A laser-induced mouse model with long-term intraocular pressure elevation

Purpose: To develop and characterize a mouse model with intraocular pressure (IOP) elevation after laser photocoagulation on the trabecular meshwork (TM), which may serve as a model to investigate the potential of stem cell-based therapies for glaucoma. Methods: IOP was measured in 281 adult C57BL/6 mice to determine normal IOP range. IOP elevation was induced unilaterally in 50 adult mice, by targeting the TM through the limbus with a 532-nm diode laser. IOP was measured up to 24 weeks post-treatment. The optic nerve damage was detected by electroretinography and assessed by semiautomatic counting of optic nerve axons. Effects of laser treatment on the TM were evaluated by histology, immunofluorescence staining, optical coherence tomography (OCT) and transmission electron microscopy (TEM). Results: The average IOP of C57BL/6 mice was 14.5±2.6 mmHg (Mean ±SD). After laser treatment, IOP averaged above 20 mmHg throughout the follow-up period of 24 weeks. At 24 weeks, 57% of treated eyes had elevated IOP with the mean IOP of 22.5±2.5 mmHg (Mean ±SED). The difference of average axon count (59.0%) between laser treated and untreated eyes was statistically significant. Photopic negative response (PhNR) by electroretinography was significantly decreased. CD45+ inflammatory cells invaded the TM within 1 week. The expression of SPARC was increased in the TM from 1 to 12 weeks. Histology showed the anterior chamber angle open after laser treatment. OCT indicated that most of the eyes with laser treatment had no synechia in the anterior chamber angles. TEM demonstrated disorganized and compacted extracellular matrix in the TM. Conclusions: An experimental murine ocular hypertension model with an open angle and optic nerve axon loss was produced with laser photocoagulation, which could be used to investigate stem cell-based therapies for restoration of the outflow pathway integrity for ocular hypertension or glaucoma. Copyright

Three-Dimensional Spectral-Domain Optical Coherence Tomography Data Analysis for Glaucoma Detection

Purpose: To develop a new three-dimensional (3D) spectral-domain optical coherence tomography (SD-OCT) data analysis method using a machine learning technique based on variable-size super pixel segmentation that efficiently utilizes full 3D dataset to improve the discrimination between early glaucomatous and healthy eyes. Methods: 192 eyes of 96 subjects (44 healthy, 59 glaucoma suspect and 89 glaucomatous eyes) were scanned with SD-OCT. Each SD-OCT cube dataset was first converted into 2D feature map based on retinal nerve fiber layer (RNFL) segmentation and then divided into various number of super pixels. Unlike the conventional super pixel having a fixed number of points, this newly developed variable-size super pixel is defined as a cluster of homogeneous adjacent pixels with variable size, shape and number. Features of super pixel map were extracted and used as inputs to machine classifier (LogitBoost adaptive boosting) to automatically identify diseased eyes. For discriminating performance assessment, area under the curve (AUC) of the receiver operating characteristics of the machine classifier outputs were compared with the conventional circumpapillary RNFL (cpRNFL) thickness measurements. Results: The super pixel analysis showed statistically significantly higher AUC than the cpRNFL (0.855 vs. 0.707, respectively, p = 0.031, Jackknife test) when glaucoma suspects were discriminated from healthy, while no significant difference was found when confirmed glaucoma eyes were discriminated from healthy eyes. Conclusions: A novel 3D OCT analysis technique performed at least as well as the cpRNFL in glaucoma discrimination and even better at glaucoma suspect discrimination. This new method has the potential to improve early detection of glaucomatous damage. © 2013 Xu et al

Gold nanorods as a contrast agent for Doppler optical coherence tomography

Purpose: To investigate gold nanorods (GNRs) as a contrast agent to enhance Doppler optical coherence tomography (OCT) imaging of the intrascleral aqueous humor outflow. Methods: A serial dilution of GNRs was scanned with a spectral-domain OCT device (Bioptigen, Durham, NC) to visualize Doppler signal. Doppler measurements using GNRs were validated using a controlled flow system. To demonstrate an application of GNR enhanced Doppler, porcine eyes were perfused at constant pressure with mock aqueous alone or 1.0×10 12 GNR/mL mixed with mock aqueous. Twelve Doppler and volumetric SD-OCT scans were obtained from the limbus in a radial fashion incremented by 30°, forming a circular scan pattern. Volumetric flow was computed by integrating flow inside non-connected vessels throughout all 12 scans around the limbus. Results: At the GNR concentration of 0.7×1012 GNRs/mL, Doppler signal was present through the entire depth of the testing tube without substantial attenuation. A well-defined laminar flow profile was observed for Doppler images of GNRs flowing through the glass capillary tube. The Doppler OCT measured flow profile was not statistically different from the expected flow profile based upon an autoregressive moving average model, with an error of -0.025 to 0.037 mm/s (p = 0.6435). Cross-sectional slices demonstrated the ability to view anterior chamber outflow ex-vivo using GNR-enhanced Doppler OCT. Doppler volumetric flow measurements were comparable to flow recorded by the perfusion system. Conclusions: GNRs created a measureable Doppler signal within otherwise silent flow fields in OCT Doppler scans. Practical application of this technique was confirmed in a constant pressure ex-vivo aqueous humor outflow model in porcine eyes. © 2014 Wang et al

SPECTRAL DOMAIN OCT DOPPLER ASSESSES AQUEOUS OUTFLOW

Glaucoma is the second leading cause of blindness worldwide, affecting approximately 67 million people and costing the US healthcare system an estimated $2.5 billion annually. The greatest risk factor for the diagnosis and progression of glaucoma is high eye pressure. All glaucoma medications and procedures are designed to reduce eye pressure, slowing disease progression and preserving vision. The eye’s aqueous humor nourishes avascular tissues in the anterior segment. It also maintains the eye’s geometry by pressurizing the globe, facilitating its ability to focus light on the retina. The balance between aqueous humor production and uptake is responsible for the pressure within the eye. Most glaucoma medications are designed to reduce pressure by increasing aqueous humor outflow, and surgeries are designed to enhance or bypass exiting outflow pathways. But, the effects of medications and procedures on a patient by patient basis on outflow remain speculative. Here, techniques for the non-invasive direct mapping and measurement of aqueous humor outflow in the living human eye are proposed. Mapping provides morphometric insights and measurements of the components of the outflow system, while the flow technique is the first to provide direct measurements of outflow, free of the assumptions plaguing other modalities. “Virtual casting” of the outflow system yields 3D maps from which terminal branches are identified. Doppler measurements quantify velocity within those branches. Total aqueous humor outflow is determined by integrating calculated flow across all identified terminal branches. These technologies can be adapted to existing FDA approved OCT clinical scanners. Clinical application of these technologies may improve the management of glaucoma by reducing the time needed to determine and implement optimal therapeutic strategies, thereby preserving vision in glaucoma patients

Reproducibility of in-vivo OCT measured three-dimensional human lamina cribrosa microarchitecture

Purpose: To determine the reproducibility of automated segmentation of the three-dimensional (3D) lamina cribrosa (LC) microarchitecture scanned in-vivo using optical coherence tomography (OCT). Methods: Thirty-nine eyes (8 healthy, 19 glaucoma suspects and 12 glaucoma) from 49 subjects were scanned twice using swept-source (SS-) OCT in a 3.5x3.5x3.64 mm (400x400x896 pixels) volume centered on the optic nerve head, with the focus readjusted after each scan. The LC was automatically segmented and analyzed for microarchitectural parameters, including pore diameter, pore diameter standard deviation (SD), pore aspect ratio, pore area, beam thickness, beam thickness SD, and beam thickness to pore diameter ratio. Reproducibility of the parameters was assessed by computing the imprecision of the parameters between the scans. Results: The automated segmentation demonstrated excellent reproducibility. All LC microarchitecture parameters had an imprecision of less or equal to 4.2%. There was little variability in imprecision with respect to diagnostic category, although the method tends to show higher imprecision amongst healthy subjects. Conclusion: The proposed automated segmentation of the LC demonstrated high reproducibility for 3D LC parameters. This segmentation analysis tool will be useful for in-vivo studies of the LC. © 2014 Wang et al

Effects of Age on Optical Coherence Tomography Measurements of Healthy Retinal Nerve Fiber Layer, Macula, and Optic Nerve Head

Purpose—To determine the effects of age on global and sectoral peripapillary retinal nerve fiber layer (RNFL), macular thicknesses and optic nerve head (ONH) parameters in healthy subjects using optical coherence tomography (OCT). Design—Retrospective, cross-sectional observational study. Participants—226 eyes from 124 healthy subjects were included. Methods—Healthy subjects were scanned using the Fast RNFL, Fast Macula, and Fast ONH scan patterns on a Stratus OCT. All global and sectoral RNFL and macular parameters and global ONH parameters were modeled in terms of age using linear mixed effects models. Normalized slopes were also calculated by dividing the slopes by the mean value of the OCT parameter for inter-parameter comparison. Main Outcome Measures—Slope of each OCT parameter across age. Results—All global and sectoral RNFL thickness parameters statistically significantly decreased with increasing age, except for the temporal quadrant and clock hours 8-10, which were not statistically different from a slope of zero. Highest absolute slopes were in the inferior and superior quadrant RNFL and clock hour 1 (superior nasal). Normalized slopes showed similar rate in all sectors except for the temporal clock hours (8-10). All macular thickness parameters statistically significantly decreased with increasing age, except for the central fovea sector, which had a slight positive slope that was not statistically significant. The nasal outer sector had the greatest absolute slope. Normalized macular slope in the outer ring was similar to the normalized slopes in the RNFL. Normalized inner ring had shallower slope than the outer ring with similar rate in all quadrants. Disc area remained nearly constant across the ages, but cup area increased and rim area decreased with age, both of which were statistically significant. Conclusions—Global and regional changes due to the effects of age on RNFL, macula and ONH OCT measurements should be considered when assessing eyes over time.National Institutes of Health (U.S.) (R01-EY13178-09)National Institutes of Health (U.S.) (R01-EY11289-23)National Institutes of Health (U.S.) (P30-EY008098

Label-free imaging of zebrafish larvae in vivo by photoacoustic microscopy

Zebrafish play an important role in biological and biomedical research. Traditional in vivo imaging methods for studying zebrafish larvae primarily require fluorescence labeling. In this work, relying on tissue intrinsic optical absorption contrast, we acquired high resolution label-free 3D images of zebrafish larvae by using photoacoustic microscopy (PAM) in vivo. The spatial resolution reaches several microns, allowing the study of microstructures in various living organs. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying various small animal models, including zebrafish larvae, Caenorhabditis elegans, frogs and drosophila larvae