Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats.

Cervical spinal cord injury impacts ventilatory and non-ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh-which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post-sigh "apnea" (i.e., the post-sigh inter-breath interval). This post-sigh inter-breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2 ) spinal cord hemisection (C2 SH) on the transdiaphragmatic pressure (Pdi ) generated during sighs and the post-sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2 SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post-sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2 SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

Enhanced Diaphragm Muscle Function upon Satellite Cell Transplantation in Dystrophic Mice.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Postanesthesia Care Unit and Anesthetic Management Outcomes Among Patients Undergoing Noncardiac Surgery: Differences by Race and Ethnicity.

PURPOSE: To investigate the association of patient race and ethnicity with postanesthesia care unit (PACU) outcomes in common, noncardiac surgeries requiring general anesthesia. DESIGN: Single tertiary care academic medical center retrospective matched cohort. METHODS: We matched 1:1 1836 adult patients by race and/or ethnicity undergoing common surgeries. We compared racial and ethnic minority populations (62 American Indian, 250 Asian, 315 Black or African American, 281 Hispanic, and 10 Pacific Islander patients) to 918 non-Hispanic White patients. The primary outcomes were: the use of an appropriate number of postoperative nausea and vomiting (PONV) prophylactics; the incidence of PONV; and the use of a propofol infusion as part of the anesthetic (PROP). Secondary outcomes were: the use of opioid-sparing multimodal analgesia, including the use of regional anesthesia for postoperative pain control; the use of any local anesthetic, including the use of liposomal bupivacaine; the duration until readiness for discharge from the PACU; the time between arrival to PACU and first pain score; and the time between the first PACU pain score of ≥4 and administration of an analgesic. Logistic and linear regression were used for relevant outcomes of interest. FINDINGS: Overall, there were no differences in the appropriate number of PONV prophylactics, nor the incidence of PONV between the two groups. There was, however, a decreased use of PROP (OR = 0.80; 95% CI: 0.69, 0.94; P = .005), PACU length of stay was 9.56 minutes longer (95% CI: 2.62, 16.49; P = .007), and time between arrival to PACU and first pain score was 2.30 minutes longer in patients from racial and ethnic minority populations (95% CI: 0.99, 3.61; P = .001). There were no statistically significant differences in the other secondary outcomes. CONCLUSIONS: The rate of appropriate number of PONV prophylactic medications as well as the incidence of PONV were similar in patients from racial and ethnic minority populations compared to non-Hispanic White patients. However, there was a lower use of PROP in racial and ethnic minority patients. It is important to have a health equity lens to identify differences in management that may contribute to disparities within each phase of perioperative care.

Neuromotor control of spontaneous quiet breathing in awake rats evaluated by assessments of diaphragm EMG stationarity.

The neuromotor control of the diaphragm muscle (DIAm) is dynamic. The activity of the DIAm can be recorded via electromyography (EMG), which represents the temporal summation of motor unit action potentials. Our goal in the present study was to investigate DIAm neuromotor control during quiet spontaneous breathing (eupnea) in awake rats by evaluating DIAm EMG at specific temporal locations defined by motor unit recruitment and derecruitment. We evaluated the nonstationarity of DIAm EMG activity to identify DIAm motor unit recruitment and derecruitment durations. Combined with assessments of root mean square (RMS) and sum of squares (SS) EMG, the durations of these phases provide physiological information about the temporal aspects of motor control. During eupnea in awake rats (n = 10), the duration of motor unit recruitment comprised 61 ± 19 ms of the onset-to-peak duration (214 ± 62 ms) of the DIAm RMS EMG. The peak-to-offset duration of DIAm EMG activity was 453 ± 96 ms, with a terminating period of derecruitment of 161 ± 44 ms. The burst duration was 673 ± 128 ms. Both the RMS EMG amplitude and the SS EMG were higher at the completion of motor unit recruitment than at the start of motor unit derecruitment, suggesting that offset discharge rates were lower than onset discharge rates. Our analyses provide novel insights into the time domain aspects of DIAm neuromotor control and allow indirect estimates of the contribution of recruitment and frequency to RMS EMG amplitude during eupnea in awake rats.NEW & NOTEWORTHY We characterized three phases of neuromotor control-motor unit recruitment, sustained activity, and derecruitment-based on statistical assessments of stationarity of the diaphragm muscle (DIAm) EMG activity in awake rats. Our findings may allow indirect estimates of the contribution of motor unit recruitment and frequency coding toward generating force and provide novel insights about the temporal aspects of DIAm neuromotor control and descending respiratory drive in unanesthetized animals.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Size-dependent differences in mitochondrial volume density in phrenic motor neurons.

Neuromotor control of diaphragm muscle (DIAm) motor units is dependent on an orderly size-dependent recruitment of phrenic motor neurons (PhMNs). Slow (type S) and fast, fatigue resistant (type FR) DIAm motor units, which are frequently recruited to sustain ventilation, comprise smaller PhMNs that innervate type I and IIa DIAm fibers. More fatigable fast (type FF) motor units, which are infrequently recruited for higher force, expulsive behaviors, comprise larger PhMNs that innervate more type IIx/IIb DIAm fibers. We hypothesize that due to the more frequent activation and thus higher energy demand of type S and FR motor units, the mitochondrial volume density (MVD) of smaller PhMNs is greater compared with larger PhMNs. In eight adult (6 mo old) Fischer 344 rats, PhMNs were identified via intrapleural injection of Alexa488-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red. PhMNs and mitochondria were imaged using multichannel confocal microscopy using a ×60 oil objective. Following optical sectioning and three-dimensional (3-D) rendering, PhMNs and mitochondria were analyzed volumetrically using Nikon Elements software. Analysis of MVD in somal and dendritic compartments was stratified by PhMN somal surface area. Smaller PhMNs (likely S and FR units) had greater somal MVDs compared with larger PhMNs (likely FF units). By contrast, proximal dendrites or larger PhMNs had higher MVD compared with dendrites of smaller PhMNs. We conclude that more active smaller PhMNs have a higher mitochondrial volume density to support their higher energy demand in sustaining ventilation.NEW & NOTEWORTHY Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

CTB-targeted protocells enhance ability of lanthionine ketenamine analogs to induce autophagy in motor neuron-like cells.

Impaired autophagy, a cellular digestion process that eliminates proteins and damaged organelles, has been implicated in neurodegenerative diseases, including motor neuron disorders. Motor neuron targeted upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue, activates autophagy in neuronal cell lines. We hypothesized that analogs of LK can be targeted to motor neurons using nanoparticles to improve autophagy flux. Using a mouse motor neuron-like hybrid cell line (NSC-34), we tested the effect of three different LK analogs on autophagy modulation, either alone or loaded in nanoparticles. For fluorescence visualization of autophagy flux, we used a mCherry-GFP-LC3 plasmid reporter. We also evaluated protein expression changes in LC3-II/LC3-I ratio obtained by western blot, as well as presence of autophagic vacuoles per cell obtained by electron microscopy. Delivering LK analogs with targeted nanoparticles significantly enhanced autophagy flux in differentiated motor neuron-like cells compared to LK analogs alone, suggesting the need of a delivery vehicle to enhance their efficacy. In conclusion, LK analogs loaded in nanoparticles targeting motor neurons constitute a promising treatment option to induce autophagy flux, which may serve to mitigate motor neuron degeneration/loss and preserve motor function in motor neuron disease.

Brain derived neurotrophic factor/tropomyosin related kinase B signaling impacts diaphragm neuromuscular transmission in a novel rat chemogenetic model.

The neuromuscular junction (NMJ) mediates neural control of skeletal muscle fibers. Neurotrophic signaling, specifically brain derived neurotrophic factor (BDNF) acting through its high-affinity tropomyosin related kinase B (TrkB) receptor is known to improve neuromuscular transmission. BDNF/TrkB signaling also maintains the integrity of antero- and retrograde communication between the motor neuron soma, its distal axons and pre-synaptic terminals and influences neuromuscular transmission. In this study, we employed a novel rat chemogenetic mutation (TrkB F616), in which a 1-naphthylmethyl phosphoprotein phosphatase 1 (1NMPP1) sensitive knock-in allele allowed specific, rapid and sustained inhibition of TrkB kinase activity. In adult female and male TrkB F616 rats, treatment with either 1NMPP1 (TrkB kinase inhibition) or DMSO (vehicle) was administered in drinking water for 14 days. To assess the extent of neuromuscular transmission failure (NMTF), diaphragm muscle isometric force evoked by nerve stimulation at 40 Hz (330 ms duration trains repeated each s) was compared to isometric forces evoked by superimposed direct muscle stimulation (every 15 s). Chronic TrkB kinase inhibition (1NMPP1 group) markedly worsened NMTF compared to vehicle controls. Acute BDNF treatment did not rescue NMTF in the 1NMPP1 group. Chronic TrkB kinase inhibition did not affect the apposition of pre-synaptic terminals (labeled with synaptophysin) and post-synaptic endplates (labeled with α-Bungarotoxin) at diaphragm NMJs. We conclude that inhibition of BDNF/TrkB signaling in TrkB F616 rats disrupts diaphragm neuromuscular transmission in a similar manner to TrkB F616A mice, likely via a pre-synaptic mechanism independent of axonal branch point failure.

Mitochondrial adaptations to inactivity in diaphragm muscle fibers.

Type I and IIa diaphragm muscle (DIAm) fibers comprise slow and fast fatigue-resistant motor units that are recruited to accomplish breathing and thus have a high duty cycle. In contrast, type IIx/IIb fibers comprise more fatigable fast motor units that are infrequently recruited for airway protective and straining behaviors. We hypothesize that mitochondrial structure and function in type I and IIa DIAm fibers adapt in response to inactivity imposed by spinal cord hemisection at C2 (C2SH). At 14 days after C2SH, the effect of inactivity on mitochondrial structure and function was assessed in DIAm fibers. Mitochondria in DIAm fibers were labeled using MitoTracker Green (Thermo Fisher Scientific), imaged in three-dimensions (3-D) by fluorescence confocal microscopy, and images were analyzed for mitochondrial volume density (MVD) and complexity. DIAm homogenate from either side was assessed for PGC1α, Parkin, MFN2, and DRP1 using Western blot. In alternate serial sections of the same DIAm fibers, the maximum velocity of the succinate dehydrogenase reaction (SDHmax) was determined using a quantitative histochemical technique. In all groups and both sides of the DIAm, type I and IIa DIAm fibers exhibited higher MVD, with more filamentous mitochondria and had higher SDHmax normalized to both fiber volume and mitochondrial volume compared with type IIx/IIb Diam fibers. In the inactive right side of the DIAm, mitochondria became fragmented and MVD decreased in all fiber types compared with the intact side and sham controls, consistent with the observed reduction in PGC1α and increased Parkin and DRP1 expression. In the inactive side of the DIAm, the reduction in SDHmax was found only for type I and IIa fibers. These results show that there are intrinsic fiber-type-dependent differences in the structure and function of mitochondria in DIAm fibers. Following C2SH-induced inactivity, mitochondrial structure (MVD and fragmentation) and function (SDHmax) were altered, indicating that inactivity influences all DIAm fiber types, but inactivity disproportionately affected SDHmax in the more intrinsically active type I and IIa fibers.NEW & NOTEWORTHY Two weeks of diaphragm (DIAm) inactivity imposed by C2SH caused reduced mitochondrial volume density, mitochondrial fragmentation, and a concomitant reduction of SDHmax in type I and IIa DIAm fibers on the lesioned side. Type I and IIa DIAm fibers were far more sensitive to inactivation than type IIx/IIb fibers, which exhibited little pathology. Our results indicate that mitochondria in DIAm fibers are plastic in response to varying levels of activity.

Cervical spinal hemisection alters phrenic motor neuron glutamatergic mRNA receptor expression.

Upper cervical spinal cord injuries (SCI) disrupt descending inputs to phrenic motor neurons (PhMNs), impairing respiratory function. Unilateral spinal hemisection at C2 (C2SH) results in loss of ipsilateral rhythmic diaphragm muscle (DIAm) EMG activity associated with lower force behaviors accomplished by recruitment of smaller PhMNs in rats. Activity during higher force, non-ventilatory behaviors that recruit larger PhMNs is minimally impaired following C2SH. We previously showed neuroplasticity in glutamatergic receptor expression in PhMN post-C2SH with changes in NMDA receptor expression reflecting functional recovery over time. We hypothesize that C2SH-induced changes in glutamatergic receptor (AMPA and NMDA) mRNA expression in PhMNs vary with motor neuron size, with more pronounced changes in smaller PhMNs. Retrogradely-labelled PhMNs were classified in tertiles according to somal surface area and mRNA expression was measured using single-cell, multiplex fluorescence in situ hybridization. Ipsilateral to C2SH, a pronounced reduction in NMDA mRNA expression in PhMNs was evident at 3 days post-injury with similar impact on PhMNs in the lower size tertile (~68% reduction) and upper tertile (~60%); by 21 days, there was near complete restoration of NMDA receptor mRNA expression across all PhMNs. There were no changes in NMDA mRNA expression contralateral to C2SH. There were no changes in AMPA mRNA expression at PhMNs on either side of the spinal cord or at any time-point post-C2SH. In summary, following C2SH there is ipsilateral reduction in PhMN NMDA mRNA expression at 3 days that is not limited to smaller PhMN recruited in the generation of lower force ventilatory behaviors. The recovery of NMDA mRNA expression by 21 days post-C2SH is consistent with evidence of spontaneous recovery of ipsilateral DIAm activity at this timepoint. These findings suggest a possible role for NMDA receptor mediated glutamatergic signaling in mechanisms supporting postsynaptic neuroplasticity at the PhMN pool and recovery of DIAm activity after cervical SCI.

Automated evaluation of respiratory signals to provide insight into respiratory drive.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals and is highly active throughout life displaying rhythmic activity. The repetitive activation of the DIAm (and of other muscles driven by central pattern generator activity) presents an opportunity to analyze these physiological data on a per-event basis rather than pooled on a per-subject basis. The present study highlights the development and implementation of a graphical user interface-based algorithm using an analysis of critical points to detect the onsets and offsets of individual respiratory events across a range of motor behaviors, thus facilitating analyses of within-subject variability. The algorithm is designed to be robust regardless of the signal type (e.g., EMG or transdiaphragmatic pressure). Our findings suggest that this approach may be particularly beneficial in reducing animal numbers in certain types of studies, for assessments of perturbation studies where the effects are relatively small but potentially physiologically meaningful, and for analyses of respiratory variability.

Frailty Transitions One Year After Total Joint Arthroplasty: A Cohort Study.

BACKGROUND: Total joint arthroplasty (TJA) is prevalent and offered to patients regardless of frailty status experiencing pain, disability, and functional decline. This study aims to describe changes in levels of frailty 1 year after TJA. METHODS: We identified a retrospective cohort of adult patients undergoing primary TJA between 2005 and 2016 using an institutional total joint registry. Associations between categorized frailty deficit index (FI) and change in FI were analyzed using linear regression models. Mortality, deep periprosthetic joint infection, and reoperation were analyzed using time to event methods. RESULTS: In total, 5341 patients (37.6% non-frail, 39.4% vulnerable, and 23.0% frail) with items necessary to determine FI at 1 year after TJA were included. Preoperatively, 29% of vulnerable patients improved to non-frail 1 year later, compared to only 11% regressing to frail. Four in 10 frail patients improved to vulnerable/non-frail. Improvements in activities of daily living (ADL) were more evident in frail and vulnerable patients, with >30% reduction in the percentage of patients expressing difficulties with walking, climbing stairs, and requiring ADL assistance 1 year after TJA. Increases in frailty 1 year after TJA were associated with significantly increased rates of mortality (hazard ratio [HR] 1.50, 95% confidence interval [CI] 1.24-1.82, P < .001), deep periprosthetic joint infection (HR 3.98, 95% CI 1.85-8.58, P < .001), and reoperation (HR 1.80, 95% CI 1.19-2.72, P = .005). CONCLUSION: Frailty states are dynamic with patient frailty shown to be modifiable 1 year after TJA. Preoperative frailty measurement is an important step toward identifying those that may benefit most from TJA and for postoperative frailty surveillance.

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.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A 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.

Quantifying mitochondrial volume density in phrenic motor neurons.

BACKGROUND: Previous assessments of mitochondrial volume density within motor neurons used electron microscopy (EM) to image mitochondria. However, adequate identification and sampling of motor neurons within a particular motor neuron pool is largely precluded using EM. Here, we present an alternative method for determining mitochondrial volume density in identified motor neurons within the phrenic motor neuron (PhMN) pool, with greatly increased sampling. NEW METHOD: This novel method for assessing mitochondrial volume density in PhMNs uses a combination of intrapleural injection of Alexa 488-conjugated cholera toxin B (CTB) to retrogradely label PhMNs, followed by intrathecal application of MitoTracker Red to label mitochondria. This technique was validated by comparison to 3D EM determination of mitochondrial volume density as a "gold standard". RESULTS: A mean mitochondrial volume density of ∼11 % was observed across PhMNs using the new MitoTracker Red method. This compared favourably with mitochondrial volume density (∼11 %) measurements using EM. COMPARISON WITH EXISTING METHOD: The range, mean and variance of mitochondrial volume density estimates in PhMNs were not different between EM and fluorescent imaging techniques. CONCLUSIONS: Fluorescent imaging may be used to estimate mitochondrial volume density in a large sample of motor neurons, with results similar to EM, although EM did distinguish finer mitochondrion morphology compared to MitoTracker fluorescence. Compared to EM methods, the assessment of a larger sample size and unambiguous identification of motor neurons belonging to a specific motor neuron pool represent major advantages over previous methods.

Peripheral Nerve Blockade with Combined Standard and Liposomal Bupivacaine in Major Lower-Extremity Amputation.

BACKGROUND AND OBJECTIVES: Optimizing perioperative analgesia for patients undergoing major lower-extremity amputation remains a considerable challenge. The utility of liposomal bupivacaine as a component of peripheral nerve blockade for lower-extremity amputation is unknown. METHODS: We conducted an observational study comparing three different perioperative analgesic techniques for adults undergoing major lower-extremity amputation under general anesthesia between 2012 and 2017 at an academic medical center: (1) no regional anesthesia, (2) peripheral nerve blockade with standard bupivacaine, and (3) peripheral nerve blockade with a mixture of standard and liposomal bupivacaine. The primary outcome of cumulative opioid oral morphine milligram equivalent utilization in the first 72 hours postoperatively was compared across groups utilizing multivariable linear regression. RESULTS: A total of 631 unique anesthetics were included for 578 unique patients, including 416 (66%) without regional anesthesia, 131 (21%) with peripheral nerve blockade with a mixture of standard and liposomal bupivacaine, and 84 (13%) with peripheral nerve blockade with standard bupivacaine alone. Cumulative morphine equivalents were lower in those receiving peripheral nerve blockade with combined standard and liposomal bupivacaine compared with those not receiving regional anesthesia (multiplicative increase 0.67; 95% CI 0.50 to 0.90; P = 0.007). There were no significant differences in opioid utilization between peripheral nerve blockade groups (P = 0.59). CONCLUSIONS: Peripheral nerve blockade is associated with reduced opioid requirements after lower-extremity amputation compared with general anesthesia alone. However, the incorporation of liposomal bupivacaine is not significantly different to blockade employing only standard bupivacaine.

How Is Surgical Risk Best Assessed? A Cohort Comparison of Measures in Total Joint Arthroplasty.

BACKGROUND: We designed this study to determine whether a Frailty Deficit Index (FI) confers added risk stratification beyond more traditional methods. The associations of preoperative scores on FI, American Society of Anesthesiologists (ASA) physical status, and Charlson Comorbidity Index (CCI) with complications after total joint arthroplasty (TJA) were compared. METHODS: Using a single institution cohort of adult patients ≥50 years undergoing primary or revision TJA from 2005 to 2016, we assessed how well the FI, CCI, and ASA scores predicted risk of mortality, infection, and reoperation. We performed 7 models for each outcome: FI, ASA, and CCI alone, FI + ASA, FI + CCI, ASA + CCI, and FI + ASA + CCI. Cox proportional hazards regression methods were used to calculate the concordance (C-) statistic, a measure of discrimination. RESULTS: Of 18,397 TJAs included, 98.9% were alive 1 year postoperatively. For mortality, all models had concordance between 0.76 and 0.79, with the FI + ASA + CCI model performing highest (C-statistic 0.79; 95% confidence interval [CI] 0.76-0.82). Unadjusted, FI had the strongest concordance (C-statistic 0.77). In FI + ASA + CCI, each increase in 1 comorbidity (of 32 total comorbidities) in the FI was significantly associated with a 12% increase in the rate of mortality (hazard ratio [HR] 1.12, 95% CI 1.07-1.17, P < .001), 10% increase in infection (HR 1.10, 95% CI 1.06-1.14; P < .001), and 6% increase in reoperation (HR 1.06, 95% CI 1.05-1.08, P < .001). CONCLUSION: Identifying at-risk patients preoperatively is crucial and may result in adjustment of postoperative care. FI was independently associated with risk of adverse outcomes following TJA even after taking into account other predictive measures.

Three Risk Stratification Tools and Postoperative Pneumonia After Noncardiothoracic Surgery.

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.