CC BY
11UDC 576.3/7:591.147.3:599.323.41:533.6.013.8
ULTRASTRUCTURAL CHANGES IN RAT THYMUS UNDER THE SYSTEMATIC EXPOSURE TO GRAVITATIONAL OVERLOADS AND GLUTARGINUM USE
G. A. Moroz1, M. A. Kriventsov2
Department of Medical Physical Culture, Sports Medicine and Physiotherapy with course of Physical Culture
2 Department of Pathological Anatomy.
Medical Academy named after S. I. Georgievsky of V. I. Vernadsky Crimean Federal University
Correspondence: 5/7, Lenin Avenue, Simferopol, 295006, Crimea Republic, Russian Federation
E-mail: [email protected]
SUMMARY
Experimental work was designed primary to study ultrastructural changes in the mature rat's thymus under systematic exposure to +Gx acceleration. Secondary task is to determine corrective potential of the Glu-targinum use (L-arginine and L-glutamic acid). Mature male Wistar rats were exposed to systematic +Gx acceleration (9 G, 10 minutes, daily, 10 times). Morphology of the thymus was investigated using transmission electron microscopy method. Analysis of changes in the thymus of the mature rats under systematic exposure to +Gx acceleration showed marked ultrastructural transformation, which is characterized by a process of accidental involution and microcirculatory disturbances caused by the systematic action of gravitational forces. Such changes were potentiated by the age-related involution. Proliferation and expanding of the connective tissue structures, replacement of the parenchyma by the adipose tissue, degeneration of the epithelial component, massive apoptotic transformation are characteristics of mature rat's thymus. Concomitant usage of the Glutarginum promotes less pronounced than in the experiments without treatment circulatory injuries and involutive changes. Systematic action of +Gx acceleration causes significant alteration of normal structural organization of the thymus of the matured rats. Using of the Glutarginum protects ultrastructural components and increases the adaptive capacity of the thymus.
УЛЬТРАСТРУКТУРНЫЕ ПРЕОБРАЗОВАНИЯ В ТИМУСЕ КРЫС ПРИ СИСТЕМАТИЧЕСКОМ ВОЗДЕЙСТВИИ ГРАВИТАЦИОННЫХ ПЕРЕГРУЗОК И ПРИМЕНЕНИИ ГЛУТАРГИНА
Г. А. Мороз, М. А. Кривенцов
РЕЗЮМЕ
Изучение ультраструктурных преобразований в тимусе крыс зрелого возраста в условиях систематического воздействия поперечно-направленных перегрузок. В качестве дополнительной цели выступало определение потенциала применения глутаргина (L-аргинина и L-глутамата) в качестве корригирующего средства. Самцов крыс линии Вистар зрелого возраста подвергали систематическому воздействию поперечно-направленных перегрузок (9 G, по 10 минут ежедневно на протяжении 10 дней). Морфологию тимуса изучали с использованием метода трансмиссионной электронной микроскопии. Анализ преобразований в тимусе крыс зрелого возраста в условиях систематического воздействия поперечно-направленных перегрузок выявил значительные изменения на ультраструктурном уровне, которые характеризовали процесс акцидентальной инволюции и расстройства микроциркуляции, вызванные воздействием гипергравитации. Подобные изменения потенцировались возрастной инволюцией тимуса. Преобразования в тимусе характеризовались пролиферацией и расширением соединительнотканных структур, замещением паренхимы жировой тканью, дегенерацией эпителиального компонента и выраженной апоптотической трансформацией. Применение глутаргина приводит к уменьшению выраженности циркулярных нарушений и инволютивных преобразований. Систематическое воздействие поперечно-направленных перегрузок приводит к существенному нарушению структурной организации тимуса зрелых крыс. Применение глутаргина способствует сохранности ультраструктурных компонентов и повышает адаптационные возможности тимуса.
Key words: thymus, ultrastructure, rat, acceleration, Glutarginum.
Modern science and technology raises the problem of the study of adaptation to different environmental factors. Area of the particular interest is the long-term impact of acceleration and space flight factors on the human and animal organisms (acceleration, vibration, weightlessness, etc.), along with development of the activities to enhance resistance of the organism to extreme
conditions. An important place among these factors occupies hypergravity. Accelerations of the different directions can occur in various periods of the flight and may pose a threat to the life and activities of the astronauts. From this point of view, one of the important aspects is studying of the methods of the protection against G-force accelerations, because pilots of the supersonic aircrafts are
exposed to intensive and long-term gravitational accelerations about +Gx / -Gx 9-12. On the other hand, preventive methods used in modern aerospace medicine, do not have sufficient effect, as proven by relatively high morbidity values among pilots [2].
The reaction of the organism to external stimuli depends on the morphological and functional state of one of the central immunogenesis organ - thymus. Thymus throughout life undergoes age-associated involution, while under stress conditions accidental involution develops [8]. At a young age, mild to moderate extreme conditions mobilize the adaptive capabilities of the organism, whereas in aging subjects with background age-associated involution long time stress can cause secondary immunodeficiency syndrome due to the rapid involution of the thymus [1]. Although there were published some articles devoted to the systemic exposure of the acceleration [5, 6], there are almost no data about age reactivity of the thymus gland to the systematic gravitational overloads. Possibility of the pharmacological correction of these maladaptive changes also remains unstudied.
The present experimental work was designed primary to study ultrastructural changes in the mature rat's thymus under systematic exposure to +Gx acceleration. Secondary task is to determine corrective potential of the Glutarginum use.
MATERIALS AND METHODS
This experiment was conducted on the male Wistar rats. Four series (2 control and 2 experimental) of male animals were used, each group consisting of 6 sexually mature Wistar rats with initial weight 260-280 g taken from a closed colony. Animals of the 1-st experimental group (E1) for 10 days were subjected to 10-minute per day exposure to +Gz acceleration with magnitude 9 G. The rats of the 2-nd experimental group (E2) also were subjected to similar hypergravity exposure with concomitant administration of Glutarginum at dose 100 mg/kg of body weight.
+Gx acceleration (direction back to chest) was modeled using the experimental centrifuge C-2/500 with a radius 0.5 meters and with a working range from 1 to 50 G. The magnitude of the modeled +Gx acceleration was 9G. Gradual onset rate and gradual decline rate were 1.4 to 1.6 G/sec and 0.6 to 0.8 G/sec, respectively. Experiment (centrifuging) was carried out on a daily basis (for 10 days), at the same time within 10 minutes. Parameters of the experimental +Gx acceleration were chosen taking into account typical exposure to hypergravity during high-speed and highly maneuverable flights [2].
Glutarginum was chosen due to its antioxidant properties and ability to inhibit lipid peroxidation at different stages, and to
participate in the maintenance of systemic and local hemodynamic parameters due to the nitric oxide formation from L-arginine L-glutamate (active pharmaceutical ingredient) [3, 4].
Rats of the 1-st control group (C1) were not exposured to acceleration. Same, rats of 2-nd control group (C2) also were not subjected to +Gz, but 30 minutes before initiation of the experiment they were administered sterile saline in an equivalent to Glutarginum dose (100 mg/kg of body weight). C1 group was used to compare results from E1 group, and C2 group - to compare results obtained from E2 group.
Animals were killed with excess ether anaesthesia on the next day after the last centrifuge run and the thymus was removed and weighed. The experiment was conducted in accordance with current bioethical norms. Further, the thoracic viscera were removed and the thymus was separated. For transmission electron microscopy the material was fixed in gluteraldehyde phosphate buffer with 1% osmium tetroxide solution, following embedding in Epon 812. Semithin sections were stained with toluidine blue, and studied under light microscope. Identification of the cell types (small, medium, large lymphocytes, macrophages, epithelial cells, mitosis, apoptotic bodies etc.) was carried out visually using semithin sections under high magnification (x1000). Ultrathin sections after Reynolds procedure for staining thin sections for electron microscopy with lead citrate were studied using transmission electron microscope TEM 125K (Sumy, Ukraine).
RESULTS
After a 10-day experimental exposure to +Gx accelerations thymus lobules were decreased. Generally, moderate atrophy with accidental involution of thymus was observed, and there was a simultaneous fall in cortical cellularity and mitotic activity of thymic lymphocytes. Some portion of the thymic tissue was substituted by adipose tissue (see Figure 1). Remaining lymphoid tissue had clear signs of the accidental involution. Capsule and connective tissue septa were thickened and oedematous. These structures contained some adipose cells and groups of the lymphocytes. Vessels of the microcirculatory bed were mostly dilated and packed by RBC with signs of perivascular oedema. Near to trabecular vessels and at the periarterial spaces were identified clusters of lymphocytes. It should be emphasized, that decreased cellularity of the thymus is the most frequently encountered histopathologic finding associated with stress-induced effects on the thymus [8]. At the same time we must realize that such changes were associated with each of apoptosis and necrosis of lymphocytes.
Figure 1. Semithin sections (1-2 ^m) of the rat thymus after 10-day exposure to +Gx accelerations. Toluidine blue staining. Magnification x1000. Notes: 1 - extended interlobular septum, 2 - apoptotic body, 3 - mast cells, 4 - adipocyte, 5 - cytoplasm of the macrophage.
Subcapsular zone of the thymus lobules looked narrowed and was represented only by 1 to 2 layers of large forms of the T-cells. Mitoses were rare. Corticomedullary region was blurred, and blood vessels and perivascular spaces were filled by lymphocytes. At the medulla lymphocyte population was characterized by a significant decrease of a relative number of the small and large forms of the lymphocytes with increasing percent of medium T-cells. Were reveals many Hassal's bodies of different sizes.
Above mentioned histopathologic changes were supported by ultramicroscopic analysis. Cells were at various stages of apoptotic transformation (chromatin hypercondensation, cytoplasm compaction). However, some cells were defined as cells with loosened cytoplasm with low electron density, in which swollen mitochondria and extended profiles of EPS were present. Some lymphocytes were characterized by the necrobiosis changes with loosening of nuclear and cell membranes, disappearance of the nucleoli and focal vacuolization of the cytoplasm. Their organelles were swollen, with signs of focal destruction of membranes. Also ultrastructural changes of the thymocytes included signs of edema, cytolysis and karyolysis. In the cytoplasm of macrophages showed active secondary lysosomes and phagocytised apoptotic bodies, which demonstrate activation of the phagocytosis. Also were occasionally revealed active plasma cells with enhanced tubule endoplasmic reticulum (see Figure 2). Ultramicroscopically, some epithelial cells were characterized by vacuolated cytoplasm, shorten tubules of the endoplasmatic reticulum, swollen mitochondria with disoriented cristae. So, epithelial cells also had ultrastructural features of dystrophic and destructive changes.
Figure 2. Active plasma cell at the corticomedullary region of the rat thymus under 10-day exposure to +Gx acceleration. TEM. Magnification x5000. 1 - nucleus of the plasma cell, 2 - nucleus of the lymphocytes, * - differentiated endoplasmic reticulum.
Some epithelial cells were characterized by the typical apoptotic changes of the nucleus (chromatin hypercondensation) and cytoplasm compaction (see Figure 3).
Figure 3. Cortex of the rat thymus under 10-day exposure to +Gx acceleration. TEM. Magnification x4000. 1 - apoptotic nucleus of the epithelial cell, 2 - nuclei of the lymphocytes.
Were shown and separate active macrophages and their clusters (see Figure 4).
Figure 4. Medulla of the rat thymus under
10-day exposure to +Gx acceleration. TEM. Magnification x3000. 1 - nucleus of the macrophage, 2 - apoptotic body, 3 - cytoplasm of the plasma cell, * - multiple lysosomes.
Therefore, revealed destructive and degenerative changes in lymphoid cells and cells of the microenvironment indicate the depletion of the cell population in all functional zones of the thymus of experimental animals exposed to +Gx acceleration.
Typical destructive ultramicroscopic changes were also found in the structures of the microvasculature of the thymus. Endothelial cells of capillaries had evidence of intracellular edema, sparse chromatin in the nucleus and fragmentation of the cell membrane. Vacuolated cytoplasm with low electronic density contained mitochondria with disrupted cristae. In some venules leached erythrocytes were observed. Blood vessels were primarily extended with features of the stasis of erythrocytes. However, some arterioles were with narrowed lumen. The wall of these vessels was thickened by a broad basal membrane and endothelial hypertrophy. Perivascular fibroblasts were characterized by the evidence of the active synthesis of the fibrous component (see Figure 5).
Figure 5. Cortex of the rat thymus under 10-day exposure to +Gx acceleration. TEM. Magnification x6000. 1 - RBCs within blood vessel, 2 - cytoplasm of the endothelial cell, 3 - nucleus of the fibroblast, * - bundles of the collagen fibers.
Perivascular space contained epithelial cells, macrophages, and sporadic plasma cells and mast cells.
On the histological slides of the thymus of rats exposed to +Gx accelerations with concomitant parenteral administration of the Glutarginum 100 mg/kg contours of the lobules were irregular. Similarly to the previous described experimental group were identified thymic lobules with partial adipose substitution. At the remaining lobules relative area of the
cortex was decreased, while relative area of the medulla was increased compared to control (C2).
Capsule and interlobular septa formed by loose connective tissue were thickened and oedematous with increasing number of the fibroblast cells and fiber component. Intracapsular and trabecular blood vessels were dilated and looked plethoric. In some cases stasis and sludge of the RBC was noted. Along the vessels was identified increased number of the mast cells on the different stages of degranulation. At the extended perivascular spaces were identified clusters of the lymphocytes. Additionally, some epithelial cells, macrophages and functionally active plasma cells were identified.
Generally, architectonics of the lymphoid tissue was disrupted. At the cortex we observed alternation of light and dark areas formed by a compact arrangement of cells. However, the cellularity although reduced relative to the control, but to a lesser degree than in the series of experiments without Glutarginum treatment. Parallel to these observations, there was noted increased phagocytic activity of the macrophages. At the same time, number of the mitotic cells was still negligible. Data from TEM analysis supported degenerative changes in epithelial cells and lymphocytes. Despite histopathological changes similar to the 1-st experimental group, in certain parts of the thymus cortex lymphocytes were generally unaffected, as evidenced by the normal nuclear-cytoplasmic ratio and by the increase in the electron density of the cytoplasm.
At the medulla total cellularity was increased, primarily due to higher number of the medium lymphocytes and well contoured epithelial cells. More often than at the control group, were revealed small and medium Hassal's bodies, some of which were at the stage of the cystoid degeneration (see Figure 6).
Figure 6. Medulla of the rat thymus under 10-day exposure to +Gx acceleration + Glutarginum. Large Hassal's body formed by the apoptotic cells with various features of lysis. TEM. Magnification x3000. 1 - cytoplasm of macrophage, 2 - apoptotic bodies.
DISCUSSION AND CONCLUSIONS
Analysis of changes in the thymus of the mature rats under systematic exposure to +Gx acceleration showed marked structural transformation, which is characterized by a process of accidental involution and microcirculatory disturbances caused by the systematic action of gravitational forces. Such changes were potentiated by the age-related involution. Proliferation and expanding of the connective tissue structures, replacement of the parenchyma by the adipose tissue, degeneration of the epithelial component are characteristics of mature rat's thymus. At the same time, histoarchitecture of the thymus was characterized by typical stress-induced changes, which are similar to changes revealed at the experiments with glucocorticoid administration [1, 7].
Concomitant usage of the Glutarginum promotes less pronounced than in the experiments without treatment circulatory injuries and involutive changes. This brings us to the conclusion that using of the Glutarginum (L-arginine L-glutamate) increases the adaptive capacity of the thymus. Immune protection properties of the Glutarginum under systematic exposure to the +Gx acceleration seem to be implemented by its membrane stabilizing activity and antioxidant effects [9].
REFERENCES
1. Aw D, Palmer DB. The Origin and Implication of Thymic Involution. Aging and Disease 2011; Vol. 2, 5:437-43.
2. Davis JR, Johnson R, Stepanek J, Fogarty JA. Fundamentals of Aerospace Medicine. 4th edition, Philadelphia: Lippincott Williams & Wilkins, 2008.
3. Egbuonu, Cemaluk AC. Sub-chronic Concomitant Ingestion of L-arginine and Monosodium Glutamate Improves Feed Efficiency, Lipid Metabolism and Antioxidant Capacity in Male Wistar Rats. Pakistan Journal of Biological Sciences 2012; Vol. 15, 6:301-05.
4. Hryb VA. Combination of Antioxidants and a Hepatoprotector in the Treatment of Patients with Diabetic Distal Symmetric Polyneuropathy [Article in Russian]. Likars'ka Sprava / Ministerstvo Okhorony Zdorov'ia Ukrainy 2010; 3-4:68-79.
5. Moroz GA. Dynamics of Relative Weight of Rats Suprarenal Glands, Thymus and Spleen Under Hypergravity. Available via http://www.researchgate.net/ publication/26622977_Dynamics_of_relative_weight_of_ rats_suprarenal_glands_thymus_and_spleen_under_ hypergravity (Accessed 14/04/2013).
6. Moroz GA. Morphofunctional Features of Thymus of Twelve-month-old Rats Exposed to Repeated Hypergravity. Available via http://www.researchgate.net/ publication/47808266_Morphofunctional_features_of_ thymus_of_twelve-month-old_rats_exposed_to_repeated_ hypergravity (Accessed 14/04/2013).
7. Norrman J, David CW, Sauter SN, Hammon HM, Blum JW. Effects of Dexamethasone on Lymphoid Tissue in the Gut and Thymus of Neonatal Calves Fed with Colostrum or Milk Replacer. Journal of Animal Science 2003; Vol. 81, 9:2322-32.
8. Pearse G. Normal Structure, Function and Histology of the Thymus. Toxicologic Pathology 2006; Vol. 34, 5:504-14.
9. Sajdel-Sulkowska EM, Kosal N, Sulkowski ZL, Lipinski B. Potential Role of Oxidative Stress in Mediating the Effect of Altered Gravity on the Developing Rat Cerebellum. Advances in Space Research: The Official Journal of the Committee on Space Research (COSPAR) 2007; Vol. 40, 9:1414-20.
