Dysregulated genes in connexin 30 deficient mice microglia [naive]. Dysregulated genes in connexin 30 deficient mice microglia [naive]

Objective: Connexins are known to compose gap junctions by a pair of hemichannels connected in a head to head configuration. Each hemichannel is composed by hexameric cluster of connexins. There are 20 members of connexins known as connexin family in mice, and are expressed on each cell surface in different fashion. Connexin 30 (Cx30) is mainly expressed on the surface of oligodendroglia and astrocyte in central nervous system (CNS), but little is known about the functional relevance in neuroinflammatory diseases. To elucidate the role of Cx30 in the pathogenesis of neuroinflammatory disease, we induced experimental autoimmune encephalomyelitis (EAE) on Cx30 knock-out (Cx30-KO) mice and analyzed the clinical and neuropathological findings with wild-type control. Methods: C57BL/6J and Cx30-KO mice > 12 weeks of age were used in this study (N > 3 in each group). EAE was induced by immunization of mice with MOG35-55 peptide emulsified in CFA at a dose of 200 μg per mouse, followed by the administration of pertussis toxin (500 ng per mouse) on days 0 and 2. Mice were sacrificed and brain, spinal cord, spleen, and optic nerve were harvested for immunohistochemical analyses at the acute and chronic phases of EAE. Mice with EAE were scored as follows: 0, no disease; 1, limp tail; 2, abnormal gait and hind limb weakness (shaking); 2.5, paralysis of one hind limb; 3, paralysis of two hind limb; 3.5, ascending paralysis (able to move around); 4, tetraplegia; and 5, moribund (death). At the onset and chronic stage of EAE, mononuclear cells were isolated and analyzed by flow cytometry to check the distinct characteristics of cellular populations in inflamed CNS lesions. Results: Initial screening of immunohistological difference revealed basic activation of microglial cells in naïve Cx30-KO mice without any behavioral phenotype. Clinical signs of EAE were ameliorated in the Cx30-KO mice than in the control group mainly during the chronic phase of disease course. Immunohistochemical analyses of the fourth lumbar segment, brain and optic nerve revealed increased number of microglia in the Cx30-KO mice. Flow cytometric analysis also confirmed the findings. In contrast, there were no significant change in astroglial or oligodendroglial phenotype. Conclusion: Microglial activation appears to be the key factor in the Cx30-KO mice EAE with alleviation of chronic disease scores. Unexpectedly, microglia were already activated in naïve CNS, indicating protective phenotypic change of microglia in Cx30-KO mice. Overall design: We have collected microglia from wild type and Cx30 deficient mice with naïve. Each group cotained 3 mice. Collected microglia were pooled together for the analysis.

### 研究目标
连接蛋白（connexins）可通过一对以头对头构型连接的半通道（hemichannels）组装成缝隙连接（gap junctions）。每个半通道由6个连接蛋白单体构成的六聚体簇组成。小鼠体内的连接蛋白家族共有20个成员，它们以不同模式表达于各类细胞表面。连接蛋白30（connexin 30, Cx30）主要表达于中枢神经系统（central nervous system, CNS）内的少突胶质细胞与星形胶质细胞表面，但目前对其在神经炎症性疾病中的功能关联尚不清楚。为阐明Cx30在神经炎症性疾病发病机制中的作用，本研究构建Cx30敲除（Cx30 knock-out, Cx30-KO）小鼠，并通过实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）造模，同时以野生型（wild-type）小鼠作为对照，对其临床及神经病理表现进行分析。

### 研究方法
本研究使用12周龄以上的C57BL/6J小鼠与Cx30-KO小鼠（每组样本量N>3）。通过向小鼠皮下注射乳化于完全弗氏佐剂（CFA）中的MOG35-55多肽（每只小鼠200 μg）进行免疫，同时于第0天和第2天腹腔注射百日咳毒素（每只小鼠500 ng）以诱导EAE模型。分别在EAE的急性期与慢性期处死小鼠，采集大脑、脊髓、脾脏及视神经组织，用于免疫组织化学分析。EAE小鼠的临床评分标准如下：0分，无疾病症状；1分，尾部松弛无力；2分，步态异常伴后肢乏力（震颤）；2.5分，单后肢瘫痪；3分，双后肢瘫痪；3.5分，上行性瘫痪（仍可自主活动）；4分，四肢瘫痪；5分，濒死状态（死亡）。在EAE发病期与慢性期，分离单核细胞并通过流式细胞术（flow cytometry）分析中枢神经系统炎症病灶内细胞群体的特征性变化。

### 研究结果
初始免疫组织化学差异筛选结果显示，未造模的Cx30-KO小鼠体内小胶质细胞（microglia）已存在基础激活状态，但未表现出任何行为学表型。与对照组相比，Cx30-KO小鼠的EAE临床症状显著减轻，该改善主要见于疾病慢性期。对第四腰椎节段、大脑及视神经的免疫组织化学分析显示，Cx30-KO小鼠体内的小胶质细胞数量显著增多，流式细胞术结果亦验证了这一发现。与之相反，星形胶质细胞与少突胶质细胞的表型未出现明显变化。

### 研究结论
小胶质细胞激活是Cx30-KO小鼠EAE慢性期临床评分降低的关键影响因素。出乎意料的是，未造模的Cx30-KO小鼠中枢神经系统内的小胶质细胞已处于激活状态，提示Cx30敲除可诱导小胶质细胞产生保护性表型改变。

### 整体实验设计
本研究从未造模的野生型与Cx30缺陷型小鼠体内分离小胶质细胞，每组各3只小鼠，将同组收集的小胶质细胞混合后进行后续分析。