Respiratory Response‐Based Intermittent Hypoxia Treatment Leads to Diaphragmatic Long Term Facilitation in Adult Rats

Abstract

Spinal cord injury (SCI) most commonly occurs at the cervical level and can lead to severe respiratory dysfunction such as paresis of the diaphragm, the primary mammalian inspiratory muscle. Intermittent hypoxia (IH) treatment (tx) can be applied to induce a prolonged increase in breathing motor output, known as long term facilitation (LTF), which is achievable following SCI in humans. However, this does not potentiate following multiple days of tx, indicating a need for optimization of existing IH protocols. Many seminal experiments demonstrating LTF in uninjured animals employ phrenic nerve recordings to measure respiratory response to moderate IH protocols consisting of 3, 5‐minute‐long periods of hypoxia (~11% O2) interspersed with periods of normoxia (~20.9% O2). This tx increases respiratory motor drive, stimulating serotonin (5‐HT) and growth factor‐dependent plasticity within the cervical spinal cord. Importantly, many studies have shown that the pattern and severity of IH dramatically affect LTF expression. Indeed, fundamental literature indicates that the details of 5‐HT‐based or other stimulation protocol are crucial for induction of plasticity across multiple phenomena, species, and preparations. In these studies, real‐time verification of the stimulus’ effect is key. Since LTF is a neuroplastic paradigm which depends on sequelae of effective stimulation of respiratory drive, we constructed an IH protocol which adapts to the animal’s real‐time respiratory response during treatment. We hypothesized that novel, respiratory response‐based (RRB) IH tx would induce a greater degree of diaphragmatic (dia) LTF than would standard fixed duration (FD) tx in uninjured adult rats. To develop RRB treatment, we first derived an initial setpoint from previous EMG/IH experiments in rats which reflected the magnitude of diaEMG amplitude increase expressed during hypoxic exposure. We then utilized this setpoint during each of three IH cycles such that the animal would qualify for progression from hypoxic to normoxic exposure by expressing a sufficiently robust diaEMG amplitude increase with respect to output measured in previous normoxic conditions. To evaluate our hypothesis, we conducted diaphragm EMG recordings in isoflurane‐anesthetized Sprague‐Dawley retired breeder rats before, during, and after three cycles of either moderate RRB or FD tx administered by nose cone. Preliminary data demonstrated that RRB treatment (n=2) resulted in LTF reaching 113.2% and 130.8% of baseline diaphragm amplitude for the two subjects at least 1‐hr. post‐IH. Further elucidation of appropriate hypoxia‐induced diaphragm amplitude setpoints is a necessary next step, followed by completion of greater numbers of both RRB and FD treatments to achieve adequate power for statistical comparison before applying RRB to the rat C2 hemisection injury model to explore the treatment’s utility in restoring breathing motor function once lost.