title: Correlation of in vivo and in vitro methods in measuring choroidal vascularization volumes using a subretinal injection induced choroidal neovascularization model.
abstract: BACKGROUND
  In vivo quantification of choroidal neovascularization (CNV) based on noninvasive optical coherence tomography (OCT) examination and in vitro choroidal flatmount immunohistochemistry stained of CNV currently were used to evaluate the process and severity of age-related macular degeneration (AMD) both in human and animal studies. This study aimed to investigate the correlation between these two methods in murine CNV models induced by subretinal injection.
  
  
  METHODS
  CNV was developed in 20 C57BL6/j mice by subretinal injection of adeno-associated viral delivery of a short hairpin RNA targeting sFLT-1 (AAV.shRNA.sFLT-1), as reported previously. After 4 weeks, CNV was imaged by OCT and fluorescence angiography. The scaling factors for each dimension, x, y, and z (μm/pixel) were recorded, and the corneal curvature standard was adjusted from human (7.7) to mice (1.4). The volume of each OCT image stack was calculated and then normalized by multiplying the number of voxels by the scaling factors for each dimension in Seg3D software (University of Utah Scientific Computing and Imaging Institute, available at http://www.sci.utah.edu/cibc-software/seg3d.html). Eighteen mice were prepared for choroidal flatmounts and stained by CD31. The CNV volumes were calculated using scanning laser confocal microscopy after immunohistochemistry staining. Two mice were stained by Hematoxylin and Eosin for observing the CNV morphology.
  
  
  RESULTS
  The CNV volume calculated using OCT was, on average, 2.6 times larger than the volume calculated using the laser confocal microscopy. The correlation statistical analysis showed OCT measuring of CNV correlated significantly with the in vitro method (R 2 =0.448, P = 0.001, n = 18). The correlation coefficient for CNV quantification using OCT and confocal microscopy was 0.693 (n = 18, P = 0.001).
  
  
  CONCLUSIONS
  There is a fair linear correlation on CNV volumes between in vivo and in vitro methods in CNV models induced by subretinal injection. The result might provide a useful evaluation of CNV both for the studies using CNV models induced by subretinal injection and human AMD studies.
authors.forename: Chuang
authors.initials: C
authors.lastname: Nie
authors.forename: Mao-Nian
authors.initials: MN
authors.lastname: Zhang
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Zhao
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Olsen
authors.forename: Kyle
authors.initials: K
authors.lastname: Jackman
authors.forename: Lian-Na
authors.initials: LN
authors.lastname: Hu
authors.forename: Wen-Ping
authors.initials: WP
authors.lastname: Ma
authors.forename: Xiao-Fei
authors.initials: XF
authors.lastname: Chen
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tie-Shan
authors.initials: TS
authors.lastname: Gao
authors.forename: Hiro
authors.initials: H
authors.lastname: Uehara
authors.affiliation: Moran Eye Center, University of Utah, Salt Lake City, Utah 84132, USA.
authors.forename: Balamurali K
authors.initials: BK
authors.lastname: Ambati
authors.affiliation: Department of Ophthalmology, The 306th Hospital of PLA, Beijing 100101, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Luo
mesh_terms: D000818:Animals; D020256:Choroidal Neovascularization / Q000473:pathology* / Q000503:physiopathology; D004195:Disease Models, Animal; D005451:Fluorescein Angiography; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D041623:Tomography, Optical Coherence