Diaphragmatic EMG Activity Research Articles

Tongue exercise-induced plasticity in upper airway EMG activity in a model of hypoglossal motor neuron loss

Motor neuron diseases (MNDs) are a group of neuromuscular disorders in which motor neurons (MNs) required for activities of daily living are lost. Patients with MNDs often develop swallowing and breathing dysfunction due to pathology of the hypoglossal (XII) axis ( e.g., XII lower motor neurons, LMNs). These co-dependent, vital functions require reciprocal roles of the tongue during swallowing, and impaired control and coordination of these opposing behaviors can lead to aspiration pneumonia and respiratory failure. Due to a lack of research, available treatments for patients with MNDs are largely palliative, and there is a critical need for therapies targeting the preservation of upper airway function and coordination. The extensive tongue muscle weakness in patients with MNDs suggests a potential role for therapeutic exercise, but suffcient evidence is lacking to conclude if tongue exercise is beneficial or harmful. A barrier in studying the impact of XII LMN degeneration and developing therapies to improve swallowing and breathing function/coordination has been the lack of an appropriate model. We therefore developed a novel rodent model of XII LMN loss using intralingual injections of cholera toxin B conjugated to saporin (CTB-SAP), which has tongue motility and swallowing deficits that are mitigated by a high-repetition/low resistance tongue exercise paradigm for muscle growth. Here, we simultaneously studied upper airway (geniohyoid) and diaphragm EMG activity with swallowing ( via videofluoroscopy swallow studies; VFSS) and breathing ( via plethysmography) to discern whether CTB-SAP induced XII LMN loss results in upper airway deficits and discoordination of swallowing and breathing functions. We hypothesized that geniohyoid (not diaphragm) EMG activity would be decreased and swallow-breathing coordination would be altered in unanesthetized and spontaneously breathing CTB-SAP rats vs. controls, and that tongue exercise would mitigate these deficits. Using custom-made headcap implants, we embedded EMG electrodes into the geniohyoid and diaphragm muscles in adult male rats prior to intralingual injection of CTB-SAP or control (CTB unconjugated to SAP) and studied chronic EMG activity during VFSS or during plethysmography studies +/- tongue exercise (N=4-6/group). Our preliminary findings show decreased geniohyoid (but not diaphragm) EMG activity as well as swallow/breathing discoordination that appears to be reversed with tongue exercise in CTB-SAP rats vs. controls. In conclusion, tongue exercise appears to enhance XII axis plasticity to improve upper airway function and coordination in the face of XII LMN degeneration. Studies are now underway to understand the underlying mechanism of tongue exercise-induced neuroplasticity, which may help guide future translational studies in patients with MNDs. R01 HL153612 and Phi Zeta. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Closed-loop electrical epidural stimulation restores ipsilesional in-phase diaphragm activity in spontaneously breathing rats after acute C2-hemisection

Epidural electrical stimulation has great promise for recovering many functions after spinal cord injury, including breathing. High-frequency stimulation of the thoracic [DiMarco and Kowalski, 2009] and cervical cord [Gonzalez-Rothi et al. 2017] recovers some phrenic and diaphragm activity. However, open-loop stimulation paradigms induce tonic activity and, thus far, have not elicited recovery of breathing function independent of the stimulation period. We have shown in C2-hemisected rats, closed-loop epidural stimulation (CLES; paced from contralateral diaphragm EMG activity) increases excitability in the respiratory motor network [Malone et al. 2022]; still, the ability of CLES to recover respiratory function following spinal cord injury has not been explored. Here, we hypothesized that CLES can elicit phasic ipsilesional diaphragm activity in C2-hemisected rats without tonic output. To test this, 3-5 month female Sprague-Dawley rats were anesthetized via inhaled isoflurane anesthetic, implanted with stimulating wire electrodes on the dorsal dura at C4 and recording electrodes bilaterally on the diaphragm, and C2-hemisected. After injury, a 15-30 minute recovery to ensure abolishment of ipsilateral diaphragm activity was followed by measurement of spinal motor evoked potentials of the diaphragm, and a baseline recording of diaphragm EMG activity. Biphasic CLES (n=3), set to an amplitude of ¼ the motor threshold, or a sham waiting period (n=3) was delivered for 20 minutes. EMG post processing was done in Spike2 using custom scripts. Stimulated animals received on average 101.8 +/- 22.8 Hz of stimulation during breaths. In stimulated animals, phasic ipsilesional diaphragm EMG activity significantly increased during stimulation (23.7 +/- 1.9 spikes per breath vs 4.32 +/- 1.9 at baseline, p < 0.005). Stimulation did not increase tonic activity of the ipsilesional diaphragm, and ipsilesional diaphragm activity stopped with cessation of stim indicating no spontaneous recovery of activity. Additionally, there was no significant recovery of sham animals during the same period (5.2 +/- 1.9 vs 4.5 +/- 1.9 spikes per breath). Peak EMG activity and modulus of the contralesional diaphragm were significantly elevated from baseline during stimulation (peak value 0.19 +/- 0.015 V vs 0.15 +/- 0.005 V baseline p < 0.05, % change modulus 34.9 +/- 5.9%, p < 0.03). This work represents the first report that CLES can increase phasic diaphragm EMG activity both ipsi- and contralaterally after acute C2-hemisection. Further study is necessary to determine if CLES can elicit similar effects in a chronic model of injury. Additionally, work to determine the mechanism underlying these changes is essential to tailor this therapy for improving breathing function in individuals with spinal cord injury. T32HL134621, R01HL153102, SPARC OT2OD023854 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuromuscular coupling of obligatory respiratory muscles to inspiratory pressure is reduced in dystrophic (mdx) mice

Neuromuscular coupling (NMC) is a recognised clinical index of inspiratory muscle function which reflects the efficiency of conversion of neural activation to inspiratory pressure. Duchenne muscular dystrophy (DMD) is characterised by respiratory insufficiency culminating in respiratory failure. We investigated inspiratory pressure generating capacity and electrical activation of obligatory inspiratory muscles in X-linked muscular dystrophy ( mdx) mice, to evaluate NMC of diaphragm, external intercostal and parasternal intercostal to inspiratory pressure across a range of behaviours in early established to late progressive disease.In urethane anaesthetised mice (1.7 g/kg i.p.), inspiratory pressure was recorded via a pressure transducer positioned in the thoracic oesophagus. Diaphragm EMG activity was recorded using needle electrodes. Studies were performed in male wildtype (WT) and mdx mice at 4, 8, 12 and 16 months old. Recordings were made at baseline and during sustained tracheal occlusion to task failure, defined as an inability to sustain peak pressure. Peak pressure was determined from the average of five successive peak efforts at the maximum response. The corresponding peak inspiratory EMG activities were determined. Two factor (age x genotype) analysis of variance was performed.Obligatory muscle EMG activities were significantly lower in mdx mice from 4 months of age. Peak inspiratory pressure was significantly greater in mdx at 4 months of age. At 8 months, peak inspiratory pressure was equivalent between WT and mdx followed by a significant decrease in peak inspiratory pressure in 12- and 16-month-old mdx. NMC was lower in mdx mice across the ventilatory and non-ventilatory range, emerging early in dystrophic disease.Reduced NMC of obligatory muscles to inspiratory pressure in mdx mice reveals a greater reliance on accessory muscles to support respiratory performance in dystrophic disease. Remarkably, this burden is adequately accommodated in early disease with a preserved capacity to generate peak inspiratory pressure. We reason that the loss of compensation afforded by accessory muscles to peak inspiratory pressure generation underpins the emergence of respiratory compromise in advanced disease. Our results may have implications for the understanding and treatment of DMD. Funded by Science Foundation Ireland SFI/19/6628 INSPIRE DMD. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tongue exercise-induced functional and structural upper airway plasticity in a rodent model of hypoglossal (XII) motor neuron loss

Motor neuron diseases (MNDs) are a group of neuromuscular disorders in which motor neurons (MNs) required for activities of daily living are lost. Patients with MNDs often develop swallowing and breathing dysfunction, leading to aspiration pneumonia, respiratory failure, and death. Available treatments for patients with MNDs are largely palliative; thus, there is a critical need for therapies targeting the preservation of upper airway function. Upper airway dysfunction in MNDs is largely attributed to degeneration of XII lower MNs (LMNs) which innervate the genioglossus muscle of the tongue and often results in progressive tongue weakness. The extensive tongue muscle weakness in patients with MNDs suggests a potential role for therapeutic exercise, but sufficient evidence is lacking to conclude if tongue exercise is beneficial or harmful. A barrier in studying the impact of XII LMN degeneration and developing therapies to improve swallowing and breathing function/coordination has been the lack of an appropriate model. We developed a novel rodent model using intralingual injections of cholera toxin B conjugated to saporin (CTB-SAP) to induce targeted loss of XII MNs and XII motor output. CTB-SAP rats have degenerative changes in the XII nerve and genioglossus, as well as decreased tongue motility and swallowing deficits that are mitigated by tongue exercise. We hypothesize that upper airway function/coordination can be improved in the face of XII LMN degeneration by tongue exercise-induced XII-tongue axis plasticity. Here, we intralingually injected adult male rats with CTB-SAP or control (CTB unconjugated to SAP), and studied the following parameters +/- tongue exercise: 1) XII motor output in anesthetized and ventilated rats via in vivo neurophysiology; and 2) structural changes via magnetic resonance imaging. Thus far, our data suggests that tongue exercise in CTB-SAP rats results in enhanced XII motor plasticity and mitigates structural airway changes (p<0.05 vs. sham-treated CTB-SAP rats). We have also started collecting pilot data to determine if upper airway (geniohyoid) EMG activity is decreased in unanesthetized and spontaneously breathing CTB-SAP rats, and if tongue exercise mitigates these deficits while diaphragm EMG activity remains unaffected +/- tongue exercise. In conclusion, tongue exercise appears to cause XII-tongue axis plasticity to improve upper airway function and coordination in the face of XII LMN degeneration. Studies are now underway to understand the underlying mechanism of tongue exercise-induced neuroplasticity, which may help guide future translational studies in patients with MNDs. Funded by: R01 HL153612 and Phi Zeta This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Basal and Hypoxic Respiratory Behaviors in Aged 6‐OHDA‐Induced SN Lesion PD Rat Model: Effects of the 5‐HT1A Receptor Agonist Buspirone

Parkinson’s disease (PD) is progressive neurodegenerative disease, with the diagnosis of idiopathic PD occurring in humans at ~60 years of age. Most studies, however, use young adult (≤3 month old) rats to investigate PD‐related abnormalities in various physiological systems, with the most commonly used experimental rat PD model being generated by unilateral injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA) into specific regions of the nigrostriatal pathway. While this model results in the loss of dopaminergic neurons and the subsequent changes in downstream neurotransmitter signaling that are characteristic of PD, it does not consider potential contributions of physical changes to the brain and neurotransmitter signaling that are part of normal aging on the progression of PD. To better understand age‐related influences and potential contributions of PD‐related abnormalities in serotonergic (5‐HT) neurotransmission on respiratory dysfunction (which is a common non‐motor symptom) in PD, we investigated basal and hypoxic (12% O2; 90s) respiratory behaviors before and after acute administration of the frequently prescribed anxiolytic 5‐HT1A receptor agonist buspirone (BUS; 0.5 mg/kg, iv) in an aged (~9 month old) female Sprague‐Dawley PD rat model produced by unilateral 6‐OHDA injection into the substantia nigra (SN). For these experiments, diaphragm EMG activity was recorded and quantified from spontaneously breathing urethane‐anesthetized rats at 2‐weeks after SN 6‐OHDA or vehicle (control) injections. We found that 6‐OHDA‐injected rats have a lower basal breathing frequency (P<0.05) due to shorter expiratory duration (TE; P<0.05) than vehicle‐injected rats, but both 6‐OHDA‐ and vehicle‐injected rats exhibit significant increases in breathing frequency (P≤0.001) and EMG amplitude (P≤0.023) during hypoxia; the magnitude of the peak frequency increase in 6‐OHDA‐injected rats was slightly greater (P=0.083). Both 6‐OHDA‐ and vehicle‐injected rats also showed similar post‐hypoxic frequency decline (P≤0.003), but only vehicle‐injected rats displayed a slight and sustained post‐hypoxic amplitude depression (P<0.1). In vehicle‐injected rats, acute BUS injection produced a progressive decrease in basal EMG amplitude (P=0.066) and a persistent increase in breathing frequency (P=0.064) by decreasing inspiratory duration (TI; P<0.001) while in 6‐OHDA‐injected rats, a transient initial increase in EMG amplitude (P=0.043) and a persistent decrease in TI (P=0.005) and TE (P=0.089) were noted. In both 6‐OHDA‐ and vehicle‐injected rats, BUS injection also slightly exaggerated the initial hypoxic frequency increase, but blunted the amplitude increase with amplitude progressively declining to near pre‐hypoxic levels before the end of the hypoxic exposure. BUS injection also shortened the duration of post‐hypoxic EMG amplitude depression in vehicle‐injected rats. These data demonstrate that some differences in basal and hypoxic respiratory behaviors exist between older 6‐OHDA‐ and vehicle‐injected rats, and that some respiratory behaviors of these rat models are differentially impacted by BUS‐induced effects on respiratory control. We suggest that this older PD rat model be further evaluated.

Distinct modulation of tracheal and laryngopharyngeal cough via superior laryngeal nerve in cat

Unilateral and bilateral cooling and bilateral transsection of the superior laryngeal nerve (SLN) were employed to modulate mechanically induced tracheobronchial (TB) and laryngopharyngeal (LPh) cough in 12 anesthetized cats. There was little effect of SLN block or cut on TB. Bilateral SLN cooling reduced the number of LPh (<50 %, p < 0.05), amplitudes of diaphragm EMG activity (<55 %, p < 0.05), and cough expiratory efforts (<40 %, p < 0.01) during LPh. Effects after unilateral SLN cooling were less pronounced. Temporal analysis of LPh showed only shortening of diaphragm and abdominal muscles burst overlap in the inspiratory–expiratory transition after unilateral SLN cooling. Bilateral cooling reduced both expiratory phase and total cough cycle duration. There was no significant difference in the average effects of cooling left or right SLN on LPh or TB as well as no differences in contralateral and ipsilateral diaphragm and abdominal EMG amplitudes.Our results show that reduced afferent drive in the SLN markedly attenuates LPh with virtually no effect on TB.

Acute intrathecal BDNF enhances functional recovery after cervical spinal cord injury in rats.

Unilateral C2 hemisection (C2SH) disrupts descending inspiratory-related drive to phrenic motor neurons and thus, silences rhythmic diaphragm muscle (DIAm) activity. There is gradual recovery of rhythmic DIAm EMG activity over time post-C2SH, consistent with neuroplasticity, which is enhanced by chronic (2 wk) intrathecal BDNF treatment. In the present study, we hypothesized that acute (30 min) intrathecal BDNF treatment also enhances recovery of DIAm EMG activity after C2SH. Rats were implanted with bilateral DIAm EMG electrodes to verify the absence of ipsilateral eupneic DIAm EMG activity at the time of C2SH and at 3 days post-C2SH. In those animals displaying no recovery of DIAm EMG activity after 28 days (n = 7), BDNF was administered intrathecally (450 mcg) at C4. DIAm EMG activity was measured continuously both before and for 30 min after BDNF treatment, during eupnea, hypoxia-hypercapnia, and spontaneous sighs. Acute BDNF treatment restored eupneic DIAm EMG activity in all treated animals to an amplitude that was 78% ± 9% of pre-C2SH root mean square (RMS) (P < 0.001). In addition, acute BDNF treatment increased DIAm RMS EMG amplitude during hypoxia-hypercapnia (P = 0.023) but had no effect on RMS EMG amplitude during sighs. These results support an acute modulatory role of BDNF signaling on excitatory synaptic transmission at phrenic motor neurons after cervical spinal cord injury.NEW & NOTEWORTHY Brain-derived neurotrophic factor (BDNF) plays an important role in promoting neuroplasticity following unilateral C2 spinal hemisection (C2SH). BDNF was administered intrathecally in rats displaying lack of ipsilateral inspiratory-related diaphragm (DIAm) EMG activity after C2SH. Acute BDNF treatment (30 min) restored eupneic DIAm EMG activity in all treated animals to 78% ± 9% of pre-C2SH level. In addition, acute BDNF treatment increased DIAm EMG amplitude during hypoxia-hypercapnia but had no effect on EMG amplitude during sighs.

Electrical Stimulation of Afferent Lumbar Sympathetic Nerve Impacts Cardio‐Respiratory Functions in Anesthetized Rats

The lumbar sympathetic chains (LSC) contain post-ganglionic vasodilator and vasoconstrictor fibers that control hindlimb vascular tone. The LSC also contain dorsal root ganglion afferent fibers whose roles in cardiorespiratory status remain poorly understood. The objective of the present study was to characterize the cardiorespiratory responses elicited by electrical stimulation (ES) of a LSC in urethane-anaesthetized male Sprague-Dawley rats (n=6) instrumented to measure/calculate mean arterial blood pressure (MAP), femoral artery blood flow (FBF) and femoral vascular resistance (FVR), brachial artery blood flow (BBF), and brachial vascular resistances (BVR), upper airway pressure (UAP), diaphragmatic EMG, respiratory frequency, inspiratory (iTV) and expiratory (eTV) tidal volumes. The right LSC was identified at the level of L4-L6 and a bipolar electrode was placed for ES (5 Hz, 0.5 msec duration, 0.5 mA amplitude for 10 sec). LSC stimulation caused a distinct pattern of cardiorespiratory responses including (a) a 12% decrease in MAP, (b) 15% decrease in ipsilateral FVR, (c) a 10% decrease in BVR (all measured at the peak decrease in MAP), (d) a 15% increase in iVT, (e) a 10% increase in eVT, (f) a15% increase in respiratory frequency, and (g) a 9% increase in diaphragmatic EMG activity. The result of this study demonstrates that ES of afferent fibers within the LSC elicits an array of cardiorespiratory changes in anesthetized rats that would be consistent with preparing the rats for exercise and for flight.

Low Dose Ampakine Stimulates Diaphragm Activity and Increases Tidal Volume following Cervical Spinal Cord Injury in Non‐Anesthetized Freely Behaving Rats

Sabhya Rana1, 2, 3, Michael D. Sunshine1, 2, 3, David D. Fuller1, 2, 3 1Department of Physical Therapy, 2Center for Respiratory Research and Rehabilitation, 3McKnight Brain Institute, University of Florida, Gainesville, FL Ampakines are allosteric modulators of AMPA receptor channel kinetics, and can stimulate breathing during hypoventilation such as following opioid overdose or in neuromuscular disorders. Our laboratory has shown that acute treatment with an ampakine, CX717 can increase inspiratory phrenic motor output in rat models of incomplete cervical spinal cord injury (SCI), when studied under anesthesia. The next step in the translational pathway to is to determine if the drugs are safe and effective in the awake animal. The goal of the current work was to evaluate the effect of two low impact ampakines (CX717 and CX1739) on diaphragm muscle activation and breathing in freely moving non-anesthetized rats following SCI. Ventilation and metabolic rate (VCO2) were measured using whole body plethysmography and diaphragm EMG was assessed using in-dwelling muscle electrodes. At 4 and 14 days following C2 spinal hemisection injury (C2Hx), rats were given a single intravenous bolus of CX717 (5 mg/kg, n=8), CX1739 (5 mg/kg, n=8) or vehicle (2-hydroxypropyl-beta-cyclodextrin; HPCD; n=8). At the 4-day time point, infusion of CX717 or CX1739 increased peak diaphragm EMG (recorded ipsilateral to C2Hx) by 40-60% compared to vehicle. However, only the CX1739 group showed an accompanying increase in tidal volume (20%). During an acute respiratory challenge with hypoxia-hypercapnia (10% O2, 7% CO2), both the CX717 and CX1739 groups showed increased diaphragm EMG compared to vehicle (65-75%); the CX1739 group showed a larger increase in tidal volume. At 14 days post injury, CX717 infusion increased the peak inspiratory ipsilateral diaphragm EMG activity (~30%) and tidal volume (~20%) during baseline breathing. In contrast, CX1739 was less effective at 14 days and produced an increase in ipsilateral diaphragm EMG activity of ~10% with no change in tidal volume. During the hypoxia-hypercapnia challenge, both the CX1739 and CX717 groups showed increased ipsi-lesional diaphragm EMG (25-35%) but no increase in tidal volume as compared to vehicle group. Neither ampakine had an impact on respiratory rate at either time point, suggesting a spinal (vs. medullary) mechanism of action. Additionally, no adverse off-target effects (e.g., changes in heart rate or hypoactive state) were apparent. Improved respiratory muscle activation is often a desirable therapeutic outcome after incomplete cervical SCI. Our data indicate that low dose, low impact ampakine treatment can increase diaphragm muscle output after cervical SCI, and therefore may provide a pharmacologic strategy that could be useful in the context of respiratory rehabilitation. The divergent response to CX717 vs. CX1739 at 4 vs. 14 days post-injury merits further study; one possibility is that ongoing neuroplastic changes in AMPA receptor expression after SCI may differentially influence the efficacy of different ampakines.

Effect of acute intermittent hypoxia on cortico-diaphragmatic conduction in healthy humans

Acute intermittent hypoxia (AIH) is a strategy to improve motor output in humans with neuromotor impairment. A single AIH session increases the amplitude of motor evoked potentials (MEP) in a finger muscle (first dorsal interosseous), demonstrating enhanced corticospinal neurotransmission. Since AIH elicits phrenic/diaphragm long-term facilitation (LTF) in rodent models, we tested the hypothesis that AIH augments diaphragm MEPs in humans. Eleven healthy adults (7 males, age = 29 ± 6 years) were tested. Transcranial and cervical magnetic stimulation were used to induce diaphragm MEPs and compound muscle action potentials (CMAP) recorded by surface EMG, respectively. Stimulus-response curves were generated prior to and 30–60 min after AIH. Diaphragm LTF was assessed by measurement of integrated EMG burst amplitude and frequency during eupnoeic breathing before and after AIH. Following baseline measurements, AIH was delivered from an oxygen generator connected to a facemask under poikilocapnic conditions (15 one minute episodes of 9% inspired oxygen with one minute room air intervals). There were no detectable changes in MEP (−1.5 ± 12.1%, p = 0.96) or CMAP (+0.1 ± 7.8%, p = 0.97) amplitudes across the stimulus-response curve. At stimulation intensities approximating 50% of the difference between minimum and maximum baseline amplitudes, MEP and CMAP amplitudes were also unchanged (p > 0.05). Further, no AIH effect was observed on diaphragm EMG activity during eupnoea post-AIH (p > 0.05). We conclude that unlike hand muscles, poikilocapnic AIH does not enhance diaphragm MEPs or produce diaphragm LTF in healthy humans.

Impact of Ampakine CX‐717 on Diaphragm EMG Activity and Breathing in Non‐anesthetized Freely Behaving Rats

Glutamatergic neurotransmission is partly mediated by ionotropic AMPA receptors, and these receptors play an important role in the neural control of breathing. Ampakines are positive allosteric modulators of AMPA receptors. These drugs can stimulate breathing during hypo‐ventilated states, such as following an opioid overdose. Our laboratory has recently shown that acute exposure to an ampakine, CX717 (15 mg/kg), can acutely stimulate neural drive to the diaphragm in anesthetized but otherwise healthy rats. The goal of the current work is to evaluate the impact of ampakine on diaphragm muscle activation in freely moving non‐anesthetized rats. Ventilation and metabolic rate (VCO2) were measured using whole body plethysmography and diaphragm EMG was assessed using intramuscular electrodes. Spinally intact rats (n=2) were given a single intravenous bolus of CX717 at four months after implantation of diaphragm EMG electrodes. Delivering CX717 at 15 mg/kg had no detectable impact on VCO2, inspiratory tidal volume or diaphragm EMG burst amplitude. In addition, at 30 minutes following IV ampakine, both animals showed a robust increase in diaphragm EMG activity, tidal volume, and the ratio of ventilation VCO2 (VE/VCO2) during an acute hypoxic challenge (5 min, 10.5% inspired O2). Qualitatively, we observed a possible sedative effect as shown of lack of movement and exploratory behavior following ampakine exposure. Our preliminary conclusion is that ampakine CX717 at a dose of 15 mg/kg has little impact on breathing in the otherwise healthy rat. Ongoing studies are evaluating impact of the vehicle (2‐hydroxypropyl‐beta‐cyclodextrin) versus ampakine using a cross‐over study design in spinally intact rats and in rats with spinal cord injury.Support or Funding Information5 R01 HL139708 02 (DDF)F31 HL145831‐01 (MDS)

Relationship (?) Between Histological and Functional Measures in Moderate Contusion SCI in Adult Rat

To create a spinal cord injury (SCI), most laboratories implement a standardized method; however, considerable variability in injury size and associated functional deficits can occur across subjects. The present study was initiated to explore relationships between the histological features of spinal cord (SC) lesion and the associated functional deficits in locomotor, respiratory, and lower urinary tract (LUT) function in a widely used rat contusion SCI model that is generated using an Infinite Horizon impactor. To this end, adult female rats (n=8) received a moderate mid‐thoracic contusion SCI (200 kdyn, T8), and the impact displacement and force were recorded. Indices of functional recovery included: (1) BBB open field locomotor scoring at 1‐week post SCI, (2) total volume of expressed urine during the first 2‐weeks post SCI, and (3) diaphragm EMG activity and reflex micturition behavior and residual urine volume recorded during an acute cystometry experiment at 4‐weeks post SCI, after which the bladder was removed and weighed. Following fixation (4% PFA) and cryoprotection of the SC, 20 μm thick horizonal sections were obtained and counterstained with cresyl violet and Cyanine R / FeCl3 to visualize cell bodies and myelin, respectively, and the section at the level of the central canal was used as the site for preliminary analysis of the extent of lesion damage and degree of white matter sparing. The lesion area ranged from 27–49% and was measured by outlining the perimeter of the lesion site using a threshold tool and then normalized (%) to the area of a standardized 7 mm rostral‐caudal length region of SC that included the injury site. Myelin thickness was measured at 3 sites at the level of the injury and at the rostral and caudal ends of the tissue (uninjured) on each side of the SC, and the injury site measurements were normalized to the average myelin thickness of the corresponding uninjured side. A spared myelin index (%) using the larger minimum myelin thickness from the right or left side of the SC was then determined and ranged from 19–91%. For generation of the SCI, impactor displacement varied from 1287 to 1728 μm, with larger displacements being associated with larger injury sites and less myelin sparing. The lesion area, however, was not a strong predictor of myelin sparing, particularly in cases with asymmetrical lesions (n=2). Both lesion area and spared myelin index were reasonable predictors of post SCI recovery of locomotor (BBB scores) and LUT (expressed urine volume) function but not in predicting the degree of dysfunction in reflex micturition (increased residual volume) and bladder hypertrophy or inspiratory burst measures and their variability. These preliminary results suggest that factors in addition to the histological characteristics of the lesion site contribute to the degree of functional deficits/recovery following SCI.Support or Funding InformationDOD CDMRP W81XWH‐17‐1‐0260; NIH NS096514; NYS DOH SCIRB C32088GG

Effects of Fluoxetine on Inspiratory Motor Activity and Chemical Control of Breathing in Spontaneously Breathing Urethane‐Anesthetized 6‐OHDA MFB‐lesioned Parkinson’s Disease Rat Model

Breathing abnormalities are a common non‐motor symptom (NMS) of Parkinson’s Disease (PD), and multiple causes have been proposed, including impaired central respiratory control. While the loss of dopaminergic neurons is the primary pathology of PD, it is becoming better appreciated that serotonergic (5‐HT) dysfunction also plays a role in the development of motor and NMS and complications in PD. To this end, recent studies addressing the role of 5‐HT in PD symptoms have revealed that activation or antagonism of specific 5‐HT receptor subtypes may have therapeutic benefit. 5‐HT neurotransmission is also known to play a critical role in control of breathing; thus, respiratory abnormalities in PD may be further complicated by the progressive 5‐HT dysfunction in PD. Ongoing work in our laboratory has been focused on characterizing the respiratory phenotype of PD using 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced preclinical rat models of PD. Here, we evaluate potential contributions of 5‐HT dysfunction by assessing the effects of acute administration of the selective 5‐HT reuptake inhibitor fluoxetine hydrochloride (FLX) on basal inspiratory motor (diaphragm EMG) activity and hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses in spontaneously breathing urethane‐anesthetized adult female rats 2‐weeks after unilateral medial forebrain bundle (MFB) 6‐OHDA or vehicle/control injection. Basal (BL) diaphragm EMG activity was recorded for ≥30 min, the HVR (12% O2, 90s) and HCVR (7% CO2, 5 min) were assessed, acute FLX (10 mg/kg, iv) was injected and allowed 30 min to exert steady‐state effects, and then the HVR and HCVR were re‐assessed. We found that in both vehicle‐ and 6‐OHDA‐injected rats, administration of FLX produced an initial increase in burst frequency (by ~30%) and amplitude (by ~15%) above BL levels within ~60 s, after which burst frequency attenuated to a new steady‐state level of ~10% above BL levels while burst amplitude returned to slightly below BL levels in vehicle‐injected rats, but attained a new steady‐state level of ~10% above BL levels in 6‐OHDA‐injected rats. The effects of FLX on HVR and HCVR frequency behaviors were negligible in both control‐ and 6‐OHDA‐injected rats although a slight attenuation of the HVR frequency increase (by ~5%) was seen. The effects of FLX on HVR and HCVR amplitude behaviors, while underpowered, showed a trend of slightly enhanced of HVR amplitude (by ~5%) and HCVR amplitude (by ~9%) in 6‐OHDA‐injected rats; however, additional experiments are needed to determine the significance of these observations. While these preliminary observations suggest that (compared to control rats) MFB‐lesioned rats exhibit a difference in FLX‐induced effects on basal inspiratory burst amplitude alterations, acute FLX administration may not be able to fully correct chemical control of breathing deficits. We suggest that additional experiments are needed to better identify specific 5‐HT mechanisms that may contribute to chemical control of breathing deficits in this PD rat model.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

Acute Fluoxetine Administration Improves Intermittent Hypoxia‐induced Inspiratory Burst Amplitude Deficits During Peak LPS‐induced Neuroinflammatory Phase in Anesthetized Spontaneously Breathing Adult Male Rats

Studies examining the impact of neuroinflammation on various aspects of neural control of breathing typically use systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) and evaluate alterations in ventilatory control during either the induction (~2–4 hr) or resolution (~24 hr) phases of the acute neuroinflammatory response. Changes in LPS‐regulated pro‐inflammatory gene/protein expression, however, have been shown to peak at ~4–6 hrs following LPS exposure, but little is known about the impact of LPS‐induced neuroinflammation on ventilatory control at this time point. Systemic LPS administration has also been shown to increase serotonin (5‐HT) transporter activity and reduce 5‐HT levels. Since numerous inspiratory motor behaviors are 5‐HT‐dependent, including some hypoxic ventilatory behaviors, LPS‐induced changes in 5‐HT could contribute to ventilatory deficits noted in LPS‐induced neuroinflammation studies. To begin to address these issues, we examined the impact of peak phase LPS‐induced neuroinflammation on inspiratory motor behaviors elicited by intermittent hypoxia (IH) in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats in both the absence and presence of acute administration of the SSRI fluoxetine hydrochloride (FLX). Beginning at ~4 hr after systemic LPS (3 mg/kg, ip) or saline (CON) treatment, basal (BL) diaphragm EMG activity was recorded for ~30 min (40% O2), FLX (10 mg/kg) or vehicle (VEH, 0.9% saline) was injected (iv) and allowed 30 min to exert steady‐state effects, and then IH exposure composed of 5 alternating 3‐min episodes of hypoxia (11% O2; 2%CO2) and hyperoxia (40% O2) was delivered, after which the rat was allowed to recover. We found that during the first 30–60s following FLX injection in both CON‐ and LPS‐treated rats, burst amplitude (by ~12%) and frequency (by ~40%) were increased, after which amplitude returned to BL levels and frequency attained a new steady‐state ~10% above BL levels; VEH injection was ineffective in altering either burst amplitude or frequency. As expected during IH exposure in CON‐treated VEH‐injected rats, a robust increase in burst frequency and amplitude were observed with the subsequent hypoxic exposures eliciting progressive augmentation of burst amplitude (PA; IH1, ~45%; IH5, ~65%). In contrast in CON‐treated FLX‐injected rats, IH‐induced amplitude effects were markedly attenuated (IH1‐IH5, ~11–21%), and similar to those seen during IH exposure in LPS‐treated VEH‐injected rats (~7–11%). In LPS‐treated FLX‐injected rats, however, an increase in both burst amplitude and PA (IH1, ~19%; IH5, ~35%) were noted. Similar IH‐induced increases in burst frequency and post hypoxic frequency decline were also observed in both LPS‐ and CON‐treated FLX‐ and VEH‐injected rats. These preliminary observations suggest that acute FLX administration may partially correct the blunted increase in burst amplitude and induce PA in response to IH exposure during peak phase in LPS‐treated rats albeit similar FLX treatment appears to impair IH‐induced amplitude effects in CON‐treated rats.Support or Funding InformationNIH NS101737; SBU Thomas Hartman Center for Parkinson’s Disease Research

Neural control of respiratory musculature in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited fatal neuromuscular disease. Despite deficits in respiratory muscle performance being well recognised, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to examine neural control of the diaphragm and upper airway musculature in dystrophin deficient mdx mice.Wild‐type (WT) and mdx mice were studied to examine diaphragm and genioglossus (GG) EMG activity using fine concentric needle electrodes in urethane (1.7mg/kg) anaesthetized spontaneously breathing mice. Basal EMG and responsiveness to chemostimulation were determined. In separate studies, phrenic and hypoglossal (XII) motor output were assessed in anaesthetized, vagotomized, paralyzed and mechanically ventilated WT and mdx mice.Diaphragm and upper airway (sternohyoid) muscle form and function were assessed. Monoamine concentrations, density of glial cells, and pro‐inflammatory gene expression were determined in the spinal cord (C3–C5) and brainstem. Diaphragm and GG EMG activities were depressed in mdx compared to WT during baseline and hypoxic‐hypercapnic challenge. Phrenic and XII nerve recordings revealed enhanced neural drive to diaphragm and GG in mdx mice during hypoxic‐hypercapnic challenge. Excitatory monoamine concentrations were elevated in mdx spinal cord (noradrenaline and serotonin) and brainstem (serotonin). Putative phrenic and hypoglossal motor nuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT. NFkB and GFAP mRNA expression were increased in mdx brainstem compared to WT, while GFAP mRNA was reduced in mdx spinal cord.Our study revealed a potentiated neural drive in phrenic and XII motor pathways in mdx mice during chemoactivation, suggesting plasticity in motor pathways facilitating breathing. Depressed EMG activity despite potentiated neural outputs, suggests compromised neuromuscular transmission, which we have also observed in respiratory EMG recordings during protracted airway obstruction (Burns et al., 2019, J Physiol). Studies of the fundamental control of breathing in models of DMD should provide a comprehensive vista of the disease, with the potential to identify novel therapeutic targets.

Inspiratory motor activity and burst‐to‐burst variability during single and repeated bout hypoxic exposures in spontaneously breathing urethane‐anesthetized adult female rats with mid‐thoracic moderate contusion SCI

Spinal cord injury (SCI) at either the cervical or thoracic level can lead to inefficient ventilation by disrupting descending inputs to respiratory motoneurons resulting in altered mechanics of breathing and a reduced ability to adequately and appropriately respond to ventilatory complications. Under these conditions, individuals with SCI may experience individual or repeated bouts of acute hypoxia that vary in severity and/or duration. Recently, the use of acute intermittent hypoxia (AIH) has been proposed as a potential therapeutic intervention to induce spinal motor plasticity and improve respiratory motor function following incomplete cervical (C2) SCI in both rodents and humans; however, much less is known about the effects of either single or repeated hypoxic exposures on respiratory motor output following mid‐thoracic SCI, which spares phrenic motoneurons. To begin to address this issue, we examined diaphragm EMG activity in response to 15‐min of hypoxia (12% O2) delivered as either a single 15‐min bout (protocol 1) or as three 5‐min bouts separated by 5‐min normoxic intervals (protocol 2) in spontaneously breathing urethane‐anesthetized naïve (control; n=6 protocol 1; n=8 protocol 2) or mid‐thoracic SCI (n=6 protocol 1; n=7 protocol 2) rats. Diaphragm EMG burst frequency and amplitude were measured and quantified at 15‐min intervals under baseline (BL) normoxic conditions and at 5‐min intervals during and following the hypoxic exposures. In addition, burst‐to‐burst frequency and amplitude variability were determined using coefficient of variation (CV) and Poincaré plot analyses from 300 consecutive EMG bursts. We found that in response to hypoxia, both SCI and control rats exhibited (1) increases in burst frequency and small increases or decreases in burst amplitude for both protocols, (2) increases in frequency CV values with the largest effect being noted in protocol 2 control rats during the first hypoxic exposure; (3) sustained increases in amplitude CV values for both protocols albeit a lower magnitude effect in SCI rats; (4) increases in short‐term (SD1) and long‐term (SD2) burst‐to‐burst frequency and amplitude that were attenuated in SCI rats compared to control rats, especially in protocol 1; and (5) post hypoxic frequency and amplitude decreases that were similar between the protocols. Both the ventilatory behaviors and burst‐to‐burst variability patterns observed in response to the repeated hypoxic exposures in protocol 2 appeared to be reproducible albeit in the first hypoxic exposure, SD1 and SD2 values for burst frequency tended to be higher and exhibit greater spread across control rats. These data demonstrate that while rats with mid‐thoracic contusion SCI exhibit similar single and repeated hypoxic exposure ventilatory behaviors, the respiratory dynamics of these behaviors exhibit reduced short‐term and long‐term burst‐to‐burst variability that are minimally affected by the pattern of the 15‐min duration of acute hypoxia.Support or Funding InformationDOD CDMRP W81XWH‐17‐1‐0260; NIH NS096514

Adenosine 2A receptor inhibition protects phrenic motor neurons from cell death induced by protein synthesis inhibition

Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.

Is non-normalized chest wall electromyogram activity a reliable index of respiratory neural drive? On the surface - yes!

Is non-normalized chest wall electromyogram activity a reliable index of respiratory neural drive? On the surface - yes!

Acute Fluoxetine (FLX) Administration Improves Inspiratory Motor Activity Response to Acute Single Bout Combined Hypoxic‐Hypercapnic Exposure in Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat at 24‐hr Post‐Lipopolysaccharide (LPS) Injection

LPS‐induced neuroinflammation has been suggested to alter serotonergic (5‐HT) neurotransmission by acting on the serotonin transporter to alter serotonin levels. In the respiratory neural control system, LPS‐induced neuroinflammation has been shown to affect multiple aspects of central ventilatory control, including 5‐HT‐dependent inspiratory motor behaviors. Ongoing work in our laboratory has been examining the impact of LPS‐induced neuroinflammation on neural control of breathing, including 5‐HT‐mediated inspiratory motor behaviors. Here, we examine the effects of acute systemic administration of the selective serotonin reuptake inhibitor (SSRI) fluoxetine hydrochloride (FLX) on basal inspiratory (diaphragm) EMG activity and the maximal chemoreceptor stimulation ventilatory response (MCVR) elicited by an acute single bout of combined hypoxic‐hypercapnic exposure in spontaneously breathing urethane‐anesthetized adult male Sprague‐Dawley rats 24‐hr after systemic LPS (3 mg/kg, ip) or vehicle (0.9% saline, ip) administration. For these experiments, basal diaphragm EMG activity was recorded for 30 min under baseline (BL) conditions (40% O2), FLX (10 mg/kg, iv) was then administered and allowed to exert its effects for 30 min, after which a 3 min MCVR (12% O2, 7%CO2) challenge was performed, followed by 30 min recovery; control experiments using vehicle (VEH; 0.9% saline) instead of FLX were also performed. We found that FLX and VEH administration were ineffective in producing significant changes in inspiratory burst amplitude or frequency in either 24‐hr post‐LPS‐ or saline‐treated rats although during the first 30–60s following FLX injection, a small transient increase (~10–15%) in burst frequency was noted for both groups. In 24‐hr post‐LPS‐ and saline‐treated rats, for the MCVR following VEH injection, most rats exhibited a sustained increase in burst amplitude (≥30% above BL) and a robust increase in burst frequency (~15–30%) that gradually returned to BL levels before the end of the challenge. In 24‐hr post‐LPS and saline‐treated rats, for the MCVR following FLX injection, a robust and maintained increase in burst amplitude (40–50% above FLX level) was also observed; however, marked differences were noted for the MCVR frequency behavior. In this case, in 24‐hr post‐saline‐treated rats, the increase in burst frequency degraded as the challenge progressed (40% above FLX level during min 1 but 97% of FLX level during min 3) while in 24‐hr post‐LPS‐treated rats, the increase in burst frequency remained relatively stable (~25% above FLX level during min 1 and ~20% above FLX level during min 3). These preliminary observations suggest that (compared to control rats), acute FLX administration following systemic LPS‐induced neuroinflammation may be capable of improving inspiratory motor deficits in hypoxic‐hypercapnic chemical control of breathing.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N‐Acetyl cysteine improves dystrophic (mdx) mouse diaphragm muscle quality and strength

Respiratory muscle weakness occurs as a consequence of dystrophin deficiency in Duchenne muscular dystrophy (DMD), with implications for breathing and airway protective behaviours. The mdx mouse model of DMD shows evidence of impaired respiratory muscle performance with attendant inflammation and oxidative stress. N‐Acetyl cysteine (NAC) is a dietary antioxidant commonly used in the treatment of respiratory disease. We sought to examine the effects of NAC intervention on respiratory system performance in the mdx mouse.Young adult male wild‐type and mdx mice were studied; a subset of mdx received 1% NAC in the drinking water for 14 days. Ventilation was assessed in conscious mice using whole‐body plethysmography. Inspiratory pressure and respiratory muscle (diaphragm and external intercostal) EMG activity were assessed in anaesthetised mice during ventilatory and non‐ventilatory behaviours. Diaphragm ex vivo force‐generating capacity, muscle structure, cytokine concentrations, glutathione status and mRNA expression were determined. Data were statistically compared by unpaired Student's t test.Diaphragm function was severely impaired in mdx, evidenced by reductions in force‐generating capacity (40–160Hz range), total muscle shortening and maximal shortening velocity compared to wild‐type. Extensive diaphragm myofibre remodelling and damage was observed for mdx, characterised by increased muscle fibrosis, immune cell filtration, muscle fibre central nucleation and embryonic myosin expression. The pro‐inflammatory cytokines IL‐1β and IL‐6 were elevated in mdx diaphragm and plasma samples. mRNA studies indicate an increase in gene expression related to muscle atrophy, regeneration and inflammation in mdx diaphragm, and a reduction in anti‐oxidant related gene expression. NAC supplementation increased mdx diaphragm force (60–160Hz), maximal shortening and shortening velocity. Collagen deposition and the infiltration of putative inflammatory cells in mdx diaphragm was decreased following NAC. Mdx diaphragm and plasma IL‐1β and IL‐6 concentrations were decreased following NAC. Glutathione reductase and peroxidase activities were increased in mdx diaphragm; both were unaffected by NAC. mRNA expression of Nrf2 was increased in mdx diaphragm and increased further with NAC supplementation. No adverse effects of NAC intervention were observed on ventilation, inspiratory pressure and EMG, and growth measures.We reveal that NAC treatment improved mdx diaphragm force‐generating capacity by way of beneficial anti‐inflammatory and anti‐fibrotic effects. These data support the potential use of NAC as an adjunctive therapy in the dystrophinopathies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.