Differential Responses of Spinal Microglia to Low-Dose LPS Delivered in the Daily Rest vs Active Phase

Abstract

Virtually all lung and neural disorders are associated with systemic and/or neuro-inflammation. Neuro-inflammation impairs important forms of neuroplasticity associated with cognitive and spinal motor learning ( e.g., respiratory motor plasticity). Microglia are resident CNS innate immune cells that regulate diverse processes including neuroinflammation and multiple forms of neuroplasticity via neuron-microglia interactions. Brain microglia exhibit profound diurnal rhythms in their response to pro-inflammatory stimuli, such as toll-like receptor 4 (TLR4) activation. Although hippocampal microglia develop a pro-inflammatory phenotype in response to TLR4 activation during the daily rest phase, little is known concerning spinal microglial responses to inflammatory stimuli across the daily rest/active cycle. Thus, we tested the hypothesis that ventral spinal microglia near the phrenic motor nucleus differentially respond to low-dose systemic lipopolysaccharide (LPS) delivered in the rest (12PM) vs active (12AM) phase. Specifically, we investigated microglial responses to systemic inflammation induced by low-dose LPS (0.1mg/kg, i.p.) in 2 populations of adult Sprague-Dawley rats (both 3-6 months old): 1) SD wildtype and 2) SD rats with humanized neuro-neutral apolipoprotein 3 (h APOE3) knock-in. All rats received intrapleural Cholera toxin B (CtB) injections 2 weeks prior to harvest to identify phrenic motor neurons. Twenty-four hours post-LPS or vehicle (0.9% saline), rats were perfused with 1X PBS and fixed with 4% PFA. Spinal tissues containing the phrenic motor nucleus (cervical 3-6) were harvested and dehydrated in 30% sucrose for immunohistochemistry. Activated microglia near phrenic motor neurons were visualized with an Iba-1 antibody and quantified and phenotyped based on morphology. Since no differences in microglia were found between wildtype and h APOE3 rats, data were combined into a single group for analyses. In vehicle-treated rats, robust Iba-1 staining near phrenic motor neurons was observed in the active, but not rest phase. In contrast, Iba-1 staining in LPS treated rats was elevated only when LPS was administered in the rest (not active) phase. After rest-phase LPS injections, microglial cell bodies were larger and had more processes compared to rest-phase controls. In the active phase, spinal microglia were similar between vehicle and LPS injected rats. Thus, spinal microglia exhibit a profound diurnal rhythm in (morphological) responses to systemic inflammation ( i.e. LPS). Further studies are needed to identify specific molecules driving time-of-day differences in spinal microglial responses to pro-inflammatory stimuli. Nevertheless, these findings increase our understanding of how diurnal rhythms impact cellular responses to physiological challenges, such as inflammation. Further, our results yield clues concerning diurnal variations in phrenic motor plasticity, since reciprocal phrenic motor neuron/microglial communication is an important regulator of phrenic motor plasticity. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.