The role of nitric oxide in the placental infection by Trypanosoma cruzi: in vitro and ex vivo study

BOLATTI ELISA; DÍAZ-LUJÁN CINTIA; TRIQUELL MARÍA FERNANDA; GUERRERO CLAUDIA E; MAZZUDULLI CINTIA; FRETES RICARDO E

Rio de Janeiro

Simposio; Simposio Internacional Conmemorativo del Centenario del Descubrimiento del Mal de Chagas; 2009

Fundación Oswaldo Cruz

Human placenta would participate actively in the control of congenital Chagas infection, which would explain, partially, the low transmission rate. The production of nitric oxide (NO) by the endothelial and inducible placental nitric oxide synthases (eNOS and iNOS respectively) could be an important part of this control. Objectives: To analyze the NOS activity in relation to the in vitro (normal placental explants and isolated trophoblast) infection by T. cruzi and in ex-vivo (placentas from chagasic mothers). Material and Methods: Ex Vivo: placentas from chagasic mothers with congenital transmission (M+RN+) (n=5), placentas from chagasic mothers without congenital transmission (M+RN-) (n=8) and normal placentas (NP) (n=3) from Pathology Services. In vitro: Chorionic villi (n=8) explants and isolated cytotrophoblast (CTB) and syncytiotrophoblast (STB) and Vero cells (control) were co-cultured with or without (control)106 T. cruzi trypomastigotes for 24h and 48h at 37 ºC. Placentas from chagasic mothers were stained for eNOS and iNOS by immuno-histochemistry, and the explants for eNOS and their intensity and area were measured (AxioVision, Zeiss). RT-PCR for eNOS and its semi quantification normalized with GADPH were studied in placental explants. NO production was analyzed in culture media. T. cruzi infection was analyzed by PCR (TCZ 1 and 2) and by microscope in H/E and Giemsa stained slices in ex vivo placentas and in in vitro explants and CTB/STB cultures (Axiovision). The parasite viability was measured in placental explants and STB/CTB culture media by the dye exclusion test. Results: Increased levels of NO in placenta-T. cruzi co-culture media. Lower expression of eNOS intensity and area (vessels and STB) in M+RN+ (+, +/-; 0,74%; 4,11% respectively) and M+RN- (++, +; 1,83%; 9,12% respectively) than NP (+++; 3,78%; 14,3%). Increased expression of eNOS (intensity and area) in the placental explants -T. cruzi co-cultures (+++; 63,33%) compared with control (+; 19,99%). The iNOS intensity and area expression of vessels and STB were higher in M+RN+ (+/-; 0,74%; 3,29%) and in M+RN- (+/-, +; 1,16%; 3,46%) than NP (-); 0,33%, 1,57%). The STB was significantly less susceptible to the T. cruzi infection (1,38 % from total infected area and 42 parasites every 100 sincicial nuclei) (p