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Karen M Joos, Robert Matthews, Ratna Prasad, Nabil Simaan, Jin H Shen; A modified histological technique for evaluation of RPE disturbance. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2226.
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Subretinal injections are increasing in frequency for vitreoretinal therapeutic delivery. We determined whether a retinal pigment epithelial (RPE) flatmount technique could be modified by the addition of RPE retrieval steps for culture to reduce artifacts.
Fresh ex-vivo porcine eyes received subretinal injections of balanced salt solution (BSS) or were undisturbed controls. The tenons and anterior segments including cornea, lens, and iris were removed. 3 ml of hyaluronidase solution (1 mg hyaluronidase/ ml of 1% HEPES in calcium and magnesium-free HBSS, filtered) was injected into each eyecup and the eyes were incubated at 37° C in 5% CO2 for 30 min. The sclera was radially incised toward the optic nerve. The vitreous and neural retina were slowly mechanically removed. The RPE/choroid/scleral pieces were fixed for 4 hours with 10% formaldehyde and then placed in PBS. The pieces were then stained for 24 hours in the dark with Alexa Fluor 680 phalloidin (ThermoFisher, 2.5% in 1% triton in HBSS), and imaged after rinsing with 0.1%triton-HBSS buffer. The segments were inverted into imaging wells and examined with a confocal microscope (LSM710, Zeiss)
Subretinal blebs were successfully produced. The hyaluronidase-phalloidin technique permitted microscopic observation of the RPE with minimal artifacts caused by the mechanical removal of the neural retina.
Hyaluronidase-phalloidin treatment in combination with leaving the RPE/choroid/scleral tissues intact may reduce the artifacts produced during preparation when a specific area of the RPE such as the site of a subretinal injection is desired for microscopic evaluation.
This is a 2020 ARVO Annual Meeting abstract.
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