Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

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 failure^1,2, are associated with acute cerebrovascular complications^3,4. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
Adult mice brain slecer matrice 3D printed	Open access file	Adult mice	Adult mice brain slicer. Printed in PLA filament.
Anti β-Actin primary antibody	Sigma-Aldrich	Clon AC-74	Antibody for loading control (Western blot)
Anti-Claudin5 primary antibody	Santa cruz Biotechnology	sc-374221	Primary antibody for tight junction protein CLDN5 of mice BBB (Western blot)
BCA protein kit	Thermo Scientific	23225	Kit for measuring protein concentration
Culture media #200 500 mL	Thermo Fisher Scientific	m200500	Culture media for placental explants
D180 CO2 incubator	RWD Life science	D180	Standard incubator to estabilize explants and culture sEVs-Nor
Evans blue dye  > 75% 10 g	Sigma-Aldrich	E2129.10G	Dye to analize blood brain barrier disruption IN VIVO
Fetal bovine serum 500 mL	Thermo Fisher Scientific	16000044	Additive growth factor for culture media 200
Himac Ultracentrifuge CP100NX	Himac eppendorf group	5720410101	Ultracentrifuge for condicioned media > 1,20,000 x g
ImageJ software	NIH		https://imagej.nih.gov/ij/download.html
Isoflurane x 100 mL	USP Baxter	212-094	Volatile inhalated anaesthesia agent for mice
Kit CellTiter 96 Non-radioactive	Promega	0000105232	In vitro assay for placental explants viability
Mouse IgG Secondary antibody	Thermo Fisher Scientific	MO 63103	Secondary antibody for CLDN5 (western blot)
NanoSight NS300	Malvern Panalytical	90278090	Nanotracking analysis of particles from placental explants condicioned media
Paraformaldehide E 97% solution 500 mL	Thermo Fisher Scientific	A11313.22	Fixative solution for brain tissue slices and intracardial perfusion (once diluted)
PBS 1 X pH 7.4 500 mL	Thermo Fisher Scientific	10010023	Wash solution for placenta explants
Peniciline-streptomicine 100x 20 mL	Thermo Fisher Scientific	10378016	Antiobiotics for placental explants culture media
ProOX C21 Cytocentric O2 and CO2 Subchamber Controller	BioSpherix	SCR_021131	CO2 regulator to induce Hypoxia in sealed chamber for sEVs-Hyp
Sodium Thiopental 1 g	Chemie	7061	humanitarian euthanasia agent
Somnosuite low flow anesthesia system	Kent Scientifics	SS-01	Isoflurane vaporizer for small rodents
Surgical Warming platform	Kent Scientifics	A41166	Warming platform for mainteinance anesthesia in mice
Syringe Filters, Polytetrafluoroethylene (PTFE), Hydrophobic, 0.22 µm, Sterile, 25 mm	Southern labware	10026	Filtration of condicioned media harvested from placental explants
Tabletop High-Speed Micro Centrifuges HITACHI himac CT15E/CT15RE	Hitachi medical systems	6020	Serial centrifugations of condicioned media < 1,20, 000 x g
Trinocular stereomicroscope transmided and reflective light 10x-160x	Center Medical	2597	Stereomicroscope to register brain slices