Abstract
Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse and facilitate axon retraction away from lesion boundaries. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects regeneration. We used the CSF1R inhibitor, PLX-5622, to deplete microglia and macrophages months after complete crush SCI in female mice. Transcriptional analyses revealed a significant inflammatory depletion within chronic SCI lesions after PLX-5622 treatment. Both transcriptional analyses and immunohistochemistry revealed that Iba1+ cells repopulate to pre-depleted densities after treatment removal. Neuronal-enriched transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with inflammatory depletion alone. Axon densities also significantly increased within the lesion after PLX-5622 treatment and after repopulation. To better examine the effect of chronic inflammation on axon regeneration, we tested PLX-5622 treatment in neuronal-specific PTEN knockout (PTEN-KO) mice. PTEN-KO was delivered using spinal injections of retrogradely transported adeno-associated viruses (AAVrg’s). PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. Axons that grew within the lesion were histologically identified as 5-HT+ and CGRP+, both of which are not robustly transduced by AAVrg’s. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI.
Significance Statement Inflammatory macrophages infiltrate the spinal cord after a spinal cord injury (SCI) and remain at elevated densities around the lesion chronically. Little is known about the consequences of sustained spinal inflammation in chronic SCI, but it is known that macrophages impair regeneration early after SCI. We hypothesized that persistent neuroinflammation may function as a sustained barrier that chronically impairs axon regeneration. Our work identifies that the chronic SCI environment actively maintains increased densities of macrophages and implicates sustained inflammation as a persistent barrier to repair. Findings from our work change our understanding of the maintenance of non-resolving inflammation in chronic SCI and identifies a need to focus on overcoming chronic inflammatory barriers that limit repair after neurotrauma.
Footnotes
No authors report any competing or conflicts of interest regarding this work.
Thank you to the Wings for Life Foundation for providing funding to support this project. Confocal microscopy was performed at the University of Kentucky’s Light Microscopy Core. pAAV-hSyn-Cre-P2A-dTomato was a gift from Rylan Larsen (Addgene viral prep # 107738-AAVrg; http://n2t.net/addgene:107738; RRID:Addgene_107738). pAAV-hSyn-mCherry was a gift from Karl Deisseroth (Addgene viral prep # 114472-AAVrg; http://n2t.net/addgene:114472; RRID:Addgene_114472).
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