Imaging resident and recruited macrophage contributions to Wallerian degeneration

Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing. EGFP+/CD64+/CD11b+/SiglecF-/Ly6G- or ECFP+/CD64+/CD11b+/SiglecF-/Ly6G- macrophages were sorted by flow cytometry from a cell suspension of three pooled sciatic nerves from Macblue x Cx3cr1egfp/+ mice (one sciatic nerve per mouse; with 3 mice pooled for one group; repeated three times) at D14 after CCI. Two populations were analyzed: (1) EGFP+/CD64+/CD11b+/SiglecF-/Ly6G- macrophages sorted from ipsi lateral nerves representing resident macrophages in injured nerves, which are represented by the 3 following samples (each being a pool of 3 mice): Res1/Res2/Res/3; and (2) ECFP+/CD64+/CD11b+/SiglecF-/Ly6G-macrophages sorted from ipsi lateral nerves representing recently recruited macrophages in injured nerves, which are represented by the 3 following samples (each being a pool of 3 mice): Res1.1/Res2.1/Res/3.1.

摘要：沃勒变性（Wallerian Degeneration, WD）是指损伤后轴突发生自主性远端变性的过程。外周神经系统（Peripheral Nervous System, PNS）中的巨噬细胞（Macrophages, MP）是调控该过程的主要细胞类群。已有研究证据提示，定居型外周神经系统巨噬细胞与造血来源巨噬细胞均可能参与该过程，但这两类细胞亚群是否行使独特功能尚不清楚。本研究结合巨噬细胞特异性荧光报告基因小鼠与坐骨神经相干反斯托克斯拉曼散射（Coherent Anti-Stokes Raman Scattering, CARS）成像技术，解析了损伤后定居型与新近招募的巨噬细胞的时空动态变化特征，并揭示了两类亚群的功能差异：招募而来的巨噬细胞负责高效脱髓鞘与髓鞘清除，而定居型巨噬细胞则参与轴突再生。本研究为靶向干预神经退行性疾病相关的细胞过程提供了新思路。
方法（本GEO数据集相关内容）：小鼠坐骨神经慢性压迫性损伤（Chronic Constriction Injury, CCI）后巨噬细胞的RNA测序：
分选得到的细胞用100μL RA1/TCEP缓冲液（NucleoSpin RNA XS试剂盒，Macherey-Nagel公司）裂解，经液氮快速冷冻后保存于-80℃，直至进行RNA提取。所有样本同步处理，RNA提取步骤（无需载体RNA，采用柱上DNase处理）严格按照试剂盒说明书操作（NucleoSpin RNA XS试剂盒，Macherey-Nagel公司）。RNA用无RNA酶水洗脱。RNA测序的cDNA文库构建采用SmartSeq方法，严格按照试剂盒说明书操作（SMART-Seq v4超低起始量RNA测序试剂盒，Clontech/TaKaRa公司）。由于细胞数量稀少，以全部洗脱得到的RNA作为逆转录起始原料，随后进行18个循环的预扩增。取1ng cDNA用于RNA测序文库构建，操作严格遵循试剂盒说明书（Nextera XT DNA文库制备试剂盒，Illumina公司）。混合后的最终样本文库在Illumina Nextseq 500测序平台上进行测序，采用MidOutPut试剂盒（测序读长为2×75bp，每轮测序产出2×130百万条读段），共进行2轮测序（分别为4重和5重索引）；双端拆分后，每个样本最终得到2×30百万条读段。
在慢性压迫性损伤后第14天（D14），通过流式细胞术从Macblue × Cx3cr1^egfp/+小鼠的3根混合坐骨神经细胞悬液中分选得到两类巨噬细胞：EGFP⁺/CD64⁺/CD11b⁺/SiglecF^-/Ly6G^-与ECFP⁺/CD64⁺/CD11b⁺/SiglecF^-/Ly6G^-巨噬细胞（每只小鼠1根坐骨神经，每组由3只小鼠的坐骨神经混合制备，实验重复3次）。本研究分析了两类细胞群：(1) 从损伤侧坐骨神经中分选得到的EGFP⁺/CD64⁺/CD11b⁺/SiglecF^-/Ly6G^-巨噬细胞，即损伤神经中的定居型巨噬细胞，对应3个样本（每个样本为3只小鼠的混合组织）：Res1、Res2、Res3；(2) 从损伤侧坐骨神经中分选得到的ECFP⁺/CD64⁺/CD11b⁺/SiglecF^-/Ly6G^-巨噬细胞，即损伤神经中的新近招募巨噬细胞，对应3个样本（每个样本为3只小鼠的混合组织）：Res1.1、Res2.1、Res3.1。