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Upon trauma, the adult murine peripheral nervous system (PNS) displays a remarkable degree of spontaneous anatomical and functional regeneration. To explore extrinsic mechanisms of neural repair, we carried out single-cell analysis of naïve mouse sciatic nerve, peripheral blood mononuclear cells, and crushed sciatic nerves at 1 day, 3 days, and 7 days following injury. During the first week, monocytes and macrophages (Mo/Mac) rapidly accumulate in the injured nerve and undergo extensive metabolic reprogramming. Proinflammatory Mo/Mac with a high glycolytic flux dominate the early injury response and rapidly give way to inflammation resolving Mac, programmed toward oxidative phosphorylation. Nerve crush injury causes partial leakiness of the blood–nerve barrier, proliferation of endoneurial and perineurial stromal cells, and entry of opsonizing serum proteins. Micro-dissection of the nerve injury site and distal nerve, followed by single-cell RNA-sequencing, identified distinct immune compartments, triggered by mechanical nerve wounding and Wallerian degeneration, respectively. This finding was independently confirmed with Sarm1 -/- mice, in which Wallerian degeneration is greatly delayed. Experiments with chimeric mice showed that wildtype immune cells readily enter the injury site in Sarm1 -/- mice, but are sparse in the distal nerve, except for Mo. We used CellChat to explore intercellular communications in the naïve and injured PNS and report on hundreds of ligand–receptor interactions. Our longitudinal analysis represents a new resource for neural tissue regeneration, reveals location- specific immune microenvironments, and reports on large intercellular communication networks. To facilitate mining of scRNAseq datasets, we generated the injured sciatic nerve atlas (iSNAT): https://cdb-rshiny.med.umich.edu/Giger_iSNAT/ .

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C high monocytes infiltrate the nerve first and rapidly give way to Ly6C negative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages ‘eat’ apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient ( Csf2 -/- ) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

Following injury to the adult mammalian central nervous system (CNS), spontaneous regeneration of damaged neurons is extremely limited. Previous work has shown that, under certain circumstances, the immune system can activate a regenerative program in injured CNS neurons. Focusing on the adult mouse visual system, I investigated how specific immune cell types and signaling pathways influence the regenerative behavior of injured retinal ganglion cells (RGCs), the projection neurons that connect the retina with the brain. Specifically, I used intraocular injection of particulate β-glucan to trigger an inflammatory response that promotes robust RGC regeneration following optic nerve injury. The main findings of my dissertation are that microglia are necessary for long-distance axon regeneration of injured RGCs, and that neutrophils are detrimental, mitigating β-glucan induced RGC repair. To demonstrate the involvement of microglia, I used a combination of pharmacological and genetic approaches to acutely ablate microglia in the adult CNS and studied the impact on β-glucan-elicited RGC regeneration. I show that microglia depletion resulted in increased leakiness of the blood retina barrier, an increase in intraocular inflammation, and large numbers of hematogenous neutrophils entering the retinal parenchyma through extravasation in post-capillary venules, resulting in microhemorrhages as demonstrated by analysis of the vitreous proteome. Single-cell analysis of vitreous leukocytes identified six subpopulations of classically activated neutrophils, highly enriched for pro-inflammatory gene products. To investigate whether blocking trafficking of immune cells into the eye is beneficial for RGC regeneration, I used a combination of genetic (Itgam-/-) and antibody (anti-CD11b, anti-Ly6G) based approaches. Blocking innate immune cell trafficking and neutrophils specifically protected the blood retina barrier, reduced microhemorrhages in the eye, and, most importantly, significantly enhanced β-glucan elicited RGC axon regeneration. Transcriptomics studies further showed that blocking immune cell trafficking does not alter the activation state but rather reduces the number of leukocytes that accumulate in the vitreous. Taken together, these studies show that protecting the inflamed vasculature augments immune-mediated axon regeneration and identifies the blood retina barrier as a therapeutic target to improve CNS regeneration.

Glioblastoma multiforme (GBM) is an aggressive central nervous system malignancy that commonly causes immune suppression in patients to avoid immune recognition and clearance. This complicates treatment options and limits the effectiveness of immunotherapy strategies. Interestingly, GBM formation can stimulate the neurogenic subventricular zone of the cerebral cortex and causes the proliferation and migration of neural stem cells (NSCs) towards the tumor. This migration reflects the NSC wound repair response following CNS injury. Studies using NSCs surgically implanted into GBM tumors showed decreased tumor growth and increased animal survival in mice; however, the mechanisms underlying these anti-tumor properties of NSCs are unknown. Here we performed co-culture proliferation assays in combination with gene expression analysis to show that there is a two-way communication between the NSCs and glioma cells that results in decreased glioma proliferation and changes to survival and apoptotic-related gene expression. These changes correlated with increased expression of tumor necrosis factor (TNF) superfamily death receptor ligands by the NSCs. NSCs also were found to express many immune-related cytokines and chemokines involved in immune signaling, suggesting a potential role in mediating anti-tumor immune responses. These results provide the first mechanistic evidence of NSC-mediated tumor suppression

Recent advances in experimental strategies that promote axon regeneration in adult mammals lay the foundation for future therapies. Reliable and unbiased quantification of regenerated axons is challenging, yet essential for comparing the efficacy of individual treatments and identification of most efficacious combinatorial therapies. Here, we introduce , a user-friendly and freely available software for the rapid quantification of regenerated axons in longitudinal nerve tissue sections. automatically identifies and traces regenerated axons, generating quantitative measurements that closely match conventional manual quantification but with significantly greater speed. Key features include length-dependent axon quantification at defined intervals from the injury site and normalization of axon density to nerve diameter to account for anatomical variability. To facilitate high-throughput analysis, the software includes an image queuing function. Additional features of allow quantification of a range of labeled cellular structures. As a proof of concept, we demonstrate accurate quantification of regenerated axons in the optic nerve, retinal ganglion cells density in retinal flat-mounts, and regenerated axon bundles in injured sciatic nerves. Collectively, we introduce a new platform that is expected to streamline and standardize regenerative outcome assessments across diverse experimental conditions and laboratories.

Upon trauma, the adult murine PNS displays a remarkable degree of spontaneous anatomical and functional regeneration. To explore extrinsic mechanisms of neural repair, we carried out single cell analysis of naïve mouse sciatic nerve, peripheral blood mononuclear cells, and crushed sciatic nerves at 1-day, 3-days, and 7- days following injury. During the first week, monocytes and macrophages (Mo/Mac) rapidly accumulate in the injured nerve and undergo extensive metabolic reprogramming. Proinflammatory Mo/Mac in the injured nerve show high glycolytic flux compared to Mo/Mac in blood and dominate the early injury response. They subsequently give way to inflammation resolving Mac, programmed toward oxidative phosphorylation. Nerve crush injury causes partial leakiness of the blood-nerve-barrier, proliferation of endoneurial and perineurial stromal cells, and accumulation of select serum proteins. Micro-dissection of the nerve injury site and distal nerve, followed by single-cell RNA-sequencing, identified distinct immune compartments, triggered by mechanical nerve wounding and Wallerian degeneration, respectively. This finding was independently confirmed with Sarm1-/- mice, where Wallerian degeneration is greatly delayed. Experiments with chimeric mice showed that wildtype immune cells readily enter the injury site in Sarm1-/- mice, but are sparse in the distal nerve, except for Mo. We used CellChat to explore intercellular communications in the naïve and injured PNS and report on hundreds of ligand-receptor interactions. Our longitudinal analysis represents a new resource for nerve regeneration, reveals location specific immune microenvironments, and reports on large intercellular communication networks. To facilitate mining of scRNAseq datasets, we generated the injured sciatic nerve atlas (iSNAT): https://cdb-rshiny.med.umich.edu/Giger_iSNAT/ Competing Interest Statement Except for Gabriel Corfas, the authors declare no competing financial or non-financial interests. Gabriel Corfas is a scientific founder of Decibel Therapeutics; he has an equity interest in and has received compensation for consulting. The company was not involved in this study.

Abstract Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first, and rapidly give way to Ly6Cnegative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages “eat” apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF deficient (Csf2-/-) mice, inflammation resolution is delayed and conditioning-lesion induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion induced neurorepair. Competing Interest Statement The authors have declared no competing interest. Footnotes * This version of the manuscript has been revised to update the co-author list.

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix- and growth factor-producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75NTR, a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75NTR exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75NTR⁻/⁻ HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75NTR signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75NTR to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix- and growth factor-producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75 super(NTR), a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75 super(NTR) exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75 super(NTR-/-) HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75 super(NTR) signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75 super(NTR) to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix– and growth factor–producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75 NTR , a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75 NTR exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75 NTR –/– HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75 NTR signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75 NTR to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.