David D. Fuller

Rehabilitation paradigms using brief hypoxia exposure can improve recovery after spinal cord injury. A low dose of ampakines, which enhance AMPA neurotransmission, coupled with brief hypoxia (A-H), uniquely evokes respiratory neuroplasticity (“phrenic motor facilitation” or pMF) via a mechanism that requires spinal NMDA, but not serotonin or adenosine receptor activation. A-H also increased diaphragm activation in rats with chronic SCI, suggesting that the A-H pairing may be useful in neurorehabilitation.

Cervical spinal cord injury directly impacts the phrenic motor neuron pool, resulting in impaired ventilation and diaphragm muscle dysfunction. Previously, we demonstrated that hyperbaric oxygen (HBO) therapy, delivered during the acute phase following SCI, can attenuate diaphragm dysfunction. However, despite the demonstrated efficacy of HBO to improve muscle function, the molecular signaling pathways regulating this effect remain unclear. These experiments test the hypothesis that the protective effects of HBO therapy are associated with upregulation of the endogenous antioxidants SOD1, SOD2 and GPX1. Adult male Sprague-Dawley rats underwent a lateral spinal contusion injury at C4 followed by 10 consecutive days of HBO (3 ATA, 100% O₂, 1 h/day). Room air-treated spinal intact and injured rats were placed in a non-pressurized chamber for equal durations. To prevent the HBO-induced increase in antioxidant expression, antisense oligonucleotides directed towards each gene of interest were administered by intraperitoneal injection daily immediately following completion of HBO exposure (10 mg/kg/day). Saline was used as both the vehicle and placebo. Our results reveal that prevention of the HBO-induced upregulation of SOD1, SOD2 and GPX1 reduces diaphragm-specific force production compared to the control, injured HBO-treated rats. This effect occurred concomitantly with increased gene expression of the inflammatory cytokines IL-6, IL-1β and TNF-α and altered redox balance in the diaphragm. Thus, these data provide evidence that the HBO-induced increase in SOD1, SOD2 and GPX1 antioxidant expression contributes, at least in part, to the therapeutic effect of HBO to preserve diaphragm function following cervical spinal cord injury.

We used a novel algorithm for breath-by-breath detection on concurrent plethysmography and diaphragm EMG recordings to identify shifts in breathing throughout the lifespan of Pompe rats. We identified a “respiratory signature” of disease progression in the Pompe rat comprising a decrease in the frequency of sniffing behaviors and increased latency from diaphragm activation to inspiratory flow during hypoxia. This signature offers new metrics to evaluate the effectiveness of potential therapies.

Treatment with a positive allosteric AMPA receptor modulator (“ampakine”) can improve respiratory muscle activation and bladder function after sub-acute (days) to chronic (weeks to months) spinal cord injury (SCI). Prior studies of SCI and excitotoxicity provide evidence that ampakines may also promote neuroprotection. We hypothesized that initiating daily low-dose treatment with the low-impact ampakine CX1739 acutely after SCI would be neuroprotective and promote recovery. Adult rats received unilateral 150 kydne C4 contusion; CX1739 (5 mg/kg, n = 12) or vehicle (hydroxypropyl beta-cyclodextrin, HPCD; n = 11) given 15 min post-SCI and daily for 14 days. Breathing was evaluated using whole-body plethysmography, and locomotion was evaluated using an open field test. Cervical spinal cords were stained with NeuN to identify neuronal soma, MCA-6H63 to identify degenerating axons, and Iba-1 to identify microglia and macrophages. No differences between the HPCD and ampakine groups were noted in neuronal counts, number of MCA-6H63 positive axons, or Iba-1 staining. Respiratory rate and tidal volume were similar between groups. Ampakine treatment, however, was associated with reduced open-field motor scores and increased relative risk of post-SCI complications. We conclude that ampakine CX1739 (5 mg/kg) given daily over 0–14 days post-SCI provides no discernible benefit, and acute ampakine treatment is contraindicated, in contrast to delayed dosing paradigms. Ampakine treatment should be reserved for the subacute and chronic SCI conditions, beyond the acute period of glutamate-related neurotoxicity. These results will be particularly important in determining the optimal timing of ampakine administration as CX1739 progresses in clinical trials.

Impaired respiratory motor output contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We investigated if expressing designer receptors exclusively activated by designer drugs (DREADDs) in the mid-cervical spinal cord could effectively stimulate phrenic motor output to increase diaphragm activation. Two primary questions were addressed: (1) does effective DREADD-mediated diaphragm activation require focal expression in phrenic motoneurons (vs. non-specific mid-cervical expression), and (2) can this method produce a sustained increase in inspiratory tidal volume? Wild-type (C57Bl/6) and ChAT-Cre mice received bilateral intraspinal (C4) injections of an adeno-associated virus (AAV) encoding the hM3D(Gq) excitatory DREADD. In wild-type mice, this produced non-specific DREADD expression throughout the mid-cervical ventral horn. In ChAT-Cre mice, a Cre-dependent viral construct was used to drive neuronal DREADD expression in the C4 ventral horns, targeting phrenic motoneurons. Diaphragm electromyograms (EMG) were recorded in isoflurane-anesthetized spontaneously breathing mice at 4–9 weeks post-AAV delivery. The DREADD ligand JHU37160 (J60) caused a bilateral, sustained (>1 hr) increase in inspiratory EMG bursting in both groups; the relative increase was greater in ChAT-Cre mice. Additional experiments in ChAT-Cre rats were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume during spontaneous breathing, assessed using whole-body plethysmography without anesthesia. Three to four months after intraspinal (C4) injection of AAV driving Cre-dependent hM3D(Gq) expression, intravenous J60 resulted in a sustained (>30 min) increase in tidal volume. Subsequently, phrenic nerve recordings performed under urethane anesthesia confirmed that J60 evoked a >200% increase in inspiratory output. We conclude that targeting mid-cervical spinal DREADD expression to the phrenic motoneuron pool enables ligand-induced, sustained increases in phrenic motor output and tidal volume. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.

Impaired respiratory motor output contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We investigated if expressing designer receptors exclusively activated by designer drugs (DREADDs) in the mid-cervical spinal cord could effectively stimulate phrenic motor output to increase diaphragm activation. Two primary questions were addressed: 1) does effective DREADD-mediated diaphragm activation require focal expression in phrenic motoneurons (vs. non-specific mid-cervical expression), and 2) can this method produce a sustained increase in inspiratory tidal volume? Wild type (C57/bl6) and ChAT-Cre mice received bilateral intraspinal (C4) injections of an adeno-associated virus (AAV) encoding the hM3D(Gq) excitatory DREADD. In wild-type mice, this produced non-specific DREADD expression throughout the mid-cervical ventral horn. In ChAT-Cre mice, a Cre-dependent viral construct was used to drive neuronal DREADD expression in the C4 ventral horn, targeting phrenic motoneurons. Diaphragm EMG was recorded in isoflurane-anesthetized spontaneously breathing mice at 4-9 weeks post-AAV delivery. The DREADD ligand JHU37160 (J60) caused a bilateral, sustained (>1 hour) increase in inspiratory EMG bursting in both groups; the relative increase was greater in ChAT-Cre mice. Additional experiments in ChAT-Cre rats were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume (VT) during spontaneous breathing, assessed using whole-body plethysmography without anesthesia. Three-to-four months after intraspinal (C4) injection of AAV driving Cre-dependent hM3D(Gq) expression, intravenous J60 resulted in a sustained (>30 min) increase in VT. Subsequently, phrenic nerve recordings performed under urethane anesthesia confirmed that J60 evoked a > 200% increase in inspiratory output. We conclude that targeting mid-cervical spinal DREADD expression to the phrenic motoneuron pool enables ligand-induced, sustained increases in phrenic motor output and VT. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.