FITC-labeled Albumin Research Articles

Pulmonary Microvascular Changes during Sepsis: Evaluation Using Intravital Videomicroscopy

A variety of pulmonary microvascular changes occur during sepsis. These include abnormal vascular reactivity, leukocyte sequestration, and leakage of protein into the alveoli. Based on intravital videomicroscopy we have developed a method to directly assess in vivo the changes that occur in the pulmonary microcirculation in a rat model of sepsis. Male Sprague–Dawley rats were assigned to control or sepsis groups. Sepsis was induced by cecal ligation and perforation. Twenty four hours later, rats were anesthetized, mechanically ventilated, and their lung prepared for intravital videomicroscopy. A specially designed transparent thoracic window was inserted into the chest wall. The dependent surface of the lung was superfused with saline solution and visualized with an inverted microscope. Vascular contractility, to phenylephrine, (PE) and hypoxia of small (15–25 μm in diameter) and medium (40–50 μm) arterioles was examined. Leukocyte traffic in the pulmonary microcirculation was studied after in vivo labeling of leukocytes with Rhodamine and visualized with fluorescence microscopy. Leak of albumin into the alveolar space was measured with FITC-labeled albumin and fluorescence microscopy. Both small and medium sized pulmonary arterioles in septic animals exhibited attenuated vascular contractility to phenylephrine, but only medium-sized arterioles displayed hypocontractility to hypoxia. Further, in septic animals there was an increase in both the number of stationary leukocytes in the pulmonary microcirculation and an increase in alveolar capillary protein leak. We conclude: (1) direct visualization of the pulmonary microvascular pressor response to hypoxia and PE in the rat is possible using this technique, (2) similar to previous in vitro studies with larger vessels, pulmonary arterioles have an attenuated contractile response to PE and hypoxia in sepsis, and (3) there is an increase in both the number of stationary leukocytes and protein leak into the alveolus in the lungs of septic animals.

Calcitonin stimulates lysosomal enzyme release and uptake in LLC-PK1 cells.

Renal tubular targeted hormones increase urinary excretion of a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG). To elucidate the mechanism of this event, the calcitonin effect on NAG handling by LLC-PK1 cells was examined. Calcitonin (1 nM to 1 microM), phorbol myristate (10 nM to 1 microM), and ionomycin (1 to 10 microM) promoted NAG release without any increase in lactate dehydrogenase release or any reduction of mitochondrial dehydrogenase activity. Treatment with 100 nM calphostin C or 50 microM KN-93 partially reversed the calcitonin effect on NAG release. Calcitonin promoted secretion of fluorescence ceramide, a reporter of protein transport from Golgi apparatus to cell surface. Calcitonin-stimulated NAG release was partially inhibited by 10 microg/ml brefeldin A, a blocker of protein transport through the Golgi apparatus. Calcitonin accelerated cellular uptake of exogenous NAG, which was inhibited by low temperature, 0.1 mM monodansyl cadaverine (receptor-mediated endocytosis inhibitor), and 10 mM mannose-6-phosphate. Furthermore, calcitonin promoted progression of intracellular membranes stained by a fluorescence membrane marker, styryl pyridinium dye, from cell periphery to perinuclear regions (commonly referred to as recycling vesicles) and increased dye release from preloaded cells. Fluorescence release from the cells pre-loaded with FITC-labeled NAG or albumin was also stimulated by calcitonin. These calcitonin effects on endocytotic and re-exocytotic pathways were inhibited by 100 nM cytochalasin D, 100 nM nocodazole, 0.1 to 1 microM bafilomycin A1, or 0.1 mM monodansyl cadaverine. Increased urinary NAG excretion has been considered to reflect renal tubular damage. However, it was demonstrated here that stimulation of secretory and recycling pathways may be an alternative mechanism for calcitonin-induced enzymuria, which will become a new indicator of renal tubular response to this hormone.

Vascular Permeability Increase as Induced by Histamine or Bradykinin is Enhanced by Advanced Glycation Endproducts (AGEs)

Advanced glycation endproducts (AGEs) may enhance vascular permeability in diabetic subjects. To test this hypothesis, AGEs were prepared in the presence of albumin (AGE-Alb). Control albumin (Alb) and AGE-Alb were then labeled with FITC (fluoresceinisothiocyanate) and injected i.v. into anesthetized hamsters at a dose of 7 mg/100 g B.W. Normal hamsters were given FITC-Alb or FITC-AGE-Alb and FITC-dextran. Vascular permeability changes were measured by direct intravital microscopy of the hamster cheek pouch preparations in fluorescent light and recorded as number of sites (=leaks) with extravasation of FITC-labeled albumin in postcapillary venules. No changes were seen during 1 hour after i.v. injection of FITC-Alb or FITC-AGE-Alb. Repeated local application of histamine 5·10 −6 M or bradykinin 5·10 −7 M to the cheek pouch for 5 min with 30-min intervals induced reversible increases in vascular permeability in all hamsters. Maximal number of leaks/cm 2 before and at 30 and 60 min after FITC-Alb-injection and histamine application was 257 ± 6 (SEM), 243 ± 6 and 231 ± 6 leaks/cm 2 in the FITC-Alb-group and 258 ± 6 (SEM), 302 ± 12 and 316 ± 11 leaks/cm 2 in the FITC-AGE-Alb-group, respectively, ( P < 0.05 at 30 and 60 min). Similar results were seen with bradykinin. Our conclusions showed that i.v.-injected AGEs augmented the histamine- and bradykinin-induced increase in vascular permeability by 34% and 46%.

Leakage responses to L-NAME differ with the fluorescent dye used to label albumin.

Nitric oxide synthase (NOS) inhibitors have been reported to increase as well as to decrease microvascular transport of macromolecules in a variety of models. This study was performed to determine whether the influence of NOS inhibition on albumin leakage was dependent on the fluorescent dyes used to label albumin. Albumin leakage was assessed in rat mesenteric venules during control conditions and after exposure to the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Albumin was labeled with any one of four dyes: FITC, sulforhodamine 101 [Texas Red (TR)], dichlorotriazinyl aminofluorescein (DTAF), or Oregon Green 514 (OG). Superfusion with L-NAME (10(-4) M) was accompanied by an increase in leakage of FITC-labeled albumin (n = 12) but not of albumin labeled with DTAF (n = 10), TR (n = 10), or OG (n = 4). In vessels perfused with both FITC- and TR-labeled albumin (n = 12), superfusion with L-NAME increased leakage of FITC- but not TR-labeled albumin. In conclusion, albumin leakage responses to L-NAME differ among various fluorescent dyes. Therefore, caution is advised in comparison of albumin leakage results that utilize different fluorescent dyes.

The Role of Microvascular Permeability in the Mechanism for Stunned Myocardium in Rats

The microvascular permeability of stunned myocardium in ratsin vivowas studied with FITC-labeled albumin (FITC–BSA). It was found that 15 and 20 min of myocardial ischemia followed by 1 hr of reperfusion resulted in myocardial stunning. The concentrations of FITC–BSA in myocardial tissue were 240.6 ± 7.8 (IS15) and 267.4 ± 7.9 (IS20) μg/g myocardium in ischemic groups, respectively, which were significantly higher than those in the control group (166.0 ± 7.9 μg/g myocardium;P< 0.01). In stunned groups, the concentrations were 224.8 ± 11.8 (MS15) and 241.7 ± 6.0 (MS20) μg/g myocardium, decreased from those in ischemic groups but still higher than those in control group by 35.4 and 45.6%, respectively. The more significant the concentration of FITC–BSA, the more serious the myocardial stunning. Electron microscopy revealed no significant vascular injury. The results suggest that the increase in microvascular permeability resulting from transient ischemia is functional and is involved in the pathogenesis of stunned myocardium.

Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

血清アルブミンを利用した薬物担体の腫よう集積性 アシル化による体内動態特性の改善

The tumor distribution and the disposition of serum albumin were investigated in mice bearing Sarcoma 180. The albumin labeled with fluoresceinisothiocyanate(FITC) were administered to the mice. The FITC-labeled albumin acylated with acid anhydrides, such as acetic anhydride, glutaric anhydride, maleic anhydride and succinic anhydride, were also administered to the mice in order to investigate the effect of chemical modification. The plasma concentration of each acylated albumin was significantly lower than that of the non-acylated albumin at 24 h after administration. The tissue distributions of the acylated albumin were also decreased compared to those of the non-acylated albumin. Especially, the accumulation of albumin in the liver and spleen was significantly reduced with the glutarylation and maleylation. Urinary excretion of albumin was significantly increased with the acylation because the degradation rates of the acylated albumins were much faster than that of non-acylated albumin. On the other hand, the acylated and non-acylated albumin were accumulated effectively in the tumor tissue in mice bearing Sarcoma 180. It was found that the tumor distribution of albumin was not impaired by the acylation. It was suggested, therefore, that the glutarylated and maleylated albumin were valuable for relative tumor-selectivity and might be utilized in the macromolecular carrier system of antitumor drugs.

Attenuating Effect of Antithrombin III on the Fibrinolytic Activation and Microvascular Derangement in Rat Gastric Mucosa

The roles for the fibrinolytic activation and disorder of coagulation in formation of gastric ulcer induced by microvascular derangement were investigated. The rat stomach was exposed and repeated electrical stimuli (RES) were applied on the small arterial wall close to the lesser curvature to induce mucosal microcirculatory disturbances. The level of tissue-type plasminogen activator (t-PA), a key enzyme for fibrinolytic activity, in the regional blood of the stomach was significantly elevated immediately after RES. At 5 min after RES, the leakage of FITC-labeled albumin and thrombus formation in the mucosal microvasculature were visually demonstrated by using an intravital microscopic system. At 30 min, hemorrhagic erosions and linear ulcers were observed in the gastric mucosa. Pretreatment with human antithrombin-III (AT-III) in the range of 0.1-10 U/kg dose-dependently attenuated both the fibrinolytic activation and microvascular alteration promoted by RES. Human AT-III also prevented RES-induced gastric mucosal injury. Thrombin inhibitory activity in the gastric vein decreased (69.0 +/- 2.1%) just after RES, and further reduced at 30 min (47.7 +/- 5.3%). The present study suggests a hypothesis that human AT-III has a preventive effect on the gastric mucosal hemorrhagic changes via attenuating the fibrinolytic activation and subsequent microcirculatory disturbances.

Oxyradical Generation from Leukocytes during Endotoxin-Induced Microcirculatory Disturbance in Rat Mesentery-Attenuating Effect of Cetraxate

By using the intravital microscope equipped with digital imaging processor, we investigated the granulocyte-mediated oxidative burst during the endotoxin-induced microvascular derangement in rat mesentery. The leukocyte behavior after the injection of acridine orange was detected by using a silicon-intensified target camera, the erythrocyte velocity was measured by using a high-speed video camera system, and the luminol-dependent photoemission was visualized by an ultrasensitive photon-counting camera in lipopolysaccharide (LPS)-treated micro-vascular beds. At 60 min after the LPS administration, a significant leakage of FITC-labeled albumin was observed along mesenteric venules under a fluorescence microscopy. The number of sticking leukocytes increased in association with the decrease in erythrocyte velocity after starting the LPS infusion. The luminol-dependent chemiluminescence in microvascular beds gradually increased over that recorded prior to LPS exposure and was fourfold higher 60 min after the start of LPS infusion. The distribution of the photoemission clearly corresponded to the venular endothelium, to which leukocytes adhered. In blood samples taken from the mesenteric vein at 60 min after the LPS administration, a decrease in the number of granulocytes and increases of total and individual chemiluminescence activities were observed. These results suggest that LPS induces oxidative burst from granulocytes on the venular endothelium. Cetraxate, an inhibitor of proteases including plasmin, significantly inhibited the leukocyte activation and prevented alterations in microvascular hemodynamics induced by LPS in vivo, whereas it had no effect on the LPS-induced oxyradical generation from adherent leukocytes in vitro. The present study demonstrates that proteases such as plasmin may play an important role in the pathogenesis of endotoxin-induced microvascular disturbances.

Immunofluorescent Studies of Urinary Casts

Immunofluorescent staining of urinary casts of patients with chronic glomerulonephritis or with acute renal failure was performed. Urinary and cast Tamm-Horsfall mucoproteins were purified by a modified McQueen's method. Rabbits were immunized by both of these materials to obtain antisera. The antigenic specificity of the prepared material was examined by Ouchterlony gel diffusion method and immunoelectrophoresis. Indirect and direct immunofluorescent staining methods were done using Tamm-Horsfall mucoprotein antiserum and FITC-GARG, FITC-labelled IgG, IgA, IgM, C3 and albumin. Hyaline casts of both chronic glomerulonephritis and acute renal failure demonstrated fluorescence with Tamm-Horsfall mucoprotein antiserum. The nature of the granular casts of chronic glomerulonephritis seemed to be different from those of acute renal failure because the granules of the former fluoresced for some serum protein fractions, but the latter fluoresced only for Tamm-Horsfall mucoprotein. The absence of fluorescence with epithelial casts for any serum protein fractions implied a different pathogenesis for epithelial casts as compared to granular casts, contrary to Lippman's assumption. A routine technique of immunofluorescent staining of urinary casts was developed. The interpretation of urinary casts should be more specific by this new technique.