Preliminary Research On The Expression And Function Of Classical MHC Class Ⅰ Molecules In Epilepsy

ObjectiveEpilepsy,one of the most common diseases in nervous system,has a complex pathogenesis which is not fully elucidated currently.Several studies demonstrate that the classical MHC classⅠmolecules are not only expressed in the immature and mature central nervous system as immune molecules and play important non-immunological functions(e.g.participate in neural development and regulate synaptic plasticity),but also are abnormally expressed in some neurological diseases.To date,it is unknown that the expression and function of classical MHC classⅠmolecules in epilepsy.This study was designed to preliminarily explore the expression,function and possible mechanism of classical MHC classⅠmolecules in epilepsy.MethodsFirstly,using Western blot,immunohistochemistry and immunofluorescence to detect the expression and clinical significance of classical MHC classⅠmolecules in human epileptic brain tissues.Secondly,adult C57BL6/J wildtype mice and MHC classⅠmolecule knockout mice were selected to establish a kainic acid(KA)induced mouse epilepsy model.The expression of MHC classⅠmolecules in the acute phase of wildtype mouse epilepsy model was tested using Western blot.Behavioral differences between wildtype and knockout mice during KA induced status epilepticus were compared.Hippocampal tissues were collected from mice at different time points during the acute phase of epilepsy,the differences in neuronal degeneration and microglial changes during the acute phase of epilepsy between wildtype and knockout mice were compared using Fluoro-Jade C staining,immunohistochemistry and RT-q PCR.Finally,the expression of synapse-related proteins in the hippocampus of wildtype and knockout mice under physiological conditions and during the acute phase of epilepsy was detected respectively using Western blot to preliminarily explore the function mechanism of MHC class I molecules in the KA induced epilepsy model.Results1.Paraffin specimens and clinical data of surgically resected brain tissues were collected from 76 patients with refractory epilepsy of different pathological types,14 patients with surgically resected fresh brain tissues and 6 autopsy control fresh brain tissues,and immunohistochemical and Western blot results showed that the expression of classical MHC classⅠmolecules was up-regulated in both neurons and microglia in various FCD and non-specific lesions(NSL).There was a weak linear correlation in the expression of MHC class I molecules between neurons and microglia.The statistical analysis showed that the expression of MHC classⅠmolecules on neurons and microglia had no linear correlation with the duration of epilepsy and the surgery age.Immunofluorescence double labeling results showed that in addition to the expression on neurons and microglia,MHC classⅠmolecules were also expressed in dysplastic neurons.2.Adult C57BL6/J wild mice and MHC class I knockout mice were used to establish a kainic acid(KA)induced mouse epilepsy model.Hippocampal tissues were collected from wild mice in the acute phase of epilepsy,and Western blot results showed that classical MHC class I expression was increased in the acute phase of the wild mouse epilepsy model.3.The behavior of mice was analyzed using Racines grading,and the behavioral results showed that compared with wildtype mice,knockout mice showed severer seizures during status epilepticus.The latency of seizure was shortened by 21.6%.The epileptogenic dose was reduced by 11.4%.4.Using FJC staining,immunohistochemistry,and RT-q PCR to detect neuronal degeneration and changes in microglia during the acute phase of KA-induced epilepsy in wild and knockout mice,the results showed that in the acute phase of KA induced epilepsy,the number of degenerated neurons and microglia,and the m RNA expression of inflammatory factors in the hippocampus were increased in both wildtype and knockout mice,and the increase was greater for knockout mice than wildtype mice at each time point.5.Western blot results showed that compared with wildtype mice,the expression of synapse-related protein Synapsin-1 and VGlu T1 in the hippocampus of knockout mice under physiological conditions was up-regulated,while the expression of VGAT was down-regulated.The VGlu T1/VGAT ratio was increased and the expression of PSD95 and Gephyrin had no changes.6.Western blot results showed that in the acute phase of KA induced epilepsy,both wildtype and knockout mice had changes in synapse-related proteins.The expression of Synapsin-1,VGlu T1 and PSD95 was greater for knockout mice compared with wildtype mice,while the expression of VGAT was lower for knockout mice compared with wildtype mice.Conclusions1.The expression of Classical MHC class I molecules was up-regulated on neurons and microglia in epileptic foci.2.MHC classⅠmolecules were involved in the onset and progression of KA induced epilepsy in mice.It had a protective effect on KA induced neuronal injury and regulated the proliferation and activation of microglia.3.MHC class I molecules may influence seizure susceptibility and the progression of epilepsy by regulating the expression of synapse-related protein.The study provides the function and the possible pathogenic mechanism of MHC classⅠmolecules in epilepsy,which is in favor of understanding the roles of MHC classⅠmolecules in neurological diseases.