Ketogenic diet attenuates kainic acid-induced hippocampal cell death by decreasing AMPK/ACC pathway activity and HSP70

The ketogenic diet (KD) prevents kainic acid (KA)-induced hippocampal cell death. There are reports that AMP-activated protein kinase (AMPK) activation regulates the intracellular signaling pathways involved in cellular survival or apoptotic cell death. In this study, we investigated the effect of the KD consumption on the expression of signaling pathway proteins AMPK and ACC, and heat shock protein (HSP) 70 in mouse hippocampus after KA treatment. Mice were fed the KD for 6 weeks and then sacrificed 48 h after KA (30 mg/kg) injection. The marked cell death found commonly in normal diet (ND)-fed mice treated with KA was not observed in the KD-fed KA-treated mice. Western blot analysis revealed that phosphorylation of AMPK and ACC was increased after KA treatment. However, phosphorylation of these proteins was reduced in those animals that received the KD. In addition, increased expression of HSP70 in the hippocampus of KA-treated mice was decreased in animals receiving the KD. These results indicate that the KD promotes neuroprotective effects through suppression of the AMPK cascade and that HSP70 is involved in neuronal cell death or oxidative stress.     

Excitotoxicity by kainate-induced seizure causes diacylglycerol kinase ζ to shuttle from the nucleus to the cytoplasm in hippocampal neurons

Diacylglycerol kinase (DGK), which consists of several isozymes, plays a pivotal role in lipid second-messenger diacylglycerol metabolism. A nuclear isozyme, DGKζ, which is translocated from the nucleus to the cytoplasm in hippocampal neurons under transient ischemic stress, is implicated in nuclear events of delayed neuronal death. Kainate (KA)-induced seizure is another model used to study excitotoxic stress. Therefore, we examined whether DGKζ is implicated in a different type of degenerative excitotoxicity in hippocampal neurons. We conducted immunohistochemical analysis of rat hippocampi after KA-induced seizures. DGKζ in hippocampal neurons shuttles from the nucleus to the cytoplasm. It never relocates to the nucleus during KA-induced seizures. Marked change in the immunoreactivity is first observed in CA1 pyramidal neurons 2 h after injection during stage 3 seizures. Immunoreactivity for DGKι remains unchanged in the cytoplasm. That for NeuN remains mostly unchanged in the nucleus. Results show that nucleocytoplasmic translocation of DGKζ also occurs in a different model of excitotoxicity that results in apoptotic neuronal death. Cytoplasmic translocation of DGKζ might be involved in early events of the apoptotic cell death pathway in hippocampal neurons under stressed conditions.     

The administration of pig brain phospholipids versus soybean phospholipids in the diet during the period of brain development in the rat results in greater increments of brain docosahexaenoic acid

Ginsenosides are known to attenuate glutamate-induced cell injuries in vitro. We investigated the in vivo effect of ginsenosides on kainic acid (KA)-induced neurotoxicity in rat hippocampus using the methods of acid fuchsin (AF) staining and heat-shock protein-70 (HSP-70) immunoreactivity to detect neuronal death and stress, respectively. Pretreatment of ginsenosides (50 or 100 mg/kg for 7 days) via intraperitoneal (i.p.) administration significantly attenuated KA (10 mg/kg i.p.)-induced cell death by decreasing AF-positive neurons in both CA1 and CA3 regions of rat hippocampus compared with KA treatment alone. Pretreatment of ginsenosides (50 or 100 mg/kg for 7 days) via i.p. administration also significantly suppressed KA-induced induction of HSP-70 in both regions of rat hippocampus. These results show that ginsenosides are effective in protecting hippocampal CA1 and CA3 cells against KA-induced neurotoxicity.     

Kainic acid induces early and transient autophagic stress in mouse hippocampus

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2-16 h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4-6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/mTOR were elevated from 6 to 16 h, and ratios of phospho-Akt/Akt were elevated at 16 h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus.     

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.     

Acupuncture suppresses kainic acid-induced neuronal death and inflammatory events in mouse hippocampus

The administration of kainic acid (KA) causes seizures and produces neurodegeneration in hippocampal CA3 pyramidal cells. The present study investigated a possible role of acupuncture in reducing hippocampal cell death and inflammatory events, using a mouse model of kainic acid-induced epilepsy. Male C57BL/6 mice received acupuncture treatments at acupoint HT8 or in the tail area bilaterally once a day for 2 days and again immediately after an intraperitoneal injection of KA (30 mg/kg). HT8 is located on the palmar surface of the forelimbs, between the fourth and fifth metacarpal bones. Twenty-four hours after the KA injection, neuronal cell survival, the activations of microglia and astrocytes, and mRNA expression of two proinflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were measured in the hippocampus. Acupuncture stimulation at HT8, but not in the tail area, significantly reduced the KA-induced seizure, neuron death, microglial and astrocyte activations, and IL-1β mRNA expression in the hippocampus. The acupuncture stimulation also decreased the mRNA expression of TNF-α, but it was not significant. These results indicate that acupuncture at HT8 can inhibit hippocampal cell death and suppress KA-induced inflammatory events, suggesting a possible role for acupuncture in the treatment of epilepsy.     

Neuroprotective effects of FK506 against excitotoxicity in organotypic hippocampal slice culture

FK506 has been originally classified as an immunosuppressant and is known to exhibit neurotrophic actions in vitro and protective effects on some neurological conditions. We investigated the neuroprotective effects of FK506 on kainic acid (KA)-induced neuronal death in organotypic hippocampal slice cultures (OHSCs). After an 18 h KA (5 μM) treatment, significantly neuronal death was detected in the CA3 region using propidium iodide staining. However, neuronal death was significantly prevented at 24 and 48 h after treatment with 0.1 μM FK506. Using cresyl violet staining, we also observed that an increased number of CA3 neurons survived in the 0.1 μM FK506 group compared to the KA only group. Based on the results of the Western blot analysis, the expressions of 5-lipoxygenase and caspase-3 were reduced 24 h after 0.1 μM FK506 treatment. The levels of superoxide dismutase (SOD) and phospho-Akt expression were increased by treatment with 0.1 μM FK506. These results suggest that FK506 may have a positive role in protecting neurons against cell death in the KA injury model of OHSCs.     

Fcγ receptors contribute to pyramidal cell death in the mouse hippocampus following local kainic acid injection

Recent studies have demonstrated the contribution of the gamma subunit of the Fc receptor of IgG (FcRγ) to neuronal death following ischemic injury and Parkinson's disease. We examined the role of FcRγ in hippocampal pyramidal cell death induced by kainic acid (KA). FcRγ-deficient mice (FcRγ−/−) and their FcRγ+/+ littermates (wild type, B6) received an injection of KA into the dorsal hippocampus. Pyramidal cell death was quantified 24 and 72 h after the injection. The number of survived pyramidal cells was significantly larger in FcRγ−/− mice than in B6 mice in both the CA1 and CA3. Immunohistochemical and immunofluorescent studies detected FcγRIIB protein in parvalbumin neurons, whereas FcγRIII and FcγRI proteins were detected in microglial cells. No activated microglial cells were detected 24 h after the KA injection in FcRγ−/− mice, whereas many activated microglial cells were present in B6 mice. The production of nitrotyrosine as well as of the inducible nitric oxide synthase and cyclooxygenase-2 proteins, increased by 16 h after the KA injection in B6 mice. In addition, tissue plasminogen activator and metalloproteinase-2 proteins increased. By contrast, the magnitude of oxidative stress and the increase in protease expression were mild in FcRγ−/− mice. Co-injection of a neutralizing antibody against FcγRll and FcγRlll with KA abolished pyramidal cell death and microglial activation. In addition, the neutralizing antibody reduced oxidative stress and expression of proteases. These observations suggested a role for FcγRllB in parvalbumin neurons as well as FcRγ in microglia in pyramidal cell death.     

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.     

Acupuncture inhibits kainic Acid-induced hippocampal cell death in mice

We examined whether acupuncture can reduce both the incidence of seizures and hippocampal cell death using a mouse model of kainic acid (KA)-induced epilepsy. ICR mice were given acupuncture once a day at acupoint HT8 (sobu) bilaterally during 2 days before KA injection. After an intracerebroventricular injection of 0.1 microg of KA, acupuncture treatment was subsequently administered once more (total 3 times), and the degree of seizure was observed for 20 min. Three hours after injection, the survival of neuronal cells and the expressions of c-Fos, c-Jun, and glutamate decarboxylase (GAD)-67 in the CA1 and CA3 were determined using immunohistochemistry and Western blotting techniques. Acupuncture reduced the severity of the KA-induced epileptic seizure and the rate of neural cell death, and it also decreased the expressions of c-Fos and c-Jun induced by KA in the hippocampus. Furthermore, acupuncture increased GAD-67 expressions in the same areas. These results demonstrated that it could inhibit the KA-induced epileptic seizure and hippocampal cell death by increasing GAD-67 expressions.     

c-JUN expression and apoptotic cell death in kainate-induced temporal lobe epilepsy

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly express immediate early gene (IEG) products, i.e., c-FOS and c-JUN, and neural stress protein, HSP72. Prolonged expression of c-JUN and c-FOS 48 hr after cerebral ischemia has been underwent delayed neuronal death. However, it is not yet clear whether IEGs actually assume the essential roles in the cell death process or simply as a by-product due to external stimuli because of the prolonged expression of c-FOS, more than one week, on intact CA2 neurons of the hippocampus in a KA-induced epilepsy model. This study investigated the relationships between prolonged expression of c-JUN and hippocampal neuronal apoptosis in a KA-induced epilepsy model. Epileptic seizure was induced in rats by a single microinjection of KA (1 microgram/microL) into the left amygdala. Characteristic seizures and hippocampal neuronal injury were developed. The expression of c-JUN was evaluated by immunohistochemistry, and neuronal apoptosis by in situ end labeling. The seizures were associated with c-JUN expression in the hippocampal neurons, of which the level showed a positive correlation with that of apoptosis. Losses of hippocampal neurons, especially in the CA3 region, were partly caused by apoptotic cell death via a c-JUN-mediated signaling pathway. This is thought to be an important component in the pathogenesis of hippocampal neuronal injury via KA-induced epilepsy.     

c-fos modulates brain-derived neurotrophic factor mRNA expression in mouse hippocampal CA3 and dentate gyrus neurons

Excess neuronal excitation by glutamate induces neuron cell death, which may contribute to the pathogenesis of acute brain injuries and neurodegenerative diseases. Our previous studies using a mouse with hippocampal c-fos gene deletion showed that c-fos regulates neuronal excitability and excitotoxicity. Moreover, a delayed induction of brain-derived neurotrophic factor (BDNF) protein expression in response to kainic acid (KA) treatment was found in c-fos mutant mice compared to wildtype controls, suggesting that c-fos is important in the temporal control of BDNF induction. To further investigate mechanisms of in vivo regulation of c-fos on BDNF expression, we studied the expression of BDNF mRNA and its colocalization with c-Fos protein in the hippocampal formation in the presence and absence of KA. By in situ hybridization, we observed that the c-fos mutant and wildtype mice exhibited similar basal expression of BDNF in the absence of KA. In contrast, the KA-induced BDNF mRNA levels were significantly different in wildtype and c-fos mutant mice in CA3 and dentate gyrus regions. Our findings indicate that c-fos regulates expression of BDNF in distinct neuron populations of the hippocampal formation in vivo.     

Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures

The role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35mg/kg, kainic acid induced convulsions in both control (75%) and interleukin-6 null (100%) mice, and caused a significant mortality (62%) only in the latter mice, indicating that interleukin-6 deficiency increased the susceptibility to kainic acid-induced brain damage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered 8.75mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal field CA1-CA3 was seen after kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal mice hippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxide synthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased, as was that for metallothionein I+II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a significant neuronal apoptosis was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1beta converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampal damage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I+II levels were lower, and those of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increased oxidative stress and neuronal death in the interleukin-6 null mice. The present results demonstrate that interleukin-6 deficiency increases neuronal injury and impairs the inflammatory response after kainic acid-induced seizures.     

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H₂O₂ resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H₂O₂ was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 μM H₂O₂ for 24 h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.     

Kainic acid-induced early genes activation and neuronal death in the medial extended amygdala of rats

The medial extended amygdala modulates pheromonal perception, influencing emotional and social behavior. As the amygdala is part of neuronal circuits that are very sensitive to excitability, its neurons are targets of seizures in temporal lobe epilepsy. It has been suggested that the hippocampus is strongly involved this pathology. There is less consistent information, however, on the effects of this disease in the amygdala. The effects of status epilepticus on the medial extended amygdala were analyzed by immunohistochemistry for neural stress and by the amino-cupric-silver technique for neuronal death in rats after kainic acid (KA) administration. Sixty adult Wistar male rats were used. Thirty animals received an injection of KA, and 30 were injected with saline. After 2, 4, 12, 24 and 48 h survival the brains were stained for Fos and FosB and for neuronal death.In the present study we show that KA induces Fos and FosB expression in neurons of the medial extended amygdala after 2, 4-48 h, with time courses that are different between them and from control animals. While Fos-IR peaks at 2-4 h post KA and then decreases, FosB-IR increases in the same period reaching its highest expression at 24-48 h. Moreover, KA injection produced massive neuronal death with a peak at 24 h. This neurodegeneration paralleled FosB-IR protein expression.These findings show that KA produces neuronal stress and activation of early genes and neuronal death in the medial extended amygdala, demonstrating the vulnerability of its neurons to the epileptogenic effects of KA.     

Role of endonuclease G in neuronal excitotoxicity in mice

Excitotoxicity is a process by which excitatory amino acids induce neuronal cell death. To what extent excitotoxicity is regulated by apoptotic molecules is currently unclear. We previously found that endonuclease G (EndoG) plays an important role in both normal apoptosis in vivo and in pre-implantation embryogenesis. To investigate whether EndoG participates in neuronal cell death, we compared EndoG expression and kainic acid (KA)-induced seizure behavior and excitotoxicity in EndoG+/- and wild-type mice. We found that EndoG expression in the hippocampus of EndoG+/- mice is reduced compared to that in the wild-type mice. The reduction of EndoG expression levels in the hippocampus did not result in altered KA-induced seizure severity in EndoG+/- mice compared to that in wild-type mice. However, both CA3 and CA1 pyramidal neurons in EndoG+/- mice are more resistant to KA-induced cell death than that in wild-type mice. These results indicate that reduced expression of EndoG in the hippocampi of EndoG+/- mice leads to resistance to excitotoxicity.     

Statin inhibits kainic acid-induced seizure and associated inflammation and hippocampal cell death

Statins are inhibitors of HMG-CoA reductase that have been recently recognized as anti-inflammatory and neuroprotective drugs. Herein, we investigated anti-excitotoxic and anti-seizure effects of statins by using kainic acid (KA)-rat seizure model, an animal model for temporal lobe epilepsy and excitotoxic neurodegeneration. We observed that pre-treatment with Lipitor (atorvastatin) efficiently reduced KA-induced seizure activities, hippocampal neuron death, monocyte infiltration and proinflammatory gene expression. In addition, we also observed that lovastatin treatment attenuated KA- or glutamate-induced excitotoxicity of cultured hippocampal neurons. These observations suggest a potential for use of statin treatment in modulation of seizures and other neurological diseases associated with excitotoxicity.     

Obesity exacerbates chemically induced neurodegeneration

Obesity is a major risk factor associated with a variety of human disorders. While its involvement in disorders such as diabetes, coronary heart disease and cancer have been well characterized, it remains to be determined if obesity has a detrimental effect on the nervous system. To address this issue we determined whether obesity serves as a risk factor for neurotoxicity. Model neurotoxicants, methamphetamine (METH) and kainic acid (KA), which are known to cause selective neurodegeneration of anatomically distinct areas of the brain, were evaluated using an animal model of obesity, the ob/ob mouse. Administration of METH and KA resulted in mortality among ob/ob mice but not among their lean littermates. While METH caused dopaminergic nerve terminal degeneration as indicated by decreased striatal dopamine (49%) and tyrosine hydroxylase protein (68%), as well as an increase in glial fibrillary acidic protein by 313% in the lean mice, these effects were exacerbated under the obese condition (96%, 86% and 602%, respectively). Similarly, a dosage of KA that did not increase glial fibrillary acidic protein in lean mice increased the hippocampal content of this protein (93%) in ob/ob mice. KA treatment resulted in extensive neuronal degeneration as determined by Fluoro-Jade B staining, decreased hippocampal microtubule-associated protein-2 immunoreactivity and increased reactive gliosis in ob/ob mice. The neurotoxic outcome in ob/ob mice remained exacerbated even when lean and ob/ob mice were dosed with METH or KA based only on a lean body mass. Administration of METH or KA resulted in up-regulation of the mitochondrial uncoupling protein-2 to a greater extent in the ob/ob mice, an effect known to reduce ATP yield and facilitate oxidative stress and mitochondrial dysfunction. These events may underlie the enhanced neurotoxicity seen in the obese mice.In summary, our results implicate obesity as a risk factor associated with chemical- and possibly disease-induced neurodegeneration.     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

Minocycline inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after treatment with kainic acid in mice

Although minocycline has been generally thought to have neuroprotective properties, the neuroprotective role of minocycline has not been investigated in the animal model of epilepsy. In this study, we investigated whether minocycline is neuroprotective against kainic acid (KA)-induced cell death through the caspase-dependent or -independent mitochondrial apoptotic pathways. Adult male ICR mice were subjected to seizures by intrahippocampal KA injection with vehicle or with minocycline. For cell death analysis, TdT-mediated dUTP-biotin nick end labeling and cresyl-violet staining were performed. Western blot analysis and immunofluorescent staining for cytochrome c and apoptosis-inducing factor (AIF) were performed. Cell death was reduced in minocycline-treated mice. Cytosolic translocation of cytochrome c and subsequent activation of caspase-3 were diminished by minocycline treatment. AIF nuclear translocation and subsequent large-scale DNA fragmentation were also reduced in minocycline-treated mice. Thus, this study suggests that minocycline inhibits both caspase-dependent and -independent apoptotic pathways and may be neuroprotective against hippocampal damage after KA treatment.