Abstract
Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a “nucleus splitting” procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe–/– and control Apoe–/– mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed −26.6 ± 9.90% (p < 0.001) and −1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.
Highlights
Vascular smooth muscle cells (VSMCs) are a crucial component of blood vessels because they regulate extracellular matrix (ECM) production, maintain mechanical homeostasis in larger vessels, and enable autoregulation in smaller vessels (Owens, 1995; Brozovich et al, 2016)
VSMC density and distribution estimates obtained from histological cross sections are potentially disturbed, as this histological technique requires fixation and sectioning of the vessel
While histology is a useful tool for assessing VSMC content of vessels, it seems ill-suited for providing accurate, quantitative information about VSMC density and distributions, especially in murine vessels
Summary
Vascular smooth muscle cells (VSMCs) are a crucial component of blood vessels because they regulate extracellular matrix (ECM) production, maintain mechanical homeostasis in larger vessels, and enable autoregulation in smaller vessels (Owens, 1995; Brozovich et al, 2016). Changes in VSMC functionality, phenotype, and content, potentially lead to changes in diameter, wall thickness, structure, and mechanical properties of the vessel (Jaminon et al, 2019). Accurate information about a vessel’s VSMC content, orientation, and distribution under wellcontrolled conditions is indispensable to improve modeling of active biomechanics of the vessel wall, thereby helping to further our understanding of the role VSMC play in vascular remodeling. VSMC density and distribution estimates obtained from histological cross sections are potentially disturbed, as this histological technique requires fixation and sectioning of the vessel. Experience shows that it is difficult to control the orientation of smaller vessels fixed in paraffin wax for sectioning, making it nearly impossible to obtain accurate VSMC orientation data from histological cross sections of small vessels. While histology is a useful tool for assessing VSMC content of vessels, it seems ill-suited for providing accurate, quantitative information about VSMC density and distributions, especially in murine vessels
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