The effect of dichloroacetate on male rat thymus and on thymocyte cell cycle

Experimental design

Male Wistar rats (gonad-intact; n = 18; 4–5 weeks old) and castrated age-matched rats (n = 18) were investigated. Gonad-intact male rats were randomly divided into three groups (n = 6 in a group): (1) control; (2) treated with DCA; and (3) treated with NaVP and DCA. In the same three groups (n = 6 per group), castrated male rats were investigated. Rat castration was performed by removing the testes at the age of 4 weeks (in the peripubertal period − 28 ± 2 days). The effect of castration and treatment on thymus in rats was assessed. The permission of the State Food and Veterinary Service of Lithuania to use experimental animals for research was obtained (2015-05-18 No. G2-28). The animals were purchased from the Animal Facility of the Lithuanian University of Health Sciences, Veterinary Academy (Kaunas, Lithuania). The animals were housed in standard colony cages with free access to food; they were housed under conditions of constant temperature (21 ± 1°C), humidity, and a light/dark cycle (12/12 h). A commercial pellet diet was provided ad libitum. The experiments were performed in compliance with the relevant laws. The animals were housed in the described conditions and acclimated for at least 5 days before the experiment.

At 5 weeks of age, rats were treated with DCA 200 mg/kg/day in drinking water and 300 mg/kg/day of NaVP plus 200 mg/kg/day of DCA (every second week, beginning with NaVP and ending with DCA) aqueous solutions for 4 weeks. The DCA and NaVP solutions were offered to animals ad libitum as their only source of drinking (in aluminum foil-wrapped bottles to avoid light decomposition). Control groups were given fresh drinking water provided ad libitum. The treatment of castrated animals was started 1 week after castration (at 5 weeks of age). After 4 weeks of treatment, animals were killed in a 70% CO₂ camera. The thymus was separated and weighted, and its one lobe (right) was taken for the thymocyte flow cytometry analysis.

NaVP and DCA were purchased from Sigma-Aldrich GmbH, Germany.

Thymocyte preparation

To minimize contamination with red cells, the carotid arteries were cut and the animals exsanguinated. Thymocytes were isolated from the thymus: the thymus was removed with forceps and cleared of blood vessels. Contaminating blood was removed by rinsing with RPMI-1640 (Biological Industries, Israel). The thymus was then minced and pipetted vigorously with a syringe several times. Large cell aggregates and connective tissue were removed by passage through two layers of surgical gauze. The resulting suspension of lymphocytes was washed free of red blood cell contamination by centrifugation two times in RPMI-1640 at 400 × g for 8 min each, and the supernatant was decanted. Thymocytes thus isolated were resuspended in RPMI-1640 supplemented with 10% (v/v) fetal calf serum (CE-Immundiagnostika GmbH, Germany) 100 µg/mL penicillin and 100 U/mL streptomycin (Sigma-Aldrich, Italy). Total counting of thymocytes was made in Fast-Read 102 chambers (Biosigma, Italy) stained with eosin (Reagena, Finland).

Flow cytometry analysis

The cell cycle analysis of control and DCA, NaVP–DCA-treated rat thymocytes was performed using an APC-BrdU flow kit (#552598) from BD Bioscience Pharmingen, US, according to the manufacturer’s instructions: thymocyte suspensions were labeled in vitro with BrdU (10 µM) added directly to the RPMI-1640 medium supplemented with fetal calf serum, penicillin, and streptomycin, for 24 h, at 37°C in a humidified atmosphere with 5% of CO_2. The cells were then fixed, permeabilized, and treated with DNase (300 µg/mL, 1 h, 37°C) before staining with anti-BrdU-APC monoclonal antibodies and the 7-AAD reagent as recommended by the manufacturer’s protocol. The cells were analyzed on a FACSCanto (Becton Dickinson, USA) for APC and 7-AAD fluorescent dye, respectively. In total, 10,000 events were counted for each sample.

Statistical analysis

Statistical analysis was performed by using the Statistical Package for the Social Sciences, version 22.0 for Windows (IBM SPSS Statistics V22.0, USA). The normality assumption of data was verified with the Kolmogorov–Smirnov test. The results are presented as the mean ± standard deviation (SD). When the normality assumptions are not met, data are expressed as a median and a range (minimum and maximum values). Differences between two independent groups were evaluated using the non-parametric Mann–Whitney U-test. The one-way ANOVA analysis was used to determine significance among the groups and post-hoc tests with Fisher’s least significant difference (LSD) were used for comparison between the individual groups. Spearman’s rank correlation coefficient (r) was used to assess relationships between thymus weight and rat body weight. Differences at the value of P <0.05 were considered significant.

The effect of castration on thymus

The mean age of rats at the end of the experiment was 60 ± 3 days and did not differ significantly among the groups. Statistically significant difference was found in the thymus weight between the intact male control and castrated male control groups (Figure 1): the thymus weight in castrated males significantly increased, i.e. castrated males showed a progressive thymus hyperplasia. No significant difference was found when comparing the rat weight of gonad-intact and castrated male rat groups (255.89 ± 48.30 and 245.12 ± 17.53; P >0.05). The weight of DCA-treated castrated rats was significantly lower than of their control (208.17 ± 22.77 and 245.12 ± 17.53; P <0.02). No statistically significant correlation was found among gonad-intact and castrated control rats’ weight and their thymus weight in all treated groups.

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Figure 1.

Investigation data of DCA and NaVP–DCA treatment effect on gonad-intact and castrated male rat thymus.

The effect of treatment on thymus

Comparing the thymus weight of the gonad-intact DCA-treated rat group with their control, the thymus weight was found statistically different—the treatment with DCA caused a significant decrease of thymus (Figure 1). A comparison of thymus weight of gonad-intact control and NaVP–DCA-treated rats showed a significant thymus weight loss after the NaVP–DCA treatment. A comparison of thymus weight between gonad-intact DCA-treated and NaVP–DCA-treated groups revealed a significantly higher thymus weight loss in the DCA-treated rat group. A moderate and statistically non-significant correlation between rat body weight and thymus weight was found in rat groups treated with DCA and in those treated with DCA and NaVP combination (P >0.05); also, statistically non-significant differences were found comparing rat weight among the gonad-intact control, DCA-treated and NaVP–DCA-treated rat groups (P >0.05).

No significant effect of DCA as well as of NaVP–DCA treatment combination was found on thymus weight of castrated rats (P >0.05). A comparison of thymus weight among castrated rats treated with DCA, treated with a DCA and NaVP combination, and respective groups of gonad-intact treated rats shows a significantly lower thymus weight in gonad-intact DCA- and NaVP–DCA-treated groups (Figure 1).