2.2. Animals and vessel preparation

Male Sprague Dawley rats (200–250 g) were purchased from the National Laboratory Animal Centre, Mahidol University, Nakhorn Pathom, Salaya, Thailand, kept under 12 h each light:dark cycle, at 22 ± 1 °C and allowed free access to standard food and water. All experimental protocols were approved by Naresuan University Animal Care and Use Committee (NUACUC, Naresuan University, Phitsanulok, Thailand, approval reference: NU-AE570619). Rats were anesthetized with 50 mg/kg sodium pentobarbital (IP) and supplemented as needed. Afterwards, thoracic aorta, primary branch of mesenteric artery and tail artery were excised and cleaned of connective tissue. Aorta and mesenteric artery were cut into rings 2–5 mm in length and suspended in water-jacketed glass baths via stainless steel loops connected to tension transducers. Rings were bathed in Krebs’ solution (mM): NaCl, 122; KCl, 5; [N-(2-hydroxyethyl) piperazine N′-(2-ethanesulfonic acid)] HEPES, 10; KH₂PO₄, 0.5; NaH₂PO₄, 0.5; MgCl2, 1; glucose, 11; and CaCl2, 1.8 (pH 7.3), at 37 °C and bubbled with air. The vessel rings were allowed to equilibrate for 1 h at a resting tension of 1 g.^22,23 In some rings, endothelium was mechanically denuded via a luminal wire. Following equilibration, each ring was tested for contractile viability by applying 10⁻⁵ M phenylephrine (PE), then endothelium function tested using 10⁻⁵ M acetylcholine (ACh) to induce more than 70% relaxation of the pre-contracted vessels.