A Simple Method for Normalization of Aortic Contractility

Abstract

Vascular contractile function changes in proliferative vascular diseases, e.g. atherosclerosis, and is documented using isolated blood vessels; yet, many laboratories differ in their approach to quantification. Some use raw values (e.g., mg, mN); others use a "percentage of control agonist" approach; and others normalize by blood vessel characteristic, e.g. length, mass, etc. A lack of uniformity limits direct comparison of contractility outcomes. To address this limitation, we developed a simple 2-step normalization method: (1) measure blood vessel segment length (mm), area (mm2) and calculate volume (mm3); then, (2) normalize isometric contraction (mN) by segment length and volume. Normalized aortic contractions but not raw values were statistically different between normal chow and high-fat diet-fed mice, supporting the practical utility and general applicability of normalization. It is recommended that aortic contractions be normalized to segment length and/or volume to reduce variability, enhance efficiency, and to foster universal comparisons across isometric myography platforms, laboratories, and experimental settings.

Keywords: Aorta; Blood vessel; Isometric contraction; Myography; Phenotypic switch; Vascular smooth muscle.

Figure 1.

Images of method for aortic parameter data acquisition. A) Digital images of blood vessel segment length (mm) acquired with digital mini-microscope camera. B) Digital image of blood vessel cross-section over-laid with digital outlines of outer and inner circumferences (NIH ImageJ). The outer area (OA) minus the inner area (IA; lumen) is the segment cross-sectional area (mm²). C) Calculation of blood vessel segment volume where, volume = length (mm) x cross-section area (mm²) in mm³

Figure 2.

Normalization of vascular contractility data. Contraction data (Raw, mN) and data normalized by aorta cross-sectional area (mN/mm²), length (mN/mm), and volume (mN/mm³) were plotted. To generate tension data, two aortic segments were exposed to contractile agents: A) phenylephrine (PE, 1 nM-10 μM); and, B) high potassium (HI K⁺; 100 mM) buffer; in a horizontal pin (DMT; 1, filled symbols), or a vertical wire (2, open symbols) organ bath system. After experimentation, length and cross-sectional area of formalin-fixed blood vessel segments were measured and the segment volume calculated (volume = length x cross-section area; see Fig. 1).

Figure 3.

Normalization of vascular contractility of aortas from C57BL/6 mice fed normal chow (NC; n=9 mice) or high fat diet (HFD; n=10 mice) for 12 weeks. Phenylephrine-induced raw contraction data (A, mN) and raw data normalized by aorta length (B, mN/mm), x-sec area (C, mN/mm²), and volume (D, mN/mm³). Values = mean ± SE. n = 9 or 10 aortas (1 aorta per 1 mouse). *, P<0.05 between NC and HFD.

Figure 4.

Normalization of vascular contractility of aortas from Sprague-Dawley wild type (WT; n=5 rats) and LDLR-KO rats (n=6 rats) fed normal chow for 1 year. Phenylephrine-induced raw contraction data (A, mN) and raw data normalized by aorta length (B, mN/mm), x-sec area (C, mN/mm²), and volume (D, mN/mm³). Values = mean ± SE. n = 5 or 6 aorta (1 aorta per rat). *, P<0.05 between WT and LDLR-KO rats.

Figure 5.

Reproducibility of aorta parameter data acquisition: intra-assay variation. The same images of a blood vessel lengthwise and in cross-section (ring) were repeatedly examined for length and cross-sectional area (x-sec area), respectively, as described above using ImageJ a total of 10 times by two different observers. Blood vessel volume (mm³) was calculated by multiplying length (mm) times x-sec area (mm²). Values = mean ± SD.

Figure 6.

Reproducibility of aorta parameter data acquisition: inter-assay variation. A) Aorta cross sectional area (mm²) measured in five separate aortas independently by two users (User 1; User 2). B) Aorta segment length (mm) measured independently by two different users in five separate aortas. C) Volume (mm³) calculated five times by two users. D) Aortic volume calculated from image analysis compared with volume derived from segment weight and aorta density (0.86 mg/L)¹. Values = mean ± SD.

Figure 7.

Intra-aortic variation in cross-sectional area. A single thoracic aorta segment was cut into five cross-sections (“donut”) and each cross-section was imaged and the area measured by two independent users (User 1; User 2). Values = mean ± SD.

Figure 8.

Resiliency of aorta parameter data acquisition over time. Comparison of aorta volumes quantified after formalin fixation of the blood vessel. A) Aorta volume measured 1 week after formalin fixation. B) Aorta volume measured 1 month after formalin fixation. C) Aorta volume measured 2 months after formalin fixation. Values = mean ± SD.