| Objectives: The aging changes in three-dimensional structure of myocardial cell connection and changes of Connexin43(Cx43),Connexin40(Cx40),N-cadherin,α-catenin expression were studied using methods such as hematoxylin eosin staining , immunohistochemistry , laser scanning confocal microscope(LSCM), and digested scanning electron microscope in order to provide morphological surport for development of embryonic heart. Methods: 5 fetal rats from 1 pregnant SD rat(pregnant 20days)and 5 postnatal male SD rats in each groups were selected : neonatal group ( 3-5days ) ; infant group(20-25days), weight 25+4g; young group (3-4months)weight 250+10g; adult group(6-8months), weight 350+10g; old group(13-15mnths), weight 450+10g. In each group, there were 5 rats. 1. After being infused with 4% polyformaldehyde through aortic,left ventricular samples of hearts were embeded in paraffin and sectioned as usual. Each piece was 5 micrometer. The results of HE staining and Immunohistochemical staining were using image analysis system to show the odds of nuclear to cytoplasm and the expressions of Cx43, Cx40 and N-cadherin in each groups. At the same time ,the mean light density of positive expression(OD/um~2) was also calculate. 2. LSCM was applied to investigate double labeling of Cx43 and Cx40 with fluorescent light in the young group. Materials were made same as above. Red light was signed to Cx43 and green light was signed to Cx40. 3. Rats from young group, adult group and old group were selected for the digested scanning electron microscopy. Cardiac samples were taken from the epicardial side as the size of 0.5×0.5×0.3um~3 and digested by 6 N NaOH in 10 minutes. Then dehydrate, dry, membrance plant were taken in turn, and then observed in scanning electron microscope. Results: 1. HE staining result: Myocardial cells shown as short rhabditis form, the nuclear were dense, muscle fibers hadn't form; Muscle fibers of the infant group grew to longitudinal, and the diameter were enlarging. While the young group and adult group rowed into tracts, the branch of the myocardial cells had increased. Light and dark striations were found; Myocardial cells in the old group were out of order, some of which had broken , the intercellular space and connective tissue had increased. 2. Immunohistochemical staining result(:1)The positive of Cx43 and Cx40 were found in myocyte membrance and within cytoplasm in the fetal and neonatal group hearts. In the infant group, they were dispersed to the transverse terminals of the myocytes, toward thedistribution within the intercalated disk, little of them were also dispersed over the cell surface. From the young to the old group, the Cx43 and Cx40 were typically located in the intercalated disk .(2)N-cadherin,α-catenin were mainly found in myocyte membrances from fetal to neonatal rat hearts. In the infant group, they were together located in myocardial cell to cell connection(similar to the place intercalated disk located),quite a few located in side to side connection. From the young to the old group, they were located safely in side to intercalated disk connection. Image analysis showed that the percentage of Cx43 and Cx40 immune positive area in rat ventricular myocardium had a progressively changement with age. 3. LSCM result: After radialized by excitated, Cx43 was signed red in cell to cell connection, Cx40 was green. When they both showed, it changed to yellow. 4.Scanning electron microscope result: Myocardium intercalated disks in the young and adult groups were composed of many foot step-like platforms. There were many finger-like processus on platforms, rowing in order. Between platforms were smooth myocyte membrance. While in the old group, the intercellular space had increased and these processus became shortness and disordered. Conclusion: 1. Research from light microscope and scanning electron microscope showed that with the aging development from the fetal group to the old group, the shape of the myocardial cells had changed from short rhabditis form to... |
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