Rats were intraperitoneally inoculated with 1×105 blood trypomastigotes of the Y strain of T. cruzi on 3rd day after pregnancy. The assays were performed on 18 days after pregnancy (14 days after infection). It is important to emphasize that because Wistar rats are normally resistant to most T. cruzi strains, we found it necessary to use relatively high inoculum (1×105 blood trypomastigotes).
Female Wistar rats weighing 180–200 g were used. Animals were randomly distributed into groups: pregnant control (PC), pregnant control treated with zinc (PCZ), pregnant infected (PI), and pregnant infected treated with zinc (PIZ). A total of 5 animals were used per group (2 animals per cage). 1 male Wistar rat was introduced into each cage and was allowed to mate with 2 females. The vaginal plug appearance was designated as being at day 1 of gestation. Rodent diet and water were available ad libitum.
Rats were treated by oral route through gavage with 20 mg/kg/day zinc sulfate (Sigma Chemical Co. MO) (da Costa et al. 2013). The treatment was started 24 h after parasite infection and was maintained until 18 days after pregnancy.
Animals were decapitated on 18th day of pregnancy with prior anesthesia using 0.25% tribromoethanol (10 mL/kg), administrated intraperitoneally.
Flow cytometry assay
Cells were dispersed from spleen by extrusion through a 70 μm nylon cell strainer, macerated in RPMI 1640 medium to produce a single cell suspension. 2×106 cells from the suspension of each organ from each experimental group were placed in 96 well round bottomed plates for flow cytometry analysis. Following Fc receptor blocking, cells were incubated with combinations of monoclonal antibodies anti-CD3-fluoresceinisotiocianate (FITC), anti-CD4-allophycocyanin (APC), anti-CD8-peridin clorofil protein (PERCP), anti-CD45RA-phycoerythrin (PE), as well as immunoglobulin isotype-matched controls. Stained cells were stored for analysis in PBS containing 0.01 mL sodium azide and 1% paraformaldehyde, in sealed tubes held in the dark. All steps were performed at 4°C. Analysis of these cells was performed using a Becton Dickinson FACScan flow cytometer with DIVA-BD software (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Spleens were aseptically removed. To prepare a single cell suspension, cells were teased out into serum-free RPMI 1640 medium. After centrifugation at 300 g for 10 min at 4°C, the pelleted cells were resuspended in RPMI 1640 containing 5% FBS (2×106 cells/mL) and added to 96 well flat bottomed plates (0.1 mL/well). The cells were subsequently stimulated with 4 μg/mL Concanavalin A (Sigma) and incubated at 37°C in a humidified 5% CO2 atmosphere for 72 h. The cells were incubated with MTT reagent (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) for 4 h, followed by lysis using acidic isopropanol and measurement at 570 nm in an ELISA reader (Sunrise Tecan).
Zinc determination in plasma samples (ICP-MS)
For zinc determination in plasma samples, a method described previously was adopted with few modifications. Briefly, samples were diluted 1:20 into a 15 mL polypropylene Falcon® tube (Becton Dickinson) with a solution containing 0.01% (v/v) Triton® X-100, 0.5% (v/v) nitric acid, and 10 µg L-1 of each one of Rh (the internal standard). Analyses were carried out with an inductively coupled plasma mass spectrometer equipped with a reaction cell (ICP-MS ELAN DRCII, PerkinElmer, SCIEX, Norwalk, CT) operating with high-purity argon (99.999; Praxaair, Brazil). Sample introduction system was composed by quartz cyclonic spray chamber and a Meinhard® nebulizer connected by Tygon® tubes to the ICP-MS’s peristaltic pump (set at 20 rpm). The ICP-MS was operated with Pt sampler and skimmer cones purchased from Perkin Elmer.
The results are expressed as mean ±SEM. A value of p <0.05 was considered statistically significant. Differences among control and infected groups were determined by one-way ANOVA with Bonferroni’s posttest. Statistical analyses were performed using Graph Pad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA).