| Epilepsy is a chronic neurologic syndrome caused by a variety of factors.Epilepsy seriously undermine the health of patients,increase the burden on individuals,families and society,and may endanger the others in some cases,such as a seizure initiation while driving.Most patients can achieve sustained seizure freedom by regular antiepileptic drugs(AEDs)therapy.However,about 30% of patients with epilepsy cannot get an ideal control after treatment of adequate trials of two appropriately chosen and used AEDs.This epilepsy is defined drug-resistant epilepsy.Frequent seizures or epileptic state destroys cognitive function of patients,especially for children who are in the development stage of neurological function.A foreign postoperative pathological study involved large cases reported that,malformation of cortical development(MCD)ranked among the three leading causes of epilepsy,and focal cortical dysplasia(FCD),the most common type of MCD,was the most common diagnosis in pediatric patients with drug-resistant epilepsy.According to the proposal of the international league against epilepsy(ILAE)in 2011,characteristics histopathological findings in isolated lesions of FCD include aberrant radial(FCD type Ia)or tangential(FCD type Ib)lamination of the neocortex,the presence of dysmorphic neurons(DNs)without(FCD type IIa)or with balloon cells(BCs,FCD type IIb).The most common subtypes in clinic are FCD Ia,IIa and IIb.The generally accepted pathologic mechanism of epilepsy is imbalance of excitation/inhibition.In brains,excitatory transmitters are released by principal cells(PCs),while the most important inhibitory transmitter,γ-aminobutyric acid(GABA),is produced by interneurons(INs).Parvalbumin(PV)expressing INs are the largest subpopulation and have the strongest inhibitory effect than any others.However,vasointestinal peptide(Vip)expressing INs are most special in physiological effect.Vip INs preferentially form synapses onto somatostatin(Sst)expressing INs,but not PCs,which is different from other INs subpopulations.By inhibiting Sst INs,Vip INs increased the gain of functional PCs indirectly.Several studies have reported that the number of PV INs decreased in FCD lesions,but there is little research data related to other INs subpopulations.In the analysis of the clinical specimens,we retrospectively analyzed both control samples and epileptic specimens pathologically diagnosed with FCD types Ia,IIa,or IIb.We quantified three major IN subpopulations,including PV,Sst and Vip positive INs across all the subgroups.Additionally,we calculated the ratio of the subpopulations of INs to the major INs(mINs)by defining the total number of the PV,Sst,and Vip INs as mINs.In the animal experiment,the effect of Vip INs on PCs in the brain slice of Vip-Cre mice was examined by combining optogenetic activation with patch clamp.The main results are as follows:1.Results based on clinical data and pathological specimens1.1 The analysis of the clinical data of FCD patients showed:(1)the ratio of patients who underwent single-lobe resection in the FCD type Ia group was lower than that in the FCD type IIb group;(2)compared with the FCD type Ia group,the percentage of male patients with FCD type II was significantly higher,and that with occipital lobe involved was lower.1.2 The quantitative analysis of INs subpopulations in the pathological specimens of FCD showed:(1)a significant increase in the ratio of Vip/mINs was observed only in the FCD type IIb group;(2)compared to the control,the density of the PV-INs in the FCD type IIb group was significantly reduced,and the ratio of PV/mINs was lower in the superficial part of the cortex of the FCD type Ia and IIb groups;and(3)the densities of the PV INs,Sst INs,and Vip INs in the FCD type Ia group were all higher than those in the FCD type IIb group.1.3 The protein level of TRPC3 was markedly elevated in FCD.Immunohistochemistry results revealed that TRPC3 staining was stronger in malformed cells and microcolumns.2.Results from animal experiments2.1(1)Results from the model preparation: although both exposed to external radiation on embryonic 16.5(E16.5),different from the best dose of the γ-ray-irradiation cortical dysplasia model of rats,which is 2.25 Gy,more appropriate dose for the X-ray-irradiation cortical dysplasia model of mouse is 1.6Gy;(2)examining the irradiated mouse model showed: the seizure threshold decreased;the cortical area of the brain was reduced and the corpora quadrigemina were exposed,discrimination of individual cortical layer is difficult in some cortical region,the linear arrangement of the pyramidal neurons in CA1 of hippocampus was interrupted in some mice,nodular heterotopia of neurons were formed in the cortex and hippocampus.2.2 Result from the brain slice of Vip-Cre mice:(1)under the differential interference contrast(DIC)microscopy,the neurons in the cortex were disorganized and no regular neuronal polarity was found;(2)single-cell recording on the fluorescent labeled cells showed an irregular spike pattern in response to weak current injection and a regular adapting firing pattern during larger depolarization;(3)when triggered by 470 nm laser light source with40 Hz frequency,recording on the pyramidal neurons in the field of the labeled cells showed an increased excitation in the MCD model group,but not in the control group,and the excitability of the triggered Vip INs increased in both the groups.In summary,our study reveals that:1.The clinical data showed that FCD type Ia causes more disruption than FCD type IIb;2.From the developmental perspective,the pathomechanism of FCD type IIb occurs during the earlier stage of the cell differentiation than that of FCD type Ia;3.Compared with the control group,the lower ratios of PV/mINs in the superficial part of the cortex in the FCD type Ia and IIb groups indicate that the relative change of the PV INs number may be involved the seizure initiation in FCD type Ia and IIb,and the higher ratio of Vip/mINs in FCD type IIb indicates that the relative change of Vip INs number may be involved the seizure initiation.4.Different from the optimal dose of 2.25 gy in rat cortical dysplasia model,that of the mouse X-ray model was 1.6 gy.A serial of examinations of this model prove that this mouse model is an ideal cortical dysplasia model.5.The results of patch clamp experiment showed that in the model of cortical dysplasia mice,the disinhibitory effect of Vip INs on pyramidal neurons was stronger than that of the control group.6.Up-regulation of TRPC3 expression might reinforce the depolarization of excitatory neurons in FCD lesions.In conclusion,we believe that the enhancement of disinhibition effect of Vip INs in MCD increases susceptibility to epilepsy,and the up-regulation of TRPC3 expression might be involved in the initiation of seizures in MCD. |
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