Animals and stress procedures
Male Sprague–Dawley rats (Rattus norvegicus
) ranging in weight from 200 to 250 g were obtained from the hatchery of Instituto Venezolano de Investigaciones Científicas (IVIC). The animals were housed individually in a room controlled for temperature, humidity and lighting. Commercial rat food and water were available ad libitum
. All manipulations followed international ethical guide [14
]. Rats were stressed by restraint in an immobilization conical tube of 50 ml (restrainer) with ventilation holes for 5 hours and for 5 consecutive days. All stress procedures occurred from 11:00 am to 4:00 pm.
Preparation of blood peripheral lymphocytes
The rats were anesthetized with ether and blood samples were taken by intracardiac puncture between 10:00 and 11:00 am in tubes with EDTA, 1.8 mg/ml. The blood was centrifuged at 1000 rpm with a vasculant rotor for 10 min at room temperature. The plasma was collected for determination of interleukins and amino acid analysis, and the layer of white cells plus some red blood cells was taken and transferred to tubes with 10 ml of isotonic saline 0.1 M sodium phosphate buffer pH 7.4 (PBS). These suspensions were placed on 3 ml of Histopaque (Sigma) (1077 g/l). After centrifugation at 2000 rpm for 30 min peripheral mononuclear cell layer was taken, washed twice with PBS and centrifuged at 1200 rpm for 10 min. To achieve enriched lymphocyte preparation with a minimal monocyte contamination. The resulting pellet was diluted with Roswell Park Memorial Institute Medium 1640 (RPMI) free of bovine serum albumin and incubated in a plastic flask for 45 min at 37° C and 5% of CO2. After the incubation, lymphocytes, which are non-adherent cells (80–90%), were dislodged from adherent monocytes, transferred to plastic tubes and washed twice. The integrity of isolated lymphocytes was determined by Trypan blue exclusion test and was greater than 90%.
Lymphocytes were cultured in 96 well plaques, in which 200,000 cells were placed in each one to a final volume of 200 µl of RPMI medium with gentamicin (100 µg/ml), L-glutamine (2 mM) and 10% fetal calf serum (Gibco BRL, Maryland). The incubation was performed at 37° C, 5% CO2 and 100% humidity for 72 h in the absence or in the presence of Con A at suboptimal concentration, 2 µg/ml. TAU (1.5–24 mM) and β–alanine (β-Ala) (0.8-50 mM) ware added to the cultures. Proliferation was measured with 3-[4,5-dimetilazol-2-il]-2,5-diphenil-tetrazolio (MTT) (Sigma, St Louis, MO) (19,20). MTT was prepared in PBS, 5 mg/ml, 20 µl was added to each well, and incubation was done for 4 h at 37°C. Then, 100 µl of solution was extracted and 100 µl of HCl 0.04 N in isopropanol was added. After mixing, the plaque was read in a GENios lector (Tecan) at 570 nm with the Program Magellan.
Measurement of plasma interleukin-1β and interleukin-10
Plasma collected for interleukin-1β (IL-1β) and interleukin-10 (IL-10) assays was stored at -80°C. The levels of IL-1β and IL-10 were measured by ELISA Endogen kits (Pierce Endogen, Cambridge, MA) following the manufacturer’s instructions. Briefly, 100 µl of samples were dispensed into 96 wells coated with rat IL-1β or IL-10 antibody and incubated for 2 hours at room temperature. After extensive washing, 100 µl of the biotinylated anti-IL-1β (or IL-10) were added to each well, and plates were incubated for 30 min at room temperature. The wells were again washed 5 times, 100 µl of Streptavidin-HRP was added and incubation was done for 30 min. 3,3´,5,5´-tetramethylbenzidine (TMB) (100 μL/well) was used as the chromogen for the colorimetric assay. The reaction was stopped by adding 100 µl/well of stop solution and the absorbance was read at 450 nm. The levels of ILs are expressed as pg/ml.
Determination of taurine
TAU in plasma and in lymphocytes was determined by high performance liquid chromatography (HPLC) with fluorescent detection employing a modified method (16). The HPLC system consisted of a Waters 2690 Separation System and a Shimadzu RF-551 fluorescent detector. A Sulpeco LC-18 column 4.6 X 100 mm, 5 µm was employed for amino acid separation. Platelet poor plasma, 300 µl, was acidified with 50 µl of 20% sulfosalicylic acid. Centrifugation was carried out at 17,000 rpm for 20 min, at 4°C, and supernatant was kept at 80°C until chromatographic analysis. Immediately before injection, 50 µl of the supernatants plus 150 µl of potassium borate buffer pH 10.4 and 200 µl of the mixture: 25 mg o-phtaldehyde, 500 µl methanol, 25 µl β-mercaptoethanol (1 g/ml), and 4.5 ml 0.4 M potassium borate buffer pH 10.4 was used for derivatization. Then, 15 µl of the derivatized preparation were injected into the chromatographic system. The levels of amino acids were calculated from the area under the curve of samples and external standards with program Millenium, and expressed as nmol/ml.
Analysis of data
Data are expressed as the arithmetic mean ± standard error of the mean (SEM). Differences were statistically analyzed using the Student's t-test. Statistical significance was considered if P < 0.05. Data management and statistical analysis were conducted employing the program Microsoft Office Excel 2007.