Myography

EDD and endothelium-independent dilation were determined ex vivo in isolated carotid arteries as previously described (Durrant et al. 2009 (link); Lesniewski et al. 2009 (link); Rippe et al.). Briefly, mice were anesthetized using isoflurane and euthanized by exsanguination via cardiac puncture. The carotid arteries were carefully excised, cannulated onto glass micropipettes and secured with nylon (11-0) suture in myograph chambers (DMT Inc.) containing buffered physiological saline solutions. The arteries were pressurized to 50 mmHg at 37° C and were allowed to equilibrate for 1 h. After submaximal preconstriction with phenylephrine (2 μmol/L), increases in luminal diameter in response to acetylcholine (ACh: 1 × 10⁻⁹ - 1 × 10⁻⁴ mol/L) with and without co-administration of the NO synthase inhibitor N-G-nitro-L-arginine methyl ester (L-NAME), (0.1 mmol/L, 30 min incubation), or the SOD mimetic, TEMPOL, (1 mmol/L, 60 min incubation), were determined. EDD also was determined in the presence of the NADPH oxidase inhibitor, apocynin, (1 mmol/L, 60 min incubation) and the exogenous BH₄ donor, sepiapterin, (1 mmol/L, 60 min incubation). Endothelium-independent dilation was determined by vasodilation in response to sodium nitroprusside (SNP: 1 × 10⁻¹⁰ - 1 × 10⁻⁴ mol/L). All dose response data are presented on a percent basis. Preconstriction was calculated as a percentage of maximal diameter according to the following formula:
$\text{Preconstriction}(\%)=({\mathrm{D}}_{\text{m}}-{\mathrm{D}}_{\text{b}})∕{\mathrm{D}}_{\text{m}}\times 100$
Because of differences in maximal carotid artery diameter between young and old animals, vasodilator responses were recorded as actual diameters expressed as a percentage of maximal response according to the following formula:
$\text{Relaxation}(\%)=({\mathrm{D}}_{\text{s}}-{\mathrm{D}}_{\text{b}})∕({\mathrm{D}}_{\text{m}}-{\mathrm{D}}_{\text{b}})\times 100$
Where D_m is maximal inner diameter at 50 mmHg, D_s is the steady-state inner diameter recorded after the addition of drug, and D_b is the steady-state inner diameter following preconstriction before the first addition of drug.
NO-dependent dilation was determined from the maximal EDD in the absence or presence of L-NAME according to the following formula:
$\text{NO-dependent dilation}(\%)={\text{Maximal dilation}}_{\mathrm{ACh}+L-\text{NAME}}-{\text{Maximum dilation}}_{\mathrm{ACh}}$