Standard Operating Procedures for Common Laboratory Animal Ocular Procedures

  • Brian C. GilgerEmail author
  • Joshua T. Bartoe
  • J. Seth Eaton
  • Ryan Boyd


In this chapter, we provide a basis for researchers, contract research laboratories, or other investigators to develop harmonized protocols for commonly performed ophthalmic procedures in laboratory animals and to assist in development of institutional standard operating procedure (SOP) documentation. Having similar protocols and SOPs among researchers and institutions will allow better comparison between studies and more efficient use of animals and enhance the quality of ocular research overall. The chapter is organized by procedure and animal species, including techniques such as intracameral, intravitreal, subconjunctival, retrobulbar, and subretinal injections.


Standard operating procedures Laboratory animal Ophthalmology Ocular toxicology 



The authors thank Justin Prater and David Culp of Powered Research for their assistance.


  1. 1.
    Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev. 2006;58(11):1131–5. Scholar
  2. 2.
    Walters TR, Lee SS, Goodkin ML, Whitcup SM, Robinson MR. Bimatoprost sustained-release implants for glaucoma therapy: 6-month results from a phase I/II clinical trial. Am J Ophthalmol. 2017;175:137–47. Scholar
  3. 3.
    Aref AA. Sustained drug delivery for glaucoma: current data and future trends. Curr Opin Ophthalmol. 2016;25(2):112–7. Scholar
  4. 4.
    Bawa G, Tkatchenko TV, Avrutsky I, Tkatchenko AV. Variational analysis of the mouse and rat eye optical parameters. Biomed Opt Express. 2013;4(11):2585–95. Scholar
  5. 5.
    Thomasy SM, Raghunathan VK, Winkler M, et al. Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium. Acta Biomater. 2014;10(2):785–91. Scholar
  6. 6.
    Kulkarni PS. The role of endogenous eicosanoids in rabbit-intraocular inflammation. J Ocul Pharmacol. 1991;7(3):227–41.CrossRefPubMedGoogle Scholar
  7. 7.
    Jampel HD, Brown A, Roberts A, Koya P, Quigley H. Effect of paracentesis upon the blood-aqueous barrier of cynomolgus monkeys. Investig Ophthalmol Vis Sci. 1992;33:165–71.Google Scholar
  8. 8.
    Allbaugh RA, Roush JK, Rankin AJ, Davidson HJ. Fluorophotometric and tonometric evaluation of ocular effects following aqueocentesis performed with needles of various sizes in dogs. Am J Vet Res. 2011;72(4):556–61. Scholar
  9. 9.
    del Amo EM, Urtti A. Rabbit as an animal model for intravitreal pharmacokinetics: clinical predictability and quality of the published data. Exp Eye Res. 2015;137:111–24. Scholar
  10. 10.
    del Amo EM, Rimpelä A-K, Heikkinen E, et al. Pharmacokinetic aspects of retinal drug delivery. Prog Retin Eye Res. 2016;57:134–85. Scholar
  11. 11.
    Del Amo EM, Vellonen KS, Kidron H, Urtti A. Intravitreal clearance and volume of distribution of compounds in rabbits: in silico prediction and pharmacokinetic simulations for drug development. Eur J Pharm Biopharm. 2015;95:215–26. Scholar
  12. 12.
    Peyman GA, Lad EM, Moshfeghi DM. Intravitreal injection of therapeutic agents. Retina. 2008;29(7):875–912. Scholar
  13. 13.
    Falavarjani KG, Nguyen QD. Adverse events and complications associated with intravitreal injection of anti-VEGF agents: a review of literature. Eye. 2013;27(7):787–94. Scholar
  14. 14.
    Hombrebueno JR, Luo C, Guo L, Chen M, Xu H. Intravitreal injection of normal saline induces retinal degeneration in the C57BL/6J mouse. Transl Vis Sci Technol. 2014;3(2):3. Scholar
  15. 15.
    Da Costa R, Röger C, Segelken J, Barben M, Grimm C, Neidhardt J. A novel method combining vitreous aspiration and intravitreal AAV2/8 injection results in retina-wide transduction in adult mice. Investig Ophthalmol Vis Sci. 2016;57(13):5326–34. Scholar
  16. 16.
    Gal-Or O, Dotan A, Dachbash M, et al. Bevacizumab clearance through the iridocorneal angle following intravitreal injection in a rat model. Exp Eye Res. 2016;145:412–6. Scholar
  17. 17.
    Dureau P, Bonnel S, Menasche M, Dufier JL, Abitbol M. Quantitative analysis of intravitreal injections in the rat. Curr Eye Res. 2001;22(1):74–7. Scholar
  18. 18.
    Xie Z, Chen F, Wu X, et al. Safety and efficacy of intravitreal injection of recombinant erythropoietin for protection of photoreceptor cells in a rat model of retinal detachment. Eye. 2012;26(1):144–52. Scholar
  19. 19.
    Merani R, Hunyor AP. Endophthalmitis following intravitreal anti-vascular endothelial growth factor (VEGF) injection: a comprehensive review. Int J Retina Vitreous. 2015;1(1):9. Scholar
  20. 20.
    Avery RL, Bakri SJ, Blumenkranz MS, et al. Intravitreal injection technique and monitoring: updated guidelines of an expert panel. Retina. 2014;34(Suppl 1):S1–S18. Scholar
  21. 21.
    Kowalski RP, Romanowski EG, Mah FS, Yates KA, Gordon YJ. Topical 0.5% moxifloxacin prevents endophthalmitis in an intravitreal injection rabbit model. J Ocul Pharmacol Ther. 2008;24(1):1–7. Scholar
  22. 22.
    Olsen TW, Feng X, Wabner K, Csaky K, Pambuccian S, Cameron JD. Pharmacokinetics of pars plana intravitreal injections versus microcannula suprachoroidal injections of bevacizumab in a porcine model. Investig Ophthalmol Vis Sci. 2011;52(7):4749–56. Scholar
  23. 23.
    Abarca EM, Salmon JH, Gilger BC. Effect of choroidal perfusion on ocular tissue distribution after intravitreal or suprachoroidal injection in an arterially perfused ex vivo pig eye model. J Ocul Pharmacol Ther. 2013;29(8):715–22. Scholar
  24. 24.
    Ozkaya A, Alkin Z, Celik U, et al. Comparing the effects of three different intravitreal injection techniques on vitreous reflux and intraocular pressure. J Ocul Pharmacol Ther. 2013;29:325–9. Scholar
  25. 25.
    Rodrigues EB, Grumann A, Penha FM, et al. Effect of needle type and injection technique on pain level and vitreal reflux in intravitreal injection. J Ocul Pharmacol Ther. 2011;27(2):197–203. Scholar
  26. 26.
    De Stefano VS, Abechain JJ, de Almeida LF, et al. Experimental investigation of needles, syringes and techniques for intravitreal injections. Clin Exp Ophthalmol. 2011;39(3):236–42. Scholar
  27. 27.
    Tansey G, Yuan P, Bungay PM, Lutz RJ, Robinson MR, et al. Preclinical evaluation of a novel episcleral cyclosporine implant for ocular graft-versus-host disease. Investig Ophthalmol Vis Sci. 2005;46(2):655–62.CrossRefGoogle Scholar
  28. 28.
    Jessen BA, MHI S, Kaur H, et al. Safety assessment of subconjunctivally implanted devices containing latanoprost in Dutch-belted rabbits. J Ocul Pharmacol Ther. 2013;29(6):574–85. Scholar
  29. 29.
    Ang M, Ng X, Wong C, et al. Evaluation of a prednisolone acetate-loaded subconjunctival implant for the treatment of recurrent uveitis in a rabbit model. PLoS One. 2014;9(5):e97555. Scholar
  30. 30.
    Tejedor J. Anesthesia for small-incision cataract surgery. In: Manual small incision cataract surgery. Cham: Springer; 2016. p. 35–47.CrossRefGoogle Scholar
  31. 31.
    Zundert VA, Kumar C, Jankovic D. Regional anesthesia in ophthalmology. In: Regional nerve blocks in anesthesia and pain therapy. Cham: Springer; 2015. p. 81–98.CrossRefGoogle Scholar
  32. 32.
    Shilo-Benjamini Y, Pascoe PJ, Maggs DJ, Kass PH, Wisner ER. Retrobulbar and peribulbar regional techniques in cats: a preliminary study in cadavers. Vet Anaesth Analg. 2013;40(6):623–31. Scholar
  33. 33.
    Accola PJ, Bentley E, Smith LJ, Forrest LJ, Baumel CA, Murphy CJ. Development of a retrobulbar injection technique for ocular surgery and analgesia in dogs. J Am Vet Med Assoc. 2006;229(2):220–5. Scholar
  34. 34.
    Raghava S, Hammond M, Kompella UB. Periocular routes for retinal drug delivery. Expert Opin Drug Deliv. 2004;1(1):99–114. Scholar
  35. 35.
    Geroski DH, Edelhauser HF. Drug delivery for posterior segment eye disease. Investig Ophthalmol Vis Sci. 2000;41(5):961–4.Google Scholar
  36. 36.
    Okada AA, Wakabayashi T, Morimura Y, et al. Trans-Tenon’s retrobulbar triamcinolone infusion for the treatment of uveitis. Br J Ophthalmol. 2003;87(8):968–71. Scholar
  37. 37.
    Sen HN, Vitale S, Gangaputra SS, et al. Periocular corticosteroid injections in uveitis: effects and complications. Ophthalmology. 2014;121(11):2275–86. Scholar
  38. 38.
    Waite D, Wang Y, Jones D, Stitt A, Raj Singh TR. Posterior drug delivery via periocular route: challenges and opportunities. Ther Deliv. 2017;8(8):685–99. Scholar
  39. 39.
    Davis FA. The anatomy and histology of the eye and orbit of the rabbit. Trans Am Ophthalmol Soc. 1929;27:400.2–441.Google Scholar
  40. 40.
    Timmers AM, Zhang H, Squitieri A, Gonzalez-Pola C. Subretinal injections in rodent eyes: effects on electrophysiology and histology of rat retina. Mol Vis. 2001;7:131–7.PubMedGoogle Scholar
  41. 41.
    Qi Y, Dai X, Zhang H, et al. Trans-corneal subretinal injection in mice and its effect on the function and morphology of the retina. PLoS One. 2015;10(8):e0136523. Scholar
  42. 42.
    Wert KJ, Skeie JM, Davis RJ, Tsang SH, Mahajan VB. Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye. J Vis Exp. 2012;(69):4286.
  43. 43.
    Park SW, Kim JH, Park WJ, Kim JH. Limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium. J Vis Exp. 2015;(102):e53030.
  44. 44.
    Westenskow PD, Kurihara T, Bravo S, et al. Performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension. J Vis Exp. 2015;(95):52247.
  45. 45.
    Bruewer AR, Mowat FM, Bartoe JT, Boye SL, Hauswirth WW, Petersen-Jones SM. Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs. PLoS One. 2013;8(4):e60218. Scholar
  46. 46.
    Komáromy AM, Varner SE, De Juan E, Acland GM, Aguirre GD. Application of a new subretinal injection device in the dog. Cell Transplant. 2006;15(6):511–9. Scholar
  47. 47.
    Xue K, Groppe M, Salvetti AP, MacLaren RE. Technique of retinal gene therapy: delivery of viral vector into the subretinal space. Eye. 2017;31(9):1308–16. Scholar
  48. 48.
    Bartuma H, Petrus-Reurer S, Aronsson M, Westman S, André H, Kvanta A. In vivo imaging of subretinal bleb-induced outer retinal degeneration in the rabbit. Investig Ophthalmol Vis Sci. 2015;56(4):2423–30. Scholar
  49. 49.
    Hirata M, Yasukawa T, Wiedemann P, et al. Fundus autofluorescence and fate of glycoxidized particles injected into subretinal space in rabbit age-related macular degeneration model. Graefes Arch Clin Exp Ophthalmol. 2009;247(7):929–37. Scholar
  50. 50.
    Monés J, Leiva M, Peña T, et al. A swine model of selective geographic atrophy of outer retinal layers mimicking atrophic AMD: a phase I escalating dose of subretinal sodium iodate. Investig Ophthalmol Vis Sci. 2016;57(10):3974–83. Scholar
  51. 51.
    Mussolino C, della Corte M, Rossi S, et al. AAV-mediated photoreceptor transduction of the pig cone-enriched retina. Gene Ther. 2011;18(7):637–45. Scholar
  52. 52.
    Koss MJ, Falabella P, Stefanini FR, et al. Subretinal implantation of a monolayer of human embryonic stem cell-derived retinal pigment epithelium: a feasibility and safety study in Yucatán minipigs. Graefes Arch Clin Exp Ophthalmol. 2016;254(8):1553–65. Scholar
  53. 53.
    Petersen-Jones SM, Komáromy AM. Dog models for blinding inherited retinal dystrophies. Hum Gene Ther Clin Dev. 2015;26(1):15–26. Scholar
  54. 54.
    Acland GM, Aguirre GD, Ray J, et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet. 2001;28:92–5. Scholar
  55. 55.
    Campochiaro PA, Lauer AK, Sohn EH, et al. Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study. Hum Gene Ther. 2017;28(1):99–111. Scholar
  56. 56.
    Ochakovski GA, Peters T, Michalakis S, et al. Subretinal injection for gene therapy does not cause clinically significant outer nuclear layer thinning in normal primate foveae. Investig Ophthalmol Vis Sci. 2017;58(10):4155–60. Scholar
  57. 57.
    Nork TM. Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque. Arch Ophthalmol. 2012;130(1):65. Scholar
  58. 58.
    Lai CM, Shen WY, Brankov M, et al. Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys. Mol Ther. 2005;12(4):659–68. Scholar
  59. 59.
    Gilger BC. Ocular pharmacology and toxicology. Arch Ophthalmol. 1967;78(4):534–62. Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Brian C. Gilger
    • 1
    Email author
  • Joshua T. Bartoe
    • 2
  • J. Seth Eaton
    • 3
  • Ryan Boyd
    • 2
  1. 1.Department of Clinical SciencesNorth Carolina State UniversityRaleighUSA
  2. 2.MPI ResearchMattawanUSA
  3. 3.Ocular Services On Demand (OSOD), LLCMadisonUSA

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