Mechanistic Differences in Neuropathic Pain Modalities Revealed by Correlating Behavior with Global Expression Profiling

Abstract

Chronic neuropathic pain is a major morbidity of neural injury, yet its mechanisms are incompletely understood. Hypersensitivity to previously non-noxious stimuli (allodynia) is a common symptom. Here, we demonstrate that the onset of cold hypersensitivity precedes tactile allodynia in a model of partial nerve injury, and this temporal divergence was associated with major differences in global gene expression in innervating dorsal root ganglia. Transcripts whose expression change correlates with the onset of cold allodynia were nociceptor related, whereas those correlating with tactile hypersensitivity were immune cell centric. Ablation of TrpV1 lineage nociceptors resulted in mice that did not acquire cold allodynia but developed normal tactile hypersensitivity, whereas depletion of macrophages or T cells reduced neuropathic tactile allodynia but not cold hypersensitivity. We conclude that neuropathic pain incorporates reactive processes of sensory neurons and immune cells, each leading to distinct forms of hypersensitivity, potentially allowing drug development targeted to each pain type.

Keywords: T cells; TrpV1; WCGNA; cold allodynia; gene expression; immune system; macrophages; neuropathic pain; tactile allodynia; transcript profiling.

Figure 1. Differences in the Onset of Cold and Tactile Allodynia

(A and B) Cold allodynia (A) develops relatively quickly post-injury, whereas tactile allodynia (B) develops at a slower pace. (C) The onset time of cold hypersensitivity is illustrated by the blue line (cold) relative to the later onset of tactile hypersensitivity illustrated by the orange line (tactile). Statistically significant differences between the values from mice after SNI and their basal measures in (A) and (B); *p < 0.01 (one-way repeated-measures ANOVA followed by Bonferroni post hoc test). There were no statistically significant differences between basal measures and values after sham surgery (gray lines, one-way repeated-measures ANOVA). Error bars indicate SEM (n = 13 or 14 per group; see Supplemental Experimental Procedures).

Figure 2. WGCNA Analysis of All Regulated Transcripts in DRG

Significantly regulated probes (moderated F-statistic, n = 1,704) were subject to WGCNA analysis to produce unbiased clusters of co-regulated transcripts representing the entire regulatory network of transcripts in the DRG following peripheral nerve injury (SNI) for 10 days sampled at least once a day over this period. The first column defines the clusters present, and the second shows heatmaps of each gene in each cluster. Blue represents low-level expression and red high-level expression. The third column gives a brief description of cluster function as defined by IPA software. Below this is the number of probes in each cluster and the p value IPA ascribed the function given. The final column gives example transcripts from each functional subdivision. See also Table S1.

Figure 3. Transcript Regulation in the DRG after SNI Follows Distinct Patterns

(A) Similarity plot of the module eigengenes of each cluster showing very little overlap in pattern regulation. (B–E) Representations of the regulation of each cluster given as singles line plots with intensity of regulation on the y axis and time on the x axis. Cluster I (B), clusters II, III, and VI (C), clusters IV and V (D), and clusters VII and VIII (E). Each cluster contains not only genes regulated in the fashion drawn but also reciprocal regulation events. See also Figures S1, S2, and S3 and Tables S1, S2, and S3.

Figure 4. Transcript Expression in the DRG Correlating with the Temporal Profile of Cold Allodynia Was Nociceptor Related, whereas That with Tactile Hypersensitivity Was Immune Cell Centric

Gene expression was correlated with the time courses of cold and tactile allodynia development using the Pearson coefficient of similarity. (A) Heatmap of the relative expression of the probes that most tightly correlate with cold allodynia onset. (B) Relative expression of the probes that most tightly correlate with tactile allodynia. (C and D) Representative functional characteristics using IPA of these cold- (C) and tactile-related (D) transcripts. (E and F) Strongest GO terms for transcripts correlated with cold (E) and tactile (F) allodynia. (G) Cross-comparison of transcripts present in each cluster with transcript lists derived from isolated DRG nociceptors and isolated macrophages/T cells. Orange represents genes contained only in the immune gene list, blue represents genes contained only in the nociceptor list, gray represents genes present in both lists, and white represents genes not contained in either list. See also Tables S4, S5, S6, S7, S8, and S9 and Figure S4.

Figure 5. Trpv1 Lineage Neuronal Deletion Mice Develop Tactile, but Not Cold, Allodynia after SNI Injury

(A) When naive mice are given the choice between two opposing temperatures, wild-type control littermate (LM) mice move toward the more ambient temperature, whereas Trpv1 DTA mice do not. Each point on the graph measures the amount of time spent on plate A in a 30-s window (y axis) when the plate was set to the temperature given on the lower x axis. The top x axis gives the temperature of the alternate plate for that time bin. (B) TrpV1 lineage DTA mice and their LM control counterparts develop tactile allodynia post-SNI. Statistically significant differences between the values from mice after SNI and their basal measures are shown (**p < 0.01). However, there was no significant difference between the two curves (two-way repeated-measures ANOVA). (C) TrpV1 lineage DTA mice develop very weak levels of cold allodynia relative to their LM controls. Statistically significant differences between the values from mice after SNI and their basal measures (**p < 0.01) and between wild-type LM controls and TrpV1 DTA mice in cold sensitivity (##p < 0.01) are shown (two-way repeated-measures ANOVA, Bonferroni post hoc test). For (A), n = 9 (TrpV1-DTA), n = 10 (LM controls); for (B) and (C), n = 8 (both groups). Error bars indicate SEM. See also Figure S5.

Figure 6. Mice Depleted of Peripheral Macrophages/Monocytes Using Clodronate Develop Delayed Tactile Allodynia but Normal Neuropathic Cold Allodynia

(A) Clodronate-treated mice develop markedly less tactile allodynia post-peripheral nerve injury than empty-liposome-treated controls. (B) Clodronate-treated mice develop significant levels of cold allodynia post-SNI. Statistically significant differences between the values from mice after SNI and their basal measure (**p < 0.01) and between mice treated with clodronate or vehicle in tactile allodynia (##p < 0.01) are shown (two-way repeated-measures ANOVA, Bonferroni post hoc test). Clodronate-treated C57BL/6 mice develop significant levels of cold allodynia post-SNI (*p< 0.05, **p<0.01), but there were no significant differences in cold allodynia between clodronate- and vehicle-treated mice (p = 0.323; two-way repeated-measures ANOVA). For (A) and (B), n = 10 clodronate, n = 13 vehicle. (C–E) Levels of myelocytes (CD45⁺CD11b⁺CD11c⁻SiglecF⁻CD3⁻) measured by FACS in blood (C) and DRG (D and E) 7 days after SNI in mice treated with clodronate or vehicle. (F) Mechanical threshold in these mice. For (C)–(F), p values are given (unpaired Student’s t test). (G and H) Iba1 immunoreactivity in the DRG from SNI mice treated with vehicle liposomes (G) or clodronate liposomes (H). Scale Bar: 100 µm. (I–K) Real-time qPCR of the macrophage/monocyte markers CD68 (I), CD11b (J), and CD163 (K) in the DRG of SNI mice treated with liposomes or clodronate. p values are given (n = 5 per group; unpaired Student’s t test). Error bars indicate SEM.

Figure 7. T and B Cell-Deficient Rag1^−/− Mice Develop Normal Neuropathic Cold Allodynia, but Not Complete Tactile Allodynia, following SNI Injury

T cell reintroduction into Rag1^−/− mice abolishes tactile sensitivity differences present between Rag1^−/− mice and wild-type control littermates (LM) but leaves cold allodynia unaltered. (A and B) Immunohistochemistry for the monocyte/macrophage marker IBA1 (red) in noninjured (A) and 7-day SNI-injured DRG in Lck Cre-zsGreen mice, which express labeled T cells (green) (B). Scale Bar: 50 µm. (C) Representative FACS plots of CD4 versus CD8 cell counts from splenic preparation showing cells positive for both markers in the wild-type and in Rag1^−/− and T mice, but not Rag1^−/− mice (top). Representative FACS plots of CD4 versus B220 counts showing the presence of B cells in wild-type LMs, but not Rag1^−/− or reconstituted Rag1^−/− mice. (D) Rag1^−/− mice develop less tactile allodynia post-SNI than their LM controls (Rag versus WT, p = 0.003; Rag versus Rag and T, p = 0.002). Reconstituted Rag1^−/− mice (Rag1^−/− and T) showed full levels of tactile sensitivity (WT versus Rag and T, not significantly different). Significant differences between the values after SNI and their basal measures are shown (**p < 0.01, Rag1^−/−mice versus WT littermates [blue #]; Rag1^−/− mice versus Rag and T [red #]; #p < 0.05, ##p < 0.01, two-way repeated-measures ANOVA, Bonferroni post hoc test). (E) Wild-type LMs, Rag1^−/−, and T cell-reconstituted mice develop similar neuropathic cold allodynia (no significant differences among the three curves). For (D) and (E), error bars indicate SEM (wild-type LM, n = 15; Rag1^−/−, n = 10; Rag1^−/− and T, n = 15).