Protective Effect of Minocycline on Hippocampus in Seizure Rats Induced by PTZ
|School||Hebei Medical University|
|Keywords||Minocycline epilepsy PTZ NMDAR1 hippocampus apoptosis|
Epilepsy is a kind of chronic disease of brain which feature is transient malfunction of central nervous system elicited by anomalous discharge of neuron. Epilepsy damage neurons part of which is apoptosis. One the other hand glutamate is the primary endogenous excitatory amino acid in brain. Released glutamate produces excitotoxicity and induces injury as one of pathologic processes in cerebral ischasemia, and NMDA receptor is involved in the excitotoxicity in eclamptic injuries of neurons by inducing proliferation and activation of microglial cells, and production of iNOS and ICE. Minocyeline is a second–generation semisynthetic tetracycline antibiotics,and possesses anti-inflammatory effects that are completely distinet from its antimicrobial from its antimicrobial action . Recent studies have shown that minocycline has remarkable neuroprotective effects in animal models of cerebral ischaemia, traumatic brain injury, Huntington’s and Parkinson’s diease. Minocycline can be rapidly and completely absorbed, and has an excellent tissue penetration into the brain and cerebrospinal fluid by passing through the blood-brain barrier. In previous studies, several neuroprotetction mechanisms of minocycline have been reported, such as inhibition of microglial activation. Mechanism of neuroprotective effect of epilepsy model, the relation of drug dose and neuroprotective effect need be elucidate. Microglial activation and excitotoxic neuronal death and be inhibited by minocycline, which may be one mechanism of the neuroprotection of minocycline. However, whether minocycline directly or indirectly inhibits NMDA receptor-mediated excitexicity in the eclamptic injury is still unclear. We obsvered the effects of the general epilepsy model,: whether minocycline directly inhibits NMDA receptor or not, whether minocycline inhibits NMDA receptor-mediated excitexicity or not, whether minocycline reduced apoptotic cell or not.Objective: We make the model of seizure rats with pentylenetetrazol, treat the rats with different dose of minocycline, detect NMDAR1 and apoptotic index in hippocampus. We study and research the effect of different dose minocycline to neuron. Meanwhile we approach whether minocycline directly or indirectly inhibits NMDA receptor mediated excitexicity in the eclamptic injury.Methods:1Animals and groups8 SD rats were selected randomly from 60 experimental rats as a normal control group. The rest 52 SD rats were injected abdominally to induce chronic-kindling models by PTZ. 32 SD rats were selected randomly from the 52 SD rats, then the 32 SD rats having build-up model rats were divided randomly completely into four groups:Normal、PTZ、MT1、MT2、MT3.2 Epileptic modelThe normal group were injected abdominally by isotonic Na chloride. The rest 52 SD rats were injected abdominally by PTZ every other day. We couldn’t consider the pattern fully successful until the rats emerged major epilepsy spontaneously.3 AdministerThe rats interfered were lavaged by minocycline in half hour after major epilepsy. The first time the rats in MT1 were given 40 mg/kg, afterward they were given 20mg/kg every 12 hour. The first time the rats in MT2 were given 90 mg/kg, afterward they were given45mg/kg every 12 hour. The first time rats in MT3 were given 180 mg/kg, afterward they were given 90mg/kg every 12 hour. PTZ and normal were given distilled water. In all 5 days.4 EEGRats anaesthetized were fixed the bench. We use unipolar lead with scalp acupuncture. We inserted acupuncture needle in the epicranium of rat’s temple . When the rat waked, we made the model with the methods above-mentioned.5 ImmunocytochemistryAfter rats were anesthetized, we perfused and fixed through the ascending aorta of heart with 4% citromint. After that, the telencephalon was taken out. Then the slides were incubated with NMDAR1 monoclone antibody, these specimens were incubated with the secondary antibody, avidin-biotin-perxidase complex. Among these approaches, we should wash these specimens with PBS, the results were visualized by using diaminobenzidine as chromogen.6 DeadendTM colorimetric TUNEL systemPerfusing、fixing、embedding and section of the steps are same as above. We dropwise TUNEL after dewaxing、protease digestioing.Results:1 All epileptic rats had epileptic attacks in higher or lower degree, proving that the method to make rats epileptic was successful.2 EEGThe electroencephalogram of the epileptic model groups appears representative sharp wave.The electroencephalogram of the normal groups is normal.3 Immunohistochemistry resultsThe normal control compares with every group whose OD was minimum obviously. There is no obviously difference in groups except the normal control.4 TUNEL resultsTo compare with the PTZ, the positive cell’s number of the MT1 decreased obviously. To compare with the PTZ, the positive cell’s number of the MT2 increased obviously. There is no obviously difference between the PTZ and MT3.Conclusion:1 The results indicated that Minocycline （45mg/kg） can suppress apoptosis, but Minocycline （90mg/kg） can strengthen apoptosis.2 Minocycline probably indirectly inhibit NMDA receptor -mediated excitexicity in the eclamptic injury.