Phrenic Motor Neuron Survival Below a Cervical Spinal Cord Injury
Authors: Holland Ashley Erin , Santiago Juliet Victoria , Allen Latoya Lynn , Asa Zachary A. , Ciesla Marissa C. , Mitchell Gordon S. , Gonzalez‐Rothi Elisa Janine 
Published: June 2021

Cervical spinal injury (cSCI) severs descending brainstem projections to spinal respiratory motor neurons, paralyzing respiratory muscles caudal to injury. Spinal hemisection at C2 (C2Hx) has frequently been used as an experimental model of cSCI to study both spontaneous and induced functional recovery of respiratory and non‐respiratory somatic motor function. One assumption implicit in these studies is that C2Hx dennervates motor neurons below the injury, but does not cause significant motor neuron pathology or cell death. However, one recent study reported substantial, bilateral motor neuron death caudal to C2Hx (Satkunendrarajah et al, 2016); in this study, motor neurons were identified by ChAT immunoreactivity in the spinal ventral horn. We have also observed that retrograde labeling with Cholera toxin B fragment (CtB) is diminished in phrenic motor neurons following C2Hx (unpublished data) when the tracer was administered post‐C2Hx. However, these observations are potentially misleading since: 1) C2Hx may diminish ChAT immunoreactivity without motor neuron cell death; and 2) retrograde CtB transport may be diminished following injury due to motor neuron inactivity below the injury, leading to the false impression that phrenic motor neurons had died. Because phrenic motor neuron cell death following cSCI would have profound implications for studies of spontaneous and/or induced phrenic motor plasticity after C2Hx, we tested the hypothesis that C2Hx has minimal impact on phrenic motor neuron cell counts when phrenic motor neurons are retrogradely labeled with CtB prior to cSCI. Phrenic motor neurons were retrogradely labeled in intact adult Sprague Dawley rats via intrapleural injections of CtB. Rats were randomly assigned to one of four groups: 1) 2 week post‐C2Hx, 2) 8 week post‐C2Hx, or 3 & 4) age matched uninjured controls. Two weeks after CtB injection, rats in the C2Hx groups underwent left lateral C2Hx injury under isoflurane anesthesia. Rats were perfused at 2 or 8 wks post‐injury, and longitudinal sections (40 μm) from C3 to C8 were processed to reveal CtB and ChAT. CtB labeled phrenic motor neurons were quantified in serial sections; no loss of phrenic motor neurons was observed at either time post‐C2Hx versus spinally intact rats (p>0.05). Analysis of ChAT immunoreactivity is ongoing; however preliminary data suggest minimal changes in phrenic motor neuron ChAT expression following injury. Although we found little evidence for phrenic motor neuron cell death caudal to C2Hx, we cannot rule out greater cell loss in other motor neuron pools. Consequently, ChAT immunoreactivity is currently underway in non‐phrenic cervical motoneurons caudal to C2Hx. These findings have important implications for studies concerning plasticity in cervical motor systems following cervical spinal injury.Support or Funding InformationSupported by: K12HD055929 (EGR), RO1HL69064 (GSM), T32HD043730 (Allen), and grants from the DoD (GSM), the American Physiological Society (AH, JVS), the McNair Scholars Foundation (JVS), and the McKnight Brain Institute (GSM, MCC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.