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Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice

Abstract

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7–14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature.

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Figure 1: Overview of the procedure.
Figure 2: Methods of 2D quantifications of the choroidal lesion.
Figure 3: FITC-dextran–labeled flat-mounted choroid at different times after laser induction.
Figure 4: Immunostaining for inflammatory cells on a flat-mounted choroid collected at different time points after laser induction.
Figure 5: Typical analyses of laser-induced CNV in mice.
Figure 6: Metabolomics analysis of sera collected from mice subjected or not to laser induction and injected or not injected with pre-miR.

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Acknowledgements

This work was supported by grants from the Fonds National de la Recherche Scientifique–FNRS (F.R.S.-FNRS, Belgium), the Foundation against Cancer (foundation of public interest, Belgium), the Fonds spéciaux de la Recherche (University of Liège), the Direction Générale Opérationnelle de l'Economie, de l'Emploi et de la Recherche from the S.P.W. (Région Wallonne, Belgium), the Interuniversity Attraction Poles Programme–Belgian Science Policy (Brussels, Belgium), the Plan National Cancer (Service Public Fédéral), the Actions de Recherche Concertées (University of Liège, Belgium). We thank S. Ormenese and G. Moraes from the GIGA Cell Imaging and Flow Cytometry facility for their support with confocal microscopy acquisitions, as well as members of the GIGA animal facility platform for their help.

Author information

Authors and Affiliations

Authors

Contributions

V.L. set up the assay CNV, collected the data and compiled the protocol with input from the authors. J.L. performed the optimization of BM transplantation and conducted the assay with knockout mice. S.H. carried out immunostaining techniques. S.B. developed the methods of quantification. M.-L.A.G. contributed to the setup of intravitreal injection. I.S. conducted the assays with mir21. N.E.S. conducted the assays with Sunitinib. P.d.T. performed metabolomics study. E.R. designed statistical analysis. J.M.F. and J.-M.R. contributed to the design of the study and critically evaluated the manuscript. A.N. designed the assays and wrote the manuscript. All authors revised the manuscript.

Corresponding author

Correspondence to Agnès Noel.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Laser burn induction.

Laser burn induction with the apparition of a bubble, which confirms the efficiency of the laser impact. (AVI 11029 kb)

Eye cleaning.

Eye is properly cleaned by removing muscles in excess and the optic nerve. Some conjunctive tissues are left close to the limbus to facilitate the eye grip during the dissection. (MP4 55859 kb)

Cornea and retina removal.

Cornea and retina are gently removed. The sclera-choroid complex is turned upside down and cut between the laser impacts. Choroid is placed on a microscopic slide, mounted medium (Vectashield) is put down and a coverslip is placed on the flat mounted choroid. (MP4 63010 kb)

3D reconstruction.

Illustration of a 3D reconstruction of laser impact after confocal imaging. (AVI 1700 kb)

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Lambert, V., Lecomte, J., Hansen, S. et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat Protoc 8, 2197–2211 (2013). https://doi.org/10.1038/nprot.2013.135

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