Isometric force measurement in mouse cerebral arteries: Establishing reference values and characterizing functional consequences of endothelial nitric oxide synthase knock-out in the basilar artery

Genetically modified mice are increasingly important tools in biomedical research. The increasing number of transgenic mouse strains available has raised the need to develop adequate tools for characterizing the functional consequences of overexpressing, knocking out, or inserting a gene. We have, therefore, adapted the methodology of isometric force measurement to study the vasoreactivity of mouse cerebral arteries in vitro. Ring segments (approximately 1 mm in length) of the basilar artery (BA) were obtained from NMRI, C57/black6 (C57B6), endothelial nitric oxide synthetase knock-out (eNOS−/−) mice and the corresponding wild-type line (eNOS+/+). The segments were transferred into organ bathes filled with a modified Krebs-Henseleit solution maintained at 37 C, and continuously gassed with a mixture of 77%/17.5%/5.5% N₂/O₂/CO₂. Contractile responses were elicited by membrane depolarisation (equimolar substitution of NaCl by KCl), or cumulative application of U46619 (a thromboxane A2-analogue) or endothelin-1 (ET-1). Relaxation was induced by applying increasing concentrations of Na-nitroprusside (SNP) or 8-bromo dibutryl-cyclic GMP (8br-cGMP) on top of stable precontraction with U46619 (1E-7 M). Contraction is given in absolute terms (mN force). Relaxation is calculated in % decrease of precontraction. Values are given as mean±SD. In NMRI mice, different levels of resting tension (RT) upon membrane depolarisation (by increasing the K+ concentration)-induced contraction were studied. In segments with a RT >1.75 mN (2.0±0.2 mN), depolarisation-induced contraction was 1.9±0.6, somewhat bigger than with lower RT (1.4±0.2 mN; contraction, 1.7±0.4 mN). Thus, RT was set to 2 mN in further experiments. Contraction induced by high K+ Krebs solution was 2.3±0.6 in NMRI, 2.3±0.6 in C57B6, 2.6±0.8 in eNOS+/+, and in eNOS−/− 3.3±1.0 (p<0.05 vs all other strains). Additional results are listed in the table 1 below with ^†p<0.1 and *p<0.05 vs. eNOS+/+. The study presents for the first time data on vascular reactivity of mouse cerebral arteries employing the isometric force measurement technique. The results indicate pronounced differences in depolaristion- and receptor-induced contraction in different mouse strains. Furthermore, SNP enhanced relaxation in eNOS−/− mice significantly, probably indicating a hypersensitivity of the sGC due to the lack of NO release from the endothelial cells. Thus, this technique allows a powerful method to study in great detail the functional consequences of a given type of genetic engineering in mouse cerebral arteries.

Table 1

		NMRI	C57B6	eNOS+/+	eNOS−/−
U46619	pD2	7.14±0.35	7.27±0.39	7.14±0.28	7.04±0.28
	EmAX	3.8±1.3	2.5±1.3	2.6±0.7	2.5±1.2
ET-1	pD2	8.62±0.39	not tested	9.04±0.40	8.78±0.51
	EmAX	3.0±1.2	not tested	1.76±0.64	2.1±1.3
SNP	pD2	5.18±0.96	4.56±0.39	4.56±0.24	5.09±0.63†
	EmAX	48.5±11.8	67.0±13.3	60.1±11.4	83.3±11.1*
cGMP	pD2	4.78±0.38	4.53±0.20	4.52±0.14	4.43±0.17
	EmAX	66.2±15.1	73.9±13.1	77.5±17.0	77.4±16.0