Spinal cord injuries (SCI) often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. Unfortunately, the untoward effects of impaired breathing are a leading cause of SCI-related death, underscoring the importance of developing strategies to restore respiratory activity. Here we show that after cervical SCI, there is upregulation of the perineuronal net (PNN) associated chondroitin sulfate proteoglycans (CSPGs) around phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with Chondroitinase ABC (ChABC) can, by itself, promote plasticity of spared tracts and restore limited activity to the paralyzed diaphragm. However, when combined with application of a peripheral nerve autograft, ChABC treatment results in lengthy regeneration of serotonergic axons and other bulbospinal fibers with remarkable recovery of diaphragm function. Following recovery and initial transection of the bridge, there occurs an unusual, overall increased tonic diaphragmatic EMG activity, suggesting considerable remodeling of spinal cord circuitry after regeneration. This is followed by complete elimination of the restored activity proving that regeneration is critical for the return of function. Overall, these experiments present a way to profoundly restore function of a single muscle following debilitating CNS trauma, through both plasticity of spared tracts and regeneration of essential pathways.

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or -inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer (ePet-EYFP) or cortical neurons were cultured on low amounts of laminin +/− relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of GAP-43 and/or β1 integrin than cortical neurons. Blocking β1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.