Table_2_The Impacts of Surgery and Intracerebral Electrodes in C57BL/6J Mouse Kainate Model of Epileptogenesis: Seizure Threshold, Proteomics, and Cytokine Profiles.docx

Intracranial electroencephalography (EEG) is commonly used to study epileptogenesis and epilepsy in experimental models. Chronic gliosis and neurodegeneration at the injury site are known to be associated with surgically implanted electrodes in both humans and experimental models. Currently, however, there are no reports on the impact of intracerebral electrodes on proteins in the hippocampus and proinflammatory cytokines in the cerebral cortex and plasma in experimental models. We used an unbiased, label-free proteomics approach to identify the altered proteins in the hippocampus, and multiplex assay for cytokines in the cerebral cortex and plasma of C57BL/6J mice following bilateral surgical implantation of electrodes into the cerebral hemispheres. Seven days following surgery, a repeated low dose kainate (KA) regimen was followed to induce status epilepticus (SE). Surgical implantation of electrodes reduced the amount of KA necessary to induce SE by 50%, compared with mice without surgery. Tissues were harvested 7 days post-SE (i.e., 14 days post-surgery) and compared with vehicle-treated mice. Proteomic profiling showed more proteins (103, 6.8% of all proteins identified) with significantly changed expression (p < 0.01) driven by surgery than by KA treatment itself without surgery (27, 1.8% of all proteins identified). Further, electrode implantation approximately doubled the number of KA-induced changes in protein expression (55, 3.6% of all identified proteins). Further analysis revealed that intracerebral electrodes and KA altered the expression of proteins associated with epileptogenesis such as inflammation (C1q system), neurodegeneration (cystatin-C, galectin-1, cathepsin B, heat-shock protein 25), blood–brain barrier dysfunction (fibrinogen-α, serum albumin, α2 macroglobulin), and gliosis (vimentin, GFAP, filamin-A). The multiplex assay revealed a significant increase in key cytokines such as TNFα, IL-1β, IL-4, IL-5, IL-6, IL-10, IL12p70, IFN-γ, and KC/GRO in the cerebral cortex and some in the plasma in the surgery group. Overall, these findings demonstrate that surgical implantation of depth electrodes alters some of the molecules that may have a role in epileptogenesis in experimental models.

颅内脑电图（intracranial electroencephalography, EEG）常被用于实验模型中癫痫发生机制与癫痫病症的研究。已知在人类及实验模型中，手术植入电极与损伤部位的慢性胶质增生和神经退行性变存在关联。然而目前尚无关于脑内电极对实验模型海马体蛋白质、大脑皮层及血浆促炎细胞因子影响的研究报道。本研究对C57BL/6J小鼠双侧大脑半球植入手术电极后，采用无偏倚、无标记蛋白质组学方法鉴定海马体中差异表达蛋白，并通过多重检测法分析大脑皮层与血浆中的细胞因子。术后7天，采用反复低剂量海人酸（kainate, KA）造模方案以诱导癫痫持续状态（status epilepticus, SE）。与未手术小鼠相比，手术植入电极可使诱导SE所需的KA剂量降低50%。于SE造模后7天（即术后14天）采集组织样本，并与赋形剂处理组小鼠进行对照比较。蛋白质组分析显示，手术组导致的差异表达蛋白（p<0.01）数量（103种，占已鉴定总蛋白的6.8%）多于仅行KA处理的未手术组（27种，占已鉴定总蛋白的1.8%）。此外，电极植入使KA诱导的蛋白表达变化数量增加了约一倍（55种，占已鉴定总蛋白的3.6%）。进一步分析表明，脑内电极植入与KA处理共同改变了与癫痫发生机制相关的蛋白表达，包括炎症通路相关蛋白（C1q系统）、神经退行性变相关蛋白（胱抑素-C、半乳糖凝集素-1、组织蛋白酶B、热休克蛋白25）、血脑屏障功能障碍相关蛋白（纤维蛋白原-α、血清白蛋白、α2巨球蛋白）以及胶质增生相关蛋白（波形蛋白、胶质纤维酸性蛋白、细丝蛋白-A）。多重检测结果显示，手术组大脑皮层中关键细胞因子如肿瘤坏死因子α（TNFα）、白细胞介素1β（IL-1β）、IL-4、IL-5、IL-6、IL-10、IL12p70、干扰素γ（IFN-γ）及KC/GRO水平显著升高，血浆中部分细胞因子水平亦出现上升。综上，本研究结果表明，在实验模型中手术植入深部电极可改变部分可能参与癫痫发生机制的分子表达。