Trypan blue not toxic for retinal pigment epithelium in vitro

PURPOSE: To investigate whether trypan blue has a toxic effect on cultured retinal pigment epithelial (retinal pigment epithelium) cells. DESIGN: Experimental study with a direct live/dead cell staining technique using fluorescent dyes. METHODS: Cultured human retinal pigment epithelium cells were exposed for 5 minutes to various concentrations of trypan blue (0.06%, 0.15%, 0.30%), and cell viability was confocally measured. RESULTS: No increased cell death was found in cultures incubated in any of the trypan blue concentrations used. CONCLUSION: These findings indicate that a short exposure of trypan blue does not have a toxic effect on cultured retinal pigment epithelium cells.     

The prioritisation of genetic screening with primary haemochromatosis as an example

In 1994, the Health Council of the Netherlands published a report entitled 'Genetic screening' which contained 12 criteria for genetic screening programmes. However, the list does not prioritize the various criteria. From the list we have selected two criteria that we consider to be the most important. Firstly, the genetic screening test should be able to discriminate between subjects who are likely to develop the disease and those who are not. Secondly, there should be an effective treatment for subjects with the genetic defect. From this point of view, for example, screening for the C282Y mutation is not a suitable approach for detecting primary haemochromatosis. Although 85-90% of the patients with this disease are homozygous for this mutation, the majority of the carriers will not develop the disease. The 12 criteria of the Health Council of the Netherlands are still applicable. However, when taking a decision as to whether or not genetic screening is useful, we recommend that priority be given to the two primary criteria.     

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation

In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free primary amino groups per 1000 amino acid residues (E/N14C). At a fixed molar ratio NHS:EDC of 0.6, the amount of heparin covalently immobilized to E/N14C increased with increasing molar ratios of EDC to heparin carboxylic acid groups (Hep-COOH), to a maximum of approximately 5-5.5 wt% at a ratio of 2. Upon incubation in cell culture medium of endothelial cells, 4 to 7% of the immobilized heparin was released during 11 days. Immobilization of increasing amounts of heparin to E/N14C progressively reduced activation of contact activation proteases. Optimal anticoagulant activity, as measured by thrombin inhibition, was obtained after heparin immobilization using a ratio of EDC to Hep-COOH of 0.2-0.4 (14-20 mg heparin immobilized per gram of collagen). Platelets deposited to (heparinized) E/N14C showed only minor spreading and aggregation, although heparin immobilization slightly increased the number of adherent platelets. The results of this study suggest that heparin immobilization to EDC/NHS-crosslinked collagen may improve the in vivo blood compatibility of this material.     

Minimal flow sevoflurane and isoflurane anaesthesia and impact on renal function

BACKGROUND AND AIM: Compound A generation and accumulation in sevoflurane anaesthesia is dependent on fresh gas flow. We investigated the extent of generation of compound A. METHODS: After Institutional Review Board approval and informed consent, patients with normal renal function were randomized to receive either sevoflurane (n = 33) or isoflurane (n = 43) minimal flow anaesthesia (0.5 L min-1) for at least 2 h under standardized conditions. Compound A concentrations were quantified and blood and urine samples were taken to assess renal involvement. Both groups were comparable. RESULTS: No significant differences concerning blood chemistry and urine measurements were found. The maximum mean compound A concentration was observed 90 min after flow reduction being 40 +/- 9 p.p.m. at a corresponding mean sevoflurane concentration of 2.1 +/- 0.5 vol%. Mean inspiratory compound A exposure was 102 +/- 33 p.p.m h-1. CONCLUSION: Compound A concentrations using 0.5 L min-1 fresh gas flow and a heated absorber were higher than previously published values using an inflow of 1 L min-1. Compound A exposure was similar to other clinical studies which did not show changes in renal and hepatic function.     

Hemin, inducer of heme-oxygenase 1, improves functional recovery from myocardial stunning in conscious dogs

OBJECTIVE: To examine the effects of pretreatment with hemin, an inducer of the potential antioxidative enzyme heme-oxygenase 1 (HO-1) or heat-shock protein 32, on myocardial stunning. DESIGN: Randomized animal study. SETTING: Animal laboratory of a university hospital. PARTICIPANTS: Chronically instrumented mongrel dogs (n = 44). INTERVENTIONS: Dogs underwent chronic instrumentation for measurement of hemodynamics and myocardial wall thickening fraction (WTF). Experiments with 12 dogs were performed on separate days in a crossover fashion: (1) 10 minutes of left anterior descending (LAD) coronary artery occlusion after application of hemin (9 mg/kg/d) for 1 week and (2) 10 minutes of LAD coronary artery occlusion without hemin pretreatment. In control experiments (n = 32), the reversible induction of HO-1, using gel electrophoresis and Western blotting, was determined. MEASUREMENTS AND MAIN RESULTS: WTF was measured as a baseline value before hemin administration and at predetermined time points until complete recovery from stunning. LAD artery occlusion caused a significant reduction in the WTF in the LAD-perfused area with and without hemin, without significant hemodynamic changes. At all time points, after 1 minute of reperfusion, the WTF as percentage of baseline values was significantly higher after hemin pretreatment (p < 0.05). Baseline WTF values were reached after 24 hours with and after >48 hours without hemin pretreatment (p < 0.05). CONCLUSION: Hemin pretreatment attenuates myocardial stunning in conscious dogs.     

Naloxone improves functional recovery of myocardial stunning in conscious dogs through its action on the central nervous system

This study tests the hypothesis that naloxone, but not its quarternarysalt, naloxone methiodide (which does not enter the centralnervous system), improves recovery from myocardial stunningin conscious dogs. Twenty dogs were chronically instrumentedfor measurement of heart rate, left atrial, aortic and leftventricular pressure (LVP), LV dP•dt_max^–1 and myocardialwall thickening fraction (WTF). Regional myocardial blood flowwas determined with coloured microspheres. Occluder around theleft anterior descending artery (LAD) allowed induction of reversibleLAD ischaemia. Each of the 20 dogs underwent two LAD ischaemicchallenges. Experiments (performed on separate days, in crossoverfashion) were: (i) 10 min of LAD occlusion after applicationof naloxone 63 µg kg^–1 or naloxone methiodide 63µg kg^–1 and (ii) occlusion without naloxone or naloxonemethiodide. WTF was measured at baseline and until completerecovery occurred. LAD ischaemia significantly reduced LAD WTFwith (mean (SD) 54 (15)% lower than baseline) and without naloxone(55 (16)% lower than baseline), without significant haemodynamicdifferences. Between 1 to 30 min of reperfusion, WTF was significantlyhigher with naloxone (P<0.05). There was no difference inWTF with or without naloxone methiodide. We conclude that naloxoneimproved recovery from myocardial stunning in conscious dogs,and that this was centrally mediated. Br J Anaesth 2001; 86: 545–9     

Students'' expectations of postregistration degree programmes

The needs of postregistration students pursuing degree-linked clinical courses have received little attention and there are few insights concerning their aspirations when they enrol on such courses. Thus the aim of this study was to explore postregistration students' perceptions of the specific needs of their patient/client group and to examine how they envisaged the course on which they had just enrolled might help them to meet these needs in addition to their own requirements for professional and personal development. Data were collected by group interview from 62 students enrolling on eight different postregistration courses, all employed in an acute hospital trust. The results were analysed inductively. They indicated that students had internalized the state of the healthcare market and were keenly aware of the need to fulfil the expectations of employers and the public, while fulfilling their own needs for education and pursuing their own professional and career trajectories. They appeared ambitious and yet appeared to demonstrate empathy for patients and their families and felt a tremendous desire to provide care of a high quality through the optimal development of technical expertise. Students' emphasis on the importance of keeping abreast of technological developments should not be lightly dismissed considering its prominent position within the acute areas where they were employed, especially as it did not replace their desire to promote the caring aspects of their work.