaryl (HMG)-CoA reductase inhibitor, has been shown to reduce inflammatory responses in vascular diseases. Thus, we investigated whether simvastatin could reduce the LPS-accelerated ischemic injury. Simvastatin (20 mg/kg) was orally administered to rats prior to cerebral ischemic insults (4 times at 72, 48, 25, and 1-h pre-ischemia). LPS was microinjected into rat corpus callosum 1 day before the ischemic injury. Treatment of simvastatin reduced the LPS-accelerated infarct size by 73%, and decreased the ischemia/reperfusion-induced expressions of pro-inflammatory mediators such as iNOS, COX-2 and IL-1ß in LPS-injected rat brains. However, simvastatin did not reduce the infiltration of microglial/macrophageal cells into the LPS-pretreated brain lesion. In vitro migration assay also showed that simvastatin did not inhibit the monocyte chemoattractant protein-1-evoked migration of microglial/macrophageal cells. Instead, simvastatin inhibited the nuclear translocation of NF-κB, a key signaling event in expressions of various proinflammatory mediators, by decreasing the degradation of IκB. The present results indicate that simvastatin may be beneficial particularly to the accelerated cerebral ischemic injury under inflammatory or infectious conditions.

Hyperthermic preconditioning severely accelerates neuronal damage in the gerbil ischemic hippocampal dentate gyrus via decreasing SODs expressions.

It is well known that neurons in the dentate gyrus (DG) of the hippocampus are resistant to short period of ischemia. Hyperthermia is a proven risk factor for cerebral ischemia and can produce more extensive brain damage related with mortality rates. The aim of this study was to examine the effect of hyperthermic conditioning (H) on neuronal death, gliosis and expressions of SODs as anti-oxidative enzymes in the gerbil DG following 5 min-transient cerebral ischemia. The animals were randomly assigned to 4 groups: 1) (N+sham)-group was given sham-operation with normothermia (N); 2) (N+ischemia)-group was given 5 min-transient ischemia with N; 3) (H+sham)-group was given sham-operation with H; and 4) (H+ischemia)-group was given 5 min-transient cerebral ischemia with H. H (39±0.5°C) was induced by subjecting the animals to a heating pad for 30 min before and during the operation. In the (N+ischemia)-groups, a significant neuronal death was observed in the polymorphic layer (PL) from 1 day after ischemia-reperfusion. In the (H+ischemia)-groups, neuronal death was also observed in the PL from 1day post-ischemia; the degree of the neuronal death was severer than that in the (N+ischemia)-groups. In addition, we examined the gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti- ionized calcium-binding adapter molecule 1 (Iba-1). GFAP(+) and Iba-1(+) glial cells were much more activated in the (H+ischemia)-groups than those in the (N+ischemia)-groups. On the other hand, immunoreactivities and levels of SOD1 rather than SOD2 were significantly lower in the (H+ischemia)-groups than those in the (N+ischemia)-groups. In brief, on the basis of our findings, we suggest that cerebral ischemic insult with hyperthermic conditioning brings up severer neuronal damage and gliosis in the polymorphic layer through reducing SOD1 expression rather than SOD2 expression in the DG.

Ischemic preconditioning maintains the immunoreactivities of glucokinase and glucokinase regulatory protein in neurons of the gerbil hippocampal CA1 region following transient cerebral ischemia.

Glucokinase (GK) is involved in the control of blood glucose homeostasis. In the present study, the effect of ischemic preconditioning (IPC) on the immunoreactivities of GK and its regulatory protein (GKRP) following 5 min of transient cerebral ischemia was investigated in gerbils. The gerbils were randomly assigned to four groups (sham­operated group, ischemia­operated group, IPC + sham­operated group and IPC + ischemia­operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia, followed by 1 day of recovery. In the ischemia­operated group, a significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) at 5 days post­ischemia; however, in the IPC+ischemia­operated group, the neurons in the SP were well protected. Following immunohistochemical investigation, the immunoreactivities of GK and GKRP in the neurons of the SP were markedly decreased in the CA1, but not the CA2/3, from 2 days post­ischemia, and were almost undetectable in the SP 5 days post­ischemia. In the IPC + ischemia­operated group, the immunoreactivities of GK and GKRP in the SP of the CA1 were similar to those in the sham­group. In brief, the findings of the present study demonstrated that IPC notably maintained the immunoreactivities of GK and GKRP in the neurons of the SP of CA1 following ischemia­reperfusion. This indicated that GK and GKRP may be necessary for neuron survival against transient cerebral ischemia.

Ischemic preconditioning protects neurons from damage and maintains the immunoreactivity of kynurenic acid in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Pyramidal neurons in region I of hippocampus proper (CA1) are particularly vulnerable to excitotoxic processes following transient forebrain ischemia. Kynurenic acid (KYNA) is a small molecule derived from tryptophan when this amino acid is metabolized through the kynurenine pathway. In the present study, we examined the effects of ischemic preconditioning (IPC) on the immunoreactivity and protein levels of KYNA following 5 min of transient forebrain ischemia in gerbils. The animals were randomly assigned to 4 groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated group, we observed a significant loss of pyramidal neurons in the CA1 stratum pyramidale (SP) at 5 days post-ischemia; however, in the IPC + ischemia-operated group, the pyramidal neurons were well protected. KYNA immunoreactivity in the SP of the ischemia-operated group was significantly altered following ischemia-reperfusion and was very low 5 days following ischemia-reperfusion. In the IPC + ischemia-operated group, however, KYNA immunoreactivity was constitutively detected in the SP of the CA1 region after the ischemic insult. We also found that the alteration pattern of the KYNA protein level in the CA1 region following ischemia was generally similar to the immunohistochemical changes observed. In brief, our findings demonstrated that IPC maintained and even increased KYNA immunoreactivity in the SP of the CA1 region following ischemia-reperfusion. The data from the present study thus indicate that the enhancement of KYNA expression by IPC may be necessary for neuronal survival following transient ischemic injury.

p63 Expression in the Gerbil Hippocampus Following Transient Ischemia and Effect of Ischemic Preconditioning on p63 Expression in the Ischemic Hippocampus.

p63 is a transcription factor of p53 gene family, which are involved in development, differentiation and cell response to stress; however, its roles in ischemic preconditioning (IPC) in the brain are not clear. In the present study, we investigated the effect of IPC on p63 immunoreactivity caused by 5 min of transient cerebral ischemia in gerbils. IPC was induced by subjecting the gerbils to 2 min of transie ischemia 1 day prior to 5 min of transient ischemia. The animals were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group and IPC + ischemia-operated-group). The number of viable neurons in the stratum pyramidale of the hippocampal CA1 region (CA1) was significantly increased by IPC + ischemia-operated-group compared with that in the ischemia-operated-group 5 days after ischemic insult. We found that strong p63 immunoreactivity was detected in the CA1 pyramidal neurons in the sham-operated-group, and the immunoreactivity was decreased with time after ischemia-reperfusion. In addition, strong p63 immunoreactivity was newly expressed in microglial cells of the CA1 region from 2 days after ischemia-reperfusion. In all the IPC + sham-operated-groups, p63 immunoreactivity in the CA1 pyramidal neurons was similar to that in the sham-operated-group, and the immunoreactivity was well maintained in the IPC + ischemia-operated-groups after cerebral ischemia. In brief, our present findings show that IPC dramatically protected the reduction of p63 immunoreactivity in the pyramidal neurons of the CA1 region after ischemia-reperfusion, and this result suggests that the expression of p63 may be necessary for neurons to survive after transient cerebral ischemia.

Ischemic preconditioning inhibits expression of Na(+)/H(+) exchanger 1 (NHE1) in the gerbil hippocampal CA1 region after transient forebrain ischemia.

The participation of Na(+)/H(+) exchanger (NHE) in neuronal damage/death in the hippocampal CA1 region (CA1) induced by transient forebrain ischemia has not been well established, although acidosis may be involved in neuronal damage/death. In the present study, we examined the effect of ischemic preconditioning (IPC) on NHE1 immunoreactivity following a 5min of transient forebrain ischemia in gerbils. The animals used in the study were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+) sham-operated-group and IPC+ischemia-operated-group). IPC was induced by subjecting animals to 2min of ischemia followed by 1day of recovery. A significant neuronal loss was found in the stratum pyramidale (SP) of the CA1, not the CA2/3, of the ischemia-operated-group at 5days post-ischemia. However, in the IPC+ischemia-operated-group, neurons in the SP of the CA1 were well protected. NHE1 immunoreactivity was not detected in any regions of the CA1-3 of the sham- and IPC+sham-operated-groups. However, the immunoreactivity was apparently expressed in the SP of the CA1-3 after ischemia, and the NHE1immunoreactivity was very weak 5days after ischemia; however, at this point in time, strong NHE1immunoreactivity was found in astrocytes in the CA1. In the CA2/3, NHE1immunoreactivity was slightly changed, although NHE1immunoreactivity was expressed in the SP. In the IPC+ischemia-operated-groups, NHE1 immunoreactivity was also expressed in the SP of the CA1-3; however, the immunoreactivity was more slightly changed than that in the ischemia-operated-groups. In brief, our findings show that IPC dramatically protected CA1 pyramidal neurons and strongly inhibited NHE1 expression in the SP of the CA1 after ischemia-reperfusion. These findings suggest that the inhibition of NHE1 expression may be necessary for neuronal survival from transient ischemic damage.

Impact of hyperthermia before and during ischemia-reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia.

Hyperthermia can exacerbate the brain damage produced by ischemia. In the present study, we investigated the effects of hyperthermia before and during ischemia-reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. The animals were randomly assigned to 4 groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 min before and during ischemia-reperfusion. In the normothermia+ischemia groups, a significant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia-reperfusion. In the hyperthermia+ischemia groups, neuronal death in the SP of the CA1 occurred at 1 day post-ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days post-ischemia. In addition, we examined activations of astrocytes and microglia using immunohistochemistry for anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia+ischemia groups than those in the normothermia+ischemia groups. Based on our findings, we suggest that an experimentally hyperthermic pre-condition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus.

Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination.

Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1(-/-) mice displayed enhanced disruption of the blood-brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8(+) T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1(-/-) mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing-remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.

Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia.

Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix­loop­helix (bHLH) transcription factors, including those that regulate differentiation and cell­cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia­reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1­4 in the gerbil hippocampus proper Cornu Ammonis regions CA1­3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1­3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non­pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non­pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia­induced delayed neuronal death.

Ischemic preconditioning protects hippocampal pyramidal neurons from transient ischemic injury via the attenuation of oxidative damage through upregulating heme oxygenase-1.

Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic injury by activating specific mechanisms. In this study, we tested the hypothesis that IPC attenuates postischemic neuronal death via heme oxygenase-1 (HO-1). Animals used in this study were randomly assigned to 4 groups; sham-operated group, ischemia-operated group, IPC plus (+) sham-operated group and IPC+ischemia-operated group. IPC was induced by subjecting gerbils to 2min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in pyramidal neurons of the hippocampal CA1 region (CA1) in the ischemia-operated groups at 5 days postischemia. In the IPC+ischemia-operated groups, CA1 pyramidal neurons were well protected. The level of HO-1 protein and its activity increased significantly in the CA1 of the IPC+sham-operated group, and the level and activity was maintained in all the time after ischemia-reperfusion compared with the ischemia-operated groups. HO-1 immunoreactivity was induced in the CA1 pyramidal neurons in both IPC+sham-operated- and IPC+ischemia-operated groups. We also found that levels or immunoreactivities of superoxide anion, 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal were significantly decreased in the CA1 of both IPC+sham-operated- and IPC+ischemia-operated groups. Whereas, treatment with zinc protoporphyrin IX (a HO-1 inhibitor) into the IPC+ischemia-operated groups did not preserve the IPC-mediated increase of HO-1 and lost beneficial effects of IPC by inhibiting ischemia-induced DNA damage and lipid peroxidation. In brief, IPC protects CA1 pyramidal neurons from ischemic injury by upregulating HO-1, and we suggest that the enhancement of HO-1 expression by IPC may be a legitimate strategy for a therapeutic intervention of cerebral ischemic damage.

Effects of ischemic preconditioning on VEGF and pFlk-1 immunoreactivities in the gerbil ischemic hippocampus after transient cerebral ischemia.

Ischemia preconditioning (IPC) displays an important adaptation of the CNS to sub-lethal ischemia. In the present study, we examined the effect of IPC on immunoreactivities of VEGF-, and phospho-Flk-1 (pFlk-1) following transient cerebral ischemia in gerbils. The animals were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+) sham-operated-group, and IPC+ischemia-operated-group). IPC was induced by subjecting gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated-group, a significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) alone 5 days after ischemia-reperfusion, however, in all the IPC+ischemia-operated-groups, pyramidal neurons in the SP were well protected. In immunohistochemical study, VEGF immunoreactivity in the ischemia-operated-group was increased in the SP at 1 day post-ischemia and decreased with time. Five days after ischemia-reperfusion, strong VEGF immunoreactivity was found in non-pyramidal cells, which were identified as pericytes, in the stratum oriens (SO) and radiatum (SR). In the IPC+sham-operated- and IPC+ischemia-operated-groups, VEGF immunoreactivity was significantly increased in the SP. pFlk-1 immunoreactivity in the sham-operated- and ischemia-operated-groups was hardly found in the SP, and, from 2 days post-ischemia, pFlk-1 immunoreactivity was strongly increased in non-pyramidal cells, which were identified as pericytes. In the IPC+sham-operated-group, pFlk-1 immunoreactivity was significantly increased in both pyramidal and non-pyramidal cells; in the IPC+ischemia-operated-groups, the similar pattern of VEGF immunoreactivity was found in the ischemic CA1, although the VEGF immunoreactivity was strong in non-pyramidal cells at 5 days post-ischemia. In brief, our findings show that IPC dramatically augmented the induction of VEGF and pFlk-1 immunoreactivity in the pyramidal cells of the CA1 after ischemia-reperfusion, and these findings suggest that the increases of VEGF and Flk-1 expressions may be necessary for neurons to survive from transient ischemic damage.