Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

被引:91
作者
Bensley, Jonathan Guy [1 ]
De Matteo, Robert [1 ]
Harding, Richard [1 ]
Black, Mary Jane [1 ]
机构
[1] Monash Univ, Sch Biomed Sci, Dept Anat & Dev Biol, Clayton, Vic 3800, Australia
基金
英国医学研究理事会;
关键词
CARDIAC-HYPERTROPHY; MYOCYTES; FETAL; HEART; NUMBER; RAT; HYPERPLASIA; CELLS;
D O I
10.1038/srep23756
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 mu m) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4', 6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2-10 mu m) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.
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页数:10
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