Gene Therapy of the Peripheral Nervous System: Celiac Ganglia

Fig. 1

Photograph of the celiac ganglia under a dissecting microscope. The celiac ganglia (circled) can be located by using the aorta, celiac artery, and superior mesenteric artery as landmarks

Keep the abdomen moist by draping the exposed tissue with sterile gauze soaked in Dulbecco’s Phosphate Buffered Saline.

Under a dissecting microscope, locate the CG (see Note 2 , Fig. 1) and remove excess connective tissue surrounding the CG (see Note 3 ).

Fill a glass micropipette pulled according to the specifications above, with a mixture of 1 μl of 1 × 10¹³ gc/ml AAV6, and 0.1 μl of 5 mM fast green (see Note 4 ). Mount the filled micropipette on a micromanipulator that is attached to a Picospritzer set at 15 ms. Keep the glass micropipette a safe distance from the CG until ready to pierce the CG (see Note 5 ).

Advance the pipette to a depth of 400 μm in the CG (see Notes 6 – 8 ). Wait 2 min, allowing the glass micropipette to make a seal with the CG. After 2 min, inject 25 % of the viral mixture into the CG by activating the Picospritzer as many times as needed (see Note 9 ). Wait 5 min before withdrawing the glass micropipette.

Move the glass micropipette to different locations around the CG and repeat step 7 until the total 1.1 μl volume is injected using four different injection sites. Be careful to inject the viral load evenly throughout the entire CG, using the fast green to track the location of the virus .

Remove the gut retractor, replace the abdominal viscera, and close the incision with 4-0 silk sutures.

10.

Give the animals analgesic (Carprofen 5 mg/kg) and antibiotic (Enrofloxacin 5 mg/kg) for 3 days post-surgery (House the animals in pairs for 4 weeks until viral expression is confirmed using immunohistochemistry) (see Note 10 ).

11.

Wipe down the workspace and any equipment used to handle AAVs with a 10 % bleach solution.

3.2 Immunohistocheical Detection of GFP

All washes and incubations are processed on a shaker at room temperature. Reduce light exposure of the samples by keepings the slides in a dark environment during washes and incubations. Each wash is 10 min.

4 weeks after the direct injection of AAV , deeply anesthetize the animals with sodium pentobarbital (50 mg/kg, ip), and then harvest the CG. Submerge the CG in 4 % paraformaldehyde for 1 day at 4 °C.

Transfer the CG to 20 % sucrose for 1 day at 4 °C, then freeze the sample at −80 °C in OCT until use.

Cryostat section (14–18 μm) the CG in a single series on to Superfrost^® plus micro slides.

Using a PAP pen outline the slides with a hydrophobic barrier.

Wash the slides three times in IB.

Incubate for with 10 % NGS (30 mins).

Incubate with anti-GFP antibody (1:1000, 1 day), diluted in 10 % NGS.

Wash three times with Tris-PBS.

Incubate with goat anti-chicken secondary antibody (1:500, 2 h) diluted in 1 % NGS.

10.

Coverslip the slides with ProLong^® Gold Antifade.

11.

Visualize the sections under a microscope capable of detecting the chromogen FITC.