Skip to main content
SpringerLink
Log in

In Vivo Evaluation of BBB Integrity in the Post-stroke Brain

  • Protocol
  • First Online:
Neural Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2616))

Abstract

The blood–brain barrier (BBB) is a dynamic interface responsible for maintaining central nervous system (CNS) homeostasis. An intact BBB protects the brain from undesired compounds and proteins from the blood; however, BBB impairment is involved in various pathological conditions including stroke. In vivo evaluation of BBB integrity in the post-stroke brain is important for investigating stroke-induced CNS pathogenesis and developing CNS-targeted therapeutic agents. In this chapter, we describe both quantitative and morphometric methods and tools to evaluate BBB integrity in vivo. These methods do not require expensive magnetic resonance imaging (MRI) and computed tomography (CT) imaging capabilities and can be conducted in research laboratories with access to a confocal microscope and fluorescence microplate reader.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Abbott NJ, Patabendige AA, Dolman DE et al (2010) Structure and function of the blood-brain barrier. Neurobiol Dis 37:13–25

    Article  CAS  Google Scholar 

  2. Alluri H, Grimsley M, Anasooya Shaji C et al (2016) Attenuation of blood-brain barrier breakdown and hyperpermeability by calpain inhibition. J Biol Chem 291:26958–26969

    Article  CAS  Google Scholar 

  3. Avsenik J, Bisdas S, Popovic KS (2015) Blood-brain barrier permeability imaging using perfusion computed tomography. Radiol Oncol 49:107–114

    Article  Google Scholar 

  4. Balda MS, Anderson JM (1993) Two classes of tight junctions are revealed by ZO-1 isoforms. Am J Phys 264:C918–C924

    Article  CAS  Google Scholar 

  5. Balkaya M, Kim ID, Shakil F et al (2021) CD36 deficiency reduces chronic BBB dysfunction and scar formation and improves activity, hedonic and memory deficits in ischemic stroke. J Cereb Blood Flow Metab 41:486–501

    Article  CAS  Google Scholar 

  6. Bernardo-Castro S, Sousa JA, Bras A et al (2020) Pathophysiology of blood-brain barrier permeability throughout the different stages of ischemic stroke and its implication on hemorrhagic transformation and recovery. Front Neurol 11:594672

    Article  Google Scholar 

  7. Berndt P, Winkler L, Cording J et al (2019) Tight junction proteins at the blood-brain barrier: far more than claudin-5. Cell Mol Life Sci 76:1987–2002

    Article  CAS  Google Scholar 

  8. Blanchette M, Daneman R (2015) Formation and maintenance of the BBB. Mech Dev 138(Pt 1):8–16

    Article  CAS  Google Scholar 

  9. Bolton SJ, Anthony DC, Perry VH (1998) Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood-brain barrier breakdown in vivo. Neuroscience 86:1245–1257

    Article  CAS  Google Scholar 

  10. Braakman HMH, Engelen M, Nicolai J et al (2018) Stroke mimics add to the phenotypic spectrum of GLUT1 deficiency syndrome. J Neurol Neurosurg Psychiatry 89:668–670

    Article  Google Scholar 

  11. Devraj K, Guerit S, Macas J et al (2018) An in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers. J Vis Exp (132):57038

    Google Scholar 

  12. Furuse M, Sasaki H, Tsukita S (1999) Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 147:891–903

    Article  CAS  Google Scholar 

  13. Gautam J, Zhang X, Yao Y (2016) The role of pericytic laminin in blood brain barrier integrity maintenance. Sci Rep 6:36450

    Article  CAS  Google Scholar 

  14. Georgieva JV, Hoekstra D, Zuhorn IS (2014) Smuggling drugs into the brain: an overview of ligands targeting transcytosis for drug delivery across the blood-brain barrier. Pharmaceutics 6:557–583

    Article  Google Scholar 

  15. Ha Park J, Yoo KY, Hye Kim I et al (2016) Hydroquinone strongly alleviates focal ischemic brain injury via blockage of blood-brain barrier disruption in rats. Toxicol Sci 154:430–441

    Article  Google Scholar 

  16. Haskins J, Gu L, Wittchen ES et al (1998) ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol 141:199–208

    Article  CAS  Google Scholar 

  17. Hirase T, Staddon JM, Saitou M et al (1997) Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110(Pt 14):1603–1613

    Article  CAS  Google Scholar 

  18. Hoffmann A, Bredno J, Wendland M et al (2011) High and low molecular weight fluorescein Isothiocyanate (FITC)-dextrans to assess blood-brain barrier disruption: technical considerations. Transl Stroke Res 2:106–111

    Article  CAS  Google Scholar 

  19. Hoffmann A, Zhu G, Wintermark M (2012) Advanced neuroimaging in stroke patients: prediction of tissue fate and hemorrhagic transformation. Expert Rev Cardiovasc Ther 10:515–524

    Article  Google Scholar 

  20. Jiang X, Andjelkovic AV, Zhu L et al (2018) Blood-brain barrier dysfunction and recovery after ischemic stroke. Prog Neurobiol 163–164:144–171

    Article  Google Scholar 

  21. Jiao H, Wang Z, Liu Y et al (2011) Specific role of tight junction proteins claudin-5, occludin, and ZO-1 of the blood-brain barrier in a focal cerebral ischemic insult. J Mol Neurosci 44:130–139

    Article  CAS  Google Scholar 

  22. Kadry H, Noorani B, Cucullo L (2020) A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS 17:69

    Article  Google Scholar 

  23. Kassner A, Merali Z (2015) Assessment of blood-brain barrier disruption in stroke. Stroke 46:3310–3315

    Article  Google Scholar 

  24. Kaur C, Ling EA (2008) Blood brain barrier in hypoxic-ischemic conditions. Curr Neurovasc Res 5:71–81

    Article  CAS  Google Scholar 

  25. Laksitorini M, Prasasty VD, Kiptoo PK et al (2014) Pathways and progress in improving drug delivery through the intestinal mucosa and blood-brain barriers. Ther Deliv 5:1143–1163

    Article  CAS  Google Scholar 

  26. Leino RL, Gerhart DZ, Van Bueren AM et al (1997) Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain. J Neurosci Res 49:617–626

    Article  CAS  Google Scholar 

  27. Liebner S, Fischmann A, Rascher G et al (2000) Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol 100:323–331

    Article  CAS  Google Scholar 

  28. Liu F, Liu Q, Yuan F et al (2019) Erg mediates downregulation of claudin-5 in the brain endothelium of a murine experimental model of cerebral malaria. FEBS Lett 593:2585–2595

    Article  CAS  Google Scholar 

  29. Lombardo SM, Schneider M, Tureli AE et al (2020) Key for crossing the BBB with nanoparticles: the rational design. Beilstein J Nanotechnol 11:866–883

    Article  CAS  Google Scholar 

  30. Lu G, He Q, Shen Y et al (2018) Potential biomarkers for predicting hemorrhagic transformation of ischemic stroke. Int J Neurosci 128:79–89

    Article  Google Scholar 

  31. Morita K, Sasaki H, Furuse M et al (1999) Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 147:185–194

    Article  CAS  Google Scholar 

  32. Nag S (2003) Blood-brain barrier permeability using tracers and immunohistochemistry. Methods Mol Med 89:133–144

    CAS  Google Scholar 

  33. Nagaraja TN, Keenan KA, Fenstermacher JD et al (2008) Acute leakage patterns of fluorescent plasma flow markers after transient focal cerebral ischemia suggest large openings in blood-brain barrier. Microcirculation 15:1–14

    Article  CAS  Google Scholar 

  34. Natarajan R, Northrop N, Yamamoto B (2017) Fluorescein Isothiocyanate (FITC)-dextran extravasation as a measure of blood-brain barrier permeability. Curr Protoc Neurosci 79:9.58.1–9.58.15

    Article  CAS  Google Scholar 

  35. Pandit R, Chen L, Gotz J (2020) The blood-brain barrier: physiology and strategies for drug delivery. Adv Drug Deliv Rev 165–166:1–14

    Article  Google Scholar 

  36. Prasad S, Sajja RK, Kaisar MA et al (2017) Role of Nrf2 and protective effects of Metformin against tobacco smoke-induced cerebrovascular toxicity. Redox Biol 12:58–69

    Article  CAS  Google Scholar 

  37. Rajsic S, Gothe H, Borba HH et al (2019) Economic burden of stroke: a systematic review on post-stroke care. Eur J Health Econ 20:107–134

    Article  CAS  Google Scholar 

  38. Rusu AD, Georgiou M (2020) The multifarious regulation of the apical junctional complex. Open Biol 10:190278

    Article  CAS  Google Scholar 

  39. Sarvari S, Moakedi F, Hone E et al (2020) Mechanisms in blood-brain barrier opening and metabolism-challenged cerebrovascular ischemia with emphasis on ischemic stroke. Metab Brain Dis 35:851–868

    Article  Google Scholar 

  40. Saunders NR, Dziegielewska KM, Mollgard K et al (2015) Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives? Front Neurosci 9:385

    Article  Google Scholar 

  41. Serlin Y, Shelef I, Knyazer B et al (2015) Anatomy and physiology of the blood-brain barrier. Semin Cell Dev Biol 38:2–6

    Article  Google Scholar 

  42. Sonoda N, Furuse M, Sasaki H et al (1999) Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: evidence for direct involvement of claudins in tight junction barrier. J Cell Biol 147:195–204

    Article  Google Scholar 

  43. Stevenson BR, Siliciano JD, Mooseker MS et al (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103:755–766

    Article  CAS  Google Scholar 

  44. Sweeney MD, Zhao Z, Montagne A et al (2019) Blood-brain barrier: from physiology to disease and Back. Physiol Rev 99:21–78

    Article  CAS  Google Scholar 

  45. Tajes M, Ramos-Fernandez E, Weng-Jiang X et al (2014) The blood-brain barrier: structure, function and therapeutic approaches to cross it. Mol Membr Biol 31:152–167

    Article  CAS  Google Scholar 

  46. Tietz S, Engelhardt B (2015) Brain barriers: crosstalk between complex tight junctions and adherens junctions. J Cell Biol 209:493–506

    Article  CAS  Google Scholar 

  47. Uldry M, Thorens B (2004) The SLC2 family of facilitated hexose and polyol transporters. Pflugers Arch 447:480–489

    Article  CAS  Google Scholar 

  48. Xie J, Shen Z, Anraku Y et al (2019) Nanomaterial-based blood-brain-barrier (BBB) crossing strategies. Biomaterials 224:119491

    Article  CAS  Google Scholar 

  49. Xu Y, He Q, Wang M et al (2019) Quantifying blood-brain-barrier leakage using a combination of evans blue and high molecular weight FITC-Dextran. J Neurosci Methods 325:108349

    Article  CAS  Google Scholar 

  50. Yang C, Hawkins KE, Dore S et al (2019) Neuroinflammatory mechanisms of blood-brain barrier damage in ischemic stroke. Am J Physiol Cell Physiol 316:C135–C153

    Article  CAS  Google Scholar 

  51. Yang L, Wang H, Shah K et al (2011) Opioid receptor agonists reduce brain edema in stroke. Brain Res 1383:307–316

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Center for Blood-Brain Barrier Research, Jerry. H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA

    Yong Zhang, Saeideh Nozohouri & Thomas J. Abbruscato

Authors
  1. Yong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Saeideh Nozohouri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas J. Abbruscato
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas J. Abbruscato .

Editor information

Editors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA

    Vardan T. Karamyan

  2. Department of Neurology, Department of Neuroscience, The University of Kentucky, Lexington, KY, USA

    Ann M. Stowe

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zhang, Y., Nozohouri, S., Abbruscato, T.J. (2023). In Vivo Evaluation of BBB Integrity in the Post-stroke Brain. In: Karamyan, V.T., Stowe, A.M. (eds) Neural Repair. Methods in Molecular Biology, vol 2616. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2926-0_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2926-0_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2925-3

  • Online ISBN: 978-1-0716-2926-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation