Injured Sciatic Nerve Atlas (iSNAT)

Description of cellular and molecular changes in the mouse sciatic nerve following crush injury Overall design: The goal was to carry out a longitudinal scRNAseq study aimed at a detailed description of the cellular and molecular changes in the mouse sciatic nerve following a mid-thigh crush injury. For comparison, we carried out scRNAseq of naïve nerve immune and peripheral blood mononuclear cells (PBMC). During cell isolation, transcription was blocked with actinomycin D. From naive nerve, immune cells were captured with an anti-CD45 magnetic bead column and processed for scRNAseq. From the same nerves, non-immune cells (CD45 negative cells) were collected and processed for scRNAseq. The resulting datasets were combined and used as baseline to describe the cellular composition and gene expression profiles at single cell resolution in the naïve sciatic nerve. Next, cohorts of mice were subjected to sciatic nerve crush injury and nerve trunks were collected at different time points during the first week following injury. Specifically, nerves were harvested at 1-day post-crush (Day1), Day3, and Day7. For a comparative analysis of the nerve micro-environment at the injury site, versus the distal nerve, 3 mm nerve segments with the injury site at the center were collected, and in addition, 3 mm nerve segments spanning from 1.5-4.5 mm distal to injury site were collected. In separate reactions, nerves were digested, cells purified over a percoll gradient, depleted of myelin and processed for scRNAseq.