Ndrg2 Expression Pattern In Hippocampus Of Epilepsy Rat Model In Various Phases

Background Epilepsy is a kind of serious nervous system desease. Its mechanism remains unclear. The main pathological changes are neuron loss, proliferation of glia and mossy fiber spouting. Many hypotheses of mechanism in epilepsy were proposed on the ground of such appearances. A hypothesis of excitatory circuit formed by mossy fiber spouting has closest relationship with this experiment. The hypothesis presumes that when seizures cause death of neurons in hilar region, mossy fiber spouting creats novel recurrent and feedforward excitatory circuit resulting in high excitability of granule cells. The abnormal mossy fiber spouting and alternation of synapse structure/function are not only the principal pathology foundations, but also the reasons of recurrent seizures.N-myc downstream-regulated gene 2 (Ndrg2), a recently discovered and multi-orientation researched gene, is involved in many physiological and pathological mechanisms including tumour genesis, tissue/cellular differentiation and stress responses. It also plays an important role in central nervous system deseases. It was reported that NGF-differentiated pheochromocytoma (PC12) cells transfected with Ndrg2 menifested neurite spouting and outgrowth in vitro, which demonstrated that Ndrg2 can promote neurite outgrowth. This phenomenon is coincident with mossy fiber spouting in epilepsy, which implies that Ndrg2 expression may upregulate and facilitate mossy fiber spouting. In addition, seizure which is a definite stressor also relates to hypoxia. Thus we proposed a hypothesis that Ndrg2 mRNA and protein expression may increase in epilepsy rat model.The aim of this study is to test this hypothesis, explore the mechanism of epileptogenesis and extend the understanding of Ndrg2 functions. Meanwhile we also research the pathologic alterations of neuron and astrocyte in epilepsy rat hippocampus during various phases after onset of status epilepticus (SE), as well as their schedule and mechanism. In order to fulfill this plan, we have to establish a stable and reliable epilepsy animal model. So we explore the method and pattern of lithium-pilocarpine induced epilepsy rat model. Methods1. Fifty-six healthy Sprague-Dawley rats were used in this study. Pretreated by lithium chloride at a dose of 3 mEq/kg i.p, the subjects were injected with pilocarpine 30mg/kg as first dose after 18~20h. Then they were administered with low dose pilocarpine (10mg/kg) every 30min until SE developed, but maximal dose was 60mg/kg. SE was terminated by diazepam after 1h. Survival rats received post-seizure care.2. The subjects were sacrificed at definite time points (4h, 24h, 7d and 6w) to sample. Rat brain tissues were dissected. Semi-quantitative RT-PCR analysis and immunofluorescence assay were used to measure Ndrg2 mRNA and protein level respectively.3. The rats were sacrificed at definite time points. Brain tissues were fixed and prepaired by common protocols. Stained by Nissl, immunohistochemistry and HE staining, hippocampus sections were observed to detect morphological features.Results1. Kindling rate of pilocarpine was 91.7% and mortality rate was 22.7%. 70.8% of experiment group were induced successfully in all. Kindling rate of male was close to that of female, but male'mortality was lower than female's (P<0.05).2. The value of Ndrg2 mRNA underwent transient peak in superactue phase. Ndrg2 protein increased significantly in latent and chronic phases.3. Neurons in hippocampus CA3 region showed acidophilic degeneration and cytoplasm was stained deeply in superacute phase (4h-post-SE). In acute phase (24h), acidophilic degeneration was most remarkable. Many neurons pyknosised with nucleoli disappeared and dendrites ruptured while astrocytes hydropsed. In latent phase (7d), massive neurons in CA3, CA1 and hilus regions necrosed or lost. Astrocytes proliferated and hypertrophied. Hippocampus formation deranged. In chronic phase (6 w) there appeared glia scar in CA3 and CA1 regions with some survived normal neurons, and granule cell layer broadened.Conclusion1. A modified kindling protocol is a simple and effective access to induce epilepsy rat model. Its advantages lie in convenience and low mortality. Male rat is more suitable to induce epilepsy model than female.2. Ndrg2 undergoes an early increase at mRNA level and delayed up-regulation at protein level in hippocampus of epilepsy rat model. The early transient up-regulation of Ndrg2 mRNA may be the consequence of stress responses mediated by glucocorticoid. Hippocampus astrogliosis induced by SE may account for delayed and long-termed up-regulation of Ndrg2 protein. Ndrg2 protein focuses on perinuclear region of astrocyte. It may play a role in stress responses and epileptogenesis.3. The pathological damages of neuron are prior to those of astrocyte in epilepsy. Both of them involve in epileptogenesis.