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An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo
用于研究胶质细胞离体激活的急性小鼠脊髓切片的制备   

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本实验方案简略版
The Journal of Neuroscience
Apr 2016

Abstract

Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord’s highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord. Here, we describe a procedure for preparing acute slices from mouse spinal cord (Garré et al., 2016). This preparation should allow further understanding of how glial cells in spinal cord respond acutely to various inflammatory challenges.

Keywords: Microglia (小胶质细胞), Astrocyte (星形胶质细胞), Spinal cord (脊髓), Neuroinflammation (神经炎症), Spina cord slices (脊髓切片)

Background

Mouse transgenic technology has been used to model different human pathologies affecting the spinal cord, many of which are characterized by local glial activation, one hallmark of neuroinflammation. A major breakthrough that has enormously increased the understanding of glial biology in health and disease is the utilization of laser scanning microscopy based techniques, such as confocal microscopy (White et al., 1987) and two-photon microscopy (Denk et al., 1990) to visualize cell structures and subcellular domains in living animals in a noninvasive fashion; for example, mice expressing genetically encoded reporters or calcium sensors have been used to image glial structures (somata and processes) and to study calcium dynamics and signaling, respectively (Davalos et al., 2005; Gee et al., 2014). In spinal cord, myelin is highly compact in the white matter of the dorsal, lateral, and ventral funiculi. In vivo structural imaging of glial cells and infiltrating immune cells has been successfully performed in the past using surgical procedures (laminectomy) that allow optical access to the dorsal spinal cord (Kim et al., 2010). However, since myelin greatly increases light scattering, imaging is limited to the superficial layers of the dorsal funiculus, masking valuable information from deeper regions such as ventral horn. We think that acute slices prepared from wild type and transgenic mice can be used in combination with high-resolution imaging techniques to offer an alternative strategy to collect structural and functional information, in vitro, from dorsal, and also lateral and ventral regions. Coronal sections interrupt ascending and descending axons and many motor axons as well. Nevertheless, the information obtained is likely to be useful in analyzing how glial cells respond acutely to inflammatory challenges in spinal cord.

Materials and Reagents

  1. Double edge razor blades (Everychina, Baili, catalog number: BP005 )
  2. Sterile 21 gauge needles (BD, catalog number: 305165 )
  3. Syringes (½ ml, 3 ml, and 20 ml)
    ½ ml (COVIDIEN, catalog number: 8881600004 )
    3 ml (BD, catalog number: 309657 )
    20 ml (BD, catalog number: 302830 )
  4. Adhesive tape
  5. Peel-a-way embedding molds (Sigma-Aldrich, catalog number: E6032 )
  6. Disposable transfer pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 336 )
  7. Six-well multidish (Thermo Fisher Scientific, catalog number: 130184 )
  8. Parafilm
  9. Coverslip (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545-88 )
  10. 70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
  11. 15 ml and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog numbers: 352095 and 352098 , respectively)
  12. Pipette tips (10 μl, 200 μl, 1,000 μl) (USA Scientific)
  13. One- to two-month-old CX3CR1EGFP/+ transgenic mice (THE JACKSON LABORATORY, catalog number: 005582 )
  14. Ketamine and xylazine (provided by NYU School of Medicine, DLAR)
  15. Isoflurane (provided by NYU School of Medicine, DLAR)
  16. 70% ethanol
  17. Low melting point agarose (Sigma-Aldrich, catalog number: A9414 )
  18. Cyanoacrylate (Instant Krazy glue)
  19. Ethidium bromide (MW: 394.3) (MP Biomedicals, catalog number: 802511 )
  20. Propidium iodide (MW: 668.4) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P3566 )
  21. Phosphate buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144 )
  22. Triton X-100, 100 ml solution (Sigma-Aldrich, catalog number: X100 )
  23. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3294 )
  24. Normal goat serum (Vector Laboratories, catalog number: S1000 )
  25. Optional: chicken anti-GFAP (EMD Millipore, catalog number: AB5541 )
  26. Mowiol® 4-88 (aqueous mounting medium) (Sigma-Aldrich, catalog number: 81381 )
  27. Tween 20
  28. Optional: alexa fluor 647-conjugate goat anti-chicken IgY - H&L (Thermo Fisher Scientific, Invitrogen, catalog number: A21449 ) secondary antibody
  29. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  30. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  31. Glucose (Sigma-Aldrich, catalog number: G7528 )
  32. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  33. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  34. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  35. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  36. HCl
  37. NaOH
  38. EDTA
  39. Paraformaldehyde (PFA, 32% solution) (Electron Microscopy Sciences, catalog number: 15714 )
  40. Artificial cerebrospinal fluid (ACSF) (see Recipes)
  41. Ca2+ and Mg2+ free-ACSF (see Recipes)
  42. 3-4% PFA, pH = 7.4 (see Recipes)

Equipment

  1. Compressed gas tank 5% CO2, 95% O2
  2. Leica vibratome and blade holder (Leica, model: VT1000 S )
  3. Standard 1000 orbital shaker (TROEMNER, catalog number: 980173 )
  4. Digital pH meter (Mettler Toledo)
  5. Hemostat clamps (World Precision Instruments, catalog number: 503736 )
  6. Forceps 12 cm long (World Precision Instruments, catalog number: 14226 )
  7. Fine dissection forceps number 5 (Roboz Surgical Instrument, catalog number: RS-4955 )
  8. SuperCut scissors (World Precision Instruments, catalog number: 14218 )
  9. Spine bone scissors (Dumont, catalog number: 15a )
  10. Digital water bath (Thermo Fisher Scientific, Fisher ScientificTM, model: Isotemp 205 )
  11. Tubing
  12. Digital scale (Mettler Toledo, model: MS104S )
  13. Micropipettes (Gilson, 0.5-2 µl, 1-10 µl , 10-200 µl , 1,000 µl )
  14. Stereo microscope with LED lights (Olympus, model: SZX10 )
  15. Zeiss-700 confocal microscope equipped with 20x objective and appropriate filters
  16. Thermistor thermometer (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: 605010100 )

Procedure

  1. Before sectioning the spinal cord
    1. Bubble (with a mixture 95% O2/5% CO2) 500 ml of normal ACSF and 50 ml of Ca2+ and Mg2+ free-ACSF for 30 min at 4 °C.
    2. Monitor pH after 30 min. pH will be close to 7.38-7.40; adjust to 7.38-7.42 if necessary.
    3. Insert one half of a razor blade in the blade holder of the vibratome and chill the stage by placing ice in the ice chamber.
  2. Deeply anesthetize mice using an intraperitoneal injection of ketamine (100 mg/kg)/xylazine (15 mg/kg).
    Note: Alternatively, isoflurane can be used to induce anesthesia.
  3. Spray the thorax and abdomen with 70% ethanol and gain access to the abdominal cavity by making a longitudinal midline cut. Open the thoracic cavity to expose the heart (use SuperCut scissors and 12 cm long forceps). (Figures 1a-1d)
  4. Hold the heart with forceps and insert a 21 gauge needle into the left ventricle.
    Note: Do not insert the needle too far, since it may damage interior heart compartments and impair circulation of fluids.
  5. Make a cut in the right atrium and perfuse mice slowly with chilled Ca2+ and Mg2+ free-ACSF saturated with 95% O2/5% CO2 through the left ventricle (20 ml/ mouse, body weight: 10-15 g).


    Figure 1. Surgical procedure for extracting the mouse spinal cord. a. A deeply anesthetized mouse is restrained on the surgical stage with adhesive tape. b and c. Midline incision used to start the laparotomy. d. Opening of thoracic cavity. e. Insertion of 21-G needle through the left ventricle for perfusion, and cutting of the right atrium. f and g. Opening of the vertebral canal and removal of the vertebral bodies cervical to lumbar by cutting the dorsal vertebral processes. h. An exposed thoracic segment of spinal cord, viewed from the ventral side.

  6. After perfusion, decapitate mice and remove the spinal column and quickly immerse it in normal ice-cold ACSF.
  7. Make two small incisions, one on each side at the cervical end. Start to remove the ventral vertebral bodies (cervical to lumbar) using special bone scissors to cut the dorsal processes on both sides (Dumont, 15a). This procedure will open the spinal canal, cut the nerves, and expose the spinal cord (Figures 1f-1h).
  8. Carefully remove the spinal cord with forceps and fine scissors and immerse it in chilled normal ACSF saturated with 95% O2/5% CO2.
    Note: Avoid stretching and pinching the cord during this procedure.
  9. Cut any remaining spinal nerve roots close to the cord with fine scissors.
  10. Maintain the spinal cord in ice-cold ACSF saturated with 95% O2/5% CO2 until embedding the cord in step 12.
  11. Dissolve low melting point agarose (4% in ACSF) by heating it in a microwave (< 1 min).
  12. Fill a disposable embedding mold (22 x 22 x 20 mm) with agarose and chill the mold on ice. Embed the thoracic or lumbar spinal cord when the agarose temperature is lower than 37 °C (Figure 2b). At this and somewhat lower temperatures agarose is semi-solid, and cords can be embedded horizontally.
  13. Keep the embedding mold on ice until the agarose becomes solid.
    Notes:
    1. Once the mold is placed on ice, agarose gelling will be completed in about 3-5 min.
    2. Steps 10-14 should not take longer than 5-10 min.
  14. Place the agarose mold as soon as possible in ice-cold ACSF saturated with 95% O2/5% CO2.
  15. Apply a small amount of cyanoacrylate to the vibratome stage, remove the agarose block from the mold and glue it onto the stage. Immerse the embedded spinal cord immediately in ice-cold ACSF (bubble with a mixture of 95% O2/5% CO2 during the entire period of sectioning). (Figures 2a-2c)


    Figure 2. The experimental setup for sectioning the spinal cord. a. Vibratome; b. A spinal cord segment placed horizontally in the agarose mold; c. The agarose cube containing the spinal cord is removed from the mold, rotated 90° and glued to the vibratome stage in the proper orientation for coronal sections.

  16. Section spinal cord in 250-300 μm slices with the Leica vibratome (VT1000 S) at 0.1 mm/sec and 70 Hz, speed and frequency of the blade in the plane of section, respectively.
    Note: Using faster speeds will prevent proper spinal cord sectioning. Before proceeding use fine brushes to remove agarose surrounding the slices.
  17. Pick slices up using transfer pipettes or fine brushes and place them in cell strainers which are immersed in a glass Pyrex beaker containing 250 ml of ACSF saturated with 95% O2/5% CO2. Allow slices to recover for 45-60 min at 35 °C.
  18. For dye uptake experiments
    1. Prepare stock solutions of 1 mM ethidium bromide (EtdBr) and 1 mM propidium iodide (PropI2) in dH2O.
    2. Bathe the slices in 10 µM EtdBr (for studying hemichannel activity) or 5 µM PropI2 (for testing cell viability) for 10 min at room temperature in ACSF saturated with 95% O2/5% CO2. The volumes of EtdBr and PropI2 added to ACSF do not change concentrations of electrolytes significantly.
      Note: This step can be conducted in a 6-well multiwell plate filled with ASCF (2 ml/well). Remove the slices from the cell strainers (see step 17) by using transfer pipettes and place them in a 6-well multiwell. Each well must be covered with Parafilm to reduce loss of O2/CO2 from the solution.
    3. Remove unbound Etd+ or PropI2 by rinsing slices 3 times with 1 ml of PBS at room temperature. Fix slices for 2 h at room temperature or overnight at 4 °C with 4% paraformaldehyde (PFA) in PBS.
  19. For immunostaining of GFAP (optional)
    1. Permeabilize PropI2 labeled and Etd+ labeled slices in Triton X-100 (1% in PBS) for 3 h in a shaker at room temperature.
    2. To block nonspecific antibody reaction, incubate slices with 0.4 ml of a blocking solution containing 5% donkey serum, 0.5% BSA, and 0.1% Triton X-100 in PBS for 0.5 h.
    3. Incubate slices with 0.4 ml of chicken anti-GFAP (1/200 in blocking solution) for 1 h.
    4. Remove antibody solution and incubate slices with 1 ml of washing solution (0.5% Tween 20, 0.1% Triton X-100, in PBS) in a shaker for 10 min. Repeat washing 3 times.
    5. Incubate slices for 1 h with goat anti-chicken conjugated Alexa-647 (1/1,000 in blocking solution).
    6. Repeat step 19d.
  20. Mount slices on coverslips using an aqueous-based mounting medium, for example, Mowiol 4-88.
  21. Take confocal images one or two days later, as convenient, with a Zeiss-700 confocal microscope equipped with a 20x objective and appropriate filters (Figure 3).
    Note: Etd+ labeling is not obviously reduced in this time period between uptake and imaging.


    Figure 3. Uptake of propidium and Etd+ by glial cells and neurons in the ventral horn of spinal cord. (Left panel) Slices were fixed overnight with 4% PFA and permeabilized, and then incubated in 5 μM PropI2 for 10 min and mounted. Motor neurons (red arrow) and microglia (green arrow) were identified by the morphology and by expression of EGFP, respectively, in acute slices prepared from CX3CR1EGFP/+ mice. In these slices, CX3CR1 and EGFP expressing cells are brain resident macrophages, a population mostly comprised of microglia. (Middle and right panels) In separate experiments, slices were maintained in ACSF saturated with 95% O2/5% CO2 for 1 h after sectioning and incubated with 5 μM PropI2 or 10 μM EtdBr for 10 min without permeabilization, rinsed and then fixed in 4% PFA before mounting. Under these conditions, there was little Prop2+ uptake in neurons (red arrow) and microglia (green arrow, middle panel). In contrast, Etd+ uptake was observed in CX3CR1EGFP/+ microglia (white arrow, Etd+ uptake plus EGFP expression), although it was rare in motor neurons (identifiable as round dark areas without small red cells, right panel, compare to left panel). The small red cells between EGFP- Etd+ cells and surrounding motoneurons are astrocytes, as was shown by GFAP immunolabeling (see Garré et al., 2016). Scale bar = 50 μm.

    Because opening of hemichannels (HCs) formed of connexin 43 (Cx43) and pannexin 1 (Px1) has been shown to be associated with enhanced Etd+ uptake in different cell culture systems (e.g., Contreras et al., 2003 and Garré et al., 2010), we used the preparation described here for evaluating HC activity in response to inflammatory challenge. Using genetic and pharmacological approaches we showed that Px1 HC opening mediated the early inflammatory response to FGF-1 and ATP. Furthermore, we identified several inflammatory mechanisms triggered by Px1 HCs (see Garré et al., 2016).

Data analysis

A complete description of statistics used for analyzing dye uptake experiments is presented in Garré et al. (2016).

Notes

  1. Since the mice used for this preparation express EGFP protein in CX3CR1 cells (microglia and perivascular macrophages), the exposure of spinal cord sections to excitation light should be minimized. It may affect the number of EGFP+ cells as well as the quality of fluorescence signals.
  2. Rapid manifestations of microglia and astrocyte activation have been observed in slices prepared from mouse cortex (Takano et al., 2014). In our hands, we have not seen obvious morphological signs of microglia or astrocyte activation up to 2 h after spinal cord sectioning, the maximum time tested. Basal TNFα level was considerably reduced in slices depleted of microglial cells (Garré et al., 2016). To improve the reproducibility of our protocol we recommend recording how long after sectioning the slices are used.
  3. If this preparation is used for electrophysiological recordings and/or for periods longer than 2 h post-sectioning, an alternative recipe for preparing enriched ACSF can be used (see Mitra and Brownstone, 2012).

Recipes

  1. Artificial cerebrospinal fluid (ACSF)
    Note: Fresh preparation is recommended.
    119.0 mM NaCl
    26.2 mM NaHCO3
    11.0 mM glucose
    2.5 mM KCl
    1.0 mM NaH2PO4
    2.5 mM CaCl2
    1.3 mM MgCl2
    Adjust pH to 7.4 using either 2 N HCl or 5 N NaOH, to lower or raise pH, respectively
    Measure osmolarity (300-310 mOsm)
    Filter the solution
  2. Ca2+ and Mg2+ free-ACSF
    119.0 mM NaCl
    26.2 mM NaHCO3
    11.0 mM glucose
    2.5 mM KCl
    1.0 mM NaH2PO4
    5.0 mM EDTA
    Adjust pH to 7.4 using either 2 N HCl or 5 N NaOH, to lower or raise pH, respectively
    Measure osmolarity (300-310 mOsm)
    Filter the solution
  3. 3-4% PFA, pH = 7.4
    Dilute the concentrated 32% PFA stock solution (Electron Microscopy Sciences) in Ca2+ and Mg2+ free PBS (Thermo Fisher Scientific) to make a solution 3-4% PFA, pH = 7.4

Acknowledgments

The protocol here was adapted from Garré et al. (2016). This work was supported by National Institutes of Health Grants NS45287 and NS55363 to M.V.L.B. and NS072238 to F.F.B., National Institutes of Health Grants GM107469 and AG048410 to G.Y. and the Research Council of Lithuania MIP-76/2015 to F.F.B.

References

  1. Contreras, J. E., Saez, J. C., Bukauskas, F. F. and Bennett, M. V. (2003). Gating and regulation of connexin 43 (Cx43) hemichannels. Proc Natl Acad Sci U S A 100(20): 11388-11393.
  2. Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., Littman, D. R., Dustin, M. L. and Gan, W. B. (2005). ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8(6): 752-758.
  3. Denk, W., Strickler, J. H. and Webb, W. W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248(4951): 73-76.
  4. Garré, J. M., Retamal, M. A., Cassina, P., Barbeito, L., Bukauskas, F. F., Saez, J. C., Bennett, M. V. and Abudara, V. (2010). FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels. Proc Natl Acad Sci U S A 107(52): 22659-22664.
  5. Garré, J. M., Yang, G., Bukauskas, F. F. and Bennett, M. V. (2016). FGF-1 triggers Pannexin-1 hemichannel opening in spinal astrocytes of rodents and promotes inflammatory responses in acute spinal cord slices. J Neurosci 36(17): 4785-4801.
  6. Gee, J. M., Smith, N. A., Fernandez, F. R., Economo, M. N., Brunert, D., Rothermel, M., Morris, S. C., Talbot, A., Palumbos, S., Ichida, J. M., Shepherd, J. D., West, P. J., Wachowiak, M., Capecchi, M. R., Wilcox, K. S., White, J. A. and Tvrdik, P. (2014). Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse. Neuron 83(5): 1058-1072.
  7. Kim, J. V., Jiang, N., Tadokoro, C. E., Liu, L., Ransohoff, R. M., Lafaille, J. J. and Dustin, M. L. (2010). Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites. J Immunol Methods 352(1-2): 89-100.
  8. Mitra, P. and Brownstone, R. M. (2012). An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse. J Neurophysiol 107(2): 728-741.
  9. Takano, T., He, W., Han, X., Wang, F., Xu, Q., Wang, X., Oberheim Bush, N. A., Cruz, N., Dienel, G. A. and Nedergaard, M. (2014). Rapid manifestation of reactive astrogliosis in acute hippocampal brain slices. Glia 62(1): 78-95.
  10. White, J. G., Amos, W. B. and Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105(1): 41-48.

简介

病理状况如肌萎缩性侧索硬化,脊髓损伤和慢性疼痛的特征在于脊髓中星形胶质细胞和小胶质细胞的活化,并已在啮齿动物中建模。 在这些动物模型中的细胞水平的体内成像由于脊髓的高度髓鞘化的功能而受到限制。 急性切片的制备可能提供一种替代和有价值的策略,从脊髓的背侧,外侧和腹侧区域体外收集结构和功能信息。 在这里,我们描述了一种从小鼠脊髓制备急性切片的过程(Garréet al。,2016)。 这个准备应该进一步了解脊髓中的神经胶质细胞是如何急剧地反应各种炎症的挑战。
【背景】已经使用小鼠转基因技术来模拟影响脊髓的不同人类病态,其中许多特征为局部胶质激活,神经炎症的一个标志。利用基于激光扫描显微技术的共焦显微镜(White et al。,1987)和双光子显微镜(Denk等,1990),大大提高了对健康和疾病中胶质生物学的认识。 )以无创的方式可视化活体动物中的细胞结构和亚细胞结构域;例如,表达遗传编码的记者或钙传感器的小鼠已被用于分别形成胶质结构(体细胞和过程)并研究钙动力学和信号传导(Davalos等人,2005; Gee等,2014)。在脊髓中,髓鞘在背侧,外侧和腹侧肌的白质中高度紧密。胶质细胞和浸润性免疫细胞的体内结构成像在过去使用允许光学接近背脊髓的外科手术(椎板切除术)成功地进行(Kim等,2010)。然而,由于髓磷脂大大增加了光散射,所以成像仅限于背侧的表皮层,从较深的区域(例如腹角)掩盖有价值的信息。我们认为从野生型和转基因小鼠制备的急性切片可以与高分辨率成像技术结合使用,以提供从体外,从背侧以及外侧和腹侧区域收集结构和功能信息的替代策略。冠状切片也会中断上升和下降的轴突以及许多运动轴突。然而,获得的信息可能有助于分析神经胶质细胞如何急性地对脊髓炎症性疾病的反应。

关键字:小胶质细胞, 星形胶质细胞, 脊髓, 神经炎症, 脊髓切片

材料和试剂

  1. 双刃刀片(Everychina,Baili,目录号:BP005)
  2. 无菌21号针头(BD,目录号:305165)
  3. 注射器(½ml,3 ml和20 ml)
    ½ml(COVIDIEN,目录号:8881600004)
    3 ml(BD,目录号:309657)
    20 ml(BD,目录号:302830)
  4. 胶带
  5. 剥离模式(Sigma-Aldrich,目录号:E6032)
  6. 一次性转移移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:336)
  7. 六井多(Thermo Fisher Scientific,目录号:130184)
  8. 石蜡膜
  9. 盖玻片(Thermo Fisher Scientific,Fisher Scientific,目录号:12-545-88)
  10. 70μm细胞过滤器(Corning,Falcon ®,目录号:352350)
  11. 15毫升和50毫升聚丙烯锥形管(Corning,Falcon ,分别),目录号:352095和352098)
  12. 移液头(10μl,200μl,1,000μl)(USA Scientific)
  13. 1至2个月龄的CX 3转基因小鼠(THE JACKSON LABORATORY,目录号:005582)
  14. 氯胺酮和赛拉嗪(由纽约大学医学院,DLAR提供)
  15. 异氟醚(由纽约大学医学院,DLAR提供)
  16. 70%乙醇
  17. 低熔点琼脂糖(Sigma-Aldrich,目录号:A9414)
  18. 氰基丙烯酸酯(Instant Krazy glue)
  19. 溴化乙锭(MW:394.3)(MP Biomedicals,目录号:802511)
  20. 碘化丙啶(MW:668.4)(Thermo Fisher Scientific,Molecular Probes TM,目录号:P3566)
  21. 磷酸盐缓冲盐水(Thermo Fisher Scientific,Gibco TM,目录号:14190-144)
  22. Triton X-100,100ml溶液(Sigma-Aldrich,目录号:X100)
  23. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3294)
  24. 正常山羊血清(Vector Laboratories,目录号:S1000)
  25. 可选:鸡抗GFAP(EMD Millipore,目录号:AB5541)
  26. Mowiol ®4-88(水性载体介质)(Sigma-Aldrich,目录号:81381)
  27. 吐温20
  28. 可选择:alexa fluor 647-共轭山羊抗鸡IgY-H& L(Thermo Fisher Scientific,Invitrogen,目录号:A21449)二抗
  29. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  30. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  31. 葡萄糖(Sigma-Aldrich,目录号:G7528)
  32. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  33. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  34. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  35. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  36. HCl
  37. NaOH
  38. EDTA
  39. 多聚甲醛(PFA,32%溶液)(Electron Microscopy Sciences,目录号:15714)
  40. 人造脑脊液(ACSF)(见配方)
  41. Ca 2 + 和Mg 2 + free-ACSF(见配方)
  42. 3-4%PFA,pH = 7.4(参见食谱)

设备

  1. 压缩气罐5%CO 2,95%O 2
  2. Leica vibratome和刀架(Leica,型号:VT1000 S)
  3. 标准1000轨道振动筛(TROEMNER,目录号:980173)
  4. 数字pH计(Mettler Toledo)
  5. 止血钳(World Precision Instruments,目录号:503736)
  6. 镊子12厘米长(世界精密仪器,目录号:14226)
  7. 精细切割钳5号(Roboz手术器械,目录号:RS-4955)
  8. SuperCut剪刀(世界精密仪器,目录号:14218)
  9. 脊骨剪刀(Dumont,目录号:15a)
  10. 数字水浴(Thermo Fisher Scientific,Fisher Scientific TM,型号:Isotemp 205)
  11. 油管
  12. 数字秤(Mettler Toledo,型号:MS104S)
  13. 微量给药(Gilson,0.5-2μl,1-10μl,10-200μl,1,000μl)
  14. 具有LED灯的立体显微镜(Olympus,型号:SZX10)
  15. 蔡司700共焦显微镜配备了20倍客观和适当的过滤器
  16. 热敏电阻温度计(SP科学软件 - 贝尔艺术产品 - H-B仪器,目录号:605010100)

程序

  1. 切断脊髓前
    1. 气泡(混合物95%O 2/5%CO 2)将500ml正常ACSF和50ml Ca 2+/Mg和Mg在4℃下30分钟的自由ACSF进行30分钟。
    2. 30分钟后监测pH值。 pH值将接近7.38-7.40;必要时调整为7.38-7.42。
    3. 将剃刀刀片的一半插入振动片的刀架中,并通过将冰放置在冰室中来冷却舞台。
  2. 使用氯胺酮(100mg/kg)/赛拉嗪(15mg/kg)腹膜内注射深度麻醉小鼠。
    注意:或者,异氟醚可用于诱导麻醉。
  3. 用70%乙醇喷洒胸部和腹部,通过纵向中线切割进入腹腔。打开胸腔暴露心脏(使用SuperCut剪刀和12厘米长镊子)。 (图1a-1d)
  4. 用镊子握住心脏,并将21号针插入左心室。
    注意:请勿将针头插入太远,否则可能会损坏内部心室并损害液体的循环。
  5. 用95%O 2饱和的冷冻Ca 2+ +和/或自由ACSF缓慢地切成右心房和灌注小鼠,/5%CO 2通过左心室(20ml /小鼠,体重:10-15g)。


    图1.提取小鼠脊髓的外科手术。 a。用胶带将手术台上的深度麻醉的小鼠限制在手术台上。 b和c。中线切口用于开始剖腹术。 d。胸腔开放e。插入21-G针穿过左心室进行灌注,切割右心房。 f和g。通过切开背脊椎过程,打开椎管并将椎体切除到腰椎。 H。从腹侧观察脊髓暴露的胸段。

  6. 灌注后,取出小鼠,取出脊柱,迅速将其浸入正常冰冷的ACSF中
  7. 做两个小切口,每一侧在颈椎端。开始使用特殊的骨头剪刀去除腹侧椎体(颈部到腰部),以切割两侧的背部过程(Dumont,15a)。该程序将打开椎管,切开神经并暴露脊髓(图1f-1h)。
  8. 用镊子和精细剪刀小心地取出脊髓,并将其浸入95%O 2/5%CO 2饱和的冷冻正常ACSF中。
    注意:在此过程中,请避免拉伸和挤压电线。
  9. 用精细剪刀切割靠近绳索的剩余脊髓神经根。
  10. 将脊髓保持在95%O 2/5%CO 2饱和的冰冷ACSF中,直到在步骤12中嵌入电线。
  11. 通过在微波(<1分钟)内加热,溶解低熔点琼脂糖(ACSF中4%)。
  12. 用琼脂糖填充一次性嵌入模具(22 x 22 x 20 mm),并将模具冷却至冰上。当琼脂糖温度低于37℃时,嵌入胸椎或腰脊髓(图2b)。在此温度稍低的条件下,琼脂糖是半固体的,并且可以将水平线嵌入。
  13. 将包埋的模具保持在冰上,直到琼脂糖变固。
    注意:
    1. 一旦模具放置在冰上,琼脂糖凝胶将在约3-5分钟内完成。
    2. 步骤10-14不应超过5-10分钟。
  14. 将琼脂糖模具尽快放在用95%O 2/5%CO 2饱和的冰冷的ACSF中。
  15. 将少量氰基丙烯酸酯加入到振动台中,从模具中取出琼脂糖块并将其粘合到载物台上。在整个切片期间,将嵌入的脊髓立即浸入冰冷的ACSF(具有95%O 2/5%CO 2混合物的气泡)。 (图2a-2c)


    图2.脊髓切片的实验设置。 a。震颤b。脊髓段水平放置在琼脂糖模具中; C。将包含脊髓的琼脂糖立方体从模具中取出,旋转90°,并以正确的方向胶合到振动台,用于冠状切片。

  16. 剖面脊髓在250-300微米的切片中,Leica vibratome(VT1000 S)分别为0.1 mm/sec和70 Hz,叶片的截面平均速度和频率。
    注意:使用更快的速度将阻止正确的脊髓切片。在继续使用精细的刷子去除切片周围的琼脂糖之前。
  17. 使用转移移液管或细刷将切片取出并将其放入细胞过滤器中,将其浸入含有250ml饱和95%O 2/5%CO 2的ACSF玻璃Pyrex烧杯中,/sub>。允许切片在35°C下恢复45-60分钟。
  18. 染料吸收实验
    1. 在dH 2 O中制备1mM溴化乙锭(EtdBr)和1mM碘化丙啶(PropI 2/2)的储备溶液。
    2. 在95%O 2饱和的ACSF中,室温下将10μMEtdBr(用于研究半通道活性)或5μMPropI 2(用于测试细胞活力)亚> 5%CO 2 。添加到ACSF中的EtdBr和PropI 2 的体积不会显着改变电解质的浓度。
      注意:该步骤可以在装有ASCF(2ml /孔)的6孔多孔板中进行。从细胞过滤器中取出切片(参见步骤17),使用转移移液管将其置于6孔多孔中。每个井都必须用Parafilm覆盖,以减少O 2 /CO 2
    3. 通过在室温下用1ml PBS冲洗切片3次,除去未结合的Etd + 或PropI 2 。在室温下将切片固定2小时或在4℃下用4%多聚甲醛(PFA)在PBS中过夜。
  19. 对于GFAP的免疫染色(可选)
    1. 在室温下在振荡器中将Triton X-100(1%PBS中)标记的片段和Etd + 标记片段在振荡器中渗透稳定。
    2. 为了阻止非特异性抗体反应,用0.4ml含有5%驴血清,0.5%BSA和0.1%Triton X-100在PBS中的封闭溶液孵育切片0.5小时。
    3. 用0.4ml鸡抗GFAP(1/200封闭溶液)孵育切片1小时
    4. 去除抗体溶液,并在振荡器中用1ml洗涤溶液(0.5%Tween 20,0.1%Triton X-100,PBS中)孵育切片10分钟。重复洗涤3次。
    5. 孵育1小时与山羊抗鸡共轭Alexa-647(1/1,000在阻塞溶液)。
    6. 重复步骤19d。
  20. 使用水基安装介质在盖玻片上安装切片,例如Mowiol 4-88。
  21. 使用一个或两天后的共焦图像,方便,使用配有20x物镜和适当过滤器的Zeiss-700共聚焦显微镜(图3)。
    注意:在摄影和成像之间的这个时间段内,Etd + 标签不会明显减少。


    图3.脊髓腹角神经胶质细胞和神经元的支持物和Etd + 的摄取。(左图)切片用4%PFA固定过夜,透化,然后在5μMPropI 2中孵育10分钟并安装。运动神经元(红色箭头)和小胶质细胞(绿色箭头)分别通过EGFP的形态学和EGFP表达分别在由CX3 EGFP/+ /老鼠。在这些切片中,CX3/CR1和表达EGFP的细胞是脑驻留巨噬细胞,大多数是由小胶质细胞组成的群体。 (中图和右图)在单独的实验中,切片在切片之后1小时保持在饱和95%O 2/5%CO 2的ACSF中,并与5μM PropI 2或10μEEtdBr 10分钟,不进行透化,漂洗,然后固定在4%PFA中,然后再安装。在这些条件下,在神经元(红色箭头)和小神经胶质细胞(绿色箭头,中间面板)中几乎没有支持物的吸收。相比之下,在CX3 EGFP/+小胶质细胞中观察到Etd + 摄取(白色箭头,Etd + 摄取加EGFP表达),尽管在运动神经元中是罕见的(可识别为没有小红细胞的右侧黑色区域,右图,与左图相比)。 EGFP - Etd + 细胞和周围运动神经元之间的小红细胞是星形胶质细胞,如GFAP免疫标记所示(参见Garré等人, 2016)。比例尺=50μm。

    由于连接蛋白43(Cx43)和pannexin 1(Px1)形成的半通道(HC)的开放已被证明与不同细胞培养系统中增强的Etd + 摄取相关(例如< em>,Contreras等人,2003和Garré等人,2010),我们使用这里描述的用于评估响应于炎症攻击的HC活性的制剂。使用遗传和药理学方法,我们显示Px1 HC开放介导对FGF-1和ATP的早期炎症反应。此外,我们确定了Px1 HCs引发的几种炎症机制(参见Garré等人,2016年)。

数据分析

用于分析染料摄取实验的统计数据的完整描述在Garré等人(2016)中提出。

笔记

  1. 由于用于该制剂的小鼠在CX3C1细胞(小神经胶质细胞和血管周围巨噬细胞)中表达EGFP蛋白,因此脊髓部分暴露于激发光应该被最小化。它可能会影响EGFP + 细胞的数量以及荧光信号的质量。
  2. 已经在从小鼠皮层制备的切片中观察到小神经胶质细胞和星形胶质细胞活化的快速表现(Takano等人,2014)。在我们的手中,脊髓切片后2小时,我们还没有看到小胶质细胞或星形胶质细胞活化的明显形态学迹象,最长时间测试。基底膜TNFα水平在切除小胶质细胞的切片中显着降低(Garré等,2016)。为了提高我们协议的重现性,我们建议您记录切片使用多长时间。
  3. 如果这种制剂用于电生理记录和/或在切片后2小时以上的时间段,可以使用制备富集的ACSF的替代方法(见Mitra和Brownstone,2012)。

食谱

  1. 人造脑脊液(ACSF)
    注意:建议采用新鲜的配方。
    119.0 mM NaCl
    26.2mM NaHCO 3
    11.0 mM葡萄糖
    2.5 mM KCl
    1.0mM NaH 2 PO 4
    2.5mM CaCl 2
    1.3mM MgCl 2
    使用2 N HCl或5 N NaOH调节pH至7.4,分别降低或提高pH值 测量渗透压(300-310 mOsm)
    过滤解决方案
  2. Ca 2 + 和Mg 2 + free-ACSF
    119.0 mM NaCl
    26.2mM NaHCO 3
    11.0 mM葡萄糖
    2.5 mM KCl
    1.0mM NaH 2 PO 4
    5.0 mM EDTA
    使用2 N HCl或5 N NaOH调节pH至7.4,分别降低或提高pH值 测量渗透压(300-310 mOsm)
    过滤解决方案
  3. 3-4%PFA,pH = 7.4
    在Ca 2+ +和/或无游离PBS(Thermo Fisher Scientific)中稀释浓缩的32%PFA储备溶液(Electron Microscopy Sciences),使溶液3-4% PFA,pH = 7.4

致谢

这里的协议改编自Garré等人。(2016)。这项工作得到美国国家卫生研究院NS45287和NS55363对M.V.L.B的支持。和NS072238给F.F.B.,National Institutes of Health Grants GM107469和AG048410给G.Y.立陶宛研究委员会MIP-76/2015至F.F.B.

参考文献

  1. Contreras,JE,Saez,JC,Bukauskas,FF和Bennett,MV(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/13130072 "target ="_ blank">连接蛋白43(Cx43)半通道的门控和调控。美国国家科学院院刊100(20):11388-11393。
  2. Davalos,D.,Grutzendler,J.,Yang,G.,Kim,JV,Zuo,Y.,Jung,S.,Littman,DR,Dustin,MLand Gan,WB(2005)。< a class = "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15895084"target ="_ blank"> ATP介导体内局部脑损伤的快速小胶质细胞反应 Nat Neurosci 8(6):752-758。
  3. Denk,W.,Strickler,JH和Webb,WW(1990)。  双光子激光扫描荧光显微镜。 科学 248(4951):73-76。
  4. Garré,JM,Retamal,MA,Cassina,P.,Barbeito,L.,Bukauskas,FF,Saez,JC,Bennett,MV and Abudara,V.(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21148774"target ="_ blank"> FGF-1诱导培养中脊髓星形胶质细胞的ATP释放,并打开pannexin和连接蛋白半通道。 > Proc Natl Acad Sci USA 107(52):22659-22664。
  5. Garré,JM,Yang,G.,Bukauskas,FF and Bennett,MV(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27122036"target ="_ blank"> FGF-1触发啮齿类动物脊髓星形胶质细胞中的Pannexin-1半通道开放,并促进急性脊髓切片中的炎症反应。 Neurosci 36(17): 4785-4801。
  6. Gee,JM,Smith,NA,Fernandez,FR,Economo,MN,Brunert,D.,Rothermel,M.,Morris,SC,Talbot,A.,Palumbos,S.,Ichida,JM,Shepherd,JD, PJ,Wachowiak,M.,Capecchi,MR,Wilcox,KS,White,JA和Tvrdik,P。(2014)。 使用"Polr2a"的神经元和胶质细胞的成像活动基因和Cre依赖性GCaMP5G-IRES-tdTomato记录小鼠。  Neuron 83(5):1058-1072。
  7. Kim,JV,Jiang,N.,Tadokoro,CE,Liu,L.,Ransohoff,RM,Lafaille,JJ and Dustin,ML(2010)。< a class ="ke-insertfile"href ="http:/www.ncbi.nlm.nih.gov/pubmed/19800886"target ="_ blank">完整脊髓和大脑皮质的双光子激光扫描显微镜成像显示皮质损伤部位免疫细胞浸润中CXCR6和神经炎症的需求。 免疫方法 352(1-2):89-100。
  8. Mitra,P.和Brownstone,RM(2012)。  用于从成年小鼠的腰椎运动神经元进行记录的体外脊髓切片制剂。神经生物学 107(2):728-741。
  9. Takano,T.,He,W.,Han,X.,Wang,F.,Xu,Q.,Wang,X.,Oberheim Bush,NA,Cruz,N.,Dienel,GA和Nedergaard,M。(2014 )。急性海马脑切片中反应性星形胶质细胞的快速表现。 Glia 62(1):78-95。
  10. White,JG,Amos,WB和Fordham,M.(1987)。通过荧光光学显微镜评估生物结构的共聚焦与常规成像。细胞生物 105(1):41-48。
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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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Garré, J. M., Yang, G., Bukauskas, F. F. and Bennett, M. V. (2017). An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo. Bio-protocol 7(2): e2102. DOI: 10.21769/BioProtoc.2102.
  2. Garré, J. M., Yang, G., Bukauskas, F. F. and Bennett, M. V. (2016). FGF-1 triggers Pannexin-1 hemichannel opening in spinal astrocytes of rodents and promotes inflammatory responses in acute spinal cord slices. J Neurosci 36(17): 4785-4801.
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