Serotonin supplementation and respiratory motor-based optimization of intermittent hypoxia procedure improve expression of long-term facilitation in adult rats
Authors: Silverstein Aaron , Alilain Warren 
Published: May 2023

Spinal cord injury (SCI) most commonly occurs at the cervical level and often renders affected individuals unable to breathe. There is no current clinical treatment capable of functionally restoring independent respiratory function once it is lost, but intermittent hypoxia (IH) is a promising therapeutic approach that can induce increased breathing motor output called long term facilitation (LTF). However, IH treatment must be improved to become standard of care. All IH protocols to date have been defined by periods of alternating exposure to pre-determined durations of low inspired oxygen (hypoxia) and normal oxygen irrespective of subjects’ response. There is significant variability of LTF expression induced by standard IH (Std-IH) treatment in many preclinical and clinical studies and the mechanism underlying some subjects’ “non-response” is unknown. Mechanistically, LTF requires intraspinal serotonin (5-HT) during IH treatment and the amount of 5-HT present within the phrenic motor nucleus predicts LTF magnitude. We first hypothesized that LTF non-response after Std-IH was due to insufficient 5-HT within the spinal cord. Second, since the amplitude of each subject’s hypoxic phrenic response during Std-IH positively correlates with LTF magnitude, we hypothesized that IH could be optimized through defining treatment by the amplitude of each animal’s hypoxic diaphragm response (HDR). To address these hypotheses, we first supplemented adult, uninjured rats with intrathecal 5-HT during each hypoxic period of Std-IH and compared LTF expression with saline-treated control. Next, we investigated whether the amplitude of the hypoxic response would still predict LTF magnitude when measured by diaphragm electromyography (diaEMG) in anesthetized, non-ventilated, and non-vagotomized rats. Then, we evaluated the efficacy of a novel respiratory motor-based IH (RMB-IH) protocol wherein the length of hypoxic exposure was defined by the magnitude of each subject’s HDR measured by diaEMG. Consistent with our hypothesis, early results demonstrated that 2/2 5-HT-treated subjects exhibited LTF averaging ~5% above baseline whereas only 1/3 saline-treated animals exhibited LTF (~6% above baseline) and the two non-responders averaged ~25% below baseline. In accordance with previous findings utilizing phrenic neurograms, our linear regression of diaEMG indicated a positive correlation of HDR and diaphragm LTF (R2 = 0.6138, p = 0.0073, n = 10). Lastly, our novel RMB-IH treatment successfully induced LTF in 2/2 subjects (~19% above baseline) while Std-IH induced LTF in only 2/5 subjects (~9% above baseline) and the three Std-IH non-responders exhibited an average amplitude decrease of ~46%. Altogether, these data contribute to a better mechanistic understanding of the factors which affect IH success and introduce promising interventions able to improve the consistency and magnitude of LTF, thus enhancing vital efforts to restore independent breathing function after SCI. NIH R01 NS101105 to Warren J. Alilain, PhD; NIH T32 AA027488 to Aaron L. Silverstein, BS BS (PIs Mark T. Fillmore, PhD and Mark A. Prendergast, PhD) This is the full abstract presented at the American Physiology Summit 2023 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.