Optimisation of high-resolution fluorescence microscopy of normal- and preeclamptic placentas

Abstract

Preeclampsia (PE) affects 3-5 % of pregnant women and may lead to maternal and/or fetal death. The main theory of PE is placental ischemia, leading to a dysfunctional placenta and clinical signs as hypertension and proteinuria in the mother. The primary aim of the thesis was to implement and optimise a method for high-resolution microscopy of placental cryo-sections. Secondary aims were to compare the morphology, total antioxidant capacity (TAC) and the oxidative stress between normal- and preeclamptic placentas. Placental tissue from the fetal and maternal side were collected from three normal pregnant women and three preeclamptic women. For each patient; eight cryo-sections were prepared, four from each side of the placenta. Two were used as negative controls investigated for autofluorescence and two were used as positive controls labelled for morphological analysis. Positive controls were labelled with CellMaskTM Orange, staining cell membranes and 4’,6 diamidino 2-phenylidole, dihydrochloride, staining nuclei. The TAC was determined by comparing the measured 3 ethylbenzothiazoline 6 sulphonic acid radical scavenging activity to an ascorbic acid standard curve. The oxidative stress was determined measuring the malondialdehyde content of the samples. Neither the normal nor the preeclamptic samples had autofluorescence affecting microscopy of the labelled sections. The method allowed visualisation of microscopic placental structures. In preeclamptic sections from the fetal side, there seemed to be more syncytial knots than in fetal sections from normal women. Bright red structures were detected in sections from the fetal side of preeclamptic samples and were not observed in normal sections. Because of their size, they were thought to be extravillous vesicles. The collection-, preservation- and labelling method was successfully implemented and is well suited for high-resolution microscopy. Although there were not found a significant difference in TAC and oxidative stress between normal- and preeclamptic placentas, neither on the fetal- or maternal side, the method is suited for placental tissue.