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Dexamethasone, Selenium, Lymphoid tissue, What&,rsquo s Known Dexamethasone is an anti-inflammatory synthetic glucocorticoid, which induces lymphoid organ atrophy. Selenium has been proved to have immunomodulatory effects and prevent immunodeficiency induced by dexamethasone. Measuring the structural properties of lymphoid organs can be effective in assessing the rate of disease recovery or progression. What&,rsquo s New We are the first to estimate quantitative structural changes in lymphoid organs by using dexamethasone as immunodeficiency and selenium as immunomodulatory agents. Lymphoid structures containing B lymphocytes are affected more than lymphoid structures containing T lymphocytes in both immunodeficiency and immunomodulatory states. IntroductionThe structural features of lymphoid organs indicate the efficient functioning of the immune system. 1, Lymphoid organ atrophy and lymphocyte apoptosis should be considered during such conditions as stress, disease, chemotherapy, and the long-term use of drugs such as glucocorticoids. These factors may induce lymphocyte apoptosis and lymphoid organ atrophy 2,, 3, and probably lead to immunodeficiency. 4, Thus, lymphoid organs undergo prominent structural changes after exposure to these factors. 5, Dexamethasone (DEX) is an anti-inflammatory synthetic glucocorticoid that induces lymphoid organ atrophy. 2,, 3, DEX has been shown to reduce the structural components of the spleen, especially the white pulp and the periarterial lymphatic sheath (PALS) zone. 3,, 6, Histomorphometric studies of lymphoid tissues demonstrated that DEX also caused not only a reduction in the thymic cortex-medulla ratio but also atrophy in lymph nodes, particularly in the paracortical zone. 3,, 7, The thymus, spleen, and lymph nodes have various components that play roles in specific immune functions. These components include the cortex and medulla of the thymus the cortex (outer and inner zones) and medulla of the lymph node and the red pulp (composed of the splenic cord and the sinusoids), white pulp (composed of the PALS zone and follicles), capsule, and trabeculae of the spleen. 8,- 10, Many herbs, bacterial toxins, and chemical elements such as selenium are immunomodulators and are suggested to prevent immunodeficiency. 11,- 13, Selenium is an essential element that induces lymphocyte proliferation and antibody response. 14,, 15, It plays an important role in alleviating lymphoid organ injury. 16, Dimensional variations in the thymus, spleen, and lymph nodes reflect the efficient functioning of these organs. Quantitative evaluations of the volume of these components can provide comparable and reliable data. Stereological methods can be used to evaluate and record quantitative structural changes in the lymphoid sub-components. 17, Briefly, the present study was conducted to quantitatively estimate the volume of the thymus, spleen, and lymph nodes and their sub-components after exposure to DEX and selenium in a rat model.Materials and MethodsThis study was conducted at Histomorphometry and Stereology Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran, in September 2016 to September 2017. The present animal experiment was carried out under the supervision of the Ethics Committee of Shiraz University of Medical Sciences (Approval No, IR.SUMS.REC.1388.S4559). Sprague-Dawley rats, weighing 230&,ndash 250 g, were obtained from the Laboratory Animal Center of Shiraz University of Medical Sciences. The rats were maintained under standard conditions and given ad libitum access to food and water. 18, Drug PreparationDEX was purchased from Osvah Pharmaceutical Company (Tehran, Iran), and selenium was prepared from Merck Company (Darmstadt, Germany).AnimalsThirty-two male rats were randomly assigned to four groups of eight. Group I (control) was given 0.5 mL/kg of normal saline intraperitoneally for three days and then orally for 30 days Group II was administered 0.4 mg/kg/d of DEX intraperitoneally for three days Group III was given 0.1 mg/kg/0.5 mL/d of selenium orally for 30 days plus 0.4 mg/kg/d of DEX intraperitoneally for three days and Group IV was administered 0.1 mg/kg/0.5 mL/d of selenium orally for 30 days. 19,, 20, At the end of the experiment, the animals were sacrificed under deep anesthesia with ether. Then, their thymus, spleen, and superficial inguinal lymph nodes were removed. The tissues were weighed on a microbalance sensitive to 0.01 g (Scaltec, Heiligenstadt, Germany). The primary volumes of the thymus, spleen, and lymph nodes were estimated by the immersion method. 21, Afterwards, the tissues were fixed in 10% buffer formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 72 hours. Stereological StudyThe orientator method was used to obtain isotropic uniform random sections. 22, To this end, the thymus, spleen, and lymph nodes were placed on a circle divided into 10 equal distances. A random number between 0 and 10 was selected, and the tissue was sectioned into two halves, with a blade in that direction. The cut surface of each half of the tissue was placed on the 0-0 direction of the second circle with 10 unequal cosine-weighted divisions, and the second cuts were made. A total of 8&,ndash 10 slabs were collected from each organ, and one circle was punched from each slab using a trocar (thickness 2). The diameters and areas of the circular pieces were measured (figure 1,). The cut surfaces of the slabs and circular pieces were embedded in soft paraffin and 5-&,micro m sections were prepared. The sections were stained by hematoxylin and eosin (H&,amp E). In the post fixing, the area of the circular piece was measured again. This method was suitable for the prevention of shrinkage.Figure 1. The photomicrograph of the rat spleen stained by hematoxylin and eosin (H&,amp E) and sectioned by the orientator method to obtain isotropic uniform random (IUR) sections is displayed. A) The spleen was placed at the center of the circle divided into equal distances. Each part of the sectioned tissue was placed on a second circle with unequal divisions. B) In this section, another number was selected and the tissue was sectioned into slabs. C) In this part, the remaining portion of the tissue was vertically placed on the same circle and sectioned into slabs in a new direction. D and E) The volumes of the different components of the thymus and spleen were estimated respectively, by the point-counting method.The volume shrinkage was calculated via the following formula, 23, volume shrinkage=1-(AA/AB)1.5where AA and AB are the areas of the circular piece after and before processing by sectioning and staining, respectively. The final volume of the tissue was estimated via the following formula, 24, Vfinal=Vprimary&,times (1-volume shrinkage)Each sample section was analyzed using a video-microscopy system that consisted of a microscope (E-200, Nikon, Tokyo, Japan) linked to a video camera (SSC, Sony, Tokyo, Japan), a computer, and a flat-screen monitor (LG, South Korea). Each parameter was estimated by examining 10&,ndash 14 microscopic fields per each thymus, spleen, and lymph node by point-counting method (figures 1, and 2,). Microscopic fields were selected by systematic random sampling method in which the slide at equal intervals along the X and Y axes were moved, using a stage micrometer. By means of a stereology software tool designed by our university, relevant grids (point grid) were overlaid on the monitor. The volume density of the different components per unit volume of the reference tissue Vv component/final was estimated via the point-counting method and the following formula, 24, Vvcomponent/ final=Pcomponent/P final where P component and P final are the points hitting the favored component and the whole tissue, respectively.Figure 2. The total volume of the lymph nodes and the volumes of their cortex and medulla were estimated by the point-counting method (500&,times , hematoxylin and eosin (H&,amp amp E]).The total volume of the components was obtained by multiplying the volume density by V final. 24, Vcomponent=Vvcomponent/reference. vfinalStatistical AnalysisThe results were analyzed using the Kruskal&,ndash Wallis test and the Dunn test. A P value of less than 0.05 was considered statistically significant. The data were analyzed using SPSS statistical software, version 15, (SPSS, Chicago, IL, USA).ResultsVolumes of the Thymus and Its Cortex and MedullaThe volumes of the thymus, thymic cortex, and thymic medulla in the DEX-treated rats were decreased by 55%, 64%, and 37%, correspondingly, on average in comparison with the control animals (P=0.001) (table 1,), indicating that the reduction in the thymic volume was associated more with the volume of the thymic cortex.GroupsControlDEXP value a,DEX+SeP value b,SeP value c,VolumeTotal (mm3)292.00&,plusmn 0.05131.50&,plusmn 0.03 0.001*,180.90&,plusmn 0.02 0.001*,290.00&,plusmn 3.00.080Cortex (mm3)201.10&,plusmn 0.0574.30&,plusmn 0.01 0.001*,134.10&,plusmn 0.02 0.001*,200.00&,plusmn 1.530.085Medulla (mm3)91.30&,plusmn 0.0157.10&,plusmn 0.02 0.001*,46.70&,plusmn 0.01 0.001*,92.00&,plusmn 1.20.121a Comparison between the normal control group and the DEX group b Comparison between the DEX+ Se group and the DEX group c Comparison between the Se group and the normal control group * A P value&,lt 0.05 was considered statistically significant. Kruskal&,ndash Wallis test and Dunn test were used to determine statistically significant differences between the groups. DEX, Dexamethasone Se, Selenium |