A Latent Propriospinal Network Can Restore Diaphragm Function After High Cervical Spinal Cord Injury

Spinal cord injury (SCI) above cervical level 4 disrupts descending axons from the medulla that innervate phrenic motor neurons, causing permanent paralysis of the diaphragm. Using an ex vivo preparation in neonatal mice, we have identified an excitatory spinal network that can direct phrenic motor bursting in the absence of medullary input. After complete cervical SCI, blockade of fast inhibitory synaptic transmission caused spontaneous, bilaterally coordinated phrenic bursting. Here, spinal cord glutamatergic neurons were both sufficient and necessary for the induction of phrenic bursts. Direct stimulation of phrenic motor neurons was insufficient to evoke burst activity. Transection and pharmacological manipulations showed that this spinal network acts independently of medullary circuits that normally generate inspiration, suggesting a distinct non-respiratory function. We further show that this “latent” network can be harnessed to restore diaphragm function after high cervical SCI in adult mice and rats

Balancing Neuroprotection with Functional Recovery: The Role of the Perineuronal Net in Preventing Excitotoxicity after Spinal Cord Injury

In spinal cord injury (SCI), initial mechanical trauma causes debilitating primary damage to neural cells and blood vessels. Following this, secondary cascades of downstream events occur, including inflammation, ischemia, and excitotoxicity — an increase in intracellular Ca2+ concentration from overactive glutamate (Glu) receptor activity leading to cell death. Additionally, there is an upregulation of the perineuronal net (PNN), a lattice-like structure of the extracellular matrix which modulates neural communication and homeostasis. The PNN is partially composed of negatively charged chondroitin sulfate proteoglycans (CSPGs). While the PNN and CSPGs can support plasticity and neuronal growth during development, after injury these ECM molecules are inhibitory to regeneration, sprouting and plasticity. However, administration of the bacterial enzyme chondroitinase ABC (ChABC) can digest these inhibitory factors and promote functional recovery. What remains unknown is the impact of removing these inhibitory factors soon after injury. We hypothesize that negatively charged CSPGs are upregulated after SCI as a neuroprotective response that attenuates excitotoxicity by acting as a sink for Ca2+. To test our hypothesis, we induced excitotoxicity by injecting rats with a threshold dose of Glu with or without ChABC utilizing the well defined respiratory motor system. 59% of SCI occurs at the cervical level, and leading causes of death and restriction of independence in these cases stem from mechanical ventilation. Therefore, we administered the dose instraspinally at the C4 level and paired treatment with intrapleural injection of cholera toxin-B to retrogradely label the phrenic motor neuron pool which innervates the diaphragm. our early findings suggest that animals treated with both Glu and ChABC had more extensive cell death. We believe this implies that following SCI, the body’s main focus is to survive and not necessarily to preserve function. CSPG upregulation could promote survival and CNS tissue preservation at the expense of plasticity and functional regeneration

Clearing Up the Phrenic Motor Neuron Survival Debate After Cervical Spinal Cord Injury

The diaphragm is the major muscle involved in breathing. Innervated by the phrenic nerve, it is controlled by phrenic motor neurons (PMNs), which receive descending inputs from the medulla. When these bulbospinal-pathways are damaged or severed in spinal cord injury (SCI), the external effects of injury are seen immediately, as the diaphragm becomes paralyzed and the individual loses the ability to breathe. However, the effect of injury on the internal circuitry, specifically PMN survival, is largely unknown. Contradictory evidence has surfaced, suggesting that there is large PMN death after injury, or conversely, that there is an absence of PMN death. However, histological techniques utilized in these studies have exposed the data to factors through which certainty cannot be guaranteed. These discrepancies are important to parse out because characterization of PMN survival is integral to studies of plasticity. The present study attempted to bridge this gap in knowledge and used XClarity clearing methods to accurately determine PMN survival after cervical SCI. XClarity transforms the tissue into a transparent medium. This allows for the whole spinal cord to be analyzed without tissue loss, as is common in other histological techniques. In this study, Sprague-Dawley rats were hemisected at the second level of the cervical spinal cord (c2Hx), which is a common experimental model of cervical SCI. Animals were divided into three groups: naïve, two weeks post-c2Hx, and five weeks post-c2Hx. Before perfusion, these animals were intrapleurally injected bilaterally with CTB-488, a retrograde tracer that labels PMNs. Depending on their group assignment, animals were perfused at five weeks post-injury, two weeks post-injury, or immediately after CTB-488 uptake. Cords were then processed with XClarity and PMN survival was characterized with Lightsheet microscopy. Analysis of PMNs is ongoing, however, preliminary data suggests that XClarity techniques are the preferable route to characterize PMN survival after injury