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. 2022 Oct 19;5(1):1105.
doi: 10.1038/s42003-022-03970-0.

scRNA-seq generates a molecular map of emerging cell subtypes after sciatic nerve injury in rats

Ditte Lovatt  1 , Alex Tamburino  2 , Alicja Krasowska-Zoladek  3 , Raul Sanoja  3   4 , Lixia Li  5 , Vanessa Peterson  5 , Xiaohai Wang  3 , Jason Uslaner  3
Affiliations

scRNA-seq generates a molecular map of emerging cell subtypes after sciatic nerve injury in rats

Ditte Lovatt et al. Commun Biol. .

Abstract

Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Classification of major cell types within rat sciatic nerve.
a Experimental outline of dissociation of sciatic nerve from naive animals and from CCI injured animals at 3, 12, and 60 days post-injury. b UMAP plot of clustered cells from all timepoints (n = 112,521 cells, n = 33 10X libraries, n = 18 animals, n = 4 timepoints). c Dot plot of scaled expression of genes differentiating major cell types and classification of their group. d Feature plot of cell type markers. Cells plotted in random order. e Heat map displaying scaled expression of top 20 differentially expressed genes among major cell types using cluster average expression values. f Proportion of cell types in scOmics data at each timepoint. g Nuclei density in nerve tissue at each timepoint quantified by histology (blue = contralateral, red = ipsilateral, gray = uninjured, individual values and mean ± SD, n = 5 technical replicates per animal, n = 3 animals, two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.0001, *p < 0.0035).
Fig. 2
Fig. 2. Identification of molecular subtypes of Schwann cells in naive and injured nerve.
a UMAP plot of Schwann cells from all timepoints (n = 16,259 cells). Top: All timepoints. Bottom: Individual timepoints. b Violin plot with boxplot overlay and outliers (dots) of markers identifying Schwann cell subtypes across all timepoints. c Dot plot of scaled expression of markers identifying subtypes. d Heat map of the distribution (percentage) of each subtype normalized to each timepoint. Mean ± SD superimposed on each cell. Left row labels and top column labels indicate cell number in cluster and timepoint, respectively. e Heat map displaying scaled expression of top 20 differentially expressed genes among Schwann cell subtypes across all timepoints. Expression is scaled. f Violin plot with boxplot overlay and outliers (dots) of specific markers identifying myelinating and Remak Schwann cell subtypes among Schwann cells. g Violin plot with boxplot overlay and outliers (dots) of specific markers identifying Repair Schwann cell subtypes among Schwann cells. h ISH of Sox10 RNA on uninjured and injured (3, 12, and 60 days post CCI) nerve. Dashed line indicates the perineurial barrier with the endoneurium above. Injured timepoints show only endoneurium and asterisks indicate ligature-induced necrosis. Scale bar, 100 µm. i Bar chart with dots of Sox10 + nuclei density in uninjured (dark gray), contralateral (light gray), ipsilateral whole area (blue), and ipsilateral proximal to necrotic area (red) (n = 3 animals, mean ± SD, two-way ANOVA with Dunnett’s multiple comparison test, *p < 0.05, **p < 0.01, ****p < 0.0001).
Fig. 3
Fig. 3. Identification of molecular subtypes of fibroblast cells in naive and injured nerve.
a Dimensional reduction plot (UMAP) of subclustered fibroblasts from all timepoints (n = 40,361 cells). Top: All timepoints. Bottom: Individual timepoints. b Heat map of the distribution of fibroblast subtypes at each timepoint (percentage, mean ± SD). Left row labels and top column labels indicate cell number in cluster and timepoint, respectively. c Violin plot with boxplot overlay and outliers (dots) of fibroblast subtype markers. d Feature plot of key markers differentiating epi-, peri- and endoneurial fibroblasts. e Dot plot of scaled expression of fibroblast subtype markers. f, g ISH of Sfrp2 (Scale bar: f, 75 µm; g, 300 µm) and i, j Gpc3 (Scale bar: i, 25 µm; j, 100 µm) in uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. The dashed boxes in (g) and (j) display the image in (f) and (i), respectively. h, k Bar chart with individual values displaying ISH data in (g) and (j) in uninjured (dark gray), contralateral nerve (light gray), ipsilateral - whole area (blue), and ipsilateral - epineurial area (red) for each timepoint (mean ± SD, n = 2–3 animals, two-way ANOVA with Sidak’s multiple comparison test, **p < 0.01, ****p < 0.0001). l, n Heatmaps of top differentially expressed genes in: l the four major fibroblast types, m naive and injured epineurial subtypes, and n naive and injured peri- and endoneurial subtypes. Normalized Z-score.
Fig. 4
Fig. 4. Identification of molecular subtypes of myeloid cells in naive and injured nerve.
a Dimensional reduction plot (UMAP) of subclustered myeloid cells (n = 32,265 cells). Top: All timepoints. Bottom: Individual timepoints. b Violin plot with boxplot overlay and outliers (dots) of cell type markers. c Heat map of the distribution of each myeloid subtype at each timepoint (percentage, mean ± SD). Left row labels and top column labels indicate cell number in cluster and timepoint, respectively. d Heat map displaying scaled expression of the top 40 differentially expressed genes across cell types from all timepoints. e Dot plot of scaled expression of genes differentiating clusters in naive and injured dominant subtypes. f Feature plot of Csf1rand Flt3 expression distribution across cells from all timepoints, respectively. g Quantification of the percentage of cells expressing (Csf1r, left) and (Flt3, right) in relevant clusters.
Fig. 5
Fig. 5. Myeloid cell types form a enveloping scar around necrotic areas.
a ISH of Cd68 in uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Column: left, epineurium; right, endoneurium. Asterisk: Necrotic area. Scale bar, 100 µm. b ISH of Siglech in endoneurium of uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. Note that at day 3 nerve does not exhibit necrosis. Scale bars: 100 µm. c, d Bar chart with individual values of ISH data in (a) and (b) in uninjured (dark gray), contralateral nerve (light gray), ipsilateral—whole area (blue), and ipsilateral—necrotic area (red) for each timepoint (mean ± SD, n = 3 animals, two-way ANOVA with Sidak’s multiple test comparison: *p < 0.05, **p < 0.01).
Fig. 6
Fig. 6. Identification of molecular subtypes of vascular cell types in naive and injured nerve.
a Dimensional reduction plot (UMAP) of subclustered vascular cells from all timepoints (n = 13,290 cells). Top: All timepoints. Bottom: Individual timepoints. b Violin plot with boxplot overlay and outliers (dots) of cell type markers. c Heat map of the distribution of each vascular subtype at each timepoint (percentage, mean ± SD). d Feature plots of vascular subtype markers. e Dot plot of scaled expression of genes differentiating clusters in among naive and injured subtypes. f Heat map displaying scaled expression of the top 20 differentially expressed genes across cell types from all timepoints displayed from cluster averages. g ISH of Des in endoneurium (left column) and epineurium (right) of uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. Scale bars: 100 µm. h ISH of Cdh5 in endoneurium (left column) and epineurium (right) of uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. Scale bars: 100 µm. i, j Quantification of ISH data in (g) and (h), respectively, displayed as bar charts with individual values: uninjured (dark gray), contralateral nerve (light gray), and ipsilateral—whole area (blue) for each timepoint (mean ± SD, n = 2–3 animals, two-way ANOVA with Sidak’s multiple comparison, *p < 0.05, ***p < 0.001). k Heat map of cell count in each cluster divided by timepoint. l Heat map of absolute count of DEGs for each cell subtype when compared among timepoints. Only cell subtypes with more than 150 cells in each group were included in the analysis.
Fig. 7
Fig. 7. Identification of molecular subtypes of lymphoid cells in naive and injured nerve.
a Dimensional reduction plot (UMAP) of subclustered lymphoid cells from all timepoints (n = 7957 cells). Top: All timepoints. Bottom: Individual timepoints. b Heat map of the distribution of each lymphoid subtype at each timepoint (percentage, mean ± SD). Left row labels and top column labels indicate cell number in cluster and timepoint, respectively. c Violin plot with boxplot overlay and outliers (dots) of cell type markers. d Feature plots of lymphoid subtype markers. e Heat map displaying scaled expression of the top 20 differentially expressed genes across cell types from all timepoints displayed from cluster averages. f ISH of Ncr1 in endoneurium and epineurium areas of uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. Scale bars: 100 µm. g Bar chart with individual values displaying number of Ncr1 + cells in uninjured (light gray), contralateral (blue), and ipsilateral (red, whole area) nerve for each timepoint (mean ± SD, n = 2–3 animals). h Percent Ncr1 + cells in ISH data in (f) in epineurial and endoneurial areas. i ISH of Cd3e in endoneurium and epineurium areas of uninjured and injured (3, 12, and 60 days post-CCI) sciatic nerve. Asterisk: Necrotic area. Scale bars: 100 µm. j Bar chart with individual values displaying number of Cd3e + cells in uninjured (light gray), contralateral (blue), and ipsilateral (red, whole area) nerve for each timepoint (mean ± SD, n = 2–3 animals).
Fig. 8
Fig. 8. Ligand–receptor interactions in naive nerve.
a Number of LR pairs between indicated cell types identified using SingleCellSignalR in naive nerve (LR > 0.05). b Circosplot of top interactions signaling from naive Schwann cells (ligand) to naive perineurial fibroblasts (receptor). c Feature plots validating cell type markers and identified LR pair in all naive cells. d Illustration of signaling between the LR pair, Cntf-Cntfr, between myelinating Schwann cells and perineurial fibroblasts. The LR score between myelinating Schwann cells and perineurial fibroblasts for Cntf and Cntfr is 0.84.
Fig. 9
Fig. 9. Ligand–receptor interactions after injury.
Number of LR interactions larger than 0.5 between the indicated two cell types after a 3 days, b 12 days, and c 60 days of CCI injury, respectively. d Heat map of scaled expression of top 100 most-variable interactions at day 12. e Subset of interactions from (d) between types of fibroblasts and Schwann cells. f Violin plot with boxplot overlay and outliers (dots) of highlighted pairs, Ngf - Ngfr and Ptn - Ptprz1, from (e). g Subset of interactions from (d) between types of fibroblasts and vascular cells. h Violin plot with boxplot overlay and outliers (dots) of highlighted pair, Timp3 - Kdr, from (g).
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References

    1. Binder A, Baron R. The pharmacological therapy of chronic neuropathic pain. Dtsch. Arztebl. Int. 2016;113:616–625. - PMC - PubMed
    1. Woolf CJ. Capturing novel non-opioid pain targets. Biol. Psychiatry. 2020;87:74–81. doi: 10.1016/j.biopsych.2019.06.017. - DOI - PMC - PubMed
    1. Smith MT, Anand P, Rice ASC. Selective small molecule angiotensin II type 2 receptor antagonists for neuropathic pain: preclinical and clinical studies. Pain. 2016;157:S33–s41. doi: 10.1097/j.pain.0000000000000369. - DOI - PubMed
    1. Shepherd AJ, et al. Macrophage angiotensin II type 2 receptor triggers neuropathic pain. Proc. Natl Acad. Sci. USA. 2018;115:E8057–E8066. doi: 10.1073/pnas.1721815115. - DOI - PMC - PubMed
    1. Yuste R, et al. A community-based transcriptomics classification and nomenclature of neocortical cell types. Nat. Neurosci. 2020;23:1456–1468. doi: 10.1038/s41593-020-0685-8. - DOI - PMC - PubMed