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Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs

器官型脑培养：研究嗜神经病毒对中枢神经系统的感染和筛选抗病毒药物的框架

Jeremy Charles Welsch Jeremy Charles Welsch
CL Claire Lionnet
CT Christophe Terzian §
BH Branka Horvat
DG Denis Gerlier
CM Cyrille Mathieu

 (§Deceased) 发布: 2017年11月20日第7卷第22期 DOI: 10.21769/BioProtoc.2605 浏览次数: 14280

评审: Kae-Jiun ChangAnonymous reviewer(s)

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Abstract

According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., 2001; Olival and Daszak, 2005). While neurological diseases are progressively documented, the underlying cellular and molecular mechanisms involved in virus infection and dissemination within the CNS are still poorly understood (Swanson and McGavern, 2015; Ludlow et al., 2016). For example, measles virus (MeV) can infect neural cells, and cause a persistent brain infections leading to lethal encephalitis from several months to years after primary infection with no available treatment (Reuter and Schneider-Schaulies, 2010; Laksono et al., 2016). The Organotypic Brain Culture (OBC) is a suitable model for the virology field to better understand the CNS infections. Indeed, it allows not only studying the infection and the dissemination of neurotropic viruses within the CNS but it could also serve as screening model of innovative antiviral strategies or molecules, such as our recently published studies about fusion inhibitory peptides and the HSP90 chaperone activity inhibitor, 17-DMAG (Welsch et al., 2013; Bloyet et al., 2016). Based on our previous work, we propose here an optimized method to prepare OBC of hippocampi and cerebellums which are suitable for small rodent models based virus studies, including mice, rats as well as hamsters at a post-natal stage, between P6 to P10. We notably took into account the stress of the slice procedure on the tissue and the subsequent cellular reactions, which is essential to fully characterize the model prior to any use in infectious conditions. With this knowledge, we propose a protocol highlighting the requirements, including potential trouble shootings of the slicing parameters, to consider the variations we observed according to the structure and animal studied. This framework should facilitate the use of OBC for better conclusive studies of neurotropic viruses.

Keywords: Organotypic brain culture (器官型脑培养) search Neurotropic viruses (嗜神经病毒) search CNS infection (中枢神经系统感染) search Brain viral dissemination (脑病毒传播) search Antiviral molecule screening (抗病毒分子筛选) search

Background

Since 1958 neurobiologists have continuously developed organotypic brain cultures (OBC) with a tremendous increase in their usage over the last two decades in the fields of neurodevelopment, neurodegenerative diseases or neuropharmacology (Bornstein and Murray, 1958; Kim et al., 2013; Humpel, 2015). In contrast, despite the advantages of this model, very few studies of virus infection, tropism or dissemination have been published (Mayer et al., 2005; Braun et al., 2006; Stubblefield Park et al., 2011). Indeed, experiments using OBC are inherently more complex to set up than classical cellular primary cultures (i.e., purified neurons or dissociated brain cultures). However, the elegance of this approach resides in the possibility to maintain major cell types in a preserved three-dimensional tissue architecture that allows studying in real time viral invasion throughout brain structures and cell subsets, in more physiological environment and without the impact of the peripheral immune system. Furthermore, since the cellular composition of the tissue is maintained, including neurons, oligodendrocytes, microglial cells and astrocytes, it becomes possible to assess and decipher the involvement and the response of each cell population during the viral infection (Lossi et al., 2009). This model also presents the advantage to reduce the animal payload compared to in vivo experiment which fits perfectly with the recommendations and regulation of animal usage in life science by the Institutional Animal Care and Use Committee (IACUC). Indeed, it is possible to generate at least 10 to 15 slices per structure and thus it allows expanding the number of tested conditions per animal. Furthermore, most of the equipment required for its implementation is easy to acquire or already available in laboratories using tissue culture approaches with interest in neuro-virology. This protocol details the preparation of cultured rodent brain slices obtained from either hippocampus or cerebellum, assessment of its viability, analysis of brain cell types, morphological rearrangements and kinetic during one-week culture. Finally, this protocol offers an example of utilization of OBC to study viral brain infection with measles virus (MeV) in rodent explants.

Materials and Reagents

  1. Sterile pipette tips, 1,000 μl (Corning, catalog number: 9032 )
  2. Sterile filtered pipette tips, 10 μl (Corning, catalog number: 4807 )
  3. Sterile Falcon 6-well flat bottom plate (Corning, Falcon®, catalog number: 353046 )
  4. Feather 81-S razor blades (Dominique Dutscher, catalog number: 711164B)
    Manufacturer: Feather Safety Razor, model: 81-S .
  5. Scalpel blades N°10 (Dominique Dutcher, catalog number: 132510 )
  6. Sterile 50 ml sterile Falcon tubes (Corning, catalog number: 430290 )
  7. Sterile Petri dishes, 35 mm (Corning, Falcon®, catalog number: 351008 )
  8. Sterile pipettes for cell culture 5 ml Falcon (Corning, Falcon®, catalog number: 356543 )
  9. Sterile Whatman paper (for the hippocampal slicing process) (GE Healthcare, catalog number: 10347510 )
  10. Sterile PTFE plate 60 x 60 x 5 mm for cerebellum slicing (ePlastics, 0.250” PTFE Sheet 12” x 12”)
  11. Sterile syringe filter with a pore size of 0.22 µm (EMD Millipore, catalog number: SLGV033RS )
  12. Sterile Millicell Cell Culture insert, 30 mm, hydrophilic PTFE, 0.4 µm (EMD Millipore, catalog number: PICM0RG50 )
  13. 96-well, white plate flat clear bottom with lid (Corning, catalog number: 3610 )
  14. Falcon 12-well flat bottom plate (Dominique Dutscher, catalog number: 064023 )
  15. Slide and coverslip
  16. Filtration unit Stericup GP Millipore, pores 0.2 µm (EMD Millipore, catalog number: SCGPU05RE )
  17. Needle (Hamilton Bell, catalog number: 6980 )
  18. Neonate rodent (mouse, rat, hamster) between postnatal day P6 to P10 (males and/or females)
    Note: Based on our experience, the sex of the animals did not affect our results, but this parameter should be considered carefully when working with other viruses than MeV.
  19. Example of virus: recombinant measles virus (IC323 strain) coding for enhanced green fluorescent protein (MeV-EGFP–1.107 pfu/ml)
  20. 70% ethanol
  21. Ketamine hydrochloride (MWI Animal Health, NDC 13985-584-10)
  22. Propidium iodide solution (Sigma-Aldrich, catalog number: P4864 )
  23. Dulbecco’s phosphate buffer saline (DPBS) 1x, w/o calcium/magnesium (Thermo Fisher Scientific, catalog number: 14190094 )
  24. AlarmarBlue® Cell Viability Reagent–Stock solution 10x (Thermo Fisher Scientific, InvitrogenTM, catalog number: DAL1025 )
  25. Anti-Glial Fibrillary Acidic Protein (GFAP) rabbit polyclonal (Agilent Technologies, Dako, catalog number: Z0334 ) used at 1/700 in BPS
  26. Anti-NeuN rabbit polyclonal (EMD Millipore, catalog number: ABN78 ) used at 1/500 in BPS
  27. Anti-calbindin D-28 K rabbit polyclonal (Swant, catalog number: CB38 ) used at 1/700 in BPS
  28. Anti-Iba1 (Wako Pure Chemical Industries, catalog number: 019-19741 ) used at 1/250 in BPS
  29. Anti-olig2 (Oligodendrocyte Lineage Transcription Factor 2) (R&D Systems, catalog number: AF2418 ) used at 1/200 in BPS
  30. Anti-rabbit IgG Fab2 Alexa Fluor® 488 (Cell Signaling Technology, catalog number: 4412S ) used at 1/750 in BPS
  31. Anti-goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A-11055 ) used at 1/750 in BPS
  32. Anti-rabbit IgG Fab2 Alexa Fluor® 555 (Cell Signaling Technology, catalog number: 4413S ) used at 1/750 in BPS
  33. Fluoprep (BioMérieux, catalog number: 75521 )
  34. Opti-MEM reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  35. RNA extraction kit NucleoSpin® RNA (MACHEREY-NAGEL, catalog number: 740955.250 )
  36. RNase Away®, 475 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 7002 )
  37. iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 170-8891 )
  38. Platinum® SYBR® Green qPCR SuperMix-UDG w/ROX (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11744500 )
  39. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-1KG )
  40. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045-1KG )
  41. Hydrochloric acid solution (HCl), 1.0 N, BioReagent, suitable for cell culture (Sigma-Aldrich, catalog number: H9892-100ML )
  42. Recombinant human insulin (Sigma-Aldrich, catalog number: 91077C-100MG )
  43. Minimum Essential Media (MEM), HEPES, GlutaMAXTM Supplement, 500 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 42360081 )
  44. Heat-inactivated horse serum, 100 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 26050070 )
  45. D-glucose cell culture grade 5 g/L (Sigma-Aldrich, catalog number: G7528 )
  46. Kynurenic acid (Sigma-Aldrich, catalog number: K3375-5G )
  47. HEPES 1 M, 100 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  48. Hibernate®-A medium (Thermo Fisher Scientific, GibcoTM, catalog number: A1247501 )
  49. Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
  50. Fetal bovine serum (FBS), 500 ml (Eurobio, catalog number: CVFSVF0001 )
  51. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787 )
  52. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  53. 1 M MgCl2 solution (see Recipes)
  54. 0.1 N NaOH solution (see Recipes)
  55. Human insulin 50 mg/ml (see Recipes)
  56. Organotypic brain culture medium (see Recipes)
  57. 10x kynurenic acid solution (see Recipes)
  58. Dissection medium (see Recipes)
  59. 8% paraformaldehyde (PFA) (see Recipes)
  60. 4% paraformaldehyde (PFA) (see Recipes)
  61. Blocking and permeabilization solution (BPS) (see Recipes)

Equipment

  1. Straight tweezers, type N°5 length 11 cm for removing the skin and the skull (Dominique Dutscher, catalog number: 005092 )
  2. Ice container
  3. 5% CO2 incubator maintained at 37 °C with humidified atmosphere (Thermo Fisher Scientific, Thermo ScientificTM, model: Series 8000 Water-Jacketed , catalog number: 3423)
  4. Biosafety cabinets
    Note: Work in a horizontal flow hood is recommended for the slices preparation and BSL2 vertical flow hood is recommended for the viral infection step with BSL2 pathogens and infection follow-up. However, if the protection glass on the BSL2 cabinet can be maintained up, the slices can be prepared the same way. Based on our experience and even if the sterility is not well preserved under these conditions, the contamination rate remains very low. In any case, the biosafety level has to be adapted depending on the virus considered (BSL2, BSL3 or BSL4) for the viral infection and follow up steps.
  5. Stainless steel dissecting scissors length 11 cm (Dominique Dutscher, catalog number: 005064 )
  6. Beaker, 250 ml
  7. McIlwain tissue chopper (Campden Instruments, model: TC752 )
  8. Pipette bulb (Fisher Scientific, catalog number: 03-448-29 )
  9. Stainless steel dissecting scissors ultra-fine length 12 cm for cutting and removing the skull (Dominique Dutscher, catalog number: 005068 )
  10. Dumont tweezers #5 for the dissection of the brain and the meninges removal, 0.1 x 0.06, Dumoxel (World Precision Instruments, catalog number: 14098 )
  11. Stainless steel forceps rounded ends length 130 mm for holding the brain during the dissection procedure and the slices separation (Dominique Dutscher, catalog number: 442256 )
  12. Lanceolate tip spatula for the midbrain removal (imLab, catalog number: NE010 )
  13. Curved tweezers type N°7 length 11 cm for the midbrain removal and harvesting the slices from the culture insert (Dominique Dutcher, catalog number: 005093 )
  14. P1000 Pipetman (Gilson, catalog number: F123602 )
  15. P20 Pipetman (Gilson, catalog number: F123600 )
  16. KOLLE needle holder for the slices separation (Hamilton Bell, catalog number: 6780 )
  17. Water bath
  18. Widefield fluorescence microscope (ZEISS, model: Axioplan 2 ) with a cooled monochrome camera (Photometrics, model: CoolSNAP HQ2 ) and a fluorescence filter set for propidium iodide (for example: excitation 550-580 nm, emission 600-660 nm)
  19. Tecan Infinite® 200 PRO series plate reader (Tecan, model: M Plex )
  20. Confocal spectral microscope (Leica, model: Leica TCS SP5 )
  21. TPersonal 48 Thermal Cycler (Analytik Jena, Biometra, model: T-Personal 48 , catalog number: 846-050-551)
  22. StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: StepOnePlusTM, catalog number: 4376600 )
  23. Stereomicroscope for dissection (Leica, model: Leica LED2000 )
  24. Fume hood
  25. -20 °C freezer
  26. -80 °C freezer

Software

  1. ImageJ (https://imagej.nih.gov/ij/ [Schneider et al., 2012]) with the plugin ‘Auto Local Threshold’ from Gabriel Landini (http://fiji.sc/Auto_Local_Threshold#Installation)
    Note: Fiji (http://fiji.sc/ [Schindelin et al., 2012]) could be used instead of ImageJ because it bundles the required plugin. Download the Macro file ‘OBC_IP_mortality.ijm’ for the analysis of the propidium iodide staining. Save the file in the ImageJ/plugins folder. ‘OBC IP mortality’ should appear in the Plugins menu.
  2. GraphPad Prism (GraphPad software–https://www.graphpad.com/scientific-software/prism/) and/or R software (https://www.r-project.org/)
  3. StepOnePlus software (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System.html)

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Welsch, J. C., Lionnet, C., Terzian, C., Horvat, B., Gerlier, D. and Mathieu, C. (2017). Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs. Bio-protocol 7(22): e2605. DOI: 10.21769/BioProtoc.2605.

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微生物学 > 微生物-宿主相互作用 > 病毒

神经科学 > 细胞机理 > 细胞分离和培养

细胞生物学 > 细胞分离和培养 > 3D细胞培养 

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Where can this macro be found OBC_IP_mortality.ijm?
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