Three-Dimensional Analysis of Collagen Lamellae in the Anterior Stroma of the Human Cornea Visualized by Second Harmonic Generation Imaging Microscopy

Morishige, Naoyuki; Takagi, Yuki; Chikama, T.–I.; Takahara, Atsushi; Nishida, Teruo

doi:10.1167/iovs.10-5657

Cited by 64 publications

(59 citation statements)

References 21 publications

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“…26–29 Other studies have examined corneal tissue independently in its native state 30–36 and in keratoconus 37,38 as well as following photochemical crosslinking (as discussed below). The results of these studies indicate that the corneal signal is optimized in the forward scattered direction, which makes sense given the cornea's transparency and the fact that light passes through the tissue to strike a monitor in forward scattered systems.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

Zyablitskaya

Takaoka

Munteanu

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PurposeSecond 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.MethodsSingle 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.ResultsTherapeutic 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.ConclusionsSecond 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.

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Section: Discussionmentioning

confidence: 99%

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

Zyablitskaya

Takaoka

Munteanu

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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“…This expression represents an average over the whole stroma. Anterior collagen fibrils are more strongly interwoven compared to posterior fibrils (Morishige et al, 2011) and may exhibit different swelling behavior. In the constant-mass coordinate system, Darcy’s law becomes

J_{V} = - \frac{K}{μ} true[\frac{normald P}{normald H_{w}} true] true[\frac{normald ξ}{normald x} false] \frac{normald H_{w}}{normald ξ}

where the first bracketed term on the right follows from Eq. (A7) and the second follows from Eq.…”

Section: A1 Swelling Coordinate ξmentioning

confidence: 99%

Section: A1 Swelling Coordinate ξmentioning

confidence: 99%

Oxygen-deficient metabolism and corneal edema

Leung

Bonanno

Radke

2011

Progress in Retinal and Eye Research

155

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Wear of low-oxygen-transmissible soft contact lenses swells the cornea significantly, even during open eye. Although oxygen-deficient corneal edema is well-documented, a self-consistent quantitative prediction based on the underlying metabolic reactions is not available. We present a biochemical description of the human cornea that quantifies hypoxic swelling through the coupled transport of water, salt, and respiratory metabolites. Aerobic and anaerobic consumption of glucose, as well as acidosis and pH buffering, are incorporated in a seven-layer corneal model (anterior chamber, endothelium, stroma, epithelium, postlens tear film, contact lens, and prelens tear film). Corneal swelling is predicted from coupled transport of water, dissolved salts, and especially metabolites, along with membrane-transport resistances at the endothelium and epithelium. At the endothelium, the Na+/K+ - ATPase electrogenic channel actively transports bicarbonate ion from the stroma into the anterior chamber. As captured by the Kedem–Katchalsky membrane-transport formalism, the active bicarbonate-ion flux provides the driving force for corneal fluid pump-out needed to match the leak-in tendency of the stroma. Increased lactate-ion production during hypoxia osmotically lowers the pump-out rate requiring the stroma to swell to higher water content. Concentration profiles are predicted for glucose, water, oxygen, carbon dioxide, and hydronium, lactate, bicarbonate, sodium, and chloride ions, along with electrostatic potential and pressure profiles. Although the active bicarbonate-ion pump at the endothelium drives bicarbonate into the aqueous humor, we find a net flux of bicarbonate ion into the cornea that safeguards against acidosis. For the first time, we predict corneal swelling upon soft-contact-lens wear from fundamental biophysico-chemical principles. We also successfully predict that hypertonic tear alleviates contact-lens-induced edema.

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“…The applications of these 2D techniques have shown potential in providing orientation distribution information to assess collagenous tissues and understand interactions between cells and the ECM. Nevertheless, we expect that 3D analysis represents more accurately the architecture of collagen fibers than 2D analysis, and may be more sensitive to subtle changes in the micro-environment [18]. For example, recent research on collagenous fibrosis has shown that 3D analysis depicts pathological changes due to fibrosis progression more precisely than 2D analysis [19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Rapid three-dimensional quantification of voxel-wise collagen fiber orientation

Liu¹,

Quinn²,

Speroni³

et al. 2015

Biomed. Opt. Express

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Abstract:Defining fiber orientation at each voxel within a 3D biomedical image stack is potentially useful for a variety of applications, including cancer, wound healing and tissue regeneration. Current methods are typically computationally intensive or inaccurate. Herein, we present a 3D weighted orientation vector summation algorithm, which is a generalization of a previously reported 2D vector summation technique aimed at quantifying collagen fiber orientations simultaneously at each voxel of an image stack. As a result, voxel-wise fiber orientation information with 4° to 5° accuracy can be determined, and the computational time required to analyze a typical stack with the size of 512x512x100 voxels is less than 5 min. Thus, this technique enables the practical extraction of voxel-specific orientation data for characterizing structural anisotropy in 3D specimens. As examples, we use this approach to characterize the fiber organization in an excised mouse mammary gland and a 3D breast tissue model. Schanne-Klein, "Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy," Microsc.

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Three-Dimensional Analysis of Collagen Lamellae in the Anterior Stroma of the Human Cornea Visualized by Second Harmonic Generation Imaging Microscopy

Abstract: Collagen lamellae in the anterior stroma of the normal human cornea are interwoven in three dimensions and adhere densely to Bowman's layer. This structure may contribute to the rigidity and curvature of the anterior portion of the cornea.

Cited by 64 publications

References 21 publications

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

Oxygen-deficient metabolism and corneal edema

Rapid three-dimensional quantification of voxel-wise collagen fiber orientation

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