Vascular Function Optimization Protocol

C57 Black 4 mice (6–10 weeks) were killed by CO₂ narcosis and aorta removed, cleaned of connective tissue and cut into 1.5 mm rings before being mounted in Mulvany-Halpern myograph organ baths containing a physiological salt solution (PSS), as we have described previously^{30 (link)}. In order to optimize the stability of vascular function over the 8-h time course, diclofenac (1 µM) and cycloheximide (1 µM) were added to the PSS to block vasoactive prostanoids and induction of vasoactive genes (e.g., NO synthase) respectively. Vessels were contracted with an EC₈₀ concentration of U46619 (10 nM). Once a stable baseline was obtained, sildenafil (10 µM), nanoMIL-89 (10 µg/ml), or Sil@nanoMIL-89 (10 µg/ml) was added to the PSS, and vascular tone monitored for 8 h. Responses in vessels incubated in PSS served as controls. Force was recorded via a PowerLab/800 (AD Instruments Ltd., UK) and analysed using Chart 6.0 acquisition system (AD Instruments Ltd., UK). All studies using animals or animal tissues/organs were conducted in accordance with UK Home Office Animals (Scientific Procedures) Act 1986.