V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Sex and APOE genotype influence respiratory function under hypoxic and hypoxic-hypercapnic conditions.

The apolipoprotein E (APOE) gene has been studied due to its influence on Alzheimer's disease (AD) development and work in an APOE mouse model recently demonstrated impaired respiratory motor plasticity following spinal cord injury (SCI). Individuals with AD often copresent with obstructive sleep apnea (OSA) characterized by cessations in breathing during sleep. Despite the prominence of APOE genotype and sex as factors in AD progression, little is known about the impact of these variables on respiratory control. Ventilation is tightly regulated across many systems, with respiratory rhythm formation occurring in the brainstem but modulated in response to chemoreception. Alterations within these modulatory systems may result in disruptions of appropriate respiratory control and ultimately, disease. Using mice expressing two different humanized APOE alleles, we characterized how sex and the presence of APOE3 or APOE4 influences ventilation during baseline breathing (normoxia) and during respiratory challenges. We show that sex and APOE genotype influence breathing during hypoxic challenge, which may have clinical implications in the context of AD and OSA. In addition, female mice, while responding robustly to hypoxia, were unable to recover to baseline respiratory levels, emphasizing sex differences in disordered breathing.NEW & NOTEWORTHY This study is the first to use whole body plethysmography (WBP) to measure the impact of APOE alleles on breathing under normoxia and during adverse respiratory challenges in a targeted replacement Alzheimer's model. Both sex and genotype were shown to affect breathing under normoxia, hypoxic challenge, and hypoxic-hypercapnic challenge. This work has important implications regarding the impact of genetics on respiratory control as well as applications pertaining to conditions of disordered breathing including sleep apnea and neurotrauma.

Functional regeneration of respiratory pathways after spinal cord injury.

Spinal cord injuries often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. The effects of impaired breathing are a leading cause of death from spinal cord injuries, underscoring the importance of developing strategies to restore respiratory activity. Here we show that, after cervical spinal cord injury, the expression of chondroitin sulphate proteoglycans (CSPGs) associated with the perineuronal net (PNN) is upregulated around the phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with chondroitinase ABC (denoted ChABC) could, by itself, promote the plasticity of tracts that were spared and restore limited activity to the paralysed diaphragm. However, when combined with a peripheral nerve autograft, ChABC treatment resulted in lengthy regeneration of serotonin-containing axons and other bulbospinal fibres and remarkable recovery of diaphragmatic function. After recovery and initial transection of the graft bridge, there was an unusual, overall increase in tonic electromyographic activity of the diaphragm, suggesting that considerable remodelling of the spinal cord circuitry occurs after regeneration. This increase was followed by complete elimination of the restored activity, proving that regeneration is crucial for the return of function. Overall, these experiments present a way to markedly restore the function of a single muscle after debilitating trauma to the central nervous system, through both promoting the plasticity of spared tracts and regenerating essential pathways.

Targeting α1- and α2-adrenergic receptors as a countermeasure for fentanyl-induced locomotor and ventilatory depression.

This study assessed the ability of α1 and α2-adrenergic drugs to decrease fentanyl-induced locomotor and ventilatory depression. Rats were given saline or fentanyl, followed by: (1) naltrexone, (2) naloxone, (3) nalmefene, (4) α1 agonist phenylephrine, (5) α1 antagonist prazosin, (6) α1D antagonist BMY-7378, (7) α2 agonist clonidine, (8) α2 antagonist yohimbine or (9) vehicle. All µ-opioid antagonists dose-dependently reversed fentanyl-induced locomotor and ventilatory depression. While the α1 drugs did not alter the effects of fentanyl, clonidine dose-dependently decreased locomotion and respiration with and without fentanyl. Conversely, yohimbine given at a low dose (0.3-1 mg/kg) stimulated ventilation when given alone and higher doses (>1 mg/kg) partially reversed (∼50 %) fentanyl-induced ventilatory depression, but not locomotor depression. High doses of yohimbine in combination with a suboptimal dose of naltrexone reversed fentanyl-induced ventilatory depression, suggestive of additivity. Yohimbine may serve as an effective adjunctive countermeasure agent combined with naltrexone to rescue fentanyl-induced ventilatory depression.

Contrasting Experimental Rodent Aftercare With Human Clinical Treatment for Cervical Spinal Cord Injury: Bridging the Translational "Valley of Death".

More than half of all spinal cord injuries (SCIs) occur at the cervical level and often lead to life-threatening breathing motor dysfunction. The C2 hemisection (C2Hx) and high cervical contusion mouse and rat models of SCI are widely utilized both to understand the pathological effects of SCI and to develop potential therapies. Despite rigorous research effort, pre-clinical therapeutics studied in those animal models of SCI sometimes fail when evaluated in the clinical setting. Differences between standard-of-care treatment for acute SCI administered to clinical populations and experimental animal models of SCI could influence the heterogeneity of outcome between pre-clinical and clinical studies. In this review, we have summarized both the standard clinical interventions used to treat patients with cervical SCI and the various veterinary aftercare protocols used to care for rats and mice after experimentally induced C2Hx and high cervical contusion models of SCI. Through this analysis, we have identified areas of marked dissimilarity between clinical and veterinary protocols and suggest the modification of pre-clinical animal care particularly with respect to analgesia, anticoagulative measures, and stress ulcer prophylaxis. In our discussion, we intend to inspire consideration of potential changes to aftercare for animal subjects of experimental SCI that may help to bridge the translational "Valley of Death" and ultimately contribute more effectively to finding treatments capable of restoring independent breathing function to persons with cervical SCI.

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.

Cervical spinal cord injury leads to injury and altered metabolism in the lungs.

High-cervical spinal cord injury often disrupts respiratory motor pathways and disables breathing in the affected population. Moreover, cervically injured individuals are at risk for developing acute lung injury, which predicts substantial mortality rates. While the correlation between acute lung injury and spinal cord injury has been found in the clinical setting, the field lacks an animal model to interrogate the fundamental biology of this relationship. To begin to address this gap in knowledge, we performed an experimental cervical spinal cord injury (N = 18) alongside sham injury (N = 3) and naïve animals (N = 15) to assess lung injury in adult rats. We demonstrate that animals display some early signs of lung injury two weeks post-spinal cord injury. While no obvious histological signs of injury were observed, the spinal cord injured cohort displayed significant signs of metabolic dysregulation in multiple pathways that include amino acid metabolism, lipid metabolism, and N-linked glycosylation. Collectively, we establish for the first time a model of lung injury after spinal cord injury at an acute time point that can be used to monitor the progression of lung damage, as well as identify potential targets to ameliorate acute lung injury.

Targeting the Microbiome to Improve Gut Health and Breathing Function After Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition characterized by impaired motor and sensory function, as well as internal organ pathology and dysfunction. This internal organ dysfunction, particularly gastrointestinal (GI) complications, and neurogenic bowel, can reduce the quality of life of individuals with an SCI and potentially hinder their recovery. The gut microbiome impacts various central nervous system functions and has been linked to a number of health and disease states. An imbalance of the gut microbiome, i.e., gut dysbiosis, contributes to neurological disease and may influence recovery and repair processes after SCI. Here we examine the impact of high cervical SCI on the gut microbiome and find that transient gut dysbiosis with persistent gut pathology develops after SCI. Importantly, probiotic treatment improves gut health and respiratory motor function measured through whole-body plethysmography. Concurrent with these improvements was a systemic decrease in the cytokine tumor necrosis factor-alpha and an increase in neurite sprouting and regenerative potential of neurons. Collectively, these data reveal the gut microbiome as an important therapeutic target to improve visceral organ health and respiratory motor recovery after SCI. Research Highlights: Cervical spinal cord injury (SCI) causes transient gut dysbiosis and persistent gastrointestinal (GI) pathology.Treatment with probiotics after SCI leads to a healthier GI tract and improved respiratory motor recovery.Probiotic treatment decreases systemic tumor necrosis factor-alpha and increases the potential for sprouting and regeneration of neurons after SCI.The gut microbiome is a valid target to improve motor function and secondary visceral health after SCI.

Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.

Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases.

In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues.

Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.

Intermittent Hypoxia Induces Greater Functional Breathing Motor Recovery as a Fixed Rather Than Varied Duration Treatment after Cervical Spinal Cord Injury in Rats.

Intermittent hypoxia treatment (IH) has been shown to improve respiratory function in both pre-clinical animal models and human subjects following spinal cord injury (SCI), historically consisting of alternating and equal intervals of hypoxic and normoxic exposure. We describe such a procedure as fixed duration IH (FD-IH) and modulation of its severity, intermittency, and post-injury time-point of application differentially affects expression of breathing motor plasticity. As such, the established IH protocol exhibits similarity to instrumental conditioning and can be described as behavioral training through reinforcement. Findings from the field of operant conditioning, a form of more advanced learning, inspire the consideration that FD-IH protocols may be improved through exchanging fixed for varied durations of hypoxia between reinforcement. Thus, we hypothesized that varied duration intermittent hypoxia treatment (VD-IH) would induce greater breathing motor recovery ipsilateral to injury than FD-IH after cervical SCI in rats. To test this hypothesis, we treated animals with VD-IH or FD-IH for 5 days at 1 week and at 8 weeks following cervical SCI, then assessed breathing motor output by diaphragm electromyography (EMG) recording, and compared between groups. At 1 week post-injury, VD-IH-exposed animals trended slightly toward exhibiting greater levels of respiratory recovery in the hemidiaphragm ipsilateral to lesion than did FD-IH-treated animals, but at 8 weeks FD-IH produced significantly greater respiratory motor output than did VD-IH. Thus, these results identify a novel sensitivity of respiratory motor function to variations in the IH protocol that may lead to development of more effective treatments following SCI.

Novel Influences of Sex and APOE Genotype on Spinal Plasticity and Recovery of Function after Spinal Cord Injury.

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 spinal cord injury (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 because of 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. Using humanized mice, we examined how the ε3 and ε4 alleles of APOE influence the efficacy of therapeutic intermittent hypoxia (IH) in inducing spinally-mediated plasticity after cervical SCI (cSCI). 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 and Mitchell, 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.

Light-induced rescue of breathing after spinal cord injury.

Paralysis is a major consequence of spinal cord injury (SCI). After cervical SCI, respiratory deficits can result through interruption of descending presynaptic inputs to respiratory motor neurons in the spinal cord. Expression of channelrhodopsin-2 (ChR2) and photostimulation in neurons affects neuronal excitability and produces action potentials without any kind of presynaptic inputs. We hypothesized that after transducing spinal neurons in and around the phrenic motor pool to express ChR2, photostimulation would restore respiratory motor function in cervical SCI adult animals. Here we show that light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory diaphragmatic motor activity. Furthermore, robust rhythmic activity persisted long after photostimulation had ceased. This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is NMDA receptor dependent. These data suggest a novel, minimally invasive therapeutic avenue to exercise denervated circuitry and/or restore motor function after SCI.

Role of Propriospinal Neurons in Control of Respiratory Muscles and Recovery of Breathing Following Injury.

Respiratory motor failure is the leading cause of death in spinal cord injury (SCI). Cervical injuries disrupt connections between brainstem neurons that are the primary source of excitatory drive to respiratory motor neurons in the spinal cord and their targets. In addition to direct connections from bulbospinal neurons, respiratory motor neurons also receive excitatory and inhibitory inputs from propriospinal neurons, yet their role in the control of breathing is often overlooked. In this review, we will present evidence that propriospinal neurons play important roles in patterning muscle activity for breathing. These roles likely include shaping the pattern of respiratory motor output, processing and transmitting sensory afferent information, coordinating ventilation with motor activity, and regulating accessory and respiratory muscle activity. In addition, we discuss recent studies that have highlighted the importance of propriospinal neurons for recovery of respiratory muscle function following SCI. We propose that molecular genetic approaches to target specific developmental neuron classes in the spinal cord would help investigators resolve the many roles of propriospinal neurons in the control of breathing. A better understanding of how spinal circuits pattern breathing could lead to new treatments to improve breathing following injury or disease.

Chronic Intermittent Hypoxia Induces Robust Astrogliosis in an Alzheimer's Disease-Relevant Mouse Model.

Sleep disturbances are a common early symptom of neurodegenerative diseases, including Alzheimer's disease (AD) and other age-related dementias, and emerging evidence suggests that poor sleep may be an important contributor to development of amyloid pathology. Of the causes of sleep disturbances, it is estimated that 10-20% of adults in the United States have sleep-disordered breathing (SDB) disorder, with obstructive sleep apnea accounting for the majority of the SBD cases. The clinical and epidemiological data clearly support a link between sleep apnea and AD; yet, almost no experimental research is available exploring the mechanisms associated with this correlative link. Therefore, we exposed an AD-relevant mouse model (APP/PS1 KI) to chronic intermittent hypoxia (IH) (an experimental model of sleep apnea) to begin to describe one of the potential mechanisms by which SDB could increase the risk of dementia. Previous studies have found that astrogliosis is a contributor to neuropathology in models of chronic IH and AD; therefore, we hypothesized that a reactive astrocyte response might be a contributing mechanism in the neuroinflammation associated with sleep apnea. To test this hypothesis, 10-11-month-old wild-type (WT) and APP/PS1 KI mice were exposed to 10 hours of IH, daily for four weeks. At the end of four weeks brains were analyzed from amyloid burden and astrogliosis. No effect was found for chronic IH exposure on amyloid-beta levels or plaque load in the APP/PS1 KI mice. A significant increase in GFAP staining was found in the APP/PS1 KI mice following chronic IH exposure, but not in the WT mice. Profiling of genes associated with different phenotypes of astrocyte activation identified GFAP, CXCL10, and Ggta1 as significant responses activated in the APP/PS1 KI mice exposed to chronic IH.

Mid-cervical spinal cord contusion causes robust deficits in respiratory parameters and pattern variability.

Mid-cervical spinal cord contusion disrupts both the pathways and motoneurons vital to the activity of inspiratory muscles. The present study was designed to determine if a rat contusion model could result in a measurable deficit to both ventilatory and respiratory motor function under "normal" breathing conditions at acute to chronic stages post trauma. Through whole body plethysmography and electromyography we assessed respiratory output from three days to twelve weeks after a cervical level 3 (C3) contusion. Contused animals showed significant deficits in both tidal and minute volumes which were sustained from acute to chronic time points. We also examined the degree to which the contusion injury impacted ventilatory pattern variability through assessment of Mutual Information and Sample Entropy. Mid-cervical contusion significantly and robustly decreased the variability of ventilatory patterns. The enduring deficit to the respiratory motor system caused by contusion was further confirmed through electromyography recordings in multiple respiratory muscles. When isolated via a lesion, these contused pathways were insufficient to maintain respiratory activity at all time points post injury. Collectively these data illustrate that, counter to the prevailing literature, a profound and lasting ventilatory and respiratory motor deficit may be modelled and measured through multiple physiological assessments at all time points after cervical contusion injury.

Rapid and robust restoration of breathing long after spinal cord injury.

There exists an abundance of barriers that hinder functional recovery following spinal cord injury, especially at chronic stages. Here, we examine the rescue of breathing up to 1.5 years following cervical hemisection in the rat. In spite of complete hemidiaphragm paralysis, a single injection of chondroitinase ABC in the phrenic motor pool restored robust and persistent diaphragm function while improving neuromuscular junction anatomy. This treatment strategy was more effective when applied chronically than when assessed acutely after injury. The addition of intermittent hypoxia conditioning further strengthened the ventilatory response. However, in a sub-population of animals, this combination treatment caused excess serotonergic (5HT) axon sprouting leading to aberrant tonic activity in the diaphragm that could be mitigated via 5HT2 receptor blockade. Through unmasking of the continuing neuroplasticity that develops after injury, our treatment strategy ensured rapid and robust patterned respiratory recovery after a near lifetime of paralysis.

MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats.

BACKGROUND: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats. HYPOTHESIS: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies. DESIGN: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluR1 and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluR1, and GluR2. RESULTS: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluR1 and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2. CONCLUSION: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.

A Latent Propriospinal Network Can Restore Diaphragm Function after High Cervical Spinal Cord Injury.

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 that 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 the 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 that 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 SCI in adult mice and rats.

Acute theophylline exposure modulates breathing activity through a cervical contusion.

Cervical spinal contusion injuries are the most common form of spinal cord injury (>50%) observed in humans. These injuries can result in the impaired ability to breathe. In this study we examine the role of theophylline in the rescue of breathing behavior after a cervical spinal contusion. Previous research in the C2 hemisection model has shown that acute administration of theophylline can rescue phrenic nerve activity and diaphragmatic EMG on the side ipsilateral to injury. However, this effect is dependent on intact and uninjured pathways. In this study we utilized a cervical contusion injury model that more closely mimics the human condition. This injury model can determine the effectiveness of therapeutic interventions, in this case theophylline, on the isolated contused pathways of the spinal cord. Three weeks after a 150 kD C3/4 unilateral contusion subjects received a 15 mg/kg dose of theophylline prior to a contralateral C2 hemisection. Subjects that received theophylline were able to effectively utilize damaged pathways to breathe for up to 2 min, while subjects treated with saline were unable to support ventilation. Through these experiments, we demonstrate that theophylline can make injured pathways that mediate breathing more effective and therefore, suggest a potential therapeutic role in the critical time points immediately after injury.