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Spinal cord injury (SCI) is a devastating and irreversible injury that impacts thousands of people each year. Most injuries occur at cervical levels, compromising the phrenic motor network, resulting in life‐threatening respiratory deficits. While there is mounting clinical and pre‐clinical evidence for spontaneous plasticity, it is limited, and life‐threatening deficits persist. One strategy for therapeutically enhancing plasticity is the use of activity‐based therapies, such as respiratory and locomotor training. Clinical studies using treadmill training have shown improved respiratory function for individuals with SCI. Therefore, the present work reverse translates these promising clinical findings back to animal models of mid‐cervical SCI to assess the effects of treadmill training on anatomical and functional respiratory plasticity after injury. Adult female Sprague‐Dawley rats (~200g) received a lateralized C3/4 contusive injury. One week post injury, the animals were blindly divided into two groups: untrained or treadmill trained (30 min, once a day, 5 days a week) for 4 weeks. Breathing behavior was measured each week using whole‐body plethysmography. After training completion, all animals were anatomically traced with retrograde and transsynaptic pseudorabies virus (PRV) to the left hemidiaphragm to label the phrenic motor network, ipsilateral to injury. Three days later, animals were anesthetized and diaphragm electromyography (dEMG) was performed to observe ipsilateral diaphragm deficits and respiratory recovery, and animals were perfused for histology. Immunohistological assessment was used to quantify interneuronal connectivity and density of descending serotonergic input to the phrenic motor network. Preliminary data demonstrated increased interneuronal connectivity with injured phrenic motor network, and increased serotonergic input to spinal networks caudal to injury. Electrophysiological assessment of diaphragm function revealed increased diaphragm activity ipsilateral to injury, after 4 weeks of training. Ongoing analyses will further elucidate the therapeutic effects of locomotor training for improving breathing post‐SCI.

Cervical spinal cord injuries (SCI), the most common SCI, compromise respiratory networks leading to life‐threatening impairments in breathing. Following a cervical injury, extensive damage occurs to the phrenic motor network (controlling the diaphragm) consisting of phrenic motoneurons and interneurons. Despite these devastating consequences, there is now significant evidence for plasticity and limited spontaneous recovery. Our ongoing work has identified spinal interneurons (SpINs), in particular the excitatory V2a subtype, as key therapeutic targets for promoting plasticity and functional recovery of the phrenic motor network. Efforts to enhance this plasticity have shown some promise, but it is likely that treatments capable of promoting repair (e.g., cell transplantation) will provide the best therapeutic outcome. Building on a long history of transplanting spinal neural precursor cells (NPCs) to promote spinal cord repair, the present work harnesses advances in stem cell engineering to enrich donor NPCs with specific subsets of stem cell derived SpINs. We hypothesize that NPCs enriched for V2a SpINs will have enhanced therapeutic efficacy over NPCs alone and promote phrenic motor recovery. We will test this hypothesis using a clinically relevant model of a cervical contusion in the adult rat, and test the functional contribution of donor V2a SpINs using inhibitory chemogenetics (hM4Di). Adult female Sprague‐Dawley rats received mid‐cervical contusion injury (Infinite Horizon impactor, intended impact force 200 kilodyne). One‐week post‐injury, aggregates of NPCs with mouse embryonic stem cell derived V2a SpINs were injected into the lesion cavity. One month post‐transplantation, animals were re‐anesthetised and traced with a retrograde, transneuronal tracer, pseudorabies virus, applied to the diaphragm muscle to assess donor‐host connectivity with the phrenic network. Three days later, animals underwent terminal diaphragm electrophysiology, at which time clozapine was injected intraspinally into the transplant to silence donor SpINs. Preliminary data revealed that clozapine‐induced suppression of donor cell activity reduced diaphragm activity ipsilateral to injury, with no measurable effect on the contralateral side. Consistent with this, immunohistochemical assessment revealed donor cells survived and synaptically integrated with the injured host phrenic network. This ongoing research offers the first in‐depth assessment of how these donor SpIN populations contribute to phrenic recovery.

Cervical spinal cord injury (SCI) often leads to damage of the phrenic motor circuit that innervates the diaphragm, resulting in life‐threatening respiratory deficits. While there is some limited spontaneous plasticity after a cervical SCI, severe respiratory deficits persist. Previous research has shown that non‐invasive respiratory training with intermittent hypoxia (IH) can enhance respiratory plasticity after SCI. There are two prominent plasticity promoting pathways associated with IH training, dependent on serotonin or adenosine. Prior work suggests there is greater activation of the serotonergic plasticity‐promoting pathway after inhibiting the adenosine pathway. Therefore, this work aims to use a clinically relevant adenosine antagonist – caffeine – in combination with IH respiratory training to further enhance the respiratory plasticity and diaphragm recovery of adult female rats after a mid‐cervical (C3/4) contusion injury. We hypothesized that 4 weeks of daily IH training primed with 8mg of caffeine, delivered 30 minutes before the training, will enhance the serotonergic plasticity‐promoting pathway to stimulate both anatomical and functional phrenic plasticity and improve respiration post‐SCI. To label phrenic circuitry, 72 hours before terminal experiments, animals were traced with a retrograde, transsynaptic tracer, pseudorabies virus (PRV), applied to the ipsilateral hemidiaphragm to label phrenic moto‐ and interneurons. Anatomical plasticity was assessed using immunohistochemistry to quantify interneuronal connectivity and density of descending serotonergic input to the phrenic motor network. Specifically, this study also investigated the connectivity of a cholinergic interneuron subpopulation within phrenic circuitry after IH training and caffeine delivery. Functional plasticity and respiratory recovery after IH training primed with caffeine were assessed with weekly whole‐body plethysmography and terminal diaphragm electromyography. The results of this study using caffeine as a primer for respiratory training can be easily translated to improve training outcomes and promote greater plasticity and recovery after SCI.