<i>In vivo</i> effect of AG on iNOS protein expression in placental tissues.

<p>Pregnant rats were injected with either 0.5 ml (0.7 ng Stx2/g body wt) or 1.5 ml of sStx2 (2 ng Stx2/g body wt) on day 15 of gestation and killed 12 h post injection. Some animals of each Stx2 concentration were treated with AG (100 µg/g body wt) 24 h before and 4 h after injection of sStx2, whereas others were treated with AG alone. Control rats were treated with PBS 24 h before and 4 h after injection of sControl. (A) Representative gel for iNOS expression. (B) Densitometric analysis of the bars. Values correspond to the mean of four different pools of five animals ± SEM. ^*P<0.001 vs 0.7 ng Stx2/g body wt, ^**P<0.05 vs 2 ng Stx2/g body wt.</p

Immunolocalization of iNOS in placental tissues.

<p>Pregnant rats were injected with 0.5 ml of sControl or sStx2 (0.7 ng Stx2/g body wt) on day 15 of gestation and killed 12 h post injection. iNOS was detected in trophoblastic giant cells (A, black arrowhead) and glycogen cells (B; black arrowhead) of placentas from control rats. An increased label of iNOS in glycogen cells (C–D, black arrowhead) was observed in placentas from Stx2-treated rats. Original magnification: A and C ×200, B and D ×1000.</p

Effects of Stx2 on pregnant rat delivery.

<p>Pregnant rats were injected with 0.05–1.5 ml of sStx2 containing 0.07–2 ng Stx2/g body wt on day 14–16 of gestation. Delivery percentages were evaluated up to 12 days post-injection (n = 6–12 rats for each concentration).</p

Nitric oxide synthase (NOS) activity in placental and kidney tissues after Stx2 injection.

<p>Pregnant rats on day 15 of gestation were injected with 0.5 or 1.5 ml of sStx2 (0.7 or 2 ng Stx2/g body wt, respectively) and killed 12 h post injection. Control rats were injected with the same volume of sControl. NOS activity was quantified by measuring the conversion of L-[¹⁴C]arginine into L-[¹⁴C] citrulline. Values corresponding to at least two separate experiments are shown. Values are expressed as the mean ± SEM (n = 6–8). ^*P<0.05 vs control; ^**P<0.01 vs control.</p

<i>In vivo</i> effects of AG on NOS activity in placental tissues.

<p>Pregnant rats were injected with 0.5 ml of sStx2 (0.7 ng Stx2/g body wt) on day 15 of gestation and killed 12 h post injection. One group was treated with AG (100 µg/g body wt) 24 h before and 4 h after sStx2 administration. Another was treated only with AG (control AG). Control rats were treated with PBS 24 h before and 4 h after administration of 0.5 ml of sControl. Values are the mean ± SEM (n = 4). ^*P<0.001 vs other experimental conditions.</p

Histology of the placentas from pregnant rats treated with AG and sStx2.

<p>Placentas from pregnant rats treated with 0.7 or 2 ng Stx2/g body wt presented necrotic areas (A and B, black arrows) as compared with controls (C). Placentas from rats treated with 100 µg AG/g body wt plus 0.7 (D) or 2 ng (E) Stx2/g body wt did not show histological changes compared with controls (C). H&E. Original magnification: ×200.</p

Macroscopic evaluation of pregnant rats treated with AG and sStx2.

<p>Uteri and fetuses from pregnant rats injected with 1.5 ml sStx2 containing 2 ng Stx2/g body wt showed intrauterine hemorrhage and fetal death 48 h after sStx2 injection (C, D). The treatment with 100 µg AG/g body wt 24 h before and 4 h after sStx2 injection caused significant reduction of the toxin effects on the feto-maternal unit (E, F). Uteri and fetuses from control rats showed normal fetal growth (A, B).</p

Western blot for iNOS protein expression in placental tissues.

<p>Pregnant rats were injected with 0.5 or 1.5 ml of sStx2 (0.7 or 2 ng Stx2/g body wt, respectively) on day 15 of gestation and killed 12 h post injection. Control rats were injected with the same volume of sControl. iNOS expression in placental tissues was detected using a specific polyclonal anti-iNOS antibody. To determine the uniformity of loading, western blots were probed with a monoclonal anti-β-actin antibody. Each value represents a pool of five animals. (A) Representative gel for iNOS expression. (B) Densitometric analysis of the bars. Values correspond to the mean of four different pools of five animals ± SEM. *P<0.01 vs controls.</p

Effect of AEA on PGE synthase activity.

<p>Uterine explants were incubated with m-AEA (100 nM), INDO (1 µM) or m-AEA plus INDO for 24 h. Results were expressed as pg PGE2/mg protein/h. ANOVA Test. a, p<0.001 vs control or INDO. n = 4. PGE₂ levels were assessed by RIA.</p

AEA mediates LPS-induced PG production.

<p>A) PGE₂ production for uterine explants incubated with LPS (1 ug/ml), m-AEA (100 nM), URB597 (1 uM), LPS plus m-AEA (100 nM) or LPS plus URB597 (1 uM) for 24 h. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs LPS; c, p<0.001 vs LPS. n = 7. B) PGF_2α production for uterine explants incubated with LPS (1 ug/ml), m-AEA (100 nM), URB597 (1 uM), LPS plus m-AEA (100 nM) or LPS plus URB597 (1 uM) for 24 h. ANOVA test. a, p<0.001 vs control; b, p<0.01 vs LPS; c, p<0.05 vs control; d, p<0.01 vs control. n = 7.</p