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0MORPHOFUNCTIONAL FEATURES OF THYMUS AT SPLENECTOMY IN HEALTHY ANIMALS
Mukhamedjanov A.Kh.
Alfraganus University https://doi. org/10.5281/zenodo. 13926436
Abstract. For a clearer understanding of morphologic shifts occurring in the thymus after splenectomy, we studied the thymus of control groups of animals. The analysis of structural features of thymus in these animals in the whole dynamics of experiments showed the absence of any pronounced changes. The main morphological properties of the thymus of control rats are summarized below.
Keywords: thymus, cortical zone, brain zone, splenectomy, mast cells, spleen.
Data on changes in the thymus during splenectomy under physiological conditions are very few and are characterized by discrepancy (G.M.Butenko et al., 1991; 1998; D.Chen et al., 1998; G.Iu.Stnichko, 1998; G.Iu.Struchko et al., 2001). At the same time, interorgan relationships "thymus-spleen" in conditions of pathology are of direct scientific and applied interest, since these organs interconnectively react to various pathological processes with disorders of the immune status of the organism (A.H. Babaeva et al., 1995). Intersystem relationships of the immune system with other systems of the organism are most clearly manifested by the example of the spleen in clinical pathology. It is well known that a number of diseases (chronic toxic viral hepatitis, liver cirrhosis, some diseases of the blood system, malaria, etc.) are often complicated by splenomegaly and hypersplenism syndrome, which are manifested in the form of pancytopenia in the blood ( N. L. H a l i t a , 1990; X. L. .K a r i m o v et al., 1995; E.A.Zotikov, 1996; F.N.Bashour et al., 2000; Y.FJiaa et al., 2001; MJBolognesi et al., 2002). Involvement of the spleen in the pathologic process in these cases is beyond doubt, however, its place and importance in the pathogenesis of these diseases remains unclear. Removal or disconnection of the spleen from the portal circulation system is considered to be one of the methods of choice for surgical treatment of hypersplenism syndrome and portal hypertension in liver cirrhosis. Nevertheless, splenectomy or splenic artery embolization, along with undoubted positive effect on blood system state, still do not completely prevent hypersplenism syndrome (H.Ben-Hur et al., 2002; Y.Nakanuma, 2001; Z.Cao et al., 2002; S.Srinivasan et al., 2003). It is probably about the fact that in conditions of liver pathology there are deeper violations of intersystem and interorgan relationships between immune and digestive systems, the clarification and correction of which is one of the necessary conditions for pathogenetic therapy (H. L. K a r i m o v et al., 1995; K. R. T u h t a e v et al., 2003; N. K. Tukhtaev et al., 1997). So far, it has not been clarified how splenectomy affects the state of immune and other systems of the organism under physiologic conditions.
Purpose of the study: The aim of the work was to find out morphological and morphometric parameters of thymus in dynamics after splenectomy in healthy animals.
Materials and methods of research: Experiments were carried out on white sexually mature male rats with body weight of 150-170 g. A total of 62 animals were used. The animals were quarantined for a week before the experiment and after exclusion of somatic and infectious diseases were transferred to the usual vivarium regime. Slaughter of both control and experimental animals was carried out on the 60th and 90th day of the experiments, in the morning hours, on an
empty stomach, under light ether anesthesia. Thymus pieces served as a material for research, which were further studied by morphological, morphometric and ultrastructural methods of research. For light-optical studies, thymus was fixed in Carnoy's or Bouin's liquids and, after appropriate wiring, embedded in paraffin. After deparaffinization, 5-7 ^m thick slices were stained with hematoxylin and eosin.
The absolute (in p,m1) and relative (in %) area of different structural and functional zones of thymus lobules and connective-tissue components were determined on the obtained preparations using a morphometric grid.
The absolute number of different cells of thymus parenchyma, as well as the number of mitotically dividing and destructive thymocytes per 1000 cells were counted. The results were expressed in absolute numbers and in percentages.
For electron microscopic studies, thymus pieces were fixed in 1.25% glutaric aldehyde solution with additional fixation in 1% osmium tetraoxide (OsO<) solution on phosphate buffer (pH-7.3). After dehydration in alcohols and absolute acetone, the slices were cast in a mixture of araldite and epon. Ultrathin sections made on an ultramicrotome LKB-4800 (Sweden) were sequentially contrasted with uranyl acetate, lead citrate and examined in electron microscopes JEM-7 and JEM- 100SX (Japan).
All digital data were processed according to the Fisher-Styodspt criteria using a software package on a Pentium II computer; differences satisfying P < 0.05 were considered reliable.
Results: The rat thymus morphologically has a pronounced lobular structure. The connective tissue capsule surrounding the thymus forms septa that divide the organ into incomplete lobules. The number of them is from 3 to 8 on the slices. The surrounding capsule and septa consist of bundles of collagen fibers, among which there are fibroblasts, fibrocytes and single tissue basophils or (mast cells). The parenchyma of each lobe of the thymus is quite clearly delineated into cortical and cerebral zones. These zones differ from each other mainly by the density of distribution of thymus lymphocytes or thymocytes (Fig. 1).
In the cortical zone, which occupies about 70-75% of the total area of the lobule, thymocytes are distributed more densely (Fig. 2). At the same time larger thymocytes or prethymocytes, which are close to prolymphocytes and large limocytes in structure, are located directly close to connective tissue septa. Among these cells one can often see thymocytes at different stages of mitotic division.
Figure 1. Thymus of the control rat. Cortical and medullary zone of the lobule. Hematoxylin and eosin staining. ca.10.
Ultrastructurally, the thymus of control rats is characterized by the presence of two main groups of cells. The first group consists of thymocytes, which include lymphocytes at different stages of differentiation. The other group of cells is represented by reticuloepithelial cells, macrophages, interdigitating cells, degenerative epithelial cells that are part of Hassall's corpuscles. Electron microscopically, thymocytes of the cortical zone represent a heterogeneous population of cells.
The results showed that in the cortical zone thymocytes are densely arranged and represented by medium and large lymphocytes, as well as cells of prolymphocyte and lymphoblast type (Fig.4). Some of these cells may be at different stages of mitotic division.
Figure 2. Thymus of the control rat. Cortical
zone. Individual reticuloepithelial cells (RECs) are seen among densely distributed thymocytes. Hematoxylin and eosin staining, ca.10.
Figure 3. Thymus of the control rat. Brain zone with Hassall's corpuscles in the center. Hematoxylin and eosin staining. ca.10.
Figure 4. Thymus of the control rat. Thymocytes of the cortical zone. Transmission electron microscopy. (TEM). Eq.11200x.
Fig. 5. Thymus of the control rat. Reticuloepithelial cell (REC) of the cortical zone. Secretory vacuoles. TEM. Eq.32000x
Between thymocytes there are outgrowths of reticuloepithelial cells, which contained individual mitochondria, bundles of tonofibrils and light secretory vacuoles with sizes from 0.5 to 1.5 p,m. Some vacuoles had small amounts of fine-grained material of high electron density (Fig. 5). Outgrowths of reticuloepithelial cells are located between thymocytes, in most cases surrounding them on all sides. Also, complexes of thymocytes with reticuloepithelial cells are quite well described in the literature and are characteristic of the thymus in almost all vertebrates. Reticuloepithelial cells in these complexes play the role of a "nurse cell", providing nourishment for differentiating thymocytes and exerting a regulatory influence on them. Studies have shown that cortical thymocytes are heterogeneous in their ultrastructure lymphoblasts and prolymphocytes. In addition to lymphoblasts and prolymphocytes in the cortical zone, the main mass of thymocytes is composed of cells identified as small and medium lymphocytes. Cell nuclei mainly contain large clumps of heterochromatin.
Among them there are many cells that by ultrastructural characteristics correspond to the nucleus. The narrow rim of the cytoplasm contains many ribosomes, single mitochondria and profiles of granular endoplasmic network. As a rule, mitoses are much less frequent in the inner part of the cortical zone than in the outer part. In deep parts of the cortical zone, macrophages are detected among thymocytes. They are characterized by irregular shape due to cytoplasmic outgrowths. The nucleus of cells is also irregularly shaped, mostly containing euchromatin. Often a nucleus is detected. The cytoplasm contains a significant number of lysosomes and phagosomes of various sizes and densities. The cytoplasm of macrophages, as a rule, is in close contact with the envelope of surrounded thymocytes. Such ultrastructural organization is characteristic of interdigitating cells of the thymus and thymus-dependent zones of peripheral organs of the immune system. Thus, the analysis of the obtained data showed the presence of two main cell populations in the thymus of control rats. The main cells of the thymic microenvironment are reticuloepithelial cells, which are characterized by certain ultrastructural features in the cortical and cerebral zones of the organ. In addition to them, interdigitating cells and macrophages participate in the creation of thymic microenvironment. Among thymocytes of superficial parts of the cortical zone, cells with ultrastructure corresponding to lymphoblasts and prolymphocytes are often detected, whereas in the corticomedullary zone and cerebral zone, thymocytes of medium and small size are mainly localized.
Thus, the rat thymus is a complex lymphoepithelial organ, where epithelial cells create a microenvironment for future T-lymphocytes and their subpopulations.
Splenectomy performed in healthy animals had no significant effect on morphologic and morphometric parameters of the rat thymus. In splenectomized animals, the lobular structure of the thymus was preserved during all periods of observation. The thymus parenchyma was clearly divided into cortical and brain zones, which had the same morphological features as in control rats (Fig.6). The cortical zone had a darker appearance due to dense distribution of thymocytes (Fig.7) In the cerebral zone thymocytes were distributed more loosely, Hassall's corpuscles were rare. Only in some cases there was a slight dilation of blood vessels and blood stasis in them. Ultrastructural studies performed after splenectomy in healthy animals revealed no significant submicroscopic changes. Thymocytes of the cortical zone are mainly represented by medium and small lymphocytes densely enough adjacent to each other (Fig.8). Outgrowths of reticuloepithelial cells containing mitochondria and vacuoles with granular material are detected between them (Fig.9). Sometimes individual macrophages and interdigitating cells with a similar above-
described structure were detected. In the same w detected on the side of cells of the cerebral zone.
Figure 6. Rat thymus on the 60th day after splenectomy. Cortical and medullary zone of the lobule. Hematoxylin and eosin staining.
ca.10.
no definite ultrastructural changes were also
Figure 7. Rat thymus on the 90th day after splenectomy. Cortical zone of the lobule. Hematoxylin and eosin staining. 06.9, ca. 10
Figure. 8. Rat thymus on the 60th day after Figure. 9. Rat thymus on the 90th day after splenectomy. Thymocytes of the cortical zone. splenectomy. REC of the cortical zone with (TEM). Eq .20000x secretory vacuoles. (TEM). Eq.3000x
Morphometric study in the dynamics of splenectomy in healthy rats showed the following. Table 1 shows that the average area of thymus lobules sections on the 40th day of experiments had a slight tendency to decrease in comparison with the control. However, the differences were statistically insignificant.
Table 1
Average area of slices of thymus lobules in the dynamics of splenectomy, performed in healthy
rats (M±t x 105 nm2)
Animal groups Observation period (days)
40 60 90
Controls n= 15 22,1±0,9 21,6±1,2 20,7±1,1
Splenectomized n=20 20,9±0,8 21,4±0,9 20,3±1,2
Table 2
Ratios of the average areas of cortical and brain zones of thymus in the dynamics of splenectomy performed in healthy rats (M±t x 105 pm2)
Animal groups Observation period (days)
40 60 90
KZ МZ RZ МZ МZ
Controls n = 15 16,2±0,4 5,9±0,1 15,9±0,5 5,71±0,3 15,6±0,7 5,14±0,2
Splenectomized n=20 15,6±0,5 5,23±0,3 16,0±0,4 5,4±0,2 15,2±0,4 5,05±0,3
Table 3.
Average density of cell location in the thymus lobule zones in the dynamics of the splenectomy performed in healthy rats (M±t x 102 cells per 105 pm2 area)
Animal groups Observation period (days)
40 60 90
МZ КZ МZ КZ МZ
Controls n= 15 17,4±0,3 9,6±0,1 16,8±0,2 9,8±0,2 17,2±0,4 9,11±0,1
Splenectomized n=20 18,6±0,4 10,2±0,3 19,1±0,1* 10,1±0,3 20,4±0,3* 10,4±0,3
Note: hereinafter the sign* indicates statistically significant differences compared to the control group at P<0.05..
Table 4
Quantitative indices of cells of different zones of thymus lobules under conditions of splenectomy on the 90th day of experiments (M±t absolute number of cells per x105 pm2)
№ Groups of living things Thymus cells Intact ^=12) Splenectomy (п=Т2)
МZ КZ МZ
1 Lymphoblasts 55,25±2,1 7,42±0,2 72,83±3,0* 10,58±0,3*
2 Large lymphocytes 136,1±3,4 11,25±0,4 157,3±4,2* 14,58±0,5*
3 Small and medium-sized lymphocytes 1396,0±18,1 722,8±15,4 1628,0±22,0* 772,8±16,0
4 Reticuloepithelial cells 60,42±1,8 172,8±5,6 64,5±1,3 175,5±6,4
5 Macrophages 12,58±0,4 6,83±0,2 14,25±0,6 8,42±0,4*
6 Interdigitating cells 25,67±0,6 15,58±0,5 27,42±0,8 18,25±0,7*
7 Granulocytes - 5,42±0,14 - 7,6±0,3*
8 Tissue basophils 13,83±0,5 6,25±0,3 15,42±0,3 8,6±0,2*
9 Plasmatic cells - 2,2±0,12 - 3,42±0,2*
10 Total cells 1700,0± 136 950,0±66,5 1980,0±118,8 1020,0±91
Table 2 shows the average areas of cortical and brain areas of thymus lobules in the dynamics of splenectomy. These parameters also had no significant differences in comparison with control animals. Somewhat different data were obtained when determining the average density of cell arrangement in thymus zones after splenectomy in healthy animals (Table 3). On the 60th day
after splenectomy, the density of cells in the cortical zone increased by 14% compared to the control, and the density of the brain zone remained without significant changes. On the 90th day of the experiments, the cell density increased even more and in the cortical zone 19% exceeded the control values. A slight increase in cell density in the cerebral zone at this time was statistically unreliable.
Conclusion. Thus, splenectomy performed in healthy sexually mature animals has no significant effect on morphological and morphometric characteristics of thymus. There is only a slight increase in the density of cell arrangement in the cortical zone, mainly due to thymocytes, an insignificant tendency to increase the number of mitoses and destructive thymocytes. It follows that under physiological conditions the spleen has minimal influence on the processes of proliferation and differentiation of T-lymphocytes in the thymus.
Table 6
Dynamics of changes in the number of destructive thymus cells in the dynamics of splenectomy performed in healthy rats (per x 103 cells, М±m)
Animal groups C Observation period (days)
40 60 90
KZ MZ KZ MZ KZ MZ
Controls n=15 24,1±1,78 12,6±0,9 26,0 ±0,4 14,0±1,2 25,3±1,7 15,3±1,2
Splenectomy n=20 27,6±2,0 13,7±1,1 24,7±1,6 12,9±1,0 27,9±1,8 16,9± 1,5
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