ABSTRACT: Sciatic nerve crush (SNC) triggers sterile inflammation within the distal nerve and de-afferented dorsal root ganglia (DRGs). In the nerve, neutrophils and pro-inflammatory Ly6Chigh monocytes appear first and rapidly give way to Ly6Clow resolving macrophages. Transcriptional profiling of injured nerve tissue identifies six macrophage subpopulations, repair Schwann cells and mesenchymal cells as the main cell types. Macrophages at the nerve crush site are distinct from macrophages associated with degenerating nerve fibers. Monocytes and macrophages in the injured nerve “eat” apoptotic cell corpses of leukocytes and thereby contribute to an anti-inflammatory milieu. Studies with chimeric mice show that following SNC few blood-derived immune cells enter DRGs. Myeloid cells in the injured nerve, but not DRGs, express the receptor for the chemokine GM-CSF. In the absence of GM-CSF, conditioning-lesion induced regeneration of DRG neuron central projections is abrogated. Thus, a carefully orchestrated immune response in the nerve is required for conditioning-lesion induced neurorepair.
Project description:Sciatic nerve crush (SNC) triggers sterile inflammation within the distal nerve and deafferented dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first, and rapidly give way to Ly6C- inflammation-resolving macrophages. Inflammation in DRGs is dominated by tissue resident macrophages, with little contribution from hematogenous leukocytes. Single-cell transcriptomics analysis of injured nerve identified six macrophage subpopulations, repair Schwann cells, and mesenchymal cells as the main cell types. Macrophages at the nerve crush site are distinct from macrophages associated with degenerating nerve fibers. Monocytes and macrophages in the injured nerve “eat” cell corpses of apoptotic leukocytes and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not DRGs, strongly express the receptor for the chemokine GM-CSF. In the absence of GM-CSF, conditioning-lesion induced regeneration of DRG neuron central projections is abolished. Thus, a carefully orchestrated immune response in the nerve is required for conditioning-lesion induced neurorepair.
Project description:We generated whole-genome gene expression profiles of dorsal root ganglion (DRG) neurons following nerve damage. DRG neurons extend one peripheral axon into the spinal nerve and one central axon into the dorsal root. The peripheral axon regenerates vigorously, while in contrast the central axon has little regenerative capacity. For this study, two groups of animals were subjected either to sciatic nerve (SN) or dorsal root (DR) crush, and at 12, 24, 72 hours and 7 days after the crush, lumbar DRGs L4, L5 and L6 were dissected and total RNA was extracted. For each time point after lesion, three biological replicate RNA samples were hybridized together with the common reference sample consisting of labeld RNA pooled from three unlesioned animals.
Project description:Severe peripheral nerve injury (PNI) often causes significant movement disorders and intractable pain. Therefore, promoting nerve regeneration while avoiding neuropathic pain, a problem that remains unsolved, is key to the clinical treatment of PNI patients. Here, we establish a novel spared nerve crush (SNC) rat model that successfully reproduces axonal regeneration and neuropathic pain after PNI. Subsequently, we obtained single-cell RNA sequencing (scRNA-seq) data from rat directly injured and indirectly injured rat dorsal root ganglion (DRG) neurons at various time points after SNC and found that the PEP1 neuronal subtype in directly injured DRG is of particular interest. Through experimental design, sc-RNA sequence processing (EDSSP) and functional verification, we identified a potential key gene, Adcyap1, that encodes a key molecule linking nerve regeneration and pain after PNI. Our study sheds new light on the intrinsic link between axonal regeneration and neuropathic pain following PNI and provides new molecular targets and ideas for therapeutic intervention.
Project description:Mice with a deletion of transcription factor Nfil3 show delayed functional regeneration after sciatic lesion compared to WT mice. To analyze differential gene expression, sciatic nerves were lesioned and nerve endings were coaptated. After two or 5 days, DRGs L4 and L5 were dissected out and gene expression was measured by microarray. Control tissue was taken from the non-injured side. We found that expression of classic regeneration associated genes was similar in WT and Nfil3 KO mice. We did identify a group of differentially expressed genes known for its role in olfactory signal transduction, but were not known as regeneration associated genes until now, which may explain the negative effects on functional recovery. Gene expression in DRGs L4 and L5 from WT and NFIL3 KO mice was analyzed at 2 and 5 days after receiving a sciatic nerve lesion. Control tissue was taken from the non-injured side. Four biological replicates were used per condition. Microarray chips were two color chips, but each channel was analyzed in single color fashion. Conditions were pseudo-randomized over chips in a balanced dye-swap setup.
Project description:Following injuryin the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. Theseastrocytes originate primarily from resident astrocytes orNG2+ oligodendrocyteprogenitor cells. However, theextentto which these cell types give rise to the lesion-filling astrocytes,andwhethertheastrocytes derived from differentcell typescontribute similarly to optic nerve regenerationremain unclear.Here we examine the distributionof astrocytes and NG2+ cellsin an optic nerve crush model.Weshow that optic nerve astrocytes partially fill the injury site over timeafter a crush injury.Viral mediated expression of a growth-promotingfactor,ciliary neurotrophic factor (CNTF),in retinal ganglion cells (RGCs) promotesaxon regeneration without altering the lesion size or the degreeof lesion-filling GFAP+ cells. Strikingly, using inducible NG2Cre driver mice, wefoundthat CNTF overexpression in RGCs increasesthe occupancy ofNG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment showsthat the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation.Lastly, we performedRNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes,including those that encode chemokines, growth factor receptors,and immune cell modulators.Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site.We discuss possible implication of theseresultsforaxon regeneration.
Project description:Axonal regeneration is enhanced by prior conditioning peripheral nerve lesions. Here we show that Xenopus dorsal root ganglia (DRGs) with attached peripheral nerves (PN-DRGs) can be conditioned in vitro, thereafter showing enhanced axonal growth in response to neurotrophins, similar to preparations conditioned by axotomy in vivo. In contrast to freshly dissected preparations, conditioned PN-DRGs show abundant neurotrophin-induced axonal growth in the presence of actinomycin D, suggesting synthesis of mRNA encoding proteins necessary for axonal elongation occurs during the conditioning period, and this was confirmed by oligonucleotide micro-array analysis.
Project description:Maladaptive changes of nerve injury–associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury–specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury–induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury–induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.
Project description:We generated whole-genome gene expression profiles of dorsal root ganglion (DRG) neurons following nerve damage. DRG neurons extend one peripheral axon into the spinal nerve and one central axon into the dorsal root. The peripheral axon regenerates vigorously, while in contrast the central axon has little regenerative capacity. For this study, two groups of animals were subjected either to sciatic nerve (SN) or dorsal root (DR) crush, and at 12, 24, 72 hours and 7 days after the crush, lumbar DRGs L4, L5 and L6 were dissected and total RNA was extracted.
Project description:Macrophages have distinct characteristics depending on their microenvironment. We performed proteomic analysis between M1 and M2 macrophages and found that cellular metabolism is the key regulator of macrophage function. We used microarray to support proteomic data between M1 and M2 macrophages. M1 macrophages are obtained using cell sorting of CD45+MHCII+CD8a-F4/80+ population from C57BL/6J bone marrow cell derived heterogenous cells under GM-CSF conditioning for 7 days. M2 macrophages are differentiated with 20% L929 cell supernatant for 7 days and sorted from CD45+F4/80+CD11b+ population.