Brainstem and Spinal Cord Pro- and Anti- Inflammatory Cytokines Across the Diurnal Cycle in Rats

Moderate acute intermittent hypoxia (mAIH), consisting of repeated episodes of low inspired oxygen concentration, elicits motor plasticity and is emerging as a promising therapy to promote functional recovery in individuals with neuromuscular injury or disease. For example, mAIH elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF) through a serotonin-dependent mechanism. However, pro-inflammatory cytokines such as IL-1β inhibit serotonin-dependent pLTF via the p38 MAPK pathway. Anti-inflammatory cytokines such as IL-4 and IL-10 inhibit the production of pro-inflammatory cytokines, although their effect on pLTF has not been investigated. Emerging evidence has identified time-of-day as an important regulator of mAIH-induced pLTF. Thus, understanding diurnal cycles in pro- and anti- inflammatory cytokine expression within the brain (i.e., medulla) and spinal cord (i.e., cervical and thoracic) regions that contain respiratory motor pools across the diurnal cycle is important to consider. Prior studies suggest that diurnal cycles in the concentration of cytokines contribute to endogenous cycles in pLTF. To investigate daily rhythms in brain and spinal inflammatory cytokine levels, we utilized sinusoidal COSINOR statistical analysis for modeling oscillations in concentrations over time. Using this model, we hypothesized that the pro-inflammatory cytokines IFN-γ, IL-1α, IL-1β, IL-2, IL-6, IL-12, and TNF-α peak in a time-of-day and tissue-specific manner, whereas the anti-inflammatory cytokines IL-4 and IL-10 have an inverse concentration distribution. We designed an R-based coding algorithm that takes raw concentration values over Circadian Zeitgeber (ZT) time, transforms them into sinusoidal concentration models, and calculates the acrophase (time of peak activity). In young (3-4 months) male Sprague-Dawley rats (n=6 each at ZT0 (lights on), ZT6, ZT12 (lights off), and ZT18), we found that cervical spinal cord cytokines exhibit a rest-active dichotomy, with IFN-γ, IL-1α, IL-1β, IL-2, and IL-4 peaking within the active phase between ZT 0-6 (0.458, 0.552, 1.26, 5.68, and 1.02 hours respectively) and IL-6, IL-10, IL-12, and TNF-α peaking in the rest phase post 18 hour mark (23.4, 18.1, 19.0, and 18.5 hours respectively). Medullary concentration peaks did not express this same dichotomy, with all cytokines peaking between hours 2 and 10 (4.93, 5.22, 4.61, 5.79, 5.20, 4.30, 4.09, 9.28, and 2.64 for IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, and TNF-α, respectively. Thoracic homogenates also did not express the dichotomy with peaks at 5.96, 6.97, 6.00, 6.40, 6.30, 11.6, 9.97, 7.91, and 6.37, respectively. We conclude that daily profiles of inflammatory cytokines differ with respect to tissue type and specific molecules. As we continue to optimize AIH as a therapeutic strategy to promote functional recovery in individuals with neuromuscular injury or disease, it is important to consider the potential implications of diurnal changes in pro- and anti-inflammatory cytokines on AIH efficacy. Supported by: HL149800 and HL148030; NIH K99HL175029 (ABM) 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.