Design–based stereological estimation of the total number of cardiac myocytes in histological sections

Abstract

Counting the total number of cardiac myocytes has not previously been possible in ordinary histological sections using light microscopy (LM) due to difficulties in defining the myocyte borders properly. To describe a method by which the total number of cardiac myocytes is estimated in LM sections using design-based stereology. From formalin-fixed left rat ventricles (LV) isotropic uniformly random sections were cut. The total number of myocyte nuclei per LV was estimated using the optical disector. Two-microm-thick serial paraffin sections were stained with antibodies against cadherin and type IV collagen to visualise the intercalated discs and the myocyte membranes, respectively. Using the physical disector in "local vertical windows" of the serial sections, the average number of nuclei per myocyte was estimated. The total number of myocyte nuclei in LV was 34.1 x 10(6) (0.08) (mean (coefficient of variation)), and the mean number of nuclei per myocyte 1.85 (0.03). Combining these estimates the total number of myocytes in LV was calculated to be 18.5 x 10(6) (0.09). This new method is applicable to a range of experiments focusing on myocyte proliferation and death.