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  1. eLife assessment

    In peripheral nerve injury, an immune response occurs to ensure debris clean-up and potential repair, however, there has not yet been a census of cell types and gene expression as these lesions undergo clearance and eventual repair. Zhao et al generate a transcriptional resource by performing scRNAseq on both the naive, injured, and repairing sciatic nerve. They identify the composition of different cell types, gene signatures, and cell-cell communication and contrast these with signatures from the blood, and compare the injured site with distal nerve segments after injury. To dissociate the immune response from injury versus Wallerian degeneration, they use SARM1 KO mice (which exhibits delayed neurodegeneration) and observe that there is still injury-induced immune influx. Overall, this is a convincing study and useful resource for the field of neuronal repair and neural-immune interactions with a clear presentation of the animals and time points, with some follow-up experiments and validation.

  2. Reviewer #1 (Public Review):

    The authors studied the dynamics of dynamic multicellular response and the cell-cell interaction networks after PNS injury. This is the first longitudinal study that has been carried out in such detail. It also includes a comparative analysis between circulating immune cells in peripheral blood, and in the injured nerve. They performed a follow-up using flow cytometry, ELISA, in situ hybridization, and immunofluorescence labeling of nerve sections. In addition, they compared the role of Wallerian degeneration in this process by using the Sarm1-/- mutant mice. The authors show how immune cells get metabolically reprogrammed after nerve injury, how the distal and proximal compartments react differentially, and how the cell interactions change during injury and resolution. The authors show great biological knowledge in the analyses of their data. This is a great resource for scientists working on the PNS or regeneration in general. To facilitate excess to their data the authors provided a web tool.

  3. Reviewer #2 (Public Review):

    In this manuscript, Zhou et al carried out a very thorough spatial and temporal transcriptomic analysis of various cellular responses in the injured sciatic nerve using single-cell RNAseq. As such, it provides a wealth of new information on how cells in the nerve respond to a crush injury, both at the injury site and distally, during the first-week post-crush. The data are technically sound and the authors validated many of the observed expression changes in specific cells using a variety of approaches such as FACS, RNAscope, and immunostaining. They also created a searchable, publicly available tool, iSNAT, that allows the exploration of changes in gene expression in the injured nerve, which will be very valuable for the research community. The authors focus particularly on immune cells in the nerve and reveal a number of interesting findings. For example, they demonstrate that monocytes and macrophages are recruited to the nerve and undergo reprogramming, initially to pro-inflammatory cells relying on glycolysis, then to inflammation-resolving cells that rely on oxidative phosphorylation. In addition, they use sarm1-/- mice, which have very delayed Wallerian degeneration, to demonstrate that independent of Wallerian degeneration, immune cells are recruited to the injury site, but minimally in the distal region. However, they find an increase in monocytes distally, suggesting that these cells fail to differentiate into macrophages in the absence of WD.

    Overall, this is a very comprehensive analysis that provides a very useful resource for the field and reveals a number of interesting new insights into the immune response in the injured peripheral nerve. These results have important implications for understanding nerve regeneration and neuropathic pain.

    As with any such study, the results are limited by the number of cells that can be analyzed and the number of sequencing reads. The authors were able to obtain a large number of most cells for analysis; however, the number of myelinating Schwann cells was fairly small, due to the need to remove myelin debris. A similar limitation has been encountered by others and does limit the ability to deeply investigate changes in Schwann cells after injury. This is particularly relevant because, as the authors bring up in their discussion, there is considerable evidence indicating that Schwann cells are involved in recruiting immune cells to the injured nerve. Thus, it was somewhat surprising that some of the signaling detected in Fig. 5 was not from Schwann cells, but this may be due to these cells being underrepresented. The authors should consider specifically examining changes in the Schwann cell profiles to determine if there is an increase in the expression of any of the known chemokines.

    Among the interesting findings that came out of their analysis was an increase in monocytes in the distal nerve of the sarm1-/- mice, suggesting that these cells are recruited prior to Wallerian degeneration (WD) but in the absence of WD, they fail to differentiate into macrophages. This finding indicates that some aspect of WD promotes the differentiation of these cells. However, the authors should confirm the increase in monocytes prior to WD in the wild-type nerve, for example at 1-day post crush. This could be done by immunostaining or FACS.

    The metabolic reprogramming observed after the injury, to a more glycolytic phenotype, is consistent with what has been observed by others for macrophages that are pro-inflammatory. However, the metabolic changes were only noted in the whole nerve at 3 dpc (Fig. 3). The authors should similarly comment on, and provide evidence for, the metabolic phenotype of the macrophages specifically in the distal nerve (Fig. 8). Are these initially pro-inflammatory and then inflammation resolving or are they always largely anti-inflammatory?

  4. Reviewer 3 (Public Review):

    The authors are to be commended on their clear presentation of the animals and time points (in table 1), their validation with ELISA, and the insightful follow-up experiments and validation. This is an important study that will be of broad interest to the field.

    However, there are key issues that must be addressed, mostly relating to a lack of basic explorative analyses on the core scRNAseq datasets found in the paper.