Author(s): Timmers AM, Zhang H, Squitieri A, GonzalezPola C
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Abstract PURPOSE: To describe a reliable and fast method for subretinal injection in rodents and to assess the effect of the procedure on retinal function and histology. METHODS: Corneas of rodents were punctured with a 28 gauge hypodermic insulin needle avoiding the lens. The injection procedure can be observed with the aid of a dissecting microscope and methylcellulose solution on the eye. A 33 gauge blunt needle was inserted into the eye through the corneal puncture and guided toward the subretinal matrix. Addition of fluorescein to the injection mixture facilitated immediate evaluation of the injection. Rat eyes were either non-injected (controls), received only a corneal puncture or were injected with fluorescent microspheres or PBS-fluorescein mixture. Retinal function and integrity were assessed through electroretinographic (ERG) analysis and postmortem histology. RESULTS: The anterior injection procedure provided a fast and simple method for subretinal injections. In rats a successful subretinal delivery was achieved in more than 90\%, with less than 5\% of the injected eyes developing cataracts. No significant differences in b-wave ERG amplitudes in rodent eyes over a five-week period were observed between non-injected control eyes and subretinally injected eyes (1 to 10 microl of PBS-fluorescein or 2 microl fluorescent microspheres). Histological analysis revealed that re-attachment of the rat retina occurred in approximately 1 day post-injection and the phagocytotic ability of RPE cells remained intact. CONCLUSIONS: This method was easily learned and required a minimum of equipment and animal preparation. With experience, 10 to 30 eyes could be injected per h. Furthermore, the injection procedure did not compromise the lens, retina or retinal pigment epithelium (RPE).
This article was published in Mol Vis
and referenced in Hereditary Genetics: Current Research