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Acute hyperbaric O 2 (HBO) therapy after spinal cord injury (SCI) can reduce inflammation and increase neuronal survival. To our knowledge, it is unknown if these benefits of HBO require hyperbaric vs. normobaric hyperoxia. We used a C4 lateralized contusion SCI in adult male and female rats to test the hypothesis that the combination of hyperbaria and 100% O 2 (i.e. HBO) more effectively mitigates spinal inflammation and neuronal loss, and enhances respiratory recovery, as compared to normobaric 100% O 2 . Experimental groups included spinal intact, SCI no O 2 therapy, and SCI + 100% O 2 delivered at normobaric pressure (1 atmosphere, ATA), or at 2- or 3 ATA. O 2 treatments lasted 1-h, commenced within 2-h of SCI, and were repeated for 10 days. The spinal inflammatory response was assessed with transcriptomics (RNAseq) and immunohistochemistry. Gene co-expression network analysis showed that the strong inflammatory response to SCI was dramatically diminished by both hyper- and normobaric O 2 therapy. Similarly, both HBO and normobaric O 2 treatments reduced the prevalence of immunohistological markers for astrocytes (glial fibrillary acidic protein) and microglia (ionized calcium binding adaptor molecule) in the injured spinal cord. However, HBO treatment also had unique impacts not detected in the normobaric group including upregulation of an anti-inflammatory cytokine (interleukin-4) in the plasma, and larger inspiratory tidal volumes at 10-days (whole body-plethysmography measurements). We conclude that normobaric O 2 treatment can reduce the spinal inflammatory response after SCI, but pressured O 2 (i.e., HBO) provides further benefit.

Impaired diaphragm activation is common in many neuromuscular diseases. We hypothesized that expressing photoreceptors in diaphragm myofibers would enable light stimulation to evoke functional diaphragm activity, similar to endogenous bursts. In a mouse model, adeno-associated virus (AAV) encoding channelrhodopsin-2 (AAV9-CAG-ChR2-mVenus, 6.12 × 10 11 vg dose) was delivered to the diaphragm using a minimally invasive method of microinjection to the intrapleural space. At 8–18 weeks following AAV injection, mice were anesthetized and studied during spontaneous breathing. We first showed that diaphragm electromyographic (EMG) potentials could be evoked with brief presentations of light, using a 473 nm high intensity LED. Evoked potential amplitude increased with intensity or duration of the light pulse. We next showed that in a paralyzed diaphragm, trains of light pulses evoked diaphragm EMG activity which resembled endogenous bursting, and this was sufficient to generate respiratory airflow. Light-evoked diaphragm EMG bursts showed no diminution after up to one hour of stimulation. Histological evaluation confirmed transgene expression in diaphragm myofibers. We conclude that intrapleural delivery of AAV9 can drive expression of ChR2 in the diaphragm and subsequent photostimulation can evoke graded compound diaphragm EMG activity similar to endogenous inspiratory bursting.

Cardiorespiratory dysfunction resulting from doxorubicin (DOX) chemotherapy treatment is a debilitating condition affecting cancer patient outcomes and quality of life. DOX treatment promotes cardiac and respiratory muscle pathology due to enhanced reactive oxygen species (ROS) production, mitochondrial dysfunction and impaired muscle contractility. In contrast, hyperbaric oxygen (HBO) therapy is considered a controlled oxidative stress that can evoke a substantial and sustained increase in muscle antioxidant expression. This HBO-induced increase in antioxidant capacity has the potential to improve cardiac and respiratory (i.e., diaphragm) muscle redox balance, preserving mitochondrial function and preventing muscle dysfunction. Therefore, we determined whether HBO therapy prior to DOX treatment is sufficient to enhance muscle antioxidant expression and preserve muscle redox balance and cardiorespiratory muscle function. To test this, adult female Sprague Dawley rats received HBO therapy (2 or 3 atmospheres absolute (ATA), 100% O2, 1 h/day) for 5 consecutive days prior to acute DOX treatment (20 mg/kg i.p.). Our data demonstrate that 3 ATA HBO elicits a greater antioxidant response compared to 2 ATA HBO. However, these effects did not correspond with beneficial adaptations to cardiac systolic and diastolic function or diaphragm muscle force production in DOX treated rats. These findings suggest that modulating muscle antioxidant expression with HBO therapy is not sufficient to prevent DOX-induced cardiorespiratory dysfunction.

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa−/−) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.

Study design Experimental Animal Study. Objective To continue validating an antibody which targets an epitope of neurofilament light chain (NF-L) only available during neurodegeneration and to utilize the antibody to describe the pattern of axonal degeneration 10 days post-unilateral C4 contusion in the rat. Setting University of Florida laboratory in Gainesville, USA. Methods Sprague Dawley rats received either a unilateral 150kdyne C4 contusion (n = 4 females, n = 5 males) or a laminectomy control surgery (n = 2 females, n = 3 males). Ten days following SCI or laminectomy, spinal cords and brainstems were processed for immunohistochemistry. Serial spinal cord and brainstem cross-sections were stained with the degeneration-specific NF-L antibody (MCA-6H63) and dual labeled with either an antibody against the C-terminus portion of NF-L (NF-L-Ct), to label healthy axons, or an antibody against amyloid precursor protein (APP), considered the current “gold standard” for identifying axonal injury. The pattern of ongoing axonal degeneration was assessed. Results Spinal cord and brainstem cross-sections from injured rats had punctate MCA-6H63 positive fibers with a pathological appearance, loss of anti-NF-L-Ct colabeling, and frequent colocalization with APP. Immunopositive fibers were abundant rostral and caudal to the lesion in white matter tracts that would be disrupted by the unilateral C4 contusion. This pattern of staining was not observed in control tissue. Conclusions The MCA-6H63 antibody labels degenerating axons following SCI and offers a tool to quantify axonal degeneration.

The spiny mouse (Acomys species) has emerged as an exciting research organism due to its remarkable ability to undergo scarless regeneration of skin wounds and ear punches. Excitingly, Acomys species demonstrate scar-free healing in a wide-range of tissues beyond the skin. In this perspective article, we discuss published findings from a variety of tissues to highlight how this emerging research organism could shed light on numerous clinically relevant human diseases. We also discuss the challenges of working with this emerging research organism and suggest strategies for future Acomys-inspired research.

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.

Cervical spinal cord contusion is the most common human spinal cord injury, yet few rodent models replicate the pathophysiological and functional sequela of this injury. Here, we modified an electromechanical injury device and characterized the behavioral and histological changes occurring in response to a lateralized C4 contusion injury in rats. A key feature of the model includes a non-injurious touch phase where the spinal cord surface is dimpled with a consistent starting force. Animals were either left intact as a control, received a non-injury–producing touch on the surface of the cord (“sham”), or received a 0.6 mm or a 0.8 mm displacement injury. Rats were then tested on the forelimb asymmetry use test, CatWalk, and the Irvine, Beatties, and Bresnahan (IBB) cereal manipulation task to assess proximal and distal upper limb function for 12 weeks. Injuries of moderate (0.6 mm) and large (0.8 mm) displacement showed consistent differences in forelimb asymmetry, metrics of the CatWalk, and sub-scores of the IBB. Overall findings indicated long lasting proximal and distal upper limb deficits following 0.8 mm injury but transient proximal with prolonged distal limb deficits following 0.6 mm injury. Significant differences in loss of ipsilateral unmyelinated and myelinated white matter was detected between injury severities. Demyelination was primarily localized to the dorsolateral region of the hemicord and extended further rostral following 0.8 mm injury. These findings establish the C4 hemi-contusion injury as a consistent, graded model for testing novel treatments targeting forelimb functional recovery.

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. nonspecific 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.