Remote delivery of rAAV-GFP to the rat brainstem through the recurrent laryngeal nerve.
OBJECTIVE
To demonstrate that a recombinant adeno-associated viral vector (rAAV) carrying the gene for green fluorescent protein (GFP) could be delivered to the rat brainstem by remote injection into the recurrent laryngeal nerve.


METHODS
rAAV-GFP is a serotype 2 adeno-associated vector containing the cDNA of GFP and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under the control of the CAG promoter (obtained from Matthew During, PhD, Thomas Jefferson Medical College). Five microliters or 10 microL of 1.4 x 109 particles/microL of rAAV-GFP were injected into the right recurrent laryngeal nerve of adult Sprague-Dawley rats. Rats were killed and perfused at 3 (n = 3) and 11 weeks (n = 3). Brainstems were removed and cryosectioned. Fluorescent in-situ hybridization (FISH) was performed on cryosections from animals killed at 3 weeks using a cDNA probe for woodchuck polyribosomal enzyme within the rAAV vector. In a third group (n = 2), Fluoro-Gold (Fluorochrome, Inc., Denver, CO) was injected into the right thyroarytenoid muscle for comparison of neuronal uptake distribution. These rats were killed and perfused at 3 weeks.


RESULTS
The presence of GFP was noted in neurons throughout the medulla of all rat brainstems after unilateral rAAV-GFP injection at both 3 and 11 weeks. In contrast to the Fluoro-Gold, GFP was noted bilaterally and outside of the nucleus ambiguus. FISH confirmed the presence of virus within neurons expressing GFP at 3 weeks.


CONCLUSIONS
Remote delivery of rAAV-GFP to the rat brainstem is possible through injection into the recurrent laryngeal nerve. This has important therapeutic implications for the future treatment of recurrent laryngeal nerve injury and neurodegenerative diseases.
Rubin
A D
AD
Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109, USA. rubinad@umich.edu
Hogikyan
N D
ND
Sullivan
K
K
Boulis
N
N
Feldman
E L
EL
D000818:Animals; D001933:Brain Stem / Q000378:metabolism*; D000229:Dependovirus; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D017919:Hepatitis B Virus, Woodchuck; D017404:In Situ Hybridization, Fluorescence; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000008:administration & dosage*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012009:Recurrent Laryngeal Nerve
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins