Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Subretinal injection in mice to study retinal physiology and disease

Nature Protocols volume 17, pages 1468–1485 (2022)Cite this article

Subjects

Abstract

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a ‘successful’ SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with ‘procedure success’ defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7–14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Applications of SRI in rodent models.
Fig. 2: Depictions of three SRI approaches.
Fig. 3: Representative images of a proper and improper SRI procedure to study the RPE phenotype.
Fig. 4: Overview of the procedure.
Fig. 5: Representative images of a successful SRI.
Fig. 6: Identification of the injection site in mouse RPE flat mounts.
Fig. 7: Follow-up of successful and improper SRI of PBS.

Similar content being viewed by others

Data availability

All supporting data are available in the figures.

References

  1. Sharma, S. et al. Pneumatic displacement of submacular hemorrhage with subretinal air and tissue plasminogen activator: initial United States experience. Ophthalmol. Retin. 2, 180–186 (2018).

    Article  Google Scholar 

  2. Herbert, E. N., Groenewald, C. & Wong, D. Treatment of retinal folds using a modified macula relocation technique with perfluoro-hexyloctane tamponade. Br. J. Ophthalmol. 87, 921–922 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Russell, S. et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849–860 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pang, J. J. et al. Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. Mol. Ther. 13, 565–572 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Ling, S. et al. Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice. Nat. Biomed. Eng. 5, 144–156 (2021).

    Article  CAS  PubMed  Google Scholar 

  6. Sun, J. et al. Protective effects of human iPS-derived retinal pigmented epithelial cells in comparison with human mesenchymal stromal cells and human neural stem cells on the degenerating retina in rd1 mice. Stem Cells 33, 1543–1553 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Kaneko, H. et al. DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature 471, 325–330 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tarallo, V. et al. DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell 149, 847–859 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dridi, S. et al. ERK1/2 activation is a therapeutic target in age-related macular degeneration. Proc. Natl Acad. Sci. USA. 109, 13781–13786 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kerur, N. et al. TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy. Invest. Ophthalmol. Vis. Sci. 54, 7395–7401 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fowler, B. J. et al. Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity. Science 346, 1000–1003 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim, Y. et al. DICER1/Alu RNA dysmetabolism induces Caspase-8-mediated cell death in age-related macular degeneration. Proc. Natl Acad. Sci. USA. 111, 16082–16087 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gelfand, B. D. et al. Iron toxicity in the retina requires Alu RNA and the NLRP3 inflammasome. Cell Rep. 11, 1686–1693 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kerur, N. et al. cGAS drives noncanonical-inflammasome activation in age-related macular degeneration. Nat. Med. 24, 50–61 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Narendran, S. et al. Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-beta induced retinal pigmented epithelium degeneration. Signal Transduct. Target Ther. 6, 149 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fukuda, S. et al. Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration. Proc. Natl Acad. Sci. USA 118, e2022751118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fukuda, S. et al. Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity. Sci. Adv. 7, eabj3658 (2011).

    Article  CAS  Google Scholar 

  18. Little, C. W. et al. Transplantation of human fetal retinal pigment epithelium rescues photoreceptor cells from degeneration in the Royal College of Surgeons rat retina. Invest. Ophthalmol. Vis. Sci. 37, 204–211 (1996).

    CAS  PubMed  Google Scholar 

  19. Peng, Y., Tang, L. & Zhou, Y. Subretinal injection: a review on the novel route of therapeutic delivery for vitreoretinal diseases. Ophthalmic Res. 58, 217–226 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Iriyama, A. et al. A2E, a component of lipofuscin, is pro-angiogenic in vivo. J. Cell Physiol. 220, 469–475 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Lyzogubov, V. V., Tytarenko, R. G., Liu, J., Bora, N. S. & Bora, P. S. Polyethylene glycol (PEG)-induced mouse model of choroidal neovascularization. J. Biol. Chem. 286, 16229–16237 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Baba, T. et al. A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection. Am. J. Pathol. 176, 3085–3097 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Narendran, S. et al. A clinical metabolite of azidothymidine inhibits experimental choroidal neovascularization and retinal pigmented epithelium degeneration. Invest. Ophthalmol. Vis. Sci. 61, 4 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shen, D., Wen, R., Tuo, J., Bojanowski, C. M. & Chan, C. C. Exacerbation of retinal degeneration and choroidal neovascularization induced by subretinal injection of Matrigel in CCL2/MCP-1-deficient mice. Ophthalmic Res. 38, 71–73 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Jo, Y. J. et al. Establishment of a new animal model of focal subretinal fibrosis that resembles disciform lesion in advanced age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 52, 6089–6095 (2011).

    Article  PubMed  Google Scholar 

  26. Georgiadis, A. et al. Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65. Gene Ther. 23, 857–862 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Xu, H. et al. Subretinal delivery of AAV2-mediated human erythropoietin gene is protective and safe in experimental diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 55, 1519–1530 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Kim, K. et al. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration. Genome Res. 27, 419–426 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Holmgaard, A. B. et al. Targeted knockout of the Vegfa gene in the retina by subretinal injection of RNP complexes containing Cas9 protein and modified sgRNAs. Mol. Ther. 29, 191–207 (2021).

    Article  CAS  PubMed  Google Scholar 

  30. Holmgaard, A., Alsing, S., Askou, A. L. & Corydon, T. J. CRISPR gene therapy of the eye: targeted knockout of Vegfa in mouse retina by lentiviral delivery. Methods Mol. Biol. 1961, 307–328 (2019).

    Article  CAS  PubMed  Google Scholar 

  31. Wu, L. et al. Subretinal gene delivery using helper-dependent adenoviral vectors. Cell Biosci. 1, 15 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Huang, R. et al. Functional and morphological analysis of the subretinal injection of human retinal progenitor cells under Cyclosporin A treatment. Mol. Vis. 20, 1271–1280 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. McGill, T. J. et al. Subretinal transplantation of human central nervous system stem cells stimulates controlled proliferation of endogenous retinal pigment epithelium. Transl. Vis. Sci. Technol. 8, 43 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Jin, Z. B. et al. Stemming retinal regeneration with pluripotent stem cells. Prog. Retin. Eye Res. 69, 38–56 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Maya-Vetencourt, J. F. et al. Subretinally injected semiconducting polymer nanoparticles rescue vision in a rat model of retinal dystrophy. Nat. Nanotechnol. 15, 698–708 (2020).

    Article  CAS  PubMed  Google Scholar 

  36. Fernandez-Bueno, I., Alonso-Alonso, M. L., Garcia-Gutierrez, M. T. & Diebold, Y. Reliability and reproducibility of a rodent model of choroidal neovascularization based on the subretinal injection of polyethylene glycol. Mol. Vis. 25, 194–203 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lambert, N. G. et al. Subretinal AAV2.COMP-Ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration. Exp. Eye Res. 145, 248–257 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Luo, L. et al. Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1. eLife 2, e00324 (2013).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Luo, L. et al. Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration. ACS Nano 7, 3264–3275 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhang, X. et al. AAV2 delivery of Flt23k intraceptors inhibits murine choroidal neovascularization. Mol. Ther. 23, 226–234 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Cao, J. et al. A subretinal matrigel rat choroidal neovascularization (CNV) model and inhibition of CNV and associated inflammation and fibrosis by VEGF trap. Invest. Ophthalmol. Vis. Sci. 51, 6009–6017 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Qiu, G. et al. A new model of experimental subretinal neovascularization in the rabbit. Exp. Eye Res. 83, 141–152 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Timmers, A. M., Zhang, H., Squitieri, A. & Gonzalez-Pola, C. Subretinal injections in rodent eyes: effects on electrophysiology and histology of rat retina. Mol. Vis. 7, 131–137 (2001).

    CAS  PubMed  Google Scholar 

  44. Qi, Y. et al. Trans-corneal subretinal injection in mice and its effect on the function and morphology of the retina. PLoS One 10, e0136523 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  45. Nickerson, J. M. et al. Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes. Methods Mol. Biol. 884, 53–69 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Parikh, S. et al. An alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach. J. Vis. Exp. https://doi.org/10.3791/54808 (2016).

  47. Muhlfriedel, R., Michalakis, S., Garcia Garrido, M., Biel, M. & Seeliger, M. W. Optimized technique for subretinal injections in mice. Methods Mol. Biol. 935, 343–349 (2013).

    Article  PubMed  CAS  Google Scholar 

  48. Park, S. W., Kim, J. H., Park, W. J. & Kim, J. H. Limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium. J. Vis. Exp. https://doi.org/10.3791/53030 (2015).

  49. Bennett, J., Anand, V., Acland, G. M. & Maguire, A. M. Cross-species comparison of in vivo reporter gene expression after recombinant adeno-associated virus-mediated retinal transduction. Methods Enzymol. 316, 777–789 (2000).

    Article  CAS  PubMed  Google Scholar 

  50. Bartuma, H. et al. In vivo imaging of subretinal bleb-induced outer retinal degeneration in the rabbit. Invest. Ophthalmol. Vis. Sci. 56, 2423–2430 (2015).

    Article  PubMed  Google Scholar 

  51. Matsumoto, H., Miller, J. W. & Vavvas, D. G. Retinal detachment model in rodents by subretinal injection of sodium hyaluronate. J. Vis. Exp. https://doi.org/10.3791/50660 (2013).

  52. Huang, P. et al. The learning curve of murine subretinal injection among clinically trained ophthalmic surgeons. Transl. Vis. Sci. Technol. https://doi.org/10.1167/tvst.11.3.13 (2022).

  53. de Melo, J. & Blackshaw, S. In vivo electroporation of developing mouse retina. Methods Mol. Biol. 1715, 101–111 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  54. Zambricki, E. A. & Dalecy, L. G. Rat sex differences in anesthesia. Comp. Med. 54, 49–53 (2004).

    CAS  PubMed  Google Scholar 

  55. Pritchett-Corning, K. R., Luo, Y., Mulder, G. B. & White, W. J. Principles of rodent surgery for the new surgeon. J. Vis. Exp. https://doi.org/10.3791/2586 (2011).

  56. Lambert, V. et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat. Protoc. 8, 2197–2211 (2013).

    Article  CAS  PubMed  Google Scholar 

  57. Wright, C. B. et al. Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice. Proc. Natl Acad. Sci. USA. 117, 2579–2587 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wang, S. et al. DDX is an essential mediator of sterile NLRC inflammasome activation. DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs. Sci. Immunol. (in press).

Download references

Acknowledgements

J.A. has received support from the UVA Strategic Investment Fund, NIH grants (R01EY028027, R01EY29799 and R01EY31039), DuPont Guerry, III, Professorship, and a gift from Mr. and Mrs. Eli W. Tullis; B.D.G. has received support from NIH grants (R01EY028027, R01EY031039 and R01EY032512), BrightFocus Foundation and the Owens Family Foundation.

Author information

Authors and Affiliations

  1. Center for Advanced Vision Science, University of Virginia School of Medicine, Charlottesville, VA, USA

    Peirong Huang, Siddharth Narendran, Felipe Pereira, Shinichi Fukuda, Yosuke Nagasaka, Ivana Apicella, Praveen Yerramothu, Xiaoyu Cai, Bradley D. Gelfand & Jayakrishna Ambati

  2. Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville, VA, USA

    Peirong Huang, Siddharth Narendran, Felipe Pereira, Shinichi Fukuda, Yosuke Nagasaka, Ivana Apicella, Praveen Yerramothu, Xiaoyu Cai, Bradley D. Gelfand & Jayakrishna Ambati

  3. Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Peirong Huang

  4. Aravind Eye Care System, Madurai, India

    Siddharth Narendran

  5. Departamento de Oftalmologia e Ciências Visuais, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

    Felipe Pereira

  6. Department of Ophthalmology, University of Tsukuba, Tsukuba, Japan

    Shinichi Fukuda

  7. Doheny Eye Institute, Los Angeles, CA, USA

    Kenneth M. Marion & Srinivas R. Sadda

  8. Department of Ophthalmology, David Geffen School of Medicine, University of California–Los Angeles, Los Angeles, CA, USA

    Srinivas R. Sadda

  9. Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, VA, USA

    Bradley D. Gelfand

  10. Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA

    Jayakrishna Ambati

  11. Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, USA

    Jayakrishna Ambati

Authors
  1. Peirong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siddharth Narendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Felipe Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shinichi Fukuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yosuke Nagasaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ivana Apicella
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Praveen Yerramothu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kenneth M. Marion
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiaoyu Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Srinivas R. Sadda
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Bradley D. Gelfand
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jayakrishna Ambati
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, P.H. and J.A.; investigation, P.H., F.P., S.N., Y.N. and S.F.; writing, P.H., F.P., S.N., K.M.M. and J.A. with assistance from I.A., P.Y., X.C., S.R.S. and B.D.G. All authors had the opportunity to discuss the results and comment on the manuscript.

Corresponding author

Correspondence to Jayakrishna Ambati.

Ethics declarations

Competing interests

J.A. is a cofounder of iVeena Holdings, iVeena Delivery Systems and Inflammasome Therapeutics, and has been a consultant for Allergan, Boehringer-Ingelheim, Immunovant, Olix Pharmaceuticals, Retinal Solutions and Saksin LifeSciences unrelated to this work. S.R.S. has been a consultant for 4DMT, Abbvie/Allergan, Apellis, Amgen, Centervue, Heidelberg, Iveric, Novartis, Optos, Oxurion, Regeneron and Roche/Genentech, received speaker fees from Novartis, Nidek, Carl Zeiss Meditec and Optos, and received research instruments from Carl Zeiss Meditec, Nidek, Topcon, Centervue, Optos and Heidelberg unrelated to this work; J.A. and B.D.G. are cofounders of DiceRx. J.A., S.N., I.A., F.P. and B.D.G. are named as inventors on patent applications filed by their university.

Peer review

Peer review information

Nature Protocols thanks Jin-Hyoung Kim, Fenghua Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Kaneko, H. et al. Nature 471, 325–330 (2011): https://doi.org/10.1038/nature09830

Tarallo, V. et al. Cell 149, 847–859 (2012): https://doi.org/10.1016/j.cell.2012.03.036

Kerur, N. et al. Nat. Med. 24, 50–61 (2018): https://doi.org/10.1038/nm.4450

Extended data

Extended Data Fig. 1 Custom needle parameters.

Detailed illustration of the needles designed for subretinal injection in rat (top) and mice (bottom).

Supplementary information

Supplementary Information

Supplementary Manual.

Supplementary Video 1

Subretinal injection procedure

Supplementary Video 2

Optical lens

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, P., Narendran, S., Pereira, F. et al. Subretinal injection in mice to study retinal physiology and disease. Nat Protoc 17, 1468–1485 (2022). https://doi.org/10.1038/s41596-022-00689-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00689-4

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing