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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A protocol is presented to evaluate whether small EVs (sEVs) isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the blood-brain barrier in nonpregnant adult female mice.

Abstract

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated with preeclampsia. The underlying mechanisms of these cerebrovascular complications remain unclear. However, they are linked to placental dysfunction and blood-brain barrier (BBB) disruption. Nevertheless, the connection between these two distant organs is still being determined. Increasing evidence suggests that the placenta releases signaling molecules, including extracellular vesicles, into maternal circulation. Extracellular vesicles are categorized according to their size, with small extracellular vesicles (sEVs smaller than 200 nm in diameter) considered critical signaling particles in both physiological and pathological conditions. In preeclampsia, there is an increased number of circulating sEVs in maternal circulation, the signaling function of which is not well understood. Placental sEVs released in preeclampsia or from normal pregnancy placentas exposed to hypoxia induce brain endothelial dysfunction and disruption of the BBB. In this protocol, we assess whether sEVs isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the BBB in vivo.

Introduction

Approximately 70% of maternal deaths due to preeclampsia, a hypertensive pregnancy syndrome characterized by impaired placentation processes, maternal systemic endothelial dysfunction, and, in severe cases, multi-organ failure1,2, are associated with acute cerebrovascular complications3,4. Most maternal deaths occur in low and middle-income countries5. However, the underlying mechanisms are still unclear despite the clinical and epidemiological relevance of cerebrovascular complications associated with preeclampsia.

Protocol

The research was carried out following the principles expressed in the Declaration of Helsinki and under the authorization of the respective Ethical Review Boards. All human participants gave their informed consent before sample collection, as reported previously25. Additionally, the Bioethics and Biosafety Committee of the Bío-Bío University approved this project (Fondecyt grant 1200250). The animal work was conducted in accordance with the cardinal principles of the three R's in th.......

Representative Results

This protocol evaluates the capacity of sEVs derived from placentas cultured in hypoxia to disrupt the BBB in nonpregnant mice. This method allows one to understand better the potential connection between the placenta and the brain in normal and pathological conditions. In particular, this method may constitute a proxy to analyze placental sEVs participation in the onset of cerebral complications in preeclampsia.

Contrary to mice injected with sEVs-Nor, mice injected with sEVs-Hyp show a progr.......

Discussion

This study unveils fresh insights into potential harm resulting from sEVs isolated from placental explants cultured in hypoxic conditions on the disruption of the rodent blood-brain barrier. The pathological mechanism involves a reduction in CLND-5 in the posterior brain region25.

Prior investigations have revealed that plasma-sEVs from individuals with preeclampsia induce endothelial dysfunction in various organs using in vitro models46.......

Acknowledgements

The authors would like to thank the researchers belonging to GRIVAS Health for their valuable input. Also, midwives and clinical staff from the Obstetrics and Gynecology Service belong to the Hospital de Chillan, Chile. Founded by Fondecyt Regular 1200250.

....

Materials

NameCompanyCatalog NumberComments
Adult mice brain slecer matrice 3D printedOpen access fileAdult miceAdult mice brain slicer. Printed in PLA filament.
Anti β-Actin primary antibodySigma-AldrichClon AC-74Antibody for loading control (Western blot)
Anti-Claudin5 primary antibodySanta cruz Biotechnologysc-374221Primary antibody for tight junction protein CLDN5 of mice BBB (Western blot)
BCA protein kitThermo Scientific23225Kit for measuring protein concentration
Culture media #200 500 mLThermo Fisher Scientificm200500Culture media for placental explants
D180 CO2 incubatorRWD Life scienceD180Standard incubator to estabilize explants and culture sEVs-Nor
Evans blue dye  > 75% 10 gSigma-AldrichE2129.10GDye to analize blood brain barrier disruption IN VIVO
Fetal bovine serum 500 mLThermo Fisher Scientific16000044Additive growth factor for culture media 200
Himac Ultracentrifuge CP100NXHimac eppendorf group5720410101Ultracentrifuge for condicioned media > 1,20,000 x g
ImageJ softwareNIHhttps://imagej.nih.gov/ij/download.html
Isoflurane x 100 mLUSP Baxter212-094Volatile inhalated anaesthesia agent for mice
Kit CellTiter 96 Non-radioactive Promega0000105232In vitro assay for placental explants viability
Mouse IgG Secondary antibodyThermo Fisher ScientificMO 63103Secondary antibody for CLDN5 (western blot)
NanoSight NS300Malvern Panalytical90278090Nanotracking analysis of particles from placental explants condicioned media
Paraformaldehide E 97% solution 500 mLThermo Fisher ScientificA11313.22Fixative solution for brain tissue slices and intracardial perfusion (once diluted)
PBS 1 X pH 7.4 500 mLThermo Fisher Scientific10010023Wash solution for placenta explants
Peniciline-streptomicine 100x 20 mLThermo Fisher Scientific10378016Antiobiotics for placental explants culture media
ProOX C21 Cytocentric O2 and CO2 Subchamber ControllerBioSpherixSCR_021131CO2 regulator to induce Hypoxia in sealed chamber for sEVs-Hyp
Sodium Thiopental 1 gChemie7061humanitarian euthanasia agent
Somnosuite low flow anesthesia systemKent ScientificsSS-01Isoflurane vaporizer for small rodents
Surgical Warming platformKent ScientificsA41166Warming platform for mainteinance anesthesia in mice
Syringe Filters, Polytetrafluoroethylene (PTFE), Hydrophobic, 0.22 µm, Sterile, 25 mmSouthern labware10026Filtration of condicioned media harvested from placental explants 
Tabletop High-Speed Micro Centrifuges HITACHI himac CT15E/CT15REHitachi medical systems6020Serial centrifugations of condicioned media < 1,20, 000 x g
Trinocular stereomicroscope transmided and reflective light 10x-160x Center Medical2597Stereomicroscope to register brain slices

References

  1. Lisonkova, S., Joseph, K. S. Incidence of preeclampsia: risk factors and outcomes associated with early- versus late-onset disease. Am J Obstet Gynecol. 209 (544), 544.e1-544.e12 (2013).
  2. Sibai, B., Dekker, G., Kupferminc, M. Preeclampsia.....

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Extracellular VesiclesBlood brain BarrierPlacentaPreeclampsiaHypoxiaCerebrovascular ComplicationsMaternal MortalityBrain Endothelial Dysfunction

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