| Objective Epileptic rat hippocampus tissue was observed morphological changes of nerve cells,the expression of apoptotic and cytokine by acupuncture at Zusanli Point,and to explore the mechanism of acupuncture in treatment of epilepsy.Method 60 SD male rats(SPF)with approximately 200-250 g weight were randomly divided into 4 groups,namely the normal group(24)and model group(36),in which the normal group also includes normal blank group and normal Zusanli group.At the same time,the model group includes model blank group and model Zusanli group.Rats in the model group were treated with chloride-pilocarpine,rats of the normal group were injected with the same amount of saline.After the success of the model,the rats were treated with acupuncture on the following day,and the samples were treated continuously after 10 d.The left hippocampus was used for HE staining to observe the morphological changes of neurons in rat hippocampus;the right part of the hippocampus is used for the preparation of single cell suspensions and the detection of cytokines to analyze the neuronal apoptosis and cytokine expression in the hippocampus.Result 1.The general situation and behavior of rat:The weight,diet and glossiness of hair of SD rats were affected by different degrees after epileptic modeling.The main effects were weight loss,diet reduction,dry hair,and seizures of different degrees.Accompanied by a certain degree of peripheral cholinergic reactions,such as salivation,blood and tears,foaming mouth and so on.Through behavioral monitoring and analysis of spontaneous epileptic seizures in rats,it was found that acupuncture at Zusanli can reduce the frequency of seizures to some extent by reducing the grade of seizures.The duration of seizures was shortened to reduce the severity of epileptic seizures,and intermittent seizures were mainly concentrated in grade ? and grade ?,and the frequency of seizures and duration of seizures began to decrease on the 7th day of treatment.There is a reversal from high-grade,high-frequency and high-duration to low-grade,high-frequency and low-duration or high-grade,low-frequency and low-duration.That is to say the whole epileptic seizure of rats after acupuncture at Zusanli point is in a state of down-grade and shortening duration.2.Hippocampal histomorphology and cell countUnder the optical microscope,the morphology of neurons in the hippocampal CA1,CA3 and DG region of the rats in each group showed changes in different degrees before and after modeling and acupuncture.The main changes were as follows:(1)Compared with the normal control group,the contour of hippocampal tissue in the model blank group was blurred and the continuity was interrupted under the low power microscope.Under the high power microscope,the neuronal cells in each region of the hippocampus were disordered,the intercellular space increased,and the number of cells decreased.Pyknosis,cytosolic condensation,nuclear dissolution,fragmentation,and the formation of a certain number of apoptotic bodies were observed.(2)After acupuncture at Zusanli point in different states,there was almost no significant change in the contours,morphology,arrangement,distribution,quantity and staining degree of hippocampal tissue in normal blank group and normal Zusanli group.However,there were obvious differences in the outline and cell distribution,arrangement,and nucleoplasm staining of the hippocampus between the model blank group and the model Zusanli group: under low power microscope,the contour of model Zusanli group was slightly clearer complete than that in model blank group.Under high power microscope,the hippocampal regions were deformed,the number of deeply stained cells,the cell fragments were obviously reduced,and the whole cells showed light staining.At the same time,the expression of positive cells in hippocampal regions was statistically analyzed by IPP software: Compared with the normal blank group,the number of positive cells in the hippocampal CA1,CA3 and DG regions of the model blank group was obviously increased,and there was a statistical difference(P<0.05);Compared with the model blank group,the number of positive cells in the hippocampal CA1,CA3,and DG regions in the model Zusanli group decreased correspondingly,and which has significant difference(P<0.05);Compared to the normal black group,the number of positive cells in hippocampal CA1 and DG decreased significantly after acupuncture treatment of normal ZuSanli(P<0.05).Although the expression of positive cells in CA3 decreased,there was not statistically significant(P>0.05);The expression of positive cells in CA1 and DG regions of the hippocampus was significant difference(P<0.05)when compared to the normal Zusanli group and model Zusanli group,whereas there was no significant expression in the CA3 region(P>0.05).3.Results of apoptosis detectionBy flow cytometry and statistical analysis,compared with the normal blank group,the early apoptosis of neurons in hippocampal tissue of rats increased after acupuncture(P<0.05),while the late apoptosis had no significant changes before and after acupuncture(P>0.05);By comparison,the hippocampal neuronal apoptosis in the model blank group was higher than the normal control group,and has statistical differences(P<0.05);when compared to the model blank group and the model Zusanli group,there was no significant difference in the early and late apoptosis of hippocampal neurons(P>0.05);Compared to the model Zusanli group and normal Zusanli group,the early and late apoptosis of hippocampal neurons was statistically significant(P<0.05).4.Cytokine expression resultsBy liquid chip detection and statistical analysis,4 cytokines related to acupuncture treatment for epilepsy were obtained,which were Leptin,RANTES,and immune cytokines IgG1 and IgG2 a,respectively:(1)The expression of Leptin and RANTES in the hippocampus of rats after acupuncture was not statistically different between the normal Zusanli group and the normal control group(P>0.05);Compared with the normal control group,the expression of RANTES in the hippocampal tissue of the model group increased significantly(P<0.05),Although the expression of Leptin was increased,there was no statistically significant difference(P>0.05);Model Zusanli group compared with the control group,the expression of Leptin and RANTES are decreased after acupuncture,and the difference was statistically significant(P<0.05);Compared to the normal Zusanli group and model Zusanli group,only the expression of RANTES difference was statistically significant(P<0.05).(2)The expression of IgG1 and IgG2 a between the normal Zusanli group and the normal control group was no significant difference(P>0.05);Compared with the normal blank group,after making the model,the contents of IgG1 and IgG2 a in the hippocampus of the model blank group increased,and the difference was statistically significant(P<0.05);The expression of IgG1 and IgG2 a in hippocampus in model Zusanli group was significantly different from that in model blank group(P<0.05);The expression of IgG2 a was significantly different between the normal Zusanli group and the model Zusanli group(P<0.05),while the expression of IgG1 was not statistically significant(P>0.05).Conclusion 1.Acupuncture at Zusanli can improve seizure degree in epileptic rats to a certain extent and reduce the damage of hippocampal neurons to treat epileptic diseases.The mechanism may be related to the decrease of Leptin,RANTES and the expression of immune cytokines IgG1,IgG2 a in hippocampus.2.In different functional states of rats,Zusanli point has specific selectivity for hippocampal histology,apoptosis and cytokine expression: Acupuncture at Zusanli has specific bidirectional regulation on the expression of cells in hippocampal CA1 and DG areas,and it has a specific regulating effect on early apoptosis of rats in normal state,and has a specific regulatory effect on the expression of RANTES and IgG2 a in rat hippocampal tissue under the epileptic state. |
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