Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.
We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 mug), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4 +/- 4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.
Ishikawa
H
H
Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Takano
M
M
Matsumoto
N
N
Sawada
H
H
Ide
C
C
Mimura
O
O
Dezawa
M
M
D000818:Animals; D019771:Axotomy; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000235:genetics; D002470:Cell Survival; D004566:Electrodes; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D049452:Green Fluorescent Proteins / Q000235:genetics; D007267:Injections; D008297:Male; D009414:Nerve Growth Factors / Q000235:genetics*; D009900:Optic Nerve / Q000601:surgery; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes; D014822:Vitreous Body
C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D049452:Green Fluorescent Proteins; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases