title: Influence of speed of sample processing on placental energetics and signalling pathways: implications for tissue collection.
abstract: BACKGROUND
  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-GSK3α and β, P-4E-BP1 and P-p70S6K were reduced by 30-65%. Stress signalling intermediates, P-eIF2α 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 ∼25% reduction in mitochondrial complex IV subunit I.
  
  
  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.
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. Electronic address: hwy20@hermes.cam.ac.uk.
authors.forename: H W
authors.initials: HW
authors.lastname: Yung
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: F
authors.initials: F
authors.lastname: Colleoni
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: D
authors.initials: D
authors.lastname: Atkinson
authors.affiliation: Department of Obstetrics and Gynaecology, University of Cambridge, Rosie Hospital, Robinson Way, Cambridge CB2 0SW, UK; National Institute for Health Research Cambridge Comprehensive Biomedical Research Centre, UK.
authors.forename: E
authors.initials: E
authors.lastname: Cook
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Murray
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK; National Institute for Health Research Cambridge Comprehensive Biomedical Research Centre, UK.
authors.forename: G J
authors.initials: GJ
authors.lastname: Burton
authors.affiliation: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK; Department of Obstetrics and Gynaecology, University of Cambridge, Rosie Hospital, Robinson Way, Cambridge CB2 0SW, UK; National Institute for Health Research Cambridge Comprehensive Biomedical Research Centre, UK.
authors.forename: D S
authors.initials: DS
authors.lastname: Charnock-Jones
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018797:Cell Cycle Proteins; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D007511:Ischemia / Q000503:physiopathology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000098:blood supply / Q000503:physiopathology*; D011247:Pregnancy; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D015852:Eukaryotic Initiation Factor-2; D010750:Phosphoproteins; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038362:Glycogen Synthase Kinase 3; D055372:AMP-Activated Protein Kinases
keywords: Ischaemia; Metabolism; Signal transduction