Mantilla, Carlos B.

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDH max ), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDH max was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDH max when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O 2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDH max ) of mitochondria, O 2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O 2 consumption and ATP production but at reduced cost of ROS formation. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O 2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O 2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O 2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O 2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O 2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O 2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O 2 consumption and ATP production. Support or Funding Information Supported by NIH grant AG44615 and HL105355.

In previous studies, we found that after cervical spinal cord hemisection at C2 (SH), neuromuscular transmission improved in the rat diaphragm muscle (DIAm). We also found that exogenous brain‐derived neurotrophic factor (BDNF) treatment enhanced neuromuscular transmission, whereas inhibiting activation of its receptor (TrkB) worsened neuromuscular transmission. We hypothesized that expression of BDNF and/or its receptor TrkB at phrenic motoneurons would increase following SH. In the present study, adult male rats underwent SH or sham surgery. Phrenic motoneurons were labeled by injection of cholera toxin B into the DIAm and retrogradely‐labeled phrenic motoneurons were sampled by laser capture microdissection (LCM). BDNF and TrkB receptor (truncated – T1 and full length – FL) mRNA were measured in LCM‐microdissected motoneurons using quantitative real‐time RT‐PCR and expressed as a ratio to ribosomal S16 RNA. We found that BDNF mRNA expression in phrenic motoneurons increased by 3 days after SH (~2‐fold). Similarly, both TrkB.T1 and TrkB.FL mRNA increased by 3 days after SH (~17‐ and ~11‐fold, respectively). It is possible that the relative expression of neurotrophins and their receptors (truncated or full‐length) may determine the time‐course of restoration of phrenic activity following SH. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.

DIAm motor units are normally recruited in an orderly fashion to accomplish different ventilatory and non‐ventilatory behaviors. Following a cervical spinal cord hemisection at C2 (SH), there is restoration of phrenic activity over time. We hypothesized that an orderly recruitment of DIAm motor units is maintained with the restoration of rhythmic DIAm electromyographic (EMG) activity following SH. A pair of electrode wires was implanted into the midcostal regions of the DIAm of adult male rats for chronic EMG recording. Three days later, these animals underwent SH surgery. We examined changes in integrated DIAm EMG activity across different motor behaviors (i.e., eupnea, hypercapnia, tracheal occlusion and fictive sneezing) assuming that DIAm EMG activity during fictive sneezing represents the maximum recruitment of DIAm motor units. As expected in controls, DIAm EMG activity increased from eupnea (~27% of max) to hypercapnia (5% CO 2; ~45% of max) to sustained tracheal occlusion (~60% of max). By 14 days after SH with restored rhythmic DIAm activity, the amplitude of EMG activity on the ipsilateral side was reduced, and the changes in EMG activity induced by increased ventilatory and non‐ventilatory drive were blunted, although an orderly recruitment of motor units was still observed. These results support a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.

Background Frailty increases risk for complications after total joint arthroplasty (TJA). Whether this association is influenced by anesthetic administered is unknown. We hypothesized that use of neuraxial (spinal or epidural) anesthesia is associated with better outcomes compared with general anesthesia, and that the effect of anesthesia type on outcomes differs by frailty status. Methods This single-institution cohort study included all patients (≥50 years) from January 2005 through December 2016 undergoing unilateral, primary and revision TJA. Using multivariable Cox regression, we assessed relationships between anesthesia type, a preoperative frailty deficit index (FI) categorized as non-frail (FI <0.11), vulnerable (FI 0.11 to 0.20), and frail (FI >0.20), and complications (mortality, infection, wound complications/hematoma, reoperation, dislocation, and periprosthetic fracture) within 1 year after surgery. Interactions between anesthesia type and frailty were tested, and stratified models were presented when an interaction (p<0.1) was observed. Results Among 18 458 patients undergoing TJA, more patients were classified as frail (21.5%) and vulnerable (36.2%) than non-frail (42.3%). Anesthesia type was not associated with complications after adjusting for age, joint, and revision type. However, in analyzes stratified by frailty, vulnerable patients under neuraxial block had less mortality (HR=0.49; 95% CI 0.27 to 0.89) and wound complications/hematoma (HR=0.71; 95% CI 0.55 to 0.90), whereas no difference in risk by anesthesia type was observed among patients found non-frail or frail. Conclusions Neuraxial anesthesia use among vulnerable patients was associated with improved survival and less wound complications. Calculating preoperative frailty prior to TJA informs perioperative risk and enhances shared-decision making for selection of anesthesia type.

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkB F616A mice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkB F616A mice were injected with 1NMPP1 ( n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O 2 /5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH 2 O during eupnea and hypoxia/hypercapnia, to ~35 cmH 2 O during sighs and tracheal occlusion, and to ~65 cm H 2 O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units. NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDH max ) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDH max activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDH max and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 μm. SDH max , quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHC Slow ), IIa (MyHC 2A ), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5–100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDH max in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia. NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.

Background: Debate surrounds the issue of whether peripheral nerve blockade or periarticular infiltration (PAI) should be employed within a contemporary, comprehensive multimodal analgesia pathway for total hip arthroplasty. We hypothesized that patients treated with a continuous posterior lumbar plexus block (PNB) would report less pain and consume less opioid medication than those treated with PAI. Methods: This investigator-initiated, independently funded, 3-arm randomized clinical trial (RCT) performed at a single high-volume institution compared postoperative analgesia interventions for elective, unilateral primary total hip arthroplasty: (1) PNB; (2) PAI with ropivacaine, ketorolac, and epinephrine (PAI-R); and (3) PAI with liposomal bupivacaine, ketorolac, and epinephrine (PAI-L) using computerized randomization. The primary outcome was maximum pain during the morning (06:00 to 12:00) of the first postoperative day (POD) on an ascending numeric rating scale (NRS) from 0 to 10. Pairwise treatment comparisons were performed using the rank-sum test, with a p value of <0.017 indicating significance (Bonferroni adjusted). A sample size of 150 provided 80% power to detect a difference of 2.0 NRS units. Results: We included 159 patients (51, 54, and 54 patients in the PNB, PAI-R, and PAI-L groups, respectively). No significant differences were found with respect to the primary end point on the morning of the first POD (median, 3.0, 4.0, and 3.0, respectively; p > 0.033 for all). Opioid consumption was low and did not differ across groups at any intervals. Median maximum pain on POD 1 was 5.0, 5.5, and 4.0, respectively, and was lower for the PAI-L group than for the PAI-R group (p = 0.006). On POD 2, maximum pain (median, 3.5, 5.0, and 3.5, respectively) was lower for the PNB group (p = 0.014) and PAI-L group (p = 0.016) compared with the PAI-R group. The PAI-L group was not significantly different from the PNB group with respect to any outcomes: postoperative opioid use including rescue intravenous opioid medication, length of stay, and hospital adverse events, and 3-month follow-up data including any complication. Conclusions: In this RCT, we found a modest improvement with respect to analgesia in patients receiving PNB compared with those receiving PAI-R, but not compared with those who had PAI-L. Secondary analyses suggested that PNB or PAI-L provides superior postoperative analgesia compared with PAI-R. For primary total hip arthroplasty, a multimodal analgesic regimen including PNB or PAI-L provides opioid-limiting analgesia. Level of Evidence: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

AbstractThe diaphragm muscle comprises various types of motor units that are recruited in an orderly fashion governed by the intrinsic electrophysiological properties (membrane capacitance as a function of somal surface area) of phrenic motor neurons (PhMNs). Glutamate is the main excitatory neurotransmitter at PhMNs and acts primarily via fast acting AMPA and N‐methyl‐D‐aspartic acid (NMDA) receptors. Differences in receptor expression may also contribute to motor unit recruitment order. We used single cell, multiplex fluorescence in situ hybridization to determine glutamatergic receptor mRNA expression across PhMNs based on their somal surface area. In adult male and female rats (n = 9) PhMNs were retrogradely labeled for analyses (n = 453 neurons). Differences in the total number and density of mRNA transcripts were evident across PhMNs grouped into tertiles according to somal surface area. A ~ 25% higher density of AMPA (Gria2) and NMDA (Grin1) mRNA expression was evident in PhMNs in the lower tertile compared to the upper tertile. These smaller PhMNs likely comprise type S motor units that are recruited first to accomplish lower force, ventilatory behaviors. In contrast, larger PhMNs with lower volume densities of AMPA and NMDA mRNA expression presumably comprise type FInt and FF motor units that are recruited during higher force, expulsive behaviors. Furthermore, there was a significantly higher cytosolic NMDA mRNA expression in small PhMNs suggesting a more important role for NMDA‐mediated glutamatergic neurotransmission at smaller PhMNs. These results are consistent with the observed order of motor unit recruitment and suggest a role for glutamatergic receptors in support of this orderly recruitment. image Cover Image for this issue: doi: 10.1111/jnc.14747.