ИЗУЧЕНИЕ МИГРАЦИОННОЙ АКТИВНОСТИ ЯДЕРНЫХ ЭРИТРОЦИТОВ И ЛЕЙКОЦИТОВ HOPLOBATRACHUS RUGULOSUS

Чан Тхи Фыонг Зунг¹, Нгуен Во Тхуан Тхань¹, Во Ван Тхань²

¹Лекторы, ²ORCID: 0000-0001-5553-0242, Лектор, аспирант, Педагогический университет г. Хошимина (Вьетнам)

ИЗУЧЕНИЕ МИГРАЦИОННОЙ АКТИВНОСТИ ЯДЕРНЫХ ЭРИТРОЦИТОВ И ЛЕЙКОЦИТОВ HOPLOBATRACHUS RUGULOSUS

Аннотация

Методом теста под агарозой изучены особенности миграционной активности ядерных эритроцитов и лейкоцитов у Hoplobatrachus rugulosus при разной температуре инкубации в опытах in vitro. Установлено, что при повышении и понижении температуры инкубации по сравнению с комнатной температурой площадь спонтанной миграции ядерных эритроцитов увеличивается и уменьшается соответственно. Для лейкоцитов данный показатель повышается при уменьшении температуры по сравнению с комнатной температурой. При всех изучаемых температурах инкубации площадь спонтанной миграции у эритроцитов была высше, чем у лейкоцитов.

Ключевые слова: миграционная активность, ядерные эритроциты, лейкоциты, лягушки.

Tran Thi-Phuong-Dung¹, Nguyen Vo-Thuan-Thanh¹, Vo Van-Thanh²

¹Lecturers, ²ORCID: 0000-0001-5553-0242, Lecturer, Postgraduate student, Ho Chi Minh City University of Education, Vietnam

INVESTIGATION OF THE MIGRATORY ACTIVITY OF HOPLOBATRACHUS RUGULOSUS NUCLEAR ERYTHROCYTES AND LEUKOCYTES

Abstract

By using method of the test under agarose, the features of erythrocyte and leukocyte migration activity in Hoplobatrachus rugulosus at different incubation temperature under condition invitro were studied. It is ascertained that raising and lowering the incubation temperature led to the increase and decrease of migratory activity, respectively, compared with room temperature. For leukocytes, this indicator was increased at reduced temperature, compared with room temperature. At all studied incubation temperatures, the area of spontaneous migration in erythrocytes was higher than that in leukocytes.

Keywords: migratory activity, nuclear red blood cells, white blood cells, frog.

Introduction

In the study of the mechanisms of adaptation, the study of the blood system might be one of the approaches, which directly or indirectly reacts to changes in the environment, objectively reflects the physiological status and allows to forecast the direction of adaptive reactions in the body.

Under the action of extreme factors on the organism, which include temperature, homeostatic constants will be changed. Hemocytes, especially white blood cells, having a high reactivity, are quickly included in the adaptation reactions. They are capable of non-specific response to alternating exposure. There are studies which report on the positive impact of high temperature on the factors of nonspecific resistance and immunegenesis.

In recent years, much attention has been paid to the study of migration and phagocytic activities of vertebrate animal blood cells [2, 6, 7, 10, 15]. The features of spontaneous migration and migration, stimulated by different substances of leukocytes under the changed functional and pathological conditions of organism were studied. The migration is one of the phases of the protective functions in the phagocytic blood cells [3–5, 8]. It’s known that the low vertebrate erythrocytes are capable of absorption of alien particles [13, 14]. However, scientific research regarding the features of migration reactions in nuclear erythrocytes and leukocytes remains little studied.

The purpose of our study was investigation of the migratory activity of Hoplobatrachus rugulosus leukocytes and nuclear erythrocytes under the action of temperature factor.

Material and methods

Experiments were carried out on frogs Hoplobatrachus rugulosus (30 individuals). The objects of the study were erythrocytes and leukocytes. The research was carried out in Ho Chi Minh City University of Education (Vietnam) at the Department of Anatomy and Physiology.

Peripheral blood taken from ether-narcotized frogs. Blood samples were from the heart. As an anticoagulant, heparin was used in a ratio of 10 units of heparin per 1 mL of blood suspension. [11]. The received blood samples were centrifuged for 10 min at a relative centrifugal force equal to 400g [10]. The leukocyte-rich lower part of the plasma and the leukocyte ring were collected. The washed and re-suspended red and white blood cells were counted in Goryaev chamber. In this work an isotonic solution (0.6% solution of NaCl) was used.

Spontaneous locomotion activity of hemocytes was evaluated in a test migration under agarose. Classic method described in many transactions [11] was used as the basic method (M. Z. Fedorova and V. N. Levin modification [8]). Hemocyte suspension containing about 300 thousand cells (were diluted with isotonic solution) was placed into the well cut out in the agarose gel applied on the object glass. The object glasses with erythrocytes and leukocytes were incubated at anaerobic conditions at temperatures of 20°C ­ in the refrigerator, 37°C ­ in thermostat, control ­ at room temperature (28^oC). One day later the cells were fixed for one hour with 10% glutaraldehyde. Then agarose was removed. Using the Romanovsky technique, hemocytes were stained with azure-eosin. The spontaneous migration areas of blood cells were determined using the software ImageJ 1.47v.

The obtained results were treated by methods of the statistical variation with the use of special software on a computer. The significance of differences was determined by Student’s t-criterion. The level of statistical significance was p≤0.05.

Results

Migratory activity of blood cells was evaluated by area of their distribution after 24 hours of incubation. Indicators of the area of spontaneous migration of Hoplobatrachus rugulosus hemocytes at different incubated temperatures are shown in the table.

Table 1 – Area of the distribution of Hoplobatrachus rugulosus blood cells after 24 hours of incubation at different temperatures, mm²

Type of cells	Incubation temperature, oC
	20	28	37
Erythrocytes	4.17±0.74 a A	5.19±0.90 b A	5.66±0.50 c A
Leukocytes	3.13 ±0.17 a B	2.78±0.03 b B	2.64±0.28 b B

Notes: ^{a, b, c} – significant difference of indicator in a single row; ^{A, B} – significant difference of indicator in a single column (p≤0.05)

The table shows that the area of distribution of erythrocytes was significantly changed by different incubated temperatures. When temperature of the incubation increases to 37^oC, the area of spontaneous migration of red blood cells increases by 9.06% compared with incubation at 28^oC. At reduced incubation temperature to 20^oC, migration area was 19/65% lower than that at temperature of 28^oC.

In leukocytes is observed the opposite pattern. At elevated temperature of the incubation, the changes of migration area of leukocyte were not observed (see the table). When temperature of the incubation reduces to 20^oC, the area of spontaneous migration increases by 12.59% compared with room temperature.

Comparative analysis of the migration area of erythrocytes and leukocytes is shown that at incubated temperatures of 20, 28 and 37^oC, migration area of red blood cells was 24.94, 46.44 and 53.36%, respectively, higher than that of white blood cells.

Discussion

It is known that for the realization of functions of phagocytes necessary to have the presence and a certain value of the “membrane reserve”, embedded in the folding of plasma membrane. The reserve of plasma membrane can be used in the formation of pseudopodia in red blood cells (except mammals), leukocytes and platelets at amoeboid movement [10, 11]. At the expense of membrane folds, passing through the narrow capillaries, phagocytes are deformed with an increase in surface area at constant volume [9-11]. The value of membrane reserve of mammal leukocytes (and especially, of human) was well studied. In nuclear red blood cells of amphibians also was revealed the presence and studied the value of “membrane reserve” that allows to implement the migration reaction and phagocytosis [12]. In studies conducted by us revealed that red blood cells of amphibians migrate.

It can be assumed, that enhancement of spontaneous locomotion of cells with increasing incubation temperature is a consequence of the activation of plasmalemma caused by thermal factor. An indirect confirmation of this is the work [5], which stated that the increase in hemocyte activity occurs not only in inflammation, but can be caused by various by nature agents.

It is known that while decreasing the ambient temperature, in the frogs is reduced body temperature and sharply limited motor activity [1]. The obtained results about migration activity of the blood cells by lowering the incubation temperature to 20°C are consistent with functional activity of animals [1]. Decrease or increase in temperature for the blood cells of functionally active animals is a factor that contributes to the activation of plasmalemma and increased motor activity of the cells.

At all studied incubation temperatures, migration area of erythrocytes was higher than that of leukocytes. This is confirmed by the work of Chernyavskikh, which states that under the same conditions of incubation, the red blood cells migrated edge of the incubation hole more active than white blood cells [9].

Список литературы / References

1. Акуленко Н.М. Сезонная динамика эритропоэза и его топографическое распределение у лягушки озерной // Запорізького національного університету. – 2008. – C. 5.
2. Галактионов В.Г. Эволюционная иммунология / В.Г. Галактионов. – М.: Академкнига. – 2005. – 408 c.
3. Ерюхин И.А. Воспаление как общебиологическая реакция: на основе модели острого перитонита / И.А. Ерюхин, В.Я. Белый, В.К. Вагнер. – Л.: Наука. – 1989. – 262 c.
4. Козинец Г.И. Кровь и инфекция / Г.И. Козинец, В.В. Высоцкий, В.М. Погорелов и др. – М.: Триада-фарм. – 2001. – 456 c.
5. Маянский А.Н. Очерки о нейтрофиле и макрофаге / А.Н. Маянский, Д.Н. Маянский. – Новосибирск: Наука. –1989. – 344 c.
6. Меньшиков И.В. Основы иммунологии. Лабораторный практикум / И.В. Меньшиков, Л.В. Бедулева. – Ижевск: Изд. Дом «Удмуртский университет». – 2001. – 136 c.
7. Мечников И.И. Лекции по сравнительной патологии воспаления / И.И. Мечников. – Москва. – 1954.
8. Федорова М.З., Левин В.Н. Спонтанная миграция нейтрофилов крови в смешанной популяции лейкоцитов и ее изменения под влиянием веществ аутоплазмы при различных функциональных состояниях организма / М.З. Федорова, В.Н. Левин // Клиническая лабораторная диагностика. –2001. – № 5. – C. 16–19.
9. Чернявских С.Д. Миграционная активность гемоцитов позвоночных животных при различной температуре // Научные ведомости Белгородского государственного университета. Серия Естественные науки. –2011. –№ 14 (3 (98)). – C. 150–154.
10. Chernyavskikh S.D. Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration / S.D. Chernyavskikh, M.Z. Fedorova, Vo Van Thanh, Do Huu Quyet // Cell and Tissue Biology. – 2012. – № 4 (6). – P. 348–352.
11. Duglas S. Investigation into Phagocytosis in the Clinical Practice / S. Duglas, P. Kui. – Moscow: Medicina. –1983. –112 p.
12. Fedorova M.Z. Comparative evaluation of morphofunctional organization of nucleated blood cells of vertebrate animals / M.Z Fedorova, S.I. Golovko, S.D. Chernyavskikh // Journal of Evolutionary Biochemistry and Physiology. – 2012. – Vol. 48, Issue 2. – P. 209–213
13. Prunesco P. Natural and experimental phagocytosis by erythrocytes in amphibians / P. Prunesco // Nature: New Biology. – 1971. – № 22 (231). – P. 143–144.
14. Prunesco P. Phagocytosis by avian red cells / P. Prunesco, C. Prunesco // The Science of Nature. – 1972. – № 1 (59). – P. 41.
15. Raffel T.R. Negative effects of changing temperature on amphibian immunity under field conditions / T.R. Raffel, R. Rohr, J.M. Kiesecker, P.J. Hudson // Functional Ecology. – 2006. – № 5 (20). – P. 819–828.

Список литературы на английском языке / References in English

1. Akulenko N.M. Sezonnaja dinamika jeritropojeza i ego topograficheskoe raspredelenie u ljagushki ozernoj [Seasonal dynamics of erythropoiesis and its topographic distribution of the lake frog] // Zaporіz'kogo nacіonal'nogo unіversitetu [Zaporizhzhya National University]. 2008. P. 5. [in Russian]
2. Galaktionov V.G. Jevoljucionnaja immunologija [Evolutionary immunology] / V.G. Galaktionov, M.: Akademkniga, 2005. 408 p. [in Russian]
3. Erjuhin I.A. Vospalenie kak obshhebiologicheskaja reakcija: na osnove modeli ostrogo peritonita [Inflammation is a general biological reaction: based on the model of acute peritonitis] / I.A. Erjuhin, V.Ja. Belyj, V.K. Vagner, L.: Nauka, 1989. 262 p. [in Russian]
4. Kozinec G.I. Krov' i infekcija [Blood and infection] / G.I. Kozinec, V.V. Vysockij, V.M. Pogorelov and others, M.: Triada-farm., 2001. 456 p. [in Russian]
5. Majanskij A.N. Ocherki o nejtrofile i makrofage [Essays about the neutrophils and macrophages]/ A.N. Majanskij, D.N. Majanskij, Novosibirsk: Nauka, 1989. 344 p. [in Russian]
6. Men'shikov I.V. Osnovy immunologii. Laboratornyj praktikum [Fundamentals of Immunology. Laboratory workshop] / I.V. Men'shikov, L.V. Beduleva, Izhevsk: Izd. Dom «Udmurtskij universitet», 2001. 136 p. [in Russian]
7. Mechnikov I.I. Lekcii po sravnitel'noj patologii vospalenija [Lectures in comparative pathology of inflammation] / I.I. Mechnikov, M., 1954. [in Russian]
8. Fedorova M.Z., Levin V.N. Spontannaja migracija nejtrofilov krovi v smeshannoj populjacii lejkocitov i ee izmenenija pod vlijaniem veshhestv autoplazmy pri razlichnyh funkcional'nyh sostojanijah organizma [Spontaneous migration of neutrophils in the blood of a mixed population of white blood cells and its changes under the influence of substances autoplasma in different functional states of an organism] // Klinicheskaja laboratornaja diagnostika [Clinical Laboratory Diagnostics]. 2001. (5). P. 16–19. [in Russian]
9. Chernjavskih S.D. Migracionnaja aktivnost' gemocitov pozvonochnyh zhivotnyh pri razlichnoj temperature [The migration activity of hemocytes of vertebrates at different temperatures] // Nauchnye vedomosti Belgorodskogo gosudarstvennogo universiteta. Serija Estestvennye nauki. [Scientific statements Belgorod State University. Series Natural sciences]. 2011. № 14 (3 (98)). P. 150–154. [in Russian]
10. Chernyavskikh S.D. Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration / S.D. Chernyavskikh, M.Z. Fedorova, Vo Van Thanh, Do Huu Quyet // Cell and Tissue Biology. 2012. № 4 (6). P. 348–352.
11. Duglas S. Investigation into Phagocytosis in the Clinical Practice / S. Duglas, P. Kui, Moscow: Medicina, 1983. 112 p.
12. Fedorova M.Z. Comparative evaluation of morphofunctional organization of nucleated blood cells of vertebrate animals / M.Z Fedorova, S.I. Golovko, S.D. Chernyavskikh // Journal of Evolutionary Biochemistry and Physiology. 2012. Vol. 48, Issue 2. P. 209–213
13. Prunesco P. Natural and experimental phagocytosis by erythrocytes in amphibians / P. Prunesco // Nature: New Biology. 1971. № 22 (231). P. 143–144.
14. Prunesco P. Phagocytosis by avian red cells / P. Prunesco, C. Prunesco // The Science of Nature. 1972. № 1 (59). P. 41.
15. 15. Raffel T.R. Negative effects of changing temperature on amphibian immunity under field conditions / T.R. Raffel, R. Rohr, J.M. Kiesecker, P.J. Hudson // Functional Ecology. 2006. № 5 (20). P. 819–828.