Transplantation of Neural Progenitors and V2a Interneurons after Spinal Cord Injury.

There is growing interest in the use of neural precursor cells to treat spinal cord injury (SCI). Despite extensive pre-clinical research, it remains unclear as to which donor neuron phenotypes are available for transplantation, whether the same populations exist across different sources of donor tissue (e.g., developing tissue vs. cultured cells), and whether donor cells retain their phenotype once transplanted into the hostile internal milieu of the injured adult spinal cord. In addition, while functional improvements have been reported after neural precursor transplantation post-SCI, the extent of recovery is limited and variable. The present work begins to address these issues by harnessing ventrally derived excitatory pre-motor V2a spinal interneurons (SpINs) to repair the phrenic motor circuit after cervical SCI. Recent studies have demonstrated that Chx10-positive V2a SpINs contribute to anatomical plasticity within the phrenic circuitry after cervical SCI, thus identifying them as a therapeutic candidate. Building upon this discovery, the present work tests the hypothesis that transplantation of neural progenitor cells (NPCs) enriched with V2a INs can contribute to neural networks that promote repair and enhance respiratory plasticity after cervical SCI. Cultured NPCs (neuronal and glial restricted progenitor cells) isolated from E13.5 Green fluorescent protein rats were aggregated with TdTomato-mouse embryonic stem cell-derived V2a INs in vitro, then transplanted into the injured cervical (C3-4) spinal cord. Donor cells survive, differentiate and integrate with the host spinal cord. Functional diaphragm electromyography indicated recovery 1 month following treatment in transplant recipients. Animals that received donor cells enriched with V2a INs showed significantly greater functional improvement than animals that received NPCs alone. The results from this study offer insight into the neuronal phenotypes that might be effective for (re)establishing neuronal circuits in the injured adult central nervous system.

Integration of Transplanted Neural Precursors with the Injured Cervical Spinal Cord.

Cervical spinal cord injuries (SCI) result in devastating functional consequences, including respiratory dysfunction. This is largely attributed to the disruption of phrenic pathways, which control the diaphragm. Recent work has identified spinal interneurons as possible contributors to respiratory neuroplasticity. The present work investigated whether transplantation of developing spinal cord tissue, inherently rich in interneuronal progenitors, could provide a population of new neurons and growth-permissive substrate to facilitate plasticity and formation of novel relay circuits to restore input to the partially denervated phrenic motor circuit. One week after a lateralized, C3/4 contusion injury, adult Sprague-Dawley rats received allografts of dissociated, developing spinal cord tissue (from rats at gestational days 13-14). Neuroanatomical tracing and terminal electrophysiology was performed on the graft recipients 1 month later. Experiments using pseudorabies virus (a retrograde, transynaptic tracer) revealed connections from donor neurons onto host phrenic circuitry and from host, cervical interneurons onto donor neurons. Anatomical characterization of donor neurons revealed phenotypic heterogeneity, though donor-host connectivity appeared selective. Despite the consistent presence of cholinergic interneurons within donor tissue, transneuronal tracing revealed minimal connectivity with host phrenic circuitry. Phrenic nerve recordings revealed changes in burst amplitude after application of a glutamatergic, but not serotonergic antagonist to the transplant, suggesting a degree of functional connectivity between donor neurons and host phrenic circuitry that is regulated by glutamatergic input. Importantly, however, anatomical and functional results were variable across animals, and future studies will explore ways to refine donor cell populations and entrain consistent connectivity.

Supraspinal respiratory plasticity following acute cervical spinal cord injury.

Impaired breathing is a devastating result of high cervical spinal cord injuries (SCI) due to partial or full denervation of phrenic motoneurons, which innervate the diaphragm - a primary muscle of respiration. Consequently, people with cervical level injuries often become dependent on assisted ventilation and are susceptible to secondary complications. However, there is mounting evidence for limited spontaneous recovery of respiratory function following injury, demonstrating the neuroplastic potential of respiratory networks. Although many studies have shown such plasticity at the level of the spinal cord, much less is known about the changes occurring at supraspinal levels post-SCI. The goal of this study was to determine functional reorganization of respiratory neurons in the medulla acutely (>4h) following high cervical SCI. Experiments were conducted in decerebrate, unanesthetized, vagus intact and artificially ventilated rats. In this preparation, spontaneous recovery of ipsilateral phrenic nerve activity was observed within 4 to 6h following an incomplete, C2 hemisection (C2Hx). Electrophysiological mapping of the ventrolateral medulla showed a reorganization of inspiratory and expiratory sites ipsilateral to injury. These changes included i) decreased respiratory activity within the caudal ventral respiratory group (cVRG; location of bulbospinal expiratory neurons); ii) increased proportion of expiratory phase activity within the rostral ventral respiratory group (rVRG; location of inspiratory bulbo-spinal neurons); iii) increased respiratory activity within ventral reticular nuclei, including lateral reticular (LRN) and paragigantocellular (LPGi) nuclei. We conclude that disruption of descending and ascending connections between the medulla and spinal cord leads to immediate functional reorganization within the supraspinal respiratory network, including neurons within the ventral respiratory column and adjacent reticular nuclei.

Enhancing neural activity to drive respiratory plasticity following cervical spinal cord injury.

Cervical spinal cord injury (SCI) results in permanent life-altering sensorimotor deficits, among which impaired breathing is one of the most devastating and life-threatening. While clinical and experimental research has revealed that some spontaneous respiratory improvement (functional plasticity) can occur post-SCI, the extent of the recovery is limited and significant deficits persist. Thus, increasing effort is being made to develop therapies that harness and enhance this neuroplastic potential to optimize long-term recovery of breathing in injured individuals. One strategy with demonstrated therapeutic potential is the use of treatments that increase neural and muscular activity (e.g. locomotor training, neural and muscular stimulation) and promote plasticity. With a focus on respiratory function post-SCI, this review will discuss advances in the use of neural interfacing strategies and activity-based treatments, and highlights some recent results from our own research.

Interdisciplinary approaches of transcranial magnetic stimulation applied to a respiratory neuronal circuitry model.

Respiratory related diseases associated with the neuronal control of breathing represent life-threatening issues and to date, no effective therapeutics are available to enhance the impaired function. The aim of this study was to determine whether a preclinical respiratory model could be used for further studies to develop a non-invasive therapeutic tool applied to rat diaphragmatic neuronal circuitry. Transcranial magnetic stimulation (TMS) was performed on adult male Sprague-Dawley rats using a human figure-of-eight coil. The largest diaphragmatic motor evoked potentials (MEPdia) were recorded when the center of the coil was positioned 6 mm caudal from Bregma, involving a stimulation of respiratory supraspinal pathways. Magnetic shielding of the coil with mu metal reduced magnetic field intensities and improved focality with increased motor threshold and lower amplitude recruitment curve. Moreover, transynaptic neuroanatomical tracing with pseudorabies virus (applied to the diaphragm) suggest that connections exist between the motor cortex, the periaqueductal grey cell regions, several brainstem neurons and spinal phrenic motoneurons (distributed in the C3-4 spinal cord). These results reveal the anatomical substrate through which supraspinal stimulation can convey descending action potential volleys to the spinal motoneurons (directly or indirectly). We conclude that MEPdia following a single pulse of TMS can be successfully recorded in the rat and may be used in the assessment of respiratory supraspinal plasticity. Supraspinal non-invasive stimulations aimed to neuromodulate respiratory circuitry will enable new avenues of research into neuroplasticity and the development of therapies for respiratory dysfunction associated with neural injury and disease (e.g. spinal cord injury, amyotrophic lateral sclerosis).