Transcriptional profiling of 5 days post LPC lesion after microglia ablation. Transcriptional profiling of 5 days post LPC lesion after microglia ablation

Following all types of neurological injury, resident macrophages are activated locally, and blood-derived macrophages are recruited. Distinguishing the role of resident and blood-derived macrophages is complicated by the difficulty in distinguishing between these cell types because they mostly express the same molecular markers in a diseased state. To begin to understand the complexity of functions of resident and blood-derived macrophages following acute injury in the central nervous system (CNS) we ablated microglia, but not infiltrating macrophages, to determine their contribution following lysolecithin-induced demyelination . Overall design: RNAseq experiment was performed on LCM collected tissue from CX3CR1CreER; Rosa-iDTR mice or CX3CR1CreER; Rosa-wt controls. Mice received tamoxifent for three days (~12-15) and then LPC demyelination of the ventral spinal cord 4-6 weeks later. Mice recieved daily diphtheria toxin injections until 5 days post-LPC when spinal cords were collected. Lesion tissue or distal control tissue was microdissected in microglia ablated and control mice. We have 3 control groups and 4 microglia ablated groups each containing 3-5 pooled mice.

各类神经系统损伤发生后，驻留巨噬细胞会在损伤局部被激活，同时血液来源的巨噬细胞会被募集至损伤部位。由于两类细胞在疾病状态下大多表达相同的分子标记物，精准区分驻留巨噬细胞与血液来源巨噬细胞的功能角色存在极大挑战。为阐明中枢神经系统（CNS）急性损伤后驻留巨噬细胞与血液来源巨噬细胞的功能复杂性，本研究通过选择性清除小胶质细胞（microglia）而非浸润性巨噬细胞，探究其在溶血卵磷脂（lysolecithin，LPC）诱导的脱髓鞘模型中的功能贡献。
整体实验设计：本研究采用RNA测序（RNAseq）技术，对经激光捕获显微切割（LCM）获取的CX3CR1CreER; Rosa-iDTR小鼠或CX3CR1CreER; Rosa-wt对照小鼠的组织样本开展检测。小鼠先接受为期3天的他莫昔芬（tamoxifen）给药（日龄约12~15日），造模4~6周后对脊髓腹侧进行溶血卵磷脂（LPC）诱导脱髓鞘处理。随后每日注射白喉毒素，直至LPC诱导后5天收集脊髓组织。分别对小胶质细胞清除组与对照组的损伤病灶组织及远端对照组织进行显微切割。本研究共设置3个对照组与4个小胶质细胞清除组，每组由3~5只小鼠的组织样本混合制备。