Spinal cord injuries can abolish both motor and sensory function throughout the body.Spontaneous recovery after injury is limited and can vary substantially between individuals.Despite an abundance of therapeutic approaches that have shown promise in preclinical models, there is currently a lack of effective treatment strategies that have been translated to restore function after SCI in the human population. We hypothesized that sex and genetic background of injured individuals could impact how they respond to treatment strategies, presenting a barrier to translating therapies that are not tailored to the individual. One gene of particular interest is APOE, which has been extensively studied in the brain due to its allele-specific influences on synaptic plasticity, metabolism, inflammation, and neurodegeneration. Despite its prominence as a therapeutic target in brain injury and disease, little is known about how it influences neural plasticity and repair processes in the spinal cord. Utilizing humanized mice, we examined how the 3 and 4 alleles of APOE influence the efficacy of therapeutic intermittent hypoxia (IH) in inducing spinally-mediated plasticity after cervical SCI. IH is sufficient to enhance plasticity and restore motor function after experimental SCI in genetically similar rodent populations, but its effect in human subjects is more variable (Golder, 2005;Hayes et al., 2014). Our results demonstrate that both sex and APOE genotype determine the extent of respiratory motor plasticity that is elicited by IH, highlighting the importance of considering these clinically relevant variables when translating therapeutic approaches for the SCI community. 4 Significance StatementThere is currently a critical need for therapeutics that restore motor and sensory function effectively after cervical spinal cord injury. Although many therapeutic approaches, including intermittent hypoxia, are being investigated for their potential to enhance spinal plasticity and improve motor outcomes after SCI, it is unknown whether the efficacy of these treatment strategies is influenced by individuals' genetic background. Here we show that APOE genotype and sex both play a role in determining the propensity for motor plasticity in humanized mice after cervical SCI. These results indicate that sex and genetic background dictate how individuals respond to therapeutic approaches, thereby emphasizing the importance of developing personalized medicine for the diverse SCI population.

Following an experimental C2 spinal cord hemisection in rats, there is a gradual spontaneous recovery of breathing function that can take place over time. Additionally, interventions at later time points are more effective after injury. What is not known is the mechanism mediating this observation. To begin to answer this question, we investigated the role of the gut microbiome after injury. Recent studies have emerged suggesting that the gut microbiome has critical implications on the proper functioning of the central nervous system (CNS). Indeed, gut dysbiosis, or a microbiome imbalance, can occur which can negatively impact the CNS. Neurotrauma, including spinal cord injury (SCI), can lead to acute gut dysbiosis and impaired recovery. It is our hypothesis that the composition of the gut microbiome is dynamic after injury with dysbiosis improving over time from an acute post‐SCI state. In this study, we build upon these initial studies and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. In order to test our hypothesis, we collected fecal samples before and up to 12 weeks after a C2 hemisection in adult female rats, in order to assess microbiome composition at various timepoints post injury. Preliminary results suggest that following cervical SCI, gut dysbiosis occurs immediately after injury but recovers by two months post injury. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on recovery of respiratory motor function and plasticity.

Each year, 17,700 Americans suffer a spinal cord injury, over half of which occur at the cervical level. These high level injuries can interrupt bulbospinal neurons that innervate the phrenic motornucleus, the origin of the phrenic nerve. Loss of these descending inputs to the phrenic nerve paralyzes the ipsilateral diaphragm, leading to breathing impairments. One approach to promote recovery of breathing function is by enhancing plasticity through strengthening of synapses or activating spared but latent pathways in the spinal cord. Activation of the latent crossed phrenic pathway can lead to a form of respiratory motor plasticity known as long term facilitation (LTF), which is characterized by a prolonged increase in breathing motor output. LTF can be induced through exposure to intermittent hypoxia (IH) or by intermittently dosing the spinal cord with serotonin (5‐HT). While a portion of the spinal cord injured population responds to IH therapy with the expected increase in respiratory output, others remain non‐responders. This inconsistency indicates that variability in the human population may influence how individuals respond to treatments that aim to enhance plasticity. Therefore, we propose that genetic diversity among the SCI population could be a key factor in determining an individual's propensity for plasticity. Apolipoprotein E (apoE) is a promising candidate gene that could be responsible for this variability because one of the apoE alleles, E4, has previously been shown to reduce synaptic plasticity by decreasing expression of glutamate receptors when compared to the E2 or E3 alleles. The present study investigates the influence of human apoE4 on respiratory motor plasticity in rats following C2 hemisection. 20 weeks after injury, rats were dosed with one isoform of the human apoE protein, E3 or E4, prior to receiving intermittent 5‐HT to induce LTF. Diaphragmatic EMG recordings demonstrated that animals exposed to human apoE3 protein exhibited an increase in diaphragmatic activity ipsilateral to the injury, but this increase was abolished in E4 dosed animals. Analysis of tissue dosed with human apoE protein indicated that apoE also modulates synaptic expression of glutamate receptors, a crucial component of LTF induction. Collectively, these experiments demonstrate ApoE4's potential to inhibit plasticity following spinal cord injury, emphasizing the importance of considering genetic diversity while developing SCI therapeutics for the human population.Support or Funding InformationNational Science Foundation Graduate Research Fellowship, University of Kentucky College of Medicine Fellowship for Excellence in Graduate Research, University of Kentucky Startup FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nearly 60% of all spinal cord injuries (SCI) occur at the cervical level. These high‐level injuries can lead to quadriplegia, autonomic dysregulation, and interruption of the descending respiratory pathways required for breathing. As respiratory motor function is required for life, it is critical to restore breathing as soon as possible after cervical SCI. Indeed, therapeutic techniques in animal studies have been successful at restoring breathing function after SCI; however, these interventions appear to be more effective at chronic than acute timepoints post‐injury. One potential cause for this observation is the impact the injury has on the gastrointestinal (GI) tract and gut microbiome, which have previously been shown to have detrimental effects on recovery outcomes, including lower limb motor function and emotional affect as indicated by increased anxiety‐like behavior. However, the impact the gut microbiome has on the recovering spinal cord at different timepoints, levels, and injury severities still needs to be fully realized. We aimed to build upon these previous findings and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. We hypothesized that cervical SCI leads to transient changes in the gut microbiome, which are most severe acutely after injury impeding functional recovery of breathing, but resolve over time and thus eventually allow for more profound recovery. To test our hypotheses, we performed left C2 hemisections on adult female rats, collected fecal samples from injured, sham, and naïve animals, and assessed microbiome composition at various timepoints pre‐ and post‐injury. Preliminary results suggest that following cervical SCI (up to 12 weeks post injury), robust differences in gut microbiome are apparent compared to non‐injured animals. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on respiratory motor function and plasticity, as well as inter‐institutional differences.

Spinal cord injury (SCI) most commonly occurs at the cervical level and can interrupt descending neural pathways, causing paralysis of the diaphragm, as well as profound breathing motor difficulties which threaten survival and greatly decrease quality of life. Intermittent hypoxia (IH) treatment is often utilized in preclinical models to attenuate breathing motor deficits resulting from cervical SCI by inducing a prolonged increase in respiratory motor output known as long term facilitation (LTF), a form of breathing motor plasticity. IH typically consists of the repeated, alternating exposure of a subject to consistent and equal 5‐minute periods of hypoxia and normoxia and thus can be fittingly termed fixed interval intermittent hypoxia (FIH). FIH exhibits similarity to the psychological construct of operant conditioning in which the increased incidence and persistence of a desired, spontaneous behavior is trained through reinforcement. As such, each interval of hypoxia can be construed as the period during which the subject responds with heightened respiratory drive and is subsequently reinforced by an interval of normoxia. Provided that IH is a form of operant conditioning, it can be optimized through application of seminal psychological findings which established variable interval schedules of reinforcement as more effective than fixed interval schedules for learning. Therefore, using the duration of the hypoxic interval as our independent variable, we hypothesize that varied interval intermittent hypoxia (VIH) treatment will induce a greater, more prolonged increase in respiratory motor output than FIH after injury. We utilized the C2 hemisection injury model in rats and treated with VIH or FIH for 5 days at 1‐week post‐injury. Following treatment, we conducted diaphragm electromyograph recordings to assess breathing motor recovery within each animal by comparing baseline activity to maximal output induced by nasal occlusion, occurring immediately after cessation of IH treatment. Preliminary results show that 1‐week post‐injury VIH treated animals exhibited recovery on average equaling 33.87±7.89% as compared to the 19.28±6.02% recovery in FIH treated animals (differences nonsignificant by unpaired t‐test, p>0.05 with uncertainty shown as standard error of the mean). These data suggest that VIH may induce more increased and prolonged recovery than FIH in post‐injury models. Ongoing work includes evaluation of VIH treatment in animals at 8 weeks after injury and will expand outcome measurement timepoints to include 1 week after cessation of treatment to interrogate the persistence of induced recovery. Additionally, further exploration will focus on the molecular markers present within the phrenic motor nucleus of the cervical spinal cord and on the development of IH paradigms based on breathing frequency.Support or Funding InformationNINDS R01NS101105 (Warren J. Alilain)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.