Background & Aims

In pathology such as chronic pain, microglia and astrocytes change states and become “reactive”. Under normal conditions, astrocytes regulate the homeostatic environment. However, little is known about how astrocytic homeostatic functions are changed in acute and chronic pain. We previously observed in a preclinical model of complex regional pain syndrome (CRPS) that spinal cord astrocytes increase expression of glial fibrillary acidic protein (GFAP) post-fracture, which is thought to indicate a reactive astrocyte state. However, short-term depletion of microglia also increases astrocytic GFAP but paradoxically reduces pain [1]. We hypothesize that microglia depletion may alter astrocyte state differently than injury, leading to a pro-homeostatic astrocyte phenotype and reduction of pain.

Methods

In this study, we used qPCR of lumbar spinal cord tissue to track the expression of genes related to astrocytic homeostatic functions in a model of the condition complex regional pain syndrome (CRPS). To model CRPS, we performed a closed tibial fracture followed by casting for three weeks. Tissue was taken at two timepoints from lumbar spinal cords from male mice: 24 hours and 5 weeks post-fracture. To look at the effect of microglia depletion/repopulation, we used a transgenic mouse model in which microglia were ablated by three daily injections of diphtheria toxin. Depletion was performed at 3 weeks post-fracture, then lumbar spinal cord tissue was collected at 5 weeks post-fracture. To determine the effects of microglia depletion in the absence of pain, we also collected tissue 24 hours after diphtheria toxin dosing in uninjured mice1.

Results

We find significant increases in RNA for glutamate transporters (Slc1a3), potassium channels (Kcnj10), glial fibrillary acidic protein (Gfap) and gap junctions (Gja1) by 24 hours post-fracture compared to uninjured mice. We find significant decreases in Slc1a3 and Kcnj10 compared to controls at the chronic, five week timepoint. We have previously shown that depletion and repopulation of microglia at 3 weeks post-fracture reduced chronic pain by 5 weeks post-fracture [1]. Interestingly, when we looked at astrocyte homeostatic genes at the 5-week post-fracture timepoint in mice where microglia were depleted at 3 weeks, there were not significant decreases in Slc1a3 or Kcnj10 compared to uninjured controls. In contrast, there are increases in RNA for GFAP and Kcnj10, but not for Gja1 or Slc1a3 by 24 hours after microglia depletion in uninjured mice.

Conclusions

Taken together, these results indicate that astrocyte function is differentially altered at acute versus chronic time points after injury, and after microglia depletion. We also show a recovery of astrocytic homeostatic genes after microglia depletion, suggesting repopulated microglia may normalize astrocyte homeostatic function. Future studies will examine astrocyte function through ex vivo calcium imaging of the intact mouse spinal cord after peripheral injury. These experiments will provide additional characterization of alterations in astrocyte function in chronic pain models as well as with microglia depletion.

References

[1] Donovan, L. J., Bridges, C. M., Nippert, A. R., Wang, M., Wu, S., Forman, T. E., Haight, E. S., Huck, N. A., Bond, S. F., Jordan, C. E., Gardner, A. M., Nair, R. V., & Tawfik, V. L. (2024). Repopulated spinal cord microglia exhibit a unique transcriptome and contribute to pain resolution. Cell reports, 43(2), 113683.

Presenting Author

Amy R Nippert

Poster Authors

Amy Nippert

PhD

University of Minnesota

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology