搜索

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   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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), Neurotropic viruses(嗜神经病毒), CNS infection(中枢神经系统感染), Brain viral dissemination(脑病毒传播), Antiviral molecule screening(抗病毒分子筛选)

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

  1. Culture plate preparation
    1. The day before slicing the brain explants, prepare 6-well culture plates by adding 1 ml of organotypic culture medium (see Recipes) into each well and place the culture inserts on the top using straight tweezers in order to reverse the hydrophobicity of PTFE and to allow a good feeding of the slices (Table 1).

      Table 1. Time duration recommended for each step


    2. Incubate the plate overnight at 37 °C in a humidified 5% CO2 atmosphere.

  2. Dissection preparation
    1. Decontaminate the walls and floor of the biosafety cabinet using 70% ethanol in water and UV irradiate for 15 min if available.
    2. Before the dissection procedure, sterilize all equipment, i.e., scissors, tweezers, PTFE plate by autoclaving and put them under the biosafety cabinet. Prepare a beaker half-filled with 70% ethanol in water in order to decontaminate the dissection material during the procedure (Table 1).
    3. Clean the tissue chopper, the razor blade and the stereomicroscope with 70% ethanol, ensure that the slicing platform is correctly decontaminated and then let everything dry. Mount the razor blade on the tissue chopper using the screw and nut and place the instruments under the biosafety cabinet and spray again with ethanol (Table 1).
    4. Place a sterilized container filled with ice under the biosafety cabinet.
    5. Prepare the dissection medium (see Recipes) as described in the ‘Recipes’ section using a sterile 50 ml conical tube. Put 6 ml of medium in the bottom of the 35 mm Petri dishes (1 Petri dish for the hippocampus, 1 Petri dish for the cerebellum) and keep the cover for the dissection. The dissection medium must be kept on ice and the entire dissection procedure must be performed at 4 °C (cold medium) to limit tissue damage and cell degeneration.
    6. Fill a Petri dish cover with 5 ml of dissection medium in order to have cold fresh medium throughout the slicing procedure.
    7. Remove the conical extremity of a 5 ml plastic pipette with scalpel blade and insert the top part with the carded cotton into the pipette bulb (defined as 5 ml truncated pipette).

  3. Dissection and slicing procedure
    Notes:
    1. Refer to the Table 1 for the duration of each step.
    2. Table 2 summarizes the troubleshooting and the proposed solution.
    1. Gently spray the neck and the head of the animal with 70% ethanol and proceed rapidly to the following step.
    2. We recommend using 6 to 10 days-old animals. Since cervical dislocation is not suitable for suckling animals, proceed with decapitation using scissors. This action must be performed by skilled accredited personnel. At this stage, it is necessary to work as quickly as possible.
      Notes:
      1. When animals give rise to a small number of babies, the suckling animals could be much bigger and the decapitation more difficult. In order to prevent potential pain and allow a more comfortable procedure for the experimenter, an anesthetic can be used. We recommend the use of ketamine at a dose of 150 mg/kg, which is an efficient activation-dependent channel blocker of N-methyl-d-aspartate (NMDA) receptors leading to a reduction of the excitotoxicity and prevent neuronal death. In combination with the kynurenic acid contained in the dissection medium, ketamine provides good neuroprotection against excitotoxicity.
      2. Brains from younger animals (between P3 to P6) are more difficult to cut into regular parallel slices because they are softer. In addition, numerous non-fully differentiated cells endowed with migration properties may alter the reproducibility of infection experiments. Brains from older animals (beyond P10 to P12) are poorly suitable because of increased cell death that can add bias to the analyses.
      3. This procedure has to follow the local animal ethics protocols and has to mention the species and strains used. Any change in the species has to be amended and validated on the ethics protocol prior to any experimentation.
    3. Hold the head between the thumb and index, and remove as much skin and flesh as possible using ultra-fine scissors.
    4. Cut the skull in a rostral way from the cavity where the top part of the spinal cord is visible to the cavity containing the olfactory bulbs using the tip of ultra-fine scissors.
    5. Remove the brain from the head by inserting the tip of the Dumont tweezers under the olfactory bulbs and lift gently until detachment from the cranium.
    6. Put the brain directly in cold dissecting medium in the 35-mm dish. At this stage, it is necessary to isolate and dissect the brain structure in dissection medium to maintain cell viability.
    7. Separate the cerebellum (slicing process–from steps C16 to C20) from the brain using a scalpel blade (Figure 1A) and conserve it in cold dissection medium during the preparation of the hippocampal slices as follows (dissecting and slicing process–from steps C8 to C15).
    8. Separate the two hemispheres along the midline (Figure 1B) and remove the midbrains (Figure 1C), holding the half brain with forceps and using the lanceolate tip spatula or alternatively a curved tweezers (with an appropriate training approximately 1 min is needed per brain). At this time, the hippocampus is visible (arrow head–Figure 1D).
    9. With a scalpel blade cut and remove the rostral part containing the olfactory bulbs (Figure 1D–about 10 sec per hemisphere).
    10. Remove the meninges using the Dumont tweezers #5. The hippocampus and cortex are ready for slicing process.
    11. Put the hippocampus face down in rostral orientation on Whatman paper (Figure 1E–about 1 min needed).
    12. Place it onto the slicing platform of the McIlwain tissue chopper (see Figure 1–arrow 2–about 1 min needed).
    13. With the 5 ml truncated pipette add one drop of dissection medium to prevent the tissue from sticking to the razor blade.
    14. Proceed rapidly to the slicing by turning on the apparatus (Figure 1E–30 sec required to slice two hippocampi).
      Note: All slicing parameters, i.e., thickness (typically between 200 and 500 µm), blade force (usually maximum force) and blade speed (typically one slice per second) should be established in preliminary tests.
    15. Remove the Whatman paper from the slicing platform and abundantly add cold dissection media on the tissue.
    16. With the lanceolate tip spatula, remove the tissue from the Whatman paper by gently sliding the tip of the spatula between the tissue and the paper.
    17. Place the tissue back in Petri dish containing cold dissection medium during the dissection of the cerebellum.
    18. In cold dissection medium and under the stereomicroscope, hold the cerebellum using forceps and gently remove meninges using Dumont tweezers #5, the inferior colliculus and brain stem (Figure 1B’).
    19. Place the cerebellum on the PTFE plate following a sagittal plan compared to the razor blade (Figure 1C’).
      Note: The rostro-caudal plane for the slicing process is not critical and the cerebellum can be sliced with the rostral or the caudal part facing the experimenter when placed on the slicing platform. However, in our experiment the caudal part of the cerebellum, as shown in Figure 1A and 1B’, was always facing the experimenter during the slicing procedure.
    20. With a P1000 pipette remove the entire medium in order to improve the cerebellum adhesion to the PTFE plate.
    21. Proceed to the slicing as described for the hippocampus (Figure 1D’).

      Table 2. Troubleshooting table



      Figure 1. Preparation of OBC from suckling rodent brain between P6 to P10. Hippocampal slice preparation (A-F, left panel). A. Collect the brain after decapitation and separate the cerebellum with a scalpel blade following the dotted line. B. Separate the two hemispheres using a scalpel blade. C. Remove the midbrain to expose the hippocampus area. D. Remove the rostral part of the brain (containing the olfactory bulbs), in order to visualize the hippocampus (black arrow head). E. Put and orientate the dissected brain in the antero-posterior axis onto the slicing platform (2) of a tissue chopper (1) with perpendicular cut plan to the razor blade (3) and cut following dotted line. F. Dissociate and transfer slices to millicell insert culture systems relying on the OBC medium. Cerebellum slice preparation (A, B’-D’, F, right panel). A. Separate the cerebellum from the rest of the brain. B’. Remove the inferior colliculi (IC) and the brain stem (BS) from the cerebellum. C’. Put cerebellum on a Teflon plate according to the antero-posterior axis, and transfer to the slicing platform of the tissue chopper. D’. Cut sagittally following the dotted line. F. Transfer in a Petri dish containing dissection medium, dissociate carefully the slices from each other and plate them on the insert culture systems.

    22. Carefully transfer the tissue into a Petri dish containing cold dissection medium.
    23. Under the stereomicroscope, separate carefully the hippocampal and/or cerebellum slices using forceps to hold the tissue and a needle mounted on KOLLE needle holder in cold dissection medium. With a good focus, mainly on the top of the tissue, the spaces between slices are easy to visualize which helps to precisely separate them (the slices separation takes 10 to 15 min for the hippocampus and only 5 min for the cerebellum).
      Note: Ensure the light source of the stereomicroscope is cold (i.e., optic fiber) to avoid cell-stress induced by overwarming of the medium during the slice dissociation.
    24. Select slices for the culture under the stereomicroscope.
      Note: The hippocampus should contain the CA (Cornu Ammonis) region and the dentate gyrus. The cerebellum should contain the deep nucleus and the different cellular layer such as the Purkinje Cell (PC) layer or the molecular layer (ML) should be visible. Refer to a brain Atlas for more details such as the Allen Brain Atlas.
    25. Plate a maximum of 4 hippocampal or 5 cerebellum slices per Millipore cell culture insert membrane using the 5 ml truncated pipette mounted on a pipette bulb. To favor the appropriate feeding and oxygenation of slices, remove as much dissection medium as possible around each brain explant after plating on the insert and incubate the slices overnight at 37 °C, 5% CO2 in a humidified atmosphere.

  4. Culture procedure
    Notes:
    1. Change the medium the day after the slicing procedure, and then every 2-3 days.
    2. Refer to the Table 1 for the duration of each step.
    1. Hold the edge of the insert from the 6-well plate with tweezers and remove old medium with a P1000 Pipetman.
    2. Put the insert back in place in the 6-well plate and add 1 ml of fresh culture medium pre-warmed at 37 °C in the water bath to avoid any thermal shock.
    3. Make sure that no air bubble is trapped between the PTFE membrane and the medium. If so, bend the 6-well plate at an angle of ~45° and gently shake it to allow the air bubbles to reach the edge of the culture insert.

  5. Viability of cultured brain explants: Cellular metabolism/mortality assessment–Alamarblue® and propidium iodide staining
    Notes:
    1. Prior to infection studies, it is extremely important to ensure that the brain explant is healthy ex vivo and determine the best time window during which the slices can be used.
    2. Based on our experience, the viability evolution is reproducible if the OBC preparation is kept identical as described in this protocol and can be assessed once for all using three independent batches of slices, for a total of 15 slices analysis per time point. However, we recommend periodically assessing the viability, at least with one of the two proposed approach, in order to make sure that the procedure is still correctly set up.
    1. Detach slices from PTFE membrane by flushing culture medium gently on the edge of the explant. 
    2. Gently transfer the tissue into a 96-well plate using a truncated 5 ml pipette.
      Note: After lengthy culture time (4 to 7 days), slices might be too adhesive to be detached without damages. The PTFE membrane of the inserts is suitable for fluorescence microscopy and does not affect the observation of the IP staining, the reading of the Alamarblue assay and the confocal analysis (no autofluoresence), hence we recommend using this type of insert to support the OBC slices.

    Propidium iodide staining

    1. In a 96-well plate, immerse slices in 200 µl of propidium iodide solution at 5 µg/ml in culture medium and incubate the plate for 45 min at 37 °C, 5% CO2 in a humidified atmosphere.
      Note: Propidium iodide enters into dying cells leading to a red-fluorescent nuclear staining. The surface density of propidium iodide labeled nuclei over the total tissue surface is used to monitor cell death and provides necessary quantification for statistical analysis (see Data analysis section). Note that there is no need to wash the slices prior to the staining.
    2. Remove the propidium iodide solution and wash slices 3 times with 200 µl of 1x DPBS (37 °C).
    3. Transfer slices into a 12-well plate and fix them with 1 ml of 4% paraformaldehyde solution (see Recipes) for 30 min at room temperature.
    4. Mount gently slices between a slide and coverslip. Make sure to not crush the tissues, especially on thick slice (300 and 500 µm).
    5. Acquire images with a widefield fluorescence microscope, at least 6 randomized fields per slice and 5 slices per time point. Use a 5x objective, to acquire large fields of view.
    6. Analyze the images in ImageJ with the provided Macro (i.e., script for ImageJ). Run the Macro (click on Plugins>‘OBC IP mortality’) and follow the instructions: first, choose the image to analyze. It will be opened in ImageJ with an enhanced display (see Figure 2). Click on the areas outside the slice (like the region outlined in yellow in Figure 2). If the region is too small or too large, the tolerance of the wand tool can be adjusted: double-click on the wand tool in the ImageJ menu (in the red frame in Figure 2), and adjust the value of the tolerance (which is 200 by default). When the region is correctly outlined, press the T key to save it in the ROI manager. Repeat for all the regions in the image. At the end, the windows should look like Figure 3.


      Figure 2. Analysis of the propidium iodide staining in ImageJ with the Macro. The windows can be arranged at the user’s convenience. The original image is on the left, its duplicate with an enhanced display on the right. The instructions are at the bottom. The tolerance of the wand tool can be adjusted by double-clicking in the red square.


      Figure 3. Second step of the propidium iodide staining analysis. The regions are saved in the ROI manager on the right, in the green frame. The user can click on ‘OK’ in the red frame.

    7. Click on ‘OK’ (see the red square in Figure 3). The Macro will do an automatic segmentation of the propidium iodide staining (with the ‘Auto Local Threshold’ plugin https://imagej.net/ Auto_Local_Threshold#Bernsen), and measure the area of this staining, and the area of the slice.
    8. When the process is finished, the propidium iodide area is displayed as a red overlay on the image and the slice area is surrounded in yellow to visually check the results of the segmentation (see Figure 4). The red overlay can be activated/deactivated by checking/unchecking the Channel 1 box in the ‘Channels’ window (see Figure 4). The numerical results (the propidium iodide area ‘Area’ and the whole slice area ‘Slice area’ in pixels, and their ratio in %) are displayed in a spread sheet which can be saved (for more details see Data analysis section point 3). Several images can be analyzed in batch and the results saved in the same file: close only the previous image and run again the Macro in the same way from step E8. The results will be added at the bottom of the spread sheet.


      Figure 4. Result of the propidium iodide staining analysis. The area of the staining is displayed in red. The area of the slice is outlined in yellow. On the bottom right, the results table can be saved with its menu File>Save As.

    Alamarblue®

    1. In a 96-well white plate, immerse slices in 200 µl of 1x Alamarblue® solution in culture medium.
    2. Incubate the plate for 2 h at 37 °C, 5% CO2 in a humidified atmosphere.
      Note: The Alamarblue® reagent becomes red proportionally to the cellular metabolism of the slices reflecting the proportion of active cells and thus the global OBC health state.
    3. Read the fluorescence emission at 580-610 nm according to the manufacturer’s protocol using a Tecan Infinite® 200 PRO series microplate reader.
    4. To analyze metabolic activity, only the animal and slice (random) factors are evaluated. We applied the Kruskal-Wallis tests since the data showed violation of the assumptions of ANOVA.
    5. Variation of mortality (Figures 5A and 5C) and metabolic activity (Figures 5B and 5D) across times (D0, D1, and D7) were analyzed using the Kruskal-Wallis test. For each day, the coefficients of variation (the ratio of standard deviation and mean) were compared between mice and hamsters using the Wilcoxon test (for more details see Data analysis section point 3) (Figure 6).
      Note: Based on our experience, we would not recommend working with OBC when one of these two parameters exceeds or varies more than 20% during the first two days since it may greatly impact the maintenance of the cell population in the slices and their chemokine response and thus the reproducibility of the results.


      Figure 5. Study of mouse and hamster OBC viability over 14 days of culture. A. Mouse or C. hamster (3 animals/group, at least 4 slices of each substructure/animal) hippocampus and cerebellum slice mortality were evaluated by propidium iodide staining and analyzed with an Axioplan Imager fluorescence microscope. B. Mouse or D. hamster (n = 3), at least 3 slices of each substructure/animal) hippocampus and cerebellum slice metabolism activity were assessed by incubation with Alamarblue reagent kit and analyzed using an Infinite 200 PRO Tecan microplate reader. The graphics show the evolution of cellular metabolism (reflecting health) over the time of the culture.


      Figure 6. Coefficients of variation analysis. Coefficients of Variation (CV) for mortality and metabolic activity were computed at each day, and globally compared between hamster and mice using the Wilcoxon test. There is no difference statistically significant between mice and hamster (P-value = 0.7987).

  6. Immunostaining of brain explants
    Note: As preliminary test, we recommend to assess the maintenance of the major cell population (Figure 7) as well as the structure preservation during the culture (Figure 8) by immunostaining before starting infection studies to exclude any variation related to the experimenter.
    1. Detach, place the slices in 12-well plates using the 5 ml truncated pipette and fix them with PFA at room temperature as described above.
      Note: In the case of infected slices, the PFA fixation can be performed directly on the culture insert. The culture medium has to be removed, and 1 ml of PFA is added below and above the culture insert in order to completely cover the slices.
    2. Proceed to the blocking and permeabilization of tissue with 1 ml of the blocking and permeabilization solution (BPS) (see Recipes) for at least 45 min at room temperature under gentle agitation.
    3. Remove the BPS and add the primary antibody (Ab) (i.e., astrocytic marker GFAP, neuronal marker NeuN or CB-28K–microglia marker Iba1 or oligodendrocyte marker Olig-2–see Figure 7) diluted in BPS and incubate for 2 h at room temperature (also possible ON at 4 °C).
      Notes:
      1. Ensure that the Ab can bind the epitope in the presence of Triton X-100 present in the BPS. If not, remove the Triton X-100 at this step and wash slices 3 times in a solution of 1x DPBS/4% FBS.
      2. By using a 48-well plate, the volume of the staining solution can be as minimal as 150 µl.
      3. For example, to highlight the hippocampal and the cerebellum structures, use respectively the neuronal marker NeuN (Figure 8A) or the Purkinje cells marker CB-28K (Figure 8B). The CB-28K decreased staining is mostly due to the Purkinje cells loss and the reorganization of the neurons into the slice. However, the apparent lower staining of the hamster CB-28K at day 7, is due to the acquisition of this image. Indeed, all the left panels of this figure are a tile reconstitution coupled with 100 µm z. Due to the irregular shape of the tissue and the mounting process, it can be difficult to keep a homogenous staining even if the exposure time is identical, since the density of the cells throughout the slice can be different.
    4. Remove the Ab and wash 3 times each for 10 min with 1x DPBS.
    5. Dilute the secondary Ab in BPS and incubate slices for at least 2 h at room temperature.
    6. Repeat the wash step.
    7. Mount the slices between slide and coverslip using fluoprep mounting medium.
      Note: The slices are still quite thick at this step; thus the immunostainings imaging have to be performed using confocal microscopy.


      Figure 7. The major cell types of the brain are present and easily detectable by immunofluorescence. A. The characteristic Purkinje cells (PC) of the cerebellum (GABAergic neurons), have been detected in mouse cerebellum slice with the Calbindin-28k marker at DIV0. The arrow head shows the soma of the PC; the arrow shows the dendritic tree of the PC and Ax designate the regrouping PC axons. B. The mouse hippocampal neurons have been stained at DIV0 using the specific neuron nuclear marker NeuN to reveal the internal organization of the hippocampus. C. The mouse hippocampal astrocytes have been stained using the specific Glial Fibrillary Astrocyte Protein (GFAP) marker at DIV0. D. The microglia have been stained at DIV7 using the specific marker Iba1 in mouse hippocampus. E. The oligodendrocytes have been stained at DIV5 using the Olig2 specific nuclear marker in mouse hippocampus. CA1: Cornu Ammonis area 1, CA3: Cornu Ammonis area 3, DG: Dentate Gyrus.


      Figure 8. Study of morphological rearrangements and kinetic of mouse or hamster OBC viability over 7 days of culture. A. Hippocampus slices from mice or hamsters were stained with anti-NeuN antibody (neurons marker) at day 0 and day 7 of culture. Pictures show a conserved general organization of the hippocampus over at least 7 days, including the Ammon’s horn (Cornu Ammonis CA) regions and dentate gyrus (DG), which showed only limited loss of cell density (white arrows) possibly related to neuron death. B. Cerebellum slices from mice or hamsters were stained with Calbindin 28K (CB 28K–Purkinje Cells marker) at day 0 and day 7 of culture. Arrows in the pictures show neuronal loss and arrow heads show an interruption of the Purkinje cells layer. Nuclei were counterstained with DAPI. Images taken at the indicated times of culture with a Leica SP5 confocal microscope show morphological evolution within slices at low (reconstructed tile–on left column for each day) or high magnification on right column for each day (20x objective in left panel of each days and 40x objective in right panel of each days corresponding to the white square) over the culture duration.

  7. Infection procedure of brain explants
    1. Dilute the virus stock in Opti-MEM medium in order to infect cultured slices (in 6-well plates) with a defined number of plaque forming units (pfu). For example, with measles virus (MeV-EGFP) 104 to 4 x 104 pfu are used. For the mock control slices, apply the same volume of Opti-MEM as for the infection.
      Note: The dose necessary for the infection should be determined experimentally for each virus/brain structure/animal model combination.
    2. Using a P20 pipette, deposit gently 2 to 10 µl of diluted viral stock on the top of the slices, and make sure that the inoculum is distributed homogeneously.
      Note: A micro-injector (with adequate equipment) can be used at this step if the study requires a single site of entry for virus dissemination studies (Ehrengruber et al., 2002).
    3. Incubate plates at 37 °C, 5% CO2 in humidified atmosphere.
    4. In case of EGFP expressing virus, the fluorescence resulting of the viral replication can be monitored using a fluorescence microscope (Figure 9).


      Figure 9. OBC infection with a recombinant measles virus expressing the EGFP. Rat cerebellum slices have been infected with 2 x 104 pfu of MeV IC323-EGFP the day of the slice preparation (DIV 0). MeV-EGFP fluorescence was observed after 5 days using an inverted fluorescence microscope (ZEISS) and camera (AxioCam, ZEISS) at low (A) or high magnification (B).

  8. Monitoring of cellular or viral gene expression by RT-qPCR
    1. Using curved tweezers, take the slice to be analyzed out of the culture insert and place it in lysis buffer RA1-10% 2-mercaptoethanol according to the manufacturer’s recommendations (RNA extraction kit–Macherey Nagel).
    2. Between treating each slice, remove RNase with RNase Away solution.
    3. Decontaminate tweezers with absolute ethanol.
    4. Rinse with RNase free water.
    5. Freeze the lysates at -80 °C and then proceed to the RNA isolation using the RNA extraction kit according to Macherey-Nagel recommendations.
    6. Prepare cDNA using iScriptTM cDNA Synthesis Kit using a TPersonal 48 Thermal Cycler.
    7. Perform the quantitative PCR on targeted cDNA, i.e., viral or cellular, using Platinum® SYBR® Green qPCR SuperMix-UDG w/ROX Kit according to manufacturer’s recommendations and StepOnePlusTM Real-Time PCR System.
    8. All results should be normalized to mRNA from a housekeeping gene such as GAPDH and analyzed as detailed elsewhere (Welsch et al., 2013).

Data analysis

  1. Analysis of immunostaining requires the use of a confocal microscope, since the OBC are relatively thick usually between 150 to 500 µm (Figures 7 and 8).
  2. For experimental design, analysis of cellular or viral gene expression requires at least 12 slices per condition and/or time point coming from separated animals, i.e., 2 slices from 3 different donors (n = 6) repeated at least 2 times (n = 12). For immunofluorescence experiment, we recommend using 1 slices from 3 different donors repeated at least 3 times (n = 9).
    Note: Regarding the viability and the structures’ evolution analyses, our results showed that the OBC from mice or hamsters (also true for rats–data not shown) lead to a similar evolution of these parameters, indicating that our protocol can be used with the main laboratory rodent species and confirming the versatility of our method.
  3. For the viability statistical analysis, the mortality and metabolic activity variables should be analyzed separately (Table 3). Concerning mortality, several factors are evaluated at different days of culture (D0, D1 and D7): animal, slices from the same animal and repeats for the same slice (i.e., several images from the same slice). Hence, the experiment has a hierarchical structure, and the model is written as:

    Mij = µ + Ai + Sj(i) + eij

    where, Mij is the mortality value of the jth slice from the ith animal, µ the general mean, Ai the effect of the ith animal, Sj(i) the effect of the jth slice from the ith animal, and eij the random effect.
    These various effects are statistically tested using the nested analysis of variance (nested ANOVA) implemented in R software as follows:

    anova(lm(formula = Mortality ~ Animal + slice %in% Animal + image %in% slice, data = file.name)).

    This analysis is performed for each day of culture (D0, D1 and D7).
    Caution: Assumptions for ANOVA should be checked (normality of the dependent variable, homoscedasticity, graphical diagnostic procedures like residuals vs. predicted values plot and normal QQ-plot of residuals). If not, the non-parametric Friedman test should be used for comparison of image differences within a slice, and slice differences within an animal, and the non-parametric Kruskal-Wallis test for animal differences.
  4. For the statistical analysis, we used GraphPad Prism and R software. Alternatively, a website called ‘Handbook of Biological Statistics’ (John H. McDonald) detailing the method and providing spreadsheets and advices for 2 to 4-level nested ANOVA is accessible at this address: http://www.biostathandbook.com/nestedanova.html. For example, if you want to analyze the time effect, use the spreadsheet dedicated to 4-level nested ANOVA and fill the column as follow: Day/animal/slice/image/fluorescence value respectively representing group/subgroup/ subsubgroup/subsubsubgroup/observation. For the analysis of the animal effect you can perform the 3-level nested ANOVA and fill the column as follow: animal/slice/image/fluorescence value respectively representing group/ subgroup/ subsubgroup/ observation (Note again that you need to respect the ANOVA assumption, and if not simply apply a Kruskall-Wallis test).

    Table 3. Statistical analysis of OBC viability

    Note: The time, animal, slice and image effects were statistically estimated according to the statistical analyses described above. P-values are reported for each test performed with ns (non-significant P > 0.05), ** P < 0.01, *** P < 0.001.

Recipes

Note: All solutions must be sterilized using a Stericup filtration unit for large volumes (organotypic culture medium, dissection media and 10x kynurenic acid) or with a syringe filter with 0.22 µm pore size.

  1. 1 M MgCl2 (1 L)
    1. Weight 95.21 g of MgCl2
    2. Bring to 1 L with distilled water
    3. Keep at room temperature up to 1 year
  2. 0.1 N NaOH (500 ml)
    1. Weight 1.99 g of NaOH
    2. Bring to 500 ml with distilled water
    3. Keep at room temperature up to 1 year
  3. Insulin solution at 50 mg/ml (2 ml)
    1. Reconstitute the totality of the vial in 2 ml of 0.005 N HCl solution
    2. Aliquot (1 ml) and store at -20 °C
  4. Organotypic brain culture medium (500 ml)
    Add all the components:
    375 ml of MEM GlutaMAX
    125 ml of heat-inactivated horse serum (store the serum at -20 °C)
    2.5 g of D-glucose
    1 ml of human insulin at 50 mg/ml (final concentration 0.1 mg/L)
    Filter to sterilize (0.22 μm) and store at 4 °C for up to 2 weeks
    Note: Addition of antibiotics is not necessary when the procedure is kept aseptic, but may be helpful to prevent contamination. When using penicillin, the concentration should be kept below 0.08 mM final concentration to avoid potential interference with synaptic activity (see Table 2).
  5. 10x kynurenic acid solution (concentrated excitotoxicity inhibitor solution) (200 ml)
    1. Dissolve 378 mg of kynurenic in 170 ml of H2O
    2. Add 20 ml of 1 M MgCl2
    3. Adjust pH at 7.4 using 0.1 N NaOH
    4. Add 1 ml of HEPES
    5. Adjust volume to 200 ml
    6. Filter to sterilize, protect from light and keep up to 2 weeks at room temperature
  6. Dissection medium (Hibernate®-A/5 g/L D-Glucose/1x Kynurenic acid) (50 ml)
    1. Prepare 500 ml of Hibernate®-A supplemented with glucose by adding 2.5 g of D-glucose
    2. Filter sterilize using a Stericup filtration unit and store at 4 °C up to 6 months
    3. The day of dissection, mix 45 ml of Hibernate®-A/5 g/L D-Glucose with 5 ml of 10x kynurenic acid
    4. Keep on ice during the dissection
  7. 8% paraformaldehyde (PFA) (100 ml)
    In a fume hood:
    1. Weigh 8 g of crystalline PFA in a beaker and add 100 ml of 1x DPBS
    2. Heat the solution until the powder is fully dissolved to 65 °C (not higher as PFA will degrade)
    3. Aliquot and store at -20 °C
    4. Dilute to 4% with 1x DPBS (make fresh prior to use)
  8. Blocking and permeabilization solution (BPS) (1x DPBS/0.3% Triton X-100/4% FBS) (50 ml)
    Add all the components:
    47.85 ml of 1x DPBS
    2 ml of FBS
    150 µl of Triton X-100
    Vortex and incubate at 37 °C in a water bath in order to help dissolving Triton X-100 for 30 min
    Store at 4 °C up to 2 weeks
    Note: Ensure that no bacteria or fungus proliferated in solution prior to use, and if so prepare a new batch.

Acknowledgments

This work was supported by French ANR NITRODEP grant (project ANR-13-PDOC-0010-01) (http://www.agence-nationale-recherche.fr) and LABEX ECOFECT (ANR-11-LABX-0048) of Lyon University, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). We are grateful to Dr. Isabelle Dussart (Université Pierre et Marie Curie, Université Paris 06, CNRS - UMR 7102, 75005 Paris, France) and Dr. Helene Clot-Faybesse (INMED, INSERM U29, Université de la Méditerranée, Marseille, France) for precious advice on OBC cultures. This protocol was adapted from Stoppini et al. (1991) with minor changes. The authors declare no conflicts or competing of interests.

References

  1. Bloyet, L. M., Welsch, J., Enchery, F., Mathieu, C., de Breyne, S., Horvat, B., Grigorov, B. and Gerlier, D. (2016). Requirement of HSP90 chaperoning in addition to phosphoprotein for folding but not for supporting enzymatic activities of measles and Nipah virus L polymerases. J Virol 90:JVI.00602-16.
  2. Bornstein, M. B. and Murray, M. R. (1958). Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of new-born rat and kitten cerebellum. J Biophys Biochem Cytol 4(5): 499-504.
  3. Braun, E., Zimmerman, T., Hur, T. B., Reinhartz, E., Fellig, Y., Panet, A. and Steiner, I. (2006). Neurotropism of herpes simplex virus type 1 in brain organ cultures. J Gen Virol 87(Pt 10): 2827-2837.
  4. Ehrengruber, M. U., Ehler, E., Billeter, M. A. and Naim, H. Y. (2002). Measles virus spreads in rat hippocampal neurons by cell-to-cell contact and in a polarized fashion. J Virol 76(11): 5720-5728.
  5. Humpel, C. (2015). Organotypic brain slice cultures: A review. Neuroscience 305: 86-98.
  6. Kim, H., Kim, E., Park, M., Lee, E. and Namkoong, K. (2013). Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology. Prog Neuropsychopharmacol Biol Psychiatry 41: 36-43.
  7. Laksono, B. M., de Vries, R. D., McQuaid, S., Duprex, W. P. and de Swart, R. L. (2016). Measles virus host invasion and pathogenesis. Viruses 8(8).
  8. Lossi, L., Alasia, S., Salio, C. and Merighi, A. (2009). Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog Neurobiol 88(4): 221-245.
  9. Ludlow, M., Kortekaas, J., Herden, C., Hoffmann, B., Tappe, D., Trebst, C., Griffin, D. E., Brindle, H. E., Solomon, T., Brown, A. S., van Riel, D., Wolthers, K. C., Pajkrt, D., Wohlsein, P., Martina, B. E. E., Baumgartner, W., Verjans, G. M. and Osterhaus, A. (2016). Neurotropic virus infections as the cause of immediate and delayed neuropathology. Acta Neuropathol 131(2): 159-184.
  10. Mayer, D., Fischer, H., Schneider, U., Heimrich, B. and Schwemmle, M. (2005). Borna disease virus replication in organotypic hippocampal slice cultures from rats results in selective damage of dentate granule cells. J Virol 79(18): 11716-11723.
  11. Olival, K. J. and Daszak, P. (2005). The ecology of emerging neurotropic viruses. J Neurovirol 11(5): 441-446.
  12. Reuter, D. and Schneider-Schaulies, J. (2010). Measles virus infection of the CNS: human disease, animal models, and approaches to therapy. Med Microbiol Immunol 199(3): 261-271.
  13. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  14. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  15. Stoppini, L., Buchs, P. A. and Muller, D. (1991). A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37: 173-182.
  16. Stubblefield Park, S. R., Widness, M., Levine, A. D. and Patterson, C. E. (2011). T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue. J Virol 85:3664-76.
  17. Swanson, P. A., 2nd and McGavern, D. B. (2015). Viral diseases of the central nervous system. Curr Opin Virol 11: 44-54.
  18. Taylor, L. H., Latham, S. M. and Woolhouse, M. E. (2001). Risk factors for human disease emergence. Philos Trans R Soc Lond B Biol Sci 356(1411): 983-989.
  19. Welsch, J. C., Talekar, A., Mathieu, C., Pessi, A., Moscona, A., Horvat, B. and Porotto, M. (2013). Fatal measles virus infection prevented by brain-penetrant fusion inhibitors. J Virol 87(24): 13785-13794.

简介

根据世界卫生组织(WHO)的统计,至少有50%的新发病毒具有致病性,可感染中枢神经系统(CNS),并诱发脑炎和其他神经系统疾病(Taylor et al。 >,2001; Olival和Daszak,2005)。虽然神经系统疾病逐渐被记录下来,但涉及病毒感染和在CNS内传播的潜在细胞和分子机制仍然知之甚少(Swanson and McGavern,2015; Ludlow等人,2016)。例如,麻疹病毒(MeV)可以感染神经细胞,并在原发感染后几个月至数年导致持续的脑感染,导致致死性脑炎,而没有可用的治疗(Reuter和Schneider-Schaulies,2010; Laksono等人。,2016)。器官型脑文化(OBC)是病毒学领域的一个合适的模型,以更好地理解中枢神经系统感染。实际上,它不仅可以研究中枢神经系统内嗜神经病毒的感染和传播,而且还可以作为创新性抗病毒策略或分子的筛选模型,如我们最近发表的有关融合抑制肽和HSP90伴侣蛋白活性抑制剂的研究, 17-DMAG(Welsch等人,2013; Bloyet等人,2016)。基于我们以前的工作,我们在这里提出了一种适用于基于小型啮齿动物模型的病毒研究的海马和小脑OBC的优化方法,包括小鼠,大鼠以及出生后阶段的仓鼠,在P6到P10之间。我们特别考虑了切片过程对组织的压力和随后的细胞反应,这对于在感染状态下使用之前充分表征模型是必不可少的。有了这些知识,我们提出了一个强调要求的协议,包括切片参数的潜在故障拍摄,考虑我们根据所研究的结构和动物观察到的变化。这个框架应该有助于使用OBC更好地研究嗜神经病毒。
【背景】自1958年以来,神经生物学家在过去二十年来在神经发育,神经退行性疾病或神经药理学领域的使用方面不断地发展了器官型脑文化(OBC)(Bornstein和Murray,1958; Kim等人, em>,2013; Humpel,2015)。相反,尽管该模型具有优势,但是已经发表了几乎没有关于病毒感染,向性或传播的研究(Mayer等人,2005; Braun等人, 2006; Stubblefield Park等,2011)。事实上,使用OBC进行的实验本身比传统的细胞原代培养物(即,纯化的神经元或分离的脑培养物)更复杂。然而,这种方法的优点在于可以将主要细胞类型保持在保存的三维组织结构中,从而允许在更多的生理环境中实时地研究贯穿脑结构和细胞亚群的病毒入侵,并且没有外周的影响免疫系统。此外,由于保持了组织的细胞组成,包括神经元,少突胶质细胞,小神经胶质细胞和星形胶质细胞,有可能在病毒感染期间评估和破译每个细胞群的参与和反应(Lossi等人, ,2009)。与体内动物实验相比,该模型还具有减少动物有效负荷的优势,该实验完全符合机构动物护理和使用委员会(IACUC)对生命科学中动物使用的建议和规定。实际上,每个结构可以产生至少10到15个切片,因此它允许扩大每只动物的测试条件的数目。此外,实施所需的大部分设备很容易获得,或已经在实验室中使用对神经病毒感兴趣的组织培养方法。该方案详细描述了从海马或小脑获得的培养的啮齿动物脑切片的制备,其生存力的评估,脑细胞类型的分析,形态重排和在一周培养期间的动力学。最后,该协议提供了利用OBC来研究啮齿动物外植体中麻疹病毒(MeV)的病毒脑感染的实例。

关键字:器官型脑培养, 嗜神经病毒, 中枢神经系统感染, 脑病毒传播, 抗病毒分子筛选

材料和试剂

  1. 无菌吸头,1,000μl(Corning,目录号:9032)
  2. 无菌过滤枪头,10μl(Corning,目录号:4807)
  3. 无菌Falcon 6孔平底板(Corning,Falcon ,目录号:353046)
  4. 羽毛81-S剃刀刀片(Dominique Dutscher,目录号:711164B)
    制造商:羽毛安全剃刀,型号:81-S。
  5. 手术刀片N°10(Dominique Dutcher,产品目录号:132510)
  6. 无菌50毫升无菌猎鹰管(康宁,目录号:430290)
  7. 无菌培养皿,35毫米(Corning,Falcon ,产品目录号:351008)
  8. 用于细胞培养的无菌移液管5ml Falcon(Corning,Falcon ,目录号:356543)
  9. 无菌Whatman纸张(用于海马切片过程)(GE Healthcare,目录号:10347510)
  10. 无菌PTFE板60 x 60 x 5 mm用于小脑切片(ePlastics,0.250“PTFE板12”x 12“)
  11. 孔径为0.22μm的无菌注射器过滤器(EMD Millipore,目录号:SLGV033RS)
  12. 无菌Millicell细胞培养插入物,30mm,亲水PTFE,0.4μm(EMD Millipore,目录号:PICM0RG50)
  13. 96孔,白色平板带盖透明底(康宁,目录号:3610)
  14. 猎鹰12孔平底板(Dominique Dutscher,目录号:064023)
  15. 幻灯片和盖玻片
  16. 过滤装置Stericup GP Millipore,孔径0.2μm(EMD Millipore,目录号:SCGPU05RE)
  17. 针(Hamilton Bell,目录号:6980)
  18. 出生后第6天至第10天(男性和/或女性)新生鼠(鼠,大鼠,仓鼠)
    注意:根据我们的经验,动物的性别并不影响我们的结果,但是在使用其他病毒而不是MeV的时候应该认真考虑这个参数。
  19. 病毒实例:编码增强型绿色荧光蛋白(MeV-EGFP-1.10×10 7 pfu / ml)的重组麻疹病毒(IC323菌株)
  20. 70%乙醇
  21. 盐酸氯胺酮(MWI Animal Health,NDC 13985-584-10)
  22. 碘化丙啶溶液(Sigma-Aldrich,目录号:P4864)
  23. Dulbecco's磷酸盐缓冲盐水(DPBS)1x,无钙/镁(Thermo Fisher Scientific,目录号:14190094)
  24. AlarmarBlue细胞活力试剂储备液10x(Thermo Fisher Scientific,Invitrogen TM,目录号:DAL1025)
  25. 在BPS 1/700使用的抗胶质纤维酸性蛋白(GFAP)兔多克隆(Agilent Technologies,Dako,目录号:Z0334)
  26. 在BPS中以1/500使用的抗NeuN兔多克隆(EMD Millipore,目录号:ABN78)
  27. 在BPS 1/700使用的抗Calbindin D-28K兔多克隆(Swant,目录号:CB38)
  28. 在BPS 1/250处使用的抗Iba1(Wako Pure Chemical Industries,目录号:019-19741)
  29. 在BPS中1/200使用的抗olig2(少突胶质细胞谱系转录因子2)(R&amp; D Systems,目录号:AF2418)
  30. 在BPS中以1/750使用的抗兔IgG Fab2 Alexa Fluor 488(Cell Signaling Technology,目录号:4412S)
  31. 在BPS中以1/750使用的抗山羊IgG(H + L)交叉吸附的第二抗体Alexa Fluor 488(Thermo Fisher Scientific,目录号:A-11055)
  32. 在BPS中以1/750使用的抗兔IgG Fab2 Alexa Fluor 555(Cell Signaling Technology,目录号:4413S)
  33. Fluoprep(生物梅里埃,目录号:75521)
  34. Opti-MEM减少血清培养基(Thermo Fisher Scientific,Gibco TM,产品目录号:31985062)
  35. RNA提取试剂盒NucleoSpin RNA(MACHEREY-NAGEL,目录号:740955.250)
  36. 475ml(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:7002)
  37. iScriptTM cDNA合成试剂盒(Bio-Rad Laboratories,目录号:170-8891)
  38. 绿色qPCR SuperMix-UDG w / ROX(Thermo Fisher Scientific,Invitrogen TM,产品目录号:11744500)
  39. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266-1KG)
  40. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045-1KG)
  41. 适用于细胞培养的盐酸溶液(HCl),1.0 N BioReagent(Sigma-Aldrich,目录号:H9892-100ML)
  42. 重组人胰岛素(Sigma-Aldrich,目录号:91077C-100MG)
  43. 最低必需培养基(MEM),HEPES,GlutaMAX TM补充物,500ml(Thermo Fisher Scientific,Gibco TM,目录号:42360081)
  44. 热失活的马血清,100ml(Thermo Fisher Scientific,Gibco TM,目录号:26050070)
  45. D-葡萄糖细胞培养级5g / L(Sigma-Aldrich,目录号:G7528)
  46. 犬尿酸(Sigma-Aldrich,目录号:K3375-5G)
  47. HEPES 1M,100ml(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  48. Hibernate-A培养基(Thermo Fisher Scientific,Gibco TM,产品目录号:A1247501)。
  49. 结晶PFA(Sigma-Aldrich,目录号:P6148)
  50. 胎牛血清(FBS),500毫升(Eurobio,目录号:CVFSVF0001)
  51. Triton TM X-100(Sigma-Aldrich,目录号:T8787)
  52. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  53. 1 M MgCl 2溶液(见食谱)
  54. 0.1N NaOH溶液(见食谱)
  55. 人胰岛素50毫克/毫升(见食谱)
  56. 器官型大脑培养基(见食谱)
  57. 10倍犬尿酸溶液(见食谱)
  58. 解剖介质(见食谱)
  59. 8%多聚甲醛(PFA)(见食谱)
  60. 4%多聚甲醛(PFA)(见食谱)
  61. 阻断和透化溶液(BPS)(见食谱)

设备

  1. 直镊子,类型N°5长度11厘米去除皮肤和头骨(多米尼克Dutscher,目录号:005092)
  2. 冰容器
  3. 5%CO 2培养箱中保持在37℃和潮湿的气氛下(Thermo Fisher Scientific,Thermo Scientific TM,型号:Series 8000 Water-Jacketed,目录号:3423) br />
  4. 生物安全柜
    注意:建议在水平流罩中进行切片制备,BSL2垂直流罩用于BSL2病原体和感染后续病毒感染步骤。但是,如果BSL2机柜上的保护玻璃可以维持,切片可以用同样的方法准备。根据我们的经验,即使在这些条件下无菌性保存得不好,污染率仍然很低。在任何情况下,生物安全水平必须根据病毒感染病毒(BSL2,BSL3或BSL4)进行调整,并采取后续步骤。
  5. 不锈钢解剖剪刀长度11厘米(多米尼克Dutscher,目录号:005064)
  6. 烧杯,250毫升
  7. McIlwain组织砍刀(Campden Instruments,型号:TC752)
  8. 吸管灯泡(Fisher Scientific,目录号:03-448-29)
  9. 不锈钢解剖剪刀超细长度12厘米切割和删除头骨(多米尼克Dutscher,目录号:005068)
  10. Dumont镊子#5用于解剖大脑和去除脑膜,0.1 x 0.06,Dumoxel(世界精密仪器公司,产品目录编号:14098)
  11. 不锈钢镊子圆形末端长度130毫米,用于在解剖过程和切片分离期间保持大脑(Dominique Dutscher,目录号:442256)
  12. Lanceolate尖抹刀去除中脑(imLab,目录号:NE010)
  13. 弯曲的镊子类型N°7长度11厘米为中脑去除并且从培养插入物收获切片(多米尼克Dutcher,编目:005093)
  14. P1000移液器(Gilson,目录号:F123602)
  15. P20 Pipetman(Gilson,目录号:F123600)
  16. 用于切片分离的KOLLE持针器(Hamilton Bell,目录号:6780)
  17. 水浴
  18. 具有冷却单色照相机(Photometrics,型号:CoolSNAP HQ2)和用于碘化丙锭的荧光过滤器组(例如:激发550-580nm,发射600-660nm)的Widefield荧光显微镜(ZEISS,型号:Axioplan 2) />
  19. Tecan Infinite <200> PRO系列读板机(Tecan,型号:M Plex)
  20. 共聚焦光谱显微镜(徕卡,型号:徕卡TCS SP5)
  21. TPersonal 48热循环仪(Analytik Jena,Biometra,型号:T-Personal 48,目录号:846-050-551)
  22. StepOnePlus TM实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:StepOnePlus TM,目录号:4376600) >
  23. 解剖的立体显微镜(徕卡,型号:徕卡LED2000)
  24. 通风柜
  25. -20°C冷冻机
  26. -80°C冰箱

软件

  1. ImageJ( https://imagej.nih.gov/ij/ [Schneider et al。 ,2012])与Gabriel Landini的插件“Auto Local Threshold”( http://fiji.sc / Auto_Local_Threshold#安装
    注意:斐济( http://fiji.sc/ 可以使用[Schindelin et al。,2012]代替ImageJ,因为它捆绑了所需的插件。下载宏文件'OBC_IP_mortality.ijm'来分析碘化丙啶染色。将文件保存在ImageJ / plugins文件夹中。插件菜单中会出现“OBC IP死亡率”。
  2. GraphPad Prism(GraphPad软件 - https://www.graphpad.com/scientific-software/prism/ )和/或R软件( https://www.r-project.org/
  3. StepOnePlus软件( https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System.html )<无线电通信/>

程序

  1. 培养板制备
    1. 切片脑外植体前一天,准备6孔培养板加入1毫升器官型培养基(见食谱)到每个井中,并放置在顶部使用直镊子培养插入,以扭转聚四氟乙烯的疏水性和允许切片的良好喂养(表1)。

      表1.每个步骤建议的持续时间



    2. 在37℃潮湿的5%CO 2气氛中将平板培养过夜。

  2. 解剖准备
    1. 如果有的话,使用70%的乙醇水溶液对生物安全柜的墙壁和地板进行净化,并照射紫外线15分钟。
    2. 在解剖过程之前,用高压灭菌器对所有的设备,即剪刀,镊子,PTFE板进行消毒,并将其放在生物安全柜的下面。准备一个用70%乙醇水溶液半杯的烧杯,以便在手术过程中去除清洁物质(表1)。
    3. 用70%的乙醇清洁组织切碎机,剃刀刀片和立体显微镜,确保切片平台正确的去污,然后让一切干燥。使用螺丝和螺母将刀片安装在组织切碎机上,然后将仪器放在生物安全柜内,再用乙醇喷雾(表1)。
    4. 在生物安全柜下放置一个装满冰块的灭菌容器。
    5. 准备解剖介质(见食谱)“食谱”部分中描述使用无菌50毫升锥形管。在35毫米培养皿底部放置6毫升培养基(1个海马培养皿,1个培养皿小脑),并保持解剖的盖子。解剖介质必须保持在冰上,整个解剖过程必须在4°C(冷的介质)进行,以限制组织损伤和细胞变性。
    6. 用5毫升解剖介质填满培养皿盖,以便在整个切片过程中获得冷的新鲜培养基。
    7. 用手术刀切除5毫升塑料移液管的圆锥形末端,然后将带有梳理棉的顶部插入移液器球管(定义为5毫升截头移液管)。

  3. 解剖和切片程序
    注意:
    1. 每个步骤的持续时间请参阅表1。
    2. 表2总结了故障排除和建议的解决方案。
    1. 用70%乙醇轻轻喷雾动物的颈部和头部,并迅速进行下一步骤。
    2. 我们建议使用6到10天的动物。由于颈椎脱臼不适合哺乳动物,应使用剪刀进行断头处理。这个行动必须由熟练的认可人员来执行。在这个阶段,有必要尽快开展工作。
      注意:
      1. 当动物产生少量的婴儿时,哺乳动物可能更大,而且斩首更加困难。为了防止潜在的疼痛并为实验者提供更舒适的手术,可以使用麻醉剂。我们推荐使用150mg / kg剂量的氯胺酮,这是一种有效的活化依赖的N-甲基-D-天冬氨酸(NMDA)受体通道阻滞剂,导致兴奋性毒性的降低并防止神经元死亡。与解剖介质中含有的犬尿酸组合,氯胺酮提供了良好的抗兴奋性神经保护作用。
      2. 来自年轻动物(P3到P6之间)的脑更难以切成规则的平行切片,因为它们更柔软。另外,许多具有迁移特性的非完全分化细胞可能改变感染实验的重现性。大龄动物的大脑(P10到P12以外)很不适合,因为增加的细胞死亡可能增加分析的偏倚。
      3. 这个程序必须遵循当地的动物伦理规程,并且必须提及使用的物种和菌株。

        在任何试验之前,物种的任何变化都必须根据道德协议进行修改和验证。
    3. 将头部握在拇指和指甲之间,用超细剪刀尽可能地去除皮肤和肉。
    4. 用超精细剪刀的尖端将头骨从可见的脊髓顶部的空腔切开到含有嗅球的空腔。
    5. 将Dumont镊子的尖端插入嗅球下方,从头部取出大脑,轻轻抬起直至从颅骨脱离。
    6. 将大脑直接置于35mm培养皿中的冷的解剖介质中。在这个阶段,有必要分离和剖析解剖介质中的大脑结构,以维持细胞活力。
    7. 如下分离小脑(从C16到C20的切片过程)与大脑的脑部(图1A),并在制备海马切片过程中在冷切割介质中保存(解剖和切片过程 - 从步骤C8到步骤C15)。
    8. 将两个半球沿着中线分开(图1B),取出中脑(图1C),用镊子夹住半脑,使用披针尖刮刀或弯曲的镊子(每个脑需要约1分钟的适当训练) 。此时,海马是可见的(箭头 - 图1D)。
    9. 用手术刀切割并移除含有嗅球的嘴嘴部分(图1D-每个半球约10秒)。
    10. 使用Dumont镊子#5去除脑膜。
      海马和皮质已准备好切片过程
    11. 将海马面朝下放在Whatman纸上的延伸方向上(图1E-大约需要1分钟)。
    12. 把它放在McIlwain组织切碎器的切片平台上(见图1-箭头2-约需1分钟)。
    13. 用5毫升截头移液器加一滴解剖介质,以防止组织粘在剃刀刀片上。
    14. 通过打开仪器快速进入切片(图1E-30秒,切片两个海马)。
      注:所有切片参数,即厚度(通常在200-500μm之间),刀片力(通常为最大力)和刀片速度(通常为每秒一片)应在初步测试中确定。

    15. 在切片平台上取下Whatman纸,并在组织上大量添加冷切片
    16. 使用披针状尖端刮刀,通过轻轻地将刮刀的尖端在组织和纸张之间滑动,从Whatman纸上取下组织。

    17. 在解剖小脑期间,将组织放回含有冷清扫介质的培养皿中。
    18. 在冷剥离介质和立体显微镜下,用钳子夹住小脑,用Dumont镊子#5,下丘和脑干(图1B')轻轻地去除脑膜。
    19. 与剃刀刀片(图1C')相比,将矢状面上的小脑置于PTFE板上。
      注意:切片过程中的rostro-caudal平面并不重要,当切片平台上放置小脑时,可以将切片的端侧或尾端部分朝向实验者。然而,在我们的实验中,如图1A和1B'所示,小脑的尾部部分在切片过程中总是面对实验者。
    20. 使用P1000移液器移除整个培养基以改善小脑对PTFE板的粘附。
    21. 继续按照海马描述的切片(图1D')。

      表2.故障排除表



      图1.从啮齿类动物的P6到P10之间哺乳的OBC制备OBC海马切片制备(A-F,左图)。 A.断头后收集大脑,用虚拟刀片用小刀片分开小脑。 B.用手术刀片分开两个半球。 C.去除中脑以暴露海马区域。 D.移除大脑的头侧部分(含有嗅球),以便可视化海马(黑色箭头)。 E.将解剖的大脑在前后轴上放置到组织切碎机(1)的切片平台(2)上并垂直切割剃刀刀片(3)并沿虚线切割。 F.依靠OBC培养基将切片分离并转移至毫微孔插入培养系统。小脑切片准备(A,B'-D',F,右图)。 A.将小脑与其余的大脑分开。 B”。去除小脑下丘(IC)和脑干(BS)。 C'。根据前后轴将小脑置于聚四氟乙烯板上,并转移至组织切碎机的切片平台。 d”。按照虚线切断。 F.转移到含有解剖介质的培养皿中,小心地将这些切片彼此分开,并将其放在插入培养系统中。

    22. 小心地将组织转移到含有冷解剖介质的培养皿中。
    23. 在立体显微镜下,使用镊子小心地分离海马和/或小脑切片,以将组织和安装在KOLLE持针器上的针头放在冷的解剖介质中。主要集中在组织的顶部,切片之间的空间易于可视化,这有助于将它们精确分开(海马分片需要10到15分钟,小脑只需要5分钟)。
      注意:确保立体显微镜的光源是冷的(即光纤),以避免在切片分离过程中因介质过热而引起细胞应激。
    24. 在立体显微镜下选择文化的切片。
      注意:海马应该含有CA(Cornu Ammonis)区域和齿状回。小脑应该包含深核,并且不同的细胞层例如浦肯野细胞(PC)层或分子层(ML)应该是可见的。有关更多详细信息,请参阅“大脑图谱”,例如 Allen Brain Atlas / A> 。
    25. 使用安装在移液器球管上的5ml截头移液管,每Millipore细胞培养插入膜最多铺放4个海马或5个小脑切片。为了有利于切片的适当进食和氧合,在插入板上铺平板后在每个脑外植体周围尽可能多地切除介质,并在37℃,5%CO 2下在湿润大气。

  4. 文化程序
    注意:
    1. 在切片过程的第二天更换介质,然后每2-3天更换一次。
    2. 每个步骤的持续时间请参阅表1。
    1. 用镊子从6孔板上夹住插入物的边缘,用P1000移液管移除旧的培养基。
    2. 将插入物放回到6孔板中,加入1ml在37℃预热的水浴中的新鲜培养基以避免热冲击。
    3. 确保PTFE膜和介质之间没有气泡。如果是这样的话,将6孔板以〜45°的角度弯曲并轻轻摇动,让气泡到达培养插件的边缘。

  5. 培养的脑外植体的存活力:细胞代谢/死亡率评估-Alamarblue®和碘化丙啶染色
    注意:
    1. 在感染研究之前,确保脑外植体在体外是健康的并且确定可以使用切片的最佳时间窗是非常重要的。
    2. 基于我们的经验,如果OBC制备保持与本方案中描述的相同,并且可以使用三个独立批次的切片进行一次全部评估,则每个时间点总共15个切片分析,可行性进展是可重现的。但是,我们建议定期评估可行性,至少采用两种方法中的一种,以确保程序仍能正确设置。

    1. 在PTFE膜上轻轻剥离培养基,切除外膜边缘的切片。&nbsp;
    2. 使用截短的5毫升移液管轻轻转移到96孔板的组织。
      注意:经过长时间的培养(4〜7天)后,切片可能太粘,不能被剥离而无损伤。插入物的PTFE膜适用于荧光显微镜,并且不影响IP染色的观察,Alamarblue测定的读取和共聚焦分析(无自荧光),因此我们推荐使用这种类型的插入物来支持OBC切片。

    碘化丙啶染色

    1. 在96孔板中,将切片浸入200μl碘化丙啶溶液(5μg/ ml)于培养基中,37℃,5%CO 2孵育45分钟,加湿大气层。
      注:碘化丙锭进入死亡的细胞导致红色荧光核染色。碘化丙啶标记的核在整个组织表面的表面密度被用于监测细胞死亡,并为统计分析提供必要的量化(参见数据分析部分)。请注意,在染色之前不需要清洗切片。
    2. 取出碘化丙锭溶液,用200μl1x DPBS(37℃)洗涤3次。
    3. 将切片转移到12孔板中,并在室温下用1ml 4%多聚甲醛溶液(参见食谱)固定它们30分钟。
    4. 在载玻片和盖玻片之间轻轻地切片。确保不要挤压组织,尤其是在厚切片(300和500μm)上。
    5. 用宽视野荧光显微镜获取图像,每个切片至少6个随机场,每个时间点5个切片。使用5倍的目标,以获得大的视野。
    6. 使用提供的Macro(ImageJ中的即,脚本)分析ImageJ中的图像。运行宏(点击插件&gt;'OBC IP死亡率')并按照说明操作:首先,选择要分析的图像。它将在ImageJ中以增强的显示方式打开(请参见图2)。点击切片外部的区域(如图2中黄色部分所示的区域)。如果区域太小或太大,可以调整棒工具的公差:双击ImageJ菜单中的棒工具(在图2中的红色框中),并调整公差的值默认为200)。当区域显示正确时,按T键将其保存在ROI管理器中。重复图像中的所有区域。最后,窗口应该如图3所示。


      图2. MacroJ ImageJ中的碘化丙啶染色分析可以方便地安排窗户。原始图像位于左侧,右侧显示增强显示。说明在底部。可以通过双击红色方块来调整魔杖工具的公差。


      图3.碘化丙啶染色分析的第二步。 这些区域保存在绿色框架右侧的ROI管理器中。用户可以点击红色框中的“确定”。

    7. 点击“OK”(见图3中的红色方块)。宏将自动分割碘化丙啶染色(使用“Auto Local Threshold”插件 https:// imagej。 net / Auto_Local_Threshold#Bernsen ),并测量这种染色的面积和切片的面积。
    8. 当过程完成时,碘化丙啶区域在图像上显示为红色覆盖层,切片区域被黄色包围,目视检查分割的结果(见图4)。可以通过在“通道”窗口中选中/取消选中通道1框来激活/取消激活红色叠加(见图4)。数据结果(碘化丙啶面积“面积”和整个切片面积“切片面积”以像素为单位,以百分比表示)显示在一张可以保存的电子表格中(更多详细信息,请参阅数据分析第3点) 。可以批量分析多个图像,并将结果保存在同一个文件中:只关闭前一个图像,并以与步骤E8相同的方式再次运行宏。结果将被添加到电子表格的底部。


      图4.碘化丙锭染色分析的结果染色区域显示为红色。切片的区域用黄色表示。在右下角,结果表格可以通过菜单文件&gt;另存为。
      保存

    的 阿尔玛蓝 的 ®

    1. 在96孔的白色平板中,将切片浸入培养基中的200μl1x Alamarblue 溶液中。
    2. 在37℃,5%CO 2下在潮湿的气氛中培养2小时。
      注意:Alamarblue试剂与切片的细胞代谢成比例地变红,反映了活性细胞的比例,因此全球OBC健康状态。
    3. 根据制造商的协议,使用Tecan Infinite 200 PRO系列酶标仪读取580-610nm的荧光发射。
    4. 为了分析代谢活动,只评估动物和切片(随机)因素。我们应用了Kruskal-Wallis检验,因为数据显示违反了方差分析的假设。
    5. 使用Kruskal-Wallis检验分析时间(D0,D1和D7)的死亡率(图5A和5C)和代谢活性(图5B和5D)的变化。对于每一天,使用Wilcoxon检验比较小鼠和仓鼠之间的变异系数(标准差和平均值的比率)(更多细节参见数据分析第3点)(图6)。
      注意:根据我们的经验,当两个参数中的一个超过或在前两天变化超过20%时,我们不推荐使用OBC,因为它可能极大地影响切片中细胞群的维持,它们的趋化因子反应以及结果的重现性。


      图5.培养14天后小鼠和仓鼠OBC活力的研究。 :一种。小鼠或仓鼠(3只动物/组,每个子结构/动物至少4片)通过碘化丙啶染色评估海马和小脑切片死亡率,并用Axioplan Imager荧光显微镜分析。 B.小鼠或仓鼠(n = 3),每个子结构/动物至少3片)通过与Alamarblue试剂盒孵育评估海马和小脑切片代谢活性,并使用Infinite 200 PRO Tecan酶标仪分析。图形显示了培养期间细胞代谢(反映健康)的演变。


      图6.变异系数分析每天计算死亡率和代谢活性的变异系数(CV),并使用Wilcoxon检验在仓鼠和小鼠之间进行全球比较。
      小鼠和仓鼠之间在统计学上没有显着差异( P <-value = 0.7987)。

  6. 脑外植体的免疫染色
    注意:作为初步试验,我们建议在开始感染研究之前通过免疫染色来评估主要细胞群的维持(图7)以及培养过程中的结构保存(图8),以排除任何与实验者。
    1. 如上所述,使用5ml截头移液管将切片放置在12孔板中并在室温下用PFA固定它们。
      注意:在感染的切片的情况下,可以直接在培养插件上进行PFA固定。培养基必须被移除,并且在培养插入物的下方和上方添加1毫升PFA以完全覆盖切片。
    2. 在室温下轻轻搅拌下,用1ml阻断和透化溶液(BPS)(见配方)进行至少45分钟的阻断和透化组织。
    3. 除去BPS并添加在BPS中稀释的一级抗体(Ab)(即,星形细胞标记物GFAP,神经元标记物NeuN或CB-28K-小胶质细胞标记物Iba1或少突胶质细胞标记物Olig-2,见图7)并在室温下孵育2小时(也可能在4°C下)。
      注意:
      1. 确保Ab能在BPS中存在的Triton X-100存在下结合表位。如果没有,在此步骤中取出Triton X-100,并在1×DPBS / 4%FBS溶液中洗涤3次。
      2. 通过使用48孔板,染色溶液的体积可以小至150μl。
      3. 例如,为了突出海马和小脑结构,分别使用神经元标记物NeuN(图8A)或Purkinje细胞标记物CB-28K(图8B)。 CB-28K染色减少主要是由于浦肯野细胞损失和神经元重组入脑片。然而,在第7天,仓鼠CB-28K的明显较低的染色是由于获得这个图像。事实上,这个图的所有左边的面板是与100μmz结合的瓷砖重构。由于组织的不规则形状和安装过程,即使曝光时间相同,也很难保持均匀的染色,因为整个切片的细胞密度可能不同。 >
    4. 取出抗体并用1×DPBS每次清洗3次,每次10分钟。
    5. 在BPS中稀释次级抗体,在室温下孵育至少2小时。
    6. 重复清洗步骤。

    7. 使用fluoprep安装介质在玻片和盖玻片之间安装切片 注意:这一步切片还是很厚的,因此免疫染色成像必须使用共聚焦显微镜进行。


      图7.大脑的主要细胞类型存在并可通过免疫荧光容易地检测到。 :一种。小鼠(GABA能神经元)的特征性Purkinje细胞(PC)在小鼠小脑切片中用Calbindin-28k标记在DIV0处检测到。箭头显示PC的体型;箭头显示PC的树状树,Ax指定重组PC轴突。 B.在DIV0使用特定的神经元核标记NeuN对小鼠海马神经元进行染色以揭示海马的内部组织。 C.小鼠海马星形胶质细胞已经使用DIV0处的特异性胶质细胞星形胶质细胞蛋白(GFAP)标记进行染色。 D.使用小鼠海马中的特异性标记Iba1将小胶质细胞在DIV7染色。 E.使用小鼠海马中的Olig2特异性核标记在DIV5染色少突胶质细胞。 CA1:Cornu Ammonis区域1,CA3:Cornu Ammonis区域3,DG:齿状回。


      图8.培养7天后小鼠或仓鼠OBC生存力的形态重排和动力学研究。 :一种。在培养的第0天和第7天,将来自小鼠或仓鼠的海马切片用抗NeuN抗体(神经元标记)染色。图片显示了至少7天的海马的保守的一般组织,包括与神经元死亡相关的仅有限的细胞密度损失(白色箭头)的阿蒙氏角(Cornu Ammonis CA)区域和齿状回(DG)。 B.小鼠或仓鼠的小脑切片在培养的第0天和第7天用Calbindin28K(CB28K-Purkinje细胞标记)染色。图中的箭头显示神经元丢失,箭头显示Purkinje细胞层的中断。细胞核用DAPI复染。用Leica SP5共焦显微镜在指定的培养时间拍摄的图像显示在每天的低(在每天的重构的瓷砖左栏中)的切片内或在每天的右列的高放大率下的切片内形态演变(每天的左图中的20x物镜在每一天的右侧面板上对应于白色方块的40倍物镜)。

  7. 脑外植体的感染过程
    1. 在Opti-MEM培养基中稀释病毒原液以感染具有限定数量的噬斑形成单位(pfu)的培养切片(在6孔板中)。例如,使用麻疹病毒(MeV-EGFP)10 4至4×10 4 pfu。对于模拟对照切片,应用与感染相同体积的Opti-MEM。
      注意:感染所需的剂量应通过实验确定每种病毒/脑结构/动物模型组合。
    2. 使用P20移液管,在切片顶部轻轻地放置2至10μl稀释的病毒原液,并确保接种物均匀分布。
      注:如果研究需要病毒传播研究的单一入口(Ehrengruber等人,2002),则可以在此步骤使用微量注射器(具有适当的设备)。

    3. 在37°C,5%CO 2的湿润环境中孵育平板
    4. 在EGFP表达病毒的情况下,可以使用荧光显微镜监测病毒复制产生的荧光(图9)。


      图9.表达EGFP的重组麻疹病毒的OBC感染大鼠小脑切片在切片当天用2×10 4 pfu的MeV IC323-EGFP感染准备(DIV 0)。使用倒置荧光显微镜(ZEISS)和照相机(AxioCam,ZEISS),在低(A)或高放大倍数(B)下5天后观察到MeV-EGFP荧光。

  8. 通过RT-qPCR监测细胞或病毒基因表达
    1. 使用弯曲的镊子,根据制造商的建议(RNA提取试剂盒-Macherey Nagel)从培养插入物中取出待分析的切片并将其置于裂解缓冲液RA1-10%2-巯基乙醇中。
    2. 在处理每个切片之间,用RNase Away溶液去除RNase。
    3. 用无水乙醇消毒镊子。
    4. 用无RNase的水冲洗。
    5. 根据Macherey-Nagel的建议,在-80℃冻结裂解物,然后使用RNA提取试剂盒进行RNA分离。
    6. 使用TPersonal 48热循环仪使用iScript RT cDNA合成试剂盒准备cDNA。
    7. 使用Platinum Green SYBR Green qPCR SuperMix-UDG w / ROX试剂盒,根据目标cDNA对病毒或细胞进行定量PCR。根据制造商的建议和StepOnePlus TM实时PCR系统。
    8. 所有的结果都应该归一化为来自管家基因(如GAPDH)的mRNA,并在其他地方详细分析(Welsch et al。 ,2013)。

数据分析

  1. 免疫染色的分析需要使用共聚焦显微镜,因为OBC相对较厚,通常在150-500μm之间(图7和8)。
  2. 对于实验设计,细胞或病毒基因表达的分析需要来自分离的动物的每个条件和/或时间点的至少12个切片,即来自3个不同供体(n = 6)的2个切片,其在至少2次(n = 12)。对于免疫荧光实验,我们推荐使用来自3个不同供体的1个切片重复至少3次(n = 9)。
    注意:关于生存能力和结构的进化分析,我们的结果显示,来自小鼠或仓鼠的OBC(对于大鼠也是如此 - 数据未显示)导致这些参数的类似的进化,表明我们的方案可以是与主要的实验室啮齿动物物种一起使用,并确认了我们的方法的多功能性。
  3. 对于生存力统计分析,死亡率和代谢活动变量应分别进行分析(表3)。关于死亡率,在不同的培养日(D0,D1和D7)评估几个因素:动物,来自同一动物的切片和对同一切片重复的(即,来自同一切片的几张图像) 。因此,这个实验有一个层次结构,模型写成:

    M ij ij =μ+ A i + S j(i)+ e ij

    其中,M ij是第i个动物的第j个切片的死亡率值,μ是总体均值,A i第i个动物的作用,S(i)第i个动作的切片效应>动物,而e 是随机效应。
    使用在R软件中实施的嵌套方差分析(嵌套ANOVA)对这些各种效应进行统计学检验,如下所示:

    (公式=死亡率〜动物+切片%%动物+图像%切片,数据=文件名))。

    这个分析是针对培养的每一天进行的(D0,D1和D7)。
    注意:应该检查方差分析的假设(因变量的正态性,同方差性,图形诊断程序如残差与预测值的关系图和正常的残差QQ曲线图)。如果不是的话,应该使用非参数的Friedman检验来比较切片内的图像差异和动物内的切片差异,以及用于动物差异的非参数Kruskal-Wallis检验。
  4. 对于统计分析,我们使用GraphPad Prism和R软件。或者,一个名为“生物统计手册”(John H. McDonald)的网站详细介绍了该方法,并提供了2至4级嵌套方差分析的电子数据表和建议,可以通过以下地址访问: http://www.biostathandbook.com/nestedanova.html 。例如,如果要分析时间效应,请使用专用于4级嵌套ANOVA的电子表格,并填写以下列:日/动物/切片/图像/荧光值分别表示组/子组/子组/子组。对于动物效应的分析,您可以执行三级嵌套方差分析,并填写如下:动物/切片/图像/荧光值分别代表组/子组/子观察/观察(再次注意,您需要尊重ANOVA假设,如果不是简单地应用Kruskall-Wallis测试)。

    表3. OBC生存力的统计分析

    注:根据上述统计分析,统计时间,动物,切片和图像效果。报告用ns进行的每个测试的P值(非显着P> 0.05),** P < 0.01,*** P < 0.001。

食谱

注意:所有溶液必须使用Stericup过滤装置进行消毒,包括大量(器官型培养基,解剖介质和10倍犬尿酸),或使用孔径为0.22μm的注射器过滤器进行消毒。

  1. 1M MgCl 2(1L)
    1. 重量为95.21克MgCl2•2%
    2. 用蒸馏水加1升
    3. 保持在室温下1年
  2. 0.1N NaOH(500ml)
    1. 重量1.99克的NaOH
    2. 带上蒸馏水至500毫升
    3. 保持在室温下1年
  3. 50毫克/毫升胰岛素溶液(2毫升)

    1. 用2毫升0.005 N HCl溶液重新整理小瓶
    2. 分装(1毫升),并在-20°C存储
  4. 器官型脑培养基(500毫升)
    添加所有组件:
    375 ml的MEM GlutaMAX
    125毫升热灭活的马血清(在-20°C储存血清)
    2.5克D-葡萄糖
    1毫升50毫克/毫升的人胰岛素(终浓度0.1毫克/升)
    过滤消毒(0.22微米),并在4°C储存长达2周
    注意:在手术过程中保持无菌状态不需要添加抗生素,但可能有助于预防污染。使用青霉素时,浓度应保持在0.08mM以下,以避免潜在的突触活性干扰(见表2)。
  5. 10×犬尿酸溶液(浓缩兴奋性毒性抑制剂溶液)(200ml)
    1. 将378毫克犬尿肽溶于170毫升H 2 O中
    2. 加入20毫升1M的MgCl 2 2/2
    3. 使用0.1N NaOH将pH调节至7.4
    4. 加1毫升的HEPES
    5. 调整容量为200毫升
    6. 过滤消毒,避光,并在室温下保持2周
  6. 解剖介质(Hibernate -A / 5g / L D-葡萄糖/ 1×犬尿酸)(50ml)
    1. 准备500毫升补充了葡萄糖的Hibernate -A加入2.5克D-葡萄糖
    2. 使用Stericup过滤装置过滤消毒,并在4°C下保存6个月。
    3. 解剖当天,混合45毫升的Hibernateâ~A/ 5克/升D-葡萄糖与5毫升的10倍犬尿酸
    4. 解剖


  7. 8%多聚甲醛(PFA)(100毫升)
    在通风橱里:
    1. 在烧杯中称取8克结晶性PFA,加入100毫升1×DPBS
    2. 加热溶液,直到粉末完全溶解到65°C(不会更高,因为PFA会降解)
    3. 分装和存储在-20°C
    4. 用1x DPBS稀释至4%(使用前新鲜)
  8. 封闭和透化溶液(BPS)(1×DPBS / 0.3%Triton X-100/4%FBS)(50ml)
    添加所有组件:
    47.85毫升的1x DPBS
    2毫升FBS
    150μl的Triton X-100
    涡旋并在37℃的水浴中孵育以帮助溶解Triton X-100达30分钟

    4°C储存至2周 注意:确保溶液中没有细菌或真菌在使用前增殖,如果有的话,准备一个新的批次。

致谢

这项工作得到了法国ANR NITRODEP资助(项目ANR-13-PDOC-0010-01)的支持( http:/ / (ANR-11-IDEX-0007)项目内的里昂大学的LABEX ECOFECT(ANR-11-LABX-0048)由法国国家研究机构(ANR)提供。感谢Isabelle Dussart博士(法国巴黎大学皮埃尔•玛丽•居里大学06,法国巴黎URS 7102,75005)和法国马赛大学的Helene Clot-Faybesse博士(INMED,INSERM U29,Universite de laMéditerranée,法国马赛) )对OBC文化的宝贵意见。该协议是从Stoppini 等改编的。 (1991)稍作改动。作者声明不存在利益冲突或竞争。

参考

  1. Bloyet,L.M.,Welsch,J.,Enchery,F.,Mathieu,C.,de Breyne,S.,Horvat,B.,Grigorov,B。和Gerlier,D.(2016)。 除了磷蛋白折叠之外,还需要HSP90伴侣支持麻疹和尼帕病毒L聚合酶的酶活性。 J Virol 90:JVI.00602-16。
  2. 伯恩斯坦,M.B。和穆雷,M.R。(1958)。 新生大鼠的生长模式,髓鞘形成,维持和变性的连续观察和小猫小脑。 J生物物理生物化学Cytol 4(5):499-504。
  3. Braun,E.,Zimmerman,T.,Hur,T.B。,Reinhartz,E.,Fellig,Y.,Panet,A。和Steiner,I。(2006)。 单纯疱疹病毒1型在脑器官培养中的神经营养作用 J Gen Virol 87(Pt 10):2827-2837。
  4. Ehrengruber,M.U.,Ehler,E.,Billeter,M.A。和Naim,H.Y。(2002)。 麻疹病毒通过细胞间的接触以极化的方式在大鼠海马神经元中传播。 / J> Virol 76(11):5720-5728。
  5. Humpel,C。(2015)。 器官型脑切片培养物:综述 神经科学 305 :86-98。
  6. Kim,H.,Kim,E.,Park,M.,Lee,E。和Namkoong,K.(2013)。 成年小鼠大脑器官形态的海马脑片培养:一种多功能的翻译神经精神药理学工具。 Prog Neuropsychopharmacol Biol Psychiatry 41:36-43。
  7. Laksono,B.M.,de Vries,R.D.,McQuaid,S.,Duprex,W.P.和de Swart,R.L。(2016)。 麻疹病毒宿主的入侵与发病机制 病毒 8( 8)。
  8. Lossi,L.,Alasia,S.,Salio,C。和Merighi,A。(2009)。 哺乳动物中枢神经系统急性切片和器官培养的细胞死亡和增殖 Prog Neurobiol 88(4):221-245。
  9. Ludlow,M.,Kortekaas,J.,Herden,C.,Hoffmann,B.,Tappe,D.,Trebst,C.,Griffin,DE,Brindle,HE,Solomon,T.,Brown,AS,van Riel, D.,Wolthers,KC,Pajkrt,D.,Wohlsein,P.,Martina,BEE,Baumgartner,W.,Verjans,GM和Osterhaus,A.(2016)。 嗜神经病毒感染是引起即时和延迟神经病理学的原因 神经病理学 131(2):159-184。
  10. Mayer,D.,Fischer,H.,Schneider,U.,Heimrich,B。和Schwemmle,M。(2005)。来自大鼠的器官型海马切片培养物中的Borna病病毒复制导致对齿状颗粒细胞的选择性损伤。 / J> Virol 79(18):11716-11723。
  11. Olival,K.J。和Daszak,P.(2005)。 新兴嗜神经病毒的生态学
  12. Reuter,D.和Schneider-Schaulies,J(2010)。 中枢神经系统麻疹病毒感染:人类疾病,动物模型和治疗方法 Med Microbiol Immunol 199(3):261-271。
  13. Schneider,C.A.,Rasband,W.S。和Eliceiri,K.W。(2012)。 NIH Image to ImageJ:25年的图像分析。 Nat Methods < / em> 9(7):671-675。
  14. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P.和Cardona,A。(2012)。 斐济:生物图像分析的开源平台。 Nat方法 9(7):676-682。
  15. Stoppini,L.,Buchs,P.A。和Muller,D。(1991)。 一种用于神经组织器官培养的简单方法 Neurosci Methods < / em> 37:173-182。
  16. Stubblefield Park,S. R.,Widness,M.,Levine,A. D.和Patterson,C. E.(2011)。在麻疹病毒感染中T细胞,白细胞介素-12和γ干扰素驱动的病毒清除脑组织。“ Virol 85:3664-76。
  17. Swanson,P.A.,2nd和McGavern,D.B。(2015)。 中枢神经系统病毒性疾病
  18. Taylor,L.H.,Latham,S.M。和Woolhouse,M.E。(2001)。 人类疾病出现的危险因素 Philos Trans R Soc Lond B Biol Sci 356(1411):983-989。
  19. Welsch,J.C。,Talekar,A.,Mathieu,C.,Pessi,A.,Moscona,A.,Horvat,B。和Porotto,M.(2013)。 通过脑渗透融合抑制剂预防致命麻疹病毒感染 Virol 87(24):13785-13794。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。