KA-induced Neuronal Death Research Articles

Lipocalin-2 Deficiency Reduces Oxidative Stress and Neuroinflammation and Results in Attenuation of Kainic Acid-Induced Hippocampal Cell Death.

The hippocampal cell death that follows kainic acid (KA)-induced seizures is associated with blood–brain barrier (BBB) leakage and oxidative stress. Lipocalin-2 (LCN2) is an iron-trafficking protein which contributes to both oxidative stress and inflammation. However, LCN2′s role in KA-induced hippocampal cell death is not clear. Here, we examine the effect of blocking LCN2 genetically on neuroinflammation and oxidative stress in KA-induced neuronal death. LCN2 deficiency reduced neuronal cell death and BBB leakage in the KA-treated hippocampus. In addition to LCN2 upregulation in the KA-treated hippocampus, circulating LCN2 levels were significantly increased in KA-treated wild-type (WT) mice. In LCN2 knockout mice, we found that the expressions of neutrophil markers myeloperoxidase and neutrophil elastase were decreased compared to their expressions in WT mice following KA treatment. Furthermore, LCN2 deficiency also attenuated KA-induced iron overload and oxidative stress in the hippocampus. These findings indicate that LCN2 may play an important role in iron-related oxidative stress and neuroinflammation in KA-induced hippocampal cell death.

Microglia attenuate the kainic acid-induced death of hippocampal neurons in slice cultures.

BackgroundStatus epilepticus‐induced hippocampal neuronal death, astrogliosis, and the activation of microglia are common pathological changes in mesial temporal lobe epilepsy (mTLE) with resistance to antiepileptic drugs. Neuronal death in mTLE gradually progresses and is involved in the aggravation of epilepsy and the impairment of hippocampus‐dependent memory. Thus, clarifying the cellular mechanisms by which neurons are protected in mTLE will significantly contribute to the treatment of epilepsy. Here, mainly using hippocampal slice cultures with or without the pharmacological depletion of microglia, we directly examined whether microglia, the resident immune cells of the brain that can act either neurotoxically or in a neuroprotective manner, accelerate or attenuate kainic acid (KA)‐induced neuronal death in vitro.MethodsHippocampal slice cultures were treated with KA to induce neuronal death in vitro. Clodronate‐containing liposomes or PLX3397 was used to deplete microglia in hippocampal slice cultures, and the effect on KA‐induced neuronal death was immunohistochemically assessed.ResultsThe loss of microglia significantly promoted a decrease in neuronal density in KA‐treated hippocampal slice cultures.ConclusionOur results suggest that microglia are neuroprotective against KA‐induced neuronal death in slice cultures.

Dexmedetomidine protects neurons from kainic acid-induced excitotoxicity by activating BDNF signaling

Glutamatergic excitotoxicity is crucial in the pathogenesis of epileptic seizures. Dexmedetomidine, a potent and highly selective α2 adrenoceptor agonist, inhibits glutamate release from nerve terminals in rat cerebrocortical nerve terminals. However, the ability of dexmedetomidine to affect glutamate-induced brain injury is still unknown. Therefore, the present study evaluated the protective effect of dexmedetomidine against brain damage by using a kainic acid (KA) rat model, a frequently used model for temporal lobe epilepsy. Rats were treated with dexmedetomidine (1 or 5 μg/kg, intraperitoneally) 30 min before the KA (15 mg/kg) intraperitoneal injection. KA-induced seizure score and elevations of glutamate release in rat hippocampi were inhibited by pretreatment with dexmedetomidine. Histopathological and TUNEL staining analyzes showed that dexmedetomidine attenuated KA-induced neuronal death in the hippocampus. Dexmedetomidine ameliorated KA-induced apoptosis, and this neuroprotective effect was accompanied by inhibited the KA-induced caspase-3 expression as well as MAPKs phosphorylation, and reversed Bcl-2 down-expression, coupled with increased Nrf2, BDNF and TrkB expression in KA-treated rats. The results suggest that dexmedetomidine protected rat brains from KA-induced excitotoxic damage by reducing glutamate levels, suppressing caspase-3 activation and MAPKs phosphorylation, and enhancing Bcl-2, Nrf2, BDNF and TrkB expression in the hippocampus. Therefore, dexmedetomidine may be beneficial for preventing or treating brain disorders associated with excitotoxic neuronal damage. In conclusion, these data suggest that dexmedetomidine has the therapeutic potential for treating epilepsy.

Neuroprotective effects of a protein tyrosine phosphatase inhibitor against hippocampal excitotoxic injury

Neuronal excitotoxicity is the neuronal cell death arising from prolonged exposure to glutamate and the associated excessive influx of ions into the cell. Sodium orthovanadate (Na3VO4,) competitively inhibits the protein tyrosine phosphatases that affect intracellular protein phosphorylation. No study has examined the role of protein tyrosine phosphatases in kainic acid (KA)-induced excitotoxic injury using sodium orthovanadate. Thus, the present study was conducted to determine the neuroprotective effects of sodium orthovanadate on KA-induced neuronal death in organotypic hippocampal slice culture. We also performed an in vivo electrophysiology study in Sprague-Dawley rats to observe the function of surviving cells after sodium orthovanadate treatment in KA-induced excitotoxicity. Rats were anaesthetized with sodium pentobarbital and KA was injected unilaterally in CA3 of the hippocampus by microinjection-cannula. Neuronal cell death, as assessed by propidium iodide uptake, was reduced by 10 and 25 μM sodium orthovanadate treatment (24 and 48 h) compared with the KA-only group. Sodium orthovanadate enhanced survival signals by increasing levels of phospho-Akt and superoxide dismutase. In addition, sodium orthovanadate treatment reduced calcineurin level for neuronal protection, which regulates activation of cellular calcium caused by KA-induced injury. In vivo results showed that sodium orthovanadate treatment elicited resistance to KA-induced behavior seizures and significantly reduced the duration of epileptiform discharges. In addition, sodium orthovanadate treatment (25 mM) significantly prevented the increase in power spectra induced by KA injection. These results suggest that sodium orthovanadate decreases the acute effects of KA, thereby inducing neuroprotective effects with reduced reactive oxygen species and cellular Ca2+. Thus, sodium orthovanadate may protect hippocampal neurons against excitotoxicity, and surviving neurons may function to reduce seizures.

Ropivacaine Protects against Memory Impairment and Hippocampal Damage in a Rat Neurodegeneration Model

Background: Ropivacaine, a long-acting amide local anesthetic agent, has been demonstrated to inhibit glutamatergic transmission. Glutamate neurotoxicity plays a pivotal role in the pathogenesis of brain disorders. The purpose of this study is to investigate the neuroprotective effect of ropivacaine against brain damage induced by kainic acid (KA), an analogue of glutamate. Methods: Rats were injected with ropivacaine (0.4 or 2 mg/kg, intraperitoneal) 30 min before KA treatment (15 mg/kg, intraperitoneal). KA-induced memory impairment was evaluated using the Morris water maze test. Extracellular hippocampal glutamate levels were assessed using high-performance liquid chromatography. Neuronal death was verified using Fluoro-Jade B and neutral red staining, and apoptosis was determined through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Western blotting was conducted to assay the levels of activated (cleaved) caspase-3 and the phosphorylation of different mitogen-activated protein kinases (MAPKs). ­Results: Ropivacaine pretreatment effectively prevented KA-induced memory impairment. KA-induced elevations of ­glutamate release in rat hippocampi were inhibited by pretreatment with ropivacaine. Histopathological and TUNEL staining analyzes showed that ropivacaine inhibited KA-induced neuronal death and apoptosis in the hippocampal CA3 region. KA-induced caspase-3 activation and MAPKs phosphorylation in the hippocampus were also reduced by ropivacaine pretreatment. Conclusions: This study ­demonstrates that ropivacaine executes a protective action against KA-induced neuronal damage and apoptosis in vivo. Protective effects may be caused by glutamate level reduction, caspase-3 activation suppression, and MAPKs phosphorylation reduction. Our findings indicate that ropivacaine can benefit prevention or treatment of glutamate excitotoxicity-related neurodegenerative diseases.

Effect of Rufinamide on the kainic acid-induced excitotoxic neuronal death in the mouse hippocampus.

Rufinamide (RUF) is a structurally unique anti-epileptic drug, used in the treatment of seizure disorders such as Lennox-Gastaut syndrome. In the present study, we investigated whether RUF protected against excitotoxic neuronal damage in the mouse hippocampal CA3 region after intraperitoneal kainic acid (KA) injection. Treatment with 25, 50 and 100mg/kg RUF significantly decreased the KA-induced neuronal death in the hippocampal CA3 region in a dose-dependent manner. In addition, 100mg/kg RUF treatment reduced the KA-induced oxidative stress-related increase of MDA level and decrease of total SOD activity in the hippocampus. KA-induced increases of pro-inflammatory cytokines, TNF-α and IL-1β, levels as well as KA-induced microglial activation were also suppressed by RUF treatment. These results indicate that RUF displays a neuroprotective effect against KA-induced excitotoxic neuronal death in the mouse hippocampus through anti-oxidant and anti-inflammatory activities.

Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.

Exclusive Activation of Caspase-3 in Mossy Fibers and Altered Dynamics of Autophagy Markers in the Mice Hippocampus upon Status Epilepticus Induced by Kainic Acid.

Epileptic seizures are generally associated with pathological changes in the hippocampus such as astrogliosis, mossy fiber sprouting, and neuronal damage. However, more than 30% of temporal lobe epilepsy in humans shows neither neuronal damage nor mossy fiber sprouting despite chronic epileptic seizures. A similar situation exists in certain commonly used strains of mice, specifically C57BL/6 and BALB/c, which exhibit epileptic seizures, but no neuronal damage upon kainic acid administration. This suggests that intrinsic factors may influence the pathological manifestations of epilepsy. Mechanisms which are behind the resistance of hippocampal cells to KA-induced neuronal death are unknown. Autophagy seems to be involved in the pathogenesis of many brain insults and to have a dual nature in neuroprotection and cell death. This study addresses the role of autophagy upon status epilepticus (SE) that has been induced by kainic acid (KA) in the C57BL/6 strain which is classified as seizure resistant. We analyzed the dynamics in the expression of autophagic and cell death markers in the hippocampus upon SE. Immunofluorescence data show that KA did not induce neuronal death in the hippocampal CA1-CA3 subfields; however, it leads to an exclusive activation of caspase-3 in the mossy fibers. We also found alterations in the expression of core proteins of the autophagic machinery. Levels of MAP1LC3, phospho-mTOR/mTOR, and Beclin 1 were significantly increased after induction of seizures. However, levels of Atg3, Atg14, Atg5-Atg12, Atg7, BAG3, Hsp70, and LAMP1 showed no significant alterations compared to controls. Although KA did not induce neuronal death, this study provides morphological and biochemical evidence that status epilepticus induced by KA activates caspase-3 in mossy fibers and induces autophagy in the C57BL/6 hippocampus. These data indicate that autophagic factors may modulate the sensitivity of pyramidal cells to KA and that autophagy may constitute a part of an endogenous neuroprotective arsenal which might be behind the resistance of C57BL/6-hippocampal cells to KA-induced neuronal death.

Luteolin protects the hippocampus against neuron impairments induced by kainic acid in rats

Glutamatergic excitotoxicity is crucial in the pathogenesis of numerous brain disorders. Luteolin, a flavonoid compound, inhibits glutamate release, however, its ability to affect glutamate-induced brain injury is unknown. Therefore, this study evaluated the protective effect of luteolin against brain damage induced by kainic acid (KA), a glutamate analog. Rats were treated with luteolin (10 or 50mg/kg, intraperitoneally) 30min before an intraperitoneal injection of KA (15mg/kg). Luteolin treatment reduced the KA-induced seizure score and elevations of glutamate levels in the hippocampus. A histopathological analysis showed that luteolin attenuated KA-induced neuronal death and microglial activation in the hippocampus. An immunoblotting analysis showed that luteolin restored the KA-induced reduction in Akt phosphorylation in the hippocampus. Furthermore, a Morris water maze test revealed that luteolin effectively prevented KA-induced learning and memory impairments. The results suggest that luteolin protected rat brains from KA-induced excitotoxic damage by reducing glutamate levels, mitigating inflammation, and enhancing Akt activation in the hippocampus. Therefore, luteolin may be beneficial for preventing or treating brain disorders associated with excitotoxic neuronal damage.

Characterization of temporal expressions of FOXO and pFOXO proteins in the hippocampus by kainic acid in mice: involvement of NMDA and non-NMDA receptors.

In the present study, we characterized the expression and role of forkhead box O (FoxO3a) in kainic acid (KA)-induced hippocampal neuronal cell death. FoxO3a and pFoxO3a expression in the CA1, CA2, and dentate gyrus regions in the hippocampus increased 0.5 and 1h after intracerebroventricular administration of KA. In addition, both FoxO3a and pFoxO3a expression in the hippocampal CA3 region increased significantly and equally for 1h but decreased gradually for 24h after KA administration. In particular, the KA-induced increases in FoxO3a and pFoxO3a expression in the hippocampal CA3 region were inhibited by pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801, dizocilpine, 1µg/5µl) or a non-NMDA receptor antagonist (CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione, 0.5µg/5µl). Furthermore, dizocilpine and CNQX produced a neuroprotective effect against KA-induced neuronal death in the CA3 region of the hippocampus. Our results suggest that FoxO3a and pFoxO3 expression is upregulated by KA. Both FoxO3a and pFoxO3a expression appear to be responsible for KA-induced neuronal death in the CA3 region of the hippocampus.

The anti-inflammatory activity of duloxetine, a serotonin/norepinephrine reuptake inhibitor, prevents kainic acid-induced hippocampal neuronal death in mice

Duloxetine (DXT), a potent serotonin/norepinephrine reuptake inhibitor, is widely used in the treatment of major depressive disorder. In the present study, we examined the effects of DXT treatment on seizure behavior and excitotoxic neuronal damage in the mouse hippocampal CA3 region following intraperitoneal kainic acid (KA) injection. DXT treatment showed no effect on KA-induced behavioral seizure activity. However, treatment with 10mg/kg DXT reduced KA-induced neuronal death in the hippocampal CA3 region at 72h after KA administration, and treatment with 20 and 40mg/kg DXT showed a noticeable neuroprotection in the hippocampal CA3 region after KA injection. In addition, KA-induced activations of microglia and astrocytes as well as KA-induced increases of TNF-α and IL-1β levels were also suppressed by DXT treatment. These results indicate that DXT displays the neuroprotective effect against KA-induced excitotoxic neuronal death through anti-inflammatory action.

Effect of pertussis and cholera toxins administered supraspinally on CA3 hippocampal neuronal cell death and the blood glucose level induced by kainic acid in mice

The effect of cholera toxin (CTX) or pertussis toxin (PTX) administered supraspinally on hippocampal neuronal cell death in CA3 region induced by kainic acid (KA) was examined in mice. After the pretreatment with either PTX or CTX intracerebroventricularly (i.c.v.), mice were administered i.c.v. with KA. The i.c.v. treatment with KA caused a neuronal cell death in CA3 region and PTX, but not CTX, attenuated the KA-induced neuronal cell death. In addition, i.c.v. treatment with KA caused an elevation of the blood glucose level. The i.c.v. PTX pretreatment alone caused a hypoglycemia and inhibited KA-induced hyperglycemic effect. However, i.c.v. pretreatment with CTX did not affect the basal blood glucose level and KA-induced hyperglycemic effect. Moreover, KA administered i.c.v. caused an elevation of corticosterone level and reduction of the blood insulin level. Whereas, i.c.v. pretreatment with PTX further enhanced KA-induced up-regulation of corticosterone level. Furthermore, i.c.v. administration of PTX alone increased the insulin level and KA-induced hypoinsulinemic effect was reversed. In addition, PTX pretreatment reduces the KA-induced seizure activity. Our results suggest that supraspinally administered PTX, exerts neuroprotective effect against KA-induced neuronal cells death in CA3 region and neuroprotective effect of PTX is mediated by the reduction of KA-induced blood glucose level.

Effect of tolbutamide, glyburide and glipizide administered supraspinally on CA3 hippocampal neuronal cell death and hyperglycemia induced by kainic acid in mice

Sulfonylureas are widely used oral drugs for the treatment of type II diabetes mellitus. In the present study, the effects of sulfonylureas administered supraspinally on kainic acid (KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30μg of tolbutamide, glyburide or glipizide for 10min and then, mice were administered i.c.v. with KA (0.1μg). The neuronal cell death in the CA3 region in the hippocampus was assessed 24h after KA administration and the blood glucose level was measured 30, 60, and 120min after KA administration. We found that i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corticosterone level and a reduction of the plasma insulin level. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced increase of plasma corticosterone level. Furthermore, i.c.v. pretreatment with tolbutamide, glyburide or glipizide causes an elevation of plasma insulin level. Glipizide, but not tolbutamide or glyburide, pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered tolbutamide, glyburide and glipizide exert a protective effect against KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of tolbutamide, glyburide and glipizide appears to be mediated by lowering the blood glucose level induced by KA.

Effects of nateglinide and repaglinide administered intracerebroventricularly on the CA3 hippocampal neuronal cell death and hyperglycemia induced by kainic acid in mice

Meglitinides (nateglinide and repaglinide) are widely used oral drugs for the treatment of type II diabetes mellitus. In the present study, the effects of meglinitides administered supraspinally on kainic acid (KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30 μg of nateglinide and repaglinide for 10 min and then, mice were administered i.c.v. with KA (0.1 μg). The neuronal cell death in the CA3 region in the hippocampus was assessed 24h after KA administration and the blood glucose level was measured 30, 60, and 120 min after KA administration. We found that i.c.v. pretreatment with repaglinide attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and hyperglycemia. However, nateglinide pretreated i.c.v. did not affect the KA-induced neuronal cell death and hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corticosterone level and a reduction of the plasma insulin level. Furthermore, i.c.v. pretreatment with repaglinide attenuated KA-induced up-regulation of plasma corticosterone level. Furthermore, i.c.v. administration of repaglinide alone increased plasma insulin level and repaglinide pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered repaglinide, but not nateglinide, exerts a protective effect against the KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of repaglinide appears to be mediated by lowering the blood glucose level induced by KA.

Neuroprotective Effects of α-Tocotrienol on Kainic Acid-Induced Neurotoxicity in Organotypic Hippocampal Slice Cultures

Vitamin E, such as alpha-tocopherol (ATPH) and alpha-tocotrienol (ATTN), is a chain-breaking antioxidant that prevents the chain propagation step during lipid peroxidation. In the present study, we investigated the effects of ATTN on KA-induced neuronal death using organotypic hippocampal slice culture (OHSC) and compared the neuroprotective effects of ATTN and ATPH. After 15 h KA (5 μM) treatment, delayed neuronal death was detected in the CA3 region and reactive oxygen species (ROS) formation and lipid peroxidation were also increased. Both co-treatment and post-treatment of ATPH (100 μM) or ATTN (100 μM) significantly increased the cell survival and reduced the number of TUNEL-positive cells in the CA3 region. Increased dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS) were decreased by ATPH and ATTN treatment. These data suggest that ATPH and ATTN treatment have protective effects on KA-induced cell death in OHSC. ATTN treatment tended to be more effective than ATPH treatment, even though there was no significant difference between ATPH and ATTN in co-treatment or post-treatment.

Proteomic Analysis of the Effect of Acupuncture on the Suppression of Kainic Acid-Induced Neuronal Destruction in Mouse Hippocampus

Kainic acid (KA) is a neurotoxin that induces epileptic seizures and excitotoxicity in the hippocampus. Acupuncture is frequently used as an alternative therapy for epilepsy, and it has been known to protect hippocampal neurons against KA toxicity. Using proteomic analysis, we investigated protein expression changes in the hippocampus following acupuncture stimulation at HT8. Eight-week-old male C57BL/6 mice (20–25 g) received acupuncture treatment at HT8 acupoint bilaterally once a day for 3 days and were then administered KA (30 mg/kg) intraperitoneally. Twenty-four hours after KA injection, neuronal survival and astrocyte activation in the hippocampus were measured, and protein expression in the hippocampus was identified by 2-dimensional electrophoresis. Acupuncture stimulation at HT8 suppressed KA-induced neuronal death and astrocyte activation in the hippocampus. We identified the changes in the expression of 11 proteins by KA or acupuncture stimulation at HT8 and found that acupuncture stimulation at HT8 normalized the expression of dihydropyrimidinase-related protein 2 and upregulated the expression of transcriptional activator protein pur-alpha, serine/threonine-protein phosphatase 5, and T-complex protein 1 subunit alpha, which are related to the survival of neurons. These results suggest that acupuncture stimulation at HT8 changes protein expression profiles in the hippocampus in favor of neuronal survival in KA-treated mice.

Induction of Per1 expression following an experimentally induced epilepsy in the mouse hippocampus

The Period1 (Per1) is a clock-oscillating gene product that plays an essential role in the generation and modulation of circadian rhythm in the suprachiasmatic nucleus (SCN) of hypothalamus. However, Per1 is also expressed in many other brain regions including cerebral cortex, hippocampus, and amygdala, suggesting that Per1 may be involved in the broader cellular functions in addition to the rhythm regulation. In this study, we found that chemical or electrical seizure-inducing stimulations regulate Per1 expression. Treatments with electric convulsive shock (ECS) or kainic acid (KA) robustly up-regulated the expressions of per1 mRNA and protein in the hippocampal formation and cerebral cortex. In consistent, we found that neuronal depolarization or KA treatment increased per1 mRNA expression in cultured primary cortical neurons. Because it has been demonstrated that Per family molecules contribute to the regulation of stress-induced cell death, we also explored the effect of Per1 overexpression on the survival of cultured neurons. However, neither basal, staurosporine- nor KA-induced neuronal death was affected by forced expression of Per1. Collectively, these results suggest that the Per1 expression is neuronal activity- and epileptogen-dependent, although its functional significance is remained to be explored.

The time-dependent effect of lipopolysaccharide on kainic acid-induced neuronal death in hippocampal CA3 region: possible involvement of cytokines via glucocorticoid

It has been reported that glucocorticoid (Gc) can induce neuronal cell toxicity in the hippocampus. In addition, we examined that serum Gc increased by restraint stress aggravated kainic acid (KA)-induced neuronal death in hippocampal CA3 region. However, the effect of other stressful stimulus like lipopolysaccharide (LPS) increasing serum Gc on KA-induced neuronal death was not elucidated until now. Thus, we examined the time course effect of LPS on KA-induced neuronal death in the hippocampal CA3 region of mice, especially to address the role of Gc and inflammatory mediators. In the present study, we found that an aggravating effect of LPS on KA-induced neuronal death was correlated with an alteration of hippocampal IL-1β mRNA level at all time points, and the serum Gc and hippocampal IL-1β mRNA level was peak at 90 min after LPS treatment (LPS 90 min) when the aggravating effect of LPS on KA-induced neuronal death was maximum. In addition, RU38486 (glucocorticoid receptor antagonist) decreased the hippocampal IL-1β mRNA level and abolished the aggravating effect of LPS on KA-induced neuronal death at LPS 90 min and 24 h. In the immunohistochemical study, we found activated and ramified microglia (OX-42) and astrocyte (GFAP) at 24 h after LPS treatment (LPS 24 h) in the hippocampus. These results suggest that Gc itself, cytokines triggered by Gc, or both appears to be involved in the LPS effect depending on LPS pretreatment time.

Kainic Acid-induced Neuronal Death is Attenuated by Aminoguanidine but Aggravated by L-NAME in Mouse Hippocampus

Nitric oxide (NO) has both neuroprotective and neurotoxic effects depending on its concentration and the experimental model. We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a selective inducible NOS (iNOS) inhibitor, on kainic acid (KA)-induced seizures and hippocampal CA3 neuronal death. L-NAME (50 mg/kg, i.p.) and/or aminoguanidine (200 mg/kg, i.p.) were administered 1 h prior to the intracerebroventricular (i.c.v.) injection of KA. Pretreatment with L-NAME significantly increased KA-induced CA3 neuronal death, iNOS expression, and activation of microglia. However, pretreatment with aminoguanidine significantly suppressed both the KA-induced and L-NAME-aggravated hippocampal CA3 neuronal death with concomitant decreases in iNOS expression and microglial activation. The protective effect of aminoguanidine was maintained for up to 2 weeks. Furthermore, iNOS knockout mice (iNOS(-/-)) were resistant to KA-induced neuronal death. The present study demonstrates that aminoguanidine attenuates KA-induced neuronal death, whereas L-NAME aggravates neuronal death, in the CA3 region of the hippocampus, suggesting that NOS isoforms play different roles in KA-induced excitotoxicity.

Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor

Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin γ1 (lamγ1) expression in the hippocampus. Lamγ1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamγ1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamγ1 KO mice. KA1 levels in tissue plasminogen activator (tPA)–KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamγ1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.