More than half of all spinal cord injuries (SCIs) occur at the cervical level, often resulting in impaired respiration. Despite this devastating outcome, there is substantial evidence for endogenous neuroplasticity after cervical SCI. Spinal interneurons are widely recognized as being an essential anatomical component of this plasticity by contributing to novel neuronal pathways that can result in functional improvement. The identity of spinal interneurons involved with respiratory plasticity post-SCI, however, has remained largely unknown. Using a transgenic Chx10-eGFP mouse line (Strain 011391-UCD), the present study is the first to demonstrate the recruitment of excitatory interneurons into injured phrenic circuitry after a high cervical SCI. Diaphragm electromyography and anatomical analysis were used to confirm lesion-induced functional deficits and document extent of the lesion, respectively. Transneuronal tracing with pseudorabies virus (PRV) was used to identify interneurons within the phrenic circuitry. There was a robust increase in the number of PRV-labeled V2a interneurons ipsilateral to the C2 hemisection, demonstrating that significant numbers of these excitatory spinal interneurons were anatomically recruited into the phrenic motor pathway two weeks after injury, a time known to correspond with functional phrenic plasticity. Understanding this anatomical spinal plasticity and the neural substrates associated with functional compensation or recovery post-SCI in a controlled, experimental setting may help shed light onto possible cellular therapeutic candidates that can be targeted to enhance spontaneous recovery.

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.

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement.

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.