The influence of kainic acid on core temperature and cytokine levels in the brain

Excitotoxic brain injury is associated with hyperthermia, and there are data showing beneficial effects of hypothermia on neurodegeneration and that hyperthermia facilitates the neurodegeneration. Cytokines are inflammatory proteins that seem to be involved in the neuroinflammation associated with epilepsy. Core temperature changes caused by the epileptogenic glutamate analogue kainic acid (KA) were investigated in relation to changes in levels of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), and the endogenous interleukin-1 receptor antagonist (IL-1ra). The temperature was measured every 10 min during the first hour, and at 90 and 120 min, and hourly until 8 h after KA-injection (10 mg/kg). The cytokines were measured in the hypothalamus, a site of temperature regulation, and in hippocampus, cerebellum, and frontal cortex. KA induced a brief hypothermia followed by hyperthermia. IL-1beta levels were increased after KA-administration in all brain regions examined and, excepting hippocampus, returned to baseline levels at 24 h. The hippocampal IL-1ra levels were significantly increased at 24 h, whereas no changes in IL-6 levels were observed. The changes in IL-1beta levels and in ratios between the levels of the three cytokines, may account for some of the temperature changes and the behavioural manifestations induced by KA.     

Brain aromatase is neuroprotective

The expression of aromatase, the enzyme that catalyzes the biosynthesis of estrogens from precursor androgens, is increased in the brain after injury, suggesting that aromatase may be involved in neuroprotection. In the present study, the effect of inactivating aromatase has been assessed in a model of neurodegeneration induced by the systemic administration of neurotoxins. Domoic acid, at a dose that is not neurotoxic in intact male mice, induced significant neuronal loss in the hilus of the hippocampal formation of mice with reduced levels of aromatase substrates as a result of gonadectomy. Furthermore, the aromatase substrate testosterone, as well as its metabolite estradiol, the product of aromatase, were able to protect hilar neurons from domoic acid. In contrast, dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone and a nonaromatizable androgen, was not. These findings suggest that aromatization of testosterone to estradiol may be involved in the neuroprotective action of testosterone in this experimental model. In addition, aromatase knock-out mice showed significant neuronal loss after injection of a low dose of domoic acid, while control littermates did not, indicating that aromatase deficiency increases the vulnerability of hilar neurons to neurotoxic degeneration. The effect of aromatase on neuroprotection was also tested in male rats treated systemically with the specific aromatase inhibitor fadrozole and injected with kainic acid, a well characterized neurotoxin for hilar neurons in the rat. Fadrozole enhanced the neurodegenerative effect of kainic acid in intact male rats and this effect was counterbalanced by the administration of estradiol. Furthermore, the neuroprotective effect of testosterone against kainic acid in castrated male rats was blocked by fadrozole. These findings suggest that neuroprotection by aromatase is due to the formation of estradiol from its precursor testosterone. Finally, a role for local cerebral aromatase in neuroprotection is indicated by the fact that intracerebral administration of fadrozole enhanced kainic acid induced neurodegeneration in the hippocampus of intact male rats. These findings indicate that aromatase deficiency decreases the threshold for neurodegeneration and that local cerebral aromatase is neuroprotective. Brain aromatase may therefore represent a new target for therapeutic approaches to neurodegenerative diseases.     

Excitotoxicity,neuroinflammation and oxidant stress as molecular bases of epileptogenesis and epilepsy-derived neurodegeneration: The role of vitamin E

Glutamate-mediated excitotoxicity, neuroinflammation, and oxidative stress are common underlying events in neurodegeneration. This pathogenic “triad” characterizes the neurobiology of epilepsy, leading to seizure-induced cell death, increased susceptibility to neuronal synchronization and network alterations. Along with other maladaptive changes, these events pave the way to spontaneous recurrent seizures and progressive degeneration of the interested brain areas.In vivo models of epilepsy are available to explore such epileptogenic mechanisms, also assessing the efficacy of chemoprevention and therapy strategies at the pre-clinical level. The kainic acid model of pharmacological excitotoxicity and epileptogenesis is one of the most investigated mimicking the chronicization profile of temporal lobe epilepsy in humans. Its pathogenic cues include inflammatory and neuronal death pathway activation, mitochondrial disturbances and lipid peroxidation of several regions of the brain, the most vulnerable being the hippocampus. The importance of neuroinflammation and lipid peroxidation as underlying molecular events of brain damage was demonstrated in this model by the possibility to counteract the related maladaptive morphological and functional changes of this organ with vitamin E, the main fat-soluble cellular antioxidant and “conditional” co-factor of enzymatic pathways involved in polyunsaturated lipid metabolism and inflammatory signaling.The present review paper provides an overview of the literature supporting the potential for a timely intervention with vitamin E therapy in clinical management of seizures and epileptogenic processes associated with excitotoxicity, neuroinflammation and lipid peroxidation, i.e. the pathogenic “triad”.     

Interleukin-1 and neuronal injury

Interleukin-1 is a pro-inflammatory cytokine that has numerous biological effects, including activation of many inflammatory processes (through activation of T cells, for example), induction of expression of acute-phase proteins, an important function in neuroimmune responses and direct effects on the brain itself. There is now extensive evidence to support the direct involvement of interleukin-1 in the neuronal injury that occurs in both acute and chronic neurodegenerative disorders. This article discusses the key evidence of a role for interleukin-1 in acute neurodegeneration - for example, stroke and brain trauma - and provides a rationale for targeting the interleukin-1 system as a therapeutic strategy.     

Caspase 3 involves in neuroplasticity,microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid

Background

The roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection.

Result

Neurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred.

Conclusions

Our results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.     

Kainic acid as a tool for the study of temporal lobe epilepsy

Temporal lobe epilepsy (limbic epilepsy, complex partial epilepsy, psychomotor epilepsy) is the most devastating form of epilepsy commonly encountered in the adult population. The attacks involve loss of consciousness, thus limiting performance of normal functions and exposing the individual to bodily injury. Moreover, long-standing or pharmacologically intractable temporal lobe epilepsy is frequently associated with the loss of neurons from the hippocampus and other brain regions (Ammon's horn sclerosis (AHS)). Unfortunately, pharmacologically intractable cases are rather common, owing to the relatively low efficacy against this condition of the available anticonvulsants. Progress in the understanding and treatment of temporal lobe epilepsy would be greatly facilitated by the availability of an animal model which reproduced the behavioral, electrographic and pathological features of this condition. Here I review evidence which indicates that the kainic acid (KA)-treated rat possesses many of the features required of such a model.     

Bruce/apollon promotes hippocampal neuron survival and is downregulated by kainic acid

Prolonged or excess stimulation of excitatory amino acid receptors leads to seizures and the induction of excitotoxic nerve cell injury. Kainic acid acting on glutamate receptors produces degeneration of vulnerable neurons in parts of the hippocampus and amygdala, but the exact mechanisms are not fully understood. We have here investigated whether the anti-apoptotic protein Bruce is involved in kainic acid-induced neurodegeneration. In the rat hippocampus and cortex, Bruce was exclusively expressed by neurons. The levels of Bruce were rapidly downregulated by kainic acid in hippocampal neurons as shown both in vivo and in cell culture. Caspase-3 was activated in neurons exhibiting low levels of Bruce causing cell death. Likewise, downregulation of Bruce using antisense oligonucleotides decreased viability and enhanced the effect of kainic acid in the hippocampal neurons. The results show that Bruce is involved in neurodegeneration caused by kainic acid and the downregulation of the protein promotes neuronal death.     

DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.     

Convulsants induce interleukin-1 beta messenger RNA in rat brain.

The effects of systemic administration of kainic acid and pentylenetetrazol on interleukin-1 beta gene expression in the rat brain was studied. After the administration of kainic acid in a convulsive dose (10 mg/kg i.p.), Interleukin-1 beta mRNA was induced intensely in the cerebral cortex, thalamus and hypothalamus, moderately in the hippocampus and weakly in the striatum, but not in the midbrain, pons-medulla and cerebellum. Pentylenetetrazol induced Interleukin-1 beta mRNA in the cerebral cortex, hypothalamus, and hippocampus with a faster time-course than kainic acid. Diazepam suppressed both the convulsion and the induction of Interleukin-1 beta mRNA produced by kainic acid. Dexamethasone suppressed the induction of Interleukin-1 beta mRNA, but did neither the convulsion nor the induction of c-fos mRNA following the injection of kainic acid. These results provide the first evidence that intensive neuronal excitation induces Interleukin-1 beta mRNA in particular regions of the brain.     

Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.     

Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures

Polyamines are ubiquitous cations that are essential for cell growth, regeneration and differentiation. Increases in polyamine metabolism have been implicated in several neuropathological conditions, including excitotoxicity. However, the precise role of polyamines in neuronal degeneration is still unclear. To investigate mechanisms by which polyamines could contribute to excitotoxic neuronal death, the present study examined the role of the polyamine interconversion pathway in kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. Treatment of cultures with N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), an irreversible inhibitor of polyamine oxidase, resulted in a partial but significant neuronal protection, especially in CA1 region. In addition, this pre-treatment also attenuated KA-induced increase in levels of lipid peroxidation, cytosolic cytochrome C release and glial cell activation. Furthermore, pre-treatment with a combination of cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) and MDL 72527 resulted in an additive and almost total neuronal protection against KA toxicity, while the combination of MDL 72527 and EUK-134 (a synthetic catalase/superoxide dismutase mimetic) did not provide additive protection. These data strongly suggest that the polyamine interconversion pathway partially contributes to KA-induced neurodegeneration via the production of reactive oxygen species.     

Does Pilocarpine-Induced Epilepsy in Adult Rats Require Status epilepticus?

Pilocarpine-induced seizures in rats provide a widely animal model of temporal lobe epilepsy. Some evidences reported in the literature suggest that at least 1 h of status epilepticus (SE) is required to produce subsequent chronic phase, due to the SE-related acute neuronal damage. However, recent data seems to indicate that neuro-inflammation plays a crucial role in epileptogenesis, modulating secondarily a neuronal insult. For this reason, we decided to test the following hypotheses: a) whether pilocarpine-injected rats that did not develop SE can exhibit long-term chronic spontaneous recurrent seizures (SRS) and b) whether acute neurodegeneration is mandatory to obtain chronic epilepsy. Therefore, we compared animals injected with the same dose of pilocarpine that developed or did not SE, and saline treated rats. We used telemetric acquisition of EEG as long-term monitoring system to evaluate the occurrence of seizures in non-SE pilocarpineinjected animals. Furthermore, histology and MRI analysis were applied in order to detect neuronal injury and neuropathological signs. Our observations indicate that non-SE rats exhibit SRS almost 8 (+/22) months after pilocarpine-injection, independently to the absence of initial acute neuronal injury. This is the first time reported that pilocarpine injected rats without developing SE, can experience SRS after a long latency period resembling human pathology. Thus, we strongly emphasize the important meaning of including these animals to model human epileptogenesis in pilocarpine induced epilepsy.     

Tissue transglutaminase overexpression in the brain potentiates calcium-induced hippocampal damage

Tissue transglutaminase (tTG) post-translationally modifies proteins in a calcium-dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild-type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid-treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid-induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.     

遗传性癫痫易感大鼠脑内NMDAR1基因表达

N-甲基-D天门冬氨酸(NMDA)受体与癫痫及癫痫易感性的形成密切相关. 以遗传性癫痫易感大鼠P77PMC为研究对象, 通过RNA印迹杂交检测,NMDA受体一型亚单位(NMDAR1)mRNA在惊厥后不同脑区表达, 结果显示: P77PMC大鼠惊厥后, 大脑皮层、海马、皮层下、下丘NMDAR1 mRNA表达呈时间依赖性增加;比较惊厥即刻与惊厥后24 h, 四个脑区NMDAR1 mRNA分别增加了111%、113%、165%和202%. 提示NMDA受体 亚单位受惊厥活动调控,并参与惊厥的发生、发展及惊厥后突触结构的重建.     

Wnt/β‐catenin signalling pathway mediated aberrant hippocampal neurogenesis in kainic acid‐induced epilepsy

Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis with massive neuronal loss and severe gliosis. Aberrant neurogenesis has been shown in the epileptogenesis process of temporal lobe epilepsy. However, the molecular mechanisms underlying aberrant neurogenesis remain unclear. The roles of Wnt signalling cascade have been well established in neurogenesis during multiple aspects. Here, we used kainic acid‐induced rat epilepsy model to investigate whether Wnt/β‐catenin signalling pathway is involved in the aberrant neurogenesis in temporal lobe epilepsy. Immunostaining and western blotting results showed that the expression levels of β‐catenin, Wnt3a, and cyclin D1, the key regulators in Wnt signalling pathway, were up‐regulated during acute epilepsy induced by the injection of kainic acids, indicating that Wnt signalling pathway was activated in kainic acid‐induced temporal lobe epilepsy. Moreover, BrdU labelling results showed that blockade of the Wnt signalling by knocking down β‐catenin attenuated aberrant neurogenesis induced by kainic acids injection. Altogether, Wnt/β‐catenin signalling pathway mediated hippocampal neurogenesis during epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis. Aberrant neurogenesis has been shown to involve in the epileptogenesis process of temporal lobe epilepsy. In the present study, we discovered that Wnt3a/β‐catenin signalling pathway serves as a link between aberrant neurogenesis and underlying remodelling in the hippocampus, leading to temporal lobe epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy.     

Effect of oxcarbazepine pretreatment on convulsive activity and brain damage induced by kainic acid administration in rats

Temporal lobe epilepsy is one of the most common types of epilepsy. Progress in the understanding and treatment of this type of epilepsy would be greatly facilitated by the availability of an animal model, which reproduced the behavioral and electrographic features of this condition. In this context, kainic acid (KA, 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine) administration causes a syndrome characterized by an acute status epilepticus and subsequent brain damage similar to that in temporal lobe epilepsy of humans. The aim of the present study was to investigate whether oxcarbazepine (10,11-dihydro-10-oxo-5 H -dibenz(b,f)azepine-5-carboxamide), an antiepileptic drug, protects against both epileptic activity and brain damage induced by KA administration. Chronically implanted adult male Wistar rats were polygraphically recorded during 10 continuous hours under 4 different conditions: a) control, b) after KA administration alone, c) after KA administration in oxcarbazepine pretreated animals and d) after the administration of oxcarbazepine alone. Animals treated with KA alone presented behavioral and electrophysiological convulsive activity as well as brain damage. Latency of seizure installation was lengthened significantly and convulsive activity was slightly reduced, however, brain damage was still present in oxcarbazepine pretreated animals. Administration of oxcarbazepine alone induced a hypnotic behavior and brain damage was also present.     

Effects of systemic administration of kainic acid on GABAergic and glutaminergic transmission in various areas of the brain

In the present study that Authors have investigated the effects of systemic injection of kainic acid on aminoacidergic transmission of different rat brain regions. Kainic acid has been used to produce an experimental model of limbic epilepsy characterized by two different phases (KA1 and KA2). Results obtained show a significant decrease of glutamic and aspartic acids (excitatory aminoacids) and glicine and taurine (inhibitory aminoacids) in both phases at hippocampal level. On the contrary GABA concentration seems to be increased.     

Clusterin (Apo J) Protects Against In Vitro Amyloid-β(1–40) Neurotoxicity

Abstract: Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-β (Aβ) plaques. Clusterin also coprecipitates with Aβ from CSF, suggesting a physiological interaction with Aβ. Given this interaction with Aβ, the goal of this study was to determine whether clusterin could modulate Aβ neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against Aβ(1–40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of Aβ(1–40) but not against H₂O₂ or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-β plaque pathology.