Male Lewis rats (6 weeks old, 140–170 g) were purchased from Charles River S.A., Spain, and were individually housed in a standard animal facility. Rats were put in individual cages to avoid cannibalism among calorie restricted animals [12]. Control rats (n = 14) had free access to an equilibrated diet (AIN-93G, Diets Inc., Pennsylvania, USA),) and water for 4 weeks. Severely calorie-restricted rats (n = 14) had daily access to 7 g of an unbalanced AIN-93G diet enriched in proteins and low in fat and carbohydrates [13] and water ad libitum for 4 weeks. This calorie restriction was equivalent to a 66% food restriction. A second group of 14 rats had daily access to 14 g of an unbalanced AIN-93G diet enriched in proteins and low in fat and carbohydrates (33% calorie restriction). The experiments were conducted in accordance with the guidelines of the International Council for Laboratory Animal Science (ICLAS). Protocols were approved by the Institutional Animals Ethics Committee. Spinal cord obtained from adult Wistar rats was homogenized in PBS buffer at a concentration of 1 g/mL to serve as an immunogen.
After 15 days of calorie restriction, each group of rats (control and moderate or severely calorie-restricted rats) were divided in two subgroups as follows: a) animals receiving complete Freund's adjuvant (n = 7); b) animals receiving complete Freund's adjuvant plus SCH (n = 7). Rats were immunized by the s.c. injection of a mixture of SCH and complete Freund's adjuvant containing Mycobacterium tuberculosis H37Ra (5 mg/mL; Difco Laboratories, Detroit, Michigan) (v/v) in a final volume of 200 μl. Animals were assessed daily for clinical signs of EAE using the following criteria: 0, normal; 0.5, loss of tonicity in distal half of tail; 1, piloerection; 2, total loss of tail tonicity; 3, hind leg paralysis; 4, paraplegia; and 5, moribund.
Animals were killed by decapitation on day 15 after immunization (7 animals per group) and blood was collected from the trunk wound in heparinized tubes and was centrifuged at 1500 × g for 15 min. The spleen, SmLN and thymus nodes were removed aseptically, weighed and placed in Petri disks containing balanced salt solution; the cells were then gently teased apart. After removing the clumps by centrifugation, the cells were suspended in sterile supplemented medium (RPMI 1640), containing 10% heat-inactivated, fetal bovine serum, 20 mM L-glutamine, 0.02 mM 2-mercaptoethanol and gentamicin (50 mg/ml), and were counted.
Mitogen assays were performed as described in detail elsewhere [14]. Splenic, SmLN or thymic cells were used at a final number of 5 × 104cells per 0.1-ml well. Control and experimental cultures were run in triplicate. Mitogens were added to the cultures at final supramaximal concentrations of 5 μg/ml. The cultures were incubated in a humidified 37°C incubator in an atmosphere of 5% CO2. After a 48 h incubation, 3H-thymidine (0.2 μCi) was added to each well in a volume of 0.02 ml. Cells were harvested 5 h later using an automated sample harvester, and the filters were counted in a liquid scintillation spectrometer. The proliferation index was estimated as the ratio between cells stimulated in the presence of mitogens and controls. Results were expressed as proliferation index/number of cells.
The relative size distributions of lymph cells in spleen, SmLN and thymus were determined by FACS analysis, as previously described [15]. For these studies, we used the following monoclonal antibodies: Anti-rat LCA (OX-33) for B lymphocytes (Serotec, Oxford, UK), anti-rat TCR alpha/beta (R7.3) for T lymphocytes (Serotec, Oxford, UK), anti-rat CD4 (OX-35) which recognize a rat T helper cell differentiation antigen (Pharmingen, San Diego, CA, USA), and anti-rat CD8a (OX-8) which recognize the reactive antigen expressed on rat T cytotoxic/suppressor cells (Pharmingen, San Diego, CA, USA). Lymphocytes, isolated as indicated above, were washed in cold PBS with 0.02% sodium azide and then incubated (3 × 105 cells/tube) with appropriate primary antibodies for 30 min at 4°C. Following two washes, the cells were incubated with 1 ml of PBS-BSA 1%, during 5 min at 4°C, washed three times, resuspended in 1% paraformaldehyde in PBS. Fluorescence intensity was analyzed by fluorescence-activated cell sorting (FACStarplus; Beckton Dickinson, Mountain View, CA). Dead cells were excluded by gating with propidium iodide.
For analyzing IFN-γ release, splenic, SmLN or thymic cells (105/100 μl) were incubated for 24 h, their media removed and, after adding fresh media including all components, they were incubated for 24 h more. Both media were collected and pooled for IFN-γ measurement. The incubations were performed in triplicates. Microscopical examination of the cell preparations used indicated that > 95% were lymph node cells. Neither treatment affected the viability of the cells. IFN-γ concentration in media culture was measured after centrifugation to remove adherent cells. An ELISA commercial kit from Endogen (Woburn, MA, USA), previously validated in our laboratory, was employed [16]. The assay was performed as follows: 100 μl of standards or unknown samples were added to each antibody-coated well, and the plates were incubated overnight at room temperature. The reaction was stopped by washing thrice with wash buffer (2% Tween 20 in 50 mM Tris, pH 3.6). The wells were incubated with 100 μl of biotynilated detecting antibody at the titter previously tested. After 1 h at room temperature the reaction was stopped by washing thrice with wash buffer. One hundred μl of streptavidin-HRP solution (in Dulbecco's phosphate-buffered saline, pH 7.4) was then added and the samples were incubated for 30 min. The reaction was stopped by adding 100 μl of 0.18 M sulfuric acid. The plates were read within 30 min in an ELISA reader set at 450 nm and 550 nm. Values were obtained by subtracting the reading at 550 nm from the reading at 450 nm, to correct for any optical defect of microtiter plate. IFN-γ release was expressed as pg/mL/48 h incubation. Sensitivity of the assay was 100 pg/mL.
Statistical analysis of results was performed by a two-way factorial analysis of variance (ANOVA) and a one-way ANOVA followed by a Bonferroni's test. P values lower than 0.05 were considered evidence for statistical significance.
Answers to all your Biology Questions
