Utilization of pectoralis minor accessory inspiratory muscles in a rodent model of respiratory motor neuron loss

Abstract

Ventilatory failure, and ultimately death, occurs in patients with neuromuscular diseases due to the loss of respiratory motor neurons (e.g., phrenic and intercostal). Prior to ventilatory failure, patients are able to maintain breathing potentially via recruitment of accessory inspiratory muscles (e.g., pectoralis minor, which are responsible for lifting the ribs upward and outward to move the chest wall following increased ventilatory demand). Since genetic rodent models of motor neuron loss develop global symptoms (e.g., dysphagia, etc.), we have developed an inducible model of only respiratory motor neuron death in order to study how breathing is impacted, and to ultimately develop targeted therapeutic interventions. Briefly, adult male rats are intrapleurally injected with cholera toxin B conjugated to saporin (CTB-SAP) that is retrogradely transported to the spinal cord, resulting in selective elimination of phrenic and intercostal motor neurons. Despite deficits in maximal ventilatory capacity and diaphragmatic output following intrapleural CTB-SAP, eupnea is maintained at 7 days (d) and 28d post-injection. Thus, we hypothesize that pectoralis minor accessory inspiratory muscle activity is increased to maintain eupnea over the course of respiratory motor neuron loss in CTB-SAP rats. To test our hypothesis, we intrapleurally injected adult male rats with bilateral CTB-SAP or control (CTB unconjugated to SAP), and 7d and 28d later studied: 1) pectoralis minor motor neuron survival using immunohistochemistry; and 2) pectoralis minor output at baseline and in response to a maximal ventilatory challenge (hypercapnia + hypoxia; max) in anesthetized, spontaneously breathing rats (n=15/group) via electromyography. Thus far, our data suggests that although the pectoralis minor motor nucleus is in close proximity to the phrenic motor nucleus, these pools are exclusive from each other and pectoralis minor motor neuron survival is unaffected following intrapleural CTB-SAP (p>0.05). Additionally, pectoralis minor muscle amplitude is significantly increased in 7d and 28d intrapleurally injected CTB-SAP rats vs. controls (p<0.05) and in 28d vs. 7d CTB-SAP rats at baseline and max (p<0.05). To test whether pectoralis minor muscles are necessary for maintaining eupnea, we evaluated breathing via whole-body plethysmography in unanesthetized adult male rats following bilateral intrapleural and intramuscular pectoralis minor muscle CTB-SAP injections vs. controls (n=3/group). Our preliminary data suggests that eupneic frequency is increased, but tidal volume is decreased in 7d CTB-SAP rats vs. controls, and our 28d experiments are currently being conducted. In conclusion, our data suggests that the pectoralis minor muscles have an independent motor pool that can become recruited to assist in maintaining eupnea following intrapleural CTB-SAP-induced respiratory motor neuron loss. This study furthers our understanding of the contribution of the pectoralis minor muscles to maintaining eupnea following respiratory motor neuron loss, and suggests that these muscles could potentially be further stimulated and harnessed to significantly prolong and/or improve breathing in patients with neuromuscular disease.