MORFOLOGI, MORFOMETRI DAN DISTRIBUSI SEL IMUNOREAKTIF INSULIN DAN GLUKAGON PADA PANKREAS TIKUS (Rattus norvegicus) OBESITAS

DELA RIA NESTI, drh. Teguh Budipitojo, M.P., Ph.D.; drh. Yuda Heru Fibrianto, MP., Ph.D.

2015 | Tesis | S2 Sain Veteriner

Gambaran histologi pankreas akibat pengaruh obesitas menggunakan tikus normal (non genetically obese) yang diinduksi obesitas dengan diet tinggi karbohidrat dan lemak belum pernah dilaporkan. Penelitian ini bertujuan untuk mengidentifikasi morfologi, morfometri dan distribusi sel imunoreaktif insulin dan glukagon dari pankreas tikus non genetically obese yang diinduksi obesitas dan membandingkannya dengan tikus normal (kontrol). Penelitian menggunakan tikus jantan jenis wistar umur 4 minggu, terdiri dari 5 ekor kelompok kontrol dan 5 ekor kelompok obesitas. Kelompok kontrol diberi pakan dengan komposisi 76% karbohidrat, 16% protein dan 8% lemak sedangkan kelompok obesitas diberi pakan dengan komposisi 47% karbohidrat, 22% protein, dan 31% lemak serta tambahan induksi peroral dari suspensi fruktosa dan kolesterol masing-masing 1%/BB dalam 2 ml Na CMC setiap hari selama 9 minggu. Eutanasi dengan kloroform dilakukan setelah pengukuran indeks obesitas, penghitungan kadar glukosa darah dan lipid. Sampel organ pankreas difiksasi dengan larutan Bouin, diblok dengan paraffin embedding dan dipotong serial dengan ketebalan 5 μm. Jaringan pankreas divisualisasikan dengan pewarnaan hematoksilin eosin, Victoria blue dan imunohistokimia metode Labeled Streptavidin-biotin (LSAB). Antibodi primer yang digunakan yaitu mouse anti-insulin (1:1500; Abcam, USA)dan rabbit anti-glucagon (1;1500; CosmoBio Co, Japan). TrekUniversal Link Kit (Biocare Medical, USA) digunakan sebagai visual detektor yang hasil pewarnaanya dianalisa secara deskriptif dan kuantitatif. Profil lipid dan glukosa kelompok obesitas menunjukkan nilai total kolesterol (160,62±3,52) mg/dl, kadar trigliserida (98,61±2,66) mg/dl, LDL (67,51±1,81) mg/dl, HDL (44,68±2,25) mg/dl dan kadar glukosa (161,43±7,61) mg/dl. Topografi pangkreas kelompok obesitas tersebar pada 3 lokasi yaitu area gastrium, duodenum, dan lien. Morfologi Pulau Langerhans kelompok obesitas berbentuk bulat, oval dan poligonal serta dikelilingi oleh serabut halus dan kapiler darah. Morfologi sel imunoreaktif insulin dan glukagon kelompok obesitas berbentuk poligonal tidak beraturan dengan inti bulat/oval dan terletak di sentral/perifer. Sel imunoreaktif insulin kelompok obesitas tersebar pada bagian tengah dan beberapa pada bagian tepi Pulau Langerhans sedangkan sel imunoreaktif glukagon dominan tersebar pada bagian tepi dan sedikit pada bagian tengah Pulau Langerhans. Penghitungan morfometri sel menunjukkan diameter Pulau Langerhans kelompok obesitas (154,38±65,56) μm, keliling Pulau Langerhans (373,51±146,69) μm, volume Pulau Langerhans 3.241.105,96 μm3, jumlah sel total/Pulau Langerhans (181,90±96,76), jumlah sel imunoreaktif insulin (113,66±60,90) dan jumlah sel imunoreaktif glukagon (46,98±25,80). Kelompok obesitas menunjukkan perbedaan dengan kontrol berupa peningkatan nilai total kolesterol, kadar trigliserida, LDL, kadar glukosa, diameter Pulau Langerhans, volume Pulau Langerhans, keliling Pulau Langerhans, jumlah sel imunoreaktf insulin/Pulau Langerhans dan jumlah sel imunoreaktif glukagon/Pulau Langerhans (P<0,05). Morfologi, morfometri dan distribusi Pulau Langerhans beserta sel imunoreaktif insulin dan glukagon pada pankreas tikus (non genetically obese) yang diinduksi obesitas menggunakan diet tinggi karbohidrat dan lemak menunjukkan adanya hiperplasia sel imunoreaktif insulin dan glukagon sebagai kompensasi peningkatan volume Pulau Langerhans.

Histology of the pancreas due to the influence of obesity using normal rats (non genetically obese) with diet-induced obesity high in carbohydrates and fats has not been reported. This study aims to identify the morphology, morphometry and distribution of Islands of Langerhans including insulin and glucagon immunoreactive cells in the pancreas of normal rats (non genetically obese) obesity induced using a diet high in carbohydrates and fats and compared with normal rats (control). This research using male rats, wistar strain, 4 weeks in age, consist of 5 rats control group and 5 rats obese group. The control group was feed with standard composition whereas the obese group was feed a high carbohydrate and fat with an additional induction of oral suspension fructose and cholesterol each doses 1%/weight homogenized in 2 ml Na CMC every day for 9 weeks. Euthanasia with chloroform used after the measurement of obesity index, counting blood glucose levels and lipid levels. Pancreas organ samples were fixed with Bouin’s solution, blocks with paraffin embedding and serial cut with a thickness of 5 μm. Pancreatic tissues were visualized by Haematoxilin eosin, Victoria blue, and immunohistochemical with Labeled Streptavidinbiotin (LSAB) staining methods. Primary antibody use mouse anti-insulin (1:1500; Abcam, USA), rabbit anti-glucagon (1:1500; CosmoBioCo., Japan) and TrekUniversal Link Kit (Biocare Medical, USA). The results were analyzed by descriptive and quantitative method. Lipid profile and glucose of obese group show total cholesterol (160.62±3.52) mg/dl, triglyceride levels (98.61±2.66) mg/dl, LDL (67.51±1.81) mg/dl, HDL (44.68±2.25) mg/dl and glucose levels (161.43±7.61) mg/dl. Topography pancreatic obese group spread in three locations (gastrium area, duodenum, and spleen). The morphology of Langerhans Island obese group are round, oval, polygonal and surroundings of fine fibers and capillary blood. Morphology of insulin and glucagon Immunoreactive cells in obese group are irregular polygonal shape with a core round/oval and located in central/peripheral. Insulin Immunoreactive cells in obese group spread in the middle and some on the edge of the island of Langerhans whereas glucagon Immunoreactive cells dominant spread on the edges and a little in the middle of the island of Langerhans.Morphometry of cells in obese group show Langerhans Island diameter (154.38±65.56) μm, Langerhans Island roving (373.51±146.69) μm, Langerhans Island volume 3,241,105.96 μm3, the total number of cells/Langerhans Island (181.90±96.76), the amount of insulin Immunoreactive cells (113.66±60.90) and the amount of glucagon immunoreactive cells (46.98±25.80). Obese group show differences with the control by increasing the value of total cholesterol, triglyceride levels, LDL, glucose levels, Langerhans Island diameter, Langerhans Island roving, Langerhans Island volume, the amount of insulin imunoreaktf cells/Langerhans Island and the amount of glucagon imunoreaktf cells/Langerhans Island (P<0.05). Morphology, morphometry and distribution of Langerhans island, insulin and glucagon immunoreactive cells in the pancreas of mice (non-genetically obese) induced obesity using a diet high in carbohydrates and fats show hyperplasia in insulin and glucagon immunoreactive cells as a compensatory increase in the volume of the island of Langerhans.

Kata Kunci : Obesitas, insulin, glukagon, Victoria blue, imunohistokimia.