Three-dimensional vascular smooth muscle orientation as quantitatively assessed by multiphoton microscopy: mouse carotid arteries do show a helix.

Smooth muscle cells (SMCs) play a pivotal role in regulating vascular tone in arteries, and are therefore an essential part of constitutive models of the artery wall. In the present study, we developed a method to quantify in 3D the orientation of SMCs in the intact artery wall. We stained cell nuclei in excised mouse carotid arteries mounted between micropipettes and imaged these in 3D using two-photon laser scanning microscopy. A clustering method was used to identify individual nuclei. Orientations of these nuclei (as a representative of the SMC orientations) were found by calculating the inertia matrix eigenvectors. Subsequently, SMC locations and orientations were converted to cylindrical and spherical coordinate systems, respectively. We found SMCs to be arranged in two distinct layers. For each of these layers, SMC orientations were described by a Bingham distribution. Distributions showed a statistically significant helical and transversal angular component in both inner and outer layers. In conclusion, this study demonstrates that SMC orientation can be quantified in 3D, and shows a distinct helical as well as transversal orientation. The acquired distribution data are essential to improve current constitutive models of the artery wall, by describing physiological SMC orientation and dispersion.