Melatonin effect on the thymus cell composition in mice with functional pinealectomy

Abstract

More and more data is accumulating about the close connection between the pineal gland and the immune system. Prolonged continuous lighting contributes to the development of light-induced functional pinealectomy, as a result of which melatonin (MT) synthesis in the pineal gland is reduced/blocked. It was found that the character of the 24-hour rhythms of the number of B- and T- cells, T-helper cells and T-suppressors/killers, the expression of CD45, CD5, CD3 and CD4 surface molecules on rat lymphocytes depends on the light regime and changes when animals are kept in conditions of constant light or constant darkness (Pelegri C. et al., 2003). Situations with functional pinealectomy or MT production inhibition lead to immunosuppression, to a decrease in adaptive cellular and humoral immune response. The use of this hormone helps to restore the immune system functions. However, the peculiarities of MT effect on the thymus cellular composition of animals under the conditions of functional pinealectomy remain not fully investigated. Thymus samples were taken to study its cellular composition using a flow cytofluorimeter. A cell suspension was prepared from the thymus by soft crushing in a glass homogenizer. Then fixation was carried out in ice 70% ethanol. Immunophenotyping of thymic CD3 hi and CD3 low T-lymphocyte subpopulations was performed by staining with CD3-APC antibodies (BioLegend Inc., USA). 3 µl of monoclonal antibody in the presence of 1% albumin was added to 50 µl of each sample. The samples were incubated at room temperature in the dark for 30 min and then washed twice with PBS. The analysis of obtained thymocytes was carried out using a CytoFlexS-100 flow cytofluorimeter (Beckman Coulter, Inc, USA). The CD3 low /CD3 hi ratio was calculated. Statistical processing was performed using the software package "Statistica12". Median values, values of the first and third quartiles, and the arithmetic mean were determined. The reliability of the differences between compared values was determined using Mann-Whitney test. The differences were considered statistically significant: at p<0.05 - *, at p<0.01 - **.