| 1. Objective:To investigate the cerebral protective and epilepsy inhibiting effects of atorvastatin on traumatic epilepsy rat through establishing traumatic epilepsy rat model and observing its behavior, electroencephalogram(EEG), hippocampus histopathology,apoptosis, the expression of caspase-3, GABA-A receptor and NMDA receptor, and changes in the content of excitatory amino acid transmitters aspartic acid(Asp) and glutamic acid(Glu) as well as inhibitory amino acid transmitters glycine(Gly) andγ-aminobutyric acid(GABA) in hippocampus tissues under atorvastatin treatment, so as to provide new ideas for early prevention and treatment of traumatic epilepsy in clinical practice.2. MethodsA total of 60 male healthy adult SD rats were randomly divided into 6 groups, with10 rats in each group: group A: normal group; group B: sham operation group; group C:model group; group D: 10mg/kg/d low-dose atorvastatin treatment group; group E:20mg/kg/d middle-dose atorvastatin treatment group; group F: 40mg/kg/d high-dose atorvastatin treatment group. One hour before model establishment, groups D, E and F were given corresponding doses of atorvastatin by gavage. One hour later, anesthesia was performed, cortical electrode was placed, baseline EEG was recorded, and Fe Cl3 was injected subcortically to establish traumatic epilepsy rat model. After operation,corresponding doses of atorvastatin were administered every day for 3d. The behavioral changes of rats were observed. The latent period of each group before the first time of epileptic discharge was recorded by computer, and the average amplitude of epileptic brain wave and the times of epileptic discharge were recorded 1h, 6h, 24 h and 72 h after operation. HE staining was used to observe the hippocampus histopathological changes of rats 72 h after establishment of model; TUNEL was used to detect the apoptosis ofhippocampus neurons; real time Western blot and immunohistochemistry were used to detect the expression of caspase-3, GABA-A receptor and NMDA receptor; high performance liquid chromatography was used to detect the expression of Asp, Glu, Gly and GABA in hippocampus tissues of each group.3. Results3.1 Behavioral manifestationThe general condition of rats in model group was significantly worse than that in normal group and sham operation group, groups D, E and F were significantly improved compared with model group, and the general condition of rats in low-dose group and middle-dose group were better than that in high-dose group. No epileptic seizures occurred in normal group and sham operation group, and Racine classification showed that the stage of epileptic seizures in groups D, E and F was significantly lower than that in group C(P<0.05).3.2 EEG manifestationBaseline EEG was recorded for all the rats, and no epileptic discharge was found.The baseline brain waves of rats in normal group and sham operation group mainly manifested as α wave and β wave, and epileptic discharge was induced in model group and groups D, E and F, which mainly manifested as sharp wave crest with high amplitude and transient episodic sharp wave, spike wave, sharp and slow wave complex,spike and slow wave complex, etc. Compared with the normal group and sham operation group, the average amplitude of EEG in model group was significantly increased(P < 0.05), while that of groups D, E and F was significantly reduced compared with the model group(P<0.05), and the latent period before the first time of epileptic discharge was significantly prolonged(P<0.05).3.3 HE staining In normal group and sham operation group, the number and structure of hippocampus cells were normal. In model group, the granular cells and pyramidal cells in hippocampus were reduced, with disordered tissue structure, irregular arrangement,swelling and deformation of cells, and karyopyknosis. Compared with model group, the hippocampus neurons in groups D, E and F were regularly arranged, with clear contour,some cells were deformed and the nucleoli were clear.3.4 TUNEL stainingIn normal group and sham operation group, TUNEL staining positive cells were dispersed in hippocampus tissues. The number of TUNEL staining positive cells in model group was significantly increased and the apoptosis index was increased(p <0.05), while the number of TUNEL staining positive cells in groups D, E and F was significantly reduced, and the apoptosis index was reduced(p<0.05).3.5 Expression of caspase-3In normal group and sham operation group, the number of caspase-3 positive cells was small, with low expression; while the expression of caspase-3 in hippocampus tissues of model group was significantly increased, and the rate of positive cells was increased(p<0.05). The expression of caspase-3 in hippocampus tissues of groups D, E and F was significantly reduced compared with the model group, and the rate of positive cells was reduced(p < 0.05). Western Blot analysis showed that the expression of caspase-3 in hippocampus tissues of model group was significantly increased(p<0.05),while that of groups D, E and F was significantly reduced compared with the model group(p<0.05).3.6 Expression of GABA-A and NMDA neurotransmitter receptorsIn normal group and sham operation group, the number of GABA-A receptor and NMDA receptor positive cells was small, with low expression, while the number of GABA-A receptor and NMDA receptor positive cells in model group was significantly increased(p<0.05); in groups D, E and F, the number of GABA-A receptor positive cells was significantly increased(p < 0.05), while the number of NMDA receptor positive cells was significantly reduced(p<0.05). Western Blot analysis showed that the expression of GABA-A receptor and NMDA receptor in hippocampus tissues of model group was significantly increased compared with normal group and shamoperation group(p<0.05); the expression of GABA-A receptor in groups D, E and F was significantly increased compared with the model group(p<0.05); the expression of NMDA receptor in groups D, E, and F was significantly reduced compared with the model group(p<0.05).3.7 Expression of amino acid transmitters Asp, Glu, Gly and GABAHigh performance liquid chromatography-fluorescence analysis showed that the levels of Asp, Glu, Gly and GABA in hippocampus tissues of normal group were low,while the levels of Asp, Glu and GABA in hippocampus tissues of model group were significantly increased(P<0.05), with no obvious increased in Gly; the levels of Asp and Glu in groups D, E and F were lower compared with model group(P<0.05), while the level of GABA was higher compared with the model group(P<0.05).Conclusion:1. Atorvastatin can inhibit the stage of epileptic seizure, prolong the latent period before epileptic seizure, inhibit the amplitude of epileptic discharge and inhibit the times of epileptic discharge.2. Atorvastatin can inhibit the expression of caspase-3, inhibit neural inflammatory response, inhibit neuron pathological injury and inhibit neuron apoptosis.3. Atorvastatin can promote the expression of GABA-A receptor, inhibit the expression of NMDA receptor, and correct the balance between excitatory and inhibitory amino acid transmitters. |
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