David D. Fuller, Ph.D - US grants

DESCRIPTION (provided by applicant): We propose to determine the effects of endogenous and exogenous female sex hormones on recovery of perhaps the most critical motor function impaired by cervical spinal cord injury (SCI): breathing. Although endogenous estrogens and progestins promote recovery following traumatic brain injury, their impact on motor recovery after SCI is unknown. Fluctuations in these hormones during the estrous cycle in female rodents suggest that potential "neuroprotective" effects may depend on the time of the injury. The first hypothesis guiding this proposal is that the estrous cycle stage and corresponding levels of progesterone (a progestin) and 17beta-estradiol (an estrogen) at the time of SCI will correlate with motor recovery (Aim 1). Administration of exogenous progesterone and 17beta-estradiol has been shown to improve locomotor recovery after SCI. However, these hormones are also powerful respiratory stimulants. Data collected by the P.I. and others indicates that respiratory stimulation after SCI strengthens existing spinal synaptic inputs to phrenic motoneurons. Thus, sex hormone therapy targeting respiratory insufficiency may combine generalized neuroprotective effects with effects specific to the phrenic motor system. Accordingly, our second hypothesis is that administration of exogenous progesterone and 17beta-estradiol after cervical SCI will improve respiratory motor recovery (Aim 2). These hypotheses will be tested using behavioral, neurophysiological, and immunoassay techniques in rats. Ventilation will be assessed in unanesthetized rats via barometric plethysmography. Phrenic output will be quantified under anesthesia, and plasma hormone levels will be measured with ELISA. These data will help establish 1) if endogenous gender hormones can influence preclinical SCI research, and 2) if progesterone and 17p-estradiol hormone therapy is an effective means of promoting persistent respiratory recovery following cervical SCI.

[unreadable] DESCRIPTION (provided by applicant): Glycogen storage disease type II (GSDII) results from deficiency of acid a-glucosidase (GAA), a lysosomal enzyme that degrades glycogen. GSDII causes cardiorespiratory failure in infants, and progressive respiratory failure in juveniles and adults. Although respiratory failure has been attributed to muscle pathology, autopsy case reports show cervical spinal glycogen accumulation and suggest motoneuron pathology. We have observed that efferent phrenic discharge, mean inspiratory airflow, and the ratio of minute ventilation to metabolic rate (VE/VCO2) are blunted in a murine GSDII model, the GAA-/- "knockout" mouse. In addition, a selective knockout mouse with normal skeletal muscle contractility and GAA expression, but no GAA in the central nervous system (CNS), also exhibits marked reductions in phrenic output and ventilation. Accordingly, CNS GAA deficiency is associated with impaired respiratory motor output, and respiratory insufficiency in GSDII may reflect both a neural and a muscular pathology. A neural mechanism is also implicated by preliminary data showing extensive cervical spinal glycogen accumulation in GAA-/- mice, particularly within retrogradely identified phrenic motoneurons. Determining the mechanisms underlying respiratory insufficiency is important because i.v. enzyme replacement (the current clinical GSDII therapy) does not target the CNS as GAA cannot cross the blood brain barrier. A promising method for targeting both muscle and the CNS is recombinant adeno-associated virus (rAAV) gene therapy. Our preliminary data indicate that rAAV effectively transfects phrenic motoneurons, and can be delivered by intraspinal or intrathoracic injection. Further, ventilation is significantly enhanced in GAA-/- mice one month following intrathoracic injection of rAAV packaged with the GAA gene (rAAV-GAA therapy). These experiments represent a unique collaboration between laboratories specializing in respiratory physiology (Fuller), gene therapy (Byrne), and neuroanatomy (Reier). We propose to test three hypotheses: 1) neural drive to the diaphragm and ventilation are attenuated in both the selective and full GAA-/- knockout mice; 2) respiratory deficits in these mice occur in parallel with glycogen accumulation in phrenic motoneurons, and 3) intraspinal and intrathoracic rAAV-GAA delivery can ameliorate spinal glycogen accumulation and enhance respiratory motor output in the selective and GAA-/- knockout mice, respectively. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuronal replacement shows promise for enhancing recovery after spinal cord injury (SCI), and is considered a major objective for stem cell-mediated spinal cord repair. Achieving effective host-graft neuronal communication, however, represents a major challenge for neuronal replacement which has not been investigated. The overall hypothesis of this proposal is that presenting newly established host-graft neuronal networks with physiologically-patterned activities will enhance functional connectivity. Based on our extensive experience with SCI modeling and both respiratory and computational neurobiology, we are proposing to test this hypothesis by transplanting rat fetal spinal cord (FSC) tissue grafts - a source of neuronal progenitors - into clinically-relevant, high cervical (C3) contusion injuries in adult rats. Preliminary transneuronal neuroanatomical tracing data show synaptic connectivity between FSC donor cells and host phrenic motoneurons and cervical interneurons. In addition, FSC grafts receive extensive serotonergic inputs from the host, and some graft neurons exhibit hypoxia-sensitive discharge patterns including apparent inspiratory- related bursting. A prerequisite anatomical-functional framework is thus in place to test our central hypothesis via the following specific aims using FSC grafts placed into C3 contusion injuries in adult rats: Aim 1) to test the hypothesis that FSC-derived neurons will become anatomically and physiologically integrated with host gray matter, and Aim 2) to test the hypothesis that training via intermittent hypoxia (IH) stimulation will enhance the anatomical and physiological integration of FSC-derived neurons. To test these hypotheses, we will use a multi-disciplinary approach including neuroanatomical studies of connectivity between the graft and host spinal cord, measurement of breathing in awake rats, and neurophysiological studies of the graft. An innovative technical feature of our proposal is that this will be the first use of microelectrode arrays to monitor graft- associated neural ensembles in the spinal cord. A FSC grafting method will be used because such grafts develop into myelinated tissue containing a large contingent of cells resembling intermediate gray matter interneurons. A unique rehabilitative paradigm - daily exposure to mild, intermittent hypoxia (IH) - will be used because it leads to spinal cord plasticity associated with persistent increases in respiratory output in spinal injured animals. Equally important, IH provides a tool to introduce or increase appropriately patterned bursting around, and possibly within, the graft. This proposal brings together unique expertise in respiratory neurophysiology, computational neurobiology, neural transplantation, and SCI in an effort to facilitate transformative advances in the understanding and treatment of SCI. PUBLIC HEALTH RELEVANCE: Respiratory compromise is a significant problem after cervical spinal cord injury. A strategy that may enhance motor recovery after spinal injury is neural replacement therapy in which cells are transplanted into the spinal cord lesion. In these experiments, we will examine if a novel rehabilitation paradigm can enhance the effectiveness of a neural transplant following spinal cord injury.

Project Summary / Abstract: Pompe disease results from mutations in the gene for acid ?-glucosidase (GAA) ? an enzyme necessary to degrade lysosomal glycogen. Early-onset disease occurs in the absence of functional GAA which leads to cardiorespiratory failure early in life. Late-onset disease is associated with reduced GAA activity and gradual progression to respiratory failure. Work from our first two grant cycles indicates neural involvement in respiratory failure in Gaa-/- mice and Pompe patients. This is relevant since the standard of care ? intravenous enzyme therapy using recombinant GAA - does not reach the central nervous system (CNS) and patients still progress to respiratory failure. Our overarching hypothesis is that adeno-associated virus (AAV) therapy is capable of restoring life-long GAA expression throughout the CNS, skeletal and cardiac muscle, thereby preserving cardiorespiratory function and prolonging life. Aim 1 focuses on AAV therapy for early-onset disease which requires early life treatments that can prevent both respiratory and cardiac failure. To better study this problem, we created a Gaa null (Gaa-/-) rat model which recapitulates the early onset phenotype with cardiorespiratory pathology and early mortality. Preliminary data indicate that neonatal AAV-GAA therapy (desmin promoter, AAV9 serotype) evokes no detectable immune response, mitigates cardiac and respiratory pathology and prevents early death. Thus, we hypothesize that a single intravenous AAV-GAA dose in young rats can drive persistent and widespread GAA expression and extend the Pompe rat lifespan. Aim 2 addresses late onset Pompe disease in which respiratory failure is the primary cause of mortality. Based on data from our first two grant cycles we hypothesize that neural directed AAV-GAA therapy in adult Pompe rats is sufficient to prevent respiratory decline and extend the lifespan. By packaging AAV-GAA with muscle (creatine kinase 8), neural (synapsin) or tissue specific (muscle and neural, desmin) promoters, and delivering the vector intrathecally, intravenously, or both, we can drive GAA expression in a manner that will determine if neural correction is necessary and sufficient to prevent decline. The aforementioned Gaa null rat will be used to test proof-of-concept for neural vs. muscle correction in the absence of endogenous GAA activity. We will also use another new Pompe rat model in which CRISPR/cas9 has been used to insert the most common human gene mutation causing late-onset Pompe disease (IVS1) into the rat genome. This is important because the IVS1 mutation leads to low but not absent GAA activity and is associated with delayed progression to respiratory failure. The proposed work is significant because current therapeutic strategies in Pompe disease only delay disease progression with eventual respiratory failure. The strategies proposed here will also contribute to the broader goal of advancing gene therapy for neurodegenerative conditions and autosomal recessive diseases.

DESCRIPTION (provided by applicant): The injured spinal cord is now recognized to have a robust capacity for neuroplasticity, and it is desirable to therapeutically harness that in ways tht will enhance respiratory outcomes after cervical spinal cord injury (SCI). Fundamental to rehabilitation and repair approaches is a basic understanding of the spinal respiratory circuit and the control of spinal respiratory neurons after chronic SCI. In principle, the injured spinal cord s essentially a new spinal cord in which neural networks and control mechanisms affecting virtually every functional domain are significantly altered. Our group recently characterized the spinal respiratory circuit anatomically after cervical SCI, but functional-anatomical correlates remain to be determined. We propose a series of experiments which will neurophysiologically define the spinal respiratory circuit after cervical SCI, examine the influence of a key neuromodulator - serotonin (5-HT) - on the circuit, and determine the impact of a promising spinal cord transplantation approach on spinal respiratory neurons and recovery of ventilation after cervical SCI. The overall hypothesis guiding this proposal is that the regulation of phrenic motoneuron (PMN) activity following chronic cervical SCI is influenced by spinal pre-phrenic interneurons and that spinal 5-HT is a critically important modulator of the spinal respiratory circuitry following chronic SCI. A rat model of high cervical SCI (lateral C2 hemisection) will be used to address three specific aims. Aim 1 will test the hypothesis that following chronic cervical SCI, PMNs retain a robust capacity for plasticity, and their bursting patterns are partly regulated by cervical interneurons. PMN and cervical interneuron activity will be measured using a multi- electrode array; anatomical and immunohistochemical methods will be used to evaluate the spinal respiratory circuit. Aim 2 will use neurophysiological, pharmacological, immunochemical, and molecular techniques to test the hypothesis that spinal 5-HT receptor activation is an integral part of phrenic motor recovery after chronic cervical SCI. Lastly, Aim 3 will test the hypothesis that transplantation of serotonergic cells can enhance or restore serotonergic modulation of spinal respiratory neurons thereby improving respiratory recovery after SCI. One week following C2 hemisection injury, adult rats will receive an intraspinal transplant of serotonergic cells derived from fetal rat raph¿ neurons. The impact of the grafts on the phrenic motor system will be assessed using behavioral, neurophysiological, pharmacological, immunohistochemical, and molecular techniques. This proposal brings together a unique and synergistic combination of expertise in respiratory neurophysiology, multi-unit recording approaches, neural transplantation, and cervical SCI modeling. Innovative aspects include: 1) the first descriptive and mechanistic studies of PMN burst patterns after chronic cervical SCI; 2) the first neurophysiological studies of respiratory-related cervical interneurons after cervical SC; 3) the use of multi- array electrodes to describe the spinal respiratory circuitry, and 4) the firs use of transplant strategies to enhance serotonergic innervation of the spinal respiratory circuit.

DESCRIPTION (provided by applicant): Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid a- glucosidase (GAA) - an enzyme necessary to degrade lysosomal glycogen. Hypoventilation is a hallmark feature of all forms of Pompe disease that has historically been attributed to respiratory muscle pathology. The experiments proposed in this R21 grant application will provide fundamental, mechanistic information about the clinical problem of respiratory insufficiency in Pompe disease. Most importantly, we propose a direct test of the hypothesis that central nervous system dysfunction is a primary contributor to respiratory insufficiency in Pompe disease. Evidence is mounting in support of this hypothesis, but definitive proof is lacking. To accomplish this goal we propose to use a site-specific Cre-Lox recombination approach to knockout the GAA gene in spinal and medullary respiratory neurons of mice while leaving skeletal and cardiac muscle gene expression unaltered. If the hypothesis is confirmed it will inform the clinical community about the underlying causes of respiratory insufficiency in Pompe patients. More importantly, confirmation of our hypothesis would necessitate a shift from the current emphasis on purely muscle directed therapies towards approaches which would impact on both muscle and neural function. Thus, overarching goal of the proposed studies is to determine if the respiratory control system becomes dysfunctional when GAA gene expression is knocked out in respiratory neurons. We also propose to compare and contrast the role of spinal motoneurons vs. medullary respiratory control neurons with regard to impaired respiratory motor output in Pompe disease. An initial clinical trial of GAA gene transfer to the diaphragm of Pompe patients is underway (ClinicalTrials.gov: NCT00976352). This work will complement the ongoing trial by examining the importance of motoneurons vs. medullary neurons to respiratory insufficiency. This is important since phrenic motoneurons can be transduced via retrograde viral transport post-diaphragm injection whereas medullary neurons will not. Thus, the clinical trial is not likely to result in transduction of medullary respiratory neurons. In developing this application we obtained a mouse colony with a floxed GAA gene. We propose to use stereotaxic and/or retrograde delivery of AAV vectors driving Cre recombinase expression to selectively knockout the GAA gene in spinal respiratory (phrenic) motoneurons (Aim 1) and brainstem respiratory control neurons (Aim 2). This work is a collaborative effort between a respiratory control scientist (Fuller), an AAV specialist and clinician working with Pompe patients (Byrne), and an AAV specialist with expertise in stereotaxic delivery (Mandel).

ABSTRACT Respiratory-related motor dysfunction is the leading cause of morbidity and mortality following cervical spinal cord injury (SCI). Respiratory impairments largely reflect impaired bulbospinal glutamatergic synaptic transmission to spinal respiratory motoneurons. Ampakines are allosteric modulators of ?-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor channel kinetics that enhance glutamatergic synaptic transmission. Since glutamatergic bulbospinal excitation of spinal respiratory motoneurons is driven in part by motoneuron AMPA receptor activation, enhancing spinal AMPA-mediated synaptic currents could increase motoneuron output. The central hypothesis guiding this proposal is that ampakines are an effective pharmacologic approach to improve breathing function after incomplete cervical SCI. Aim 1 will test the hypothesis that acute delivery of ampakines stimulates breathing after incomplete cervical SCI, and does so by facilitating synaptic transmission in spared respiratory motor pathways in the spinal cord. These experiments will determine effective dose, safety profile and primary site of action (e.g., medullary vs. spinal). Studies will be done after both acute and chronic cervical SCI; minute ventilation and respiratory muscle electromyogram (EMG) activity will be evaluated in unanesthetized rats; phrenic nerve activity will be evaluated in anesthetized rats. Aim 2 moves beyond direct stimulation of breathing to test the hypothesis that ampakines increase the capacity for neuroplasticity in the phrenic motor circuit. Preliminary data indicate that ampakine pre-treatment greatly enhances phrenic motor plasticity induced by spinal, serotonin receptor agonist administration. Based on these data, we propose a detailed cellular model to explain the impact of ampakines on serotonin- dependent phrenic motor plasticity. Additional preliminary data show that ampakine pretreatment causes dramatic increases in phrenic motor facilitation induced by acute intermittent hypoxia (AIH). We focus on AIH since it is well-established to induce spinal, serotonin-dependent phrenic motor plasticity, and has proven to be a simple and safe neurorehabilitation approach in humans with SCI. Thus, low-dose ampakines may be useful to enhance the impact of other neurorehabilitation modalities. Hypotheses derived from the cellular model will be tested by cervical spinal delivery of serotonin receptor antagonists and/or siRNAs targeting downstream signaling molecules. A comprehensive series of outcome measures will include neurophysiological studies of phrenic output, neurochemical evaluation of signaling pathways, and assessment of respiratory capacity in awake rats. We suggest that the proposed work is significant because of the need for strategies to improve motor function in patients with SCI. Innovative aspects include: 1) the use of ampakines to stimulate breathing after cervical SCI; 2) the use of ampakines increase respiratory neuroplasticity and functional recovery, and 3) the first cellular model to explain the impact of ampakines on spinal respiratory neuroplasticity.

DESCRIPTION (provided by applicant): The Interdisciplinary Training Program in Rehabilitation and Neuromuscular Plasticity (NMPT) at the University of Florida (UF) was initiated in 2003. The overall goal of this program is to help build a critical capacity of well- trained rehabilitation scientists capable of conducting translational neuromuscular plasticity research. This predoctoral training program is unique in that it emphasizes the interaction and joint training of rehabilitation clinicians and basic science trainees. Candidates are selected fro a pool of outstanding students with diverse backgrounds and are admitted by one of three graduate programs: Interdisciplinary Biomedical Sciences, Rehabilitation Sciences or Applied Physiology and Kinesiology. The program capitalizes on several existing strengths including a core of well-established and productive rehabilitation investigators, outstanding research facilities and integrated interdisciplinary centers, an exemplary record of collaboration, strong institutional commitment, and a culture of successful mentorship in rehabilitation at multiple levels, including graduate students, postdoctoral fellows and junior faculty. The NMPT program is a well-defined, closely mentored program with clearly established training objectives and an effective evaluation process. Upon entering the program, each Trainee prepares an individualized training plan under the guidance of an experienced Faculty Mentor and Translational Research Co-Advisor. The individualized plan consists of a structured didactic program, specialized courses, journal clubs and seminars, laboratory research and multiple scientific dissemination experiences. Trainees learn cutting edge research methodologies and acquire extensive research experience, while building a solid foundation in research design. Trainees also benefit from integration in strong Collaborative Translational Research Partnerships around five central themes. Over the past ten years the NMPT program has successfully graduated seventeen graduate students and 94% of NMPT graduates have continued on in postdoctoral positions. The trainees that have graduated have accumulated an excellent record of scholarly productivity, with an average of over 6 peer-reviewed manuscripts. These former trainees are now well-positioned to make contributions in the field of neuromuscular plasticity.

ABSTRACT In response to NINDS PA-18-358 (?exploratory and innovative projects? related to neurological disorders), we propose a 2-yr project to develop a novel method to electrically stimulate the spinal cord. The purpose is to restore respiratory muscle activation and breathing during acute or chronic hypoventilation associated with opioid overdose or neurotrauma. Based on deep brain stimulation data, the method is called temporal interference (T-I) stimulation. The premise is to target the spinal cord with two, high frequency, but low amplitude electrical waveforms. The waveforms are delivered at kilohertz frequencies that are well above values that directly stimulate neurons. The frequency of the two waveforms is offset by a small amount (e.g. 1- 5 Hz), and where the two electrical fields sum in the tissue, neuronal populations are recruited in phase with the offset. Our overall hypothesis is that T-I stimulation can be used to target energy to the ventral cervical spinal cord to regulate diaphragm activation with minimal off target effects. For this application, we conducted proof-of-concept preliminary experiments using rat models of opioid overdose and cervical spinal cord injury (SCI). We initially examined T-I stimulation delivered via simple sub-cutaneous neck electrodes following acute opioid overdose. The rationale is that a rapid and easily applied method to sustain breathing could be useful to sustain ventilation in emergency clinical situations. Remarkably, T-I stimulation delivered using sub- cutaneous leads in the neck region was able to evoke diaphragm motor recruitment, and the discharge could be regulated by varying the offset frequency between the two stimulus waveforms. To our knowledge, no prior methods have been able to induce rhythmic diaphragm contractions i.e., ?pacing? with a minimally invasive stimulation approach. Additional preliminary experiments focused on epidural stimulation of the cervical spinal cord. The rationale is that epidural stimulation is gaining traction as a means of restoring somatic and/or autonomic motor function after SCI and shows promise for activating the diaphragm. Electrodes on the mid- cervical dorsal epidural surface were used to deliver two waveforms at KHz frequencies. The T-I dual wave epidural stimulation was remarkably effective at activating and regulating diaphragm motor units. Compared to single wave epidural stimulation, we predict that T-I will offer advantages including 1) improved efficacy after incomplete and/or chronic lesions; 2) ability to produce a wide range of diaphragm motor unit recruitment patterns with more natural ?burst? envelope, and 3) ability to more focally target the stimulus to the ventral horn. The overall hypothesis will be tested by evaluating and optimizing T-I stimulation delivered via subcutaneous (Aim 1) or epidural electrodes (Aim 2). The project is innovative since dual waveform T-I stimulation has not previously been explored as a means of activating the respiratory muscles. PI Dr. Fuller has extensive experience with rodent models of SCI and Co-I Dr. Otto is a biomedical engineer with 20+ years of experience in electrical stimulation of the central nervous system including electrode design.

Abstract The Interdisciplinary Training Program in Rehabilitation and Neuromuscular Plasticity (NMPT) at the University of Florida (UF) was initiated in 2003. The overall goal of this program is to help build a critical capacity of highly-trained rehabilitation scientists capable of conducting translational neuromuscular plasticity research. This predoctoral training program is unique in that it emphasizes the interaction and joint training of rehabilitation clinicians and basic science Trainees. The NMPT program draws students from five thriving PhD programs in the biomedical sciences, and our Faculty are active members of multiple Centers and Institutes devoted to distinct aspects of neuromuscular plasticity and rehabilitation. The program capitalizes on existing UF strengths including a core of well-established and highly productive rehabilitation investigators, outstanding research facilities, an exemplary record of collaboration, extraordinary institutional commitment, and a culture of successful mentorship in rehabilitation at multiple levels, including graduate students, postdoctoral fellows and junior faculty. Upon entering the program, each Trainee prepares an individualized training plan under the guidance of an experienced Faculty Mentor and Translational Research Co-Advisor. The individualized plan consists of a structured didactic program, specialized courses, journal clubs and seminars, laboratory research and multiple scientific dissemination experiences. The NMPT program is a well-defined, closely monitored program with clearly established training objectives and an effective evaluation process. We have a well- defined management structure that includes a Program Director, Curriculum Coordinator, a Translational Science Advisor, an Internal Steering Committee and an External Advisory Board. The NMPT program has been highly successful since its inception in 2003 with 25 of 29 graduates (86%) in academic or clinical research positions and 4 in industry research or government biomedical fields. Over the last cycle (2014- present), we graduated 10 NMPT Trainees. These Trainees have published an average of 12 PubMed- indexed manuscripts and all have obtained academic, research or biomedical positions. The NMPT program is achieving our goal of training scientists capable of engaging in translational rehabilitation research and sustaining independently funded research programs.

ABSTRACT Hyperbaric oxygen (HBO) therapy involves brief (?1 hr) exposure to pressurized oxygen at ?3 ATM and is used frequently for wound healing and decompression sickness. Our preliminary data and literature reports have led to the central hypothesis that HBO, delivered in the acute phases (days to weeks) after cervical spinal cord injury (SCI), attenuates diaphragm atrophy and dysfunction, reduces cervical spinal cord pathology, and improves respiratory neuromuscular recovery. The proposed mechanistic link between HBO therapy and attenuation of both muscular and neural pathology after SCI is oxidative stress. Preliminary data demonstrate that cervical contusion injury leads to substantial increases in ROS in the diaphragm and atrophy. Preliminary testing also showed that 1 hr HBO therapy for 10 days decreased diaphragm ROS formation and increased diaphragm antioxidant capacity. The HBO therapy also considerably attenuated the atrophy and contractile impairments that occurred after cervical contusion. In regards to spinal neuropathology, secondary damage (i.e., pathology that develops after the initial trauma) impairs motor recovery. Preliminary histological and molecular data demonstrate a neuroprotective impact of HBO with reduction in secondary damage in the contused cervical spinal cord. This includes attenuated neuronal loss with reduced expression of apoptotic markers and reduced inflammation after HBO therapy. Since oxidative stress contributes to secondary damage, we predict that HBO- induced upregulation of antioxidant expression underlies these effects. Collectively, the preserved diaphragm function and attenuated cervical pathology lead to our overall hypothesis that respiratory recovery will be improved by HBO therapy. Aim 1 will test the hypothesis that HBO therapy during acute through sub-acute phases after cervical SCI reduces diaphragm atrophy and improves contractility. The hypothesis will be tested with histological, molecular and functional evaluation of the diaphragm. To test oxidative mechanisms, antisense oligonucleotides will be used to block translation of specific antioxidants during HBO therapy. To determine if antioxidant mechanisms are sufficient to explain the HBO therapeutic effects, we will overexpress specific antioxidants using adeno-associated virus (AAV). Aim 2 will test the hypothesis that the neuroprotective impact of HBO therapy during acute through sub-acute phases after cervical SCI leads to improved phrenic motor recovery. The hypothesis will be tested with histological, molecular, and neurophysiological methods (direct phrenic nerve recordings and diaphragm electromyography). As in Aim 1, mechanistic studies will utilize antisense oligonucleotides and AAV strategies to modulate antioxidant formation in the spinal cord. Co-PI Dr. Smuder is an expert in diaphragm biology and mechanisms of atrophy. Co-PI Dr. Fuller has extensive experience in preclinical SCI models of respiratory dysfunction. Consultant Dr. Dean is an authority on HBO.