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Development of thymic medulla was examined on consecutive gestational days (GD) in Wistar rats. Medullary thymic epithelial cells (TEC) were identified by immunocytochemical localisation of neuron-specific enolase (NSE). Organisation of thymic medullary architecture was determined by interaction of thymocytes with NSE-positive TEC, that led to formation of lymphoepithelial complexes (GD 19), in which the cells exhibited proliferative activity or traits of apoptosis. The studies indicated that differentiation events and organisation of thymic medulla require stage-specific interactions between TEC and thymocytes.

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We investigated age-related changes in immunocytochemical localisation of cytokeratin 16 (CK16) in thymuses of female Wistar rats at various stages of adult life (months 1, 3, 6, 12). Within the 1 st month of life, distribution of CK typical for individual subsets of thymic epithelial cells (TEC) was observed. The most numerous CK16+ TEC were observed in the outer region of medulla, in the outer cells of Hassall's corpuscles and in the superficial epithelial layer neighbouring the connective tissue of the capsule, septa and vessels of the thymus. In the 3rd month of life, increased intensity of CK16 reaction in superficial TEC was accompanied by increased numbers of CK 16+ TEC in the outer region of the medulla. Age-related alterations in the distribution of the studied markers were evident beginning from the 6th month of life and involved increased expression of CK16 in the superficial layer of TEC, which at the interface with the septa formed stratified epithelium. In parallel, decreased numbers of CK16+ TEC were observed in the outer region of the medulla. Changes in CK16+ TEC distribution of a similar type developed in 12-month old rats and they probably reflected altered functions of some TEC populations and decreased or increased biological activity of other TEC populations.

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Alterations in the thymic epithelial cell activity were analysed during pregnancy and lactation in Wistar rats by examining the presence and in situ distribution of lymphoepithelial complexes formed by thymic nurse cells (TNC). TNC were identified in paraffin sections by their expression of MHC class II antigens, CD54 molecule and a neuromarker, protein gene product 9.5 (PGP9.5). On the first days of pregnancy (gestational days, GD) the number of PGP9.5+ TNC was found to decrease abruptly. On GD 14, a transient increase was noted in the number of PGP9.5+, MHC+, CD54+ TNC. Another increase was observed in the course of lactation, when the weight of the thymus reached the lowest values. While the increase in TNC numbers during lactation may be linked to the process of reconstruction of the thymic lymphoid population, the augmented activity of lymphoepithelial interactions on GD14 may be associated with thymic engagement in pregnancy-induced immune processes.

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In this paper we describe ultrastructure of the peritoneal membrane from single peritoneal biopsies collected from chronically dialysed rats with spontaneous peritonitis. The results were compared with those obtained in chronically dialysed animals without peritonitis. In rats with peritonitis, peritoneum was much thicker than in peritonitis-free animals. The increased thickness of the peritoneum during peritonitis was due to infiltration of the submesothelial tissue with oedematous fluid and to the presence of huge amount of cells in the stroma. The connective tissue cells were accumulated just underneath the peritoneal surface. In deeper parts of the interstitium, infiltrating acute inflammatory cells were present (lymphocytes, polymorphonuclear cells: neutrophils and eosinophils). Inversely, the increased thickness of the peritoneum in peritonitis-free animals was mainly due to enhanced amounts of collagen. Additionally, in rats with peritonitis, the surface was often denuded of mesothelial cells. The damaged mesothelial cells that detached from the peritoneal surface were also found. In conclusion, the morphological changes observed in rats with peritonitis are similar to those reported in humans, thus the model of peritonitis in dialysed rats can be used for the study of peritoneal remodeling during peritoneal dialysis complicated by peritonitis.

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The kinetics of thymic epithelial cell development was examined in Wistar strain rats between 13th and 21st days of foetal life. The studies were based on immunocytochemical localisation of cytokeratin 16 (CK 16), Ki67 and on ultrastructural observations of thymus development. Expression of CK16 in individual groups was evaluated using the Micro Image v.4.0 software. In order to monitor changes in CK16 expression in individual days of foetal life, their results were subjected to statistical analysis, demonstrating: (1) correlation between CK16 expression and duration of foetal life, (2) most pronounced CK16 expression on the 16th day of foetal life, (3) typical localisation of CK16-positive cells in individual days of foetal life. The morphological observations suggest that individual subpopulations of epithelial cells differ in their kinetics of proliferative activity.

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We investigated first stages of thymic medulla organisation in foetuses of Wistar strain rats. between 13th and 17th days of foetal life (GD). Medullary cells were identified by immunocytochemical localisation of neuron-specific enolase (NSE) as well as by traits of ultrastructure. The first thymic medullary precursor cells which were reactive for NSE were at first spread all over the thymic primordium. In the period of thymus colonisation by lymphoid cells, the following stages were distinguished in medulla organisation: (1) migration of NSE+ cells to the central portion of the thymus (GD 14-15), (2) small medullary epithelial patches, distributed within the thymus (GD 16), and (3) expansion of medullary patches into medullary compartment (GD 17). At the second and third stages of the medulla organisation, an increase in the number of NSE+ cells, followed by differentiation of their ultrastructure and increase in their biological activity were observed. We conclude that formation of medullary architectural pattern is controlled by interactions between maturing epithelial cells and developing lymphoid cells and by angiogenesis in the region.

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The model to estimate peritoneal function in chronically dialysed rats was previously presented by us. The aim of the paper is to report the findings obtained in electron microscopy of peritoneal biopsies from Wistar rats dialysed for 1 month with high glucose dialysis solution. In control animals, thin mesothelial cells were covered with microvilli. The submesothelial tissue was composed of sparse bundles of parallelly oriented collagen fibers with a few resting cells. In chronically dialysed rats, mesothelial cell layer was thicker and cells were fully packed with intracellular structures, mainly secretory granules with a homogeneous content. The submesothelial tissue was expanded due to the increased amount of collagen fibers, oedema and increased amount of submesothelial cells which were activated. The use of electron microscopy to study the peritoneum in dialysed rats is an excellent supplement to the chronic functional model of peritoneal dialysis in rats.

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The possible involvement of endogenous vasoactive intestinal peptide (VIP), cholecystokinin (CCK) and neurotensin (NT) in the control of thymocyte proliferation has been investigated in vivo in the immature rat. For this task, we have studied the effects of the administration of selective antagonists of the receptors of the three neuropeptides on the mitotic index (% of metaphase-arrested cells after vincristin injection) of thymocytes. Both CCK- and TN-receptor antagonists were ineffective. In contrast, two VIP receptor antagonists (VIP-As) enhanced the mitotic index of thymocytes. VIP reversed the effect of VIP-As, but when administered alone it did not alter the mitotic activity of thymocytes. In light of these findings, we conclude that endogenous VIP exerts a maximal tonic inhibitory influence on the basal proliferative activity of rat thymocytes, while endogenous CCK and NT do not play a relevant modulatory role in this process.

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OBJECTIVE: We previously put forward an experimental model for evaluating peritoneal function in chronically dialyzed rats. In the present paper, we show the morphologic alterations detected by electron microscopy in the peritonea of chronically dialyzed rats. MATERIALS AND METHODS: The study was done in male Wistar rats. Animals were divided into two groups: control (non dialyzed) rats and dialyzed rats [intraperitoneally exposed to Dianeal 3.86% (Baxter Healthcare Corporation, Deerfield, IL, U.S.A.) for 1 month]. At the end of the study, the rats were humanely killed by bleeding, and samples of the visceral peritoneum covering the liver were taken from 3 rats in each group. The samples were examined by transmission electron microscopy (TEM). RESULTS: In control rats, the peritoneum was composed of flattened mesothelial cells covering the thin layer of the submesothelial connective tissue (ST), containing few fibroblasts. In dialyzed animals, a dramatic expansion of the ST was found. We saw compartmentalization of the ST, with changed morphology of the fibroblasts, altered organization of collagen fibers, and changes in the cells infiltrating the ST. Apart from the fibroblasts, mast cells were relatively numerous. CONCLUSIONS: Our work underlines the capabilities of morphology studies in an animal model for assessing peritoneal dialysis fluid biocompatibility.

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The study attempted to define characteristics of thymic epithelial cells within rat thymus based on the expression of neuroendocrine markers. Using an immunohistochemical approach, the following markers were localised: protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE) and chromogranin A (ChA). It was shown that cells displaying immunostaining typical for individual markers reside in distinct regions of the thymus and represent subtypes within various populations of thymic epithelial cells. An immunoreactivity for PGP 9.5 was found exclusively in a subtype of cortical epithelial cells, located mostly within the inner zone of the cortex. On the other hand, NSE represented a marker of most epithelial cells located in the medulla. Few such cells which were negative for NSE proved positive for ChA. Among the cells with a strong reaction for NSE some cells also manifested a positive reaction for ChA. While the pattern of neuroendocrine marker distribution may reflect functional properties of thymic epithelial cells which might be different within distinct areas of the thymus, the differential expression of individual markers seems to reflect biological activity of the cells and/or distinct stages of their differentiation.

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Cholecystokinin (CCK) is a multifunctional regulatory peptide, which acts through two main subtypes of receptors, named CCK-A and CCK-B. Evidence indicates that CCK modulates cell proliferation in various tissues in a paracrine manner, and proofs are available of the presence of CCK in both adrenal glands and thymus. Hence, we have investigated the possible mitogenic action of this peptide on these two tissues, by evaluating the /1000 of metaphase-arrested cells after vincristin injection (mitotic index). The systemic administration of CCK (three subcutaneous injections of 20 nmol/kg, 28, 16 and 4 h before the sacrifice) increased the mitotic index in both the outer adrenal and thymus cortexes of immature (20-day-old) rats and the enucleated adrenal gland of adult (2-month-old) animals at day 5 and 8 of regeneration. The simultaneous administration of equimolar doses of a selective CCK-A receptor antagonist blocked the effect of CCK, while a CCK-B antagonist was ineffective. These findings indicate that CCK exerts a marked CCK-A-mediated proliferogenic effect on both adrenal cortex and thymus in the rat, the physiological relevance of which, however, remains to be demonstrated. In fact, the administration of the CCK-A antagonist alone was ineffective, thereby casting doubts on the role played by endogenous CCK in the maintenance and stimulation of adrenal and thymus growth.

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Endothelins (ETs) are a family of vasoactive peptides widely distributed in the body systems, where they exert pleiotropic biological effects, acting through two main subtypes of receptors, named ETA and ETB. Evidence indicates that ET-1 plays a permissive role in the development of neural crest-derived structures, among which are the epithelial cells of the thymus. These cells are known to control proliferation and differentiation of thymocytes, a process requiring adequate levels of glucocorticoids. Therefore, we have investigated the effects of ET-1, that binds both ETA and ETB receptors, on thymocyte proliferation in monolaterally adrenalectomized rats with contralateral enucleated adrenal at day 4 and 8 of regeneration, when glucocorticoid production is very low and, respectively, rather normal. Metaphase index (percentage of metaphase arrested cells) of thymocytes is the lowest at day 4 of regeneration, and markedly rose at day 8, thereby confirming the need of sizable levels of circulating glucocorticoid for the maintenance of a normal rate of thymocyte proliferation. ET-1 markedly increased the mitotic index of thymocytes at both times of adrenal regeneration. At day 8 of regeneration, the ETA-receptor antagonist BQ-123 markedly lowered mitotic index of thymocytes, and annulled its ET-1-evoked raise. Conversely, the ETB-receptor antagonist BQ-788 was ineffective. Collectively, these findings clearly indicate that endogenous ETs, through the activation of ETA receptors, are involved in the maintenance and stimulation of thymocyte proliferation in the adult rat, thereby playing a possibly important role in the modulation of the immune-system functions.

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Endothelins (ETs) are a family of vasoactive peptides widely distributed in the body systems, where they carry out major autocrine/paracrine regulatory functions, acting through two main subtypes of receptors (ETA and ETB). Evidence suggests that ETs play a permissive role in the development of neural crest-derived craniofacial structures, among which the thymus. Therefore, we have investigated whether ETs regulate thymocyte proliferation in the adult rat ET-1 (which binds both ETA and ETB receptors) increased the mitotic index (% of metaphase-arrested cells) in the thymus cortex, while ET-3 (which preferentially binds ETB) and the selective ETB-receptor agonists BQ-3020 and IRL-1620 did not. The ETA-receptor antagonists BQ-123 and BQ-610, but not the ETB-receptor antagonist BQ-788, abolished the ET-1 effect. Moreover, BQ-123 and BQ-610, when administered alone, evoked a significant decrease in the mitotic index. Collectively, these findings clearly indicate that endogenous ETs, through the activation of ETA receptors, are involved in the maintenance and stimulation thymocyte proliferation in the adult rat, thereby playing a possible important role in the modulation of the immune-system functions.

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Thymic nurse cells are defined in vitro as multicellular complexes of epithelial cells and thymocytes. Although these structures have been implicated in the intrathymic differentiation of thymocytes, little is known about the biology of this cell complex and about the occurrence of the cells in the thymus in situ. Therefore, to clarify the matter, in this review we have presented characteristics of epithelial cells capable of forming complexes with thymocytes, in light of the literature data and the experience of the authors. The structure of cells within the complexes allowed us to distinguish three types of thymic nurse cells. Three-dimensional reconstruction of the thymus and observations employing TEM and SEM demonstrated the presence of distinct types of complexes in various topographic regions of the thymus. Where possible, the functional relevance of the morphological data was analyzed.

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In consecutive serial paraffin sections centers of gravity were defined for individual cross-sections of thymic medulla of the rat. Coordinates of the centers provided grounds for a three-dimensional reconstruction of thymic medulla using for the purpose computer techniques. In all cases the medulla exhibited continuity throughout the organ and showed dendritic character with up to IVth order branching. Moreover, analysis of thymic medulla structure allowed to exclude existence of a structure which would correspond to thymic lobuli. Apart from volume and area of the cortex and the medulla, lengths of the medulla and of its branches were determined. Statistical analysis of the results demonstrated that length of individual types of medulla branches was the least variable character of the thymus while the most variable one included area of the cortex and the medulla and their volume. All parameters describing volume and area of the thymic cortex/thymic medulla were found to correlate with each other. No significant relation was observed between the parameters on one hand and length of the medulla on the other.

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In the present study, frequency of thymocyte divisions was analyzed in the rat thymic cortex, as related to (1) distance from thymic capsule and (2) the daily cycle. Frequency of thymocyte divisions was estimated in successive 10 micron(s) thick layers of the cortex. An evident daily cycle was detected in the thymic cortex with increase in the number of cell divisions around 8 pm.

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The study aimed et determining morphological relations between thymic cells and thymocytes. The studies were performed on 16 days old rats of Wistar strain. The material was processed for ultrastructural studies in a routine manner. For 50 thymocytes computer reconstruction was performed which allowed to define cell volume, volume of cell nucleus and of condensed chromatin. The value characterizing three-dimensional morphology of thymocytes provided grounds for analysis using graph theory. The stereological methods allowed to examine microarchitecture of individual thymic zones. Thymocytes in contact with dendritic cells or with macrophages were found to differ in morphology from thymocytes with no such contacts. The results indicated direct effect of dendritic cells and of macrophages on thymocytes.

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The study aimed et defining morphological relationships between thymic stroma cells and thymocytes. The studies were conducted on 16-days old Wistar strain rats. The material was routinely processed for ultrastructural examination. On grounds of serial section analysis, types of contacts of 83 thymocytes were established, for each cell total area and areas of contact with other cells were estimated. Employing stereological techniques microarchitecture of individual thymic zones was studied. The results point to a direct influence of thymic stroma cells on thymocytes.

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Thymic nurse cell complexes (TNC-c) were isolated from thymuses of BDF1 mice at pre-determined intervals during the 12-week latency period that precedes the development of leukemias. T-cell leukemias were induced by a single i.v. injection of 50 mg/kg of methylnitrosourea (MNU). In order to clarify processes taking place in TNC-c, the complexes of mice after MNU injection were compared with TNC-c of age-matched control mice, with respect to their number per thymus, the distribution of TNC-c according to their size (the number of intra-TNC thymocytes reflects the type of TNC-c), the number of intra-TNC thymocytes that undergo DNA synthesis, and the phenotype of thymocytes inside TNC-c. During the latency period of leukemogenesis, the effects of MNU were shown to involve, in addition to changes in number of TNC-c, a decrease in the number of thymocytes incorporating labeled thymidine, viz., the number of dividing cells, thus affecting the size distribution of TNC-c types. Intra-TNC thymocytes of control mice were heterogeneous in their phenotype and represented cells at varying stages of their maturation cycle. MNU administration was followed by selective differentiation of thymocytes within TNC-c to Lyt 1-thymocytes in some and to Lyt 2-thymocytes in others. Lyt 1 and Lyt 2 being specific antigens expressed by thymocytes.

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