June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
A modified histological technique for evaluation of RPE disturbance
Author Affiliations & Notes
  • Karen M Joos
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
    Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, United States
  • Robert Matthews
    Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Ratna Prasad
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Nabil Simaan
    Mechanical Engineering, Computer Science, Otolaryngology, Vanderbilt University, Nashville, Tennessee, United States
  • Jin H Shen
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Karen Joos, Vanderbilt (P); Robert Matthews, None; Ratna Prasad, None; Nabil Simaan, Vanderbllt (P); Jin Shen, Vanderbilt (P)
  • Footnotes
    Support  NIH/NEI 5R01EY028133-02; Joseph Ellis Family and William Black Research Funds, NEI Core Grant 6P30EY08126 to Vanderbilt Vision Research Center, Unrestricted Departmental Grant from Research to Prevent Blindness, Inc., NY.
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2226. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : 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.

Methods : 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)

Results : 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.

Conclusions : 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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×