title: Ultrasound microbubbles enhance recombinant adeno-associated virus vector delivery to retinal ganglion cells in vivo.
abstract: OBJECTIVE
  The aim of this study was to investigate whether ultrasound-mediated microbubble destruction enhances the transduction efficiency of recombinant adeno-associated virus (rAAV)-mediated enhanced green fluorescent protein (EGFP) gene into retinal ganglion cells (RGCs) of rats and whether it causes relevant adverse effects.
  
  
  METHODS
  Thirty-two adult Sprague-Dawley rats were divided into four groups with different ultrasound powers, and retinal flat mounts and hematoxylin and eosin staining sections were made for optimization of parameters. A further 70 adult Sprague-Dawley rats were divided into four groups randomly. The first group (group A) was used as a normal control with 10 rats, and the remaining rats were evenly divided into groups B, C, and D. Each group included 20 rats. Groups B and C received rAAV-encoding EGFP gene (rAAV(2)-EGFP) in phosphate-buffered saline without and with ultrasound to the retina, respectively. Group D received microbubbles and rAAV(2)-EGFP mixture and ultrasound to the retina. The injection approach was intravitreal injection for all eyes. After 21 days, RGCs were labeled retrogradely with Fluoro-Gold. After 28 days, retinal flat mounts, frozen sections, and pathologic sections were assessed in each group. Expression of EGFP reporter gene was observed on laser confocal microscopy and evaluated according to average optical density and transfected RGC rate. To evaluate adverse effects with retinal flat mounts, labeled RGCs were counted, and retinal pathologic sections were examined.
  
  
  RESULTS
  When ultrasound parameters (frequency, 0.3 MHz; power, 0.5 W/cm(2); total time, 60 seconds [irradiation time, 5 seconds; interval time, 10 seconds; four times]) were selected, EGFP expression was stronger, and retinas were not damaged. In the second part of the experiment, RGCs were labeled with Fluoro-Gold successfully. Green fluorescence can be observed in labeled RGCs in groups B to D. While average optical density and transfected RGC rate in group D were the highest compared to the other groups, no significant reduction in RGC number was detected with retrograde labeling. No obvious damage was observed with pathologic sections.
  
  
  CONCLUSIONS
  Ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGCs in rats, and it may represent a novel gene delivery method in gene therapy for glaucomatous optic neuroprotection.
authors.affiliation: Department of Ophthalmology, Second Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing 400010, China.
authors.forename: Wenyue
authors.initials: W
authors.lastname: Xie
authors.forename: Su
authors.initials: S
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Su
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wang
authors.forename: Yuanyi
authors.initials: Y
authors.lastname: Zheng
authors.forename: Yi
authors.initials: Y
authors.lastname: Fu
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D066298:In Vitro Techniques; D008297:Male; D045423:Microbubbles; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000528:radiation effects* / Q000821:virology*; D013010:Sonication / Q000379:methods*; D014162:Transfection / Q000379:methods*