A comparative study of leukocyte adhesion to the endothelium of the thoracic aorta and left carotid artery in rats has been performed after administration of two hyperlipidemic diets for 15 days, proinflammatory agents (thrombin, lipopolysaccharide and zymosan activated serum) and plasma expanders [dextran, polyvinylpyrrolidone (PVP), rat albumin and several bovine albumins from different sources]. Leukocytes adhered to the endothelium were. demonstrated in surface preparations by esterase activity. Activation of circulating leukocytes was measured by nitroblue tetrazolium reduction and luminol enhanced chemiluminescence. Both hyperlipidemic diets produced, in all rats, more leukocyte adhesion in the aorta than in the carotid artery. All proinflammatory agents produced at 1 h, increases in leukocyte adhesion - which in all rats were greater in the carotid artery than in the aorta - and leukocyte activation, which was higher at 3 h than at 1 h. Dextran, PVP, bovine albumins 103700 and A-4503 at 18 h produced slight increases in leukocyte adhesion in the aorta but not in the carotid artery. Rat albumin and bovine albumin A-7906 determined an intense leukocyte adhesion at 18 h which was not preferential to either vessel. Adhesion produced by A-7906 was maximal at 12 h and partially inhibited by dexamethasone. This last albumin produced leukocyte activation at 3 h and was sequestered 5 min after administration, reaching normal values at 1 h. Albumins 103700 and A-4503 neither activated leukocytes nor were sequestered after administration.

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead(R) blue and red, 0.5 and 1 mu m particle size, were inadequate because they were insufficiently colored to be identified individually at 400 x. Fluoresbrite(TM) YG 0.25 and 0.50 mu m at doses of 0.2 and 2 x 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B-R (MbB) at 0.3 m1/100 g, red iron at 2 x 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite(TM) indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.

Endothelium is a monocellular layer very prone to mechanical injury when vessels are handled in vitro. Injury is due to 1) spontaneous vessel shortening and collapse after excision, which produces partial detachment of the endothelium from the matrix (Lewis et al., 1991), and 2) obligated manipulations such as the washing off of blood and periadventitial tissue removal. Apart from this type of mechanical damage, the use of solutions inadequate from a physiological point of view may also produce metabolic injury. Morrison et al. (1976, 1977) reported losses of endothelium in rabbit aorta when solutions without oxygen or nut~ents were utilized. In a previous work (Gabaldon and Capdevila, 1991), we studied the effects of different washing solutions and of several techniques for removal and cleaning of the rat aorta on endothelial integrity determined by silver staining of the interendothelial junctions. As prostacyclin is produced by both the endothelium and the medial layer of the aorta (Ts’Ao et al., 1979; Goldsmith, 1982; Boeynaems et al., 1985), loss of endothelial integrity in preparation may affect diffusion of the reaction product and alter the overall production of prostacyclin by the vessel. The aim of the present paper is to establish the effect of aortic processing on endothelial integrity and prostacyclin synthesis, utilizing I) different solutions for washing of the aorta; 2) different techniques for removal and cleaning of the aorta; and 3) mild in situ

Leukocyte adhesion and other injury parameters have been studied in the aortic endothelium of Sprague-Dawley rats in two situations: (1) spontaneous pathology in conventional rats with antibodies to Mycoplasma pulmonis and/or Kilham or Sendai viruses, and (2) intravascular coagulation by thrombin administration in SPF rats. Adhesion (esterase (+) leukocytes/mm2) in SPF rats was 8 +/- 5 (n = 12). Adhesion in 38% of the conventional rats was 54 +/- 27 (n = 8), half of them being non-analyzed and the rest having antibodies to M. pulmonis and/or Kilham rat virus. In 19 rats with antibodies to M. pulmonis and/or Kilham or Sendai viruses, AgNO3 and hematoxylin staining of the aortic endothelium showed an increase in leukocyte adhesion, and the presence of argyrophilic cells, stigmata and granularity - severe endothelial lesions being observed in some cases. Adhesion in rats after 0.25, 1, 3 and 6 h of thrombin administration (30 units/100 g) was not different from controls. Adhesion after 24 h was 108 +/- 53 (n = 10) and 60 +/- 59 (n = 10), and 22 +/- 20 (n = 10) in rats treated with thrombin plus heparin or hirudin, respectively. Thrombin produced endothelial lesions at all times studied, and these included membrane blebs, platelet and erythrocyte adhesion and alterations in the pattern of endothelial esterase activity.

Assessment of endothelial integrity is an obligatory step in many pharmacological studies. Integrity of endothelium is affected by manipulations performed during the removal and cleaning of the vessel and by some of the silver-staining techniques utilized for demonstrating interendothelial junctions. When aortas were cleaned of periadventitial tissue in cold Tris-saline (once separated from the animal) by untrained personnel, only 45% of the endothelium was preserved. When cleaning was performed in situ by trained personnel while flushing with cold Krebs-Ringer-6% albumin, over 95% was left intact. AgNO3-staining performed before fixation produced a 50% loss of endothelium when using NH4Br and (NH4)2S as developers. AgNO3-staining performed after fixation produced over 95% recuperation of endothelium when 2% glutaraldehyde, 150 mM NaCl, 40 mM phosphate buffer, pH 7.4, were utilized as initial fixative, NH4Br and (NH4)2S being equally effective as developers. Chloride ions were necessary to intensify silver lines. Several patterns of deendothelization were produced by mechanical and chemical injury with saponin, NH4Br and (NH4)2S. In all cases, hematoxylin staining was employed as an auxiliary technique to interpret images of injured endothelium. Presence of albumin protected the endothelium from mechanical damage.

Comments on techniques for characterizing leukocytes adhered to the aortic endothelium of the rat are given. Alpha-naphthyl acetate esterase positive leukocytes were studied by optical microscopy of en face intima-media preparations. Results indicate 1) 1% paraformaldehyde-2% glutaraldehyde is a better fixative than formalin-calcium or 4% paraformaldehyde with or without 1.5 mM CaCl2; the latter produces distortion of leukocytes, endothelial desquamation and enzymate inhibition, 2) washing the aorta with phosphate-buffered saline for 90 sec prior to fixation-perfusion produces a notable decrease in the number of leukocytes adhered, 3) diazotized parasaniline is better than fast blue RR salt as coupling agent in the esterase reaction, and 4) counterstaining with 1% methyl green for 1 min, before or after the esterase reaction, is not adequate because of limited contrast and the heavy staining of smooth muscle. Counterstaining with Gill's hematoxylin No. 3 for 90 sec is adequate only when done before the esterase reaction. Inhibition of endothelial esterase activity by hematoxylin decreases background, favors contrast of adhered leukocytes and makes it possible to observe nucleus-cytoplasm relations.

Adhesion of leukocytes to the aortic endothelium was studied in specific pathogen-free (SPF) and conventional rats and in SPF rats with diet-induced hypercholesterolemia. Nonspecific esterase activity with alpha-naphthyl acetate as substrate was used to characterize the adhered cells. Phagocytic activity was determined by injecting i.v. 0.1-0.4 ml/100 g doses of Monastral blue B (MbB). Adhesion in SPF rats was 8 +/- 4 esterase (+) cells/mm2. Adhesion in conventional rats was of the same order except in 2 cases with antibodies to Mycoplasma pulmonis and Kilham rat virus, where adhesion was 44 and 68 esterase (+) cells/mm2, respectively. For all MbB doses studied, phagocytic activity arose in a percentage of the adherent cells, ranging from 5 to 85%. Rats fed the hyperlipidic diet for 15 days developed severe hypercholesterolemia and adhesion was drastically increased to 200-700 esterase (+) cells/mm2. Results indicate that: (1) spontaneous pathology in rats may produce an increased adhesion of leukocytes to the endothelium, and (2) phagocytic activity is only expressed in a fraction of the esterase (+) cells adhered to the endothelium.