AGE-RELATED CHANGES IN THE STRUCTURAL, HYDRATION AND CHEMICAL PARAMETERS OF VISCERAL LYMPH NODES

ABSTRACT

The concept of the lymphatic region changes the point of view about the stereotypical changes in the structure of lymph nodes. Using a set of methods (morphological, thermogravimetric, X-ray fluorescence analysis), the structure, hydration and trace element profile of visceral lymph nodes in aging were evaluated. Without excluding the general signs of aging, the regional specificity of the mesenteric and tracheobronchial lymph nodes is shown. Each of these lymph nodes differs in the dimension of the structures of the cortical and medullary matter belonging to the T - and B-zones, the redistribution of free and bound water fractions during dehydration, the formation of an individual trace element profile with a general deficiency of trace elements. The observed changes have an age-induced cause, a region-dependent nature and indicate a weakening of the drainage and immune function of the lymph nodes that have undergone senile changes.

Keywords: lymph node, morphology, trace elements, gerontology.

INTRODUCTION

The problem of aging and the issue of health and quality of life of older people remains relevant today due to the characteristic of morphological changes on the part of different organs and systems of the body. Aging is an inevitable process accompanied by disorganization of the structure and function of many organs, including lymph nodes belonging to the lymphatic system [1-3]. Lymph nodes perform an important function in providing drainage and forming an immune response in the region, and they are indicators of the state of the internal environment [1, 4]. At the same time, the prognostic significance of assessing the state of the compartments of regional lymph nodes in their relationship with the drained areas remains one of the main research directions in the morphophysiology of aging.

In the lymphatic region, water, microelement and immune homeostasis depends on the lymph nodes in different periods of life. It is the lymph nodes that are an important element of fluid and trace element metabolism in the lymphatic regions of the body. Age-related changes in the fluid and trace element balance are accompanied by a violation of the mechanism of adaptive structural rearrangements and the immune response [5-7], which is directly related to the lymph nodes. Visceral lymph nodes, which are subject to age-related transformation, are actively involved in this. Those things actualizes the research of the morphophysiology of the lymphatic (lymphoid) system as a life support system associated with endoecological safety and the need to increase nonspecific resistance in aging [1, 3, 4].

The aim of the research is to study the structural organization of visceral lymph nodes with their water and trace element composition during aging.

MATERIALS AND METHODS

The experiment on animals and their removal from the experiment were carried out in accordance with the principles of bioethics, the rules of laboratory practice set out at the Geneva Conference (1971), as well as in the documents "On the humane treatment of experimental animals" (Ministry of Health of the USSR No. 775 of 12.08.1977), "International recommendations for conducting biomedical research using animals" (1985) and in accordance with the order of the Ministry of Health of the Russian Federation No. 267 of 19.06.2003. The study was approved by the NIIKEL Ethics Committee (Protocol No. 126 of 30.11.2016).

The experiment involved 80 white male Wistar rats with conditional allocation of age groups - "young" (3-5 months) and " old " (1.5–2 years). The animals received a standard diet (extruded compound feed) with free access to water and were controlled. Visceral lymph nodes (mesenteric and tracheobronchial) belonging to different lymphatic regions were selected as the object of the study.

Histological examination of lymph nodes. The collected lymph nodes were fixed in 10% neutral formalin. This was followed by the classical scheme of wiring and pouring the material into paraffin, followed by the preparation of histological sections. Histological sections were stained with hematoxylin-eosin, a trichromic dye according to C. Masson and subjected to morphometry.

The thermogravimetric method is used to assess hydration with the determination of different water fractions (total, free and bound) and the coefficients of hydration and density in the lymph nodes [8, 9]. The principle of the method is to dry the lymph node at a constant temperature with the registration of changes in the weight of the organ on the analytical balance. Knowing the volume of the lymph node and the area of its structures, the volume of fluid falling on a particular structure of the lymph node was calculated in accordance with the Cavalieri–Aker–Glagolev principle.

The content of trace elements (Se, Mn, Fe, Cu, Zn) in the lymph nodes was determined by X-ray fluorescence analysis using synchrotron radiation (X-ray diffraction). In the work, the equipment of the CCP "SCS" was used on the basis of the Novosibirsk LSE UNU / "VEPP-4 – VEPP-2000 Complex" at the Institute of Nuclear Physics SB RAS, Novosibirsk [10].

Statistical processing of the research results was carried out using the Excel program and StatPlus Pro, AnalystSoft Inc. The arithmetic mean was calculated with the determination of its standard error. The Student's t-test was applied to assess the level of statistical significance of differences (p ≤ 0.05) under the condition of a normal distribution or close to it.

RESULTS AND DISCUSSION

The general pattern of aging is the involution of lymphoid tissue with a minimization of the size of structural and functional zones, an increase in connective tissue, a decrease in the area of secondary lymphoid nodules in the lymph nodes, regardless of their localization, in comparison with young animals. At the same time, there is a formation of regional specifics of the internal structure of lymph nodes, which determines their functional specialization during aging.

Mesenteric lymph node. With age, the structure of the mesenteric lymph node belonging to the intestinal lymphatic region is reorganized (Table 1). An increase in connective tissue in the mesenteric lymph node is indicative. Senile changes relate to the structural and functional zones of the cortical substance. One of the signs of aging is considered to be a decrease in the size of the interstitial part [4, 11], but this is not always the case. Morphometry revealed that the interstitial part of the cortex (cortical plateau) decreases by 2.1 times in the mesenteric lymph node. The paracortical region retains its size, but its structure is depleted of cells, demonstrating the syndrome of cellular emptying. With age, the area of lymphoid nodules with a germinal center (B-zone) decreases by 1.9 times to a certain level of 5%, which corresponds to their low activity during aging (Table 1). The area of the structures of the mesenteric lymph node medulla changes. Aging leads to the dominance of the B-dependent zone, represented by brain cords, and a uniform 1.9-fold narrowing of the sinus system with the exposure of the reticular stroma of the mesenteric lymph node.

Structural reorganization of the mesenteric lymph node is associated with dehydration and changes in the lymphatic drainage mechanism during aging. A 32.7% decrease in the volume of total water in the mesenteric lymph node is characteristic in comparison with young animals (Table 2). At the same time, the transport capacity of the lymph node changes due to a 2-fold decrease in the free fraction of water – the volume of free fluid (lymph) in the sinus system, which correlates with its area (Tables 1, 2). At the same time, the volume of the bound water fraction remains at the same level in the lymph node, which is important for preserving the functions and viability of cells and tissues [8, 9]. The decrease in the water saturation of lymph nodes is associated with the deposition and decrease in the transit of lymph volume through the sinus system in old animals, the value of increased hydration coefficients (0.83±0.06) and density (0.82±0.07 mg/mm3) indicates the dehydration of the mesenteric lymph node associated with the age-related transformation of the microanatomic organization.

With age, the microelement homeostasis also changes, which determines the size of the compartments and the immune function of the lymph nodes. The formation of a trace element profile is characterized by a decrease in the iron content by 1.4 times and zinc, selenium, copper by 1.2 times, with a tendency to increase manganese by 1.2 times in comparison with young animals (Table 3). This forms a qualitatively different trace element profile with a deficient content of most trace elements in old age.

The morphofunctional provision of the mesenteric lymph node characterizes the weakening of its drainage and immune functions, which is due to the involution of lymphoid tissue, dehydration and deficiency of most trace elements with an age-induced cause [4, 12].

Table 1.

The area of the main structures of the lymph nodes in young and old animals, %

Note: in tables 1-3 *P1-2, 3-4 < 0.05; ° P1–3, 2-4 < 0.05 – the level of statistical significance of the differences

Table 2.

Indicators of total water and its structural fractions in the lymph nodes of young and old animals, %

Table 3.

The content of trace elements in the lymph nodes of young and old animals, %

Tracheobronchial lymph node. With age, the structure of the tracheobronchial lymph node belonging to the bronchopulmonary region is reorganized (Table 1). To a greater extent, they are subject to sclerotic changes and replacement of lymphoid tissue with connective or adipose tissue. The structures of the cortical substance, including the cortical plateau and the paracortex, tend to decrease in comparison with young animals. The area of lymphoid nodules with a germinative center (B-zone) of the tracheobronchial lymph node significantly decreases by 2.3 times, causing their low activity of cell proliferation during aging (Table 1). There was a 1.2-fold decrease in the structures of the medulla, including the brain cords and the brain sinus. In the tracheobronchial lymph node, the size of the area of the interstitial part of the cortex, brain cords (B-zone), paracortex (T-zone) against the background of narrow lymphatic sinuses determines a weakened immune response of a mixed type with limited drainage function during aging.

A sign of an aging tracheobronchial lymph node is the process of dehydration (Table 3). This is reflected by a decrease in the volume of total water by 7.28% and by 1.7 times the volume of the free fraction of water in comparison with young animals. The fact of a decrease in the volume of free fluid (lymph) correlates with the size of the sinus system, which indicates a low level of functioning of the lymphatic drainage mechanism in the tracheobronchial lymph node. At the same time, the volume of the bound fraction of water in the lymph node increases by 1.2 times. The water saturation of the tracheobronchial lymph node is reflected by the coefficients of hydration (2.42±0.08) and density (1.30±0.17 mg/mm3), which indicate significant dehydration against the background of senile transformation of the lymph node structure.

Age-related structural and hydration parameters of the lymph node are combined with the formation of a certain microelement profile. The level of iron and copper content did not change in the lymph node. There was a decrease in zinc (by 1.2 times), selenium (by 1.5 times) and an increase in manganese (by 1.3 times) with aging. We assume that the formed trace element profile reflects the change in the size of compartments due to a decrease in the proliferation of lymphoid cells and, consequently, the function of the lymph node during aging.

A comparative analysis of visceral lymph nodes determined the peculiarity of the manifestation of aging in accordance with the implementation of the principle of the regional determinant of the concept of the lymphatic region [4]. It was found that during aging, lymph nodes have distinctive features depending on their localization (regional specificity), based on the quantitative and qualitative characteristics of individual structural and functional zones, hydration and trace element profile.

Belonging to the lymphatic region determines the structural relationships within the lymph node during aging. The involution of lymphoid tissue occurs in a non-uniform manner. Thus, the mesenteric lymph node is distinguished by the small size of the cortical plateau with enlarged structures of the medulla, and the tracheobronchial lymph node, on the contrary, is characterized by a wide interstitial area of the cortex (cortical plateau) with reduced sizes of structures of the medulla (Table 1). The minimum size of lymphoid nodules is common for lymph nodes.

The localization of the lymph node is decisive in the formation of the morphological variant of the structure, based on the predominance of thymus-dependent and/or thymus-independent zones, the size of the sinus system, and, as a result, its functional specialization, associated in the tracheobronchial lymph node with the performance of immune function (B-zone), and in the mesenteric lymph node – simultaneously with drainage and immune functions.

Despite the general dehydration of visceral lymph nodes during aging, they have their own characteristics (Table 2). The tracheobronchial lymph node is distinguished by low hydration parameters, at which the free water fraction decreases and the bound water fraction increases during aging. In the mesenteric lymph node, the hydration indicators remain relatively high with the predominance of the free fraction of water.

With a deficient trace element profile, the content of trace elements is ambiguous in the lymph nodes of old animals (Table 3). In the mesenteric lymph node, there is a high concentration of zinc, selenium, which exceeds by 1.2-1.4 times its concentration in the tracheobronchial lymph node (p < 0.01). In the tracheobronchial, the amount of manganese content is 1.2 times greater than in the mesenteric lymph node. The content of iron and copper at the same level in the mesenteric and tracheobronchial lymph nodes. This forms qualitatively different microelement profiles of lymph nodes with a deficient content of most trace elements in old age.

The emergence of the concept of the lymphatic region [4] changes the point of view about the stereotypical changes in the structure of lymph nodes, indicating the need to study their regional specificity in aging. It is not always possible to link the structural and functional insufficiency of lymph nodes and the low proliferative capacity of lymphoid cells with the mechanism of aging at the level of the lymphatic region. The structure of the lymph nodes changes with age, showing signs of their involution against the background of an increase in the connective tissue component. But the involutive processes occur unevenly in the lymph nodes from different areas of the body [1, 3, 11]. Based on the belonging to the lymphatic region, each lymph node differs from each other in the size of individual structural and functional zones, the degree of hydration and individual trace element profile.

The implementation of the principle of regional determinant allowed us to find a connection between the microanatomic organization of lymph nodes with the level of hydration and the content of trace elements in relation to the elderly and senile age, when the main functions of visceral lymph nodes are reduced. In our opinion, the visceral lymph nodes are in a state of functional tension due to insufficient reactivity of the lymphoid tissue in old age [13, 14]. It is obvious that the lymph nodes react by changing their structure to changes in the water and trace element balance in the lymphatic region, provoked by the aging of the body. Senile dehydration of the lymph node is associated with the redistribution of water fractions. As a rule, the free fraction of water can decrease and the fraction of bound water can increase. This may indicate a certain degree of tension of compensatory and adaptive mechanisms with a possible weakening and further depletion of the drainage function of the lymph nodes. At a late stage of ontogenesis, the function of lymph nodes is not provided with a sufficient content of trace elements. An increase in the manganese content is considered as a protection factor against peroxidation and dehydration [15]. Without denying the general signs of aging, regional signs were identified for the lymph nodes, which are associated with the peculiarity of the lymphatic regions that are in different contact with the external environment. This allows you to maintain the function of senile lymph nodes, although it is reduced due to age..

Thus, it is the belonging to different regions that determines the age-related features of the microanatomic organization, hydration and trace element balance of the lymph nodes, which allows for a comparative analysis to identify lymph nodes with a predominance of a certain function. To maintain the structural organization of lymph nodes, certain hydration conditions and trace elements are necessary for the differentiation and proliferation of lymphoid cells involved in the formation of functionally significant compartments of lymph nodes subjected to senile changes [3, 11]. All this is region-dependent, reflecting the peculiarity of the lymphatic region.

CONCLUSION

Aging leads to the formation of structural-hydration and microelement features of visceral lymph nodes, which have a region-dependent character in accordance with the concept of the lymphatic region.

Visceral lymph nodes have common signs of aging: a) an increase in connective tissue with a minimization of the structures of the cortical substance and an increase in the structures of the medulla against the background of dehydration and the development of a deficiency of most trace elements, which indicates a decrease in drainage-detoxification and immune functions.

With aging, lymph nodes have distinctive signs depending on their localization (regional specificity), based on structural-hydration and microelement parameters: a) in the mesenteric lymph node, the area of the interstitial part of the cortex is reduced and the area of the medulla is increased due to brain cords with narrowed lymphatic sinuses against the background of dehydration with a decrease in the total and free fraction of water; in the deficient trace element profile, there is an increased concentration of selenium and zinc; b) the tracheobronchial lymph node is characterized by an increased size of the cortical plateau and a decrease in the area occupied by the brain cords and sinus; against the background of dehydration with an increased volume of bound water fraction; a high content of manganese is characteristic in a deficient trace element profile.

References:

1. Бородин Ю.И. Лимфатическая система и старение // Фундаментальные исследования. – 2011. – № 5. – С.11–15.
2. Hadamitzky С., Spohr H., Debertin A.S. Age-dependent histoarchitectural changes in human lymph nodes: an underestimated process with clinical relevance? // J. Anat. – 2010. – V.216. – P.556–562.
3. Gorchakova O., Kolmogorov Yu., Gorchakov V., Demchenko G. Interrelation of trace elements and the structural organization of lymph nodes at young and senile age // Archiv Euromedica. – 2020. – Vol. 10. – Num. 2. – P.22–25. http://dx.doi.org/10.35630/2199-885X/2020/10/2.6
4. Бородин Ю.И., Горчакова О.В., Суховершин А.В., Горчаков В.Н. Концепция лимфатического региона в профилактической лимфологии. – LAP LAMBERT Academic Publishing, 2018. – 73 c.
5. Кудрин А.В., Скальный А.В., Жаворонков А.А., Скальная М.Г., Громова О.А. Иммунофармакология микроэлементов. – М.: Изд-во КМК, 2000. – 537 с.
6. Павлова Т.В., Куликовская В.Ф., Павлова Л.А. Клиническая и экспериментальная морфология. – М.: ООО «Медицинское информационное агентство», 2016. – 256 с.
7. Steiger T.K., Weiskopf N., Bunzeck N. Iron Level and Myelin Content in the Ventral Striatum Predict Memory Performance in the Aging Brain // J. Neurosci. – 2016. – Mar 23. – V. 36(12). – P.3552–3558. doi: 10.1523/JNEUROSCI.3617-15.2016.
8. Старовойтова Н.В., Фаращук Н.Ф., Янкович И.В. Особенности процессов гидратации в крови детей различных соматических типов // Вестник Смоленской медицинской академии. – 2000. – № 3. – С.13–15.
9. Фаращук Н.Ф., Рахманин Ю.А. Вода – структурная основа адаптации. – Москва-Смоленск, 2004. – 177 с.
10. Piminov P.A. Synchrotron Radiation Research and Application at VEPP-4. Physics Procedia. – 2016. – Vol. 84. – P.19–26. DOI:10.1016/j.phpro.2016.11.005
11. Popov V.V., Demchenko G.A., Gorchakova O.V., Gorchakov V.N. Structural features of lymph nodes in different lymphatic regions at the natural and accelerated aging // European Journal of Natural History. – 2020. – № 4. – P.19–23.
12. Василенко А.М., Шарипова М.М. Дефицит микроэлементов и проблема коморбидности. Микроэлементы в медицине. – 2019. Том 20(1). С.4–12. DOI: 10.19112/2413-6174-2019-20-1-4-12].