Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 ± 0.7 percent in 2-month-old Cx (N = 5) vs 13.4 ± 0.3 percent (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 ± 0.9 percent in 2-month-old Cx (N = 5) vs 15.5 ± 0.3 percent (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 ± 1.2 percent in 10-month-old vs 9.8 ± 0.9 percent in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 ± 0.2 percent in 10-month-old vs 18.6 ± 0.7 percent in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats.

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 +/- 0.7% in 2-month-old Cx (N = 5) vs 13.4 +/- 0.3% (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 +/- 0.9% in 2-month-old Cx (N = 5) vs 15.5 +/- 0.3% (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 +/- 1.2% in 10-month-old vs 9.8 +/- 0.9% in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 +/- 0.2% in 10-month-old vs 18.6 +/- 0.7% in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.

Neonatal castration affects intrathymic kinetics of T-cell differentiation and the spleen T-cell level.

To test putative interdependence in the ontogenesis of the hypothalamic-pituitary-gonadal and thymic-lymphatic axes, thymocyte differentiation and maturation was examined in neonatally castrated (Cx) adult rats. In the hypercellular thymi of Cx rats, the proportion of the least mature CD4(-)CD8(-)TCRalphabeta(-) triple negative (TN) thymocytes was reduced, while the proportions of all downstream double positive (DP) subsets (TCRalphabeta(-), TCRalphabeta(low) and TCRalphabeta(high)) were increased when compared with neonatally sham-castrated (Sx) adult rats. This suggested an accelerated thymocyte transition from the TN to DP TCRalphabeta(low) developmental stage accompanied by an increased positive/ reduced negative thymocyte selection. The increased thymocyte surface density of Thy-1, which is implicated in thymocyte hyposensitivity to negative selection, in Cx rats further supports the previous assumption. The finding that the proportions of both single positive (SP) TCRalphabeta(high) thymocyte subsets were reduced, while their numbers were increased (CD4(+)CD8(-)) or unaltered (CD4(-)CD8(+)), coupled with results demonstrating an increased level of CD4(-)CD8(+) cells without changes in that of CD4(+)8(-) cells in the spleen indicate: (i) accelerated differentiation and maturation of the positively selected DP TCRalphabeta(high) thymocytes towards CD4(-)8(+) TCRalphabeta(high) cells followed by increased emigration of the mature cells and (ii) decelerated differentiation and maturation towards CD4(+)8(-)TCRalphabeta(high) cells in Cx rats. Furthermore, the unaltered proportion of intrathymically developing CD4(+)CD25(+)Foxp3(+) regulatory cells in Cx rats, in light of putative hyposensitivity of thymocytes to negative selection suggesting reduced elimination of autoreactive cells, may provide a firm basis for understanding the reasons behind increased susceptibility of Cx rats to autoimmune disease induction.

Long-term beta-adrenergic receptor blockade increases levels of the most mature thymocyte subsets in aged rats.

Age-related increase in the density of thymic noradrenergic fibres and noradrenaline (NA) concentration is proposed to be associated with thymic involution and altered thymopoiesis. To test this hypothesis thymocyte differentiation/maturation and thymic structure were studied in 18-month-old male Wistar rats subjected to 14-day-long propranolol (P) blockade of beta-adrenoceptors (beta-ARs). The treatment primarily resulted in changes in the T-cell receptor (TCR)-dependent stages of thymopoiesis, which led to an increase in both the relative and absolute numbers of the most mature single positive (SP) CD4(+)CD8(-) (including cells with the CD4(+)CD25(+) regulatory phenotype) and CD4(-)CD8(+) TCRalphabeta(high) thymocytes. Accordingly, in the thymi of these rats an increase in both numerical density and absolute number of medullary thymocytes encompassing mainly the most mature SP cells was found. These findings, together with an increase in the thymocyte surface expression of the regulatory molecule Thy-1 (CD90) (implicated in negative regulation of TCRalphabeta-dependent thymocyte selection thresholds) in the same rats, may suggest increased positive/reduced negative thymocyte selection. Collectively, the results indicate that a decline in thymic efficiency in generating both conventional and regulatory T cells, and consequently in immune function, in aged rats may be, at least partly, attenuated by long-term blockade of beta-ARs with P.

Morphometrical characteristics of age-associated changes in the thymus of old male Wistar rats.

In order to provide a morphometrical description of the changes in the aged rat thymus and to relate them to apoptotic and proliferative activity of thymocytes, the thymuses from 3- and 18-month-old male Wistar rats and the percentages of bromodeoxyuridine-incorporating and apoptotic cells in cultures of thymocytes were assessed by stereological analysis and flow cytometry, respectively. In old rats the volume of lymphoepithelial thymic tissue is markedly reduced, reflecting a sharp decrease in the total number of thymocytes. A reduction in the proliferative capacity of thymocytes and increase in their susceptibility to apoptosis are, most likely, primarily responsible for a 7-fold reduction in thymic cellularity in old animals. Furthermore, only the volume of cortical compartment was affected by aging, while that of medulla, despite of reduced cellularity, was not significantly altered. The loss of functional tissue in aged thymus is compensated by a substantial increase in the volume of inter-lobular connective and adipose tissue, so the thymic weight remained unaltered in old rats. These results suggest that thymus of aged Wistar rats exhibits morphological characteristics similar to those found in aged human thymus and thus may serve as an animal model for further investigations of thymus-related changes in immunological aging.

Characterization of thymocyte phenotypic alterations induced by long-lasting beta-adrenoceptor blockade in vivo and its effects on thymocyte proliferation and apoptosis.

Adult male Wistar rats were subjected to propranolol (P, 0.40 mg/100 g/day) or saline (S) administration (controls) over 14 days. The expression of major differentiation molecules on thymocytes and Thy-1 (CD90) molecules, which are shown to adjust thymocyte sensitivity to TCRalphabeta signaling, was studied. In addition, the sensitivity of thymocytes to induction of apoptosis and concanavalin A (Con A) signaling was estimated. The thymocytes from P-treated (PT) rats exhibited an increased sensitivity to induction of apoptosis, as well as to Con A stimulation. Furthermore, P treatment produced changes in the distribution of thymocyte subsets suggesting that more cells passed positive selection and further differentiated into mature CD4+ or CD8+ single positive (SP) TCRalphabeta(high) cells. These changes may, at least partly, be related to the markedly increased density of Thy-1 surface expression on TCRalphabeta(low) thymocytes from these rats. The increased frequency of cells expressing the CD4+25+ phenotype, which has been shown to be characteristic for regulatory cells in the thymus, may also indicate alterations in thymocyte selection following P treatment. Inasmuch as positive and negative selections play an important role in continuously reshaping the T-cell repertoire and maintaining tolerance, the hereby presented study suggests that pharmacological manipulations with beta-AR signaling, or chemically evoked alterations in catecholamine release, may interfere with the regulation of thymocyte selection, and consequently with the immune response.

Chronic alpha1-adrenoreceptor blockade produces age-dependent changes in rat thymus structure and thymocyte differentiation.

In order to examine the influence of chronic alpha1-adrenergic receptor (alpha1-AR) blockade on the thymus structure and T-cell maturation, peripubertal and adult male rats were treated with urapidil (0.20 mg/kg BW/d; s.c.) over 15 consecutive days. Thymic structure and phenotypic characteristics of the thymocytes were assessed by stereological and flow cytometry analysis, respectively. In immature rats, treatment with urapidil reduced the body weight gain and, affecting the volume of cortical compartment and its cellularity decreased the organ size and the total number of thymocytes compared to age-matched saline-injected controls. The percentage of CD4+8- single positive (SP) thymocytes was decreased, while that of CD4-8+ was increased suggesting, most likely, a disregulation in final steps of the positively selected cells maturation. However, alpha1-AR blockade in adult rats increased the thymus weight as a consequence of increase in the cortical size and cellularity. The increased percentage of most immature CD4-8- double negative (DN) cells associated with decreased percentage of immature CD4+8+ double positive (DP) thymocytes suggests a decelerated transition from DN to DP stage of T-cell development. As in immature rats, the treatment in adult rats evoked changes in the relative numbers of SP cells, but contrary to immature animals, favoring the maturation of CD4+8- over CD4-8+ thymocytes. These results demonstrate that: i) chronic blockade of alpha1-ARs affects both the thymus structure and thymocyte differentiation, ii) these effects are age-dependent, pointing out to pharmacological manipulation of alpha1-AR-mediated signaling as potential means for modulation of the intrathymic T-cell maturation.