Application of stereological analysis of cell volume to isolated myocytes in culture with and without adrenergic innervation

To comprehend structural changes in cardiac myocytes accompanying adrenergic innervation, it is essential that a three dimensional analysis be performed. To date, biological studies which utilize stereological methods have been limited to cells in tissue and in organs. Our laboratory has utilized current stereological techniques for measuring absolute volumes of individual myocytes in primary culture. Cell volumes are calculated for two distinct groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons (Figure 1).Cardiac myocytes are cultured from the ventricular apices of newborn rats. Cells are plated directly onto tissue culture dishes with or without preplated explants from the paravertebral thoracolumbar sympathetic chain. On day four cultures are photographed and marked for one-to-one cell location. Following conventional fixation and embeddment in eponate-12, the cells are relocated and mounted for microtomy. The cells are completely sectioned at 120nm in their parallel orientation to the surface of the dish (Figure 2). Serial sections are collected on formvar coated slotted grids and are recorded in sequence.