Development of a Phrenic Nerve Injury Model in Rats to Study Axonal Regeneration and Compensatory Respiratory Neuroplasticity
Authors: Ross Ashley , Kelly Mia N. , Perim Raphael R. , Mitchell Gordon S. 
Published: April 2020

Phrenic nerve injury (PNI) is an unintended consequence of some clinical procedures, including heart and lung transplants. Injury can occur in multiple ways, including an accidental stretching, heating via an electrocautery device or (less often) crushing the phrenic nerve. These types of phrenic nerve damage can severely impair function of the ipsilateral diaphragm, causing respiratory impairments. Currently, the strategy to resolve phrenic nerve injuries is to wait for spontaneous recovery, leaving patients compromised for prolonged periods. Thus, there is a need to study PNI and mechanisms of recovery to guide new treatments that would accelerate this process. When studying regrowth after peripheral nerve injuries, it is essential to report complete and sustained loss of axons and, therefore, function. Although nerve crush injury models have been described, there is high variability in the completeness of injury between published methods. Thus, the purpose of this study was to: 1) develop a ventral approach to perform reproducible PNI; 2) identify a surgical tool that produces a robust and persistent loss of phrenic nerve and ipsilateral diaphragm muscle function; and 3) monitor acute compensatory responses in diaphragm and intercostal muscle activities. We monitored the functional extent of phrenic nerve crush injury via ipsilateral diaphragm EMG recordings in anesthetized, spontaneously breathing rats (n=6). Since we are also interested in mechanisms of compensation immediately following injury, we monitored compensatory responses in contralateral diaphragm EMGs (n=6) and bilateral intercostal muscle EMGs (n=1). We performed the nerve crush on the right phrenic nerve since: 1) the right phrenic nerve has ~20% more axons than the left (Song et al., 1999); and 2) during human lung transplantation, the right phrenic nerve is injured more often (Sheridan et al., 1995). PNI induced with a serrated, micro‐mosquito hemostat crush (n=1) resulted in spared nerve activity, as shown by an initial decrease in phrenic activity followed by immediate recovery of 50% compared to baseline. PNI induced with Dumont 3C forceps (n=5) induced a sustained decrease in phrenic activity (>1 hour). A major goal of this project is to study variability in compensation from spared nerve activity/function. Since it is well documented that the contralateral diaphragm compensates for loss of ipsilateral diaphragm function, we were interested in mechanisms of compensation during a PNI. We observed an average increase of 15% in activity in the contralateral diaphragm immediately after phrenic nerve crush (n=3). Additionally, we observed a 30% increase in ipsilateral intercostal activity (n=1). Arterial blood samples taken 2, 30, and 60 minutes post‐crush (n=3) indicated increasing PaCO2 over time post‐PNI (~55 mmHg immediately post‐crush to ~70 mmHg one hour post‐crush). Increasing PaCO2 may recruit silent but spared neural pathways in the affected nerve and/or recruit activities in the contralateral diaphragm and accessory muscles. This model of PNI will enable studies concerning cellular mechanisms promoting phrenic nerve regeneration post‐injury, and new ways to accelerate that growth.

Support or Funding Information

NIH R01 HL148030, T32 HL134621 (MNK), and the UF McKnight Brain Institute.