| BACKGROUND&AIMS In 1863 , Von Recklinghausen found that there are some pores between some adjoining mesothelial cells under the light microscopy. He named these pores as the peritoneal lymphatic stomata (LS) and thought that it is a direct absorptive way in the peritoneal cavity by LS. In 1977, Tsilibary approved mouse LS by electron microscopy. Since then, other scholars also found LS in various animals. In 1990, Li found the LS in humans for the first time and considered that LS are relatively stable, which has absorptive function from the peritoneal cavity.LS is the opennings of lymphatics on the peritoneal mesothelium. The peritoneal cavity is connected with subperitoneal lymphatics through LS, which is the main absorptive passage of liquid^ grains and cells in physiologic and pathologic condition.Many scholars have paid much attention to study on the structure and function of LS. But so far, there are few papers concerning the research of the development of LS. In our experiment, it was applied to study on development of peritoneal LS in mouse by using SEM, TEM, computer image processing and enzyme-histochemistry. The experimental results will *Supported by the National Natural Science Foundation of China (No. 39970381)5provide important information for LS functional research and clinical applications.MATERIALS & METHODS 1, Animal breeding and groupings:NIH mice were used in the experiment, and a male mouse was caged with two females. All animals for the experiment was maintained in animal laboratory with air-condition and given standard forage. In the experiment, the presence of a vaginal plug was noted as embryonic day 1 (EDI). The diaphragms were removed in ED 13 - ED 15^ ED 18 -, postnatal days (PD1) -, PD5> PD 10 and adult mice. 2> Preparation for TEM: the diaphragmatic peritoneum were cut in 1 mm3 and fixed in 2.5% glutaraldehyde solution for 4-6 hours, postfixed in 1% OsO4 for 1 hour, at 4癈. After block-staining in 2% uranyl acetate , dehydration in a graded ethanol series and via propylen oxide embedding in Epon 812 were performed. Semithin section were made for identification and orientation of the mesothelium. Thin section were prepared on a LKB 2088 ultramicrotome, double stained with uranyl acetate and lead citrate and examined in Phlips EM410 TEM by 60Kv. 3^ Preparation for SEM: the diaphragmatic peritoneum were fixed in 2.5% glutaraldehyde solution for 4-6 hours, postfixed in 1% OsO4 (Ih), at 4癈. dehydration in a graded series of ethanol, CC>2 critical point drying ,mounting on aluminium stubs and sputter-covering with gold or platinium were made. Specimens were examined in Hitachi S-260 SEM by 20KV. 4, Computer image progressing : the SEM image can be stored in computer hard disk with the video frequency signal of simulative image transformed to figure image by samlping-block. We applied the Elescope 1.0 image processing software to analyse the SEM image. The system can automaticlly or half-automaticlly analyse the SEM images according image greyscale. The area of CMC and6its percentage in the peritoneum, the area and number of LS were accounted. 5% enzyme-histochemistry: (1) frozen sections: the fresh diaphragm was cut in rectangle, washed by o.lM phosphate-bufered saline(PBS), and frost sections were made at the thickness 7um; (2) double stained with 5' -Nase-Alphase : The sections of diaphragm were incubated for 60 minutes in a lead-based standard medium (O.lmol/L Tris-maleic acid buffer 42ml, 5 ' -AMP 20mg, 2.5% bitter salt 5ml, sucrose 3g, 2% lead nitrate 3ml) for 5' -Nase reaction at 37~38癈. After rinsing in distilled water, the diaphragm was immersed in 1% yellow ammonium sulfate solution for 1 min. After washed by distilled water two or three times, the diaphragm was incubated in reactive solution two (including naphthol AS-BI 20mg, DMF 0.5ml, O.lmol/L Tris-muriatic acid buffer 50ml, Fast blue B salt 50mg) for two hours at 37-38癈. Then the diaphragm was mounted with resinene and examined under light microscopy. 7> observation of the development...
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