Special Issue Collagen Structural Changes in Rat Tarsus After Crosslinking Sruti S. Akella1,*,JuanLiu1,2,YuanMiao1,3, Roy S. Chuck1, Anne Barmettler1,and Cheng Zhang1 1 Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY, USA 2 Department of Ophthalmology, Ning Xia Eye Hospital, Yinchuan, China 3 Aier School of Ophthalmology, Central South University, Changsha, China Correspondence: Cheng Zhang, Purpose: Surgery is the standard treatment for floppy eyelid syndrome, but crosslink- Department of Ophthalmology and ing (CXL) tarsus has recently been proposed as an alternative. To the best of our Visual Sciences, Montefiore Medical knowledge, this study is the first to use second-harmonic generation (SHG) microscopy Center, 3332 Rochambeau Avenue, to examine tarsal collagen ex vivo before and after photo-activated crosslinking. To Centennial Building, Third Floor, quantify crosslinking, this study examined fluorescence recovery after photobleaching Bronx, NY 10467, USA. e-mail: (FRAP), which indirectly measures tissue stiffness. [email protected] Methods: Anne Barmettler, Department of Upper eyelid tarsal plates were dissected from 21 Sprague–Dawley rats (total Ophthalmology and Visual Sciences, of 42 tarsal plates). Six normal plates were sent for histopathology and SHG imaging; the Montefiore Medical Center, 3332 remaining 36 were crosslinked with phosphate-buffered saline (PBS) alone or riboflavin in PBS (concentrations of 0.1%, 0.3%, and 0.5%). Tissues were irradiated with 365-nm Rochambeau Avenue, Centennial 2 Building, Third Floor, Bronx, NY ultraviolet A light (power, 0.45 mW/cm ) for 30 minutes and immediately underwent 10467, USA. e-mail: SHG microscopy. Stiffness was indirectly measured with FRAP using fluorescein isothio- [email protected] cyanate (FITC)–dextran. Received: June 23, 2020 Results: SHG imaging of normal tarsus showed that the organization of collagen Accepted: November 18, 2020 bundles is complex and varies greatly depending on location. After crosslinking with Published: April 29, 2021 high-concentration riboflavin (0.5%), collagen fibers showed clear structural changes, becoming more densely packed and wavier compared to control. FRAP half-time Keywords: crosslinking; tarsus; to fluorescence recovery was significantly increasedP ( < 0.05), indirectly indicating floppy eyelid syndrome increased tissue stiffness. No structural changes were observed after crosslinking with Citation: Akella SS, Liu J, Miao Y, lower riboflavin concentrations of 0.1% and 0.3%. Chuck RS, Barmettler A, Zhang C. Conclusions: Collagen structural changes in rat This is the first report of SHG microscopy used to image tarsus collagen tarsus after crosslinking. Transl Vis before and after crosslinking. These results highlight collagen structural changes, with Sci Technol. 2021;10(5):3, effects on tissue stiffness indirectly confirmed byFRAP. https://doi.org/10.1167/tvst.10.5.3 Translational Relevance: Collagen fibers in the tarsus may be a therapeutic target for crosslinking in order to treat symptomatic floppy eyelid syndrome. The current conventional treatment for FES is surgi- Introduction cal; a large piece of tarsus is removed in order to tighten the upper eyelid and prevent eversion. However, Eyelid tarsus is a complex biomechanical tissue the recurrence rate after surgical resection may be that serves as the main support for the eyelid.1 Struc- as high as 40%.2 Furthermore, many patients with tural alterations in the tarsus can cause various disease floppy eyelid syndrome suffer from obstructive sleep conditions such as entropion, ectropion, and floppy apnea, metabolic syndrome, or high blood pressure, eyelid syndrome (FES). These can, in turn, lead to all of which may make for a poor surgical candidate.2 intense patient discomfort, ocular surface complica- In these patients, a nonsurgical treatment could be tions, and loss of vision. These acquired disorders are optimal. thought to be a result of alterations in collagen and Recently, tarsal crosslinking (CXL), or the process elastin, which are the major components of the tarsal of forming strong covalent bonds between neighbor- plate.1 ing collagen fibrils, has been explored in animal and Copyright 2021 The Authors tvst.arvojournals.org | ISSN: 2164-2591 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from tvst.arvojournals.org on 09/30/2021 Crosslinking Rat Tarsus TVST |SpecialIssue|Vol.10|No.5|Article3|2 cadaver models as an alternative treatment to stiffen tarsus in FES.3–6 Crosslinking is already well known to Methods most ophthalmologists as a treatment for keratoconic corneas. In the US Food and Drug Administration- A total of 42 upper eyelid tarsal plates were approved Dresden protocol, corneas incubated with harvested from 21 Sprague–Dawley rats. The rat was riboflavin are exposed to ultraviolet A (UV-A) radia- chosen as the experimental model for two reasons: tion. The process generates reactive oxygen species freshtissueisreadilyavailablefromtherat,andthe that crosslink collagen fibers and ultimately stiffen the layers of the rat eyelid histologically appear similar to cornea, halting the ectatic process. Because crosslink- those of the human eyelid.13 Six of these specimens ing ultimately works by creating bonds between colla- were immediately sent for conventional histopathology gen, hypothetically any collagen-rich structure could (n = 3) or imaging with SHG microscopy (n = 3) also be targeted. To this point, the theory behind as normal control. The remaining 36 tarsal plates tarsus crosslinking arises from previous histopatholog- were randomly crosslinked using control (PBS) and ical studies of floppy eyelids that showed that colla- one of three riboflavin concentrations. We chose gen fibers accumulate as elastin degrades.7,8 This is 0.1% as the starting concentration because it is the likely a compensatory mechanism by which excess same solution used in the Dresden corneal proto- collagen is produced in order to counteract overload, col. Using this as a starting point, we hypoth- prevent fatigue, and resist the abnormal force generated esized that the tarsus would require a stronger by an increasingly floppy eyelid. Theoretically, tarsal concentration compared to the cornea, so 0.3% and crosslinking would target and strengthen this excess 0.5% concentrations were added. Then, 20 tarsal collagen, thereby alleviating symptoms of FES. If this plates were sent for SHG imaging and FRAP; is the case, crosslinking should alter the microstruc- 16 tarsal plates were examined with histopathology ture of collagen, and indeed Zyablitskaya et al.9 showed (Fig. 1). All animal experiments adhered to the ARVO that scleral CXL is seen microscopically as increased Statement for the Use of Animals in Ophthalmic and “waviness” of collagen fibers. Vision Research. To test the efficacy of CXL, previous papers have focused on histopathological and tensile load studies Sample Preparation of the tarsus; however, conventional histopathology requires a certain amount of tissue processing. Extrap- Twenty-one Sprague–Dawley rats (Charles River olating from the work done by Li et al.,10 this process- Laboratories, Wilmington, MA) were euthanized at ing may potentially alter tissue at a microscopic level. 6 weeks of age, and a total of 42 upper eyelids were Ideally, a method to examine the tissue without stain- immediately harvested en bloc from the medial to the ing or fixation would be used. One such method is lateral canthus. To isolate tarsal plates for study, the second-harmonic generation (SHG) microscopy, which lids were dissected to remove the skin and orbicularis, is a coherent nonlinear process in which two incident along with the attachments of the levator aponeuro- photons interact with tissue at a specific plane and are sis and Müller’s muscle, without altering the posterior directly converted into a single photon without absorp- conjunctiva. The lid margin with skin and hair follicles tion or emission. Computer software is able to capture was left untouched (Fig. 2). Irradiation and imaging this conversion at different planes and generate a three- took place within 3 hours after euthanasia. dimensional image of collagen fibers. Without tissue staining or fixation, the collagen fibers can be viewed Irradiation ex vivo with minimal tissue manipulation. In this study, SHG microscopy was used to image Individual tarsal plates were soaked in either rat tarsus collagen before and after photo-activated PBS (control) or one of three different concentra- crosslinking. Finally, SHG microscopy was coupled tions (0.1%, 0.3%, or 0.5%) of riboflavin (riboflavin with a separate technique called fluorescence recov- 5-phosphate salt hydrate powder; Merck KGaA, ery after photobleaching (FRAP), which measures Darmstadt, Germany) in PBS for 30 minutes. Next the the diffusion of fluorescently tagged molecules within tarsal plates were exposed to a beam of UV-A radiation live tissues and has been shown previously to corre- (Analytik Jena, Jena, Germany) with a peak wavelength late with tissue stiffness in mouse sclera.11,12 We of 365 nm at a distance of 5 cm. The total radia- hypothesized that SHG is a more sensitive measure- tion density of the UV-A light source was measured ment of CXL effects and therefore will detect 5 cm away from the light source with two independent changes at lower energy levels than other described meters,