A novel set-up for the ex vivo analysis of mechanical properties of mouse aortic segments stretched at physiological pressure and frequency

Abstract

Key points: Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats min^-1 ). Active vessel tone was shown to have surprisingly large effects on isobaric stiffness. The effect of structural vessel wall alterations was confirmed using a genetic mouse model. This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics.

Abstract: Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min^-1 . The technique enables us to derive pressure-diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10 Hz), the Peterson modulus (E_P ; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80-120 mmHg to 100-140 mmHg, while, at normal pressure, E_P increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening.

Keywords: VSMC tone; arterial stiffness; basal nitric oxide; cyclic stretch.

Figure 1. Schematic diagram of the ROTSAC

The aortic segment was mounted between two metal hooks in an organ bath. (1) A current source was used to control the distension force and clamp frequency (10 Hz) of the force–length transducer. Force and displacement were measured by the transducer (2) and acquired at 1 kHz by a PowerLab DAQ device (3). Diameter, length and force were used to calculate the pressure that would exist in an equilibrated vessel segment with the given dimensions and wall stress (4).

Figure 2. Bland–Altman analysis of geometrical parameters

Comparison between effective diameter (D _eff) (A and B) and length (L _eff) (C and D) and the calculated parameters (D _calc and L _calc) derived from the displacement of the needle and the diameter–length relationship, respectively. The dashed lines represent the bias and 95% limits of agreement.

Figure 3. Processing of the data obtained from a compliant and stiff vessel segment

The graphs show examples of a WT aortic segment, either fully contracted using 1 μm PE and 300 μm l‐NAME (stiff), or in Krebs–Ringer solution (compliant). Pressure (continuous line) and diameter (dashed line) are plotted against time (A). As shown, segments were stretched between 80 and 120 mmHg at a stretch frequency of 10 Hz. When pressure and diameter are plotted against each other, a typical hysteresis loop was obtained (B). Compliant (black) and stiff (grey) vessel segments are shown to illustrate the differences in distension with similar pressure changes (A) and the difference in slope of the pressure–diameter loops (B).

Figure 4. Isobaric vessel parameters under conditions with different VSMC tone and pressure

Diastolic diameter (D ₀) (A), relative distension (B), compliance (C) and Peterson modulus (E _P) (D) were measured at physiological stretch frequency (10 Hz), either at normal pressure (80–120 mmHg) or at high pressure (100–140 mmHg). KR, Krebs–Ringer; PE, 1 μm phenylephrine; PE+LN, 1 μm phenylephrine + 300 μm l‐NAME. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 (effect of VSMC tone, vs KR), ^††† P < 0.001 (effect of pressure, vs. 80‐120 mmHg), two‐way ANOVA with repeated measures for pressure factor, Bonferroni post hoc test (n = 5).

Figure 5. Comparison between WT mice (n = 5) and mice with a heterozygous mutation of the Fbn1 gene (n = 4)

The aorta was dilated, as indicated by increased diastolic diameter (D ₀) (A). Relative distension (B) and compliance (C) were significantly lower and the Peterson modulus (E _P) was significantly increased (D). Measurements were done at physiological pressure range (80–120 mmHg) and stretch frequency (10 Hz). Line and error bars represent median and interquartile range, respectively. ^* P < 0.05, Mann–Whitney U test.

Figure 6. Geometrical and biomechanical parameters of eNOS^−/− mice aortic segments

Increased aortic stiffness was confirmed in eNOS^−/− mice (n = 5). Although diastolic diameter (D ₀) was similar to wild‐type (WT) mice (n = 5) (A), relative distension (B), compliance (C) and the Peterson modulus (E _P) (D) were significantly different. Measurements were done at a physiological pressure range (80–120 mmHg) and stretch frequency (10 Hz). Line and error bars represent median and interquartile range, respectively. ^** P < 0.01, Mann–Whitney U test.

Figure 7. Effect of repetitive stretch on elastin fibre integrity and E_P

Orcein‐stained section of aortic segments of WT mice after being mounted in the ROTSAC set‐up at physiological pressure. The integrity of the elastin fibres after 4 h of stretching at 10 Hz (A) was similar to when no stretch was applied (B). E _P at 80–120 mmHg was unchanged (P = 0.97, paired t‐test) (C) after 4 h of stretching in a group of randomly selected mice with different baseline values of E _P (WT, n = 3; eNOS^−/−, n = 5; P = 0.97, paired t test). Scale bar: 100 μm. [Colour figure can be viewed at wileyonlinelibrary.com]