Harvesting, Isolation, and Functional Assessment of Primary Vagal Ganglia Cells

Eric Dubuis1, Megan Grace1, Michael A. Wortley1, Mark A. Birrell1, Maria G. Belvisi1

1 Respiratory Pharmacology Group, Pharmacology and Toxicology Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.15
DOI:  10.1002/0471141755.ph1215s62
Online Posting Date:  October, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Airway sensory nerves play an important defensive role in the lungs, being central in mediating protective responses like cough and bronchoconstriction. In some cases, these responses become excessive, hypersensitive, and deleterious. Understanding the normal function of airway nerves and phenotype changes associated with disease will help in developing new therapeutics for treating chronic obstructive pulmonary disease and chronic cough. Guinea pigs, and to a lesser extent ferrets, are commonly employed for studying the cough reflex because they have a cough response similar to humans. While rats and mice do not exhibit a cough response, they do possess sensory nerves that respond to the same range of tussive stimuli as guinea pigs and humans. Described in this unit are protocols for harvesting guinea pig, mouse, and rat sensory nerve cell bodies to assess molecular and functional changes associated with pulmonary disease, and to identify new targets for therapeutic intervention. Curr. Protoc. Pharmacol. 62:12.15.1‐12.15.27. © 2013 by John Wiley & Sons, Inc.

Keywords: neuron; vagal ganglia; retrograde labeling; harvesting; primary culture

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: In Vivo Retrograde Labeling of Airway Neurons
  • Basic Protocol 2: Vagal Ganglia Harvesting from Guinea Pig
  • Alternate Protocol 1: Vagal Ganglia Harvesting from Mice and Rats
  • Basic Protocol 3: Enzymatic Isolation of Neurons from Vagal Ganglia
  • Support Protocol 1: Functional Assessment of Primary Cultured Vagal Ganglia Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: In Vivo Retrograde Labeling of Airway Neurons

  Materials
  • 0.5 mg/ml DiI (DiIC 18 or 3 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate; see recipe) in 0.9% sterile saline
  • Mice, rats, or guinea pigs
  • Isoflurane (Isoflo; Abbott) suitable for vaporization
  • Balance suitable for weighing animals
  • Inhaled anesthetic system, e.g., Vaportec Series 3 Vaporizers (Burtons Medical Equipment Ltd.)
  • Pipet with sterile tips (1‐ml for guinea pigs, 100‐µl for mice)
  • Needle for intratracheal administration of dye; for rats, use 15‐ to 18‐G blunt‐end needle, bent ∼30°, 10 mm from the end (a bent modified metal oral dosing gavage needle is employed; see Fig. A for scaled picture)
  • 1‐ml syringe
  • Small operating table/bench (angled to ∼15°)

Basic Protocol 2: Vagal Ganglia Harvesting from Guinea Pig

  Materials
  • Euthatal (or equivalent, 200 mg/ml pentobarbitone) in a syringe with a needle attached (21‐G for guinea pigs, 23‐G for rats, and 25‐G for mice)
  • 70% ethanol in a spray‐bottle
  • Sterile Hanks’ balanced salt solution without calcium, magnesium, and phenol red (HBSS; see recipe), keep on ice
  • Institutionally approved laboratory disinfectant solution
  • Sharps waste container
  • Set of sterile instruments (World Precision Instruments; see Fig. ):
    • Two pairs of straight Mayo dissecting scissor (14 cm)
    • Two pairs of straight‐tip iris scissor (10 cm)
    • Dissecting scissors straight with one sharp tip and one blunt tip (12.5 cm)
    • Liston bone cutting forceps (14 cm)
    • Curved blunt forceps (iris 14 cm serrated)
    • Pliers with serrated jaws (12 cm), optional
    • Three fine forceps no. 5
    • Corneal scissor with curved blades (blade 3‐4 mm, tip 100 µm or less)
  • Sterile disposable sample tube (≈7 ml)
  • Appropriate animal waste container
NOTE: All tools should be sterilized in an autoclave. All solutions should be filter‐sterilized using a 0.2‐µm filter. A separate set of sterile instruments must be used each time to conduct the dissection. If more than one animal is to be dissected, use one sterile set of tools per animal.

Alternate Protocol 1: Vagal Ganglia Harvesting from Mice and Rats

  Materials
  • Dissected ganglia in HBSS (see protocol 2 or protocol 3Alternate Protocol), on ice
  • Poly‐D‐lysine‐coated fluorodishes (sterile 35‐mm glass tissue culture dish with cover, WPI Ltd.)
  • HBSS (see recipe), ice cold and room temperature
  • Laminin (see recipe)
  • Papain solution (see recipe)
  • Papain activation solution (PAS)
  • Collagenase type IV/dispase II solution (see recipe)
  • F12 medium (see recipe)
  • Percoll (see recipe)
  • Sterile L15 medium (see recipe)
  • Complete F12 medium (see recipe)
  • Class II biological hood
  • Sterile gloves
  • 15‐ml conical tubes
  • 37°C water bath
  • 2‐ml microcentrifuge tubes
  • Dissecting dishes
  • Dissecting microscope
  • Fine forceps no. 5, sterile
  • Corneal scissors with fine curved blade (blade: 3 to 4 mm, tip: ≤100‐µm)
  • Petri dishes
  • Sterile glass culture dish (∼5‐cm diameter)
  • Centrifuge
  • Box of disposable sterile plastic pipets
  • Rocker
  • Set of five glass Pasteur pipets with successively smaller tip sizes (ranging from 1 to 0.3 mm)
  • 37°C, 5% CO 2 cell culture incubator

Basic Protocol 3: Enzymatic Isolation of Neurons from Vagal Ganglia

  Materials
  • Fura‐2AM (see recipe)
  • Culture medium
  • Cells in fluorodish (see protocol 4)
  • Inverted epifluorescence microscope (e.g., Zeiss Axiovert 200, Carl Zeiss Microscopy), mounted on an anti‐vibration table (e.g., TMC 63–500 Series table, Scientifica)
  • Incubation chamber (e.g., XL‐3 incubator, Carl Zeiss Microscopy)
  • Epi‐illuminator with a stable light source (e.g., HBO 50 100 W mercury/xenon light arc lamp, Carl Zeiss Microscopy)
  • Tungsten‐halogen lamp with housing and heat filter (HXP 100, Carl Zeiss Microscopy)
  • Air objective lens with medium magnification, 10× and 20× (e.g., 20× LD Plan‐Neofluar KORR air objective for a Zeiss microscope with a 0.4 aperture and 7.9 mm focal distance, Carl Zeiss Microscopy)
  • Excitation and emission filters and a set of dichromatic mirrors appropriate for the fluorescent dyes employed (BP 335/7, BP 387/11, BS 410, and BP 520/10 for Fura2, and BP 531/40, BS 565, and BP 593/40 for DiI, Carl Zeiss Microscopy); to record intracellular calcium with ratiometric dye (i.e., Fura‐2) the microscope should be equipped with a filter wheel or rotating prism such as the OptoScan monochromator (Cairn Research Ltd.) to rapidly change the excitation light
  • Set of shutters to control the light exposition time during and between experiments (Carl Zeiss Microscopy)
  • Cooled, fast‐acquisition CCD camera (e.g., EM‐CCD C9100‐02 camera driven by Simple‐PCI software, Hamamatsu)
  • A computer with sufficient data storage capacity and software to control the positioning of filters, shutters, and image acquisition system
  • Software to record image data (SimplePCI, Hamamatsu)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., and Struhl, K. (eds.). 2013. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Barnes, P.J. 1996. Neuroeffector mechanisms: The interface between inflammation and neuronal responses. J. Allergy Clin. Immunol. 98:S73‐S81; discussion S81‐S73.
  Bautista, D.M., Jordt, S.E., Nikai, T., Tsuruda, P.R., Read, A.J., Poblete, J., Yamoah, E.N., Basbaum, A.I., and Julius, D. 2006. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124:1269‐1282.
  Belvisi, M.G. 2003a. Airway sensory innervation as a target for novel therapies: An outdated concept? Curr. Opin. Pharmacol. 3:239‐243.
  Belvisi, M.G. 2003b. Sensory nerves and airway inflammation: Role of A delta and C‐fibres. Pulm. Pharmacol. Ther. 16:1‐7.
  Belvisi, M.G., Dubuis, E., and Birrell, M.A. 2011. Transient receptor potential A1 channels: Insights into cough and airway inflammatory disease. Chest 140:1040‐1047.
  Birrell, M.A., Belvisi, M.G., Grace, M., Sadofsky, L., Faruqi, S., Hele, D.J., Maher, S.A., Freund‐Michel, V., and Morice, A.H. 2009. TRPA1 agonists evoke coughing in guinea pig and human volunteers. Am. J. Respir. Crit. Care Med. 180:1042‐1047.
  Canning, B.J. 2008. The cough reflex in animals: Relevance to human cough research. Lung 186:S23‐S28.
  Canning, B.J. 2010. Afferent nerves regulating the cough reflex: Mechanisms and mediators of cough in disease. Otolaryngol. Clin. North Am. 43:15‐25.
  Canning, B.J. and Spina, D. 2009. Sensory nerves and airway irritability. Handb. Exp. Pharmacol. 139‐183.
  De Alba, J., Raemdonck, K., Dekkak, A., Collins, M., Wong, S., Nials, A.T., Knowles, R.G., Belvisi, M.G., and Birrell, M.A. 2010. House dust mite induces direct airway inflammation in vivo: Implications for future disease therapy? Eur. Respir. J. 35:1377‐1387.
  Geppetti, P., Patacchini, R., Nassini, R., and Materazzi, S. 2010. Cough: The emerging role of the TRPA1 channel. Lung 188:S63‐S68.
  Grace, M., Birrell, M.A., Dubuis, E., Maher, S.A., and Belvisi, M.G. 2012. Transient receptor potential channels mediate the tussive response to prostaglandin E2 and bradykinin. Thorax 67:891‐900.
  Slatko, B.E., Kieleczawa, J., Ju, J., Gardner, A.F., Hendrickson, C.L., and Ausubel, F.M. 2011. “First generation” automated DNA sequencing technology. Curr. Protoc. Mol. Biol. 96:7.2.1‐7.2.28.
  Springer, J., Geppetti, P., Fischer, A., and Groneberg, D.A. 2003. Calcitonin gene‐related peptide as inflammatory mediator. Pulm. Pharmacol. Ther. 16:121‐130.
  Stevenson, C.S. and Belvisi, M.G. 2008. Preclinical animal models of asthma and chronic obstructive pulmonary disease. Exp. Rev. Respir. Med. 2:631‐643.
  Taylor‐Clark, T.E., Nassenstein, C., and Undem, B.J. 2008. Leukotriene D4 increases the excitability of capsaicin‐sensitive nasal sensory nerves to electrical and chemical stimuli. Br. J. Pharmacol 154:1359‐1368.
  Texier, I., Goutayer, M., Da Silva, A., Guyon, L., Djaker, N., Josserand, V., Neumann, E., Bibette, J., and Vinet, F. 2009. Cyanine‐loaded lipid nanoparticles for improved in vivo fluorescence imaging. J. Biomed. Opt. 14:054005.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library