Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro

Fluids and Barriers of the CNS

Marie-Magdeleine Ruchoux⁵  Marie-Pierre Dehouck³  Roméo Cecchelli³  Jean Plouët⁶  Michel Franck¹  Catherine Godfraind⁴  Lucie Dehouck³  Fabien Gosselet³  Maxime Culot³  Elodie Vandenhaute² 

[1] Ecole Nationale Vétérinaire de Lyon, Laboratoire de Zootechnie, F-69280, Marcy l’Etoile, France;Present address: Mannheim Medical Faculty, University of Heidelberg, Childrens Hospital, Theodor-Kutzer-Ufer 1-3, D-68167, Mannheim, Germany;UArtois, LBHE, F-62300, Lens, France;Département de neuropathologie, Clinique Saint-Luc, ULB, 1200, Bruxelles, Belgium;CEA, DSV/DRM, Groupe d'Innovation Diagnostique et Thérapeutique sur les Infections à Prions, 18 Route du Panorama, BP 6, 92265, Fontenay aux Roses Cedex, France;Inserm, U 689, Centre de Recherche Cardiovasculaire Inserm Lariboisière, 75475, Paris Cedex 10, France

关键词: Porcine stress syndrome;    Perivascular spaces;    Pericytes;    Neurovascular unit;    Endothelial permeability;    Blood–brain barrier;   

Others  :  807247
DOI  :  10.1186/2045-8118-9-11

 received in 2011-12-13, accepted in 2012-05-08,  发布年份 2012

【 摘 要 】

Background

The function of pericytes remains questionable but with improved cultured technique and the use of genetically modified animals, it has become increasingly clear that pericytes are an integral part of blood–brain barrier (BBB) function, and the involvement of pericyte dysfunction in certain cerebrovascular diseases is now emerging. The porcine stress syndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH) in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms, including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to the human syndrome. Symptoms are due to an enhanced calcium release from intracellular stores. These conditions are associated with a point mutation in ryr1/hal gene, encoding the ryanodine receptor, a calcium channel. Important blood vessel wall muscle modifications have been described in PSS, but potential brain vessel changes have never been documented in this syndrome.

Methods

In the present work, histological and ultrastructural analyses of brain capillaries from wild type and ryr1 mutated pigs were conducted to investigate the potential impairment of pericytes, in this pathology. In addition, brain pericytes were isolated from the three porcine genotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/hal alleles, respectively), and tested in vitro for their influence on the permeability of BBB endothelial monolayers.

Results

Enlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to a partial or total detachment of the astrocytic endfeet. These spaces were electron lucent and sometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level, brain pericytes did not seem to be affected because they showed regular morphology and characteristics, so we aimed to check their ability to maintain BBB properties in vitro. Our results indicated that pericytes from the three genotypes of pigs had differing influences on the BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able to maintain low BBB permeability.

Conclusions

Electron microscopy observations demonstrated brain capillary modifications in PSS condition, but no change in pericyte morphology. Results from in vitro experiments suggest that brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition at the ultrastructural level, are not able to maintain BBB integrity in comparison with pericytes from wild-type animals.

【 授权许可】

   
2012 Vandenhaute et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Fujii J, Otsu K, Zorzato F, de Leon S, Khanna VK, Weiler JE, O'Brien PJ, MacLennan DH: Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 1991, 253:448-451.
• [2]Franck M, Figwer P, Godfraind C, Poirel MT, Khazzaha A, Ruchoux MM: Could the pale, soft, and exudative condition be explained by distinctive histological characteristics? J Anim Sci 2007, 85:746-753.
• [3]Ledbetter MW, Preiner JK, Louis CF, Mickelson JR: Tissue distribution of ryanodine receptor isoforms and alleles determined by reverse transcription polymerase chain reaction. J Biol Chem 1994, 269:31544-31551.
• [4]Giannini G, Conti A, Mammarella S, Scrobogna M, Sorrentino V: The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J Cell Biol 1995, 128:893-904.
• [5]Zlokovic BV: The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 2008, 57:178-201.
• [6]Bonkowski D, Katyshev V, Balabanov RD, Borisov A, Dore-Duffy P: The CNS microvascular pericyte: pericyte-astrocyte crosstalk in the regulation of tissue survival. Fluids Barriers CNS 2011, 8:8. BioMed Central Full Text
• [7]Katyshev V, Dore-Duffy P: Pericyte coculture models to study astrocyte, pericyte, and endothelial cell interactions. Methods Mol Biol 2012, 814:467-481.
• [8]Tigges U, Welser-Alves JV, Boroujerdi A, Milner R: A novel and simple method for culturing pericytes from mouse brain. Microvasc Res 2012. Epub ahead of print
• [9]Kim JH, Yu YS, Kim DH, Kim KW: Recruitment of pericytes and astrocytes is closely related to the formation of tight junction in developing retinal vessels. J Neurosci Res 2009, 87:653-659.
• [10]Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, Kataoka Y, Niwa M: Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol 2007, 27:687-694.
• [11]Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, Tanaka K, Niwa M: A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. J Neurosci Res 2009, 54:253-263.
• [12]Berezowski V, Landry C, Dehouck MP, Cecchelli R, Fenart L: Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood–brain barrier. Brain Res 2004, 1018:1-9.
• [13]Takata F, Dohgu S, Yamauchi A, Sumi N, Nakagawa S, Naito M, Tsuruo T, Shuto H, Kataoka Y: Inhibition of transforming growth factor-beta production in brain pericytes contributes to cyclosporin A-induced dysfunction of the blood–brain barrier. Cell Mol Neurobiol 2007, 27:317-328.
• [14]Dehouck MP, Meresse S, Delorme P, Fruchart JC, Cecchelli R: An easier, reproducible, and mass-production method to study the blood–brain barrier in vitro. J Neurochem 1990, 54:1798-1801.
• [15]Carson FL, Martin JH, Lynn JA: Formalin fixation for electron microscopy: a re-evaluation. Am J Clin Pathol 1973, 59:365-373.
• [16]Cecchelli R, Dehouck B, Descamps L, Fenart L, Buee-Scherrer VV, Duhem C, Lundquist S, Rentfel M, Torpier G, Dehouck MP: In vitro model for evaluating drug transport across the blood–brain barrier. Adv Drug Deliv Rev 1999, 36:165-178.
• [17]Siflinger-Birnboim A, Del Vecchio PJ, Cooper JA, Blumenstock FA, Shepard JM, Malik AB: Molecular sieving characteristics of the cultured endothelial monolayer. J Cell Physiol 1987, 132:111-117.
• [18]Plouet J, Moukadiri H: Specific binding of vasculotropin to bovine brain capillary endothelial cells. Biochimie 1990, 72:51-55.
• [19]Armulik A, Abramsson A, Betsholtz C: Endothelial/pericyte interactions. Circ Res 2005, 97:512-523.
• [20]Ozerdem U, Grako KA, Dahlin-Huppe K, Monosov E, Stallcup WB: NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 2001, 222:218-227.
• [21]Vandenhaute E, Dehouck L, Boucau MC, Sevin E, Uzbekov R, Tardivel M, Gosselet F, Fenart L, Cecchelli R, Dehouck MP: Modelling the neurovascular unit and the blood–brain barrier with the unique function of pericytes. Curr Neurovasc Res 2011, 8:258-269.
• [22]Dehouck B, Dehouck MP, Fruchart JC, Cecchelli R: Upregulation of the low density lipoprotein receptor at the blood–brain barrier: intercommunications between brain capillary endothelial cells and astrocytes. J Cell Biol 1994, 126:465-473.
• [23]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 2007, 6:650-661.
• [24]Hamm S, Dehouck B, Kraus J, Wolburg-Buchholz K, Wolburg H, Risau W, Cecchelli R, Engelhardt B, Dehouck MP: Astrocyte mediated modulation of blood–brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts. Cell Tissue Res 2004, 315:157-166.
• [25]Nag S: Morphology and properties of astrocytes. Methods Mol Biol 2011, 686:69-100.
• [26]Schreurs MP, Houston EM, May V, Cipolla MJ: The adaptation of the blood–brain barrier to vascular endothelial growth factor and placental growth factor during pregnancy. FASEB J 2012, 26:355-362.
• [27]Esser S, Wolburg K, Wolburg H, Breier G, Kurzchalia T, Risau W: Vascular endothelial growth factor induces endothelial fenestrations in vitro. J Cell Biol 1998, 140:947-959.
• [28]Wardlaw JM: Blood–brain barrier and cerebral small vessel disease. J Neurol Sci 2010, 299:66-71.
• [29]Peppiatt CM, Howarth C, Mobbs P, Attwell D: Bidirectional control of CNS capillary diameter by pericytes. Nature 2006, 443:700-704.
• [30]Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom P, Strittmatter K, et al.: Pericytes regulate the blood–brain barrier. Nature 2010, 468:557-561.
• [31]Daneman R, Zhou L, Kebede AA, Barres BA: Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature 2010, 468:562-566.
• [32]Winkler EA, Bell RD, Zlokovic BV: Central nervous system pericytes in health and disease. Nat Neurosci 2011, 14:1398-1405.
• [33]Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T: A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem 2004, 89:503-513.
• [34]Thanabalasundaram G, Schneidewind J, Pieper C, Galla HJ: The impact of pericytes on the blood–brain barrier integrity depends critically on the pericyte differentiation stage. Int J Biochem Cell Biol 2011, 43:1284-1293.
• [35]Barres BA: The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 2008, 60:430-440.