Effect of a transdermal film with erythropoietin on lymphocyte death and free radical oxidation in blood of rats with experimental thermal injury

Abstract

Aim. Studying the effect of erythropoietin (EPO) as a component of the transdermal film (TDF) on lymphocyte apoptotic death and free radical oxidation in blood of rats with experimental thermal injury (TI) (trauma). Methods. Experiments were performed on 90 white mongrel male rats weighing 220±20 g. The IIIA grade TI with a burn area of 3.5% body surface was induced under general anesthesia by immersing the interscapular region of the skin in purified water at 98-99ºС. EPO (42 IU/cm²) as a component of the 12 cm² TDF was applied daily, immediately after the TI. The animals were randomized to three groups: group 1 (n=10), intact control; group 2 (n=40), TI with application of an aseptic dressing to the burn area; group 3 (n=40), TI with application of the EPO-containing TDF to the burn area. The study was conducted at days 3, 5, 8, and 14 after TI. Lymphocytes were isolated from blood using the ficoll-verographin density gradient (1.077) and their apoptosis was evaluated by staining the cells with fluorochrome-conjugated Annexin V (Annexin-5-FITC) and 7-aminoactinomycin D (7-AAD). The cells were differentiated into intact cells, cells with early apoptosis signs, cells with late apoptosis signs, and partially necrotized cells. Concentration of lipid peroxidation (LP) products was measured spectrophotometrically; superoxide dismutase (SOD) activity was measured in plasma and catalase activity was measured in plasma and red blood cells. Results. TI increased the count of lymphocytes of the Annexin-5-FITC+/7-AAD (early apoptosis signs) and Annexin-5-FITC+/7-AAD+ (late apoptosis or necrosis signs) phenotypes, and concentrations of dienic conjugates, ketodienes, conjugated trienes, and Schiff bases in heptanol and isopropanol phases of plasma and lymphocyte lipid extracts. Plasma and red cell catalase activities were increased whereas plasma SOD activity was decreased. Application of EPO as a component of the TDF decreased the amount of lymphocytes with early and late apoptosis signs and the contents of dienic conjugates, conjugated ketodienes and trienes, Schiff bases in plasma and lymphocytes, and catalase activity in plasma and erythrocytes, and increased the plasma activity of SOD. Conclusion. The use of EPO as a component of TDF in TI reduced lymphocyte apoptosis, activity of LP processes, and increased the plasma activity of SOD compared to the group of animals with TI without the EPO treatment.