Impact of extended normobaric hyperoxia on spinal microglia in the acutely injured vs intact spinal cord

Spinal cord microglia are activated within minutes of spinal cord injury (SCI), contributing to neuroinflammation. After acute SCI, daily treatment with 1-hr exposure to either hyper- or normobaric 100% O 2 can reduce proinflammatory gene expression and attenuate immunohistological markers of spinal inflammation (PMID: 38110993, 35152735). We evaluated microglia in the injured spinal cord after a normobaric hyperoxia (N-H) exposure paradigm of increased duration relative to prior studies. We hypothesized that the N-H paradigm would reduce microglial inflammatory activity. Adult male Sprague-Dawley rats received a 150-Kd lateralized C4 spinal contusion injury. The N-H group (n=9) received 100% O 2 for 3 hours, twice per day, for the first 7 days after SCI. Sham rats (n=8) was exposed to room air. Spinal intact rats were also treated with N-H (n=9) or sham (n=8). Tissue was harvested on day 7 after SCI and CD11b microbeads were used to isolate microglia from rostral segments (C2-C3), the injury epicenter (C4) or caudal (C5-C6) and analyzed by flow cytometry (n=4/group) or were used for RNA extraction and qRT-PCR for gene expression analysis (n=4-5/group). At the injury, microglia from sham SCI rats had elevated levels of the inflammatory molecules CD14 (1.9-fold) and CD45 (2-fold) and decreased levels of MHCII (OX-6, 0.7-fold); values were similar in the N-H group. Analysis of microglial mRNA with qRT-PCR revealed lesion epicenter upregulation of SPP1 (>3000-fold), APOE (>200-fold), CD14 (>150-fold), CXCL17 (>90-fold) and IL1β (3-fold). Caudal to SCI, microglia had increased SPP1 (>60-fold), CD14 (> 6-fold) and IL1β (2-fold). Rostral to SCI, SPP1 was increased >180-fold. Expression of these genes in the N-H group was similar in the rostral cord and epicenter. However, in the caudal spinal cord IL1β (3.8-fold), CD14 (>20-fold) and CXCL17 (>20-fold) were increased after N-H. In general, the O 2 treatment had an opposite impact on spinal intact rats with 2- and 4-fold decreased inflammatory gene expression in N-H vs. sham group. In additional experiments, cytokines were evaluated in spinal homogenates using Q-Plex ELISA (same spinal segments as above). In sham treated SCI rats IL1β protein was elevated in all regions (1.3 – 2.5-fold) whereas IL6 and TNFα were decreased by 40%. Values were similar in the N-H group. In spinal intact rats, O 2 treatment decreased IL6 (by 30%) and TNFα (by 40%) but IL1α and ILβ were unaffected. In summary, in adult rats with C4 contusion a daily 2x3 hour N-H paradigm did not reduce spinal neuroinflammation assessed on day 7 post-SCI. These findings do not support our hypothesis and we conclude that this increased duration N-H protocol does not offer further benefits, at least regarding the neuroinflammatory response. Our data also reveal differences in the sensitivity of the intact vs. injured spinal cord to 100% O 2 exposure. Funding: R01NS139422 (DDF and PB) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.