Vascular Ring Preparation and Pharmacological Studies

The rats were anesthetized with pentobarbital (60 mg/kg, i.p.). The descending thoracic aorta was excised by midline incision, cleaned of fat and connective tissues, with care taken not to stretch the vessel excessively or to disturb the luminal surface of the rings, to ensure the integrity of the endothelium. The aorta was then cut into small rings (3-5 mm in width) and suspended between two wire stirrups in a jacketed organ bath containing 50 ml of normal Krebs physiological solution of the following composition (mM): NaCl 118.2, KCl 4.7, CaCl₂·2H₂O 2.5, KH₂PO₄ 1.2, MgCl₂ 1.2, glucose 11.7. The bathing solution was bubbled continuously with a mixture of 95% oxygen and 5% carbon dioxide at 37°C. The rings were suspended with a resting tension of 0.8 g for at least 60 min then reduced to 0.6 g with constant changing of Krebs solution (every 15 min) to prevent accumulation of metabolites that could otherwise lead to misinterpretation of results [10]. The isometric tension was recorded by the force-displacement transducer (Model 7004; Ugo Basil Varese, Italy) connected to Data capsule Model 17400 for the isometric contractions. The rings were then progressively stretched to an optimal tension of 1 g and allowed to equilibrate for 60 min. Following the equilibration period, the aortic rings were allowed to achieve maximal tension by repeated exposure (each for 5 min) to isotonic potassium chloride solution (high K⁺ 60 mM). The rings that showed vasoconstriction response induced by phenylephrine (PE) (10^–7 M) were studied. PE was then washed out, and the tension was returned to the baseline levels before the administration of the drugs. The aortic ring was denuded to remove the endothelial layer in some preparations by inserting a pair of forceps into the lumen of each artery and gently rotating it. The viability of each aortic ring was validated by pre-contraction of PE (10^–7 M) and relaxed by ACh (10^–5 M) just before the experiment commenced. Relaxation of ≥ 70% indicated the presence of a functional or intact endothelial layer while the lack of relaxation indicated the successful removal of the layer.