The role of extracellular calcium in ischemia/reperfusion injury in skeletal muscle

Ischemia-reperfusion injury to skeletal muscle, following an acute arterial occlusion is a significant cause of morbidity and mortality. The purpose of this study is to examine the role of extracellular calcium in the production of cellular necrosis following a prolonged period of normothermic ischemia. Bilateral canine gracilis muscles were made ischemic for 4.5 to 5 hr. The control muscle had normal blood reperfusion (ionized Ca2+ 1.2 mM). The treated muscle was perfused for 30 min with an oxygenated solution (ionized Ca2+ 0.11 mM) containing free radical scavengers followed by normal blood perfusion. Necrosis was determined by nitroblue tetrazolium staining after 48 hr of reperfusion. Total muscle Ca2+ was measured by atomic absorption spectrometry. Pre- and postischemic muscle Ca2+ levels were similar (2.8 +/- 0.4 vs 3.2 +/- 0.8 nmole/mg protein, n = 13, P greater than 0.1). After 30 min of reperfusion the treated muscle Ca2+ was 2.4 +/- 0.4 compared to control levels of 8.6 +/- 0.8 nmole/mg protein (P less than 0.001). Total tissue calcium returned to normal at 60 min in viable muscle, but continued to accumulate in necrotic tissue. However, the delay in initial muscle Ca2+ influx was not associated with increased overall salvage of muscle 78 +/- 9% vs 77 +/- 8% necrosis, (P greater than 0.1). In conclusion we could not demonstrate a protective effect of reduced extracellular Ca2+ during early reperfusion, and it negated our previously demonstrated beneficial effects of free radical scavengers. It was shown however that the early ability to extrude intracellular calcium was associated with significant salvage of muscle tissue.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Carbon dioxide recruitment threshold as a predictor of successful weaning in chronic obstructive pulmonary disease patients: A pilot study

Abstract Several methods have been used to predict successful weaning and extubation among chronic obstructive pulmonary disease (COPD) patients. The objective of this study is to determine whether carbon dioxide recruitment threshold (PCO₂RT) can be used as adjunct to conventional weaning parameters to predict early weaning and successful extubation. Twelve COPD patients who were ready to be extubated based on conventional weaning parameters were divided into group A ( n = 7) and group B ( n = 5). Group A were those patients with better weaning parameters and hence a higher probability of successful extubation as compared to group B. Carbon dioxide apnoeic threshold (PCO₂AT) was obtained by hyperventilating the patient using an increment of two breaths per min until apnoea occurs. At this point, the PCO₂AT or the PaCO₂ during said apnoeic period was recorded. A dead space of 150 cc is then added to the circuit until the patient starts to breathe as evidenced by the sensitivity trigger indicator. The PCO₂ obtained at this period is termed PCO₂RT. After weaning for 30 min on a T-tube, another arterial blood gas is determined and this is called the PCO₂SB or the CO₂ level after 30 min on spontaneous breathing. If the PCO₂SB-PCO₂RT difference is high with a sensitivity of 85.71% and specificity of 100% vs sensitivity of 57.14% and specificity of 60% using the conventional weaning parameters. Thus an increase in PCO₂SB at 30 min T-tube is indicative of impending respiratory pump failure and that other causes of failure to wean must be investigated.     

Pulsed mode versus near-continuous mode delivery of diode laser photocoagulation for high-risk retinopathy of prematurity.

OBJECTIVE: To compare structural and functional outcomes and efficiency of diode laser photocoagulation for retinopathy of prematurity (ROP) when delivered in a pulsed mode versus a near-continuous mode. METHODS: A retrospective study was conducted of 138 patients who underwent diode laser photocoagulation for threshold ROP using either pulsed or near-continuous delivery. Laser-related complications and structural and functional outcomes were analyzed. Prospectively, time efficiency and total energy used were evaluated in nine infants with bilateral symmetric high-risk prethreshold ROP in which one eye of each infant was randomized to pulsed and the fellow eye to near-continuous delivery. RESULTS: There was no significant difference between groups with regards to prevalence of posterior disease (Zone 1 or posterior Zone 2) (p = 0.11), postoperative vitreous haze (p = 0.60), postoperative complications (p = 0.38), retinal detachment (p = 0.90), strabismus (p = 0.73), amblyopia (p = 0.69), or refractive error (p = 0.95). Mean time for treatment was 23 minutes using pulsed delivery versus 14 minutes per eye with near-continuous delivery (p < 0.001). The mean total power used per eye with pulsed mode delivery was 1.5 x 10(5) W versus 1.1 x 10(5) W with near-continuous delivery (p = 0.015). CONCLUSIONS: No differences in complications, functional outcome, or structural outcome were found between using pulsed mode and near-continuous mode diode laser delivery for high-risk ROP. Near-continuous laser delivery, in our hands, was more time-efficient and used less total power.     

Mechanism of uptake and retention of F-18 BMS-747 158-02 in cardiomyocytes: a novel PET myocardial imaging agent

Background BMS-747158-02 is a novel fluorine 18-labeled pyridazinone derivative designed for cardiac imaging. The uptake and retention mechanisms of F-18 BMS-747158-02 in cardiac myocytes were studied in vitro, and the biodistribution of F-18 BMS-747158-02 was studied in vivo in mice. Methods and Results Fluorine 19 BMS-747158-01 inhibited mitochondrial complex I (MC-I) in bovine heart submitochondrial particles with an IC₅₀ of 16.6±3 nmol/L that was comparable to the reference inhibitors of MC-1, rotenone, pyridaben, and deguelin (IC₅₀ of 18.2±6.7 nmol/L, 19.8±2.6 nmol/L, and 23.1±1.5 nmol/L, respectively). F-18 BMS-747158-02 had high uptake in monolayers of neonatal rat cardiomyocytes (10.3%±0.7% of incubated drug at 60 minutes) that was inhibited by 200 nmol/L of rotenone (91%±2%) and deguelin (89%±3%). In contrast, an inactive pyridaben analog, P-0 (IC₅₀ value>4 μmol/L in MC-1 assay), did not inhibit the binding of F-18 BMS-747158-02 in cardiomyocytes. Uptake and washout kinetics for F-18 BMS-747158-02 in rat cardiomyocytes indicated that the time to half-maximal (t_1/2) uptake was very rapid (approximately 35 seconds), and washout t_1/2 for efflux of F-18 BMS-747158-02 was greater than 120 minutes. In vivo biodistribution studies in mice showed that F-18 BMS-747158-02 had substatial myocardial uptake (9.5%±0.5% of injected dose per gram) at 60 minutes and heart-to-lung and heart-to-liver ratios of 14.1±2.5 and 8.3±0.5, respectively. Positron emission tomography imaging in the mouse allowed clear cardiac visualization and demonstrated sustained myocardial uptake through 55 minutes. Conclusions F-18 BMS-747158-02 is a novel positron emission tomography cardiac tracer targeting MC-I in cardiomyocytes with rapid uptake and slow washout. These characteristics allow fast and sustained accumulation in the heart.