Hyperbaric Oxygen Therapy after Mid-Cervical Spinal Contusion Injury.

Hyperbaric oxygen (HBO) therapy is frequently used to treat peripheral wounds or decompression sickness. Evidence suggests that HBO therapy can provide neuroprotection and has an anti-inflammatory impact after neurological injury, including spinal cord injury (SCI). Our primary purpose was to conduct a genome-wide screening of mRNA expression changes in the injured spinal cord after HBO therapy. An mRNA gene array was used to evaluate samples taken from the contused region of the spinal cord following a lateralized mid-cervical contusion injury in adult female rats. HBO therapy consisted of daily, 1-h sessions (3.0 ATA, 100% O2) initiated on the day of SCI and continued for 10 days. Gene set enrichment analyses indicated that HBO upregulated genes in pathways associated with electron transport, mitochondrial function, and oxidative phosphorylation, and downregulated genes in pathways associated with inflammation (including cytokines and nuclear factor kappa B [NF-κB]) and apoptotic signaling. In a separate cohort, spinal cord histology was performed to verify whether the HBO treatment impacted neuronal cell counts or inflammatory markers. Compared with untreated rats, there were increased NeuN positive cells in the spinal cord of HBO-treated rats (p = 0.004). We conclude that HBO therapy, initiated shortly after SCI and continued for 10 days, can alter the molecular signature of the lesioned spinal cord in a manner consistent with a neuroprotective impact.

AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice.

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.

Anatomy and physiology of phrenic afferent neurons.

Large-diameter myelinated phrenic afferents discharge in phase with diaphragm contraction, and smaller diameter fibers discharge across the respiratory cycle. In this article, we review the phrenic afferent literature and highlight areas in need of further study. We conclude that 1) activation of both myelinated and nonmyelinated phrenic sensory afferents can influence respiratory motor output on a breath-by-breath basis; 2) the relative impact of phrenic afferents substantially increases with diaphragm work and fatigue; 3) activation of phrenic afferents has a powerful impact on sympathetic motor outflow, and 4) phrenic afferents contribute to diaphragm somatosensation and the conscious perception of breathing. Much remains to be learned regarding the spinal and supraspinal distribution and synaptic contacts of myelinated and nonmyelinated phrenic afferents. Similarly, very little is known regarding the potential role of phrenic afferent neurons in triggering or modulating expression of respiratory neuroplasticity.

Automated Gait Analysis Through Hues and Areas (AGATHA): A Method to Characterize the Spatiotemporal Pattern of Rat Gait.

While rodent gait analysis can quantify the behavioral consequences of disease, significant methodological differences exist between analysis platforms and little validation has been performed to understand or mitigate these sources of variance. By providing the algorithms used to quantify gait, open-source gait analysis software can be validated and used to explore methodological differences. Our group is introducing, for the first time, a fully-automated, open-source method for the characterization of rodent spatiotemporal gait patterns, termed Automated Gait Analysis Through Hues and Areas (AGATHA). This study describes how AGATHA identifies gait events, validates AGATHA relative to manual digitization methods, and utilizes AGATHA to detect gait compensations in orthopaedic and spinal cord injury models. To validate AGATHA against manual digitization, results from videos of rodent gait, recorded at 1000 frames per second (fps), were compared. To assess one common source of variance (the effects of video frame rate), these 1000 fps videos were re-sampled to mimic several lower fps and compared again. While spatial variables were indistinguishable between AGATHA and manual digitization, low video frame rates resulted in temporal errors for both methods. At frame rates over 125 fps, AGATHA achieved a comparable accuracy and precision to manual digitization for all gait variables. Moreover, AGATHA detected unique gait changes in each injury model. These data demonstrate AGATHA is an accurate and precise platform for the analysis of rodent spatiotemporal gait patterns.

Neuropathology in respiratory-related motoneurons in young Pompe (Gaa(-/-)) mice.

Respiratory and/or lingual dysfunction are among the first motor symptoms in Pompe disease, a disorder resulting from absence or dysfunction of the lysosomal enzyme acid α-glucosidase (GAA). Here, we histologically evaluated the medulla, cervical and thoracic spinal cords in 6 weeks old asymptomatic Pompe (Gaa(-/-)) mice to determine if neuropathology in respiratory motor regions has an early onset. Periodic acid-Schiff (PAS) staining indicated glycogen accumulation was exclusively occurring in Gaa(-/-) hypoglossal, mid-cervical and upper thoracic motoneurons. Markers of DNA damage (Tunel) and ongoing apoptosis (Cleaved Caspase 3) did not co-localize with PAS staining, but were prominent in a medullary region which included the nucleus tractus solitarius, and also in the thoracic spinal dorsal horn. We conclude that respiratory-related motoneurons are particularly susceptible to GAA deficiency and that neuronal glycogen accumulation and neurodegeneration may occur independently in early stage disease. The data support early therapeutic intervention in Pompe disease.

Power spectral analysis of hypoglossal nerve activity during intermittent hypoxia-induced long-term facilitation in mice.

Power spectral analyses of electrical signals from respiratory nerves reveal prominent oscillations above the primary rate of breathing. Acute exposure to intermittent hypoxia can induce a form of neuroplasticity known as long-term facilitation (LTF), in which inspiratory burst amplitude is persistently elevated. Most evidence indicates that the mechanisms of LTF are postsynaptic and also that high-frequency oscillations within the power spectrum show coherence across different respiratory nerves. Since the most logical interpretation of this coherence is that a shared presynaptic mechanism is responsible, we hypothesized that high-frequency spectral content would be unchanged during LTF. Recordings of inspiratory hypoglossal (XII) activity were made from anesthetized, vagotomized, and ventilated 129/SVE mice. When arterial O2 saturation (SaO2) was maintained >96%, the XII power spectrum and burst amplitude were unchanged for 90 min. Three, 1-min hypoxic episodes (SaO2 = 50 ± 10%), however, caused a persistent (>60 min) and robust (>400% baseline) increase in burst amplitude. Spectral analyses revealed a rightward shift of the signal content during LTF, with sustained increases in content above ∼125 Hz following intermittent hypoxia and reductions in power at lower frequencies. Changes in the spectral content during LTF were qualitatively similar to what occurred during the acute hypoxic response. We conclude that high-frequency content increases during XII LTF in this experimental preparation; this may indicate that intermittent hypoxia-induced plasticity in the premotor network contributes to expression of XII LTF.

Abrupt changes in pentobarbital sensitivity in preBötzinger complex region, hypoglossal motor nucleus, nucleus tractus solitarius, and cortex during rat transitional period (P10-P15).

On postnatal days P10-P15 in rat medulla, neurotransmitter receptor subunit composition shifts toward a more mature phenotype. Since medullary GABAARs regulate cardiorespiratory function, abrupt alterations in GABAergic synaptic inhibition could disrupt homeostasis. We hypothesized that GABAARs on medullary neurons become more resistant to positive allosteric modulation during P10-P15. Medullary and cortical slices from P10 to P20 rats were used to record spontaneous action potentials in pre-Botzinger Complex (preBötC-region), hypoglossal (XII) motor nucleus, nucleus tractus solitarius (NTS), and cortex during exposure to pentobarbital (positive allosteric modulator of GABAARs). On P14, pentobarbital resistance abruptly increased in preBötC-region and decreased in NTS, but these changes in pentobarbital resistance were not present on P15. Pentobarbital resistance decreased in XII motor nucleus during P11-P15 with a nadir at P14. Abrupt changes in pentobarbital resistance indicate changes in GABAergic receptor composition and function that may compensate for potential increased GABAergic inhibition and respiratory depression that occurs during this key developmental transitional period.

Protecting motor networks during perinatal ischemia: the case for delta-opioid receptors.

Perinatal ischemia is a common clinical problem with few successful therapies to prevent neuronal damage. Delta opioid receptor (DOR) activation is a versatile, evolutionarily conserved, endogenous neuroprotective mechanism that blocks several steps in the deleterious cascade of neurological events during ischemia. DOR activation prior to ischemia or severe hypoxia is neuroprotective in spinal motor networks, as well as cortical, cerebellar, and hippocampal neural networks. In addition to providing acute and long-lasting neuroprotection against ischemia, DOR activation appears to provide neuroprotection when given before, during, or following the onset of ischemia. Finally, DORs can be upregulated by several physiological and experimental perturbations. Potential adverse side effects affecting motor control, such as respiratory depression and seizures, are not well established in young mammals and may be mitigated by altering drug choice and method of drug administration. The unique features of DOR-dependent neuroprotection make it an attractive potential therapy that may be given to at-risk pregnant mothers shortly before delivery to provide long-lasting neuroprotection against unpredictable perinatal ischemic events.