June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Second Harmonic Generation Microscopy of Rat Scleral Remodeling by Collagenase and Reparative Collagen Mimetic Peptides
Author Affiliations & Notes
  • Daniel E. Savage
    University of Rochester David and Ilene Flaum Eye Institute, Rochester, New York, United States
    Center for Visual Science, Rochester, New York, United States
  • Jiaxi Zhou
    Center for Visual Science, Rochester, New York, United States
  • James Andrew Germann
    Center for Visual Science, Rochester, New York, United States
  • Robert O. Baratta, MD
    Stuart Therapeutics, Florida, United States
  • Brian J Del Buono
    Stuart Therapeutics, Florida, United States
  • Eric Schlumpf
    Stuart Therapeutics, Florida, United States
  • Susana Marcos
    Center for Visual Science, Rochester, New York, United States
    University of Rochester David and Ilene Flaum Eye Institute, Rochester, New York, United States
  • Footnotes
    Commercial Relationships   Daniel Savage None; Jiaxi Zhou None; James Germann None; Robert Baratta, MD Stuart Therapeutics, Code E (Employment), Stuart Therapeutics, Code I (Personal Financial Interest), Stuart Therapeutics, Code O (Owner), Stuart Therapeutics, 2018248297, 2020286248, BR1120190210563, Code P (Patent); Brian Del Buono Stuart Therapeutics, Code E (Employment), Stuart Therapeutics, Code I (Personal Financial Interest), Stuart Therapeutics, Code O (Owner); Eric Schlumpf Stuart Therapeutics, Code E (Employment), Stuart Therapeutics, Code I (Personal Financial Interest), Stuart Therapeutics, Code O (Owner), Stuart Therapeutics, 2018248297, 2020286248, BR1120190210563, Code P (Patent); Susana Marcos Stuart Therapeutics, Code F (Financial Support)
  • Footnotes
    Support  P30 Core Grant EY001319-46, Unrestricted grant Research to Prevent Blindness (Flaum Eye Institute, University of Rochester, NY), Additional funding from Stuart Therapeutics
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 849. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Daniel E. Savage, Jiaxi Zhou, James Andrew Germann, Robert O. Baratta, MD, Brian J Del Buono, Eric Schlumpf, Susana Marcos; Second Harmonic Generation Microscopy of Rat Scleral Remodeling by Collagenase and Reparative Collagen Mimetic Peptides. Invest. Ophthalmol. Vis. Sci. 2023;64(8):849.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Myopia has been shown to be associated with increased scleral protease activity [Zhao, et al., 2018]. Protease activity results in structural changes that can be visualized using Second Harmonic Generation (SHG) microscopy. Collagen mimetic peptides (CMPs) may act as reparative agents in restoring collagen organization and structure after collagenase digestion [Baratta, et al., 2022]. This work tests the ability of CMPs to reorganize digested collagen in rat sclera. A reversal of collagenase digestion in scleral tissue could provide a basis for new therapeutics targeting myopia progression.

Methods : Fresh scleral tissue was obtained from 3 albino rats euthanized by KCl injection. The rat eyes were dissected, and the extra-scleral tissue was scraped off. Bare sclerae from 5 rat eyes were imaged using a custom-built SHG microscope similar to that described previously [Germann, et al., 2020]. 200 μm x 200 μm en face images were taken at 5 μm intervals through the juxtapapillary sclerae. The tissue samples were then partially digested in a 200 μg/mL collagenase solution (TDzyme) for 30 min. The samples were subsequently rinsed with a balanced saline solution (BSS) and re-imaged at approximately the same locations. Finally, the samples were soaked in a 294 nM type 1 CMP solution (Stuart Therapeutics) for 30 minutes prior to undergoing BSS rinsing and final SHG microscopy. The collagen arrangement at each tissue depth was quantified using a previously validated order coefficient (OC) [Germann, et al., 2018].

Results : SHG microscopy of the untreated scleral tissue showed a high degree of organization. Collagenase treatment resulted in a subjective straightening of the collagen fibers and a widening of the inter-fiber spacing with a statistically significant reduction of the OC (p<0.05). Treatment with CMPs effected subjective collagen reorganization but did not significantly increase the OC from the digested scleral collagen (p < 0.05).

Conclusions : SHG microscopy showed subjective CMP-induced reorganization of rat scleral collagen after collagenase application. The changes are consistent with inhibition of the collagen enzymatic digestion. Additional work relating quantitative measurements of collagen density and organization with scleral biomechanical measurements following CMP treatment will give further insights on its potential as an alternative to treat progressive myopia.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

×
×

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.

×