Up-regulations Of CRH And CRHR1 In The Epileptogenic Tissues Of Patients With Intractable Infantile Spasms

Infantile spasms(IS)are an age-specific epileptic syndrome with specific epileptic spasms and hypsarrhythmia on electroencephalogram(EEG),and a poor mental developmental prognosis and lacking treatment options.Excessive endogenous corticotropin-releasing hormone(CRH)in infant brain might result in IS.But,the data from human IS of the theory is limited.ACTH and cortisol were observed low regulated,further study suggested ACTH displayed antiepileptic effect by inhibited CRH expression in the amygdala.Many animal experiments show that CRH can induce epileptic spasm,increase the excitability of the immature brain.Therefore,excessive expression of CRH in brain were thought results in infantile spasm,but the data about brain tissue of patients with IS still was lack.CRH mainly has two receptor substyes in the brain,CRHR1 and CRHR2,CRH is more affinity to CRHR1 than CRHR2,CRHR1 is the important receptors for CRH effect.CRHR1 and CRHR2 belong to G protein coupled receptor family,after combined with CRH,triggered cAMP and IP3 and then activated intracellular protein kinase A and PKC signaling pathways.PKC is close to epileptic seizures,and electrophysiological studies in rats hippocampal neurons have found that the activation of PKC has an important relationship with epilepsy susceptibility,involved in the occurrence and maintaining mechanism of epilepsy.The role of CRH,CRHR1 and downstream signal factors in IS still was unclear,in this study we detected the CRH,CRHR1 expression in the epileptogenic tissues of patients with IS and control,and further analyzed the change of the important downstream signal factors PKC and PKA,to understand their role in infantile spasms.The surgical specimens of 17 refractory infant spasm and 6 autopsy control cortex were included in the study.Real-time PCR was used to detect the expression of CRH,CRHR1 and CRHR2 mRNA in epileptogenic tissues and autopsy samples,and further Western blot and immunohistochemistry were used to examined the protein expression and cellular localization of CRH,CRHR1.Because CRHR1 involved in the PKA and PKC signaling pathways,so we also studied the CRHR1 downstream signal moleculars PKA and PKC expression in infantile spasm and control group.The results was following:The expression of CRH,CRHR1 and CRHR2 in epileptogenic tissues of IS and the control group1.Real-time PCR and Western Blot results showed in mRNA and protein level,CRH,CRHR1 expression were obviously increased compared to the control,and CRHR2 were no significantly difference between the epileptogenic tissues of IS and control group.We further analyze CRHR1 downstream moleculars PKA and PKC,the results indicated the expression of PKC was obviously higher in the epileptogenic tissues of IS than control group,and PKA was not obvious difference between the epileptogenic tissues of IS and control group.2.The immunohistochemical analysis results showed that CRH and CRHR1 immunoreactivity(IR)were increased significantly in the epileptogenic tissues of IS compared to the control group,CRH is mainly distributed in neurons,CRHR1 distributed mainly in neurons,also expressed in glial cells.Immunofluorescence double showed the CRH IR were co-labeled with NF200 in neurons in the epileptogenic tissues of IS,not with GFAP in astrocytes and HLA in microglia.CRHR1 positive neurons,astrocytes and microglia were respectively co-expressed with NF200,GFAP and HLA.PKC IR was increased significantly in the epileptogenic tissues of IS compared with the control.All above results suggested CRH and CRHR1 and downstream signaling molecules PKC might involve in the epileptic spasm of IS.But the specific targets still need further study.