Research in experimental medicine. Zeitschrift fur die gesamte experimentelle Medizin einschliesslich experimenteller Chirurgie最新文献

The combination of confocal laser scan microscopy and the pH-sensitive fluorescent dye BCECF allowed us to record simultaneously intracellular pH, cell viability and relative cell volume. pH was measured by using the pH-sensitive excitation wavelength at 488 nm and the pH-independent excitation wavelength at 442 nm to obtain ratio images. Cell volume was traced by measuring fluorescence dye concentration at 442 nm. Isolated villus tip rat duodenal enterocytes were exposed to 20 mM NH4Cl, sodium free, or 1 mM amiloride buffer. Sodium free buffer and amiloride buffer acidified the cells. Cell volume did not change in sodium free buffer, or NH4Cl exposure, but amiloride led to an increase in cell volume of 20%. After acidification of the duodenal cells, amiloride buffer increased cell volume by almost 50%. These studies revealed that cell volume regulation during pH changes in short-living cells could easily be detected by confocal microscopy and BCECF.

A sonographic examination of the lung has so far been impossible because of sound reflection. In conjunction with video-assisted thoracoscopic surgery, lung sonography would be helpful to make up for the lack of direct palpation. Animal experiments with pigs were performed to find out whether lung sonography becomes possible following bronchoalveolar flooding with a suitable liquid. The lung was filled with whole electrolyte solution through the left leg of a double-lumen endotracheal tube after resorption atelectasis (method 1) or compressive atelectasis (method 2). As an alternative, liquid perfluorocarbon was used (method 3). Under atelectasis, the lung thus flooded was investigated by ultrasound applied transpleurally and endobronchially. The first results proved that lung flooding is possible if certain prerequisites are fulfilled. Perfluorocarbon flooding led to total sound absorption which prevented sonography, whereas flooding with whole electrolyte solution made complete lung sonography possible, making visible the intrapulmonary vessels, bronchi and peribronchial lymphatic nodes. Measurements proved that the unilateral flooding caused no significant changes in the arterial and central venous pressure nor in transcutaneous oxygen saturation.

A flavonoid, Hesperidin was evaluated for its ability to inhibit tumour initiation by a polycyclic aromatic hydrocarbon and tumour promotion by a phorbol ester in the skin of CD-1 mice. Subcutaneous application of Hesperidin did not inhibit 7,12-dimethylbenz(a)anthracene-induced tumour initiation but did inhibit 12-O-tetradecanoyl-13-phorbol acetate-induced tumour promotion. Results provide evidence for a potential chemopreventive activity of Hesperidin.

We tested the hypothesis that preventing cyclic GMP degradation with zaprinast, (a selective cyclic GMP-phosphodiesterase inhibitor) would produce a blunted reduction in myocardial O2 consumption in renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy. Four groups of anesthetized open-chest New Zealand white rabbits (n = 26) were utilized. Either vehicle or zaprinast (3 x 10(-3) M) was applied topically to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. The 1K1C rabbits had a greater heart weight-to-body weight ratio (2.94 +/- 0.08 g/kg) than controls (2.58 +/- 0.17). Systolic blood pressure was higher in 1K1C (102 +/- 9 mm Hg) than in controls (86 +/- 3). Zaprinast significantly and similarly increased cyclic GMP in both control (3.90 +/- 0.47 to 4.66 +/- 0.89 pmol/g) subepicardium (EPI) and (5.08 +/- 0.69 to 7.06 +/- 1.36) subendocardium (ENDO) and 1K1C hearts (5.53 +/- 0.61 to 7.48 +/- 1.51 EPI and 6.48 +/- 0.42 to 8.88 +/- 1.08 ENDO). Myocardial O2 consumption (ml O2/min/ 100 g) was significantly lower in controls treated with zaprinast (EPI: 8.8 +/- 0.1; ENDO: 9.5 +/- 1.9) than in controls treated with vehicle (EPI: 13.6 +/- 1.3; ENDO: 16.2 +/- 2.9). This effect was diminished in 1K1C rabbits treated with zaprinast (EPI: 10.3 +/- 2.4; ENDO: 11.2 +/- 2.6) compared with the vehicle-treated 1K1C group (EPI: 13.3 +/- 1.2; ENDO: 14.5 +/- 2.4). There was a similar increase in myocardial cyclic GMP after treatment with zaprinast, but a greater depression of myocardial O2 consumption in control animals than in 1K1C after treatment with zaprinast. This suggested that the reduction in myocardial O2 consumption, related to increases in cyclic GMP caused by cyclic GMP-phosphodiesterase blockade, was less in 1K1C cardiac hypertrophy.

A novel model for the investigation of the microcirculation in synovial tissue of the mouse knee joint is presented. The mouse knee joint was exposed on a specially designed plexiglass stage with a slight flexion. After partial resection of the skin, the patella tendon was cut transversally, which allowed for visualization of the "Hoffa's fatty body", an intraarticular fatty tissue containing synovial cells on the interior surface of the joint. An intravital fluorescence microscope was adjusted to observe the microcirculation of this intraarticular synovial tissue without opening of the joint capsula. For staining of the plasma, fluorescein isothiocyanate (FITC)-dextran was used, and for the staining of leukocytes rhodamine 6G was used. The tissue investigated presents with a high-density honeycomb-like capillary network, containing some postcapillary venules and a few arterioles. The following parameters were assessed off-line using a computer-assisted microcirculation analysis system: flow and diameter of arterioles and postcapillary venules, as well as functional capillary density. Moreover, leukocyte-endothelial cell interaction was quantified by counting the number of rolling cells and cells adhering to the endothelium in postcapillary venules. As an indication of endothelial leakage, macromolecular extravasation was also assessed. To validate the model, we investigated these parameters at three time points during an observation period of 60 min. There was no change in functional capillary density, nor in vessel diameter after 60 min of observation. Moreover, there was neither a change in the number of rolling cells, nor in the number of cells adhering to the endothelium nor in extravasation of FITC-dextran, thus indicating the stability of the preparation. The new model allows the quantitative analysis of the intraarticular microcirculation of the synovial fatty tissue in vivo. It provides insight into the dynamics of synovial microcirculation and leukocyte-endothelial cell interaction in acute or chronic joint inflammation.

Impairment of intestinal nutritive perfusion and accumulation of inflammatory cells in the intestinal microvasculature are well-known sequelae of mesenteric ischemia/reperfusion, sepsis, and shock. However, the molecular mechanisms underlying these alterations are still not fully understood. The mouse is particularly suitable for the study of these mechanisms since in this species the involvement of, for example, adhesion receptors or pro-/anti-adhesive mediators can be selectively investigated by the use of monoclonal antibodies or gene-targeted strains. The aim of our present study was, therefore, to establish a model to investigate the microcirculation in the mouse small intestine. Under anesthesia by inhalation of isoflurane-N2O, Balb/c mice (n = 16) were laparotomized, and a segment of the jejunum was exteriorized for intrvital fluorescence microscopy. Using FITC-dextran (MW 150,000) as a plasma marker, functional capillary density (FCD) of both the intestinal mucosa and muscle layer was analyzed. Nutritive perfusion was homogeneous in both compartments with values for FCD of 512 +/- 15 cm-1 in mucosa and 226 +/- 21 cm-1 in the muscle layer. No significant changes were observed throughout the observation period of 2 h (FCD values at the end of the observation period: 524 +/- 31 cm-1 and 207 +/- 7 cm-1 in mucosa and muscle, respectively). Besides capillary perfusion, leukocyte-endothelial cell interaction was analyzed in postcapillary venules of the intestinal submucosa using rhodamine-6G as an in vivo leukocyte stain. Under physiological conditions only a few white blood cells were found rolling along or firmly adherent to the microvascular endothelium (number of rolling leukocytes 1 +/- 0.2 cells/mm per second; number of adherent leukocytes: 18 +/- 7 cells/mm2). In a separate group rhodamine-6G-labeled syngeneic platelets were infused to analyze platelet-endothelial cell interactions quantitatively in vivo. Platelets rolled along or attached to the endothelium in a manner similar to leukocytes. However, in contrast to leukocytes the interactions were not restricted to venules, but were also observed in small arterioles. The newly established model allows for the visualization and quantitative assessment of both nutritive perfusion and platelet/leukocytendothelial cell interactions within the distinct layers of the mouse small intestine. Using this model in combination with gene-targeted mice or monoclonal antibodies it is possible to investigate the molecular mechanisms of intestinal inflammation reactions.

Moderately elevated homocysteine concentrations, reflecting deficiency of some nutritional factors required for homocysteine metabolism (folate, vitamin B-6, vitamin B-12) and/or less severe genetic defects, are common in the general population. Several studies have indicated the role of homocysteine as an independent risk factor for vascular disease. A pilot study published recently suggested that plasma homocysteine levels increase during weight reduction in slightly overweight, otherwise healthy subjects (group A). We examined a comparable group of 13 overweight subjects (group B) using a standardised caloric intake and defined vitamin supplementation (Medyn: folate 0.2 mg/ vitamin B-68.0 mg/ vitamin B-120.010 mg three times the day orally) to determine the effect of weight reduction on serum homocysteine levels and to compare the results with those of the pilot study. Mean body weight declined from 87.0 +/- 20.2 to 84.2 +/- 20.1 kg (P < 0.05) in group A and 85.7 +/- 11.3 to 82.5 +/- 9.9 kg (P = 0.049) in group B. Serum homocysteine levels rose from 7.9 +/- 2.0 to 8.7 +/- 2.3 mumol/l (P < 0.0001) in group A and decreased from 8.19 +/- 1.73 to 7.35 +/- 0.88 mumol/l (P = 0.0022) in group B. No correlation was found between the changes in body weight and in homocysteine levels (r = 0.02 in group A, r = 0.18 in group B). Additionally, no correlation was found between serum folate levels and changes in homocysteine levels (r = 0.03 in group A, r = 0.09 in group B). The results suggest that an adequate oral vitamin-supplementation protects against increased homocysteine production during weight reduction.

Association between lipids and renal disease has been reported recently. Its pathogenic mechanisms remain unknown. The aims of this study were to establish: (1) if a cholesterol-rich diet, alone or associated with nephrectomy, produces nephropathy; and (2) if a treatment with omega-3 polyunsaturated fatty acids (PUFA) reduces glomerulosclerotic lesions. Sixty Sprague-Dawley rats were randomized in two different groups: (A) sham operated rats and (B) uninephrectomized rats. Rats in both groups were divided into three subgroups (A1-3, B1-3) according to the diet they were fed: normal chow diet, cholesterol-rich diet (4.5%) or cholesterol-rich diet supplemented with omega-3 PUFA. Twenty weeks later, serum creatinine, creatinine clearance, serum cholesterol, triglycerides, albumin, proteinuria, mesangial cell score and focal glomerulosclerosis were assessed. Results showed that a cholesterol-rich diet significantly increased serum cholesterol, proteinuria and glomerular lesions and decreased creatinine clearance, especially in nephrectomized rats. Glomerular lesions, serum cholesterol and proteinuria ameliorated when cholesterol-rich diet was supplemented with PUFA. Hypertension was noticed only in nephrectomized rats following a normal chow diet. Simple correlation analysis showed that glomerulosclerosis correlated with renal weight, blood creatinine, cholesterol and proteinuria. In spite of some significant differences in urinary prostaglandins, no correlation with glomerular lesions was found. Multiple logistic regression analysis showed that cholesterol and proteinuria were independent risk factors for induction of glomerular sclerosis. In conclusion, a diet rich in cholesterol induces glomerulosclerosis, especially if it is associated with unilateral nephrectomy. Omega-3 PUFA administration reduces serum cholesterol, proteinuria and glomerular injury.

Unlabelled: The administration of protamine to neutralize the circulating heparin is common practice in cardiovascular surgery. The use of this drug is sometimes associated with hemodynamic alterations of varying degree and intensity (systemic hypotension, pulmonary hypertension and even cardiogenic shock). An intrinsic action of protamine has been suggested to be the cause of these vascular reactions. This action is blocked when protamine forms a complex with heparin, although in other cases it appears that the heparin-protamine complex is the factor responsible for these hemodynamic alterations. The aim of this experimental study was to characterize the vasodilatory action of protamine on the systemic circulation, determining whether or not it is dose-dependent; to analyze the role of endothelium; and to evaluate whether this vasodilatory effect is modified by the presence of heparin.

Materials and methods: The abdominal aorta was dissected from eight New Zealand rabbits and then sectioned into vascular rings for study in an organ chamber. Mechanical disruption of endothelium was performed on some rings (n = 14). Once submaximal contraction was reached (ClK 80 mM), protamine sulfate with a final concentration in the organ chamber of 80-400 micrograms/ml was added to one of the groups (n = 12). In the second group (n = 12), equal concentrations of protamine were tested in the presence of heparin at a final concentration of 100 U/ml.

Results: The mean vasodilatation reached in the group of rings exposed only to protamine was 95.4 +/- 1.5% with respect to the submaximal contraction induced with ClK. In the second study group, the rings were exposed to protamine at equally increasing concentrations (80-400 micrograms/ml) but with the presence of heparin in the organ chamber. The mean vasodilatation in this group was 90 +/- 1.5. No statistically significant differences in vasodilatation were found between this group and the protamine without heparin group. On the other hand, in the endothelium-denuded rings (n = 14) exposed to isolated protamine and to protamine-heparin, no vasodilatory response was observed.

Conclusion: Our results show that the administration in vitro of protamine induces endothelium-dependent vasodilatation of the systemic circulation. Likewise, this relaxing effect mediated through endothelium is not blocked when protamine forms a complex with heparin in comparable concentrations of both drugs. Based on these preliminary findings, we believe that in high-risk patients the prevention of systemic vasodilatation and cardiovascular collapse produced by protamine should move towards the use of other substances that can neutralize the anticoagulant effect of heparin or towards pre-medication guidelines that prevent these secondary effects in the case of protamine administration.

We evaluated the effects of oral administration of E4021 (100 mg/kg/day), a type V phosphodiesterase inhibitor, on immunoreactivities of endothelin-1, endothelin receptors, and nitric oxide synthases in pulmonary arteries in a rat model of pulmonary hypertension. Immunoreactivities of endothelin-1 and endothelial nitric oxide synthase were observed significantly less frequently, together with significant reduction of right ventricular overload and medial thickening in rats treated with E4021 than in the control with monocrotaline on day 28. The levels of plasma endothelin-1 and serum nitrite and nitrate were significantly lower in rats that received E4021 than in the control with monocrotaline. Oral administration of E4021 modulates endogenous immunoreactivities of endothelin-1 and endothelial nitric oxide synthase with the improvement or right ventricular overload and medial thickening.