YI 1.7 Transmural Quantification of Murine Vascular Smooth Muscle Cell Density Distribution from 3D Microscopy Images

Abstract

Purpose

Investigating the biomechanical role of smooth muscle cells (SMCs) in arteries requires knowledge of their structural distributions. Compared to histology, 3D microscopy offers non-destructive ex vivo imaging under realistic conditions [1]. Robust 3D segmentation of SMCs, however, is challenging. We propose a method for automatic SMC quantification, and assessed its potential using a murine SMC apoptosis model.

Methods

After euthanasia, carotid arteries (control and with induced SMC apoptosis: SM22α-hDTR [2]) were excised and mounted between micropipettes (Figure A). Nuclei were stained with SYTO41. Arteries were imaged using two-photon microscopy [1], while stretched to in vivo length and pressurised to 100 mmHg (Figure B). Image stacks were processed as follows: 1) deconvolution; 2) nuclei segmentation using vesselness filtering [3,4] (Figure C); 3) cylindrical coordinate system identification; 4) splitting of coincident nuclei, based on cores defined from groups of neighbouring voxels with similar orientations [3] (Figure D and E); 5) cylindrical coordinate system re-identification; and 6) cell density-distribution quantification (Figure F). Segmentation performance was assessed by comparing with manual cell counts.

Results

Figure E demonstrates the method’s ability to split undersegmented coinciding nuclei. Cell counts were lower in SM22α-hDTR than in control; algorithm-derived counts were comparable to manual (Figure F). The control sample showed multiple SMC layers, while the SM22α-hDTR sample showed a single SMC layer (Figure F), which was confirmed visually.

Conclusion

We developed a precise tool to quantify SMC distributions in ex vivo murine arteries, to facilitate quantitative modelling of SMC biomechanics. We intend to expand the current approach to address cell orientation, shape, and size.