Longitudinal Assessment of Outcome Measures of Diaphragm Motor Unit Connectivity As Biomarkers of Phrenic Motor Unit Degeneration and Compensation

Abstract

Phrenic motor neurons (MNs) form the final link from the central nervous system (CNS) to the diaphragm muscle. Ventilatory function is modulated by the CNS via groups of muscle fibers each controlled by a single MN (motor units). Processes such as injury, aging, and neurodegeneration can result in losses of MNs, and their motor unit (MU) counterparts, but MNs and MUs have some capacity for compensation through mechanisms such as axonal sprouting and collateral sprouting to regain connectivity with denervated muscle. Motor unit number (motor unit number estimation, MUNE), MU size (single motor unit potential amplitude, SMUP), and summated muscle excitation (compound muscle action potential amplitude, CMAP) have been used clinically and preclinically for decades to track MU connectivity but efforts have largely focused on limb muscles. We aimed to apply these measures to a model of phrenic MN degeneration (intrapleural injection of cholera toxin B fragment conjugated to saporin, CTB-SAP).We hypothesized that CTB-SAP-induced phrenic motor neuron death would result in loss of diaphragm MU number and compensatory increases in MU size. Under isoflurane anesthesia, rats were bilaterally, intrapleurally injected with CTB-SAP (25 μg) to target phrenic MNs and extra CTB (25 μg) to label surviving phrenic MNs, and control rats received unconjugated CTB (25 μg) and SAP (25 μg). Diaphragm CMAP, SMUP, and MUNE were acquired bilaterally and averaged at baseline/pre-injection (n=14), as well as 7 (n=14), 14 (n=14), 21 (n=6), and 28 (n=14) days post-injection. No baseline differences between groups were found. MUNE showed a significant change for time (p<0.05), treatment (p<0.001), and interaction time x treatment (p<0.01) with ~40% reduction of MUNE in CTB-SAP rats. Average SMUP of CTB-SAP rats demonstrated a significant change with time (p<0.05) and treatment (p<0.01), but no significant interaction with ~50-60% increase in SMUP amplitude. CMAP showed no significant change. Our results show a significant ~40% loss of MUs in CTB-SAP rats, similar to previous studies on morphological MN counts. In parallel with MU loss, MUs enlarged (increased SMUP), consistent with compensatory increases of MU size likely through MU collateral sprouting. In conclusion, our findings suggest that the use of diaphragm MU electrophysiological measures can provide insights into the onset and progression of CTB-SAP-induced pathology concerning the loss of phrenic MU integrity. Hence, these biomarkers can be used to investigate mechanisms of phrenic motor neuron degeneration and compensation, and to expedite the translation of experimental findings to clinical testing. Supported by a Spinal Cord Injury and Disease Research grant. 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.