The Role Of TSP2 In Glioma Related Epileptogenesis

Objective:Patients with glioma often have seizures,and a considerable number of patients with glioma associated with epilepsy show resistance to conventional antiepileptic drugs,which seriously affects the quality of life of patients.The reasons of seizures in patients with brain tumors are multifactorial.Existing studies believe that the tumor itself and the peritumoral microenvironment may jointly determine the epileptic phenotype of patients with brain tumors.The possible pathophysiological changes include mutations in tumor cell genes,neural network and neuromodulation abnormalities,glial cell dysfunction,blood supply and vascular permeability changes,accompanied by peritumoral tissue edema,and a large number of inflammatory factors and metabolically active molecules released around the tumor,these factors may jointly promote seizures in patients with brain tumors.Thrombospondins（TSP）is a kind of oligomeric extracellular matrix polysaccharide protein that mediates cells and cells,cells and extracellular matrix in various pathophysiological processes such as angiogenesis,inflammation,osteogenesis,cell proliferation and apoptosis.Recent studies have found that TSP may induce the expression of excitatory synapses in a variety of neurological disease models.The studies have found that epidemic growth factor（EGF）of TSP,as an auxiliary subunit of voltage-gated calcium channels（VGCC）,can be combined with the domain of von Willebrandbrand Factor（v WF-A）ofα2δ1 to jointly promote excitatory synaptogenesis.The current research on the mechanism of epilepsy mainly includes the theory of imbalance of neurotransmitter and its receptor,the theory of imbalance of excitability and inhibitory synapse,the theory of dysfunction of ion channels,etc.Given the existing research on TSP,we speculate that the binding of TSP derived from glioma to its receptorα2δ1 may promote the formation and remodeling of excitatory synapses around the tumor,causing the imbalance of“excitement-inhibition”in the peritumoral cortex,which in turn leads to the occurrence of secondary epilepsy in glioma patients.Methods:1.C6 glioma cells were cultured in vitro,and transplanted into the rat cortex with stereotactic microinjection.The NSS behavioral scores,HE staining,immunohistochemical staining,and cortical EEG recordings in vivo were used to evaluate whether the model rat was successfully constructed and available for the study of the mechanism of tumor-related epilepsy.2.Western Blot and immunohistochemical staining were used for the analysis of the expression level of TSP2 in model rats.Overexpression or knockout of the TSP2 gene in C6glioma cells,and then immunofluorescent double-labeling staining,Golgi staining,transmission electron microscopy,cortical EEG recordings in vivo were performed to analyze the effects of expression level of TSP2 in C6 glioma cells on the changes of synaptic plasticity and peritumoral cortical excitability.3.Western Blot and immunohistochemical staining were used to analyze the differential expression of TSP2 receptor and its downstream signaling molecules（α2δ1,Rac1）in the peritumoral cortex of model rats;Cortical EEG recordings in vivo were further performed to analyze the effect of Gabapentin,the antagonist of TSP2/α2δ1 pathway,on the excitability of the peritumoral cortex.Results:Ⅰ.Construction and evaluation of cortical C6 glioma model rats.The NSS score showed that the scores in the model group was significantly higher than that in the sham group on the 10th day after the C6 glioma cells injected,and then,the NSS score in the model group tended to be stable.Gross anatomical specimens and HE staining of brain tissue showed that C6 glioma cells formed a tumor mass in rat cortex,and a series of pathological changes occurred around the tumor.Immunohistochemical staining showed that the expression of Neu N,MAP2 and GFAP changed in peritumoral area.The EEG in vivo showed an increase in the frequency of epileptiform discharges in peritumoral cortex.Ⅱ.the role of TSP2 in the peritumoral cortex of C6 glioma model rats.Western Blot and immunohistochemical staining results showed that TSP2 expression in the tumor center of C6 glioma model rats was significantly up-regulated.Immunofluorescent staining showed that synaptophysin expression was upregulated in peritumoral area.Immunofluorescent staining,Golgi staining,and transmission electron microscopy showed that the expression of excitatory synapses,synaptic spine density,and synaptic density increased in peritumoral area of C6^WT rats.Recordings of cortical EEG in vivo showed that more epileptic discharges appeared around the tumor of C6^WT rats.After overexpression or knockout of the TSP2 gene in glioma cells,the expression of synapses and levels of epileptiform discharges upregulated and downregulated,respectively.However,changes in the expression levels of TSP2 in glioma cells had no effect on the expression of peritumoral inhibitory synapses.Ⅲ.Possible downstream molecular mechanisms for TSP2 to mediate pathophysiological changes around the tumor.Western Blot results showed that theα2δ1,downstream signal molecule of TSP2,was upregulated at the periphery of the tumor.Immunofluorescent staining results confirmed this result.Rac1,the downstream signal molecule ofα2δ1,was also up-regulated.Gabapentin,a kind of antagonist of TSP2/α2δ1,was used to intervene in model rats,and the results showed that Gabapentin reduced the degree of epileptiform discharges in the peritumoral cortex.The evidence above suggests that TSP2 may mediate the process of excitatory synaptogenesis in the peritumoral cortex through theα2δ1/Rac1 pathwayConclusions:1.TSP2 is up-regulated in the center of glioma-related epilepsy model rats2.TSP2 derived from glioma may cause upregulation of the number of excitatory synapses and abnormalities of synaptic structure through theα2δ1/Rac1 signaling pathway,which in turn leads to increased excitability of peritumoral cortex.3.Gabapentin,a kind of antagonist of the TSP2/α2δ1 pathway,can partially reduce the excitability of the peritumoral cortex,and may be a new target for the prevention of glioma-related epilepsy.