Study On Neuroprotective Effect Of Minocycline Against Limbic Seizure-induced Hippocampus Damage In Rats

Epilepsy is one of the most common neurological disorders characterized by chronic and unpredictable onset of recurrent seizures. Although treatment strategies have been dramatically advanced in the past decade, 30% epileptic patients continue to experience seizures. In the subset of these patients, temporal lobe epilepsy is the most common type. Because temporal lob and hippocampal structures are very important in memory formulating and affective reacting, so that progressive cognitive decline is always observed in these patients. Recent experimental and human studies have shown that selective neurodegeneration in hippocampus is the substantial reason. Seizures induced inflammation and cytokines secretions in hippocampus participate in neuronal excitability and/or glial scar formation, therefore exacerbate the disorder.Minocycline is a tetracycline derivative with broad-spectrum antibiotic effect. In the past few years it has been shown to have remarkably neuroprotective and anti-inflammative qualities in models of Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis and cerebral ischemia. But whether or not it has protective effect against seizure-induced neurodegeneration and inflammation in hippocampus has not been explored. Here we induced limbic seizures in rats by intraamygdaloid injection of kainic acid to investigate Minocycline's therapeutic effect.Part I Neuronal protective effect of Minocycline against limbic seizures induced hippocampal neurodegeneration in rats.Focal limbic seizures were induced by injecting kainic acid into right amygdaloid in rats, and were terminated after 40 min by diazepam (30 mg/kg) administration. Then the animals were randomly divided into Minocycline treated group (MT group) and non-treated group (kainic acid group, KA group). Animals either in KA group or in MT group were sacrificed at 2h, 4h, 8h, 24h, 48h and 7d (each group: n=6) following diazepam administration. A sham-operated control group (saline control group, SC group) was established by injecting saline into right amygdala, and the animals were sacrificed at4h, 24h, 48h and 7d (each group: n=6). Cresyl-violet staining were performed to evaluate morphologic changes of neurons in hippocampus and to calculate survival neuron numbers in CA3 region. The result showed that morphologic appearance and survival numbers of neurons in hippocampus had no obviously difference between MT groups and KA groups at the first three time-points (i.e. 2h, 4h and 8h). Twenty-four hours after limbic seizures were ceased; the number of survival neuron in CA3 region was markedly higher in MT group than in KA group, although it showed a decreased tendency both in MT group and in KA group. At 7d we interestedly found that the pyramid neuron number in ipsilateral CA3 region was reduced by 90.28% in KA group, but was only 48.55% in MT group. Moreover, pyramid neuron loss was also observed in ipsilateral CA1 region and contralateral CA3 region in KA groups, but relatively limited only in ipsilateral CA3 region in MT groups. The results suggested that Minocycline can distinctively inhibit neuron loss and has potential neuroprotective effect against hippocampal damage after KA treatment. This effect was more significant on chronic persistent neuron loss, perhaps was in the respect of apoptotic neuron degeneration.Part II Effect of Minocycline on Seizure-induced Neuronal Apoptotic Pathway in Hippocampus.In order to verify our hypothesis that Minocycline protects neuron in respect of antiapoptosis, we performed TUNEL staining to lable apoptotic neurons in hippocampal CA3 region for exploring whether there is difference between KA group and MT group on 24h and 48h after seizures were ceased. Then immunohistochemical staining and western-blotting were performed to detect Bcl-2 and Bcl-xl, two important anti-apoptotic factors that modulate mitochondria apoptosis pathway. Western-blotting for pro-caspase-3 (33kD) and caspase-3 fragmentation (17kD) are also performed for evaluating apoptotic degree. The result showed that positive TUNEL staining neuron can be seen both in KA groups and in MT groups. In MT group positive neuron are mainly located in CA3-A sub-region, but relatively dispersed in whole CA3 region in KA groups. TUNEL positive neuron numbers in MT group are decreased 54.79% and 55.67% compare with KA group at 24h and 48h correlatively. Immunohistochemical staining show that content of Bcl-2 in pyramidal neuron in CA3 region are significantly elevatedafter Minocycline treated, but no change has been observed with regard to Bcl-xl. Western-blotting result show that, in MT group the level of caspase-3 cleaved fragments was down-regulated associated with up-regulation of anti-apoptosis protein Bcl-2. The up-regulation of Bcl-2 was observed as early as 2 hours after seizures were ceased, reached peak level at 24 hours, and lasted to 72 hours. Conversely, Bcl-xl was not up-regulated obviously, difference can only be observed between MC group and normal control group at 48h after seizure. From these data we conclude that Minocycline can reduce cleavage of caspase-3 and inhibit activation of mitochondria-dependent apoptotic signaling pathway after acute injury induced by seizure. This effect is contributed to up-regulation of Bcl-2. Anti-apoptosis protein Bcl-2 is possibly therapeutic target in treatment of brain injury associated with seizures.Part III Ani-inflammation Effect of Minocycline in Hippocampus after Limbic Seizures in rats.Seizure induces inflammation in hippocampus and inflammative cytokines or gial-cytosis, on the other hand, exacerbate epileptic attacks. In this part we explored Minocycline's anti-inflammative effct in limbic seizure model in rats. Using inmmunohistochemistry staining, we investigate the distribution of activated astrocyte and microcyte in bilateral hippocampuses. Using ELISA, we investigated the changes in the expression of proinflammatory cytokines EL-1β and TNF-α . We found that limbic seizures rapidly activate astrocyte. Two hours after seizures were ceased, fat dark stained amoebic GFAP positive cells were observed bilateral both in MT group and in KA group. There was no difference between Minocycline treated and non-treated groups. Microglial activation was first observed at 8h after seizure. Minocycline can partialy prevent epileptic induced activation of microglia. IL-1β and TNF- α were significantly enhanced by seizure. IL-1 β concentration increased by 3.43 fold (P<0.003) in KA group at 24h, but only increased 60% in MT group (no significant). TNF- α were elevated earlier and even notable than EL-1 β . TNF-α concentration increased by 15.2 fold at 4h, and by 57.93 fold at 24h in KA group (P<0.003). Minocycline can significantly inhibit the elevatory tendency. Our result suggests that Minocycline can inhibit seizure-induced inflammation by depressing microglial activation. It would be a hopefult therapy againstseizure induced inflammation.