IMMUNOGISTOCHEMICAL ANALYSIS OF PEYER’S PATCHES OF THE ALBINO RATS’ SMALL INTESTINE IS NORMAL

Hryn V. H.

MORPHOLOGY

Scentific article

Peyer’s patches are the integration of a number of individual lymphoid nodules, each of which usually distinguished zones with their predominant localization in the T- and B-lymphocytes. In addition, their key structure in which the initiation of immune reactions is carried out is their apical sections, a polarized monolayer of different types of enterocytes, among which mainly absorption, goblet and so-called M-cells. The aim of this study was to carry out an immunohistochemical analysis and clarify areas of primary localization of the main types of immunocompetent cells of Peyer’s patches of the albino rats small intestine is normal. Object and methods of research. 30 mature albino male rats weighted 200,0±20,0 g were involved into the study. For the study used portions of the small intestine, which are the Peyer’s patches. To study the immunohistochemical (IHC) features of Peyer’s patches, the material was fixed with 10% neutral formalin for 24 h, embedded in paraffin, sections were prepared with a thickness of 4 μm, which were applied to highly adhesive Super Frost glasses and dried at a temperature of 37°C for 18 hours. The unmasking heat treatment was performed by the method of boiling sections in citrate buffer (pH 6.0). For visualize primary antibody detection system used Ultra Vision Quanto Detection Systems HRP Polymer (Thermo scientific). It was used as the chromogen DAB (diaminobenzidine). Assessment of cell composition and immune cell responses in Peyer’s patches, their lymphoid nodules, and lymphoid-associated epithelium was detected by expression of T- and B-cell differentiation clusters (CD3, CD79), plasma cell marker (CD38), macrophage marker (CD68 (KP1)), a dendritic cell marker (CD23), an epithelial cell marker of PAN-cytokeratin (Cytokeratin PAN AE1/AE3). Used primary monoclonal antibodies (moAb) firm Thermo scientific (Germany), Ready-to-Use. Results were calculated using the eyepiece grid Avtandilov a 10 randomly selected fields at X400 magnification. Evaluation IHC label was determined by the distribution. The extent of the label into account for the percentage of positively stained in brown color of the membranes (and the cytoplasm – for CD68) cells by the total number of cells in the visual field. The complex morphological studies carried out on microscope Primo Star (Carl Zeiss) using a AxioCam program (ERc 5s). Results of work. As a result, immunohistochemistry Peyer’s patches of the small intestine intact animals received generalized data on the quantitative ratio therein between main populations of immunocompetent cells. Conclusions. The bulk of immunocompetent cells were B-lymphocytes (about 47%) and T-lymphocytes (about 35%), while plasmacytes, macrophages and dendritic cells accounted for about 5% for each of them. But it was not possible to obtain not only information about the number of lymphoid-associated epithelium of the Peyer’s patch M cells, but even proved fruitless pursuit of their elective clearer identification.

immunohistochemical method, Peyer’s patches, lymphoid-associated epithelium, M-cells, dendritic cells, plasmocytes, B- and T-lymphocytes.

1. Dillon A, Lo DD. M Cells: Intelligent Engineering of Mucosal Immune Surveillance. Front. Immunol. 2019;10:1499. DOI: 10.3389/ fimmu.2019.01499
2. Bykov AS, Karaulov AV, Tsomartova DA, Kartashkina NL, Goryachkina VL, Kuznetsov SL, i dr. M-kletki – odin iz vazhnykh komponentov v initsiatsii immunnogo otveta v kishechnike. Infektsiya i immunitet. 2018;8(3):263-72. Dostupno: https://doi.org/10.15789/2220-7619-2018- 3-263-272 [in Russian].
3. Kanaya T, Ohno H. The Mechanisms of M-cell Differentiation. Biosci Microbiota Food Health. 2014;33(3):91-7. DOI: 10.12938/bmfh.33.91
4. Da Silva C, Wagner C, Bonnardel J, Gorvel JP, Lelouard H. The Peyer’s Patch Mononuclear Phagocyte System at Steady State and during Infection. Frontiers in immunology. 2017;8:1254. DOI: 10.3389/fimmu.2017.01254 Available from: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC5630697/
5. Morikawa M, Tsujibe S, Kiyoshima-Shibata J, Watanabe Y, Kato-Nagaoka N, Shida K, et al. Microbiota of the Small Intestine Is Selectively Engulfed by Phagocytes of the Lamina Propria and Peyer’s Patches. PloS one. 2016;11(10):e0163607. DOI: 10.1371/journal.pone.0163607 Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049916/
6. Lelouard H, Fallet M, de Bovis B, Méresse S, Gorvel JP. Peyer’s patch dendritic cells sample antigens by extending dendrites through M cellspecific transcellular pores. Gastroenterology. 2012;142(3):592-601. Available from: https://www.sciencedirect.com/science/article/abs/pii/ S0016508511016374
7. Hryn VH, Kostylenko YuP, Korchan NA, Lavrenko DA. Structural form of the follicle-associated epithelium of peyers’ patches of the albino rats’ small intestine. Georgian medical news. 2019;9(294):118-23. Available from: http://www.geomednews.org/shared/issues/med294.pdf
8. Hryn V. Internal structure of the lymphoid nodules of the peyer’s patches of small intestine in albino rats. Georgical Medical News. 2019;11(296):122-6. Available from: https://www.ncbi.nlm.nih.gov/pubmed/31889718
9. Rybakova AV, Makarova MN. Sanitarnyy kontrol’ eksperimental’nykh klinik (vivariyev) v sootvetstvii s lokal’nymi i mezhdunarodnymi trebovaniyami. Mezhdunarodnyy vestnik veterinarii. 2015;4:81-9. Dostupno: https://rucont.ru/efd/379080 [in Russian].
10. Dobrelya NV, Boytsova LV, Danova IV. Pravova baza dlya provedennya etychnoyi ekspertyzy doklinichnykh doslidzhenʹ likarsʹkykh zasobiv z vykorystannyam laboratornykh tvaryn. Farmakolohiya ta likarsʹka toksykolohiya. 2015;2:95-100. Dostupno: http://www.irbis-nbuv. gov.ua/cgi-bin/irbis_nbuv/cgiirbis_64.exe?I21DBN=LINK&P21DBN=UJRN&Z21ID=&S21REF=10&S21CNR=20&S21STN=1&S21FMT=ASP_ meta&C21COM=S&2_S21P03=FILA=&2_S21STR=flt_2015_2_16 [in Ukrainian].
11. Svitlychnyy O, Berehelya I. Administratyvnyy zakhyst tvaryn, yaki vykorystovuyutʹsya v naukovykh eksperymentakh, navchalʹnomu protsesi ta vyrobnytstvi biolohichnykh preparativ, vid zhorstokoho povodzhennya. Pidpryyemnytstvo, hospodarstvo i pravo. 2017;2:150-4. Dostupno: http://www.irbis-nbuv.gov.ua/cgi-bin/irbis_nbuv/cgiirbis_64.exe?I21DBN=LINK&P21DBN=UJRN&Z21ID=&S21REF=10&S21CNR=20&S21STN =1&S21FMT=ASP_meta&C21COM=S&2_S21P03=FILA=&2_S21STR=Pgip_2017_2_30 [in Ukrainian].
12. Avtandilov GG. Osnovy kolichestvennoy patologicheskoy anatomii [monografiya]. M.: Meditsina; 2002. 240 s. Dostupno: https://www. mmbook.ru/catalog/patanatomija-gistologija-morfologija/105036-detail [in Russian].
13. Fal’chuk YL, Markov AG. Izucheniye bar’yernykh kharakteristik epiteliya peyyerovykh blyashek krysy. Vestnik SPbGU. Seriya 3: Biologiya. 2015;3(3):75-86. Dostupno: http://proxy.library.spbu.ru:2110/item.asp?id=24307669 [in Russian].
14. Al’berts B, Dzhonson A, L’yuis D. Molekulyarnaya biologiya kletki: v 3-kh tomakh. M.: Izhevsk: NITS «Regulyarnaya i khaoticheskaya dinamika», Institut komp’yuternykh issledovaniy. 2013;III. 1028 s. Dostupno: http://chembaby.com/wp-content/uploads/2015/12/MBK3.pdf [in Russian].
15. Kucharzik T, Lügering N, Schmid KW, Schmidt MA, Stoll R, Domschke W. Human intestinal M cells exhibit enterocyte – like intermediate filaments. Gut. 1998 Jan;42(1):54-62. DOI: 10.1136/gut.42.1.54
16. Madara JL. Functional morphology of Epithelium of the Small Intestine. Comprehensive Physiology. Handbook of Physiology, The Gastrointestinal System, Intestinal Absorption and Secretion. 2011;19:83-120. Available from: https://doi.org/10.1002/cphy.cp060403
17. Neutra MR, Mantis NJ, Kraehenbuhl JP. Collaboration of epithelial cells with organized mucosal lymphoid tissues. Nature Immunology. 2001;2:1004-9. DOI: 10.1038/ni1101-1004

«Bulletin of problems biology and medicine» Issue 1 (155), 2020 year, 292-296 pages, index UDK 616.341:599.323.4

10.29254/2077-4214-2020-1-155-292-296