| Background Epilepsy is a common neurological disease. A substantial number of epileptic patients (20%～30%) continue to have seizures in spite of adequate treatment with antiepileptic drugs. The mechanisms explaining why some patients are responsive and others prove resistant to antiepileptic drugs are poorly understood. It has been reported that overexpression of P-glycoprotein and other efflux transporters, such as multidrug resistance-associated proteins, lung-resistance protein, in the cerebrovascular endothelium, perivascular astrocytes, and parenchymal cells in the region of the epileptic focus, may lead to drug resistance in epilepsy. Despite long-term study, the progress of understanding its molecular mechanism has been limited. It has been reported that mitochondrial toxin, 3-nitropropionic acid could evoke the seizures. Recently some researchers find the changes of mitochondrial type Mn-superoxide dismutase and mitochondrial rieske protein in epilepsy. Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of epilepsy. In contrast to the short-term alterations of mitochondria in epilepsy, it remains unclear whether and exactly how cerebral mitochondria change during long-term epileptic conditions. And studies of mitochondria in epilepsy have been limited mostly to chemical models in which it is difficult to differentiate the consequences of seizures from those of convulsants. So we selected pharmacoresisitant epileptic models from electrical amygdala kindled rats by administering phenytoin. To better understand the molecular details of mitochondrial dysfunction in pharmacoresistance, it would be highly valuable to know the changes of mitochondrial proteins. We searched for the new target for the intractable epilepsy through the technique of subcellular proteomics.Objective We created the temporal epileptic animal model through the chronic electrical stimulation in amygdala and got the refractory group through the administration of phenytoin; Explore the mechanism of refractory epilepsy through the technique of mitochondrial proteomics; Observe the expression of Voltage-dependent anion channels (VDAC) and discuss the role of it in the process of refractory epilepsy; To investigate the energy and ultrastructure changes. Observe the emotional, behavioral, and cognitive disorders in refractory epilepsy, and discuss the mechanism on the formation of refractory epilepsy.Methods Create the temporal epileptic animal model through the electrical stimulation in amygdala (intensity:400μA, wave space: 1ms, frequency:60Hz, duration:1s) and observe the successful rate. EEG was recorded and the pathologic changes were observed; By using two-dimensional gel electrophoresis coupled with mass Spectrometry, we undertook a global analysis of proteins expression in pharmacoresistant epileptic model selected by phenytoin through the chronic electrical amygdala-kindled rats. Some identified proteins were further investigated using Western blot and immunohistochemistry. Energy contents were measured by high performance liquid chromatography. The correlation analyses were performed between VDACs and the energy charge. Electron microscope was used to observe the ultrastructure; Observe the emotional, behavioral, and cognitive disorders in refractory epilepsy through open filed test, catch-against test and Morris maze test.Results The sucessful kindling rate was 71.59%. 26.38% was phenytoin-resistant. High-wave-spikes and spontaneous seizures were observed. Neurons were damaged and dispersed; Nineteen differential proteins (P<0.05) were found: Aconitase, Glutamate dehydrogenase 1, Malate dehydrogenase, NADH dehydrogenase 1 alpha subcomplex 10, ATP synthase alpha subunit precursor, Glyceraldehyde-3-phosphate dehydrogenase, Triosephosphate isomerase-1, Similar to pyruvate kinase isoenzyme, ATP binding cassette, subfamily B, member 10, Phospholipase A2, Serine proteinase inhibitor, serpin, Heat shock protein 70, Uncoupling protein, Tubulin beta 2, Aldose A, Cofilin 1, Aldehyde dehydrogenase, Voltage-dependent anion channel 1,Voltage-dependent anion channel 2. We identified VDAC1 with a 2.82-fold increased level (P<0.05) and VDAC2 with a 3.97-fold decreased level (P<0.05) in the hippocampus of phenytoin-resistant rats compared to those of phenytoin-sensitive rats, which was confirmed by Western blot analysis and immunohistochemistry. The correlation coefficient of energy charge with VDAC1 and VDAC2 respectively was -0.67 (P<0.05) and 0.81 (P<0.05). Vascular damaged mitochondria and apoptosis neurons were observed through electron microscope. The refractory group had emotional, behavioral, and cognitive disorders, which showed the obvious tremor reflection, decreased open filed activity(P<0.05), increased catch-refusion(P<0.05), long escape latency(P<0.05), decreased swim time(P<0.05) and cross times(P<0.05) in the pool.Conclusion We successfully created the refractory epileptic animal model; Many mitochondrial proteins are involved in the process of refractory epilepsy; The increase of VDAC1 and the decrease of VDAC2 play an important role during the process, which provides new molecular evidence in understanding the mechanism of refractory epilepsy; The refractory group had emotional, behavioral, and cognitive disorders, which perhaps had the relationship with mitochondrial damages and energy disorders due to the changes of VDACs.
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