Experimental Study Of Thoracic Duct Ligation's Impact On Domestic Rabbit's Body Weight, Blood Sugar And The Organizational Structure Of Pancreatic Tissue

Object:Immunohistochemistry and immunocytochemistry studies have shown that islets are mainly composed of four types of neuroendocrine cells, namely B, A, D and PP cells.They are able to respectively synthesize andsecretemonoamines and peptide hormones such as insulin, glucagon, somatostatin and pancreatic polypeptide. The number of B-cells is the largest. The insulin which is the secretions of B-cells, as the only hypoglycemic hormone in the body, is the only hormone which can prompt the compound of glycogen,fat,and protein at the same time as well. It plays a very important role in maintaining the relative stability of blood sugar. Insulin function is roughly the same in different species of animals. Insulin is a protein hormone which is the product of islet B-cells stimulated by the endogenous or exogenous substances (such as glucose, lactose, ribose, glucagon, etc.),and is made of A, B two peptide chains. After the compound in ribosome of B-cell rough endoplasmic reticulum, insulin stores in the B-cells with the form of ? particles.In the traditional view, insulin and other peptide hormones enter the blood through the capillary wall, and need the well-working of hepatic portal vein .During that time, the lymphatic system does not involve in insulin absorption and transport. However, some scholars hold that lymphatic and endocrine cells have the close relationship. According to the organizational structure features of lymphatic capillaries, whose diameter and permeability is far greater than capillary. So they hold that Biological macromolecules insulin and other peptide hormones are easier to enter the lymphatic capillaries to transfer. Besides, whether collateral circulation could be established soon after ligation of thoracic duct, there are still a different conclusion. If the pancreatic lymphatic flow is blocked, would it affect the pancreatic structure and glucose metabolism ? Based on this view, in this study, through the surgical method of thoracic duct ligation on domestic rabbit, prepare experimental animal models of pancreatic lymph stasis, implement conventional HE staining, Congo red staining and amylin peptide immunohistochemistry on pancreatic tissue, carry on light microscope and electron microscope observation.Amylin peptide ,also named as diabetes-related peptide, is polypeptide which is made of 37 amino acids. Amylin peptide and insulin co-store in secretory granules of islet cells. Some studies also show that amylin peptide has a toxic effect on islet cell membrane, can also induce insulin resistance. In the occurrence and development process of type 2 diabetes, the changes of insulin secretion from normal or over-developed to reduce have relations with insulin-amyloid protein (amylin peptide) deposition. So the appearance of amylin peptide deposition in pancreas is pathology of patients with type 2 diabetes. The amount of islet cell replaced by amylin peptide deposition determines the development of type 2 diabetes or severity evaluation. We can observe the experimental animals'morphological changes of the organizational structure of pancreatic, compare the blood glucose and weight of normal and experimental animals by light and electron microscopy. These experiments can provide basis in morphology for discussing insulin and other peptide hormones'transporting way, amylin peptide deposition mechanism in pancreas.Methods:1 experimental animal and drawing materials: Choose twenty healthy adult domestic rabbits (provided by the Experimental Animal Center of Hebei Medical University), regardless of sex. Divide into two groups randomly: experimental (operation) group and control (sham-operated)group. For clearly showing chylocyst and thoracic duct in abdominal operation, learn from the method that gastric perfusion vegoil leads to the thoracic duct dilatation and displaying milky, poure edible oil 5-10 Ml into domestic rabbits'stomach,. About 3 hours later, thoracic duct and chylocyst filling is the most obvious. Then use 6% chloral hydrate (5ml/kg) ear vein anesthesia. Because thoracic duct wall is very thin, individual stripping ligation are easy to peel injury. So adopt thoracic duct ligation method in clinical esophageal cancer surgery. The cluster ligation of thoracic duct and surrounding tissue avoids thread split which is the result of individual thoracic duct ligation. Moreover, owing to lower position of thoracic duct injection, neck ligation easily cause rupture of pleural Top , further leads to pneumothorax or death of experimental animals due to venous bleeding. Therefore, carry on ligation under the abdominal diaphragm and the start department of thoracic duct. The sham-operated group do not ligate , just make thoracic duct full of free. After the operation, experimental animals are fed conventionally, measured blood sugar of sharp-eared peripheral blood weekly (fast 8 hours before measurement), measured body weight. Two groups of rabbits are observed changes in body weight and blood sugar. Six months after surgery, animals were killed under 6% chloral hydrate in the ear vein under deep anesthesia. Quickly obtain the rabbits'pancreatic tissue samples for light microscopy and electron microscopy sample preparation.2 Sample preparation and observation of light microscope: Through hematoxylin-eosin staining and light microscope, congo red staining and light microscope, amylin peptide immunohistochemistry and light microscope towards tissue slices of paraffin-embedded pancreatic specimens to show subtle structural changes in pancreatic tissue of two groups of animals.3 Sample preparation and observation of TEM: Through conventional TEM sample preparation, ultra-thin tissue sections of pancreatic specimens and TEM observation to reveal the ultrastructural changes in pancreas (especially pancreatic insulin) of experimental animals.Results:1 Compared with the control group, blood sugar of rabbits in experimental group gradually increase after the operation, difference is significant(p <0.05). The average weight of rabbits in experimental group is lower than the control group, difference is significant(p <0.05). Sixteen weeks after the surgery, hyperglycemia of 2 cases of animal in the experimental group is apparent, up to 7.5-8.2 mmol / L, and associated with tinea, hind multiple ulcers, weight loss significantly. The experimental group compared with the control group rabbits, pancreas look pale, dark, like fatty tissue.2 Light microscope observation(1) In the HE staining of histological sections, the experimental group significantly widen pancreatic lobular gap, accumulation of fatty tissue appears in the connective tissue gap. A large number of cytoplasm in acinar cells and islet cells show vacuolar state.(2) In the Congo red staining of pancreatic tissue sections, pancreatic lobular tissue space widens significantly. Connective tissue proliferates apparently. A large number of adipose tissue accumulate. Islets show congo Red staining vermilion color reaction.(3) The light microscopic observation of amylin peptide immunohistochemical staining sections demonstrates that the experimental group of animals and its surrounding islets,shows amylin peptide immunoreactive strong positive brown color reaction.3 TEM observationTEM observation of pancreatic tissue ultrathin section demonstrates that the experimental group animals'islet cell gap widens, and even pipeline structure which is pool-like and connects with each other among cells forms. Within the cell gap, a large number of lipid droplets-like substance, amyloid material , and even fine fiber group forms.Conclusion:(1) Thoracic duct ligation may lead to pancreatic lymph stasis, and the changes of experimental rabbits'body weight, blood sugar and organizational structure of pancreatic. These changes may be related to transport barriers of peptide hormones such as lipid and insulin.(2) After the surgical ligation of thoracic duct,experimental pancreatic lymph stasis and the changes in the organizational structure of pancreas show that lymphatic collateral circulation can not be established soon. Therefore, the normal lymphatic flow plays an important role for the maintenance of pancreatic endocrine function.