April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Evaluation of Alternative Methods to Quantify Ocular Angiogenesis in Mice
Author Affiliations & Notes
  • K. Paes
    Ophthalmology, Lexicon Pharmaceuticals, Inc, The Woodlands, Texas
  • E. Wang
    Ophthalmology, Lexicon Pharmaceuticals, Inc, The Woodlands, Texas
  • R. Wudali
    Ophthalmology, Lexicon Pharmaceuticals, Inc, The Woodlands, Texas
  • D. S. Rice
    Ophthalmology, Lexicon Pharmaceuticals, Inc, The Woodlands, Texas
  • Footnotes
    Commercial Relationships  K. Paes, Lexicon Pharmaceuticals, Inc., F; E. Wang, Lexicon Pharmaceuticals, Inc., F; R. Wudali, Lexicon Pharmaceuticals, Inc., F; D.S. Rice, Lexicon Pharmaceuticals, Inc., F.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4949. doi:
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      K. Paes, E. Wang, R. Wudali, D. S. Rice; Evaluation of Alternative Methods to Quantify Ocular Angiogenesis in Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4949.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Gene manipulations or therapeutic agents aimed at the vasculature often employ ocular tissue analysis from mouse preclinical models. Standard methodology to quantify effects on the vasculature is often very labor intensive. Therefore, we established several alternative techniques to study physiological and pathological angiogenesis in mouse models using rapamycin as a control agent.

Methods: : Physiological retinal angiogenesis (DRA): Neonatal C57BL/6J mice were administered 4mg/Kg Rapamycin (s.c.) or vehicle once daily from birth to postnatal day (PD)4. On PD5 mice were euthanized and eyes were collected for wholemount retinal vessel staining with Isolectin-IB4. Wholemount retinas were subjected to a digital analysis (AngioSys) of vascular morphology.Pathological retinal angiogenesis (OIR): C57BL/6J mouse pups were exposed to 75% oxygen from PD7-12. Pups were returned to room air and received 4mg/Kg rapamycin (i.p.) or vehicle once daily from PD12-16. On PD17 pups were euthanized and eyes enucleated for retinal wholemount vascular staining (anti-NG2 combined with isolectin IB4) and histological analysis of vaso-obliteration and neovascularization.Laser-induced choroidal neovascularization (CNV): Rupture of Bruch’s membrane was induced in adult C57BL/6J mice via focal laser photocoagulation. Rapamycin was administered (i.p.) at 4mg/Kg once daily through seven days post CNV induction. Angiography was performed at day seven to evaluate the extent of vascular leakage from CNV lesions. Animals were then euthanized and eyes were collected to prepare choroidal flat-mounts and stained with various vascular markers. Confocal imaging and morphometry (Imaris) was performed on flat-mounted tissues to evaluate effects of rapamycin on the lesions.

Results: : Rapamycin treatment in the DRA model resulted in smaller retinal areas (13%, p<0.0001), reduced vascular length (77%, p<0.0001) and branching (85%, p<0.0001) under physiological conditions when compared to vehicle treated neonates. In the OIR model, rapamycin treated pups exhibited a much larger area of vaso-obliteration (43%, p=0.0002), significantly smaller neovascular area and (73%, p=0.0006) fewer pathological neovascular nuclei (76%, p<0.0001) compared to vehicle treated animals. Treatment of Rapamycin in the CNV model resulted in decreased neovascular leakage from lesion sites (65%, p<0.0001) and small lesions (57%, p<0.0001) relative to vehicle treated animals.

Conclusions: : The methodology employed in this study enables a thorough evaluation of physiological and pathological vasculature affected by systemic treatment of rapamycin.

Keywords: imaging/image analysis: non-clinical • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • neovascularization 

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