Experimental Study On The Effects Of Lymphatic Stasis On The Functions And Tissue Structures Of The Pancreas, Liver And Kidney In Rabbits.

Diabetes mellitus (DM) refers to a number of disorders that involve in relative lack of insulin secretion or a deficiency of insulin function, that share the cardinal characteristic feature if elevated blood glucose levels, of which type 2 (type 2 diabetes mellitus, T2DM) has the highest incidence, accounts for over 95% of the patients. To explore the pathogenesis of T2DM, researchers at home and abroad has conducted a lot of research work with animal models. There are many ways to establish experimental animal models of diabetes, but blood and urine glucose levels are the major index to judge whether the animal models are established successfully or not.Lymphatic system is an important part of the circulatory system, which consists of lymphatic channels, lymph tissues and lymph organs. It plays a critical role in physiological and pathological processes, such as, in absorbing and delivering macromolecules (e.g. long-chain fatty acids, protein and peptide hormones), in spreading inflammation and transferring cancer cells and etc.Insulin is mainly composed of peptide hormones. People have different views on its delivery ways after insulin was secreted from B cells and then transferred into the interstitial space. Most scholars believe that after insulin entered into capillaries, it transferred through the blood directly. Are there any other alternative ways for insulin to transfer under Control physiological conditions except through the vascular system? Many researchers reported that insulin presented in the thoracic duct in different experimental subjects. These results suggested that the lymphatic system might be an important delivery access of insulin after insulin was secreted into interstitial fluid. The way, which insulin was secreted into lymphatic vessels and then entered into blood circulation, transfered to target organs, target tissues and target cells of all the body, might play a great role in avoiding the"the first passing effect"of the liver. Whether insulin and other macromolecules enter into capillaries directly or they are absorbed through lymphatic capillaries and then into blood circulation indirectly, whether that the blocked pancreatic lymph return influence the delivery of insulin need to have a further study. Meanwhile, the influence of blocked lymphatic return on the function and tissue structures of liver and kidney needs further study, too.The experiment was divided into 5 parts, that is, establishing experimental animal models through ligating abdominal thoracic duct in rabbits to create lymphatic drainage block; observing the tissue morphology of the pancreas, liver and kidney of model animals and the controls with light and electron microscopy; examining the blood glucose, lipids, insulin, the functions of the liver and kidney of the model animals and the controls with biochemical and radioimmunoassay; exploring the effects of lymphatic stasis on the tissue structures and the function of the pancreas, liver and kidney of the model animals. The experimental contents and results are the followings:Part 1 Establishing animal models with lymphatic stasisin rabbits and exploring its relationships with type 2 diabetes mellitusObjective: To establish experimental animal models of type 2 diabetes mellitus low close streptozotocin (STZ) was often selected to inject into abdominal cavity for several times combined with special dietary approaches. The pathogenesis and clinical features of the experimental animals induced with high sugar and fatty diet combined with intraperitoneal injection of low chose STZ was similar to that of the patients with type 2 diabetes mellitus. It's easy to perform in this way and has a high success rate, which is considered an ideal experimental approach in the research work of diabetes. However, the animal models established in this way belong to type 1 diabetes mellitus and its difficult to replicate type 2 diabetes animal models with the gradual destruction of islet cells. Some scholars believe that the lymphatic system is an important access to transfer polypeptide hormone insulin. The development of type 2 diabetes may be related to the delivery of insulin and the block of pancreatic lymphatic return. In order to explore the relationships between the block of pancreatic lymphatic return and the abnormal delivery of insulin of type2 diabetes, we established the experimental animal models to observe the fat accumulation within the pancreas in the model rabbits and examine the changes of blood glucose and etc. to search for an ideal approach to establish animal models of diabetes.Methods: 20 10-month-old rabbits (From the Experimental Animal Center of Hebei Medical University), male and female in half, raised separately, were divided randomally into the model group (operation) and the controls (sham-operation). The animals were fed with 5 to 10 ml vegetable oil before performing legation of thoracic duct 2 to 3 hours, and then making anesthesia through intraperitioneal injection with 6% Chloral hydrate (5 ml/kg).Under the direction of surgical microscope, the thoracic ducts of the controls were fully free instead of ligating while the thoracic ducts of the model group were completely exposed at crura of diaphragm of abdominal cavity and fastened. The model animal operated were fed conventionally. Six months later they were conducted anesthesia through intraperitioneal injection with 6% chloral hydrate (5ml/kg) to determine the fat accumulation conditions. The blood glucose was tested after fasting 12 hours with Lifescan instrument from United States, the test paper from Uni-check.Results:1. The changes of fat in abdominal cavity before and after the models established: (1) While creating animal models, the abdominal cavity was opened and found that the fat in the abdominal cavity was less visible, the mesentery was clear and transparent, the pancreas was light red, there were less fats around the kidneys which were not enveloped by fats. (2) After succeeding in creating the models for 6 months, the abdominal cavity was opened again to observe the related changes. The results showed the fact that the fats in abdominal cavity were greatly increased. The fatly tissues in mesentery thickened. It was hard to identify the pancreatic tissue, some of which were almost replaced by fatty tissues. Fatty tissues around the kidneys thickened and increased remarkably and covered the kidneys.2. The changes of blood glucose before and after the models established: (1) The blood glucose was greatly elevated and there was a significant difference statistically before and after thoracic duct ligation in the model groups. (2) The blood glucose level in the model group was higher than that of the controls, there was significant difference between the two groups. (3) The changes of the blood glucose in the controls were not remarkably and there was no statistical difference.Conclusions: The experiment animal models of pancreatic lymphatic stasis can be established by applying abdominal surgery to fasten thoraces duct in rabbits. The results show that a great deal of fats are deposited in the abdominal cavity, the blood glucose level is increated which are similar with the predisposing process of type 2 diabetes indicial course.Part 2 Observation on the morphological changes of tissue structures of pancreas induced by ligating thoracic duct in rabbitsObjective: The lymphatic system plays an extremely important role in the absorbing and delivering process of lipids and proteins and macromolecules (such as insulin) polypeptide fatty acids and other peptide hormones as well as in the physiological and pathological conditions of the diffusing of inflammation and transferring of cancer cells. To explore the blocked effects of lymph stasis on the influence of fine and ultra-structures of the pancreatic tissue we created the experimental animal models with pancreatic lymph stasis through ligating thoracic duct in abdominal cavity in rabbits.Methods: 20 10-month-old rabbits were selected and divided randomly into the model group (operation) and the control group (sham-operation). After animal models established for 6 months, some specimens from the tissue of pancreas were embedded with paraffin to make pathologic sections. Some sections were stained with HE and Congo red to observe the pathologic change, some were stained with the pancreatic islet amyloid polypeptide (PIAP)-immunohistochemistry. Some specimens from the tissue of pancreas were prepared into ultrathin sections and observed with TEM.Results:1. The sections with conventional HE staining were examined under light microscopy. The results indicated the fact that: (1) in the controls, the sizes of islets of pancreas were different, the islet cells were distributed like clusters and scattered in pancreatic acinar or located in connective tissues of pancreatic lobule; the larger catheters, blood vessels and lymphatic vessels in pancreas were often accompanied with connective tissues. (2) In the model group, a lot of lipid droplets presented in islet cells, even vacuolated cells could be observed; lipid droplets also could be seen in acinar cells, even showed vacuolar degeneration and other changes; the space of connective tissues in pancreas was enlarged and widened, the fatty tissues were heaped together, the channels of lymphatic vessel alike were expanded.2. The sections with Congo red staining were determined under light microscopy. The results showed the fact that: (1) In the controls , there was not marked staining in or around islets of pancreas; (2) In the model group, amyloidal-like depositions presented in islets of pancreas, islets appeared dark red by Congo red staining; the spaces of pancreatic lobule were widened and the proliferations of connective tissue were increased significantly.3. Observations of immunohistochemical staining under light microscopy. The results of frozen sections with immunohistochemical staining showed the fact that positive products of pancreas, islet cells and their surrounding tissues were commonly stained into brown color. Compared with the controls, the tissue sections of the pancreas in the model group were stained dark brown. The positive immunoreactions of pancreatic islet amyloid polypeptide were enhanced markedly.4. Observations of ultrathin tissue sections under electron microscopy. (1) In the controls, the results indicated that islet cells arranged closely, various organelles were rich in the cells, different shapes and sizes of secretary granules were obvious and visible. (2) In the model group, different sizes and electron density of secretary granules were seen in islet cells; the halo (pancreatic islet amyloid polypeptide considered to exist in the area) of some secretary granule was obviously enlarged in islet B cells; some organelles in the islet cells became sparse, cell membrane became unclear, chromatin gathered on one side, even occurred nuclear condensation like cell dilapidated, the space of islet cell was widened, the lipid droplet-like and amyloidal clumps were seen in the enlarged space of islet cells, even formed fibril-like structure; In addition, the obvious lipid accumulation as well as some red blood cells outside blood vessels were often seen in the space of pancreatic tissues or in the space if widened islet cells.Conclusions: The thoracic duct fastened with surgery plays a certain role in the structure if the pancreas in the rabbits, such as lymphangiectasia, connective tissue space widened, fat accumulation and morphological changes of peptide deposition; the structural changes of the pancreas induced by experimental lymphatic stasis is bound to affect the exocrine of the pancreas and the endocrine of the pancreatic islets.Part 3 Effects of thoracic duct fastened on the functions and the structure of liver tissues in rabbitsObjective: The liver is just like a bio-processing factory in the human body, it produces a number of macromolecules, while the lymphatic system is an important returning channel for the macromolecules to pass through. If lymphatic stasis occurred, the returning way of some macromolecules would be affected. To determine the effects of thoracic duct fastened on the functions of the structure of the liver tissues, we established the experimental animal models by blocking the returning of lymphocytes with thoracic duct legation.Methods: 20 10-month-old rabbits were selected and randomly divided into the model group (operation) and the control group (sham-operation). After animal models established for six months, some specimens from the tissue of the liver were embedded with paraffin to make pathologic sections with HE and immunohistochemistry staining to observe the pathologic changes under light microscopy. Before and after establishing the models for 6 months, the blood taken from femoral vein of the experimental animal was conducted centrifugation at 3000 rpm to get the serum to examine the functions of the livers, the experimental animals were applied anesthesia with 6% chloral hydrate (6 ml/kg) through abdominal cavity.Results:1. The results of liver function: TBil, DBil, ALT, AST markedly increaseed in model rabbit, and albumin markedly decreased (P<0.05).2. Observations of the liver of the rabbits with naked eyes: In the controls, the appearance of the livers were Control, presented light red and smooth capsule. The sizes of the livers in the experimental animal with thoracic duct ligation increasing remarkably, the capsules were tensed and the edges rounded, the livers became softened and covered with fatty tissues.3. The sections with conventional HE staining were examined under light microscopy. The results indicated the fact that: (1) in the controls, the tissue structures of the liver in the rabbits were seen clearly and sharply, the structures of hepatic lobules were Control, the liver cells arranged orderly, the sinus cable structures were clear, a small amount of fatty vacuoles combined with loosening of liver cells were seen in some liver cells, the endothelia cells and liver megoxycytes hepatic sinusoid, the interlobular weins, arteries and bile ducts were mainly located around the hepatic duct, reticular fibers and fatty cells were seen scattered in the space of hepatic sinusoid. Necrosis of the liver cells was not found. (2) in the model group established animal models for 3 months indicated that the tissue structures were seen clearly, the structures of the liver lobules were Control, the numbers of the fatty cells increased and the sizes of the liver cells became larger, moderate fatty degenerstion of the liver cells were found swelled and the space of the liver sinnsoid became narrowly. (3) in the model group after establishing the animal models for six months showed that the numbers of the fatty cells increased and the sizes of the liver cells became larger, the fat capsule formations and moderate and severe fatty degeneration were seen, a small ammount of nuclear necrosis was found in the liver cells. The inflammatory cells were seen in the liver cells, and the majorities were lymphocytes, while megoxycytes were not increased. The structures of liver tissues were damaged, the abControl structures of the hepatic lobules were seen, but the phony lobules were not found.4. The results of the model animal with immunohistochemistry staining showed that the leptin immunoreactivities in the liver tissnes of the controls were mainly located in the cell membrane of the livers. Compared with the controls, the liver steatosis and fibrosisrea of the leptin expression were obviously increased. There was a significant difference between 2 groups.5. The results of ultrathin tissue sections under electron microscope indicated the fact that: 1). In the controls, the cell membrane structures of the livers were seen clearly, hepatocyte nuclease were in the centre of the cells which appeared round or oval, the nucleolus located nearly or in the central part; there were many organelles in the cytolipin, especially the rough endoplasmic reticulum and mitochondria, endoplasmic reticulum appeared tubular and vesicular structures, surrounded by a large number of glycogen granules; the shapes and the sizes of the mitochondrion were nearly the same and presented round or oval, the matrixes were rich in the liver cells and the ridge were less; Golgi complex and lysosomes were seen in the cytoplasm near the liver bile canaliculi, there were no or a few lipid droplets in the hepatic stellate cells; there were not any in flammatory cells and fibrous tissue proliferation in the liver cells. 2). In the model group, the abControl ultrastructure of the liver cells were seen, and the cords of the liver cells arranged disorderly, the liver cells became swelling and round, the electron density of the celll membrane was reduced; the nuclear membrane presented tortuous and the order part protruded, the spaces of nucleolus were expanded slightly; the sizes of the nuclears were shrunk, the nuclear chromatins were condensed into a mass, some nuclear membranes were unclear or disappeared completely, the nucleus heterochromatin increased, the mitochondrias of the cytoplasm presented different sizes and shapes, most of them presented round or oval, some of them presented irregular shape, swelling, shrinking or damaging in the outer membrane were visible, some super-large mitochondria presented in some cells; the endoplasmic reticulum were enlarged, some even degranulated; the lipid droplets in different sizes were seen in the cytoplasms, some were heaped together, some lipid droplets extruded the nucleus; some of hepatic sinusoids be capillarized; the fibroblasts and a large number of collagen fibers were seen in the liver cells; the space of hepatic sinusoid was obviously enlaeged and collagen fibers were found.Conclusions: Blocking thoracic duct can result in the changes of functions and morphology in the liver of rabbits. As times goes on, hepatic steatosis accompanied with inflammatory cell infiltration could be seen in the livers. The fatty liver may be developed into non-alcoholic fatty liver disease, liver fibrosis and cirrhosis.Part 4 Effects of blocking thoracic duct on the functions and tissue structures of the Kidney in rabbitsObjective: To explore the effects of lymphatic stasis on the functions and tissue structures of the experimental animal models in rabbits by blocking thoracic duct.Methods: 20 10-month-old rabbits were selected and divided into the model group (operation) and the control group (sham-operation). After animal models established for six months, some specimens from the tissues of the kidneys were embedded with paraffin to make pathologic sections with H E and immunohistochemistry staining to observe the pathologic changes under light microscopy, some sections of the kidney-tissues were made to be examined under electron microscopy. Before and after establishing the models for 6 months, the blood taken from femoral vein of the model animals was conducted centrifugation at 3000rpm/min to get the serum to examine the functions of the kidneys, the model animals were applied anesthesia with 6% chloral hydrate (6 ml/kg) through abdominal cavity.Results:1. The results of the functions of the kidney: BUN and S-Cr markedly increaseed in model rabbit (P<0.05). 2. The results of sections with conventional HE staining in the contrls indicated that renal glomeruli were visible in the rabbits of the model group, the spaces of connection tissue were widened, the fatty tissues heaped up, the structures of lymphatic-like channels were expanded.3. The results of observations on the glomerular amyloid polypeptide with immunohistochemical approach showed that the expressions of the rabbits of the model group were higher than that of the control group. There was a significant difference between the two groups.4. Under the electron microscopy, the results showed the fact that in the rabbits of the model group the renal glomeruli were shrunk, the mesangium was broadened, capillary basement membrane showed diffuse or noclular thickening, the amyloid fibers with irregular arrangement were seen in the capillary basement membrane and mesangial area, its diameter was 8-10 nm. Some disordered fibrosis were seen in the subendothelium and subepithelial membrane in the basement membranes, nurse cells (Sertoli's cells) were marged together, the amyloid fibers were found in the renal tubular basement membrane and the tunica as well as in the capillary wall and the vessel wall of interstitial tissues, most of the endothelial cells were fallen away from the blood vessels. The microvilli on the surface of tubular epithelial cell were irregularly arranged, some of them were damaged. Some amyloid fibers were distributed in basement membrane in ribbon or flake-like state.Conclusions: 1. The vacuole changed in the renal glomeruli and tubular resulted from ligating the thoracic duct in rabbits. 2. Ligating the thoracic duct can result in the damage of renal functions in rabbits.Part 5 Effects of ligating the thoracis duct on the lipid and glucose metabolism in rabbitsObjective: To explore the effects of lymph stasis on the lipid and glucose metabolism in rabbits.Methods: 20 10-month-old rabbits were selected and divided randomly into the model group (operation) and the control group (sham- operation). The blood glucose, lipid and insulin of the animals in the model and control groups were examined before establishing the models. After the models established, the blood glucose of the model rabbits fasted for 12 hours was detected every week. After the models established for 6 months, the blood glucose lipid and insulin were determined.Results:1. The blood glucose level of the fasted rabbits: The results indicated that the blood glucose of the rabbits before the thoracic duct fasteened was lower than that of the rabbits after the thoracic duct fasttened, there was a significant difference between the two times.2. The results of the insulin level of the fasted rabbits: The results showed that the insulin level of the rabbits fastened thoracic duct was lower than that of the controls, the difference was statistically significant.3. The levels of serum free fatty acids(FFA), trilycerides(TG) and very low density lipoprotein(VLDL) in the model group was higher obviously than that of the controls. There was a significant difference between the two groups (P<0.01). There was not a significant difference between the two groups concerning the serum cholesterol levels.Conclusions: Ligation of thoracic duct can lead to abnormal metabolism of the blood glucose in rabbits. The collateral circulation of the lymphatic system can not be established quickly and completely after ligating the thoracic duct. The advantages and disadvantages of preventive ligating the thoracic duct still need to have a further study.