Title: Plasticity induced recovery of breathing occurs at chronic stages after cervical contusion

: Severe mid-cervical contusion injury causes profound deficits throughout the respiratory motor system which last from acute to chronic time points post injury. Here we use chondroitinase ABC (ChABC) to digest chondroitin sulphate proteoglycans (CSPGs) within the extracellular matrix (ECM) surrounding the respiratory system at both acute and chronic time points post injury to explore whether augmentation of plasticity can recover normal motor function. We demonstrate that, regardless of time post injury or treatment application, the lesion cavity remains consistent showing little regeneration or neuroprotection within our model. However, through electromyography (EMG) recordings of multiple inspiratory muscles, we show that application of the enzyme at chronic time points post injury initiates the recovery of normal breathing in previously paralysed respiratory muscles. This reduced the need for compensatory activity throughout the motor system. Application of ChABC at acute time points recovered only modest amounts of respiratory function. To further understand this effect, we assessed the anatomical mechanism of this recovery. Increased EMG activity in previously paralyzed muscles was brought about by activation of spared bulbospinal pathways through the site of injury and/or sprouting of spared serotonergic fibres from the contralateral side of the cord. Accordingly, we demonstrate that alterations to the ECM and augmentation of plasticity at chronic time points post cervical contusion can cause functional recovery the respiratory motor system and reveal mechanistic evidence of the pathways which govern this effect. The present study shows the long-term functional deficits caused by unilateral mid-cervical contusion injury. We demonstrate that, while endogenous plasticity and compensatory function are evident, these are not sufficient to facilitate a robust return of function. We hypothesised that enhancement of plasticity through an exogenously applied treatment strategy would facilitate recovery of respiratory motor function following contusion injury. Indeed, this study provides the first direct comparison of a plasticity inducing treatment strategy for respiratory motor dysfunction at both acute and chronic time points following cervical contusion injury. Here we show that ChABC treatment applied at chronic stages following cervical contusion injury can mediate recovery of ‘normal’ activity within the previously compromised ipsilateral hemidiaphragm, reducing the need for compensatory activity in the other muscles. Alternatively, acute application of the drug only marginally aids recovery of respiratory motor function. The mechanism of this recovery is, in part, mediated through serotonergic sprouting of neuronal pathways. At chronic time points post contusion, this treatment induced recovery partially occurs through the contused respiratory fibres but is also governed through the activation of latent pathways, or sprouting, modulatory axons from the undamaged contralateral side of the spinal cord. These data show that enhancement of plasticity can facilitate recovery of the respiratory motor system following severe mid-cervical contusion if applied at chronic time points following trauma.