ISSN 20790570, Advances in Gerontology, 2014, Vol. 4, No. 1, pp. 16–21. © Pleiades Publishing, Ltd., 2014. Original Russian Text © L.A. Obukhova, V.B. Vais, L.E. Bakeeva, S.V. Sergeeva, N.G. Kolosova, 2013, published in Uspekhi Gerontologii, 2013, Vol. 26, No. 2, pp. 229–235.
Structural and Functional Basis of Accelerated Involution of the Thymus in OXYS Rats L. A. Obukhovaa, V. B. Vaisb, L. E. Bakeevab, S. V. Sergeevac, and N. G. Kolosovac a
Novosibirsk National Research State University, ul. Pirogova 2, Novosibirsk, 630090 Russia email:
[email protected] bBelozerskii Institute PhysicalChemical Biology, Moscow State University, Vorob’evy Gory, 1, Moscow, 119991 Russia cInstitute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, pr. Akad. Lavrent’eva 10, Novosibirsk, 630090 Russia Abstract—Involution of the thymus is one of the most expressed indices of aging of the immune system. The mechanisms of involution of the thymus are relatively well studied, whereas the reasons and mechanisms of accelerated involution of the thymus are less known. We have previously reported that premature aging in OXYS rats is associated with accelerated involution of the thymus. The aim of the present study was to exam ine morphofunctional conditions of epithelial cells in the thymus of OXYS rats. Immunohistochemical study revealed that in 3.5monthold OXYS rats, i.e. in the initial period of agerelated involution, the network of epithelial cells in the thymus was reduced, and the volume and area of the surface of epithelial cells in the cor tical substance was lower compared to control Wistar rats. Electron microscopy found strong changes in the ultrastructure of epithelial cells, such as shrinkage of the cytoplasm volume, a clear decrease in the size and number of secretory vacuoles, and multiple autophagosomes and phagolysosomes. Our data show that a pos sible mechanism of the reduction of the epithelial network in the thymus of OXYS rats is an enhancement of autophagy, probably related to specific mitochondria dysfunction in these rats. The fact of agerelated retar dation of autophagy in some tissues is well known. In spite to this, based on the data from OXYS rats, we sup pose that longterm deviation of intensity of this process from the physiological level may be directed not only to its attenuation but also to its activation and thus results in degenerative modifications of organs and the for mation of the progeroid phenotype of the body. Keywords: involution of the thymus, autophagy, premature aging, OXYS rats DOI: 10.1134/S2079057014010081
INTRODUCTION
In a man, involution of the thymus starts at the age of one year and appears in the form of a slow reduction of the volume of epithelial space. In the sexual matu ration period, this process accelerates and slows again after 30, when its rate is about 1% per year. Similar changes are observed in most of mammals [16, 24]. A decrease in the epithelial space of the thymus is accompanied by an accumulation of adipose cells in the cortical and medulla layers, perivascular space, capsula, and interlobular partitions. Recent studies [29] have reported that an increase in the number of adipose cells in the thymus is not associated with infil tration, as has been supposed previously, but is a result of strongly controlled epithelialmesenchymal modi fications, during which epithelial cells initially trans form to fibroblasts and then to adipose cells [12]. Adi pose cells promote involutionassociated changes pro ducing factors such as oncostatin M, LIF, IL6, sex hormones, and glucocorticoids suppressing thymus functions [17]. Substitution of the specific microenvi ronment of the thymus with adipose tissue results in a decreased production of Tlymphocytes. However, in
Involution of the thymus is one of the mostly expressed features of aging [9]. The thymus is a pri mary lymphoid organ where Tlymphocytes are devel oped from hematopoietic precursor cells. It has highly differentiated and very complex stroma consisting of a tridimensional network of epithelial cells and con nected fibroblasts, macrophages, and interdigitating and endothelial cells, which create a microenviron ment providing for the development of thymocytes [3, 7, 28]. During aging, the epithelial space of the thymus becomes smaller, the production of native Tlymphocytes capable of novel antigens decreases, and thus the vulnerability to various bacterial and viral infections increases, vaccination efficacy drops, and the risk of the development of malignant neoplasia grows. The epithelium of the thymus plays a crucial role in the elimination of autoresponsive Tcells [8]; therefore, the probability of the development of autoimmune diseases increases with age. A decrease in the regulatory and morphogenetic functions of Tlym phocytes is a key feature of the pathogenesis of degener ativedystrophic diseases specific for aging [4, 19]. 16
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spite of its involution, the thymus contains some func tionally active lymphoid tissue until senility. A sequence of morphological and functional changes in the thymus is described relatively well [5, 9, 16]. The reasons and mechanisms of premature or accelerated thymus involution are less studied. How ever, recovery of its structure and functions is a basis of strategies aimed at retarding aging of the immune sys tem and providing healthy longevity [1, 16, 19]. Accelerated thymus involution may be induced by many internal and external reasons and be mediated by any of its structural elements. For development and maintenance of the functional condition of the thy mus, continuous interactions of thymocytes with their microenvironment, consisting of epithelial cells, den dritic cells, vascular endothelium, fibroblasts, and ele ments of the extracellular matrix, are necessary [7]. Wang et al. [30] have reported that rapidly aging mice of BXD 8, 18, and 32 strains exhibited a block of the staged transition of double negative thymocytes and their transition to the stage of double positive cells. Data from other authors demonstrate that accelerated involution may be caused by an impaired thymus microenvironment [2, 6, 25]. According to Orthman et al. [22], thymus involution is a result of the accumu lation of a number of molecular impairments in both developing thymocytes and thymic epithelial cells. Li et al. [15] supposed that accelerated thymus involution in DBA/2 mice is related to decreased proliferative activity, elevated apoptosis of thymocytes, and disar rangement of the epithelial network and connective tissue stroma. We found that accelerated thymus involution was related to premature aging in OXYS rats. In these ani mals, protective treatment with mitochondrial antiox idant SkQ1 beneficially influenced morphofunctional indices in the thymus [21]. In OXYS rats, specific fea tures of the thymus are inborn hypoplasia, lower peak values of weight, size, and cellularity, imbalance of Tlymphocyte subpopulations, and accelerated age related involution [21] associated with unknown rea sons. Our previous study on apoptosis of thymocytes, which we considered as a possible mechanism of hypoplasia and accelerated thymus involution in OXYS rats, did not reveal any substantial changes. Thus, we supposed that accelerated thymus involution in OXYS rats is mainly related to impairments in its epithelial elements. The aim of the present study was to examine the morphofunctional condition of epithe lial cells in the thymus of 3.5monthold OXYS rats. In rats of this age, postnatal thymus growth ends and age related involution starts. MATERIALS AND METHODS Animals. This study was performed in male OXYS and Wistar rats at the Common Use Center “Genebank of Laboratory Animals,” Institute of Cytology and Genetics, Siberian Branch, Russian ADVANCES IN GERONTOLOGY
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Academy of Sciences, in accordance with the “Rules for Studies Involving Laboratory Animals.” Rats were housed five per a cage of 57 × 36 × 20 cm in size at a temperature of 22 ± 2°C under conditions of artificial light/dark cycle (12 h/12 h) and had free access to water and food, the standard granulated feeding for laboratory animals (“Chara”, ZAO “Assortiment Agro”, Russia). At the end of experiment, the animals were anaesthetized with ester and decapitated. Study on expression of Bax and Bcl2 proteins. Thymus tissue was collected in liquid nitrogen and stored at –70°C until use. The samples were homoge nized in a RIPA lysis buffer containing proteases inhibitors. Homogenates were centrifuged at 12000 g and 4°C for 10 min. The protein concentration in the samples was measured using a “BioRad protein assay” kit. Aliquots containing 40 µg of protein were resus pended in a buffer prior to loading onto SDSPAAG. Prior to loading, the samples were heated at 95°C. Proteins were separated in 15% SDSPAGE and transmitted onto nitrocellulose membrane (Amer sham) using a Mini TransBlot Electrophoretic Trans fer Cell (BioRad). The membranes were blocked with 5% skimmed milk in a phosphate buffer (PBST, pH 7.5) and then incubated for 1 h with mouse mono clonal antibodies against Bcl2 (BD Biosciences Pharmingen) or Bax (Santa Cruz Biochemicals, Inc., Santa Cruz, CA, United States) at a dilution of 1 : 500. Binding of the primary antibodies with the studied proteins was detected using secondary rabbit antibod ies against mouse IgG conjugated with horse reddish peroxidase at a dilution of 1 : 10000 for 1 h. For the detection of immunoreactive bands, the membranes were incubated with a luminescent substrate using a ECL Western Blot Detection System kit (Amersham, Inc., Arlington Heights, IL, United States). Signals were registered using an Xray film Kodak XAR5 Xray. Images were analyzed densitometrically. Immunohistochemical study. This experiment was performed in 3.5monthold OXYS and Wistar rats. Staining of fixed frozen 20µm sections of the thymus was performed using primary rabbit polyclonal anti body against rat cytokeratins (Abcam, cat. no. 34951) and secondary donkey antirabbit Cy3conjugated antibody (Jackson Immunoresearch Laboratory, cat. no. 711165152) according to the manufacturer’s protocol. Micropreparations were studied using a laser scanning LSM500 microscope at the Common Use Center, Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences. Analysis of images and morphometry were performed using “3D for LSM” software with technical assistance by I.B. Belan. We measured the surface area of epithelial cells in the standard sample of 300056 µm3 representing a square optical field of 15876 µm2 consisting of 70 optical sec tions of 0.27 µm in thickness along the Zaxis. The data were analyzed using oneway analysis of variances (ANOVA) and Statistica 6.0 software. Post hoc comparisons of group means were performed using the
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RESULTS AND DISCUSSION C
M
Fig. 1. Network of epithelial cells in the cortical (C) and medullar (M) substances of the rat thymus. Immunohis tochemical staining using primary polyclonal antibody against rat cytokeratins and secondary Cy3conjugated antibody. Confocal laser scanning microscopy, lens ECPlanNeofluar 20×/0.5.
least significant differences (LSD) test. The differ ences were considered as significant at p < 0.05. Electron microscopy. This experiment was per formed in 3.5monthold OXYS and Wistar rats. For the electron microscopy study, the samples were fixed with 3% glutaric aldehyde dissolved in a buffer, pH 7.4, at 4°C for 2 h. Then they were postfixed in 1% osmium tetroxide dissolved in the same buffer for 1.5 h and dehydrated in ethanol solutions with increasing con centrations (70% ethanol saturated with uranyl acetate). The samples were put into epoxy resin Epon812. Serial ultrathin sections were prepared using a Leica Ultracut ultramicrotome (“Leica”, Austria) and stained with lead according to the Reinolds method [10]. The sections prepared were viewed and photo graphed using a HU11B electron microscope (“Hita chi”, Japan) [23]. Volume and surface area of epithelial cells in the standard sam ple of the cortical substance of the thymus (300056 µm3) in 3.5monthold OXYS and Wistar rats, M ± m Group Wistar, n = 6 OXYS, n = 4 p
Surface area Volume of epithelial of epithelial cells, µm2 cells, µm3 63398 ± 2884 43939 ± 5810 0.01
310418 ± 10080 216552 ± 26156 0.005
Immunohistochemical Staining and Confocal Microscopy of Epithelial Cells of the Thymus In order to study a tridimensional network of epi thelial cells in the thymus, we applied polyclonal anti bodies against cytokeratins with a wide spectrum of action, which interacted with cytokeratins of cortical and medullar epithelial cells. Cortical and medullar tissues were well differentiated in the stained sections, in accordance with the features of their microanatomy (Fig. 1). In Wistar rats, cortical epithelial cells were oriented transversely to thymus capsula and had long thin branches that contacted the branches of adjacent cells and formed a network with small loops of hexa gonal form, which are similar to a honeycomb. Med ullar epithelial cells had more massive plain and wide branches forming a complex mazelike network in the medullar substance. In general, OXYS rats had similar networks of epithelial cells; however, in the cortical substance, the cells were larger to some extent and the network looked thinner (Fig. 2). These observations were supported by quantitative data from image anal ysis. In OXYS rats, the volume and surface area of epi thelial cells in the standardized sample of the cortical substance were significantly lower compared to those observed in Wistar rats (table). In the medullar sub stance, these indices were similar in both strains of rats although in OXYS rats, we observed trends toward their decreases. Electron Microscopy Study of Epithelial Cells in the Thymus In the thymus of OXYS rats, epithelial cells were smaller in size due to strong shrinkage of the cyto plasm. Their nuclei were also smaller than usual and electrondense, and they were of irregular form, with crude chromatin masses located along the internal surface of the nuclear membrane and also seeded everywhere in the nucleus. The nucleoli were poorly seen (Fig. 3). Electrondense cytoplasm formed long and thin branches going between adjacent lympho cytes far from the body. The size and number of secretory vacuoles were smaller or completely absent. In the peri nuclear area and branches, we observed a lot of autopha gosomes and phagolysosomes containing inside cellular organelles at various stages of degradation. Usually, these organelles were mitochondria, secretory vacuoles, and, frequently, myelinlike masses. In the area of autophagy, we observed enlarged cisterns of endoplasmic reticulum without ribosomes and multiple small vacuoles and vesicles probably originating from the cisterns of endoplasmic reticulum. In the perinuclear area and more frequently in the branches, we observed thick batches of intermediate filaments. In addition to destructed cells, cells and fragments of cells with rela tively normal structure were also observed. ADVANCES IN GERONTOLOGY
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(a)
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(b)
Fig. 2. Network of epithelial cells in the cortical substance of the thymus of Wistar (a) and OXYS (b) 3.5monthold rats. Immu nohistochemical staining using primary polyclonal antibody against rat cytokeratins and secondary Cy3conjugated antibody. Confocal laser scanning microscopy, lens PlanApochromat 63×/1.4 Oil DIC.
Content of Proteins Regulating Apoptosis Bax and Bcl2 in the Thymus Studies on the contents of proteins Bax and Bcl2 in the protein extract from the thymuses of 10dayold Wistar and OXYS rats using immunoblotting did not reveal any significant differences between the strains. However, a ratio Bax/Bcl2 in OXYS rats was signifi cantly higher compared to Wistar rats (1.4 ± 0.1 and 1.1 ± 0.1, respectively, p < 0.037), indicating that cells of OXYS rats were slightly more vulnerable to apopto sis compared to Wistar rats. Studies on the proteins of apoptosis control were performed using total protein extracts from the thymus; therefore, our conclusion on the higher vulnerability of cells to apoptosis is gen eral to both lymphoid and epithelial cells of the thy mus. In 2monthold animals, i.e., at the start of thy mus involution, the levels of Bax or Bcl2 proteins expression and the ratio of Bax/Bcl2 were similar in both Wistar and OXYS rats. Agerelated thymus involution is a complex, multi factor, and genetically programmed process, in which a gradual substitution of thymocytes and epithelial cells with adipose tissue occurs. The rate of agerelated involution depends on the initial size of the thymus, which in its turn depends on genotype, as has been demonstrated in animals of var ious strains [10]. Specifically, DBA/2 mice exhibit higher vulnerability to infections, weaker immune response, smaller thymus size, and increased its age related involution [15]. On the contrary, in female transgenic MCL1 mice, the thymus is larger and its agerelated involution is slower [11]. We previously ADVANCES IN GERONTOLOGY
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found that in 10dayold OXYS rats, the weight and volume of the thymus were lower compared to Wistar rats. The reasons for thymus hypoplasia in OXYS rats remain unclear; however, we suppose that the higher vulnerability to apoptosis of thymus cells in 10day old OXYS rats may influence thymus development. Probably, this is a mechanism of long term effects on postnatal growth and the rate of agerelated involu tion, because the differences in thymus sizes were revealed in OXYS and Wistar rats at the start of age
1 µm Fig. 3. Epithelial cell in the cortical substance of the thy mus of OXYS rat. Multiple phagolysosomes in the perinu clear region and cytoplasmic branches are observed. Mag nification, ×7000.
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related involution and involution developed more rap idly. During the period of 2–14 months, the volumes of the thymuses decreased by factors of 6 and 12.5, the volumes of the cortical thymus substance declined by factors of 10.7 and 22, and volumes of the medullar thymus substance decreased by factors of 6 and 15 in Wistar and OXYS rats, respectively [21]. Morphologi cal signs of thymus involution in OXYS rats substan tially differed from those observed in the rats with nor mal physiological aging. In Wistar rats, we observed a classical pattern of agerelated thymus involution caused by a gradual substitution of its parenchyma with adipose cells, whereas in OXYS rats, the volume of adipose tissue was substantially lower and fibrosis became a predominate process [21]. These changes may be well explained from the point of modern knowledge on the mechanisms of adipose transforma tion of the thymus, which involve epithelialmesen chymal modifications and are in accordance with the data by other authors, who have demonstrated that involution of atrophied or hypoplasic thymus was not associated with an elevated number of adipose cells [29]. We found that the network of epithelial cells in 3.5monthold OXYS rats was reduced. The volume and surface area of epithelial cells in the cortical sub stance were significantly decreased and in the med ullar tissue, they tended to be lower. Electron micro scopy studies supported the results of immunohis tochemical experiments. The size of epithelial cells was reduced due to cytoplasm shrinkage. The presence of multiple autophagosomes and phagolysosomes in epithelial cells indicates that reduction of the epithe lial network in the thymus is mediated by autophagy. Autophagy is associated with lysosomemediated degradation of some cytoplasmic proteins and organelles and their repeated use for the synthesis of new macro molecules. Autophagy is a physiological, vitally important process, which is necessary for maintenance of cellular homeostasis, elimination of damaged struc tures, maintenance of cellular metabolism under the conditions of deficit of energy and nutrients, and perfor mance by the cell of some specific functions (for exam ple, during immune reactions) [14]. Autophagy plays an important role in the processes of development, differ entiation, and cell renewal [18]. Autophagy enhance ment in prematurely aging OXYS rats may be caused by a mitochondrial dysfunction that is specific for this strain of rats and the need for their continual removal. Autophagy is a genetically programmed, finely controlled process, for which either an enhancement or weakening may result in pathological changes in cells [13]. On the one hand, the enhancement of autophagy is a mechanism of cell survival; on the other hand, it may lead to a loss of mass limit by the cell, its degeneration, and death. Epithelial cells of the thymus exhibit a high constitutive level of autophagy. The importance of this is not clearly understood. It is sup posed that this mechanism is involved in the negative
and positive selection of thymocytes and/or in responses of thymus epithelium to various stresses [20, 26, 27]. However, under physiological conditions, the basal level of autophagy does not cause a strong decrease in the volume of cellular cytoplasm found here in OXYS rats. Autophagy is a dynamic process; therefore, we cannot make a final conclusion on enhanced autophagy based on the static electron microscopy study. The accumulation of autophago somes and lysophagosomes in the cell may be caused by an inhibition of autophagy at the stage of fusion of autophagosome and lysosome or enzymatic degrada tion of the content of phagolysosomes related to lyso somes defects [13]. However, the other study [26] revealed that inhibition of autophagy in epithelial cells of the thymus was associated with accumulation of autophagosomes but did not result in a decrease in the size of the thymus and the development of involution. CONCLUSIONS We can conclude that destruction of the epithelial network in the thymus of OXYS rats starts long before agerelated thymus involution. It starts from the inborn hypotrophic changes. In young adult animals, autophagy enhances and finally, results in degenera tion and cell death. Fibrosis specifically contributes to modification of the thymus structure. Atrophy of the lymphoid tissue may be a secondary event. REFERENCES 1. Aspinall, R. and Mitchell, W., Reversal of ageassoci ated thymic atrophy: treatments, delivery, and side effects, Exp. Gerontol., 2008, vol. 43, no. 7, pp. 700– 705. 2. Cheng, L., Guo, J., Sun, L., et al., Postnatal tissuespe cific disruption of transcription factor FoxN1 triggers acute thymic atrophy, J. Biol. Chem., 2010, vol. 285, no. 8, pp. 5836–5847. 3. Cuddihy, A.R., Ge, S., Zhu, J., et al., VEGFmediated crosstalk within the neonatal murine thymus, Blood, 2009, vol. 113, no. 12, pp. 2723–2731. 4. Derhovanessian, E., Solana, R., Larbi, A., and Pawelec, G., Immunity, aging and cancer, Immunol. Aging, 2008, vol. 5, no. 11. doi: 10.1186/17424933511 5. Dooley, J. and Liston, A., Molecular control over thy mic involution: from cytokines and microRNA to aging and adipose tissue, Eur. J. Immunol., 2012, vol. 42, pp. 1073–1079. 6. Gillard, G.O., Dooley, J., Erickson, M., et al., Aire dependent alterations in medullary thymic epithelium indicate a role for Aire in thymic epithelial differentia tion, J. Immunol., 2007, vol. 178, pp. 3007–3015. 7. Gordon, J. and Manley, N.R., Mechanism of thymus organogenesis and morphogenesis, Development, 2011, vol. 138, no. 18, pp. 3865–3878. 8. Griesemer, A.D., Sorenson, E.C., and Hardy, M.A., The role of the thymus in tolerance, Transplantation, 2010, vol. 90, no. 5, pp. 465–474. ADVANCES IN GERONTOLOGY
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No. 1
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STRUCTURAL AND FUNCTIONAL BASIS OF ACCELERATED INVOLUTION 9. Gruver, A.L., Hudson, L.L., and Sempowski, G.D., Immunosenescence of aging, J. Pathol., 2007, vol. 211, no. 2, pp. 144–156. 10. Gui, J., Mustachio, L.M., Su, D.M., and Craig, R.W., Thymus size and agerelated thymic involution: early programming, sexual dimorphism, progenitors and stroma, Aging Dis., 2012, vol. 3, no. 3, pp. 280–290. 11. Gui, J., Morales, A.J., Maxey, S.E., et al., MCL 1 increases primitive thymocyte viability in female mice and promotes thymic expansion into adulthood, Int. Immunol., 2011, vol. 23, pp. 647–659. 12. Kvell, K., Varecza, Z., Bartis, D., et al., Wnt4 and LAP2alpha as pacemakers of thymic epithelial senes cence, PLoS One, 2010, vol. 5, no. 5, p. e10701. doi: 10.371/journal.pone.0010701 13. Levine, B. and Kroemer, G., Autophagy in the patho genesis of disease, Cell, 2008, vol. 132, no. 1, pp. 27–42. 14. Levine, B., Mizushima, N., and Virgin, H., Autophagy in immunity and inflammation, Nature, 2011, vol. 469, no. 7330, pp. 323–335. 15. Li, L., Hsu, H.C., William, G.E., et al., Cellular mechanism of thymic involution, Scand. J. Immunol., 2003, vol. 57, pp. 410–422. 16. Lynch, H.E., Goldberg, G.L., Chidgey, A., et al., Thy mic involution and immune reconstitution, Trends Immunol., 2009, vol. 37, no. 7, pp. 366–373. 17. De Mello Coelho, V., Bunbury, M., Rangel, L.B., et al., Fat storing multilocular cells expressing CCR5 increase in the thymus with advancing age: potential role for CCR5 ligands on the differentiation and migration of preadipocytes, Int. J. Med. Sci., 2010, vol. 7, no. 1, pp. 1–4. 18. Mizushima, N. and Levine, B., Autophagy in mamma lian development and differentiation, Nat. Cell. Biol., 2010, vol. 12, no. 9, pp. 823–830. 19. Naylor, P.H., Quadrini, K., Garaci, E., et al., Immuno pharmacology of thymosin alpfa1 and cytokine synergy, Ann. N.Y. Acad. Sci., 2007, vol. 1112, pp. 235–244. 20. Nedjic, J., Aichinger, M., Emmerich, J., et al., Autophagy in thymic epithelium shapes the T cell rep ertoire and is essential for tolerance, Nature, 2008, vol. 455, pp. 396–400.
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21. Obukhova, L.A., Skulachev, V.P., and Kolosova, N.G., Mitochondria targeted antioxidant SkQ1 inhibits age dependent involution of the thymus in normal and senes cenceprone rats, Aging, 2009, vol. 1, no. 4, pp. 1–13. 22. Ortman, C.L., Dittmar, K.A., Witte, P.L., and Le, Ph.T., Molecular characterization of the mouse involved thy mus: aberrations in expression of transcription regula tors in thymocyte and epithelial compartments, Int. Immunol., 2002, vol. 14, no. 7, pp. 813–822. 23. Reynolds, E., The use of lead citrate at high pH as an electron opaque stain in electron microscopy, J. Cell Biol., 1963, vol. 17, pp. 208–212. 24. Steinman, G.G., The involution of the aging human thymic epithelium is independent of puberty: a mor phometric study, Scand. J. Immunol., 1985, vol. 22, pp. 563–575. 25. Su, D. and Manley, N.R., Stagespecific changes in fetal thymocyte proliferation during the CD4–8– to CD4+8+ transition in wild type, Rag I–/–, and Hoxa3, PaxI mutant mice, BMC Immunol., 2002, vol. 3, no. 12. doi: 10.1186/14712172312 26. Sukseree, S., Mildner, M., Rossiter, H., et al., Auto phagy in the thymic epithelium is dispensable for the development of self tolerance in a novel mouse model, PLos One, 2012, vol. 7, no. 6, p. e38933. doi: 10.1371/journal.pone.0038933 27. Uddin, M.N., Nishio, N., Ito, S., et al., Autophagic activity in thymus and liver during aging, Age, 2012, vol. 34, no. 1, pp. 75–85. 28. Van Ewijk, W., Holländer, G., Terhorst, C., and Wang, B., Stepwise development of thymic microenvironments in vivo is regulated by thymocyte subsets, Development, 2000, vol. 127, pp. 1583–1591. 29. Youm, Y.H., Yang, H., Sun, Y., et al., Deficient ghrelin receptormediated signaling compromises thymic stro mal cell microenvironment by accelerating thymic adi posity, J. Biol. Chem., 2009, vol. 284, no. 11, pp. 7058– 7077. 30. Wang, X., Hsu, H.C., Wang, Y., et al., Phenotype of genetically regulated thymic involution in young BXD RI strains of mice, Scand. J. Immunol., 2006, vol. 64, no. 3, pp. 287–294.
Translated by M. Stepanichev
