Патохистолошка и имунохистохемиска анализа на антителата во колон кај стаорци со предизвикан колитис и третирани со микроинкапсулиран синбиотик

Abstract

Inflammatory bowel disease with its two forms (ulcerative colitis and Crohn’sdisease) presents a worldwide health problem in the human and animal population. The disrupted wellbeing and the expensive treatment for the disease, imposes the need for finding an alternative preventive measures. That’s why the worldwide attention is focused on the use of functional food with probiotics, prebiotics and sinbiotics for prevention to this as well as other diseases. The poor understanding of the etiology and the pathogenesis of the disease as well as the need for styduing the properties of different probiotics and their influence on the immunological system were the goals of this doctoral dissertation. This work presents the results from the investigation of the structural, histochemical and immunohistochemical changes in the colon, treated with new functional product in experimental model of colitis induced with threenitrobensene sulfonic acid (TNBS). The investigation was performed on female Wistar rats with body weight of 180-250 g and age of 10 – 14 weeks, devided in five groups. The materials (colon) are fixated in neutral formalin, embaded in parafin and cut by mictotom on 3-5 μм thick slides. The hematoxilin and eosin (H.E.) and Periodic Acid Schiff (PAS)methods are used for staining of the slides, and for the immunohistochemical method En Vision Kit (Dako ChemMate, Denmark) is used with the following primary antibodies: monoclonalrabbit-anti-human CD3 for T-lymphocites (DAKO), monoclonalmouse-anti-human CD79αcy and CD20cy for B-lymphocites (DAKO), monoclonalmouse-anti-human CD68 and monoclonalmouse-anti-human MAC387 (DAKO) for macrophages. For the purpose of the investigation, the rats were treated with ayran (Zdravje Radovo, Makedonija) containing microencapsulated synbiotic (once and twice per day) and ayran with blank microparticles. The microparticles were prepared of alginate and chitosan, while the symbiotic was composed of probiotic Lactobacillus casei 01 and the prebiotic inulin enriched with oligofructose, mixture of oligofructose with polymerization degree 2-8 and long-chain inulin fraction (DP 10-60), Synergy 1. The application of TNBS (30 mg/kg) caused changes to the colon, adequate for simulation of the Crohn’s disease. From patomorphologic aspect, the changes of the colon are in the form of hyperemic areas, ulcerations, decrease in the number of goblet cells and the amount of mucin, as well as infiltration with macrophages, lymphocites and leukocites. The immunohistochemical method shows that the concentration of the CD3+ T- lymphocites is significantly higher in the positive control, distributed in all the layers of the colon: the mucosa (if present), the submucosa, muscularis and in some cases, the serosa as well as the peripheral layer of the lymphoid aggregates. The rats in the positive control group had higher concentration of CD20 и CD79α-positive cells compared to the negative control group, distributed trough the whole structure of the colon: the mucosa (if present), the submucosa, muscularis and in some cases, even the serosa as well as the celtral area of the lymphoid aggregates. The number of the CD79α-positive cells was significantly higher compared to the CD20-posutive B-cells. The number of CD68 и MAC387-positive macrophages in the colitic group was significantly increased in the deep lamina propria, between the crypts and near the crypts. The results from this examination showed that the ayran containing microencapsulated synbiotic (without diference between the groups treated once and twice per day) has a great therapeutic potential. The use of monoclonal antybodies for the cell of the immunological system pruve that the anti-inflammatory property of the ayran with microencapsulated synbiotic is due to improvement of the epithelial barier and the decrease of the inflammatory infiltrate composed of T and B-lymphocies, plasma cells and tissue macrophages. The immunohistochemical method allows for simulataneus observation of the tissue changes and the distribution of immunological cells in it.