Hidmark Asa S, Nawroth Peter P, Fleming Thomas
Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg.
Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg; Institute for Diabetes and Cancer IDC Helmholtz Center Munich, Germany & Joint Heidelberg-IDC Translational Diabetes Program; German Center for Diabetes Research (DZD).
J Vis Exp. 2017 Dec 6(130):56538. doi: 10.3791/56538.
Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.
外周神经系统（PNS）中的驻留神经免疫细胞对于维持健康神经中的神经元完整性至关重要。PNS的免疫细胞会受到损伤和疾病的影响，进而影响神经功能和再生能力。神经元免疫细胞通常通过免疫荧光（IF）进行分析。虽然IF对于确定免疫细胞在神经中的位置至关重要，但IF只是半定量的，并且该方法仅限于可同时分析的标记数量以及表面表达程度。在本研究中，流式细胞术用于定量分析单个小鼠坐骨神经或背根神经节（DRG）中的白细胞浸润情况。使用DAPI进行单细胞分析，并同时分析几种蛋白质的表面或细胞内表达。按照此方案处理的一只小鼠的两条坐骨神经均产生了≥30,000个单核事件。通过CD45表达确定的坐骨神经中白细胞比例约为坐骨神经总细胞含量的5%，在DRG中约为5 - 10%。尽管此方案主要关注PNS内的免疫细胞群体，但流式细胞术能够同时测量多种标记的灵活性意味着神经中存在的其他细胞群体，如施万细胞、周细胞、成纤维细胞和内皮细胞，也可以使用此方法进行分析。因此，该方法为研究对PNS的全身影响提供了一种新手段，例如神经毒理学和神经病变的遗传模型或在慢性疾病（如糖尿病）中的应用。
