Jetton, Emily Hope (2004-11). The development of processing methods for a quantitative histological investigation of rat hearts. Master's Thesis.
Thesis
In order to understand the mechanical functions of the cardiac muscle it is important to first understand the microstructure of the tissue. Young et al. (1998) realized that quantitative three-dimensional information about the ventricular myocardium is necessary to analyze myocardial mechanics. They developed a technique using confocal fluorescence laser scanning microscopy to obtain three-dimensional images. While this method worked well in rebuilding the myocardial tissue image by image, it was quite extensive and costly. Costa et al. (1999) developed a method that was used to perform three-dimensional reconstruction as well. Their method, while less expensive and much less time consuming, required sheet assumptions and did not look directly at the cross-fiber plane. From Dr. Criscione's previous work on canines (Ashikaga et al., 2004), we found that the sheet structure can be accurately determined from cross-fiber sections without making any sheet assumptions. We have now expanded on those ideas and created a method to perform the quantitative histological investigation of the rat hearts in a way that is both timely and cost effective. We developed a processing method that preserves the orientation of the fiber and sheet angles. This method was carried out using plastic embedding since the dehydration process used in paraffin embedding has a tendency to grossly distort tissue. Once the heart was fixed in formalin, we then removed the septum and sliced it several times vertically. This allowed us to image the tissue at several depths and find an average fiber angle for each slice. Next, the specimen was hardened, and the sheet orientation was evaluated using polarized light. Once both fiber and sheet angles were obtained from several depths within the septum, we then constructed a three-dimension model of the wall. This method was both cost effective and less time consuming than previous ones and will be a method that can be used in the future to compare the myocardial tissue of diseased and healthy rat hearts so that we may better understand the mechanical functions of the heart as it remodels due to disease.
