Skip to main content
SpringerLink
Log in

Towards the development of a human in vitro model of the blood–brain barrier for virus-associated acute encephalopathy: assessment of the time- and concentration-dependent effects of TNF-α on paracellular tightness

Experimental Brain Research volume 239, pages 451–461 (2021)Cite this article

Abstract

The pathogenesis of virus-associated acute encephalopathy (VAE) involves brain edema caused by disruption of the blood–brain barrier (BBB). We aimed to develop an in vitro VAE model using an in vitro BBB model, to evaluate the dynamics of vascular dysfunction caused by tumor necrosis factor (TNF)-α. A co-culture model, consisting of Transwell®-grown human brain microvascular endothelial cells and pericytes, was treated with serially diluted TNF-α. Transendothelial electrical resistance (TER) was measured using cellZscope®. A permeability assay, using fluorescein isothiocyanate-conjugated sodium or dextran, was performed. Changes in claudin-5 localization and expression after TNF-α treatment were observed using immunofluorescence staining and western blot analysis. The TER decreased and permeability increased after TNF-α treatment; recovery time was dependent on TNF-α concentration. Claudin-5 was delocalized after TNF-α treatment and recovered in a TNF-α concentration-dependent manner. The expression of claudin-5 decreased 24 h after the TNF-α treatment and completely recovered 48 h after TNF-α treatment. Claudin-5 delocalization was likely associated with vascular hyperpermeability. To conclude, we evaluated vascular endothelial cell permeability and injury in VAE using an in vitro BBB model treated with TNF-α. This system can be useful for developing novel therapeutic strategies for VAE and designing treatments that target vascular permeability.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Code availability

Not applicable.

References

  • Ali C, Nicole O, Docagne F, Lesne S, MacKenzie ET, Nouvelot A, Buisson A, Vivien D (2000) Ischemia-induced interleukin-6 as a potential endogenous neuroprotective cytokine against NMDA receptor-mediated excitotoxicity in the brain. J Cereb Blood Flow Metab 20:956–966

    Article  CAS  Google Scholar 

  • Almutari MM, Gong C, Xu YG, Chang Y, Shi H (2016) Factors controlling permeability of the blood-brain barrier. Cell Mol Life Sci 73:57–77

    Article  Google Scholar 

  • Anda T, Yamashita H, Khalid H, Tsutsumi K, Fujita H, Tokunaga Y, Shibata S (1997) Effect of tumor necrosis factor-alpha on the permeability of bovine brain microvessel endothelial cell monolayers. Neuro Res 19:369–376

    Article  CAS  Google Scholar 

  • Asano Y, Yoshikawa T, Kajita Y, Ogura R, Suga S, Yazaki T, Nakashima T, Yamada A, Kurata T (1992) Fatal encephalitis/encephalopathy in primary human herpesvirus-6 infection. Arch Dis Child 67:1484–1485

    Article  CAS  Google Scholar 

  • Aslam M, Ahmad N, Srivastava R, Hemmer B (2012) TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cld5 promoter in mouse brain endothelial cells. Cytokine 57:269–275

    Article  CAS  Google Scholar 

  • Capaldo CT, Nusrat A (2009) Cytokine regulation of tight junctions. Biochem Biophys Acta 1788:864–871

    Article  CAS  Google Scholar 

  • Castro Dias M, Coisne C, Lazarevic I, Baden P, Hata M, Iwamoto N, Francisco DMF, Vanlandewijck M, He L, Baier FA, Stroka D, Bruggmann R, Lyck R, Enzmann G, Deutsch U, Betsholtz C, Furuse M, Tsukita S, Engelhardt B (2019) Claudin-3-deficient C57BL/6J mice display intact brain barriers. Sci Rep 9:203

    Article  Google Scholar 

  • Castro Dias M, Coisne C, Baden P, Enzmann G, Garrett L, Becker L, Hölter SM; German Mouse Clinic Consortium, Hrabě de Angelis M, Deutsch U, Engelhardt B (2019) Claudin-12 is not required for blood-brain barrier tight junction function. Fluids Barriers CNS 16:30

    Article  Google Scholar 

  • Clark IA, Alleva LM, Vissel B (2010) The roles of TNF in brain dysfunction and disease. Pharmacol Ther 128:519–548

    Article  CAS  Google Scholar 

  • de Vries HE, Blom-Roosemalen MC, van Oosten M, de Boer AG, van Berkel TJ, Breimer DD, Kuiper J (1996) The influence of cytokines on the integrity of the blood-brain barrier in vitro. J Neuroimmunol 64:37–43

    Article  Google Scholar 

  • Deli MA, Descamps L, Dehouck MP, Cecchelli R, Joó F, Abrahám CS, Torpier G (1995) Exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induced a delayed increase of permeability and cytoplasmic stress fiber formation of actin. J Neurosci Res 41:717–726

    Article  CAS  Google Scholar 

  • Dohgu S, Takata F, Yamauchi A, Nakagawa S, Egawa T, Naito M, Tsuruo T, Sawada Y, Niwa M, Kataoka Y (2005) Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production. Brain Res 1038:208–215

    Article  CAS  Google Scholar 

  • Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777–1788

    Article  CAS  Google Scholar 

  • Furuse M, Sasaki H, Fujimoto K, Tsukita S (1998) A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occluding in fibroblasts. J Cell Biol 143:391–401

    Article  CAS  Google Scholar 

  • Hartmann C, Zozulya A, Wegener J, Galla HJ (2007) The impact of glia-derived extracellular matrices on the barrier function of cerebral endothelial cells: an in vitro study. Exp Cell Res 313:1318–1325

    Article  CAS  Google Scholar 

  • Hewitt KJ, Agarwal R, Morin PJ (2006) The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 6:186

    Article  Google Scholar 

  • Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T (2004) A pericyte-derived angiopoietin-1 multimeric complex induces occluding gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem 89:503–513

    Article  CAS  Google Scholar 

  • Hosoya M, Nunoi H, Aoyama M, Kawasaki Y, Suzuki H (2005) Cytochrome c and tumor necrosis factor-alpha values in serum and cerebrospinal fluid of patients with influenza-associated encephalopathy. Pediatr Infect Dis J 24:467–470

    Article  Google Scholar 

  • Ichiyama T, Nishikawa M, Yoshitomi T, Hayashi T, Furukawa S (1998) Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 in cerebrospinal from children with prolonged febrile seizures. Comparison with acute encephalitis/encephalopathy. Neurology 50:407–411

    Article  CAS  Google Scholar 

  • Karampatsas K, Osborne L, Seah ML, Tong CYW, Prendergast AJ (2018) Clinical characteristics and complications of rotavirus gastroenteritis in children in east London: A retrospective case-control study. PLoS ONE 13:e0194009

    Article  Google Scholar 

  • Kasai T, Togashi T, Morishima T (2000) Encephalopathy associated with influenza epidemics. Lancet 355:1558–1559

    Article  CAS  Google Scholar 

  • Kawada J, Kimura H, Ito Y, Hara S, Iriyama M, Yoshikawa T, Morishima T (2003) Systemic cytokine responses in patients with influenza-associated encephalopathy. J Infect Dis 188:690–698

    Article  CAS  Google Scholar 

  • Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, Blasig IE (2008) Structure and function of claudins. Biochim Biophys Acta 1778:631–645

    Article  CAS  Google Scholar 

  • Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA, Said HM (2004) TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol 286:G367–G376

    Article  CAS  Google Scholar 

  • Ma TY, Boivin MA, Ye D, Pedram A, Said HM (2005) Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol 288:G422–G430

    Article  CAS  Google Scholar 

  • Mark KS, Miller DW (1999) Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-alpha exposure. Life Sci 64:1941–1953

    Article  CAS  Google Scholar 

  • McKenzie JA, Ridley AJ (2007) Roles of Rho/Rock and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213:221–228

    Article  CAS  Google Scholar 

  • Megard I, Garrigues A, Orloski S, Jorajuria S, Clayette P, Ezan E, Mabondzo A (2002) A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation. Brain Res 927:153–167

    Article  CAS  Google Scholar 

  • Miyazaki K, Hashimoto K, Sato M, Watanabe M, Tomikawa N, Kanno S, Kawasaki Y, Momoi N, Hosoya M (2017) Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy. Pediatr Res 81:942–947

    Article  CAS  Google Scholar 

  • Mizuguchi M (1997) Acute necrotizing encephalopathy of childhood: a novel form of acute encephalopathy prevalent in Japan and Taiwan. Brain Dev 19:81–92

    Article  CAS  Google Scholar 

  • Mizuguchi M, Yamanouchi H, Ichiyama T, Shiomi M (2007) Acute encephalopathy associated with influenza and other viral infections. Acta Neurol Scand 115:45–56

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Morita H, Hosoya M, Kato A, Kawasaki Y, Suzuki H (2005) Laboratory characteristics of acute encephalopathy with multiple organ dysfunctions. Brain Dev 27:477–482

    Article  Google Scholar 

  • Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, Kataoka Y, Niwa M (2007) Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol 27:687–694

    Article  CAS  Google Scholar 

  • Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, Tanaka K, Niwa M (2009) A new blood-brain model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int 54:253–263

    Article  CAS  Google Scholar 

  • Neuhaus W, Wirth M, Plattner VE, Germann B, Gabor F, Noe CR (2008) Expression of claudin-1, claudin-3 and claudin-5 in human blood-brain mimicking cell line ECV304 is inducible by glioma-conditioned media. Neurosci Lett 446:59–64

    Article  CAS  Google Scholar 

  • Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660

    Article  CAS  Google Scholar 

  • Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Cell Biol 2:285–293

    Article  CAS  Google Scholar 

  • Yamashita T, Sawamoto K, Suzuki S, Suzuki N, Adachi K, Kawase T, Mihara M, Ohsugi Y, Abe K, Okano H (2005) Blockade of interleukin-6 signaling aggravates ischemic cerebral damage in mice: possible involvement of Stat3 activation in the protection of neurons. J Neurochem 94:459–468

    Article  CAS  Google Scholar 

  • Zlokovic BV (2008) The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57:178–201

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Naoki Tomikawa for the useful discussion and advice. We are also grateful to the laboratory’s technical staff, Ken Honzumi, Sakurako Norito, and Mieko Tanji, for their excellent technical skills.

Funding

The study was supported by a grant from the Grants-in-Aid for Scientific Research (KAKENHI, Grant No. 17K10120).

Author information

Authors and Affiliations

  1. Department of Pediatrics, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima, 960-1295, Japan

    Hajime Maeda, Koichi Hashimoto, Hayato Go, Kyohei Miyazaki, Masatoki Sato, Yukihiko Kawasaki, Nobuo Momoi & Mitsuaki Hosoya

  2. Department of Pediatrics, Sapporo Medical University School of Medicine, 17, Minami 1-jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido, 060-8556, Japan

    Yukihiko Kawasaki

Authors
  1. Hajime Maeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Koichi Hashimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hayato Go
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kyohei Miyazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masatoki Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yukihiko Kawasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nobuo Momoi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mitsuaki Hosoya
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Hajime Maeda, Kyohei Miyazaki, Hayato Go, Koichi Hashimoto, and Mitsuaki Hosoya. The first draft of the manuscript was written by Hajime Maeda and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hajime Maeda.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

Not applicable.

Additional information

Communicated by Sreedharan Sajikumar.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maeda, H., Hashimoto, K., Go, H. et al. Towards the development of a human in vitro model of the blood–brain barrier for virus-associated acute encephalopathy: assessment of the time- and concentration-dependent effects of TNF-α on paracellular tightness. Exp Brain Res 239, 451–461 (2021). https://doi.org/10.1007/s00221-020-05985-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00221-020-05985-7

Keywords

search

Navigation