The Response Of Astrocytes And Relationship With Neurons In The Hippocampus Of Rats With Epilepsy Kindled By Pentylenetetrazole

Epilepsy, one of chronic neurological disorders, affects about 1% of the world population. It is a common condition in the brain characterized by repetitively occurring seizures caused by abnormal discharging of neurons. Repetitive convulsions seriously interfere the life of epileptics and their families. There are one third of epileptic activity that could not be controlled in clinic. Glial cells take up 90% number of the total cells in the central nervous system. Not only do glial cells play a structural, trophic, and protective role to neurons, but also they have an excitative quality. Glial cells can actively talk to neurons. They may integrate information and regulate neuronal activity. So they are a part of the communication network within the central nervous system. One of common pathological changes in epileptic focus is proliferation of glias. Therefore, the role of glia involved in epileptogenesis could not be missed.Objective: In this study, the responses of neurons and astrocytes in hippocampi of the Pentylenetetrazole (PTZ)-kindled epileptic rats were investigated. The effects of carbenoxolone (CBX), a gap junction blocker, on the expression of Cx43, Cx32 in the reactived astrocytes and neurons, and onelectroencephalogram(EEG) of PTZ-kindled epileptic rats. We are going to explore the roles of glias in epileptogenesis.Methods:The experimental epilepsy model of the kindled rat was established by intraperitoneal injection of a dose of PTZ under threshold every day.Immunohistochemistry method was used to observe the response of astrocytes and neurons, including expression of GFAP-Li, Cx43-Li, Fos-Li, Cx43-Li in hippocampi of the PTZ-kindled epileptic rats.The immunohistochemical staining of neurons and astrocytes, EEG, as well as behaviors was studied after injecting CBX into the lateral ventricle of the PTZ-kindled epileptic rats. Western blot was performed to detect the content of Cx43 and Cx32.Ultrastructure between activated astrocytes and neurons in hippocampi was observed by immune-electron-microscopic staining method.Video- EEG was used to analyze changes of brain wave induced by CBX in the PTZ-kindled epileptic rats.The primary cultured neurons and astrocytes were treated with PTZ stimulation. Immunofluorescence was used to study the expression of Cx43 and Cx32 in neurons and astrocytes. By means of atomic force microscope we observed cellular membrane and membrane protein in astrocytes and neurons.Results:(1) Spontaneous seizures were appeared in PTZ-kindled epileptic rats. The anti-Fos, anti-GFAP, anti-Cx43 and anti-Cx32 immunohistochemmical staining had increased significantly in hippocampi of PTZ-kindled epileptic rats. The activated astrocytes showed GFAP-Li and Cx43-Li staining, and activated neurons showed Fos-Li and Cx32-Li staining. The increase of GFAP-Li astrocytes increased in the early phase of kindling process, and intensified in following kindling process. There was no difference in GFAP-Li staining between the V seizure rats and the spontaneous seizures rats. An intimate relationship between activated astrocytes and neurons was presented by the anti-Fos/GFAP-Li and anti-Cx43/Cx32-Li double staining.. The astrocytescould actively participate in epileptogenesis.(2) The numbers of NMDAR₂-Li, Fos-Li and Cx32-Li neurons, the expression of Cx43-Li, as well as the spontaneous seizures decreased in PTZ-kindled rats treated with CBX. However, GFAP-Li astrocytes did not change.(3) EEG recordings of the PTZ-kindled epileptic rats displayed various seizure waveforms. Such as random spikes, polyspikes, slow waves, rhythmic spikes, the spike and wave complexes. The rhythmic spikes or the spike and wave complexes are obvious properties of the PTZ-kindled epileptic rats. After treated with CBX, the rhythmic spike and wave complexes decreased, meanwhile, the amplitude of background rhythm enlarged and slow waves increased.(4) By immune electron microscope staining method, we found the existence of gap junction in astrocytes, or in neurons, and Cx43-Li hemichannel. Synaptic complex was composed of a synapse and an astrocyte process inserting synaptic cleft. The number of excitatory synapses increased in PTZ- kindled rats.(5) Cx43-Li granules increased in the cultured astrocytes and Cx32-Li increased apparently in the cultured neurons after treated with PTZ stimulation. The whole shape of astrocytes or neurons by PTZ stimulating was not different from control cells under atomic fordce microscope. By amplifying a small part of surface cell, we found the structure of agglomerate granules assembled denser than that of the control cells.Conclusions:(1) The plastic changes of astrocytes and neurons appeared in hippocampi of PTZ-kindled epileptic rats. The activated astrocytes have intimate relationship with the activated neurons, and actively participated in epileptogenesis induced by PTZ kinkding.(2) After CBX treatment, spontaneous seizures, activity of neurons, and the expression of Cx32-Li and Cx43-Li decreased in PTZ-kindled epileptic rats. It is suggested that gap junction and hemichannels closely involve in...