Cervical Spinal Contusion Alters NKCC1 and KCC2 Expression in Phrenic Motor Neurons

Abstract

Spinal chloride‐dependent synaptic inhibition plays a key role in coordinating breathing. Chloride‐dependent synaptic inhibition requires neuronal chloride gradients established by the balance of the membrane‐bound chloride co‐transporters NKCC1 and KCC2. Spinal transection disrupts the NKCC1/KCC2 balance, diminishing chloride gradients in neurons below the injury, potentially contributing to the spasticity in many spinal injury patients. It is not known if similar disruptions in the NKCC1/KCC2 balance occur in respiratory motor neurons below incomplete cervical contusion injuries. Such disruptions could have dual effects, partially reversing respiratory muscle paralysis, but with the disadvantage of dis‐coordination/spasticity in respiratory muscles. Here, we tested the hypothesis that incomplete, cervical spinal contusion injuries disrupt NKCC1/KCC2 regulation in identified phrenic motor neurons. Further, since some of the same kinases regulating NKCC1 and KCC2 membrane trafficking and gene expression are activated by acute intermittent hypoxia (AIH), we tested the hypothesis that repetitive AIH normalizes membrane NKCC1 and KCC2 expression in phrenic motor neurons after cervical contusion injuries. One week after unilateral C2 contusions (135KD; Infinite Horizons) or sham surgeries, rats were exposed to either normoxia or repetitive AIH (10, 5 min episodes per day, 5 min intervals; 3 days per week) for 4 weeks. All rats received intrapleural injections of Cholera toxin B fragment (CTB) to identify phrenic motor neurons. Immunofluorescence and confocal microscopy were used to reveal NKCC1 and KCC2 protein within CTB‐positive phrenic motor neurons; NKCC1 and KCC2 immuoreactivity in phrenic motor neurons was assessed with a semi‐automated quantification algorithm implemented in MATLAB. First, CTB‐positive phrenic motor neuron somata were identified. Then, optical density was assessed in the cytosol and the presumptive phrenic motor neuron membrane. After unilateral C2 contusions: 1) NKCC1 level at the cell membrane relative to the cytosol increased after cervical spinal contusion both ipsi‐ and contralateral to the injury; and 2) KCC2 levels at the cell membrane relative to the cytosol marginally decreased contralateral to injury (p = 0.07) mainly due to increased cytosolic KCC2 expression on both sides. Repetitive AIH had no significant effects on either protein versus normoxic controls. Thus, unilateral C2 contusion injury exerts complex effects on the NKCC1/KCC2 balance in phrenic motor neurons five weeks post‐injury. These changes may enable at least partial compensation for loss of inspiratory drive to phrenic motor neurons since diminished chloride gradients should increase motor neuron excitability and preserve tidal volume. However, compensation may come at the cost of respiratory muscle coordination, such as sporadic diaphragm activation during expiration. Increased cytosolic KCC2 and decreased cytosolic NKCC1 expression suggest that regulatory mechanisms are working to restore a normal NKCC1/KCC2 balance at this time post‐injury.Support or Funding InformationDoD CDMRP SC120226 and NIH HL69064