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PurposePilot study to evaluate adverse events and intraocular pressure (IOP)-lowering of a novel, noninvasive glaucoma procedure, femtosecond laser, image-guided, high-precision trabeculotomy (FLIGHT).DesignProspective, nonrandomized, single-center, interventional, single-arm clinical trial.ParticipantsEighteen eyes from 12 patients with open-angle glaucoma.MethodsEighteen eyes from 12 patients underwent FLIGHT, creating a single channel measuring 500-μm wide by 200-μm high through the trabecular meshwork and into Schlemm's canal. Adverse events, IOP, and other parameters were evaluated out to 24 months.Main outcome measuresOutcomes were the rates and types of adverse events and the rate of postprocedure best-corrected visual acuity loss (≥ 2 lines) compared with baseline. Efficacy outcomes were reduction in mean intraocular pressure (IOP) with respect to baseline and the percentage of eyes with a ≥ 20% reduction in IOP.ResultsEighteen eyes from 12 patients were enrolled in the study; 11 patients (17 eyes) returned at 24 months. There were no serious adverse events related to the laser treatment. Well-defined channels were clearly visible at 24 months by gonioscopy and anterior segment OCT, with no evidence of closure. At 24 months, the mean IOP was reduced by 34.6% from 22.3 ± 5.5 to 14.5 ± 2.6 mmHg (P < 5e-5), with an average of 2.0 ± 1.2 hypotensive medications compared with 2.2 ± 1.1 at baseline (P = 0.22). Fourteen out of the 17 study eyes (82.3%) achieved a ≥ 20% reduction in IOP at 24 months when compared with baseline.ConclusionThe FLIGHT system demonstrated a favorable safety profile in this initial pilot study, with no device-related serious adverse events. The channels appeared patent at 24 months, indicating medium-term durability.Financial disclosuresProprietary or commercial disclosure may be found after the references.

PurposeThe purpose of this study was to investigate femtosecond laser trabeculotomy (FLT) in a clinically relevant manner (i.e., delivering the surgical laser beam through the cornea of the intact, human anterior segment to create channels from the anterior chamber into the Schlemm's canal) and to investigate the effect of this treatment on intraocular pressure in perfused human anterior segments.MethodsPerfused human anterior segments (15 eyes) received either FLT treatment (n = 8) or a sham-treatment (n = 7). Intraocular pressure (IOP) in the perfused samples was recorded before and after treatment. Spectral domain optical coherence tomography, second harmonic generation imaging, and transmission electron microscopy were used to investigate the FLT channels.ResultsThe FLT group (n = 7, 1 eye excluded) had a statistically significant reduction in mean IOP of 20.2% from baseline after treatment (5.06 ± 1.46 mm Hg to 4.04 ± 1.63 mm Hg; P < 0.0005), whereas the control group (n = 7) remained statistically unchanged (7.72 ± 3.45 mm Hg to 7.78 ± 3.51 mm Hg; P < 0.71). Imaging confirmed that the channels traversed the entire trabecular meshwork into the Schlemm's canal.ConclusionsThis study has provided the first direct evidence supporting the feasibility of clinically applicable, noninvasive femtosecond laser trabeculotomy for the treatment of glaucoma. Various imaging modalities revealed minimal collateral damage to adjacent issues.Translational relevanceThis work demonstrates noninvasive femtosecond laser trabeculotomy in a laboratory setting that is clinically relevant.

PurposeThis study describes a femtosecond laser (FS) approach to machine corneal epithelial microchannels for enhancing riboflavin (Rf) penetration into the cornea prior to corneal crosslinking (CXL).MethodsUsing a 1030-nm FS laser with 5- to 10-µJ pulse energy, the corneal epithelium of slaughterhouse rabbit eyes was machined to create 2-µm-diameter by 25-µm-long microchannels at a density of 100 or 400 channels/mm2. Rf penetration through the microchannels was then determined by applying 1% Rf in phosphate-buffered saline for 30 minutes followed by removal of the cornea and extraction from the central stromal button. Stromal Rf concentrations were then compared to those obtained using standard epithelial debridement or 0.01% benzalkonium chloride (BAK) to disrupt the epithelial barrier.ResultsMicrochannels formed using a 5-µJ/pulse at a density of 400 channels/mm2 achieved a stromal Rf concentration that was 50% of that achieved by removal of the corneal epithelium and imbibing with 1% Rf. Stromal Rf levels were also equal to that of debrided corneas soaked with 0.5% Rf, threefold higher than those soaked with 0.1% Rf, and twofold higher than corneas soaked in BAK without epithelial debridement. Organ culture of treated corneas showed a normal corneal epithelium following FS machining while BAK-treated corneas showed extensive epithelial and stromal damage at 24 hours posttreatment.ConclusionsFS corneal epithelial machining can be used to enhance penetration of Rf into the stroma for corneal CXL.Translational relevanceThe creation of epithelial microchannels allows for stromal Rf concentrations high enough to perform true transepithelial crosslinking.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function.Statement of significanceThis work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

PurposePrevious studies indicate that there is an axial gradient of collagen lamellar branching and anastomosing leading to regional differences in corneal tissue stiffness that may control corneal shape. To further test this hypothesis we have measured the axial material stiffness and quantified the collagen lamellar complexity in ectatic and mechanically weakened keratoconus corneas (KC).MethodsAcoustic radiation force elastic microscopy (ARFEM) was used to probe the axial mechanical properties of the cone region of three donor KC buttons. 3 Dimensional second harmonic generation microscopy (3D-SHG) was used to qualitatively evaluate lamellar organization in 3 kC buttons and quantitatively measure lamellar branching point density (BPD) in a separate KC button that had been treated with epikeratophakia (Epi-KP).ResultsThe mean elastic modulus for the KC corneas was 1.67 ± 0.44 kPa anteriorly and 0.970 ± 0.30 kPa posteriorly, substantially below that previously measured for normal human cornea. 3D-SHG of KC buttons showed a simplified collagen lamellar structure lacking noticeable angled lamellae in the region of the cone. BPD in the anterior, posterior, central and paracentral regions of the KC cornea were significantly lower than in the overlying Epi-KP lenticule. Additionally, BPD in the cone region was significantly lower than the adjacent paracentral region in the KC button.ConclusionsThe KC cornea exhibits an axial gradient of mechanical stiffness and a BPD that appears substantially lower in the cone region compared to normal cornea. The findings reinforce the hypothesis that collagen architecture may control corneal mechanical stiffness and hence corneal shape.

The purpose of this study was to develop and test a nonlinear optical device to photoactivate riboflavin to produce spatially controlled collagen crosslinking and mechanical stiffening within the cornea. A nonlinear optical device using a variable numerical aperture objective was built and coupled to a Chameleon femtosecond laser. Ex vivo rabbit eyes were then saturated with riboflavin and scanned with various scanning parameters over a 4 mm area in the central cornea. Effectiveness of NLO CXL was assessed by evaluating corneal collagen auto fluorescence (CAF). To determine mechanical stiffening effects, corneas were removed from the eye and subjected to indentation testing using a 1 mm diameter probe and force transducer. NLO CXL was also compared to standard UVA CXL. The NLO CXL delivery device was able to induce a significant increase in corneal stiffness, comparable to the increase produced by standard UVA CXL.

PurposeTo determine whether riboflavin-induced collagen crosslinking (CXL) could be precisely achieved in the corneal stroma of ex vivo rabbit eyes using nonlinear optical excitation with a low numerical aperture lens and enlarged focal volume.SettingGavin Herbert Eye Institute, University of California Irvine, Irvine, California, USA.DesignExperimental study.MethodsThe corneal epithelium was removed and the corneas were soaked in 0.5% riboflavin solution. Using a 0.1 numerical aperture objective, a theoretical excitation volume of 150 μm × 3 μm was generated using 1 W of 760 nm femtosecond laser light and raster scanned with 4.4 μm line separation at varying effective speeds over a 4.50 mm × 2.25 mm area. Corneal sections were examined for collagen autofluorescence.ResultsCollagen autofluorescence was enhanced 2.9 times compared with ultraviolet-A (UVA) CXL. Also, increasing speed was linearly associated with decreasing autofluorescence intensity. The slowest speed of 2.69 mm/s showed a mean of 182.97 μm ± 52.35 (SD) long autofluorescent scan lines axially in the central cornea compared with 147.84 ± 4.35 μm for UVA CXL.ConclusionsDecreasing dwell time was linearly associated with decreasing autofluorescence intensity, approaching that of UVA CXL at a speed of 8.9 mm/s. Using an effective speed of 8.9 mm/s, nonlinear optical CXL could be achieved over a 3.0 mm diameter area in fewer than 4 minutes. Further development of nonlinear optical CXL might result in safer, faster, and more effective CXL treatments.Financial disclosureNone of the authors has a financial or proprietary interest in any material or method mentioned.

PurposeThe biomechanical properties of the cornea have an important role in determining the shape of the cornea and visual acuity. Since the cornea is a nonhomogeneous tissue, it is thought that the elastic properties vary throughout the cornea. We aim to measure a map of corneal elasticity across the cornea.MethodsAn acoustic radiation force elasticity microscope (ARFEM) was used to create a map of corneal elasticity in the human cornea. This ARFEM uses a low frequency, high intensity acoustic force to displace a femtosecond laser-generated microbubble, while using a high frequency, low intensity ultrasound to monitor the position of the microbubble within the cornea. From the displacement of the bubble and the magnitude of the acoustic radiation force, the local value of corneal elasticity is calculated in the direction of the displacement. Measurements were conducted at 6 locations, ranging from the central to peripheral cornea at anterior and posterior depths.ResultsThe mean anterior elastic moduli were 4.2 ± 1.2, 3.4 ± 0.7, and 1.9 ± 0.7 kPa in the central, mid, and peripheral regions, respectively, while the posterior elastic moduli were 2.3 ± 0.7, 1.6 ± 0.3, and 2.9 ± 1.2 kPa in the same radial locations.ConclusionsWe found that there is a unique distribution of elasticity axially and radially throughout the cornea.

To investigate the role of collagen structure in corneal biomechanics, measurement of localized corneal elasticity with minimal destruction to the tissue is necessary. We adopted the recently developed acoustic radiation force elastic microscopy (ARFEM) technique to measure localize biomechanical properties of the human cornea. In ARFEM, a low-frequency, high-intensity acoustic force is used to displace a femtosecond laser-generated microbubble, while high-frequency, low-intensity ultrasound is used to monitor the position of the microbubble within the cornea. Two ex vivo human corneas from a single donor were dehydrated to physiologic thickness, embedded in gelatin and then evaluated using the ARFEM technique. In the direction perpendicular to the corneal surface, ARFEM measurements provided elasticity values of E = 1.39 ± 0.28 kPa for the central anterior cornea and E = 0.71 ± 0.21 kPa for the central posterior cornea in pilot studies. The increased value of corneal elasticity in the anterior cornea correlates with the higher density of interweaving lamellae in this region.