title: Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.
abstract: 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.
authors.affiliation: Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Ishikawa
authors.forename: M
authors.initials: M
authors.lastname: Takano
authors.forename: N
authors.initials: N
authors.lastname: Matsumoto
authors.forename: H
authors.initials: H
authors.lastname: Sawada
authors.forename: C
authors.initials: C
authors.lastname: Ide
authors.forename: O
authors.initials: O
authors.lastname: Mimura
authors.forename: M
authors.initials: M
authors.lastname: Dezawa
mesh_terms: 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
chemical_list: 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