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

Background Counting the total number of cardiac myocytes has not previously been possible in ordinary histological sections using light microscopy (LM) due to difficulties in de. ning the myocyte borders properly. Aim To describe a method by which the total number of cardiac myocytes is estimated in LM sections using design-based stereology. Materials and methods 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-mu m-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. Results 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). Conclusion This new method is applicable to a range of experiments focusing on myocyte proliferation and death.