Citations

Cardiopulmonary bypass (CPB) is associated with significant postoperative morbidity, but its effects on the neuromuscular system are unclear. Recent studies indicate that even relatively short periods of mechanical ventilation result in significant neuromuscular effects. Carbon monoxide (CO) has gained recent attention as therapy to reduce the deleterious effects of CPB. We hypothesized that 1) CPB results in impaired neuromuscular transmission and reduced diaphragm force generation; and 2) CO treatment during CPB will mitigate these effects. In adult male Sprague-Dawley rats, diaphragm muscle-specific force and neuromuscular transmission properties were measured 90 min after weaning from normothermic CPB (1 h). During CPB, either low-dose inhaled CO (250 ppm) or air was administered. The short period of mechanical ventilation used in the present study (∼3 h) did not adversely affect diaphragm muscle contractile properties or neuromuscular transmission. CPB elicited a significant decrease in isometric diaphragm muscle-specific force compared with time-matched, mechanically ventilated rats (∼25% decline in both twitch and tetanic force). Diaphragm muscle fatigability to 40-Hz repetitive stimulation did not change significantly. Neuromuscular transmission failure during repetitive activation was 60 ± 2% in CPB animals compared with 76 ± 4% in mechanically ventilated rats ( P < 0.05). CO treatment during CPB abrogated the neuromuscular effects of CPB, such that diaphragm isometric twitch force and neuromuscular transmission were no longer significantly different from mechanically ventilated rats. Thus, CPB has important detrimental effects on diaphragm muscle contractility and neuromuscular transmission that are largely mitigated by CO treatment. Further studies are needed to ascertain the underlying mechanisms of CPB-induced neuromuscular dysfunction and to establish the potential role of CO therapy.

Rewarming patients after profound hypothermia may result in acute heart failure and high mortality (50–80%). However, the underlying pathophysiological mechanisms are largely unknown. We characterized cardiac contractile function in the temperature range of 15–30°C by measuring the intracellular Ca2+concentration ([Ca2+]i) and twitch force in intact left ventricular rat papillary muscles. Muscle preparations were loaded with fura-2 AM and electrically stimulated during cooling at 15°C for 1.5 h before being rewarmed to the baseline temperature of 30°C. After hypothermia/rewarming, peak twitch force decreased by 30–40%, but [Ca2+]iwas not significantly altered. In addition, we assessed the maximal Ca2+-activated force (Fmax) and Ca2+sensitivity of force in skinned papillary muscle fibers. Fmaxwas decreased by ∼30%, whereas the pCa required for 50% of Fmaxwas reduced by ∼0.14. In rewarmed papillary muscle, both total cardiac troponin I (cTnI) phosphorylation and PKA-mediated cTnI phosphorylation at Ser23/24 were significantly increased compared with controls. We conclude that after hypothermia/rewarming, myocardial contractility is significantly reduced, as evidenced by reduced twitch force and Fmax. The reduced myocardial contractility is attributed to decreased Ca2+sensitivity of force rather than [Ca2+]iitself, resulting from increased cTnI phosphorylation.

Object In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. Methods The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. Results The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA. Conclusions By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

ABSTRACTPasteurella (Mannheimia) haemolyticaleukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate thatP. haemolyticaLkt binds to bovine CD18, the common subunit of all β2 integrins. We designed experiments with the following objectives: to identify which member of the β2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced β2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml;P< 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P< 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

Background and Purpose— The purpose of this study was to determine whether implanting exogenous fibroblasts on platinum coils could enhance intra-aneurysmal fibrosis. Hypotheses included: (1) fibroblast-coated (FBC) platinum coils can improve angiographic results after embolization; and (2) FBC platinum coils can accelerate histological healing of embolized aneurysms. Methods— Experimental aneurysms in rabbits were embolized with control platinum coils (n=18) or FBC coils (n=18). Subjects were euthanized at 14 days, 1 month, 3 months and 6 months after implantation. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0 to 12) based on neck and dome healing. Results— Histology total scores and fibrosis ratio at 14 days were significantly greater in the FBC coil group compared with controls (6.6±1.9 versus 2.5±1.1, 1.2±0.6% versus 0.2±0.3%, respectively; P =0.0090). Cavities embolized with FBC coils showed cellular proliferation and thrombus organization, with an endothelialized membrane bridging the neck. There were no differences between groups in the later timepoints. The FBC coil group showed radiographic stability in 11 (61%) cases, coil compaction in 2 (11%) cases, and progressive occlusion in 5 (28%) cases. No progressive occlusion was seen in controls; 3 (17%) of 18 control cases exhibited coil compaction ( P =0.0546). Conclusions— FBC coils can accelerate early histological healing compared with control coils in the rabbit aneurysm model.

Background Volatile anesthetics produce bronchodilation in part by depleting sarcoplasmic reticulum Ca stores in airway smooth muscle (ASM). Other bronchodilatory drugs are known to act via cyclic nucleotides (cyclic adenosine 3',5'-cyclic monophosphate, cyclic guanosine 3',5'-cyclic monophosphate). Intracellular Ca regulation in ASM involves plasma membrane Ca influx, including that triggered by sarcoplasmic reticulum Ca depletion (store-operated Ca entry [SOCE]). The authors hypothesized that anesthetics and bronchodilatory agents interact in inhibiting SOCE, thus enhancing ASM relaxation. Methods In enzymatically dissociated porcine ASM cells imaged using fluorescence microscopy, sarcoplasmic reticulum Ca was depleted by 1 microm cyclopiazonic acid in 0 extracellular Ca, nifedipine, and potassium chloride (preventing Ca influx through L-type channels and SOCE). Extracellular Ca was rapidly reintroduced to selectively activate SOCE in the presence or absence of 1 minimum alveolar concentration (MAC) halothane, isoflurane, or sevoflurane. Anesthetic interference with SOCE regulation by cyclic nucleotides was examined by activating SOCE in the presence of (1) 1 microm acetylcholine, (2) 100 microm dibutryl cyclic adenosine 3',5'-cyclic monophosphate, or (3) 100 microm 8-bromo-cyclic guanosine 3',5'-cyclic monophosphate. Results SOCE was enhanced by acetylcholine, whereas volatile anesthetics and both cyclic nucleotides partially inhibited Ca influx. Preexposure to 1 or 2 MAC anesthetic (halothane > isoflurane > sevoflurane) inhibited SOCE. Only halothane and isoflurane inhibited acetylcholine-induced augmentation of Ca influx, and significantly potentiated cyclic nucleotide inhibition such that no influx was observed in the presence of anesthetics and cyclic nucleotides. Conclusions These data indicate that volatile anesthetics prevent sarcoplasmic reticulum refilling by inhibiting SOCE and enhancing cyclic nucleotide blunting of Ca influx in ASM. Such interactions likely result in substantial airway relaxation in the presence of both anesthetics and bronchodilatory agents such as beta agonists or nitric oxide.

Abstract Previous studies have suggested that the proinflammatory cytokine, TNF-α, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca2+ responses to agonist stimulation. The present study examined the effects of TNF-α on Ca2+ influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca2+ entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-α treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-α treatment also increased both the peak and plateau intracellular Ca2+ concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-α treatment on SOCE and agonist-induced intracellular Ca2+ concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-α treatment may result in increased myocyte activation due to altered Ca2+ influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Abstract Rationale: Hypercapnic respiratory failure because of inspiratory muscle weakness is the most important cause of death in chronic obstructive pulmonary disease (COPD). However, the pathophysiology of failure of the diaphragm to generate force in COPD is in part unclear. Objectives: The present study investigated contractile function and myosin heavy chain content of diaphragm muscle single fibers from patients with COPD. Methods: Skinned muscle fibers were isolated from muscle biopsies from the diaphragm of eight patients with mild to moderate COPD and five patients without COPD (mean FEV1 % predicted, 70 and 100%, respectively). Contractile function of single fibers was assessed, and afterwards, myosin heavy chain content was determined in these fibers. In diaphragm muscle homogenates, the level of ubiquitin-protein conjugation was determined. Results: Diaphragm muscle fibers from patients with COPD showed reduced force generation per cross-sectional area, and reduced myosin heavy chain content per half sarcomere. In addition, these fibers had decreased Ca2 + sensitivity of force generation, and slower cross-bridge cycling kinetics. Our observations were present in fibers expressing slow and 2A isoforms of myosin heavy chain. Ubiquitin-protein conjugation was increased in diaphragm muscle homogenates of patients with mild to moderate COPD. Conclusions: Early in the development of COPD, diaphragm fiber contractile function is impaired. Our data suggest that enhanced diaphragm protein degradation through the ubiquitin-proteasome pathway plays a role in loss of contractile protein and, consequently, failure of the diaphragm to generate force.

Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2 + /− vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca 2+ ([Ca 2+ ] i ) regulation. We examined cAMP content in vascular smooth muscles from Pkd2 + /− mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2 + /− vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca 2+ ] i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3 H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c , and activated caspase 9 and 3 analyses). The low [Ca 2+ ] i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2 + /− VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca 2+ ] i in wild-type VSMCs. The [Ca 2+ ] i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.