Background & Aims

Astrocytes are key regulators of neuronal signaling in physiological conditions and can drive neuroinflammation in pathological states, such as chronic pain. Spinal cord astrocytes become reactive in various mouse models of injury; however, we lack a nuanced understanding of how astrocytes may be functioning differently throughout injury progression. Complex Regional Pain Syndrome (CRPS), a chronic pain condition that develops following limb fracture, is an ideal model for studying the temporal response of astrocytes to injury because it consists of two phases: an acute phase characterized by peripheral inflammation followed by a refractory chronic phase characterized by central sensitization.1,2 By examining astrocyte gene expression changes in these distinct post-injury phases, we can better understand the transition from acute to chronic pain and uncover novel astrocyte-specific therapeutic targets.

Methods

As a model of CRPS, we performed right hindlimb closed tibial fracture followed by cast-immobilization for 3-weeks in adult male and female mice. We utilized translating ribosome affinity purification (TRAP) technology to isolate and sequence the astrocyte translatome at 3-weeks, 5-weeks, and 7-weeks post-fracture. Specifically, we utilized inducible CreERT2 driven by the pan-astrocytic promoter Aldh1l1 to induce HA-tag expression on translating ribosomes only in astrocytes (Aldh1l1-CreERT2; Rpl22HA). Raw sequencing data were passed through nf-core RNAseq (v3.12.0) pipeline and normalized using variance-stabilizing transformation. We then performed weighted gene co-expression network analysis (WGCNA) to identify gene modules, or networks of genes with coordinated expression patterns. Modules were correlated to traits of interest (such as post-injury timepoint) and significantly associated modules were further investigated.

Results

WGCNA captured eight gene modules that were significantly correlated to post-injury timepoints. Of these significant modules, five modules correlated with acute (3-week) CRPS and three modules correlated with chronic (5- and 7-week combined) CRPS, indicating acute and chronic phase-specific astrocyte response profiles. We then examined individual genes within each module by quantifying Gene Significance (GS), or the correlation between a gene and the timepoint of interest. Genes in a significant module with high absolute value of GS were identified as highly important to that module. For example, the canonical astrocyte connexin gene Gja1 was identified as a highly important gene significantly correlated with acute CRPS, and thus warrants further exploration as a driver of the acute injury-induced astrocyte response.

Conclusions

Using an unbiased data-reduction analysis tool, we have identified time-specific gene expression changes in astrocytes in acute and chronic CRPS, implicating these cells as potential regulators of CRPS development and maintenance. Our future studies will further explore candidate genes as potential therapeutic targets in CRPS. Overall, our findings represent the first comprehensive investigation of temporal astrocyte gene changes in chronic pain.

References

1. Birklein F, Ibrahim A, Schlereth T, Kingery WS. The Rodent Tibia Fracture Model: A Critical Review and Comparison With the Complex Regional Pain Syndrome Literature. J Pain. 2018;19(10):1102.e1-1102.e19. doi:10.1016/j.jpain.2018.03.018
2. Bruehl S, Maihöfner C, Stanton-Hicks M, et al. Complex regional pain syndrome: Evidence for warm and cold subtypes in a large prospective clinical sample. Pain. 2016;157(8):1674-1681. doi:10.1097/j.pain.0000000000000569

Presenting Author

Janelle Siliezar-Doyle

Poster Authors

Janelle Siliezar-Doyle

Bachelor of Science

Stanford University

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology