A practical three-dimensional reconstruction method to measure the volume of the sexually-dimorphic central nucleus of the medial preoptic area (MPOC) of the rat hypothalamus.
Published estimates of the volume of the sexually-dimorphic central nucleus of the medial preoptic area (MPOC) have been quite variable both within and between laboratories. To obtain MPOC volume, most experimenters began with a two-dimensional (2-D) approach. They outlined the MPOC on each of several individual sections; then they added up the area contained on each section and multiplied the total by the section thickness. A 3-D reconstruction approach, although promising, has been somewhat impractical until recently due to the requirements for highly specialized software and massive computing support. Here, we describe the application of commercially-available PC-based software to measure MPOC volume by 3-D reconstruction. Male and female Sprague-Dawley rats, 24 or 50 days of age, were perfusion-fixed with 10% neutral phosphate-buffered formaldehyde. Following processing and embedding, a series of 20-microm sagittal paraffin sections were cut and mounted onto slides. After staining with cresyl violet, they were digitized using a microscope-mounted video camera connected to a frame-grabber in a Pentium-class computer (MCID-M5+). In addition to the MPOC, the anterior commissure, fornix, paraventricular nucleus, medial division of the bed nucleus of the stria terminalis, third ventricle and the bed nucleus of the anterior commissure were identified on the screen image and outlined using a computer mouse. These outlines were then aligned and rendered in 3-D with a solid overlay. The additional areas, such as anterior commissure, form landmarks within 3-D space to improve the accuracy with which the MPOC may be located and outlined. The reconstruction provides a striking illustration of the geometric relations between the structures of the anterior hypothalamus in the male and female rat. Moreover, the volumes determined from the overlays were reproducible between repeated studies in our laboratory. Our volume measurements confirm the sexual dimorphism previously reported for MPOC volumes, and provide a relatively quick, accurate and reliable protocol that should be useful in future experimental studies of environmental estrogenic compounds.