A method for three-dimensional quantification of vascular smooth muscle orientation: application in viable murine carotid arteries

When studying in vivo arterial mechanical behaviour using constitutive models, smooth muscle cells (SMCs) should be considered, while they play an important role in regulating arterial vessel tone. Current constitutive models assume a strictly circumferential SMC orientation, without any dispersion. We hypothesised that SMC orientation would show considerable dispersion in three dimensions and that helical dispersion would be greater than transversal dispersion. To test these hypotheses, we developed a method to quantify the 3D orientation of arterial SMCs. Fluorescently labelled SMC nuclei of left and right carotid arteries of ten mice were imaged using two-photon laser scanning microscopy. Arteries were imaged at a range of luminal pressures. 3D image processing was used to identify individual nuclei and their orientations. SMCs showed to be arranged in two distinct layers. Orientations were quantified by fitting a Bingham distribution to the observed orientations. As hypothesised, orientation dispersion was much larger helically than transversally. With increasing luminal pressure, transversal dispersion decreased significantly, whereas helical dispersion remained unaltered. Additionally, SMC orientations showed a statistically significant (p < 0.05) mean right-handed helix angle in both left and right arteries and in both layers, which is a relevant finding from a developmental biology perspective. In conclusion, vascular SMC orientation (1) can be quantified in 3D; (2) shows considerable dispersion, predominantly in the helical direction; and (3) has a distinct right-handed helical component in both left and right carotid arteries. The obtained quantitative distribution data are instrumental for constitutive modelling of the artery wall and illustrate the merit of our method.