JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Medicine

Forstyrrelse af musens blod-hjerne-barriere af små ekstracellulære vesikler fra hypoxiske humane moderkager

Published: January 26, 2024
doi:
10.3791/65867
, , , , , , , , ,
1Vascular Physiology Laboratory, Department of Basic Sciences,Universidad del Bío-Bío, Chillan, 2Nursery School, Health Faculty,Universidad Santo Tomás, Los Angeles, 3Obstetrics and Gynecology Department,Herminda Martin Clinical Hospital, Chillan, 4Faculty of Medicine,Universidad Católica de la Santísima Concepción, Concepcion, 5Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences,Universidad de Concepción, Chillan, 6Group of Research and Innovation in Vascular Health (GRIVAS Health), Chillan

Summary

En protokol præsenteres for at evaluere, om små EV’er (sEV’er) isoleret fra placentaeksplanter dyrket under hypoxiske forhold (modellering af et aspekt af præeklampsi) forstyrrer blod-hjernebarrieren hos ikke-gravide voksne hunmus.

Abstract

Cerebrovaskulære komplikationer, herunder cerebralt ødem og iskæmisk og hæmoragisk slagtilfælde, udgør den førende årsag til moderdødelighed forbundet med præeklampsi. De underliggende mekanismer bag disse cerebrovaskulære komplikationer forbliver uklare. Men, de er forbundet med placenta dysfunktion og blod – hjerne barriere (BBB) forstyrrelse. Ikke desto mindre er forbindelsen mellem disse to fjerne organer stadig ved at blive bestemt. Stigende beviser tyder på, at moderkagen frigiver signalmolekyler, herunder ekstracellulære vesikler, i moderens cirkulation. Ekstracellulære vesikler er kategoriseret efter deres størrelse, med små ekstracellulære vesikler (sEV’er mindre end 200 nm i diameter) betragtes som kritiske signalpartikler under både fysiologiske og patologiske tilstande. I præeklampsi er der et øget antal cirkulerende sEV’er i moderens cirkulation, hvis signalfunktion ikke forstås godt. Placenta-sEV’er frigivet i præeklampsi eller fra normale graviditetsplacentas udsat for hypoxi inducerer hjerneendoteldysfunktion og forstyrrelse af BBB. I denne protokol vurderer vi, om sEV’er isoleret fra placentaeksplanter dyrket under hypoxiske forhold (modellering af et aspekt af præeklampsi) forstyrrer BBB in vivo.

Introduction

Ca. 70% af moderens dødsfald på grund af præeklampsi, et hypertensive graviditetssyndrom præget af nedsatte placentationsprocesser, maternel systemisk endoteldysfunktion og i alvorlige tilfælde multiorgansvigt 1,2 er forbundet med akutte cerebrovaskulære komplikationer 3,4. De fleste mødredødsfald forekommer i lav- og mellemindkomstlande5. Imidlertid er de underliggende mekanismer stadig uklare på trods af den kliniske og epidemiologiske relevans af cerebrovaskulære komplikationer forbundet med præeklampsi.

På den anden side er ekstracellulære vesikler (EV’er) (diameter ~ 30-400 nm) essentielle mediatorer af intercellulær kommunikation mellem væv og organer, herunder maternal-placenta interaktion6. Ud over proteiner og lipider på den ydre overflade transporterer EV’er last indeni (proteiner, RNA og lipider). EV’er kan kategoriseres i (1) exosomer (diameter ~ 50-150 nm, også kaldet små EV’er (sEV’er)), (2) mellemstore / store EV’er og (3) apoptotiske legemer, som adskiller sig efter størrelse, biogenese, indhold og potentiel signalfunktion. EV’ernes sammensætning bestemmes af de celler, de stammer fra, og sygdomstype7. Syncytiotrophoblastafledte EV’er udtrykker placenta alkalisk fosfatase (PLAP)8,9, som detekterer placentae-afledte cirkulerende små EV’er (PDsEV’er) under graviditet. PLAP hjælper også med at skelne ændringer i PDsEVs last og deres virkninger i præeklampsi versus normotensive graviditeter 10,11,12,13,14,15.

Placenta er blevet anerkendt som den nødvendige komponent i patofysiologien af præeklampsi16 eller cerebrale komplikationer forbundet med denne sygdom 17,18,19. Men hvordan dette fjerne organ kan fremkalde ændringer i hjernens cirkulation er ukendt. Da sEV’er spiller afgørende roller i celle-til-celle-kommunikation på grund af deres evne til at overføre bioaktive komponenter fra donor- til modtagerceller 6,20,21, har et stigende antal undersøgelser forbundet placenta sEV’er med generering af maternal endoteldysfunktion21,22,23,24, herunder hjerneendotelceller25,26hos kvinder med præeklampsi. Således kan kompromiset med hjernens endotelfunktion føre til forstyrrelse af blod-hjerne-barrieren (BBB), en kritisk komponent i cerebrovaskulære komplikationer forbundet med præeklampsi 3,27.

Ikke desto mindre rapporterede prækliniske fund ved hjælp af rottecerebrale kar udsat for serum hos kvinder med præeklampsi28 eller humane hjerneendotelceller udsat for plasma hos kvinder med præeklampsi29, at cirkulerende faktor (er) inducerer forstyrrelse af BBB. På trods af flere kandidater med potentiale til at skade BBB til stede i moderens cirkulation under præeklampsi, såsom forhøjede niveauer af proinflammatoriske cytokiner (dvs. tumornekrosefaktor)18,28 eller vaskulære regulatorer (dvs. vaskulær endotelvækstfaktor (VEGF))29,30,31 eller oxidative molekyler såsom oxiderede lipoproteiner (oxo-LDL)32,33, blandt andre34, ingen af dem etablerer en direkte forbindelse mellem moderkagen og BBB. For nylig har sEV’er isoleret fra hypoxiske placentas vist evnen til at forstyrre BBB hos ikke-gravide hunmus25. Da placenta sEV’er kan bære de fleste af de anførte cirkulerende faktorer med kapacitet til at forstyrre BBB, betragtes sEV’er som egnede kandidater til at forbinde den skadede placenta, være bærer af skadelige cirkulerende faktorer og forstyrre BBB i præeklampsi.

Denne protokol giver os mulighed for at undersøge, om sEV’er isoleret fra placentaeksplanter dyrket under hypoxiske forhold kan forstyrre BBB hos ikke-gravide hunmus som en proxy for forståelse af patofysiologien af cerebrale komplikationer under præeklampsi.

Protocol

Forskningen blev udført efter principperne i Helsingfors-erklæringen og under bemyndigelse fra de respektive etiske undersøgelsesudvalg. Alle menneskelige deltagere gav deres informerede samtykke før prøveindsamling, som rapporteret tidligere25. Derudover godkendte Udvalget for Bioetik og Biosikkerhed ved Bío-Bío Universitet dette projekt (Fondecyt grant 1200250). Dyrearbejdet blev udført i overensstemmelse med kardinalprincipperne for de tre R’er i brugen af dyr i forsøg<sup class="xref"…

Representative Results

Denne protokol evaluerer kapaciteten af sEV’er afledt af placentas dyrket i hypoxi for at forstyrre BBB hos ikke-gravide mus. Denne metode gør det muligt for en bedre at forstå den potentielle forbindelse mellem moderkagen og hjernen under normale og patologiske forhold. Især kan denne metode udgøre en proxy til analyse af placenta sEVs deltagelse i begyndelsen af cerebrale komplikationer i præeklampsi. I modsætning til mus injiceret med sEVs-Nor, viser mus injiceret med sEVs-Hyp et prog…

Discussion

Denne undersøgelse afslører frisk indsigt i potentiel skade som følge af sEV’er isoleret fra placentaeksplanter dyrket under hypoxiske forhold om forstyrrelsen af gnaverblod-hjernebarrieren. Den patologiske mekanisme indebærer en reduktion i CLND-5 i den bageste hjerneregion25.

Tidligere undersøgelser har vist, at plasma-sEV’er fra personer med præeklampsi inducerer endoteldysfunktion i forskellige organer ved hjælp af in vitro-modeller <sup class="xref"…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne vil gerne takke forskerne i GRIVAS Health for deres værdifulde input. Også jordemødre og klinisk personale fra obstetrik og gynækologi service tilhører Hospital de Chillan, Chile. Grundlagt af Fondecyt Regular 1200250.

Materials

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
DOWNLOAD MATERIALS LIST

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. Lancet. 365 (9461), 785-799 (2005).
  3. Hammer, E. S., Cipolla, M. J. Cerebrovascular dysfunction in preeclamptic pregnancies. Curr Hypertens Rep. 17 (8), 64 (2015).
  4. Okanloma, K. A., Moodley, J. Neurological complications associated with the preeclampsia/eclampsia syndrome. Int J Gynaecol Obstet. 71, 223-225 (2000).
  5. Frias, A. E., Belfort, M. A. Post magpie: how should we be managing severe preeclampsia. Curr Opin Gynecol Obstet. 15 (6), 489-495 (2003).
  6. Familari, M., Cronqvist, T., Masoumi, Z., Hansson, S. R. Placenta-derived extracellular vesicles: Their cargo and possible functions. Reprod Fertil Dev. 29 (3), 433-447 (2017).
  7. Montoro-Garcia, S., Shantsila, E., Marin, F., Blann, A., Lip, G. Y. Circulating microparticles: new insights into the biochemical basis of microparticle release and activity. Basic Res Cardiol. 106, 911-923 (2011).
  8. Germain, S. J., Sacks, G. P., Sooranna, S. R., Sargent, I. L., Redman, C. W. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J Immunol. 178 (9), 5949-5956 (2007).
  9. Tannetta, D., Masliukaite, I., Vatish, M., Redman, C., Sargent, I. Update of syncytiotrophoblast derived extracellular vesicles in normal pregnancy and preeclampsia. J Reprod Immunol. 119, 98-106 (2017).
  10. Collett, G. P., Redman, C. W., Sargent, I. L., Vatish, M. Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules. Oncotarget. 9 (6), 6707-6717 (2018).
  11. Cooke, W. R., et al. Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5′-tRNA halves. Biochem Biophys Res Commun. 518 (1), 107-113 (2019).
  12. Gill, M., et al. Placental syncytiotrophoblast-derived extracellular vesicles carry active nep (neprilysin) and are increased in preeclampsia. Hypertension. 73 (5), 1112-1119 (2019).
  13. Kandzija, N., et al. Placental extracellular vesicles express active dipeptidyl peptidase IV; levels are increased in gestational diabetes mellitus. J Extracell Vesicles. 8 (1), 1617000 (2019).
  14. Motta-Mejia, C., et al. Placental vesicles carry active endothelial nitric oxide synthase and their activity is reduced in preeclampsia. Hypertension. 70 (2), 372-381 (2017).
  15. Sammar, M., et al. Reduced placental protein 13 (PP13) in placental derived syncytiotrophoblast extracellular vesicles in preeclampsia – A novel tool to study the impaired cargo transmission of the placenta to the maternal organs. Placenta. 66, 17-25 (2018).
  16. Burton, G. J., Woods, A. W., Jauniaux, E., Kingdom, J. C. Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta. 30 (6), 473-482 (2009).
  17. Warrington, J. P., et al. Placental ischemia in pregnant rats impairs cerebral blood flow autoregulation and increases blood-brain barrier permeability. Physiological Reports. 2 (8), e12134-e12134 (2014).
  18. Warrington, J. P., Drummond, H. A., Granger, J. P., Ryan, M. J. Placental Ischemia-induced increases in brain water content and cerebrovascular permeability: Role of TNFα. Am J Physiol Regul Integr Comp Physiol. 309 (11), R1425-R1431 (2015).
  19. Johnson, A. C., et al. Magnesium sulfate treatment reverses seizure susceptibility and decreases neuroinflammation in a rat model of severe preeclampsia. PLoS ONE. 9 (11), e113670 (2014).
  20. Escudero, C. A., et al. Role of extracellular vesicles and microRNAs on dysfunctional angiogenesis during preeclamptic pregnancies. Front Physiol. 7, 1-17 (2016).
  21. Salomon, C., et al. Placental exosomes as early biomarker of preeclampsia: Potential role of exosomalmicrornas across gestation. J Clin Endocrinol Metab. 102 (9), 3182-3194 (2017).
  22. Knight, M., Redman, C. W., Linton, E. A., Sargent, I. L. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. Br J Obstet Gynaecol. 105 (6), 632-640 (1998).
  23. Gilani, S. I., Weissgerber, T. L., Garovic, V. D., Jayachandran, M. Preeclampsia and extracellular vesicles. Curr Hypertens Rep. 18 (9), 68 (2016).
  24. Dutta, S., et al. Hypoxia-induced small extracellular vesicle proteins regulate proinflammatory cytokines and systemic blood pressure in pregnant rats. Clin Sci (Lond). 134 (6), 593-607 (2020).
  25. Leon, J., et al. Disruption of the blood-brain barrier by extracellular vesicles from preeclampsia plasma and hypoxic placentae: attenuation by magnesium sulfate. Hypertension. 78 (5), 1423-1433 (2021).
  26. Han, C., et al. Placenta-derived extracellular vesicles induce preeclampsia in mouse models. Haematologica. 105 (6), 1686-1694 (2020).
  27. Amburgey, O. A., Chapman, A. C., May, V., Bernstein, I. M., Cipolla, M. J. Plasma from preeclamptic women increases blood-brain barrier permeability: role of vascular endothelial growth factor signaling. Hypertension. 56 (5), 1003-1008 (2010).
  28. Cipolla, M. J., et al. Pregnant serum induces neuroinflammation and seizure activity via TNFalpha. Exp Neurol. 234 (2), 398-404 (2012).
  29. Bergman, L., et al. Preeclampsia and increased permeability over the blood brain barrier – a role of vascular endothelial growth receptor 2. Am J Hypertens. 34 (1), 73-81 (2021).
  30. Torres-Vergara, P., et al. Dysregulation of vascular endothelial growth factor receptor 2 phosphorylation is associated with disruption of the blood-brain barrier and brain endothelial cell apoptosis induced by plasma from women with preeclampsia. Biochim Biophys Acta Mol Basis Dis. 1868 (9), 166451 (2022).
  31. Schreurs, M. P., Houston, E. M., May, V., Cipolla, M. J. The adaptation of the blood-brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy. FASEB J. 26 (1), 355-362 (2012).
  32. Schreurs, M. P., Cipolla, M. J. Cerebrovascular dysfunction and blood-brain barrier permeability induced by oxidized LDL are prevented by apocynin and magnesium sulfate in female rats. J Cardiovasc Pharmacol. 63 (1), 33-39 (2014).
  33. Schreurs, M. P. H., et al. Increased oxidized low-density lipoprotein causes blood-brain barrier disruption in early-onset preeclampsia through LOX-1. FASEB J. 27 (3), 1254-1263 (2013).
  34. Escudero, C., et al. Brain vascular dysfunction in mothers and their children exposed to preeclampsia. Hypertension. 80 (2), 242-256 (2023).
  35. Russell, W. M. S., Burch, R. L. The principles of humane experimental technique. Universities Federation of Animal Welfare. , (1959).
  36. Miller, R. K., et al. Human placental explants in culture: approaches and assessments. Placenta. 26 (6), 439-448 (2005).
  37. Troncoso, F. A. J., Herlitz, K., Ruiz, F., Bertoglia, P., Escudero, C. Elevated pro-angiogenic phenotype in feto-placental tissue from gestational diabetes mellitus. Placenta. 36 (4), 2 (2015).
  38. Zhang, H. C., et al. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Stem Cells Dev. 21 (18), 3289-3297 (2012).
  39. Thery, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3 (Unit 3), 22 (2006).
  40. Carroll, R. W., et al. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria. PLoS One. 5 (10), e13124 (2010).
  41. Wu, J., et al. Transcardiac perfusion of the mouse for brain tissue dissection and fixation. Bio Protoc. 11 (5), e3988 (2021).
  42. Walchli, T., et al. Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain. Nat Protoc. 10 (1), 53-74 (2015).
  43. Wang, H. L., Lai, T. W. Optimization of Evans blue quantitation in limited rat tissue samples. Sci Rep. 4, 6588 (2014).
  44. Morita, K., Sasaki, H., Furuse, M., Tsukita, S. Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol. 147 (1), 185-194 (1999).
  45. Lara, E., et al. Abnormal cerebral microvascular perfusion and reactivity in female offspring of reduced uterine perfusion pressure (RUPP) mice model. J Cereb Blood Flow Metab. 42 (12), 2318-2332 (2022).
  46. Chang, X., et al. Exosomes from women with preeclampsia induced vascular dysfunction by delivering sflt (soluble fms-like tyrosine kinase)-1 and seng (soluble endoglin) to endothelial cells. Hypertension. 72, 1381-1390 (2018).
  47. Smarason, A. K., Sargent, I. L., Starkey, P. M., Redman, C. W. The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro. BJOG. 100 (10), 943-949 (1993).

Tags

Extracellular VesiclesBlood-brain BarrierPlacentaPreeclampsiaHypoxiaCerebrovascular ComplicationsMaternal MortalityBrain Endothelial Dysfunction
check_url/65867?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Sandoval, H., León, J., Troncoso, F., de la Hoz, V., Cisterna, A., Contreras, M., Castro, F. O., Ibañez, B., Acurio, J., Escudero, C. Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas. J. Vis. Exp. (203), e65867, doi:10.3791/65867 (2024).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image