Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord

Abstract

Background: CRPS is a painful, debilitating, and often-chronic condition characterized by various sensory, motor, and vascular disturbances. Despite many years of study, current treatments are limited by our understanding of the underlying mechanisms. Little is known on the molecular level concerning changes in gene expression supporting the nociceptive sensitization commonly observed in CRPS limbs, or how those changes might evolve over time.

Results: We used a well-characterized mouse tibial fracture/cast immobilization model of CRPS to study molecular, vascular and nociceptive changes. We observed that the acute (3 weeks after fracture) and chronic (7 weeks after fracture) phases of CRPS-like changes in our model were accompanied by unique alterations in spinal gene expression corresponding to distinct canonical pathways. For the acute phase, top regulated pathways were: chemokine signaling, glycogen degradation, and cAMP-mediated signaling; while for the chronic phase, the associated pathways were: coagulation system, granzyme A signaling, and aryl hydrocarbon receptor signaling. We then focused on the role of CcL2, a chemokine that we showed to be upregulated at the mRNA and protein levels in spinal cord tissue in our model. We confirmed its association with the nociceptive sensitization displayed in this model by demonstrating that the spinal but not peripheral administration of a CCR2 antagonist (RS504393) in CRPS animals could decrease mechanical allodynia. The spinal administration of CcL2 itself resulted in mechanical allodynia in control mice.

Conclusions: Our data provide a global look at the transcriptional changes in the spinal cord that accompany the acute and chronic phases of CRPS as modeled in mice. Furthermore, it follows up on one of the top-regulated genes coding for CcL2 and validates its role in regulating nociception in the fracture/cast model of CRPS.

Figure 1

Physiological and behavioral changes in CRPS mice. CRPS mice display increased temperature (A) and edema (B) on the affected hindpaw at 3 weeks post-fracture. In addition, they show signs of mechanical allodynia (C) and decreased weight bearing (D) for up to 7 weeks after fracture. **p<0.01, *** p<0.001. n=8/group. Errors bars=S.E.M.

Figure 2

Canonical pathway analysis. CRPS affects transcriptional programs unique to each of the acute and chronic timepoints. All pathways were scored and ranked according to Ingenuity Pathway Analysis using Fisher’s exact test. The dotted line indicates the threshold value of p<0.05.

Figure 3

Transcriptional pathway analysis and validation of transcript mRNA expression. IPA identified interacting networks affecting cell signaling. Up-regulated transcripts are marked with red (A). SPRR1a, GAL, and PDYN transcripts were validated by quantitative PCR. The dotted line indicates control measures (B). *** p<0.001. n=4/group. Errors bars=S.E.M.

Figure 4

Increase in spinal CcL2 mRNA and protein levels in CRPS mice. 3 weeks following fracture, CRPS mice show a significant increase in spinal CcL2 levels at the mRNA (A) and protein (B) levels, compared to both control (*) and the 7-week timepoint (#). *** p<0.001. n=4/group. Errors bars=S.E.M.

Figure 5

Amelioration of mechanical allodynia in CRPS mice following intrathecal, but not intraplantar, administration of the CCR2 antagonist, RS504393. Intrathecal RS504393 (3 μg) resulted in increased mechanical thresholds in the affected paw compared to vehicle-treated (*) and control mice (#). This was true for both the 3- (A) and 7-week (C) timepoints. Intraplantar injections failed to show any efficacy (B,D). * p<0.05; ** p<0.01.n=8/group. Errors bars=S.E.M.