title: Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera.
abstract: Second harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea. Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects.
  
  
  
  Single sub-Tenon's (sT) injections (400 μL) using SMG (40-400 mM) were made at the equatorial 12 o'clock position of the right eye of cadaveric rabbit heads (n = 16 pairs). After 3.5 hours, confocal microscopy (CM) was performed using 860 nm two-photon excitation and 400 to 450 nm emission. Pixel density and fiber bundle "waviness" analyses were performed on the images. Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. Comparison experiments with riboflavin photochemical crosslinking were done.
  
  
  
  Therapeutic tissue crosslinking localization studies indicated that crosslinking changes occurred at the site of injection and in adjacent sectors. Second harmonic generation signals revealed large fibrous collagenous bundled structures that displayed various degrees of waviness. Histogram analysis showed a nearly 6-fold signal increase in 400 mM SMG over 40 mM. This corresponded to a ΔTm = 13°C for 400 mM versus ΔTm = 4°C for 40 mM. Waviness analysis indicated increased fiber straightening as a result of SMG CXL.
  
  
  
  Second harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG. These changes provide insights into the macromolecular changes that are induced by therapeutic crosslinking technology and may provide a method to evaluate connective tissue protein changes induced by scleral crosslinking therapies.
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Mariya
authors.initials: M
authors.lastname: Zyablitskaya
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Takaoka
authors.affiliation: Confocal and Specialized Microscopy Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York, United States.
authors.forename: Emilia L
authors.initials: EL
authors.lastname: Munteanu
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nagasaki
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Trokel
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Paik
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000008:administration & dosage*; D004195:Disease Models, Animal; D007267:Injections; D018613:Microscopy, Confocal; D009216:Myopia / Q000175:diagnosis / Q000188:drug therapy* / Q000378:metabolism; D011817:Rabbits; D012521:Sarcosine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D012590:Sclera / Q000187:drug effects / Q000473:pathology*
chemical_list: D003432:Cross-Linking Reagents; C020420:monomethylolglycine; D003094:Collagen; D012521:Sarcosine