Mantilla, Carlos B.

Motor units of the diaphragm muscle (DIAm) are recruited in an orderly fashion across ventilatory and non‐ventilatory tasks. Spinal hemisection at C2 (C2HS) blocks descending ipsilateral input to phrenic motoneurons and causes DIAm paralysis. With time after C2HS, there is spontaneous recovery of phrenic nerve activity ipsilateral to the injury. We hypothesized that recruitment order of DIAm motor units is maintained following recovery of DIAm electromyographic (EMG) activity post‐C2HS. DIAm EMG activity in adult male rats was recorded chronically using a pair of electrode wires implanted into the midcostal region bilaterally. DIAm EMG activity was strongly correlated to transdiaphragmatic pressure (Pdi) measured using esophageal and gastric balloons across different motor behaviors (Eupnea, hypoxia‐hypercapnia (10%O 2 and 5%CO 2 ), tracheal occlusion, fictive sneezing and bilateral supramaximal phrenic nerve stimulation). Fictive sneezing produced forces comparable to those elicited by phrenic nerve stimulation, and thus DIAm EMG activity during fictive sneezing was considered maximal. Absence of DIAm EMG activity was verified at the time of C2HS and 3 days (3D) post‐C2HS. Over time, a subset of animals displayed functional recovery of rhythmic DIAm EMG activity ipsilateral to C2HS, although DIAm EMG activity was reduced when compared to pre‐injury levels. EMG activity increased across ventilatory and non‐ventilatory tasks associated with increasing drive in C2HS animals displaying functional recovery, supporting a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury.

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS. Supported by NIH grant AR51173.

Myasthenia gravis is a disease of the neuromuscular junction (NMJ) associated with autoimmune destruction of the motor end‐plate. Inhibitors of acetylcholine (ACh) esterase are commonly used to mitigate symptoms by increasing ACh availability at the NMJ. Brain‐derived neurotrophic factor (BDNF) improves neuromuscular transmission in the rat diaphragm muscle (DIAm) by maintaining ACh release with repeated activation. We hypothesized that BDNF will mitigate the deficit of neuromuscular transmission in a passive transfer model of myasthenia gravis. Female Lewis rats were injected with vehicle, McAb1 (that does not induce myasthenia), and a myasthenogenic McAb3 antibody. Neuromuscular transmission failure (NMTF) was determined in DIAm‐phrenic nerve preparations by comparing the isometric force evoked by phrenic nerve stimulation and that evoked by superimposed direct muscle stimulation. After 2 min of repetitive stimulation, neuromuscular transmission was significantly impaired in myasthenic animals (NMTF: 80%) compared to vehicle and McAb1 animals (NMTF: ~60%). Exogenous BDNF treatment significantly improved neuromuscular transmission in all groups and NMTF in McAb3‐treated rats was no longer different from control or McAb1‐treated animals. We conclude that BDNF improves neuromuscular transmission in adult myasthenic rats. Supported by a Career Development Award from Mayo Clinic.

Neuregulin (NRG) is important for expression of acetylcholine (ACh) receptors during development of the neuromuscular junction (NMJ). NRG treatment significantly increases ACh‐induced currents in hippocampal neurons, but it is not known if NRG exerts a similar effect at NMJs. Diaphragm muscle (DIAm)‐phrenic nerve preparations from 7‐ and 14‐day old rats were used to determine the effects of NRG (heregulin β177–244) on neuromuscular transmission and DIAm contractile properties. Neuromuscular transmission failure (NMTF) was assessed by the difference in force elicited by repetitive phrenic nerve stimulation (0.2 ms pulses in 40 Hz, 330 ms trains) and superimposed, intermittent direct DIAm stimulation (1 ms pulses in 40 Hz, 330 ms trains applied every 15 seconds). NMTF was higher in 7‐day old compared to 14‐day old rats (76% vs. 65%; p<0.05). Exogenous NRG treatment significantly improved neuromuscular transmission in 7‐day old rats (NMTF: 52%; p<0.05), but not in 14‐day old rats (65%). Isometric DIAm contractile properties were not affected by NRG treatment in either age group. These results indicate that NRG has important synaptic effects during early postnatal development of the DIAm. Whether NRG effects on neuromuscular transmission result from acute changes in AChR expression or alternative mechanisms remains to be explored. Supported by NIH grant AR51173 and a Career Development Award from Mayo Clinic.

The balance between protein synthesis and degradation determines if there is a net loss (atrophy) or net gain (hypertrophy) in muscle mass. Recent studies suggest that PI3K‐dependent activation of Akt is an important regulator of both protein synthesis (via Erk and mTOR) and degradation (via the transcription factor FoxO1). Unilateral denervation (DNV) of the diaphragm muscle (DIAm) increases protein synthesis at 3d and degradation by 5d post‐DNV such that protein breakdown becomes evident by 5d after DNV. We hypothesized that DIAm DNV results in time‐dependent changes in Akt activation, leading to initial mTOR and subsequent FoxO1 activation. Akt, Erk, and mTOR phosphorylation and FoxO1 nuclear translocation were measured by Western blot analyses. At 1h and 6h post‐DNV, pAkt/Akt and pErk/Erk decreased. pErk/Erk increased at 1d and 3d, and pAkt/Akt increased at 5d post‐DNV. Although mTOR phosphorylation and expression increased 5d post‐DNV, the ratio of phospho‐mTOR/mTOR did not change at any time point post‐DNV. Nuclear translocation of FoxO1 increased 1d post‐DNV. Taken together, these results indicate that concomitant changes in PI3K/Akt, Erk, mTOR, and FoxO1 activity all contribute to, but do not completely account for, the time‐dependent changes in protein balance observed after DNV. Regulation of other signaling pathways, e.g., AMPK, and/or trophic influences, e.g., neuregulin, must be considered.

Skeletal muscle growth is achieved by a net increase in muscle protein synthesis, decreased protein degradation or both. The Akt and mTOR signaling transduction pathways regulate both protein synthesis and degradation. We previously reported that the nerve‐derived growth factor neuregulin (NRG) increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt‐dependent manner. In the present study, we report that basal protein degradation in the diaphragm muscle is also significantly reduced by NRG treatment (23%). The NRG‐induced reduction of basal protein degradation is abrogated by pretreatment with inhibitors of PI3K (LY294002, 50 μM), mTOR (rapamycin, 100 nM) or MEK (PD98059, 50 μM). In fact, inhibition of any of these pathways alone is sufficient to increase basal protein degradation by greater than 30%. Importantly, NRG treatment nullifies the increase in protein degradation induced by PI3K or MEK inhibition, but not that induced by mTOR inhibition. Thus, unlike NRG effects on protein synthesis, the NRG‐induced decrease in protein degradation is abrogated not only by inhibition of the PI3K/Akt pathway, but also by inhibition of the MAP kinase. Based on these results, we suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, and that mTOR is a critical regulator of NRG effects on protein balance in skeletal muscle. Supported by NIH grant AR51173.

Autophagy maintains cellular homeostasis by the elimination of damaged proteins or organelles. Our overarching hypothesis is that autophagy contributes to aging‐related neuromuscular dysfunction. The mechanisms regulating autophagy flux are promising therapeutic targets to mitigate the effects of neuromuscular dysfunction across conditions such as aging. In previous studies, we found that autophagy flux is reduced in early old age (18‐month old mice; 90% survival), with progressive autophagosome accumulation in old age evident by increased LC3 and p62 protein expression in cervical motor neurons (24‐month old mice; 75% survival). Neurotrophins acting through the tropomyosin‐related kinase receptor subtype B (TrkB) are critical to the maintenance of neuromuscular function including neuromuscular transmission in the diaphragm muscle and generation of maximal transdiaphragmatic pressure (Pdi), both in young and aged mice. Specifically, inhibition of TrkB signaling impairs maximal Pdi generation whereas activation of TrkB signaling blunts failure in neuromuscular transmission. We hypothesize that inhibiting TrkB kinase activity reduces autophagy flux resulting in increased LC3 and p62 expression, like the aging effects at phrenic motor neurons. TrkB F616A mice at 18‐ and 24‐ months of age, harboring a mutation that induces susceptibility to 1NMPP1‐mediated TrkB kinase inhibition, were randomized to receive 1‐week treatment with vehicle or 1NMPP1 (n=6 per age and treatment group). Protein expression of the autophagy markers LC3 and p62 in cervical spinal cord (CSC) segments C2‐C6 was analyzed by Western blotting. There was a significant effect on LC3 and p62 expression of age, but not of 1NMPP1 treatment, and no age*treatment interaction. LC3B expression increased by ~50% and p62 expression increased by ~30% in 24 month‐old compared to 18 month‐old mice. These results are consistent with previous reports that there is an aging‐related impairment of autophagy and accumulation of autophagosomes; however, inhibiting TrkB kinase activity does not modulate autophagy in these age groups. Understanding the role of TrkB signaling in mediating changes in autophagy and age‐related neuromuscular dysfunction will be important in advancing potential therapeutic applications of TrkB agonists.

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.

TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkB F616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Background Postoperative in-hospital pneumonia is a serious complication. This study aims to investigate the association between 3 preoperative stratification tools (American Society of Anesthesiologists Physical Status [ASA-PS] score, Charlson Comorbidity Index [CCI], and Rockwood Frailty Deficit Index [FI]) and risk for postoperative pneumonia. Methods We identified adult patients who developed postoperative pneumonia following noncardiothoracic surgery under general anesthesia, between January 1, 2016 and December 31, 2017. Patients with postoperative pneumonia were 1:1 matched to control subjects based on age, sex, and the exact type of operations. Medical records were reviewed to identify variables that may be associated with risk for developing postoperative pneumonia. Analyses adjusted for clinical characteristics were performed using the conditional logistic regression, taking into account 1:1 matched set case-control study design. Results We identified 211 cases of postoperative pneumonia, and all 3 tested stratification tools were associated with increased risk: ASA-PS (after all adjustments of American Society of Anesthesiologists (ASA) III, odds ratio 4.17 [95% confidence interval 1.74-10.01]; ASA > III 24.03 [6.54-88.32]), CCI (CCI values > 3, 1.29 [1.02-1.63] per unit CCI score), and frail FI score 3.25 (1.45-7.27). Because of incomplete intake documentation, the FI could not be calculated in 57 (13.5%) patients, but these “unknown frailty” patients were also at increased risk for postoperative pneumonia, 3.15 (1.29-7.72). Discussion Three commonly used stratification indices (ASA-PS score, CCI, and FI) were associated with increased risk for postoperative pneumonia. Patients unable to complete intake form to calculate the FI were also at increased risk.