Jingjing REN, Mingwei YANG, Genqiang YAN, Jianjun JIANG, Pengyan WANG

In vitro Model of the Blood-brain Barrier Established by Co-culture of Primary Sheep Cerebral Microvascular Endothelial and Astrocyte Cells

Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. It plays a primary role in a selective diffusion barrier at the level of the cerebral microvascular endothelium. Here, we have developed and characterized an in vitro model of the blood-brain barrier by seeding primary brain microvascular endothelial cells (BMECs) and astrocyte cells (ACs) from sheep on a polyester Transwell cell culture membrane with 0.4-μm pores, and conducted transepithelial electrical resistance measurements (TEER), leakage tests and assays for the permeability of horseradish peroxidase (HRP). The results of TEER and 4-h leakage assay showed that the new syngeneic BBB model had a characteristic of BBB in vivo. The HRP permeability test showed that the BBB model had good barrier function. Results showed that our model may be a valuable tool in the study of the mechanisms of brain invasion mediated by internalins of Listeria monocytogenes.

Primer Koyun Serebral Mikrodamar Endotelyal ve Astrosit Hücrelerinin Birlikte Kültüre Edilmesiyle Oluşturulmuş In vitro Kan Beyin Bariyeri Modeli

Kan beyin bariyeri beyin mikrodamar endotelyal hücreleri ve nörovasküler birimin komşu elementleri tarafından oluşturulur ve devamlılığı sağlanır. Kan beyin bariyeri serebral mikrovasküler endotel seviyesinde seçici difüzyon bariyeri olarak önemli bir rol oynar. Bu çalışmada 0.4- μm gözeneğe sahip polyester Transwell hücre kültürü membranına koyun primer beyin microdamar endotel hücreleri ve astrosit hücrelerinin ekimiyle in vitro kan beyin bariyeri oluşturuldu. Karakterizasyonu transepitelyal elektrik direnç ölçümleri, sızıntı testleri ve horseradish peroksidaz geçirgenliği testleri ile gerçekleştirildi. Transepitelyal elektrik direnç ölçümleri ve 4-h sızıntı testleri sonuçları yeni oluşturulan singeneik kan beyin bariyeri modelinin in vivo kan beyin bariyeri özelliklerine sahip olduğunu gösterdi. Horseradish peroksidaz geçirgenliği testi yeni oluşturulan kan beyin bariyerinin iyi bariyer fonksiyonlarına sahip olduğunu gösterdi. Elde edilen sonuçlar oluşturulan Listeria monocytogenes internalinleri tarafından oluşturulan beyin invazyonunun mekanizmasının çalışılmasında bu modelin yararlı olabileceğini göstermektedir.

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Tian X, Brookes O, Battaglia G: Pericytes from mesenchymal stem cells as a model for the blood-brain barrier. Sci Rep, 7, 39676, 2017. DOI: 10.1038/srep39676

Scism JL, Laska DA, Horn JW, Gimple JL, Pratt SE, Shepard RL, Dantzig AH, Wrighton SA: Evaluation of an in vitro, co-culture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources. In Vitro Cell Dev Biol Anim, 35 (10): 580-592, 1999. DOI: 10.1007/s11626-999- 0096-3

Schroeter ML, Müller S, Lindenau J, Wiesner B, Hanisch UK, Wolf G, Blasig IE: Astrocytes induce manganese superoxide dismutase in brain capillary endothelial cells. Neuroreport, 12 (11): 2513-2517, 2001. DOI: 10.1097/00001756-200108080-00045

Gaillard PJ, Voorwinden LH, Nielsen JL, Ivanov A, Atsumi R, Engman H, Ringbom C, de Boer AG, Breimer DD: Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci, 12 (3): 215-222, 2001. DOI: 10.1016/S0928- 0987(00)00123-8

Kaisar MA, Sajja RK, Prasad, S, Abhyankar VV, Liles T, Cucullo L: New experimental models of the blood-brain barrier for CNS drug discovery. Expert Opin Drug Discov, 12 (1): 89-103, 2017. DOI: 10.1080/17460441.2017.1253676

Rapôso C, Odorissi PAM, Oliveira ALR, Aoyama H, Ferreira CV, Verinaud L, Fontana K, Ruela-de-Sousa RR, da Cruz-Höfling MA: Effect of Phoneutria nigriventer, venom on the expression of junctional protein and P-gp efflux pump function in the blood–brain barrier. Neurochem Res, 37 (9): 1967-1981, 2012. DOI: 10.1007/s11064-012-0817-y

Kim KS: Microbial translocation of the blood-brain barrier. Int J Parasitol, 36 (5): 607-614, 2006. DOI: 10.1016/j.ijpara.2006.01.013

Hanada S, Fujioka K, Inoue Y, Kanaya F, Manome Y, Yamamoto K: Cell-based in vitro blood-brain barrier model can rapidly evaluate nanoparticles’ brain permeability in association with particle size and surface modification. Int J Mol Sci, 15 (2): 1812-1825, 2014. DOI: 10.3390/ ijms15021812

Wuest DM, Lee KH: Optimization of endothelial cell growth in a murine in vitro blood-brain barrier model. Biotechnol J, 7 (3): 409-417, 2012. DOI: 10.1002/biot.201100189

Burek M, Salvador E, Förster CY: Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model. J Vis Exp, 66, e4022, 2012. DOI: 10.3791/4022

Steiner O, Coisne C, Engelhardt B, Lyck, R: Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation. J Cereb Blood Flow Metab, 31 (1): 315-327, 2011. DOI: 10.1038/jcbfm.2010.96

Thomsen LB, Burkhart A, Moos T: A triple culture model of the blood-brain barrier using porcine brain endothelial cells, astrocytes and pericytes. PLoS One, 10 (8): e0134765, 2015. DOI: 10.1371/journal. pone.0134765

Pieper C, Pieloch P, Galla HJ: Pericytes support neutrophil transmigration via interleukin-8 across a porcine co-culture model of the blood-brain barrier. Brain Res, 1524, 1-11, 2013. DOI: 10.1016/j. brainres.2013.05.047

Wilhelm I, Fazakas C, Krizbai IA: In vitro models of the blood-brain barrier. Acta Neurobiol Exp, 71 (1): 113-128, 2011.

Bobilya DJ: Isolation and Cultivation of Porcine Astrocytes. In, Milner R (Ed): Astrocytes. Methods in Molecular Biology (Methods and Protocols), Vol 814, 12-135, Humana Press, 2012.

Bernas MJ, Cardoso FL, Daley SK, Weinand ME, Campos AR, Ferreira AJG, Hoying JB, Witte MH, Brites D, Persidsky Y, Ramirez SH, Brito MA: Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier. Nat Protoc, 5 (7): 1265-1272, 2010. DOI: 10.1038/ nprot.2010.76

Gelbíčová T, Pantůček R, Karpíšková R: Virulence factors and resistance to antimicrobials in Listeria monocytogenes serotype 1/2c isolated from food. J Appl Microbiol, 121 (2): 569-576, 2016. DOI: 10.1111/jam.13191

Seveau S, Pizarro-Cerda J, Cossart P: Molecular mechanisms exploited by Listeria monocytogenes during host cell invasion. Microbes Infect, 9 (10): 1167-1175, 2007. DOI: 10.1016/j.micinf.2007.05.004

Pagliano P, Ascione T, Boccia G, De Caro F, Esposito S: Listeria monocytogenes meningitis in the elderly: epidemiological, clinical and therapeutic findings. Infez Med, 24 (2): 105-111, 2016

Shimojima Y, Ida M, Nakama A, Nishino Y, Fukui R, Kuroda S, Hirai A, Kai A, Sadamasu K: Prevalence and contamination levels of Listeria monocytogenes in ready-to-eat foods in Tokyo, Japan. J Vet Med Sci, 78 (7): 1183-1187, 2016. DOI:10.1292/jvms.15-0708

Cossart P, Lebreton A: A trip in the “New Microbiology” with the bacterial pathogen Listeria monocytogenes. Febs Lett, 588 (15): 2437- 2445, 2014. DOI: 10.1016/j.febslet.2014.05.051

Gründler T, Quednau N, Stump C, Orian-Rousseau V, Ishikawa H, Wolburg H, Schroten H, Tenenbaum T, Schwerk C: The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood–cerebrospinal fluid barrier. Microbes Infect, 15 (4): 291-301, 2013. Doi: 10.1016/j. micinf.2012.12.005

Jeong S, Kim S, Buonocore J, Park J, Welsh CJ, Li J, Han A: A three-dimensional arrayed microfluidic blood-brain barrier model with integrated electrical sensor array. IEEE Trans Biomed Eng, 65 (2): 431-439, 2018. DOI: 10.1109/TBME.2017.2773463

Liu C, Chen K, Lu Y, Fang, Z, Yu GR: Catalpol provides a protective effect on fibrillary Aβ1-42-induced barrier disruption in an in vitro model of the blood-brain barrier. Phytother Res. 1-9, 2018. DOI: 10.1002/ptr.6043

Tao-Cheng JH, Nagy Z, Brightman MW: Tight junctions of brain endothelium in vitro are enhanced by astroglia. J Neurosci, 7 (10): 3293- 3299, 1987.

Haseloff RF, Blasig IE, Bauer HC, Bauer H: In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro. Cell Mol Neurobiol, 25 (1): 25-39, 2005. Doi: 10.1007/s10571-004-1375-x

Abbott NJ: Dynamics of CNS barriers: Evolution, differentiation, and modulation. Cell Mol Neurobiol, 25 (1): 5-23, 2005. Doi: 10.1007/s10571- 004-1374-y

Wolff A, Antfolk M, Brodin B, Tenje M: In vitro blood-brain barrier models-an overview of established models and new microfluidic approaches. J Pharm Sci, 104 (9): 2727-2746, 2015. DOI: 10.1002/jps.24329

Abbott NJ, Dolman DE, Drndarski S, Fredriksson SM: An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes. Methods Mol Biol, 814, 415-430, 2012. DOI: 10.1007/978- 1-61779-452-0_28

Cecchelli R, Berezowski V, Lundquist S, Culot M, Renftel M, Dehouck MP, Fenart L: Modelling of the blood–brain barrier in drug discovery and development. Nat Rev Drug Discov, 6 (8): 650-661, 2007. Doi: 10.1038/ nrd2368

Parkes I, Chintawar S, Cader MZ: Neurovascular dysfunction in dementia-human cellular models and molecular mechanisms. Clin Sci, 132 (3): 399-418, 2018. DOI: 10.1042/CS20160720

Wang Y, Wang N, Cai B, Wang GY, Li J, Piao XX: In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells. Neural Regen Res, 10 (12): 2011-2017, 2015. Doi: 10.4103/1673-5374.172320

Abbott NJ, Rönnbäck L, Hansson E: Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 7 (1): 41-53, 2006. Doi:10.1038/nrn1824

Hou H, Zhang G, Wang H, Gong H, Wang C, Zhang X: High matrix metalloproteinase-9 expression induces angiogenesis and basement membrane degradation in stroke-prone spontaneously hypertensive rats after cerebral infarction. Neural Regen Res, 9 (11): 1154-1162, 2014. Doi: 10.4103/1673-5374.135318