Alterations in Cytoskeleton and Mitochondria in the Development and Reversal of Steatosis in Human Hepatocytes.

BACKGROUND & AIMS: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. METHODS: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer, in which we fully optimized reagent dosing specifically for human hepatocytes. RESULTS: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network, leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. CONCLUSIONS: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Mechanism of pulmonary immunosuppression: extrapulmonary burn injury suppresses bacterial endotoxin-induced pulmonary neutrophil recruitment and neutrophil extracellular trap (NET) formation.

Pulmonary immunosuppression often occurs after burn injury (BI). However, the reasons for BI-induced pulmonary immunosuppression are not clearly understood. Neutrophil recruitment and neutrophil extracellular trap (NET) formation (NETosis) are important components of a robust pulmonary immune response, and we hypothesized that pulmonary inflammation and NETosis are defective after BI. To test this hypothesis, we established a mouse model with intranasal LPS instillation in the presence or absence of BI (15% of body surface burn) and determined the degree of immune cell infiltration, NETosis, and the cytokine levels in the airways and blood on d 2. Presence of LPS recruited monocytes and large numbers of neutrophils to the airways and induced NETosis (citrullinated histone H3, DNA, myeloperoxidase). By contrast, BI significantly reduced LPS-mediated leukocyte recruitment and NETosis. This BI-induced immunosuppression is attributable to the reduction of chemokine (C-C motif) ligand (CCL) 2 (monocyte chemoattractant protein 1) and CCL3 (macrophage inflammatory protein 1α). BI also suppressed LPS-induced increase in IL-17A, IL-17C, and IL-17E/IL-25 levels in the airways. Therefore, BI-mediated reduction in leukocyte recruitment and NETosis in the lungs are attributable to these cytokines. Regulating the levels of some of these key cytokines represents a potential therapeutic option for mitigating BI-mediated pulmonary immunosuppression.-Sakuma, M., Khan, M. A. S., Yasuhara, S., Martyn, J. A., Palaniyar, N. Mechanism of pulmonary immunosuppression: extrapulmonary burn injury suppresses bacterial endotoxin-induced pulmonary neutrophil recruitment and neutrophil extracellular trap (NET) formation.

An ALPHA7 Nicotinic Acetylcholine Receptor Agonist (GTS-21) Promotes C2C12 Myonuclear Accretion in Association with Release of Interleukin-6 (IL-6) and Improves Survival in Burned Mice.

The role of interleukin-6 (IL-6) in physiological processes and disease is poorly understood. The hypothesis tested in this study was that selective alpha7 acetylcholine receptor (α7AChR) agonist, GTS-21, releases IL-6 in association with myonuclear accretion and enhances insulin signaling in muscle cells, and improves survival of burn injured (BI) mice. The in vitro effects of GTS-21 were determined in C2C12 myoblasts and 7-day differentiated myotubes (myotubes). The in vivo effects of GTS-21 were tested in BI wild-type (WT) and IL-6 knockout (IL6KO) mice. GTS-21 dose-dependently (0 µM, 100 µM, and 200 µM) significantly increased IL-6 levels in myoblasts and myotubes at 6 and 9 h. GTS-21-induced IL-6 release in myotubes was attenuated by methyllycaconitine (α7AChR antagonist), and by Stat-3 or Stat-5 inhibitors. GTS-21 increased MyoD and Pax7 protein expression, myonuclear accretion, and insulin-induced phosphorylation of Akt, GSK-3ß, and Glut4 in myotubes. The glucose levels of burned IL6KO mice receiving GTS-21 decreased significantly compared with sham-burn IL6KO mice. Superimposition of BI on IL6KO mice caused 100% mortality; GTS-21 reduced mortality to 75% in the IL6KO mice. The 75% mortality in burned WT mice was reduced to 0% with GTS-21. The in vitro findings suggest that GTS-21-induced IL-6 release from muscle is mediated via α7AChRs upstream of Stat-3 and -5 pathways and is associated with myonuclear accretion, possibly via MyoD and Pax7 expression. GTS-21 in vivo improves survival in burned WT mice and IL6KO mice, suggesting a potential therapeutic application of α7AChR agonists in the treatment of BI.

Role of Elevated Fibrinogen in Burn-Induced Mitochondrial Dysfunction: Protective Effects of Glycyrrhizin.

INTRODUCTION: Skeletal muscle wasting and weakness with mitochondrial dysfunction (MD) are major pathological problems in burn injury (BI) patients. Fibrinogen levels elevated in plasma is an accepted risk factor for poor prognosis in many human diseases, and is also designated one of damage-associated molecular pattern (DAMPs) proteins. The roles of upregulated fibrinogen on muscle changes of critical illness including BI are unknown. The hypothesis tested was that BI-upregulated fibrinogen plays a pivotal role in the inflammatory responses and MD in muscles, and that DAMPs inhibitor, glycyrrhizin mitigates the muscle changes. METHODS: After third degree BI to mice, fibrinogen levels in the plasma and at skeletal muscles were compared between BI and sham-burn (SB) mice. Fibrinogen effects on inflammatory responses and mitochondrial membrane potential (MMP) loss were analyzed in C2C12 myotubes. In addition to survival, the anti-inflammatory and mitochondrial protective effects of glycyrrhizin were tested using in vivo microscopy of skeletal muscles of BI and SB mice. RESULTS: Fibrinogen in plasma and its extravasation to muscles significantly increased in BI versus SB mice. Fibrinogen applied to myotubes evoked inflammatory responses (increased MCP-1 and TNF-α; 32.6 and 3.9-fold, respectively) and reduced MMP; these changes were ameliorated by glycyrrhizin treatment. In vivo MMP loss and superoxide production in skeletal muscles of BI mice were significantly attenuated by glycyrrhizin treatment, together with improvement of BI survival rate. CONCLUSIONS: Inflammatory responses and MMP loss in myotubes induced by fibrinogen were reversed by glycyrrhizin. Anti-inflammatory and mitochondrial protective effect of glycyrrhizin in vivo leads to amelioration of muscle MD and improvement of BI survival rate.

Ultrasound-Guided Regional Anesthesia for Pediatric Burn Reconstructive Surgery: A Prospective Study.

Pediatric patients face multiple reconstructive surgeries to reestablish function and aesthetics postburn injury. Often, the site of the harvested graft for these reconstructions is reported to be the most painful part of the procedure and a common reason for deferring these reconstructive procedures. This study in pediatric burn patients undergoing reconstructive procedures examined the analgesia response to local anesthetic infiltration versus either a single ultrasound-guided regional nerve block of the lateral femoral cutaneous nerve (LFCN) or a fascia iliaca compartment block with catheter placement and continuous infusion. Nineteen patients were randomized to one of three groups (infiltration, single-shot nerve block, or compartment block with catheter) and received intraoperative analgesia intervention. Postoperatively, visual analog scale pain scores were recorded-for pain at the donor site-every 4 hours while awake-for 48 hours. This nonparametric data was analyzed using a two-way ANOVA, Friedman's test, and Kruskal-Wallis test, with significance determined at P < 0.05. The analysis demonstrated that the patients in the regional anesthesia groups were significantly more comfortable over the 48 hour hospital course than the patients in the control group. The patients receiving a single-shot block of the LFCN were more comfortable on postoperative day (POD) 0 while the catheter patients were more comfortable on POD 1 and POD 2. There was not a statistically significant difference in opioid requirements in any group. Regional anesthetic block of the LFCN, with or without catheter placement, provides an improved postoperative experience for the pediatric patient undergoing reconstructive surgery with lateral/anterolateral skin graft versus local anesthesia infiltration of donor site. For optimal comfort throughout the postoperative period, an ultrasound-guided block with continuous catheter may be beneficial.

Midazolam exacerbates morphine tolerance and morphine-induced hyperactive behaviors in young rats with burn injury.

Midazolam and morphine are often used in pediatric intensive care unit (ICU) for analgesia and sedation. However, how these two drugs interact behaviorally remains unclear. Here, we examined whether (1) co-administration of midazolam with morphine would exacerbate morphine tolerance and morphine-induced hyperactive behaviors, and (2) protein kinase C (PKC) would contribute to these behavioral changes. Male rats of 3-4 weeks old were exposed to a hindpaw burn injury. In Experiment 1, burn-injured young rats received once daily saline or morphine (10mg/kg, subcutaneous, s.c.), followed 30min later by either saline or midazolam (2mg/kg, intraperitoneal, i.p.), for 14 days beginning 3 days after burn injury. In Experiment 2, young rats with burn injury were administered with morphine (10mg/kg, s.c.), midazolam (2mg/kg, i.p.), and chelerythrine chloride (a non-specific PKC inhibitor, 10nmol, intrathecal) for 14 days. For both experiments, cumulative morphine anti-nociceptive dose-response (ED50) was tested and hyperactive behaviors such as jumping and scratching were recorded. Following 2 weeks of each treatment, ED50 dose was significantly increased in rats receiving morphine alone as compared with rats receiving saline or midazolam alone. The ED50 dose was further increased in rats receiving both morphine and midazolam. Co-administration of morphine and midazolam also exacerbated morphine-induced hyperactive behaviors. Expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor and PKCγ in the spinal cord dorsal horn (immunohistochemistry; Western blot) was upregulated in burn-injured young rats receiving morphine alone or in combination with midazolam, and chelerythrine prevented the development of morphine tolerance. These results indicate that midazolam exacerbated morphine tolerance through a spinal NMDA/PKC-mediated mechanism.

A single injection of botulinum toxin decreases the margin of safety of neurotransmission at local and distant sites.

BACKGROUND: We tested the hypothesis that a single injection of botulinum toxin not only has local, but also distant effects on muscle function, biochemistry, and pharmacodynamics of atracurium. METHODS: Botulinum toxin (2.5 U) was injected into the tibialis muscle of anesthetized rats (n = 26). The contralateral side with no injection served to study distant effects. Control animals (n = 25) received a saline injection. Neuromuscular function, pharmacology, and expression of acetylcholine receptors (nAChRs) were evaluated in the tibialis at 0, 4, and 16 days after injection and in comparison with saline- injected controls. RESULTS: On day 4, botulinum toxin caused complete paralysis of the tibialis, while its contralateral side showed a decrease in absolute twitch tension (1.8 N [1.6; 1.9] vs 3.0 N [2.8; 3.1], Newton, P < 0.05). On day 16, muscle weakness was only present on the toxin-injected side where absolute twitch tension was decreased (0.6 N [0.6, 0.7] vs 3.4 N [3.1, 3.7], P < 0.05). Tibialis mass was decreased on the toxin-injected side at day 4 (1.46 mg/g [1.43, 1.48] vs 1.74 mg/g [1.72; 1.75], P < 0.05) and on day 16 (0.78 mg/g [0.76, 0.79] vs 1.73 mg/g [1.69; 1.77], P < 0.05). Effects distant from the site of injection were seen on day 16, when muscle atrophy was also present in the adjacent gastrocnemius and soleus muscles. Normalized to tibialis mass, specific twitch tension (tension/g muscle) was reduced on the contralateral side at day 4 and on the toxin-injected side at day 16 in relation to saline controls. At day 16, an increased sensitivity to atracurium was seen on the toxin-injected side, evidenced as a decreased ED(50) (0.23 mg/kg [0.13, 0.33] vs 0.72 mg/kg [0.63, 0.82], P < 0.05) and a lower infusion rate (38 µL/kg/min [32, 43] vs135 µL/kg/min [126, 144], P < 0.05), together with a reduced plasma concentration requirement of atracurium (0.5 µg/mL [0.4, 0.7] vs 4.5 µg/mL [3.8, 5.2], P < 0.05) to achieve a steady state 50% reduction in baseline (absolute) twitch tension. ED(50) of atracurium was also decreased on the contralateral side at day 16 in relation to saline controls. The nAChRs in the tibialis were increased on the toxin-injected side to 123 fmol/mg [115, 131] vs 28 fmol/mg [25, 29] (P < 0.05) in time-matched saline-injected controls at day 4 and to 378 [341, 413] vs 27 fmol/mg [25, 29] (P < 0.05) at day 16. CONCLUSIONS: Botulinum toxin has local and distant effects on muscle. The decrease in specific twitch tension indicates that the muscle atrophy alone cannot explain the functional changes; neuromuscular transmission is also impaired. An increased sensitivity to atracurium on the toxin-injected side, despite up-regulation of nAChRs, seems unique to botulinum toxin.

Nociceptive behavior following hindpaw burn injury in young rats: response to systemic morphine.

OBJECTIVE: Develop a burn injury model in young age rats. BACKGROUND: Management of pain after burn injury in pediatric patients is an unresolved clinical issue. METHODS: A burn injury model in young rats of 3-4 weeks old was developed by briefly immersing the dorsal part of the right hindpaw in a hot water bath (85°C) for 12 seconds under pentobarbital anesthesia. RESULTS: Burn injury, but not sham control, induced nociceptive behaviors (mechanical allodynia, thermal hyperalgesia) when examined on post-injury day 2, 4, and 7. In burn-injured rats, there was the upregulated expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor, Akt1, Akt2, and protein kinase C γ (PKCγ), but downregulated expression of neuronal nitric oxide synthase (NOS), inducible NOS, and glycogen synthase kinase-3ß, within the spinal cord dorsal horn ipsilateral to burn injury. Moreover, intraperitoneal administration of a clinically available NMDA receptor antagonist dextromethorphan (30 mg/kg, once daily × 7 days beginning on day 7 after burn injury) attenuated mechanical allodynia and thermal hyperalgesia in burn-injured rats. Different from our previous finding in adult burn-injured rats; however, burn injury in young rats of this age did not spontaneously shift the morphine antinociceptive response curve to the right within the dose range used in the study when exposed to morphine for the first time, suggesting that the development of intrinsic tolerance to morphine antinociception may be different from adult rats following burn injury. CONCLUSIONS: Our data suggest that this model may be used to explore the mechanisms of burn injury-induced nociception in young rats and to differentiate the sequelae from burn injury between adult and young rats under certain experimental conditions.

Increased sensitivity to a nondepolarizing muscle relaxant in a patient with acquired neuromyotonia.

Neuromyotonia is a disorder of hyperexcitability of the peripheral nerve. It has electromyographic features of spontaneous, continuous, irregularly occurring doublets, or multiplets of motor unit potential discharges. Neuromyotonia is characterized by both myokymic and neuromyotonic discharges. To our knowledge, this is the first report in the literature to assess the sensitivity of skeletal muscle to a nondepolarizing muscle relaxant drug, rocuronium, in a woman with acquired neuromyotonia. She had a past medical history notable for prolonged postoperative paralysis following anesthesia. The patient showed increased sensitivity to the neuromuscular effects of rocuronium. This increase in sensitivity may be explained by downregulation of acetylcholine receptors in response to chronic high agonist (acetylcholine) concentrations. If patients with neuromyotonia receive anesthesia, we recommend that smaller doses of a nondepolarizing muscle relaxant be administered, accompanied by monitoring of neuromuscular function, so as to provide optimal muscle relaxation while avoiding overdose and prolonged postoperative recovery.

Measurement of muscle strength in the intensive care unit.

Traditional (indirect) techniques, such as electromyography and nerve conduction velocity measurement, do not reliably predict intensive care unit-acquired muscle weakness and its clinical consequences. Therefore, quantitative assessment of skeletal muscle force is important for diagnosis of intensive care unit-acquired motor dysfunction. There are a number of ways for assessing objectively muscle strength, which can be categorized as techniques that quantify maximum voluntary contraction force and those that assess evoked (stimulated) muscle force. Important factors that limit the repetitive evaluation of maximum voluntary contraction force in intensive care unit patients are learning effects, pain during muscular contraction, and alteration of consciousness.The selection of the appropriate muscle is crucial for making adequate predictions of a patient's outcome. The upper airway dilators are much more susceptible to a decrease in muscle strength than the diaphragm, and impairment of upper airway patency is a key mechanism of extubation failure in intensive care unit patients. Data suggest that the adductor pollicis muscle is an appropriate reference muscle to predict weakness of muscles that are typically affected by intensive care unit-acquired weakness, i.e., upper airway as well as extremity muscles. Stimulated (evoked) force of skeletal muscles, such as the adductor pollicis, can be assessed repetitively, independent of brain function, even in heavily sedated patients during high acuity of their disease.

In vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized, ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation. RESULT & CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.