CC BY
64
UDC 616.61-076.5:616-001.18/.19
MORPHOFUNCTIONAL CHARACTERISTICS OF THE MAST CELL POPULATION OF THE LIVER OF WHITE RATS BY DEEP IMMERSION HYPOTHERMIA (EXPERIMENTAL RESEARCH)
Altai State Medical University, Barnaul
Institute of Molecular Pathology and Pathomorphology of the Federal Research Center for Fundamental and Translational Medicine, Novosibirsk
A.Yu. Dolgatov1, I.P. Bobrov1, A.V. Lepilov1, N.G. Kryuchkova1, A.A. Alymova1, E.L. Lushnikova2, O.P. Molodykh2
The research objective was the estimation of the morphofunctional activity of mast cells of rat liver by hypothermia. The research was conducted on 20 Wistar rats. Hypothermia was modeled by means of immersion of the animals, caged individually, into the water of 5°C at the ambient temperature of 7°C. The criteria of the termination of the impact was rectal temperature 20-25 °C, that showed the deep stage of hypothermia. During the experiment, the animals were divided into 4 groups. The rats of the 1st group were killed immediately after the termination of cold impact, the animals of the 2nd group were killed 2 days after the experiment, the rats of the 3rd group were examined in 7 days and the 4th group animals were studied after 14 days of cold impact. The results of the research showed that the cold stress had an expressed influence on mast cells of rat liver. On the 2nd day of the experiment, the morphofunctional activity of mast cells increased, which coincided with the onset of regeneration of hepatic cells. On the 7th day (in the period of highest activity of adaptive processes) the quantative and morphometric parameters of mast cells were the highest. On the 14th day of the experiment, the activity of mast cells sufficiently decreased. Thus, hypothermia is a potent activator of the morphofunctional activity of tissue mast cells. Interstitial mast cells can be important factors in the activation of regenerative and adaptive processes in the liver in response to the damaging effect of the cold factor. Key words: hypothermia, liver, mast cells, adaptation.
Despite numerous studies devoted the death of people from cold, the diagnosis of cold death and fatal hypothermia continues to be relevant. At present, many aspects of the effect of cold on the human body have been thoroughly studied, and diagnostic and differential diagnostic criteria for death from hypothermia have been developed, but a whole view on compensatory-adaptive processes and adaptation of the organism to the cold factor in pathological anatomy and medical science is currently missing.
According to modern concepts, mast cells (MC) are the key link in the influence on metabolic processes, being of an important biological significance in tumor growth, regeneration, adaptation to stress and hypoxia [1, 2, 3, 4, 5, 6, 7 , 8, 9, 10].
The mast cell population in the tissues of the body is an independent regulatory system. This statement is confirmed by the fact that MC of various tissues of experimental animals are systematically included in the adaptive processes under the influence of extreme factors on it. At the same time, only a few works are devoted to the study of the value of MC by hypothermia. Thus, it has been shown that exposure to cold factor causes an increase in the number of MC and their degranulating forms in the skin, skeletal muscles and mesentery of the intestine of experimental animals [11, 12, 13].
The research objective was to study the effect of deep immersion hypothermia on the mor-
phofunctional activity of the mast cell population of the rat liver in the experiment.
Materials and methods
The study was performed on 25 Wistar rats. The hypothermia was modeled by placing animals in individual cages in water at a temperature of 5 °C at an ambient temperature of 7 °C. The criterion for the termination of exposure was the achievement by the animals of a rectal temperature of 20-25 °C, which corresponded to a deep degree of hypothermia. The exposure time was individual and averaged 40±5 min. During the experiment, the rats were divided into 4 groups. Animal of the 1st group (n = 5) were derived from the experiment by decapitation immediately after the termination of cooling, animals of the 2nd group (n = 5) - after 2 days, animals of the 3rd group (n = 5) - after 7 days, and animals of the 4th group (n = 5) - after 14 days. A morphological study of the liver of intact animals (n = 5), which served as a control group, was also conducted.
For histological examination, liver samples were fixed in 10% neutral formalin, then the material was wired on a TISSUE-TEK VIPTM6 machine (Sakkura, Japan), embedded in Histomix paraffin (TISSUE-TEK TEC 5 paraffin filling station, Sakkura, Japan). Sections 5-7 ^m thick were made on a Accu-Cut SRM rotary microtome (Sakku-ra, Japan), stained with hematoxylin and eosin
in a TISSUE-TEK Prisma automat (Sakkura, Japan) and placed under the film in a TISSUE-TEK Film automat (Sakkura, Japan). MC were revealed by toluidine blue (BioVitrum, Russia). Microscopic photographs of MC in the liver were obtained using a Nikon Eclipse E200 microscope (Japan) with a VIDI CAM digital video camera (Russia) at the zoom x 400. The distribution density of MC was calculated in five fields of view by the microscope zoom x 400 in the Image Tool. 3. The index of degranulation of TK (IDTK) was calculated (the percentage of cells in the state of degranulation of the total number of TK). The morphometry of MC was performed using the licensed morpho-metric program "VideoTest-Morphology 5.2". Statistical processing of the data was performed using the statistical package Statistica 6.0.
Results and discussion
The results of the study showed, that in the liver of rats of the control group, MC were located in the connective tissue of the portal tracts and along the sinusoids. They were small and round in shape (Figure 1). The number of cells varied from 1 to 2, their average number was 1.2 ± 0.2 in five fields of view by x 400 zoom. The MC area was 34.0 ± 0.7 |jm2. The granules were mainly located compactly in the cytoplasm of MC. The number of compact forms of MC was 83.2 ± 0.3%. The phenomenon of degranulation was noted in a small number of cells. DIMC (degranulation index) was equal to 16.8 ± 0.5% (Table 1).
Immediately after exposure to a single deep immersion hypothyroidism, MC in the liver tissue looked small, rounded or elongated, located alone in the portal tracts, localized near the vessels and bile ducts (Figure 2). The distribution density of MC was 4.0 ± 0.5 in five fields of view, the cell area was 40.2 ± 3.2 ^m2. Compact forms of MC equaled 84.5% ± 5.1. DIMC was 15.5% ± 5.1 (Table 1).
» - ♦ Jk
J ¿i* jri-
Jt* ^ET
■0 / ^ * r » % >'
£
i, gj .. *
tJ
Figure 1 - Small number of small mast cells in the portal tracts of the liver of rats of the control group (shown by arrows). Staining by toluidine blue. Zoom x 400.
Figure 2 - Increase in the number of mast cells in the portal tracts of the rat liver immediately after conducting deep hyperthermia (shown by arrows).
Staining by toluidine blue. Zoon x 400.
On the 2nd day after a single immersion hypothermia, MC in the liver were located in groups and singly in the portal tracts, in the stroma of the liver, near the vessels and bile ducts. MC looked polymorphic, with elongated cells predominating (Figure 3). The distribution density of MC at this time of the experiment was 10.2 ± 1.4, the area of MC - 51.5 ± 1.9 |jm2. Compact forms of MC were 75.8% ± 0.9. DIMC was equal to 24.2% ± 0.9 (Table 1).
Figure 3 - 2 days after hypothermia, the number of mast cells in the portal tracts of the rat liver increases, the cells are enlarged in size, polymorphic (shown by arrows). Staining by toluidine blue. Zoon x 400.
On the 7th day after hypothermia, MC were located in groups and singly in the portal tracts, near the vessels, the bile ducts and in the connective tissue stroma. At this period of the experiment, by their morphological characteristics, MC differed significantly from the cells of the previous study: they had large sizes, elongated and irregularly shaped cells prevailed (Figure 4). The distribution density of MC at this time of the experiment was 10.8±0.9, their area increased to 73.3±3.2 ^m2.
Compact forms of MC were 67.2% ± 1.9. DIMC was equal to - 32.8% ± 0.9 (Table 1).
Figure 4 - 7 days after hypothermia, the number of mast cells in the portal tracts of the rat liver is high, the cells are of large size (shown by arrows). Staining by toluidine blue. Zoon x 400.
On the 14th day of the experiment, MC were located in the portal tracts, mainly singly, near the vessels and bile ducts. In comparison with
Conclusion
The results of the study showed that a single deep immersion hypothermia had a significant impact on the morphofunctional activity of liver MC in experimental animals. The number and size of MC began to increase immediately after exposure to hypothermia, and after 7 days, during the active regeneration and adaptation of hepato-cytes, the number of MC and their morphometric parameters were the highest, and the number of de-granulating forms of these cells was maximum. By the 14th day of the experiment, the content of MC in the liver remained at the same level, but the area of MC and DIMC significantly decreased. The increase in the amount of MC in the liver tissue of animals on the 7th day of the experiment may be due to their migration from other organs. An 24
the previous experiment, the size of MC decreased. Round-shaped cells prevailed (Figure 5). The distribution density of MC at this time of the experiment was 9.0±1.4, the cell area was 57.9±2.3 ^m2. Compact forms of MC were 84.9% ± 3.1. DIMC was 15.1% ± 3.1 (Table 1).
a • <
Figure 5 - 14 days after hypothermia, the number of mast cells in the portal tracts of the rat liver decreases (shown by arrows). Staining by toluidine blue. Zoon x 400.
increase in the morphofunctional activity of MC in the posthypothermic period leads to an acceleration of regenerative processes in the liver tissue after the damage caused by exposure to the cold factor. Thus, hypothermia is a potent activator of the activity of the interstitial liver MC. Interstitial MC serve as important stimulators of hepatocyte regeneration in the process of adaptation of the liver to hypothermia.
References
1. A.F. Lazarev, I.P Bobrov, T.M. Cherdantse-va, V.V. Klimachev V.M. Brjuhanov, A.M. Avdaljan, V.A. Lubennikov, V.J. Gervald. Mast cells and tumor growth. Siberian Journal of Oncology. 2011; 4: 59-63.
2. Varricchi G., Galdiero M.R., Loffredo S., Marone G., Iannone R., Gianni Marone G., Granata
Table 1
Quantitative and morphometric characteristics of mast cells, depending on the duration of the experimental single deep immersion hypothermia
Mast cell parameters
Term of experiment MC number Compact MC number (%) DIMC MC area (^m2)
Control 1,2±0,2* 83,2±0,3 16,8±0,5 34,0±0,7*
Immediately after hypothermia 4,0±0,5* 84,5±5,1* 15,5±5,1* 40,2±3,2*
2 days after hypothermia 10,2±1,4* 75,8±0,9* 24,2±0,9* 51,5±1,9*
7 days after hypothermia 10,8±0,9 67,2±1,9* 32,8±0,9* 73,3±3,2*
14 days after hypothermia 9,0±1,4 84,9±3,1* 15,1±3,1* 57,9±2,3*
Note * - data are significant by <0.05.
F. Are mast cells masters in cancer? Front. Immunol. 2017; 8: 1-13.
3. Maciel T.T., Moura I.C., Hermine O. The role of mast cells in cancers. F1000Prime Reports. 2015; 7: 1-6.
4. Cherdantsev T.M., Bobrov I.P., Avdalyan A.M., Klimachev V.V., Kazartsev A.V., Kryuchko-va N.G., Klimachev I.V., Myadelets M.N., Lepilov A.V., Lushnikova E.L., Molodykh O.P. Mast cells in kidney cancer: clinical and morphological relationships and prognosis. Bulletin of experimental biology and medicine. 2017; 6: 768-773.
5. Kazarcev A.V., Cherdanceva T.M., Bobrov I.P., Lazarev A.F., Klimachev V.V.I. Intratumoral stromal mast cells in renal cell carcinoma: clinical and morphological comparisons. Russian journal of Ocnology. 2017; 1: 21-25.
6. Bobrov I.P., Cherdantsev T.M., Kryuch-kova N.G., Lepilov A.V., Lazarev A.F., Avdalyan A.M., Klimachev V.V., Myadelets M.N., Kazartsev A.V., Malinina E.I. Diagnostic and prognostic value of mast cell research in kidney cancer. Modern problems of science and education. 2017; 3: Science-educa-tion.ru
7. Artashyan O.S., Yushkov B.G. Participation of mast cells in the process of reparative regeneration of the liver. Journal of Ural Medical Academic Science. 2011; 2(1): 9-10.
8. Artashyan O.S., Yushkov B.G., Khramtso-va Yu.S. Morphological aspects of the participation of mast cells in the formation of a common adaptation syndrome. Tavricheskiy Mediko-Biologicheskiy Vestnik. 2012; 15(3): 22-25.
9. Artashyan O.S., Yushkov B.G., Khramtso-va Yu.S. Participation of mast cells in process of re-parative regeneration of the thyroid gland. Journal of Ural Medical Academic Science. 2013; 2: 9-10.
10. Klimin V.G., Artashyan O.S., Yushkov B.G. Mast-cells in hypoxia. Journal of Ural Medical Academic Science. 2006; 1: 45-48.
11. Lutsenko M.M., Lutsenko M.T. Morpho-functional characteristic of intestinal mesenterium on the general cooling of the organism. Bulletin Physiology and Pathology of Respiration. 2010; 37: 56-59.
12. Lychova N.A., Shakhmatov E.E., Kiselev V.E., Bobrov E.P., Lepilov A.V., Dolgatov A.U. Pathomorphology of skeletal musculator and mi-crocirculatory vascular bed in experimental hypo-thermism. Russian Journal of Forensic Medicine. 2017; 2: 12-16.
13. Bobr O.A., Myadelets O.D., Dubrovsky V.V. The dynamics of the population of mast cells of the wound process in rats subjected to hypo-biotic conditions (fasting, hypothermia). Vestnik VGMU. 2006; 4: 1-10.
Contacts
Corresponding author: Dolgatov Andrey Yur-yevich, Candidate of Medical Sciences, Associate Professor of the Department of forensic medicine
and pathological anatomy named after professor
VN. Kryukov with the course of FVE of the Altai
State Medical University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 408439.
E-mail: adolgatov@yandex.ru
Author information
Bobrov Igor Petrovich, Senior researcher of the Morphological Laboratory of the Center for Medical and Biological Research of the Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 669927. E-mail: ig.bobrov2010@yandex.ru
Lepilov Aleksandr Vasilyevich, Doctor of Medical Sciences, Head of the Department of forensic medicine and pathological anatomy named after professor VN. Kryukov with the course of FVE of the Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. Email: lepilov@list.ru
Kryuchkova Natalia Gennadyevna, Assistant Professor of the Department of forensic medicine and pathological anatomy named after professor VN. Kryukov with the course of FVE of the Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. Email: cruckova@yandex.ru
Alymova Yekaterina Yevgenyevna, 5 year student
of ASMU, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 408439.
E-mail: papasha199614@mail.com
Lushnikova Yelena Leonidovna, Doctor of Biological Sciences, Professor, Director of the Institute of Molecular Pathology and Pathomorphology, Head of the Department of Molecular Cell Biology and Morphology, Head of the Laboratory of Cytology and Cell Biology, Novosibirsk. 639117, Novosibirsk, ul. Timakova, 2. Tel.: +7 (383) 334-80-03. E-mail: pathol@inbox.ru
Molodykh Olga Pavlovna, Doctor of Biological Sciences, Professor, Head of the Laboratory of Mechanisms of Pathological Processes of the Institute of Molecular Pathology and Pathomorphology, Novosibirsk.
639117, Novosibirsk, ul. Timakova, 2. Tel.: +7 (383) 334-80-03. E-mail: pathol@inbox.ru
