Nicotine protects against the dexamethasone potentiation of kainic acid-induced neurotoxicity in cultured hippocampal neurons

To elucidate the neuroprotective effect of nicotine, we investigated whether nicotine may attenuate dexamethasone potentiation of kainic acid-induced neurotoxicity. Primary hippocampal culture was pre-treated with nicotin for 24 h followed by dexamethasone (10⁻⁴ M) for 24 h. Then, cultures were exposed with kainic acid (10⁻⁴ M) and cellular viability was determined by LDH effluxmetry. Nicotine pre-treatment (10⁻⁹−10⁻⁷ M) dose-dependently attenuated dexamethasone potentiation of kainic acid-induced neurotoxicity. These results may support the epidemiological data suggesting a neuroprotective effect of cigarette smoking on Alzheimer's disease or Parkinson's disease.     

The role of early Ca influx in the pathogenesis of delayed neuronal death after brief forebrain ischemia in gerbils

To examine the role of calcium influx in the early phase after brief forebrain ischemia and subsequent delayed neuronal cell death in the hippocampus,⁴⁵Ca autoradiography and electron microscopic cytochemistry, by a combined oxalate-pyroantimonate method, were carried out in gerbil brains after 5 min bilateral common carotid arterial occlusion. Further, neuronal during the ischemic and postischemic periods was determined by conventional or immunohistochemical staining for microtubule-associated protein 2 (MAP2) with and without calcium-entry blockers.⁴⁵Ca autoradiography showed a high peak of calcium in the hippocampus at 5 min of recirculation. Electron cytochemical microscopy also demonstrated accumulation of intracellular calcium pyroantimonate deposits in the neuronal cells in all regions. At 30 min of reperfusion, amounts of calcium in the hippocampus returned to the control levels, and intracellular dense calcium pyroantimonate deposits were reduced in these areas. Loss of the reaction for MAP2 was noted in the medial CA1 of the hippocampus immediately after 5 min ischemia and at 5 and 30 min after reperfusion. MK-801 (10 mg kg⁻¹, anN-methyl-d-aspartate (NMDA) receptor antagonist, injected intraperitoneally 1 h before ischemia, suppressed the early increase of calcium in the forebrain and neuronal cell necrosis in the CA1. However, neither injection of MK-801 30 min after reperfusion nor preischemic treatment with 0.5 mg kg⁻¹ Nicardipine, voltage-sensitive calcium channel antagonists, prevented neuronal death. In immunohistochemical staining for MAP2, the ischemic lesion in the medial CA1 maintained after 5 min ischemia and the subsequent early reperfusion period in the untreated brains was protected by the preischemic injection of 10 mg kg⁻¹ MK-801, but was not restored by the injection of 0.5 mg kg⁻¹ Nimodipine or 1 mg kg⁻¹ Nicardipine. In conclusion, it is suggested that an early excess of calcium influx could be caused mainly by excitatory amino acid overload through NMDA receptor-mediated calcium channels during the ischemic and early postischemic periods.     

Melatonin attenuates the changes in polyamine levels induced by systemic kainate administration in rat brains

Systemically administered kainate has been demonstrated to induce neuronal damage and changes of the levels of biochemical substances related to neurotoxicity. Polyamines are thought to be important in the generation of edema and neuronal cell loss associated with various type of excitotoxicity. Melatonin exerts potent free radical scavenging, antioxidant, and neuroprotective properties. This study was designed to estimate the effect of exogenous melatonin administration on the changes of polyamine levels in rat brains after systemic administration of kainate. Kainate [10 mg/kg, intraperitoneally (i.p.)] was injected into the rats to produce excitotoxicity. Melatonin (15 mg/kg, i.p.) was administered 1 h before, immediately after, and 1 h after kainate treatment. We examined the polyamine [putrescine (PU), spermidine (SD) and spermine (SM)] levels in the cerebral cortex and hippocampus and neuronal density in the hippocampal CA1 and CA3 subsectors in brain sections. PU levels were increased 8 and 24 h after kainate treatment and the administration of melatonin attenuated these changes. Only minor changes were noted in the levels of the polyamine SD and SM after the kainate treatment. In histology, neuronal injuries in the hippocampal CA1 and CA3 subsectors were examined 3 days after kainate treatment and melatonin reduced the kainate-induced neuronal injuries. Our results show that melatonin inhibits the polyamine responses in the cerebral cortex and hippocampus following kainate-induced excitotoxicity and PU may be responsible for the protective effect of melatonin against kainate-induced excitotoxicity.     

Picolinic acid modulates kainic acid-evoked glutamate release from the striatum in vitro

Since picolinic acid, a tryptophan metabolite yielded by the kynurenine pathway, selectively attenuates quinolinic and kainic acid excitotoxicity that is dependent on the presence of a glutamatergic afferent input, it was hypothesized that this agent may inhibit the presynaptic release of glutamate. Using superfused rat striatal slices, this study examined the potential of picolinic acid, and related pyridine monocarboxylic acids, to modify kainic acid-induced glutamate release. Kainic acid (0.25, 0.5 and 1.0 mM) stimulated the release of glutamate, an effect which was calcium dependent and was attenuated in the presence of the kainate/AMPA receptor antagonist, 6,7-dinitroquinoxalene-2,3-dione (500 μM). Picolinic acid significantly decreased glutamic acid release evoked by exposure of striatal slices to 1 mM kainate in the presence of calcium. The inhibitory action of picolinic acid on kainate-induced release was also shared by nicotinic and isonicotinic acid. In the absence of external calcium, kainic acid-induced glutamate release was significantly reduced by approximately 65%. Under this condition, picolinic acid (100 μM) failed to influence kainic acid-induced release. Picolinic acid (100 μM) itself increased glutamate release by 35% over basal release. While the ability of picolinic acid to inhibit excitotoxin-induced release supports the notion that it may act presynaptically to modify excitotoxicity, lack of structural specificity in its action tends to cast doubt on this mechanism of action.     

Protective effects of tetramethylpyrazine on kainate-induced excitotoxicity in hippocampal culture

Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb, Chuanxiong. This study focuses on the protective effect of tetramethylpyrazine in kainate-induced excitotoxicity in rat hippocampus. Primary neuronal cultures raised from cells isolated from the hippocampi of 7-day old rats were treated with kainate (75-450 microM) for 12, 24, and 48 h. Our results revealed that kainate induced neuronal damage in a dose- and time-dependent manner, reaching maximal damage at 150 microM and 24 h and persisted for higher doses and 48 h. In addition, 1 h of kainate (150 microM) treatment led to significant generation of free radicals and reduction of mitochondrial membrane potential (MMP) which persisted for > or = 4 h on continued exposure. Ten minutes pretreatment with 1 or 5 microM tetramethylpyrazine dose dependently and significantly attenuated the kainate-induced damage. Taken together, the results suggest that multiple mechanisms including protection of mitochondria, decrease in free radical generation and scavenging of free radicals might be involved in TMP's protection against kainate induced cell toxicity.     

Localization of aromatase and 5α-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus

Experiments were designed to identify the neural cell type(s) responsible for the aromatization and 5α-reduction of androgens in the rat hypothalamus. Primary cultures of fetal rat hypothalamic cells, which had enhanced neuronal morphology, were treated at various times after plating with kainic acid (KA), a neurotoxic agent which selectively destroys neuronal cells. Neuronal morphology was disrupted in a time (0–6 days)- and dose (10⁻⁴–10⁻² M)-dependent fashion after KA treatment, with no apparent change in the appearance of the flattened, underlying non-neuronal cells. KA treatment for 4 days decreased aromatization by 94% in a dose-dependent fashion (10⁻⁴–10⁻² M KA), while 5α-reduction declined by no more than 25%. A 6-day time course with 10⁻³ M KA showed a dramatic decline in aromatization and no alteration in 5α-reduction. In control experiments, substance P, a neuronal peptide, declined after KA treatment while the activity of glutamine synthetase, a glial enzyme, did not change. We conclude from these results that aromatase is localized primarily to neuronal cells in the hypothalamus while 5α-reductase is confined primarily to non-neuronal cells.     

Kainic acid-induced seizures and brain damage in the rat: Role of calcium homeostasis

Seizure activity induced by kainic acid (KA) and subsequent neuronal death are thought to be associated with an increase in cytoplasmic free calcium ([Ca²⁺]_i) and can be prevented by N-methyl-D-aspartate (NMDA) antagonists. In addition to influx through receptor operated Ca²⁺ channels the increase in [Ca²⁺]_i may be the result of an increased influx through voltage-operated calcium channels and/or release from intracellular deposits. It was therefore investigated whether compounds other than NMDA antagonists with known actions on the intracellular Ca²⁺ homeostasis had any protective effect against KA-induced neuronal death. Voltage-operated calcium channels in the cell membrane were blocked with the L-type ion channel antagonist, Nimodipine (1.0 mg/kg), and release of Ca²⁺ from internal stores was prevented with Dantrolene (10 mg/kg). Animals from two control groups injected with kainate (8 mg/kg) exhibited a survival rate of 67 and 53%, respectively. Countings of neurons in dorsal hippocampus showed subtotal or total loss in the CA1 and CA3 subregions. There were no significant differences concerning seizure and survival rates in the groups injected with kainate and treated with Dantrolene or Nimodipine and the control groups. The group treated with Dantrolene showed no neuropathological changes in the hippocampal CA3 region and only slight changes in the CA1 region, while the neuron loss in the Nimodipine group did not differ from that of its control group. The results emphasize the importance of Dantrolene-sensitive Ca²⁺ release from intracellular stores for the development of seizureinduced neuronal death. © 1995 Wiley-Liss, Inc.     

Sensitive indicators of injury reveal hippocampal damage in C57BL/6J mice treated with kainic acid in the absence of tonic-clonic seizures

Sensitive indices of neural injury were used to evaluate the time course of kainic acid (KA)-induced hippocampal damage in adult C57BL/6J mice (4 months), a strain previously reported to be resistant to kainate-induced neurotoxicity. Mice were injected systemically with saline or kainate, scored for seizure severity (Racine scale), and allowed to survive 12 h, one, three, or seven days following which they were evaluated for neuropathological changes using histological or biochemical endpoints. Most kainate-treated mice exhibited limited seizure activity (stage 1); however, cupric-silver and Fluoro-Jade B stains revealed significant damage by 12 h post-treatment. Immunohistochemistry and immunoassay of glial fibrillary acidic protein and lectin staining revealed a strong treatment-induced reactive gliosis and microglial activation. Immunostaining for immunoglobulin G revealed a kainate-induced breach in the blood-brain barrier. Nissl and hematoxylin stains provided little information regarding neuronal damage, but revealed the identity of non-resident cells which infiltrated the pyramidal layer. Our data suggest sensitive indicators of neural injury evaluated over a time course, both proximal and distal to treatment, are necessary to reveal the full extent of neuropathological changes which may be underestimated by traditional histological stains. The battery of neuropathological indices reported here reveals the C57BL/6J mouse is sensitive to excitotoxic neural damage caused by kainic acid, in the absence of tonic-clonic seizures.     

Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice

Interpretation of data from gene targeting studies can be confounded by the inherent traits of the background inbred strains used in the generation of transgenic and null mutant mice. We have therefore compared the behaviour and response to CNS injury of four inbred strains commonly used in molecular genetic studies to produce models of neurological disease. Adult, male 129/Ola, BALB/c, C57BL/6 and FVB/N mice (2–4 months) were initially subjected to behavioural tests that comprised a neurological examination, determination of motor function and cognitive testing in the Morris water maze. Also the response to CNS injury following an acute kainic acid (KA) challenge (30 mg kg⁻¹, i.p.) was determined. The 129/Ola and BALB/c strains showed significant motor deficits when compared with the C57BL/6 and FVB/N strains. In contrast, only the FVB/N strain showed evidence of apparent cognitive impairments in the water maze as evidenced by increased pathlengths to locate the escape platforms and impaired performance in a probe trial. In addition, the FVB/N strain showed the most severe seizure response and mortality rate (62%) following administration of KA (30 mg kg⁻¹, i.p.). These behavioural changes were also associated with a greater degree of cell body and synaptophysin loss in the pyramidal CA3 hippocampal cell layer and astrogliosis 72-h post-dose. These data suggest that the FVB/N strain may not be the most suitable background strain for the development of new transgenic mice for the study of genes implicated in the learning and memory process.     

Antioxidant activity of the organotellurium compound 3-[4-(N,N-dimethylamino)benzenetellurenyl]propanesulfonic acid against oxidative stress in synaptosomal membrane systems and neuronal cultures

Antioxidant activities of 3-[4-(N,N-dimethylamino) benzenetellurenyl]propanesulfonic acid sodium salt (NDBT) were evaluated in solution, red blood cells, synaptosomal membranes, and cultured hippocampal neuronal cells after exposure to peroxynitrite (ONOO⁻) and hydroxyl radicals. The organotellurium compound NDBT possesses significant activity towards hydrogen peroxide and/or the hydroxyl radical in solution, demonstrated by inhibition of hydroxylation of terephthalic acid. In addition, the compound displayed great antioxidant abilities as shown by: reduction of ONOO⁻-induced 2,7-dichlorofluorescein (DCF) fluorescence in synaptosomes; complete prevention of lipid peroxidation in synaptosomes caused by OH radicals (TBARS), and significant prevention of protein oxidation caused by ONOO⁻ and OH, indexed by the levels of protein carbonyls in synaptosomes and neuronal cells. The presence of the compound abolished neuronal cell death caused by ONOO⁻. Further, the compound was effective in preventing the oxidative changes in synaptosomal membrane protein conformation and crosslinking (EPR spin labeling). Finally, the organotellurium molecule attenuated peroxynitrite-induced, luminol-dependent chemiluminescence in red blood cells — an index of cellular oxidation. These findings demonstrate the great potential of the antioxidant and are consistent with the notion that NDBT may have a role to play in modulating oxidative stress in neurodegenerative disorders, including Alzheimer’s disease.