Effects of scorpion venom heat-resistant peptide on the hippocampal neurons of kainic acid-induced epileptic rats

Scorpion venom is a Chinese medicine for epilepsy treatment, but the underlying mechanism is not clear. Scorpion venom heat-resistant peptide (SVHRP), a peptide isolated from the venom of Buthus martensii Karsch, has an anti-epileptic effect by reducing seizure behavior according to a modified Racine scale. The present study aimed to investigate the molecular mechanism of SVHRP on temporal lobe epilepsy. The hippocampus and hippocampal neurons from kainic acid-induced epileptic rats were treated with SVHRP at different doses and duration. Quantitative RT-PCR and immunoblotting were used to detect the expression level of brain-derived neurotrophic factor (BDNF), neuropeptide Y (NPY), cAMP-response element binding protein (CREB), stromal interaction molecule (STIM), and calcium release-activated calcium channel protein 1 (ORAI1). In the hippocampal tissues and primary hippocampal neuron cultures, SVHRP treatment resulted in increased mRNA and protein levels of BDNF and NPY under the epileptic condition. The upregulation of BDNF and NPY expression was positively correlated with the dose level and treatment duration of SVHRP in hippocampal tissues from kainic acid-induced epileptic rats. On the other hand, no significant changes in the levels of CREB, STIM, or ORAI1 were observed. SVHRP may exhibit an anti-epileptic effect by upregulating the expression of BDNF and NPY in the epileptic hippocampus.

Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-alpha, -beta I, -beta II, and -delta in microglia following kainic acid-induced seizures

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC alpha, beta I, beta II and delta, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.

Role of gamma-aminobutyric acid B (GABA B) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA B) receptors in hippocampal cell death induced by KA (0.1 microgram) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA B receptors antagonist, 20 microgram) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca2+ /calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA B receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity.

Increasing evidence supports the role of excitotoxicity in neuronal cell injury. Thus, it is extremely important to explore methods to retard or reverse excitotoxic neuronal injury. In this regard, certain dietary compounds are beginning to receive increased attention, in particular those involving phytochemicals found in medicinal plants in alleviating neuronal injury. In the present study, we examined whether medicinal plant extracts protect neurons against excitotoxic lesions induced by kainic acid (KA) in female Swiss albino mice. Mice were anesthetized with ketamine and xylazine (200 mg and 2 mg/kg body wt. respectively) and KA (0.25 microg in a volume of 0.5 microl) was administered to mice by intra hippocampal injections. The results showed an impairment of the hippocampus region of brain after KA injection. The lipid peroxidation and protein carbonyl content were significantly (P < 0.05) increased in comparison to controls. Glutathione peroxidase (GPx) activity (EC 1.11.1.9) and reduced glutathione (GSH) content declined after appearance of excitotoxic lesions. As GPx and GSH represent a major pathway in the cell for metabolizing hydrogen peroxide (H2O2), their depletion would be expected to allow H2O2 to accumulate to toxic levels. Dried ethanolic plant extracts of Withania somnifera (WS), Convolvulus pleuricauas (CP) and Aloe vera (AV) dissolved in distilled water were tested for their total antioxidant activity. The diet was prepared in terms of total antioxidant activity of plant extracts. The iron (Fe3+) reducing activity of plant extracts was also tested and it was found that WS and AV were potent reductants of Fe3+ at pH 5 5. CP had lower Fe3+ reducing activity in comparison to WS and AV. Plant extracts given singly and in combination 3 weeks prior to KA injections resulted in a decrease in neurotoxicity. Measures of lipid peroxidation and protein carbonyl declined. GPx activity and GSH content were elevated in hippocampus supplemented with WS and combination of WS + CP + AV. However, when CP and AV were given alone, the changes in the GPx activity and GSH content were not significant. Although the major factors involved in these properties of phytochemicals remain to be specified, the finding of this study has suggested that phytochemicals present in plant extracts mitigate the effects of excitotoxicity and oxidative damage in hippocampus and this might be accomplished by their antioxidative properties.

Calcium/Calmodulin Kinase II Activity of Hippocampus in Kainate-Induced Epilepsy

This study investigated calcium/calmodulin kinase II (CaMKII) activity related to long-standing neuronal injury of the hippocampus in kainate (KA)-induced experimental temporal lobe epilepsy. Epileptic seizure was induced by injection of KA (1 g/L) dissolved in phosphate buffer (0.1 M, pH 7.4) into the left amygdala. Clinical seizures, histopathologic changes and CaMKII activity of the hippocampus were evaluated. Characteristic early limbic and late seizures were developed. Hippocampal CaMKII activity increased significantly 4 and 8 weeks after intra-amygdaloid injection of KA, when late seizures developed. The histopathologic changes of the hippocampus included swelling of neuronal cytoplasm with nuclear pyknosis and loss of neurons in CA3 during this period. The increased activity of CaMKII may correlate with appearance of distant damage in the hippocampus. The above results indicate that intra-amygdaloid injection of KA produces excitatory signals for ipsilateral CA3 neurons in the hippocampus and that subsequently increased levels of CaMKII in postsynaptic neurons induce neuronal injury via phosphorylation of N-methyl-D-aspartate type glutamate receptor.

c-JUN Expression and Apoptotic Cell Death in Kainate-Induced Temporal Lobe Epilepsy

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly ex-press 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 (1g/l) 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.

Changes of gastroduodenal peroxidase activity of rats by cerebellar modulation.

The effect of vestibulo-cerebellar lesion and its stimulation by rotation on gastric and duodenal peroxidase activity of rats was studied. Vestibulocerebellar lesion by kainic acid produced gastroduodenal ulceration and peroxidase activity of these tissues were decreased. Mucosal thickness of gastric and duodenal tissue were also decreased. It was observed that when vestibulo-cerebellar lesioned rats were subjected to vestibular stimulation, the peroxidase activity was increased together with increased mucosal thickness of gastric and duodenal tissue. At the same time, it was noted that the severity of ulceration was decreased. We conclude that the study of peroxidase activity is a sensitive and potentially useful estimate of gastric and duodenal injury produced by cerebellar lesion that can be valuable in assessing ulcerogenesis and healing.

Effect of cerebellar modulation on rat gastric secretion and enterochromaffin-like cell.

Effect of cerebellar lesion and vestibular stimulation (VS) on the activity and alternation of ECL-cells along with changes in gastric volume and acid secretion was studied. The results suggest that cerebellar lesion caused increased gastric volume and acid secretion and tended to decrease ECL-cell density. On the other hand VS of nodular lesioned rats resulted in decrease of above parameter which became marked only after 21 days of nodular lesion.

Domoic acid induces neurotoxicity and ip3 mobilization in cultured cells of embryonic chick retina

Domoic acid (DOM), 1 to 50 µM, a glutamate agonist responsible for several neurological effects such as loss of memory and confusion, induced the death of cultured neurons of chick embryonic retina, in a concentration- and Ca2+ -dependent manner. This effect was blocked by 100 µM CNQX, a competitive antagonist of the non-NMDA receptor, but not by 10 µM MK-801, a non-competitive antagonist of the NMDA receptor. DOM also induced inositol triphosphate (ip3) accumulation 4 to 7 times above basal levels. This effect was also dependent on external Ca2+ and was entirely blocked by 100 µM CNQX, but not by 10 µM MK-801. These results suggest that DOM interaction with non-NMDA glutamate receptors mediates signal transduction with ip3 accumulation and cell death