Fig 2: Validation of the transcriptome data by single cell based quantitative High Content Screening (HCS) microscopy focusing on selected signaling-relevant proteins.(A) Triple staining of the neuronal marker UCHL1 and two different TRPV1 antibodies derived from rabbit and goat, respectively, to facilitate the analysis of various targets. The staining intensities obtained with both TRPV1 antibodies correlated significantly (Spearmans ? = 0.96, p<2.2e-16). (B-E, G) Co-labeling of TRPV1 and CART (B), Nos1 (C), KChIP1 (D), KChIP2 (E), and CaMKIIa (G). Plots of respective controls are shown in S1 Fig. (F) Average fluorescence intensities of TRPV1 and the indicated targets in TRPV1-negative (grey) and -positive (black) neurons. Signal intensities of all analyzed targets were significantly higher within the TRPV1(+) population (n = 3 with>3000 analyzed neurons per experiment, paired two-tailed t-tests).

Fig 3: Alleviation of the injury-induced pain phenotype by HA hydrogel implantation and by morphine treatment.(A) (i) Alleviation of the injury-induced pain phenotype by HA hydrogel implantation and by morphine treatment. (i) Low-dose morphine (2 mg/kg) injection 30 min before the Hargreaves test resulted in significantly higher withdrawal latency times than in injured rats without morphine. (ii) Low-dose morphine injection before the von Frey test resulted in significantly higher 50% withdrawal thresholds than in injured rats or the morphine treatment. (iii) Low-dose morphine injection before the tail-flick test resulted in significantly lower withdrawal latency times than in the injured rats without morphine. (B) (i) In the Hargreaves test, withdrawal latency times were significantly higher in the HA hydrogel–treated injury group (and the control group) than in the untreated injury group. (ii) In the von Frey test, 50% withdrawal thresholds were significantly higher in the HA hydrogel–treated injury group (and the control group) compared to the untreated injury group. (iii) In the tail-flick test, withdrawal latency times were significantly lower in the HA hydrogel–treated injury group (and the control group) than in the untreated injury group. (iv) Post-mortem, expression of genes encoding molecular markers of nociception substance P (Tac1) and c-Fos was significantly lower in the HA hydrogel–treated injury group (and the control) than in the untreated injury group. (C) (i) Confocal microscopy demonstrated the presence in AF and NP tissues, particularly in the untreated injury group, of the GAP43 protein (yellow label) for nerve ingrowth, the sensory neuropeptide CGRP (purple label), and TRPV1 (green label). (ii) The NGF receptor Trk-A (red label) was evident in AF and NP tissues. (iii) The quantified percentage volume fractions of GAP43, CGRP, TRPV1, and Trk-A were significantly higher in the untreated injury group than in the sham control group or the HA hydrogel–treated injury group in both AF and NP tissues. *P < 0.05, significant difference between groups, by one-way ANOVA. n = 10 for (A) and (B), and n = 4 for (C). Data are means ± SEM. Area fraction data were normalized to the total area. Scale bars, 50 µm.

Fig 4: PGD2 is a paracrine mediator synthesized in myelinated large-diameter neurons that acts on TRPV1(+) neurons.(A) Dose-dependent induction of RII phosphorylation in sensory neurons after 1 min stimulation with PGD2 (EC50 = 377 nM, n = 3,>2000 neurons/condition; one-way ANOVA with Bonferroni's multiple comparisons test). (B) PGD2 did not induce pRII in non-neuronal cells of the same cultures shown in A. (C) Time course of RII phosphorylation indicating long-lasting effects of PGD2 (10 µM) on sensory neurons. (D) Stimulation with PGD2 also results in phosphorylation of the ERK1/2 measured in the same cultures shown in D. (E) Representative experiment demonstrating that induction of RII phosphorylation is enhanced in TRPV1(+) neurons (total of 3664 neurons). Plots of respective controls are shown in S2 Fig. (F) Fold changes of pRII intensities in TRPV1(-) (grey bars) and TRPV1(+) (black bars) neurons after 1 min stimulation with 10 µM PGD2 (n = 3,>2000 neurons/condition, one-way ANOVA with Bonferroni's multiple comparisons test). (G) Co-labeling of TRPV1 and PTGDS revealing that PTGDS is expressed in neurons lacking TRPV1 (total of 9951 neurons, also refer to S2 Fig. for control plots). (H) Co-labeling of NF200 and PTGDS showing that PTGDS(+) neurons express NF200 (total of 12966 neurons, also refer to S2 Fig. for control plots).(I) Size distribution of PTGDS(+) (green), NF200(+) (red), and all sensory neurons (black) indicating that PTGDS(+) neurons are larger than other neurons. (J) Suggested pathway of interneuronal communication between subgroups of sensory neurons. Large-diameter mechanosensitive neurons express PTGDS resulting in the production of PGD2, which activates DP1 receptors present on nociceptive neurons expressing TRPV1.

Fig 5: Transcriptome analysis reveals novel pain targets enriched in TRPV1(+) neurons.(A) qPCR quantification of TRPV1 mRNA levels in samples hybridized on microarrays (n = 4, p<0.001, One-way ANOVA). (B) Relative expression levels of TRPV1 mRNA determined by microarray hybridizations (n = 4, p<0.001, One-way ANOVA with Bonferroni's multiple comparisons test). (C) Correlation matrix of the expression level of all genes (normalized fluorescence intensities) detected using micorarrays. Clustering of overall gene expression is visible between RTX and capsaicin treated samples. (D) qPCR quantification of TRPV1 mRNA levels in samples used for RNA-Seq (n = 3, p<0.001, paired two-tailed t-test). (E) Relative expression levels of TRPV1 mRNA determined by RNA-Seq (n = 3, p<0.001, One-way ANOVA). (F) Overview of the number of target transcripts identified by microarray hybridizations and RNA-Seq. Raw fluorescence intensities of microarrays were background corrected, log2 transformed, normalized, and filtered for expressed genes (Illumina detection p-value <0.01 in at least one of the samples, see Material and Methods sections for details). Sequencing was performed using a 5500xl SOLiD System resulting in 664 million reads in total (see Material and Methods sections for details). The reads were filtered to remove ribosomal RNA, tRNAs, and vector sequences. The remaining reads mapped to 16590 genes of the reference genome (rn5). Read counts were transformed to RPKM values (Reads per kilo base per million), normalized, and filtered to remove weakly expressed transcripts (RPKM>0.1). P-values of differentially expressed genes identified with both methods were adjusted for multiple testing with Benjamini and Hochberg's method, adjusted p-values <0.05 were considered significant.