Clearing Up the Phrenic Motor Neuron Survival Debate After Cervical Spinal Cord Injury

Abstract

The diaphragm is the major muscle involved in breathing. Innervated by the phrenic nerve, it is controlled by phrenic motor neurons (PMNs), which receive descending inputs from the medulla. When these bulbospinal-pathways are damaged or severed in spinal cord injury (SCI), the external effects of injury are seen immediately, as the diaphragm becomes paralyzed and the individual loses the ability to breathe. However, the effect of injury on the internal circuitry, specifically PMN survival, is largely unknown. Contradictory evidence has surfaced, suggesting that there is large PMN death after injury, or conversely, that there is an absence of PMN death. However, histological techniques utilized in these studies have exposed the data to factors through which certainty cannot be guaranteed. These discrepancies are important to parse out because characterization of PMN survival is integral to studies of plasticity. The present study attempted to bridge this gap in knowledge and used XClarity clearing methods to accurately determine PMN survival after cervical SCI. XClarity transforms the tissue into a transparent medium. This allows for the whole spinal cord to be analyzed without tissue loss, as is common in other histological techniques. In this study, Sprague-Dawley rats were hemisected at the second level of the cervical spinal cord (c2Hx), which is a common experimental model of cervical SCI. Animals were divided into three groups: naïve, two weeks post-c2Hx, and five weeks post-c2Hx. Before perfusion, these animals were intrapleurally injected bilaterally with CTB-488, a retrograde tracer that labels PMNs. Depending on their group assignment, animals were perfused at five weeks post-injury, two weeks post-injury, or immediately after CTB-488 uptake. Cords were then processed with XClarity and PMN survival was characterized with Lightsheet microscopy. Analysis of PMNs is ongoing, however, preliminary data suggests that XClarity techniques are the preferable route to characterize PMN survival after injury