Effects Of Long-lasting High-expression Of BDNF In Ventrolateral Periaqueductal Gray On Hippocampal Neurons In Epileptic Rats

1.Objective: A rat model of status epilepticus was built by intraperitoneal injection of lithium chloride-pilocarpine(Li-Pilo).The adeno-associated virus vector introduces brain-derived neurotrophic factor(BDNF)into vl PAG,and preliminarily investigates the sustained high expression of BDNF in vl PAG On the effect of hippocampal neurons in epileptic rats,further study the effect of vl PAG on epilepsy.2.Method:(1)75 male SD rats were randomly divided into virus group,empty virus group and saline group.Using brain stereotactic technology,the vl PAG area was injected with BDNF and green fluorescent protein(Green fluorescent protei,GFP)gene recombinant Adeno-associated virus(AAV),negative adeno-associated virus(NAAV)and normal saline(Normal saline,NS),after one week,status epilepticus(SE)were caused by intraperitoneal injection of lithium chloride-pilocarpine each.Every group was randomly divided into SE24 h,7d,14 d,21d and30 d.HE and Nissl staining were used to observe the histological morphology of vl PAG and hippocampal neurons,and Tunel reagent was used to detect neuronal apoptosis.(2)Another 45 SPF male SD rats were randomly divided into virus group,empty virus group and normal saline group.Using brain stereotactic technology,AAV,negative adeno-associated virus NAAV and normal saline NS were injected into the vl PAG area respectively,and one week later,SE was induced by intraperitoneally injecting pilocarpine.And Western Blot(WB)was used to detect the expression of BDNF at 7d,14 d,and 21 d after the introduction of each group.(3)45 healthy SPF male SD rats were divided into virus group,empty virus group,and saline group randomly.Repeat the above steps.After reaching SE,using the video surveillance(8 h/day)to observe the frequency,duration of seizure and the severity for2 weeks.(4)24 SPF healthy male SD rats were randomly divided into virus group,empty virus group,and normal saline group.Repeat the above steps and start the water maze experiment 7 days after reaching SE3.Result:(1)HE staining: Normal rat neurons are tightly arranged,the cell morphology is complete,the cytoplasm is transparent,the nucleus is round or oval,the nucleolus is clear,and the chromatin is evenly distributed.Compared with the empty virus and normal saline group,most of the neurons with clear edges were seen in the virus group,and the number of neurons with complete morphology increased significantly.In SRSs,the damage tends to decrease,but the neuron arrangement is loose and the structure is disordered(2)Nissl staining: Nissl staining can reflect the function of nerve cells to synthesize proteins.Compare with the empty virus group and the saline group,there are more Nissl bodies in the hippocampus of the virus group.(3)Tunel: TUNEL staining can reflect neuronal apoptosis.Compared with the empty virus group and the normal saline group,the tunel-positive cell has decreased.(4)WB analysis: Compared with the other two groups,the protein expression of the virus group increased significantly on 7d,14 d,and 21 d.As time increased,the expression level increased sequentially(5)Behavior: Compared with the empty virus group and the normal saline group,the average seizure frequency,duration and severity of the virus group carrying the BDNF gene were significantly reduced(P<0.05)Compared with the empty virus group and the normal saline group,the ability of learning and memory of the rats carrying the BDNF gene virus group have improved.4.Conclusion:This study shows that the continuous expression of brain-derived neurotrophic factor in vl PAG has a protective effect on hippocampal neurons of epileptic rats and can inhibit the occurrence and development of epilepsy.