SUMMARY 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 which 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 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 which 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 spinal cord injury in adult mice and adult rats.

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Translating spinal cord injury (SCI) therapies which promote axonal regeneration from preclinical animal models into the human population is challenging. One potential obstacle is that human genetic predispositions may limit the efficacy of such experimental treatments. The clinically relevant ApoE4 (E4) allele, present in about 14% of the human population, corresponds to an increased incidence of Alzheimer's disease—however, its role in recovery from SCI is poorly understood despite suggestive data implicating its involvement. Indeed, two clinical studies found that SCI individuals with the E4 allele had less motor recovery than individuals without the allele despite longer time in rehabilitation. ApoE4 may mediate this diminished recovery by limiting regeneration and sprouting. Robust regeneration is energy intensive and requires efficient mitochondria, and studies have shown that ApoE4 impairs mitochondrial function. Given these mitochondrial deficits, we hypothesize that ApoE4 can impair regeneration and sprouting. To test this hypothesis, we investigated the impact of ApoE4 on sprouting and neurite outgrowth. In our experiments, we cultured dorsal root ganglia neurons from mice expressing human ApoE isoforms—ApoE2 (E2), ApoE3 (E3), or ApoE4—under the control of the endogenous mouse ApoE promoter. We then analyzed differences in 1) neurite complexity and 2) robustness of outgrowth between genotypes. In two of three independent experiments, E4 neurons had decreased neurite outgrowth and decreased neurite branching compared to E2 and E3 neurons. Data from the Spot Assay, an in vitro model of the glial scar and CNS regeneration, also suggest that chondroitin sulfate proteoglycans may inhibit regeneration in E4 neurons to an even greater extent than in E3 neurons. In addition, with preliminary in vivo data, we are beginning to characterize serotonergic sprouting after lateral C2 hemisection in mice of each ApoE genotype. Since outgrowth, sprouting, and regeneration all partially mediate recovery after CNS injury, impairments in these processes can adversely affect recovery. These foundational studies address not only the possible genetic influence of ApoE4 on recovery from CNS injury, but also a critical gap in knowledge—whether there is a genetic contribution underlying responses to treatment in SCI individuals.Support or Funding InformationUniversity 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.

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.

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.