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Adhesion and transmigration of leukocytes into arterial walls occurs after vascular injury and may play a role in the development of atherosclerosis and restenosis. This protocol presents a simple, rapid method for quantifying leukocyte adhesion to artery segments ex vivo. The procedure involves isolating leukocytes from rabbit whole blood and labelling with the gamma-emitting isotope 51Cr. Labelled leukocytes are added to open rings of subclavian artery taken from the same rabbit. After gamma counting, percentage leukocyte adhesion can be calculated with reference to a sample containing a quantity of labelled leukocytes equivalent to that which was added to the artery. Leukocyte adhesion was increased by L-NAME, thrombin and increasing incubation time and decreased by low temperatures. In addition, leukocyte adhesion was found to be increased following a vascular stretch injury performed in vitro. This protocol offers a number of advantages: the rapidity of the leukocyte isolation and labelling; the small quantity of leukocytes required; the ability to use autologous leukocytes; the applicability to whole arteries and arteries injured in vitro or in vivo, allowing the effects of vascular injury on leukocyte adhesion to be studied.

Many of the currently available techniques for quantifying leukocyte adhesion require monolayers of cells and are therefore unsuitable for use in ex vivo arterial tissue. Here we describe a rapid method to measure adhesion of leukocytes to intact artery strips and to determine the effect of artery injury on adhesiveness of leukocytes with and without activation. Leukocytes were isolated from rabbit blood, labelled with 51Cr, and added to the luminal face of the left and right subclavian arteries derived from the same animal. In some experiments the endothelium was removed before addition of leukocytes and in another series of experiments the artery was injured by inflating a balloon catheter within the lumen in vitro before leukocyte addition. After washing, the adhesion of labelled leukocytes was quantified by gamma counting. To determine localization of the leukocytes, some arteries were fixed in situ and examined microscopically, with confirmation of leukocyte identification by enzyme cytochemistry. The adhesion of leukocytes increased progressively during 60 min and was inhibited by reducing the temperature to 4 degrees C. Adhesion was increased by the nitric oxide synthase inhibitor L-NAME. Stretching the artery wall in vitro using a balloon catheter increased leukocyte adhesion within 1 h after injury. In contrast, this did not occur following simple arterial denudation. Histological examination of stained en face preparations and transverse sections of the subclavian arteries revealed loosely adherent granulocytic leukocytes on the endothelial surface. This technique is straightforward and allows accurate and rapid measurement of autologous leukocyte adhesion to normal and pathologically altered arteries ex vivo.

Ventricular arrhythmias were studied in rat isolated hearts subjected to coronary artery occlusion and reperfusion. Free radicals in the perfusate were detected by continuous flow luminol-enhanced chemiluminescence. Administration of purine (2.3 mM) and xanthine oxidase (0.12 U ml-1 min-1) did not significantly modify the severity of reperfusion-induced arrhythmias but did generate free radicals. No free radical generation was detected during the period of coronary artery occlusion or reperfusion. Superoxide dismutase (SOD) 20-80 U ml-1 did not alter the severity of reperfusion arrhythmias but, in the presence of 80 U ml-1 SOD, occlusion-induced arrhythmias were augmented. SOD did not produce any effect on haemodynamics at the concentrations tested. Ventricular arrhythmias and cardiac haemodynamics were also not significantly changed by the combination of scavengers, SOD (10 U ml-1), catalase (100 U ml-1) and mannitol (20 mM). These data suggest that the superoxide free radical is unlikely to be the primary cause of reperfusion induced arrhythmias in rat isolated hearts subjected to regional ischaemia.

The effects of a range of free-radical scavenging drugs on luminol-enhanced chemiluminescence (CL) generated by porcine leukocytes, following activation by two nonreceptor-mediated stimulants, phorbol myristate acetate (PMA; a protein kinase activator) and ionomycin (a cation ionophore), and by xanthine plus xanthine oxidase (X-XO), have been examined. Superoxide dismutase (0.1 units/mL) and catalase (50 units/mL) inhibited X-XO, but they were ineffective in leukocyte suspensions except at concentrations 500 times and 20 times higher. Sodium azide (10(-5) to 10(-3) M) caused a marked inhibition in CL production in activated leukocytes, but not of X-XO CL. The antioxidants, glutathione (10(-3) M) and L-ascorbic acid (10(-3) M) were ineffective in activated leukocytes, but caused total inhibition of X-XO-induced CL. Mannitol (100 mM) had no effect on chemiluminescence in either system. Captopril (10(-3) M) produced an inhibition of CL in both systems and this inhibition was significantly modified by pH. Thus, the present study has established a standard screening procedure for the assessment of free-radical scavenging activity using activated porcine leukocytes and xanthine-xanthine oxidase.

Studies on such injury processes as atherosclerosis, angioplasty, and restenosis, have shown an impairment of relaxations mediated by endothelium-derived relaxing factor (EDRF). Increasingly, the rabbit carotid artery is being used as the vessel of choice in such studies, but a definite protocol for the assessment of endothelial dysfunction or denudation has not been developed. Using isolated carotid artery rings, we have obtained reproducible dose-response curves in endothelially intact and denuded vessels from normally fed rabbits to a variety of vasoconstrictors and endothelium-dependent and -independent vasodilators. Endothelium-dependent vasodilators (acetylcholine (1 x 10(-8)-1 x 10(-6) M), carbachol (1 x 10(-8)-5 x 10(-6) M), substance P (0.01-100 nM), and A23187 (1 x 10(-8)-1 x 10(-7) M) relaxed the arteries in a concentration-dependent manner but produced no relaxation in denuded vessels. Endothelium-independent nitric oxide (NO) donors [Sin-1 (1 x 10(-8)-1 x 10(-6) M)] and sodium nitroprusside (1 x 10(-8)-1 x 10(-6) M)) relaxed both intact and denuded vessels to a similar degree (slight augmentation of the relaxation induced in denuded vessels was not significant), demonstrating that denuded vessels did not have an impaired reactivity to NO. Concentration response curves to the vasoconstrictors [5-HT (1 x 10(-7)-1 x 10(-4) M) and KCl (15-60 mM)] were produced in intact vessels and it was shown that similar contraction was produced by 1 x 10(-6) M 5-HT in intact and denuded vessels. This indicated that the vessels retained contractile ability following denudation.(ABSTRACT TRUNCATED AT 250 WORDS)