Phrenic Motor Neuron Survival Caudal to C2 Hemisection

Abstract

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 both respiratory and non‐respiratory somatic motor function after cSCI. 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that retrograde labeling with Cholera toxin B fragment (CtB) is diminished in phrenic motor neurons following C2Hx (unpublished data). However, these observations are potentially misleading since: 1) C2Hx may diminish ChAT immunoreactivity without motor neuron cell death; and 2) retrograde transport of CtB may be diminished when administered following an injury, due to the relative inactivity of motor neurons below the injury, giving 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 if phrenic motor neurons are retrogradely labeled with CtB PRIOR to spinal injury. 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 C2Hx, 2) 8 week C2Hx, or 3) age matched, spinal intact controls. One week following the CtB injection, rats in the C2Hx groups underwent a left lateral C2Hx injury under isoflurane anesthesia. Rats were then perfused at 2 or 8 weeks post‐injury, and longitudinal sections (40 μm) from C3 to C8 were processed to reveal CtB, ChAT and NeuN. CtB labeled phrenic motor neurons were quantified using a custom MATLAB code. Preliminary analyses suggest minimal loss of phrenic motor neurons at either time post‐C2Hx relative to spinal intact rats. Assessment of ChAT and NeuN immunoreactivity in phrenic motor neurons after C2Hx is ongoing. 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. These findings have important implications for studies concerning motor plasticity following cervical spinal injury.Support or Funding InformationSupported by: T32HD043730, K12HD055929 (EGR), RO1HL69084 (GSM), and grants from the DoD (GSM), and the McKnight Brain Institute (GSM, MCC)