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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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

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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

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Materials

Basic Protocol 1: In Vivo Retrograde Labeling of Airway Neurons

Materials

0.5 mg/ml DiI (DiIC₁₈ 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% CO₂ 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)

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Figures

Figure 12.15.1 Anatomical differences between the rat/mouse and guinea pig. (A ) Real‐size picture of the curved intratracheal cannula needed to dose the rats with the retrograde dye DiI. The value of the angle used to bend the cannula is given in degrees and the vertex is indicated by the cross of the dash lines located 13 mm from the tip of the cannula. (B ,C ) Diagram of the vagal ganglia in guinea pig (left panel) and mouse/rat (right panel).

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Figure 12.15.2 Overview of the tools needed for the dissection and harvesting of vagal ganglia.

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Figure 12.15.3 Muscle removal from head and neck section for guinea pigs. (A ‐C ) Lateral and dorsal views of the step‐by‐step removal of muscles needed to facilitate the opening of the skull and the extraction of the ear bone.

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Figure 12.15.4 Skull opening and removal of the brain. (A ) Dorsal view of step‐by‐step cut and removal of the skull bones. (B ) Removal of parietal and interparietal bones. (C ) Lateral view of the brain and optical chiasma section. (D ) Dorsal view of cuts of the medulla connected nerves and the spinal cord.

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Figure 12.15.5 Ear bones extraction and vagal ganglia harvesting. (A ) Cuts required in the surrounding bones to free the ear bone without dismantling the skull. (B ) 3‐D schematic showing how to handle the body while extracting the ear bone and dorsal view showing the position of the forceps during the extraction. (C ) Schematic of vagal ganglia (nodose and jugular) harvesting in guinea pig after extraction of the ear bone. Inset shows more in details how the jugular and nodose ganglia are located near the occipital bone.

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Figure 12.15.6 Adjustments in muscle and bone removal for mice and rats. (A ) Lateral schematic of muscle removal from rat neck and head. (B ) Dorsal schematic of bones removal from the top of rat skull.

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Figure 12.15.7 Schematic of equipment classically needed to perform epifluorescence recordings.

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Figure 12.15.8 Steps to follow to load neurons with Fura2‐AM.

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Figure 12.15.9 Functional assessment of an airway‐specific neuron. (A ) (1) images of neurons recorded in bright‐field differential interference contrast (DIC) microscopy mode. (2) DiI fluorescence with the light intensity expressed by a black (no light) to red (highest light) color code. (3) Combined image of DiC and DiI recordings. (4) Typical light intensity observed for Fura2 bound to calcium at rest. (5) Typical light intensity for unbound Fura2 at rest. The scale indicated on top of panel (1) is a 50‐µm scaled bar. (B ) Ratio of the light intensity emitted by calcium‐bound Fura2/unbound Fura measured by respectively exposing the neuron to λ = 340 nm and λ = 380 nm. Fluorescence scale is indicated on the left. Drug perfusions are indicated below the trace by black arrows. Time scale is given below the trace by a black bar. The pictures below the trace represent the light intensities recorded over time (examples taken as indicated with numbers on the trace).

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Literature Cited

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