Fogarty, Matthew J.

Mid‐cervical spinal cord contusion injury results in tissue damage, disruption of spinal pathways, and motoneuron loss. Phrenic motoneurons located in C3–5 segments of the cervical spinal cord innervate the diaphragm muscle (DIAm), and unilateral C4 contusion results in loss of 40–50% of motoneurons ipsilateral to the injury (~25% of the total motoneuron pool). Over time after unilateral C4 spinal cord contusion injury, DIAm electromyography (EMG) increases both contralateral and ipsilateral to the side of injury, suggesting compensation due to an increased activation of the surviving motoneurons. However, whether C4 contusion impairs the ability of the DIAm to accomplish higher force motor behaviors is unknown. Transdiaphragmatic pressure (Pdi) was measured across motor behaviors over time after unilateral C4 contusion injury. Maximum Pdi (Pdi max ) was elicited by bilateral phrenic nerve stimulation at 7 days post‐injury. We hypothesized that Pdi max is reduced following C4 mid‐cervical contusion injury, which will constrain the Pdi generated during different motor behaviors. Since ventilatory behaviors of the DIAm require <50% Pdi max , we further hypothesized that Pdi generated during ventilatory behaviors of the DIAm is not impaired after unilateral C4 mid‐cervical spinal cord contusion injury. In support of our hypothesis, we observed that Pdi max was reduced by ~25% after C4 mid‐cervical spinal cord contusion injury compared to a laminectomy control group. This decrease in Pdi max is consistent with the extent of phrenic motoneuron loss following contusion injury. We also found that during both eupnea (quiet breathing) and breathing stimulated by 10% O 2 (hypoxia) and 5% CO 2 (hypercapnia), Pdi generation was unimpaired by C4 mid‐cervical spinal cord contusion injury, again consistent with the lower force requirement of these ventilatory motor behaviors. Prior to injury, Pdi generated during airway occlusion was ~40% of Pdi max . One day following contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 cm H 2 O to ~20 cm, but this reduction was completely reversed by 7 days post‐injury. The reduction in Pdi amplitude at one day post‐injury cannot be attributed to the ~25% loss of phrenic motoneurons, and thus, may reflect a disruption of input to phrenic motoneurons or acute inflammation of their surrounding milieu. Over time after injury, changes in the balance between inhibition and excitation may result in recovery of higher‐force behaviors. Support or Funding Information Funded by NIH R01‐HL096750 and T32‐HL105355 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Glycine (Gly) is a key inhibitory neurotransmitter at phrenic motor neurons (PhMN). In development, Gly is excitatory, transitioning to inhibitory action in the postnatal period. In adult conditions such as amyotrophic lateral sclerosis (ALS), excessive excitatory signaling onto motor neurons results in excitotoxic motor neuron death. However, it is not known how an imbalance of neurotransmission early in development impacts PhMNs at maturity. This is a knowledge gap, as some congenital hypertonia conditions exhibit an imbalance in neurotransmitter signaling, with these individuals suffering significant morbidity and mortality from respiratory disorders. Using an animal model of congenital hypertonia ( spa mouse [B6.Cg‐ Glrb spa /J] with a Gly receptor mutation), we evaluated PhMN and motor unit physiology. Similar to previous work in other Gly mutants, we hypothesized that spa mice will have excessive developmental PhMN loss with preserved PhMNs having a smaller somal size compared to wildtype (WT) mice. We also hypothesized that fewer PhMNs in spa mice will result in an increased diaphragm (DIAm) innervation ratio (number of DIAm fibers innervated by a PhMN) and increased DIAm neuromuscular transmission failure (NMTF). S pa and WT mice from our colony, ages 6 to 16 weeks, underwent unilateral retrograde labeling of PhMNs via rhodamine phrenic nerve dip. After three days, mice were euthanized, perfused with 4% paraformaldehyde, cervical spinal cord excised and processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging. Absolute PhMN counts were obtained using mosaic images of adjacent serial sections. Somal size measurements (i.e. surface area) were obtained using ImageJ. DIAm force was determined from DIAm strip preparations stretched to optimal length, and force was evoked by phrenic nerve stimulation at 40 Hz in 330 ms duration trains repeated each s (33% duty cycle) for 120 s. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in H&E stained cross‐sections. S pa mice had 30% fewer PhMNs compared to WT ( P =0.005) with somal size being 22% smaller in spa mice ( P =0.019). Spa mice had 20% NMTF during the initial stimulus train compared to 8% in WT mice with spa mice having 82% NMTF and WT having 40% NMTF at end of stimulation. Spa mice had a 39% greater innervation ratio of DIAm motor units compared to WT. Developmental PhMN loss and somal size is significantly disrupted in congenital hypertonia due to impaired Gly signaling and may impact performance of high‐force expulsive DIAm activities. In addition, the increased innervation ratio of DIAm motor units in spa mice, likely contributes to the NMTF due to branch point failure. Overall, this results in less effective airway defense and straining behaviors in congenital hypertonia. Our next step is to identify crucial developmental milestones when excessive PhMN loss occurs and the impact on large versus small PhMNs.

Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

There are notable respiratory deficits related to congenital diaphragmatic hernia (CDH), yet the contribution, if any, of frank diaphragm muscle weakness to CDH is unexplored. Here, we use the well-established Nitrofen teratogen model to induce CDH in rat pups, followed by diaphragm muscle contractility and morphological assessments. Our results show diaphragm muscle weakness in conjunction with reduced muscle fiber density and size, contributing to CDH morbidity.

Individuals with motor control deficits, including cerebral palsy (CP) often have respiratory impairments. Glycine-receptor mutant spa mice have early-onset hypertonia, and limb motor impairments, similar to individuals with CP. We hypothesized that in the diaphragm of spa mice, disruption of glycinergic inputs to MNs would result in increased phrenic–DIAm neuromuscular transmission failure. Pathophysiologic abnormalities in neuromuscular transmission may contribute to respiratory dysfunction in conditions where early developmental MN loss or motor control deficits are apparent.

Respiratory dysfunction is a leading cause of morbidity and mortality in individuals with cerebral palsy (CP). In children and adults with CP, movement and physical function is always affected. Yet, many clinicians overlook potential for impaired movement and function of the diaphragm muscle (DIAm) in individuals with CP. Since individuals with pre-existing respiratory disorders are at greater risk for respiratory complications if they contract COVID-19, understanding potential risks to individuals with CP is important. In this review we present research on respiratory function and DIAm force generation in children with CP. We compare this clinical work to basic science research investigating phrenic motor neuron and DIAm motor unit dysfunction in an animal model with CP symptoms, the spa mouse. Finally, we integrate the clinical and basic science work in respiratory function in CP, discussing potential for individuals with CP to have severe respiratory symptoms from COVID-19.

Abstract The total motor neuron (MN) somato‐dendritic surface area is correlated with motor unit type. MNs with smaller surface areas innervate slow (S) and fast fatigue‐resistant (FR) motor units, while MNs with larger surface areas innervate fast fatigue‐intermediate (FInt) and fast fatigable (FF) motor units. Differences in MN surface area (equivalent to membrane capacitance) underpin the intrinsic excitability of MNs and are consistent with the orderly recruitment of motor units (S > FR > FInt > FF) via the Size Principle. In amyotrophic lateral sclerosis (ALS), large MNs controlling FInt and FF motor units exhibit earlier denervation and death, compared to smaller and more resilient MNs of type S and FR motor units that are spared until late in ALS. Abnormal dendritic morphologies in MNs precede neuronal death in human ALS and in rodent models. We employed Golgi‐Cox methods to investigate somal size‐dependent changes in the dendritic morphology of hypoglossal MNs in wildtype and SOD1 G93A mice (a model of ALS), at postnatal (P) day ~30 (pre‐symptomatic), ~P60 (onset), and ~P120 (mid‐disease) stages. In wildtype hypoglossal MNs, increased MN somal size correlated with increased dendritic length and spines in a linear fashion. By contrast, in SOD1 G93A mice, significant deviations from this linear correlation were restricted to the larger vulnerable MNs at pre‐symptomatic (maladaptive) and mid‐disease (degenerative) stages. These findings are consistent with excitability changes observed in ALS patients and in rodent models. Our results suggest that intrinsic or synaptic increases in MN excitability are likely to contribute to ALS pathogenesis, not compensate for it.

AbstractCharacterization of growth and survival of mice displaying early onset hypertonic symptoms is critical as these animals are important for research investigating mechanisms and treatments of pediatric conditions associated with hypertonia, such as cerebral palsy. Currently, most animal models of cerebral palsy reproduce risk factors for developing this condition, with most failing to develop the physical symptoms or failing to survive in the postnatal period. The B6.Cg‐Glrbspa/J (Gly receptor mutation) transgenic mouse (spa mouse), displays symptoms of early onset hypertonia, though little has been reported on growth and survival, with no reports of growth and survival since genotyping became available. We found that the majority of spa mice display symptoms by P14‐P16. Of mice surviving to weaning, only ~9% were spa mice. By weaning age, spa mice had significantly lower weights than their heterozygote and wild‐type littermates. Of mice that died after weaning and prior to use in experiments or being culled, 48% were spa mice. The poor growth and decreased survival of spa mice across multiple developmental and adult ages resembled the varied survival rates observed in humans with mild or severe cerebral palsy. The understanding of the expected survival of these mice is helpful for planning breeding and animal numbers for experiments. Due to the symptoms and timing of symptom onset, spa mice will be valuable in uncovering mechanisms and long‐term effects of early onset hypertonia in order to move toward interventions for these conditions.

Neuromotor control of diaphragm muscle is dependent on the orderly recruitment of phrenic motor neurons (PhMNs) in a size‐dependent manner. Type S and FR motor units comprise smaller, more easily recruited PhMNs that innervate fatigue resistant, lower force‐producing type I and type IIa muscle fibers. Type FInt and FF motor units, comprise larger, rarely recruited PhMNs innervating the more fatigable, higher force‐producing type IIx and/or IIb muscle fibers. The activity and energy demand upon PhMNs of type S and FR motor units is greater than that of PhMNs from type FInt and FF units, due to the high duty cycle (~35%) of ventilation. Accordingly, activity of type FInt and FF motor units is required for higher force behaviors that are performed intermittently over much shorter durations. These include the expulsive/straining maneuvers during coughing, sneezing, defecation and parturition. The energy demand in both PhMNs and diaphragm muscle fibers is met by mitochondria. To preserve homeostasis, mitochondrial functions are regulated by dynamic, continuous cycles of fusion and fragmentation. We hypothesized that mitochondrial density and mitochondrial fusion will be greater in smaller PhMNs, due to their more frequent activation during ventilation. In six adult (6‐month old) Fischer 344 rats, PhMNs were retrogradely labeled by intrapleural injection of Alexa488‐conjugated CTB. Three days later, cervical transdural infusion of MitoTracker Red was performed every 10 min for 1.5 h under anesthesia. Spinal cord tissue was collected following transcardial perfusion with 4% paraformaldehyde, sectioned longitudinally at 70 μm, and mounted onto slides. Two‐channel sequential confocal z‐stack (0.50 μm step size) mosaics of PhMNs and mitochondria were acquired using a 60x oil immersion objective and analyzed using Elements software (Nikon). The surface areas of PhMNs were measured and divided into tertiles. The lower tertile of PhMNs (comprising type S and FR motor units) had greater mitochondrial volume densities, greater mitochondrial aspect ratios and form factors and greater mean individual mitochondrial volumes than upper tertile PhMNs (comprising type FInt and FF motor units). We further characterized the differences in mRNA expression of mitochondrial dynamics associated proteins (Mfn2 and Drp1) in large vs. small PhMNs using fluorescent in‐situ hybridization techniques. Our results suggest that PhMNs of type S and FR motor units have increased mitochondrial density, supporting their high energy demand to sustain ventilation. Their increased mitochondrial fusion may underlie their resilience to PhMN loss in aging and disease. Support or Funding Information Supported by a National Institutes of Health grants R01‐AG044615

Congenital diaphragmatic hernia (CDH) occurs in approximately 1 in 2,500 live births; it is associated with high rates of neonatal mortality, especially in cases with severe pulmonary hypoplasia and pulmonary arterial hypertension. Treatments have focused on hernia repair and ameliorating lung hypoplasia, with the precise pathophysiological mechanisms of CDH remain unknown. We investigated whether or not diaphragm muscle (DIAm) weakness is apparent in CDH, evaluating DIAm force generation and ventilatory function in a Nitrofen rat model. 7 pregnant rats received 100 mg of Nitrofen in 2 mL of olive oil at 9 days gestational age and rat pups were collected at birth. Ventilatory function was assessed in isolated plethysmography chambers (Buxco). Following these functional assessments, neonatal pups were euthanized, and the DIAm with its rib attachments excised. The presence or absence of a CDH was confirmed and a DIAm strip was obtained either from control pups, or from the unaffected side in CDH pups. The isometric force generation of these DIAm strips and their fatigueability was assessed using field stimulation of muscle at various frequencies (5–100 Hz) and normalized to DIAm cross‐sectional area. Statistical significance was determined using a Student’s t‐test or Mann‐Whitney u test at the P <0.05 level. A marked reduction in both neonatal respiratory rate (−42%) and respiratory minute ventilation (−43%) was observed in CDH pups compared to controls ( P <0.01). We observed reduced DIAm twitch (−40%), maximum tetanic (−72%) and residual force following fatigue (−69%) in CDH pups compared to controls ( P <0.05). CDH neonatal rat pups exhibit DIAm weakness and ventilatory disturbances. Based on previous modelling of ventilatory function, it is possible that DIAm weakness contributes to the poor ventilation, as CDH DIAm forces following fatigue were less than control groups. This work has important considerations when assessing novel treatment outcomes in clinical CDH.