Stereological study of the diabetic heart of male rats

Abstract

The present study aimed to quantitatively compare the normal and diabetic hearts of rats using stereological methods. Diabetic and control rats received streptozotocin (60 mg/kg) and no treatments, respectively. On the 56(th) day, the hearts were removed and their total volume was estimated using isotropic Cavalieri method. The total volume of the connective tissues and vessels, total length and diameter of the vessels, total number of cardiomyocytes nuclei, and the mean volume of the cardiomyocytes were estimated, as well. In comparison to the control animals, 60 and 43% increase was observed in the total volume of the connective tissue and microvessels of the diabetic rats, respectively (P<0.05). The percent of the vessel profiles with the diameter of 2-4 µm was decreased, while the percent of the vessel profiles with the diameter of 4.1-8 µm was increased in the diabetic hearts (P<0.05). No significant difference was found in the vessels with more than 8 µm diameters. The total number of the cardiomyocytes' nuclei and the number-weighted mean volume were respectively decreased by 37 and 64% in the diabetic group (P<0.01). A significant difference was observed between the two groups concerning the left ventricle volume to body weight ratio as an index for ventricular hypertrophy (P<0.05), while no difference was found regarding the right ventricle to body weight ratio. It can be concluded that diabetes can induce structural changes, including loss and/or atrophy of the cardiomyocytes, accompanied with increase in the connective tissue in the rats' hearts.

Keywords: Stereology; diabetes; heart.

Figure 1

A. The heart is removed. B. Isector method. The heart is embedded in a spherical agar block. C. Starting at a random position outside the heart, the tissue is totally cut into slabs to obtain eight to twelve slabs (here 8) with a known thickness (T). D. Using the point-counting method, the area of each slab is estimated. E. The arrow shows the right upper corner of the cross which is considered as a point.

Figure 2

Point-counting method. To estimate the volume density of the connective tissue and vessels, the number the points hitting the structure is divided by the total points.

Figure 3

Disector method for estimating the numerical density of the nuclei of the cardiomyocytes. The nucleus (arrow) which is located inside the frame and does not touch the left and lower borders of the frame is counted. The nucleus should not be appeared in the reference section (left) but in the look-up section (right). To estimate the mean volume of the cardiomyocytes, the intercept length (dotted line) is determined in the cells sampled by the disector.

Figure 4

Length density and diameter of the vessels. The profiles of the vessels (arrow) which are located inside the frame and do not touch the left and lower borders of the frame are counted. The diameter of the vessels is estimated from the broadest diameter orthogonal to the longest axis of the vessels which approximately touched the center of the vessels.

Figure 5

Microscopic evaluation of the diabetic heart (B) shows more connective tissue and atrophic changes of the cardiomyocytes in comparison to the control group (A).