Pattern sensitivity of ampakine-hypoxia interactions for evoking phrenic motor facilitation in anesthetized rat.

Repeated hypoxic episodes can produce a sustained (>60 min) increase in neural drive to the diaphragm. The requirement of repeated hypoxic episodes (vs. a single episode) to produce phrenic motor facilitation (pMF) can be removed by allosteric modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors using ampakines. We hypothesized that the ampakine-hypoxia interaction resulting in pMF requires that ampakine dosing precedes the onset of hypoxia. Phrenic nerve recordings were made from urethane-anesthetized, mechanically ventilated, and vagotomized adult male Sprague-Dawley rats during isocapnic conditions. Ampakine CX717 (15 mg/kg iv) was given immediately before (n = 8), during (n = 8), or immediately after (n = 8) a 5-min hypoxic episode (arterial oxygen partial pressure 40-45 mmHg). Ampakine before hypoxia (Aprior) resulted in a sustained increase in inspiratory phrenic burst amplitude (i.e., pMF) reaching +70 ± 21% above baseline (BL) after 60 min. This was considerably greater than corresponding values in the groups receiving ampakine during hypoxia (+28 ± 47% above BL, P = 0.005 vs. Aprior) or after hypoxia (+23 ± 40% above BL, P = 0.005 vs. Aprior). Phrenic inspiratory burst rate, heart rate, and systolic, diastolic, and mean arterial pressure (mmHg) were similar across the three treatment groups (all P > 0.3, treatment effect). We conclude that the presentation order of ampakine and hypoxia impacts the magnitude of pMF, with ampakine pretreatment evoking the strongest response. Ampakine pretreatment may have value in the context of hypoxia-based neurorehabilitation strategies.NEW & NOTEWORTHY Phrenic motor facilitation (pMF) is evoked after repeated episodes of brief hypoxia. pMF can also be induced when an allosteric modulator of AMPA receptors (ampakine) is intravenously delivered immediately before a single brief hypoxic episode. Here we show that ampakine delivery before hypoxia (vs. during or after hypoxia) evokes the largest pMF with minimal impact on arterial blood pressure and heart rate. Ampakine pretreatment may have value in the context of hypoxia-based neurorehabilitation strategies.

Targeting drug or gene delivery to the phrenic motoneuron pool.

Phrenic motoneurons (PhrMNs) innervate diaphragm myofibers. Located in the ventral gray matter (lamina IX), PhrMNs form a column extending from approximately the third to sixth cervical spinal segment. Phrenic motor output and diaphragm activation are impaired in many neuromuscular diseases, and targeted delivery of drugs and/or genetic material to PhrMNs may have therapeutic application. Studies of phrenic motor control and/or neuroplasticity mechanisms also typically require targeting of PhrMNs with drugs, viral vectors, or tracers. The location of the phrenic motoneuron pool, however, poses a challenge. Selective PhrMN targeting is possible with molecules that move retrogradely upon uptake into phrenic axons subsequent to diaphragm or phrenic nerve delivery. However, nonspecific approaches that use intrathecal or intravenous delivery have considerably advanced the understanding of PhrMN control. New opportunities for targeted PhrMN gene expression may be possible with intersectional genetic methods. This article provides an overview of methods for targeting the phrenic motoneuron pool for studies of PhrMNs in health and disease.

Current avenues of gene therapy in Pompe disease.

PURPOSE OF REVIEW: Pompe disease is a rare, inherited, devastating condition that causes progressive weakness, cardiomyopathy and neuromotor disease due to the accumulation of glycogen in striated and smooth muscle, as well as neurons. While enzyme replacement therapy has dramatically changed the outcome of patients with the disease, this strategy has several limitations. Gene therapy in Pompe disease constitutes an attractive approach due to the multisystem aspects of the disease and need to address the central nervous system manifestations. This review highlights the recent work in this field, including methods, progress, shortcomings, and future directions. RECENT FINDINGS: Recombinant adeno-associated virus (rAAV) and lentiviral vectors (LV) are well studied platforms for gene therapy in Pompe disease. These products can be further adapted for safe and efficient administration with concomitant immunosuppression, with the modification of specific receptors or codon optimization. rAAV has been studied in multiple clinical trials demonstrating safety and tolerability. SUMMARY: Gene therapy for the treatment of patients with Pompe disease is feasible and offers an opportunity to fully correct the principal pathology leading to cellular glycogen accumulation. Further work is needed to overcome the limitations related to vector production, immunologic reactions and redosing.

Oxygen therapy attenuates neuroinflammation after spinal cord injury.

Acute hyperbaric O2 (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% O2 (i.e. HBO) more effectively mitigates spinal inflammation and neuronal loss, and enhances respiratory recovery, as compared to normobaric 100% O2. Experimental groups included spinal intact, SCI no O2 therapy, and SCI + 100% O2 delivered at normobaric pressure (1 atmosphere, ATA), or at 2- or 3 ATA. O2 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 O2 therapy. Similarly, both HBO and normobaric O2 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 O2 treatment can reduce the spinal inflammatory response after SCI, but pressured O2 (i.e., HBO) provides further benefit.

Chemogenetic activation of hypoglossal motoneurons in a mouse model of Pompe disease.

Pompe disease is a lysosomal storage disease resulting from absence or deficiency of acid α-glucosidase (GAA). Tongue-related disorders including dysarthria, dysphagia, and obstructive sleep apnea are common in Pompe disease. Our purpose was to determine if designer receptors exclusively activated by designer drugs (DREADDs) could be used to stimulate tongue motor output in a mouse model of Pompe disease. An adeno-associated virus serotype 9 (AAV9) encoding an excitatory DREADD (AAV9-hSyn-hM3D(Gq)-mCherry, 2.44 × 1010 vg) was administered to the posterior tongue of 5-7-wk-old Gaa null (Gaa-/-) mice. Lingual EMG responses to intraperitoneal injection of saline or a DREADD ligand (JHU37160-dihydrochloride, J60) were assessed 12 wk later during spontaneous breathing. Saline injection produced no consistent changes in lingual EMG. Following the DREADD ligand, there were statistically significant (P < 0.05) increases in both tonic and phasic inspiratory EMG activity recorded from the posterior tongue. Brainstem histology confirmed mCherry expression in hypoglossal (XII) motoneurons in all mice, thus verifying retrograde movement of the AAV9 vector. Morphologically, Gaa-/- XII motoneurons showed histological characteristics that are typical of Pompe disease, including an enlarged soma and vacuolization. We conclude that lingual delivery of AAV9 can be used to drive functional expression of DREADD in XII motoneurons in a mouse model of Pompe disease.NEW & NOTEWORTHY In a mouse model of Pompe disease, lingual injection of adeno-associated virus (AAV) serotype 9 encoding DREADD was histologically verified to produce transgene expression in hypoglossal motoneurons. Subsequent intraperitoneal delivery of a DREADD ligand stimulated tonic and phase tongue motor output.In a mouse model of Pompe disease, lingual injection of adeno-associated virus (AAV) serotype 9 encoding DREADD was histologically verified to produce transgene expression in hypoglossal motoneurons. Subsequent intravenous delivery of a DREADD ligand stimulated tonic and phase tongue motor output.

Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[18F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Feasibility of transcutaneous spinal direct current stimulation combined with locomotor training after spinal cord injury.

STUDY DESIGN: Feasibility study, consisting of random-order, cross-over study of a single intervention session, followed by a parallel-arm study of 16 sessions. OBJECTIVES: To investigate the feasibility of a novel combinatorial approach with simultaneous delivery of transcutaneous spinal direct current stimulation (tsDCS) and locomotor training (tsDCS + LT) after spinal cord injury, compared to sham stimulation and locomotor training (sham + LT), and examine preliminary effects on walking function. SETTING: Clinical research center in the southeastern United States. METHODS: Eight individuals with chronic incomplete spinal cord injury (ISCI) completed the two-part protocol. Feasibility was assessed based on safety (adverse responses), tolerability (pain, spasticity, skin integrity), and protocol achievement (session duration, intensity). Walking function was assessed with the 10 m and 6 min walk tests. RESULTS: There were no major adverse responses. Minimal reports of skin irritation and musculoskeletal pain were consistent between groups. Average training peak heart rate as percent of maximum (mean(SD); tsDCS + LT: 66 (4)%, sham + LT: 69 (10)%) and Borg ratings of perceived exertion (tsDCS + LT: 17.5 (1.2), sham + LT: 14.4 (1.8)) indicate both groups trained at high intensities. Walking speed gains exceeded the minimal clinically important difference (MCID) in three of four who received tsDCS + LT (0.18 (0.29) m/s) and one of four in sham + LT (-0.05 (0.23) m/s). Gains in walking endurance exceeded the MCID in one of four in each group (tsDCS + LT: 36.4 (69.0) m, sham + LT: 4.9 (56.9) m). CONCLUSIONS: Combinatorial tsDCS and locomotor training is safe and feasible for individuals with chronic ISCI, even those with considerable walking impairment. Study outcomes support the need to investigate the efficacy of this approach.

Gene delivery to the hypoglossal motor system: preclinical studies and translational potential.

Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are often inadequate, and the tongue is a readily accessible target for therapeutic gene delivery. In this regard, gene therapy specifically targeting the tongue motor system offers two general strategies for treating lingual disorders. First, correcting tongue myofiber and/or hypoglossal (XII) motoneuron pathology in genetic neuromuscular disorders may be readily achieved by intralingual delivery of viral vectors. The retrograde movement of viral vectors such as adeno-associated virus (AAV) enables targeted distribution to XII motoneurons via intralingual viral delivery. Second, conditions with impaired or reduced tongue muscle activation can potentially be treated using viral-driven chemo- or optogenetic approaches to activate or inhibit XII motoneurons and/or tongue myofibers. Further considerations that are highly relevant to lingual gene therapy include (1) the diversity of the motoneurons which control the tongue, (2) the patterns of XII nerve branching, and (3) the complexity of tongue muscle anatomy and biomechanics. Preclinical studies show considerable promise for lingual directed gene therapy in neuromuscular disease, but the potential of such approaches is largely untapped.

Phrenic afferent activation modulates cardiorespiratory output in the adult rat.

Phrenic afferents project to brainstem areas responsible for cardiorespiratory control and the mid-cervical spinal cord containing the phrenic motor nucleus. Our purpose was to quantify the impact of small- and large-diameter phrenic afferent activation on phrenic motor output. Anesthetized and ventilated rats received unilateral phrenic nerve stimulation while contralateral phrenic motor output and blood pressure were recorded. Twelve currents of 40-Hz inspiratory-triggered stimulation were delivered (20 s on, 5 min off) to establish current response curves. Stimulation pulse width was varied to preferentially activate large-diameter phrenic afferents (narrow pulse width) and recruit small-diameter fibers (wide pulse width). Contralateral phrenic amplitude was elevated immediately poststimulation at currents above 35 µA for wide and 70 µA for narrow pulse stimulation when compared with animals not receiving stimulation (time controls). Wide pulse width stimulation also increased phrenic burst frequency at currents ≥35 µA, caused a transient decrease in mean arterial blood pressure at currents ≥50 µA, and resulted in a small change in heart rate at 300 µA. Unilateral dorsal rhizotomy attenuated stimulation-induced cardiorespiratory responses indicating that phrenic afferent activation is required. Additional analyses compared phrenic motor amplitude with output before stimulation and showed that episodic activation of phrenic afferents with narrow pulse stimulation can induce short-term plasticity. We conclude that the activation of phrenic afferents 1) enhances contralateral phrenic motor amplitude when large-diameter afferents are activated, and 2) when small-diameter fibers are recruited, the amplitude response is associated with changes in burst frequency and cardiovascular parameters.NEW & NOTEWORTHY Acute, inspiratory-triggered stimulation of phrenic afferents increases contralateral phrenic motor amplitude in adult rats. When small-diameter afferents are recruited, the amplitude response is accompanied by an increase in phrenic burst frequency, a transient decrease in mean arterial blood pressure, and a slight increase in heart rate. Repeated episodes of large-diameter phrenic afferent activation may also be capable of inducing short-term plasticity.

Ampakine pretreatment enables a single hypoxic episode to produce phrenic motor facilitation with no added benefit of additional episodes.

Repeated short episodes of hypoxia produce a sustained increase in phrenic nerve output lasting well beyond acute intermittent hypoxia (AIH) exposure (i.e., phrenic long-term facilitation; pLTF). Pretreatment with ampakines, drugs which allosterically modulate AMPA receptors, enables a single brief episode of hypoxia to produce pLTF, lasting up to 90 min after hypoxia. Here, we tested the hypothesis that ampakine pretreatment would enhance the magnitude of pLTF evoked by repeated bouts of hypoxia. Phrenic nerve output was recorded in urethane-anesthetized, mechanically ventilated, and vagotomized adult male Sprague-Dawley rats. Initial experiments demonstrated that ampakine CX717 (15 mg/kg iv) caused an acute increase in phrenic nerve inspiratory burst amplitude reaching 70 ± 48% baseline (BL) after 2 min (P = 0.01). This increased bursting was not sustained (2 ± 32% BL at 60 min, P = 0.9). When CX717 was delivered 2 min before a single episode of isocapnic hypoxia (5 min, [Formula: see text] = 44 ± 9 mmHg), facilitation of phrenic nerve burst amplitude occurred (96 ± 62% BL at 60 min, P < 0.001). However, when CX717 was given 2 min before three, 5-min hypoxic episodes ([Formula: see text] = 45 ± 6 mmHg) pLTF was attenuated and did not reach statistical significance (24 ± 29% BL, P = 0.08). In the absence of CX717 pretreatment, pLTF was observed after three (74 ± 33% BL at 60 min, P < 0.001) but not one episode of hypoxia (1 ± 8% BL at 60 min, P = 0.9). We conclude that pLTF is not enhanced when ampakine pretreatment is followed by repeated bouts of hypoxia. Rather, the combination of ampakine and a single hypoxic episode appears to be ideal for producing sustained increase in phrenic motor output.NEW & NOTEWORTHY Pretreatment with ampakine CX717 created conditions that enabled an acute bout of moderate hypoxia to evoke phrenic motor facilitation, but this response was not observed when ampakine pretreatment was followed by intermittent hypoxia. Thus, in anesthetized and spinal intact rats, the combination of ampakine and one bout of hypoxia appears ideal for triggering respiratory neuroplasticity.

Case Studies in Neuroscience: Neuropathology and diaphragm dysfunction in ventilatory failure from late-onset Pompe disease.

Pompe disease (PD) is a neuromuscular disorder caused by a mutation in the acid alpha-glucosidase (GAA) gene. Patients with late-onset PD retain some GAA activity and present symptoms later in life, with fatality mainly associated with respiratory failure. This case study presents diaphragm electrophysiology and a histological analysis of the brainstem, spinal cord, and diaphragm, from a male PD patient diagnosed with late-onset PD at age 35. The patient was wheelchair dependent by age 38, required nocturnal ventilation at age 40, 24-h noninvasive ventilation by age 43, and passed away from respiratory failure at age 54. Diaphragm electromyography recorded using indwelling "pacing" wires showed asynchronous bursting between the left and right diaphragm during brief periods of independent breathing. The synchrony declined over a 4-yr period preceding respiratory failure. Histological assessment indicated motoneuron atrophy in the medulla and rostral spinal cord. Hypoglossal (soma size: 421 ± 159 µm2) and cervical motoneurons (soma size: 487 ± 189 µm2) had an atrophied, elongated appearance. In contrast, lumbar (soma size: 1,363 ± 677 µm2) and sacral motoneurons (soma size: 1,411 ± 633 µm2) had the ballooned morphology typical of early-onset PD. Diaphragm histology indicated loss of myofibers. These results are consistent with neuromuscular degeneration and the concept that effective PD therapy will need to target the central nervous system, in addition to skeletal and cardiac muscle.NEW & NOTEWORTHY This case study offered a unique opportunity to investigate longitudinal changes in phrenic neurophysiology in an individual with severe, ventilator-dependent, late-onset Pompe disease. Additional diaphragm and neural tissue histology upon autopsy confirmed significant neuromuscular degeneration, and it provided novel insights regarding rostral to caudal variability in the neuropathology. These findings suggest that a successful treatment approach for ventilator-dependent Pompe disease should target the central nervous system, in addition to skeletal muscle.

Diaphragm Pacing and a Model for Respiratory Rehabilitation After Spinal Cord Injury.

BACKGROUND AND PURPOSE: Cervical spinal cord injury (CSCI) can cause severe respiratory impairment. Although mechanical ventilation (MV) is a lifesaving standard of care for these patients, it is associated with diaphragm atrophy and dysfunction. Diaphragm pacing (DP) is a strategy now used acutely to promote MV weaning and to combat the associated negative effects. Initial reports indicate that DP also may promote neuromuscular plasticity and lead to improvements in spontaneous diaphragm activation and respiratory function. These outcomes suggest the need for reevaluation of respiratory rehabilitation for patients with CSCI using DP and consideration of new rehabilitation models for these patients and their unique care needs. SUMMARY OF KEY POINTS: This article discusses the rationale for consideration of DP as a rehabilitative strategy, particularly when used in combination with established respiratory interventions. In addition, a model of respiratory rehabilitation and recovery (RRR) is presented, providing a framework for rehabilitation and consideration of DP as an adjuvant rehabilitation approach. The model promotes goals such as respiratory recovery and independence, and lifelong respiratory health, via interdisciplinary care, respiratory training, quantitative measurement, and use of adjuvant strategies such as DP. Application of the model is demonstrated through a description of an inpatient rehabilitation program that applies model components to patients with CSCI who require DP. RECOMMENDATIONS FOR CLINICAL PRACTICE: As DP use increases for patients with acute CSCI, so does the need and opportunity to advance rehabilitation approaches for these patients. This perspective article is a critical step in addressing this need and motivating the advancement of rehabilitation strategies for CSCI patients. (See Video Abstract, Supplemental Digital Content, available at: http://links.lww.com/JNPT/A348).

Hyperbaric Oxygen Treatment Following Mid-Cervical Spinal Cord Injury Preserves Diaphragm Muscle Function.

Oxidative damage to the diaphragm as a result of cervical spinal cord injury (SCI) promotes muscle atrophy and weakness. Respiratory insufficiency is the leading cause of morbidity and mortality in cervical spinal cord injury (SCI) patients, emphasizing the need for strategies to maintain diaphragm function. Hyperbaric oxygen (HBO) increases the amount of oxygen dissolved into the blood, elevating the delivery of oxygen to skeletal muscle and reactive oxygen species (ROS) generation. It is proposed that enhanced ROS production due to HBO treatment stimulates adaptations to diaphragm oxidative capacity, resulting in overall reductions in oxidative stress and inflammation. Therefore, we tested the hypothesis that exposure to HBO therapy acutely following SCI would reduce oxidative damage to the diaphragm muscle, preserving muscle fiber size and contractility. Our results demonstrated that lateral contusion injury at C3/4 results in a significant reduction in diaphragm muscle-specific force production and fiber cross-sectional area, which was associated with augmented mitochondrial hydrogen peroxide emission and a reduced mitochondrial respiratory control ratio. In contrast, rats that underwent SCI followed by HBO exposure consisting of 1 h of 100% oxygen at 3 atmospheres absolute (ATA) delivered for 10 consecutive days demonstrated an improvement in diaphragm-specific force production, and an attenuation of fiber atrophy, mitochondrial dysfunction and ROS production. These beneficial adaptations in the diaphragm were related to HBO-induced increases in antioxidant capacity and a reduction in atrogene expression. These findings suggest that HBO therapy may be an effective adjunctive therapy to promote respiratory health following cervical SCI.

Intermittent Hypoxia Enhances Functional Connectivity of Midcervical Spinal Interneurons.

Brief, intermittent oxygen reductions [acute intermittent hypoxia (AIH)] evokes spinal plasticity. Models of AIH-induced neuroplasticity have focused on motoneurons; however, most midcervical interneurons (C-INs) also respond to hypoxia. We hypothesized that AIH would alter the functional connectivity between C-INs and induce persistent changes in discharge. Bilateral phrenic nerve activity was recorded in anesthetized and ventilated adult male rats and a multielectrode array was used to record C4/5 spinal discharge before [baseline (BL)], during, and 15 min after three 5 min hypoxic episodes (11% O2, H1-H3). Most C-INs (94%) responded to hypoxia by either increasing or decreasing firing rate. Functional connectivity was examined by cross-correlating C-IN discharge. Correlograms with a peak or trough were taken as evidence for excitatory or inhibitory connectivity between C-IN pairs. A subset of C-IN pairs had increased excitatory cross-correlations during hypoxic episodes (34%) compared with BL (19%; p < 0.0001). Another subset had a similar response following each episode (40%) compared with BL (19%; p < 0.0001). In the latter group, connectivity remained elevated 15 min post-AIH (30%; p = 0.0002). Inhibitory C-IN connectivity increased during H1-H3 (4.5%; p = 0.0160), but was reduced 15 min post-AIH (0.5%; p = 0.0439). Spike-triggered averaging indicated that a subset of C-INs is synaptically coupled to phrenic motoneurons and excitatory inputs to these "pre-phrenic" cells increased during AIH. We conclude that AIH alters connectivity of the midcervical spinal network. To our knowledge, this is the first demonstration that AIH induces plasticity within the propriospinal network.SIGNIFICANCE STATEMENT Acute intermittent hypoxia (AIH) can trigger spinal plasticity associated with sustained increases in respiratory, somatic, and/or autonomic motor output. The impact of AIH on cervical spinal interneuron (C-IN) discharge and connectivity is unknown. Our results demonstrate that AIH recruits excitatory C-INs into the spinal respiratory (phrenic) network. AIH also enhances excitatory and reduces inhibitory connections among the C-IN network. We conclude that C-INs are part of the respiratory, somatic, and/or autonomic response to AIH, and that propriospinal plasticity may contribute to sustained increases in motor output after AIH.

Airway smooth muscle dysfunction in Pompe (Gaa-/- ) mice.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.

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.

High-frequency epidural stimulation across the respiratory cycle evokes phrenic short-term potentiation after incomplete cervical spinal cord injury.

C2 spinal hemilesion (C2Hx) paralyzes the ipsilateral diaphragm, but recovery is possible through activation of "crossed spinal" synaptic inputs to ipsilateral phrenic motoneurons. We tested the hypothesis that high-frequency epidural stimulation (HF-ES) would potentiate ipsilateral phrenic output after subacute and chronic C2Hx. HF-ES (300 Hz) was applied to the ventrolateral C4 or T2 spinal cord ipsilateral to C2Hx in anesthetized and mechanically ventilated adult rats. Stimulus duration was 60 s, and currents ranged from 100 to 1,000 µA. Bilateral phrenic nerve activity and ipsilateral hypoglossal (XII) nerve activity were recorded before and after HF-ES. Higher T2 stimulus currents potentiated ipsilateral phasic inspiratory activity at both 2 and 12 wk post-C2Hx, whereas higher stimulus currents delivered at C4 potentiated ipsilateral phasic phrenic activity only at 12 wk (P = 0.028). Meanwhile, tonic output in the ipsilateral phrenic nerve reached 500% of baseline values at the high currents with no difference between 2 and 12 wk. HF-ES did not trigger inspiratory burst-frequency changes. Similar responses occurred following T2 HF-ES. Increases in contralateral phrenic and XII nerve output were induced by C4 and T2 HF-ES at higher currents, but the relative magnitude of these changes was small compared with the ipsilateral phrenic response. We conclude that following incomplete cervical spinal cord injury, HF-ES of the ventrolateral midcervical or thoracic spinal cord can potentiate efferent phrenic motor output with little impact on inspiratory burst frequency. However, the substantial increases in tonic output indicate that the uninterrupted 60-s stimulation paradigm used is unlikely to be useful for respiratory muscle activation after spinal injury.NEW & NOTEWORTHY Previous studies reported that high-frequency epidural stimulation (HF-ES) activates the diaphragm following acute spinal transection. This study examined HF-ES and phrenic motor output following subacute and chronic incomplete cervical spinal cord injury. Short-term potentiation of phrenic bursting following HF-ES illustrates the potential for spinal stimulation to induce respiratory neuroplasticity. Increased tonic phrenic output indicates that alternatives to the continuous stimulation paradigm used in this study will be required for respiratory muscle activation after spinal cord injury.

Peripheral nerve and neuromuscular junction pathology in Pompe disease.

Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of 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.

Correcting Neuromuscular Deficits With Gene Therapy in Pompe Disease.

OBJECTIVE: We have recently reported on the pathology of the neuromuscular junction (NMJ) in Pompe disease, reflecting disruption of neuronal and muscle homeostasis as a result of glycogen accumulation. The aim of this study was to examine how the alteration of NMJ physiology contributes to Pompe disease pathology; we performed molecular, physiological, and histochemical analyses of NMJ-related measures of the tibialis anterior muscles of young-, mid-, and late-stage alpha-glucosidase (GAA)-deficient mice. METHODS: We performed intramuscular injection of an adeno-associated virus (AAV)9 vector expressing GAA (AAV9-hGAA) into the tibialis anterior muscle of Gaa(-/-) mice at early, mid, and severe pathological time points. We analyzed expression of NMJ-related genes, in situ muscle force production, and clearance of glycogen in conjunction with histological assessment of the NMJ. RESULTS: Our data demonstrate that AAV9-hGAA is able to replace GAA to the affected tissue and modify AChR mRNA expression, muscle force production, motor endplate area, and innervation status. Importantly, the degree of restoration for these outcomes is limited by severity of disease. Early restoration of GAA activity was most effective, whereas late correction of GAA expression was not effective in modifying parameters reflecting NMJ structure and function nor in force restoration despite resolution of glycogen storage in muscle. INTERPRETATION: Our data provide new mechanistic insight into the pathology of Pompe disease and suggest that early systemic correction to both neural and muscle tissues may be essential for successful correction of neuromuscular function in Pompe disease. Ann Neurol 2015;78:222-234.