Influence of speed of sample processing on placental energetics and signalling pathways: Implications for tissue collection

Introduction: The placenta is metabolically highly active due to extensive endocrine and active transport functions. Hence, placental tissues soon become ischaemic after separation from the maternal blood supply. Ischaemia rapidly depletes intracellular ATP, and leads to activation of stress-response pathways aimed at reducing metabolic demands and conserving energy resources for vital functions. Therefore, this study aimed to elucidate the effects of ischaemia ex vivo as may occur during tissue collection on phosphorylation of placental proteins and kinases involved in growth and cell survival, and on mitochondrial complexes. Methods: Eight term placentas obtained from normotensive non-laboured elective caesarean sections were kept at room-temperature and sampled at 10, 20, 30 and 45 min after delivery. Samples were analyzed by Western blotting. Results: Between 10 and 45 min the survival signalling pathway intermediates, P-AKT, P-GSK3 alpha and beta,P-4E-BP1 and P-p70SEK were reduced by 30-65%. Stress signalling intermediates, P-eIF2 alpha increased almost 3 fold after 45 min. However, other endoplasmic reticulum stress markers and the Heat Shock Proteins, HSP27, HSP70 and HSP90, did not change. Phosphorylation of AMPK, an energy sensor, was elevated 2 fold after 45 min. Contemporaneously, there was an similar to 25% reduction in mitochondrial complex IV subunit I. Discussion and conclusions: These results suggest that for placental signalling studies, samples should be taken and processed within 10 min of caesarean delivery to minimize the impact of ischaemia on protein phosphorylation. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.