Establishing Of Experimental Rabbit Model With Lymph Silted Up And Its Influence To Hepatic Tissue Structure

Objective:Normal lymph drainage of the organs,which is very important for maintaining the relative stabilization of the organ homeostasis. Realizing the normal lymph drainage; and the influence of lymph drainage obstruction for tissue structure of organ and metabolism of body means important physiological and pathological Significance. As is known to all, lymphatic vessels is a kind of one-way drainage vessel systems, mainly recycles macromolecular substances such like liquid, fat and protein, etc. from the various organs and tissues; and infuses into the blood as lymph form, and with the function of clearing cell debris and pathogens. About half of the body lymph root from in the organs in the peritoneal cavity, especially the small intestinal and liver. If we just ligate the thoracic duct, the protein and fat substance from the liver and small intestinal are blocked, whether it will affects the transportion of protein and fat and the substance metabolism seriously? Will it establish a branch cycle quickly? As for these questions now, there is no a unified understanding in academia. Regarding the different views precent, this experiment is trying to approach the experimental lymph silted up, to induce the changes of the hepatic tissue, to provide experimental evidence through the animal hepatic tissue changes by ligating the thoracic duct of the rabbit.Methods:1. Experimental Animal and MaterialsFive-months-old rabbits, body weight: 2.5-3.0 kg, male or female, experiment on 15 rabbits, the other five is for control (sham operation); feed with lipid food 2-3 hours before experiment, intravenous injection on the edge of the ear for anesthesia with 6% chloral hydrate (5ml/kg), acting thoracic duct ligation in the abdomen. After operation, all the rabbits were fed as usually, draw the materials after 3 months and 6 months, which for the hepatic tissue of the light and electron microscope observation.2. Sample preparation and light-microscope observationParaffin embeds with hepatic tissue slice, HE staining and microscope observation; frozen tissue slice, carry out the 5'-nucleotidase and alkaline phosphatase (5'-Nase-ALPase) histochemical double staining and light microscope observation; which show the fine structure of the hepatic tissue and the distribution characteristics of vascular system in the rabbit liver.3. Sample preparation and scanning electron microscope observationAccording to the hepatic tissue sample of the 5'-Nase-ALPase histo-chemical double staining, after dried completely, no need for metal spraying, using scanning electron microscope, observe the secondary electron image and back scattering electron image, which show the vessels inside the hepatic tissue and the distribution characteristics of the lymphatic vessels.4. Immunohistochemical staining and light microscope observationTo compare the hepatic tissue slice of the experimental rabbits, carrying out the leptin immunohistochemical staining, observing the leptin and its receptor expressition with light microscope, showing the leptin immunological positive reactive cells of the rabbit hepatic tissue.5. Preparation and observation for the transmission electron microscope samplesPrepare the hepatic tissue sample for transmission electron microscope observation, on the ultra-thin tissue section of rabbit liver, which show the ultra-structure characteristics of the rabbit hepatic tissue, and the influence on the hepatic tissue ultra-structure of the experimental lymph silted up.Results:1. Through HE staining light microscope observation on the paraffin embedding tissue slice, compared with the experimental rabbit hepatic tissue, which is very clear, there is a central vein along with the long axis in the centre of the hepatic lobule, hepatic cords and sinusoid take the central vein as the centre, which is in a radial arrangement. Three concomitant channels are obviously seen in the portal areas and gate areas, they are interlobular vein, interlobular artery and interlobular bile duct. However, it is difficult to distinguish the lymphatic vessels and tissue interstice. The normal structure of the rabbit hepatic tissue is damaged, a large number of hepatocyte, especially hepatocyte near to the central veins, which show obvious lipid substance deposition and vacuolated changes; the lipid substance deposition of the hepatic lobule is in fan-shaped distribution, a large number of hepatocytes are damaged, some of the hepatic sinusoids vanish.2. Frozen sections of the enzyme histo-chemical double staining with light microscope observation shows the similarities between the rabbit hepatic tissue structure characteristics and HE staining section, the lymphatic vessels in the hepatic tissue shows a sepia positive reaction of 5'-Nase, the wall is a little bit thinner, the lumen is bigger and irregular, only seen inside the hepatic interstitial tissue; blood vessel is in blue staining and shows a alkalinous phosphatase positive reaction, the lumen is small and mostly round.3. Enzyme histo-chemical double staining sections with scanning electron microscope observation shows, because there is a great deal of deposition of lead reactive substance, which makes the interlobular bile ducts, artery, veins and lymphatic vessels, showing a back scattering image, it is not easily to distinguish the vessels in the hepatic tissue, the bright back scattering electron image mainly appears in the portal areas.4. Observation on the frozen sections with immuno-histochemical staining by light microscope, and compared with the leptin immune positive reactive cells of the rabbit hepatic tissue, mainly distributed in the hepatocyte cell membrane, fat cell membrane, fat-storing cell membrane and their cytoplasm. The leptin immune reactive positive cells were similar to the compared control group, the leptin immune positive reaction is enhanced, especially those obvious the hepatocytes with lipid deposition, mainly shows a strong leptin immune positive reaction. 5. Observation on the ultra-section of rabbit hepatic tissue with transmission electron microscope, the hepatocytes are closely arranged on the rabbit hepatic tissue, all kinds of the organelles are abundant in the hepatocytes, different form and size of granules are obviously seen. Besides, the endothelial cells of the hepatic sinusoid, kupffer cell,fat-storing cell and their ultra-structure characteristics can be observed. However, on the experimental rabbit hepatic tissue, the ultra-structural changes of the hepatocyte is most obvious; that is mitochondrial swelling, mass deposition of the lipid droplet, depression of the membrane electron density; and the heterochromatin in the hepatocyte nucleus, or even the clumping and karyopyknosis of the chromatin will occur. Hepatic interstitial tissue is becoming broader, connective tissue hyperplasia and the deposition of the fat and protein inside the interstitial space, or even forms a blob structure of collagen fibril.Conclusion:1. The lymph of the liver mainly reflows through the thoracic duct; the thoracic duct of the surgical ligation in the abdomen will establish an experimental animal mode of hepatic lymph silted up;2. The experimental hepatic lymph silted up influence on the transport of the protein and fat substance in the liver, which will cause the deposition of the lipoprotein and other macro-molecular substances, and induce or lead to ultra-structure changes of the hepatic tissue.3. There are leptin immune positive reactive cells in the rabbit hepatic tissue, the immune reaction will enhance when the lymph silted up happens, which indicates that the liver may be the main leptin generate (fat-storing cells) and active (hepatocytes) the organ.4. After the ligation of thoracic duct of the rabbit, the hepatic lymphatic collateral circulation will not establish immediately, which indicates that lymph silted up is related to some diseases such like fatty liver.