To visualize and quantify conventional outflow directly in its anatomic location.
We obtained fluorescein canalograms in six porcine whole eyes and six porcine anterior segment cultures. Eyes were ...perfused with a constant pressure of 15 mmHg using media containing 0.017 mg/ml fluorescein. Flow patterns were visualized using a stereo dissecting microscope equipped for fluorescent imaging. Images were captured every 30 seconds for 20 minutes for time lapse analysis. Anterior chamber cultures were imaged again on day three of culture. Canalograms were first analyzed for filling time per quadrant. We then wrote a program to automatically compute focal flow fits for each macropixel and to detect convergent perilimbal flow patterns with macropixels grouped into 3 equal-radial width rings around the cornea. A generalized additive model was used to determine fluorescence changes of individual macropixels.
The resulting imaging algorithm deployed 1024 macropixels that were fit to determine maximum intensity and time to fill. These individual fits highlighted the focal flow function. In whole eyes, significantly faster flow was seen in the inferonasal (IN) and superonasal (SN) quadrants compared to the superotemporal (ST) and inferotemporal (IT) ones (p<0.05). In anterior chamber cultures, reduced flow on day 1 increased in all quadrants on day 3 except in IT (p<0.05). Perilimbal ring analysis uncovered convergent perilimbal flow.
An algorithm was developed that analyzes regional and circumferential outflow patterns. This algorithm found flow patterns that changed over time and differ in whole eyes and anterior segment cultures.
To study the anterior-posterior lamina cribrosa deformation (LCD) and the scleral canal expansion (SCE) produced by an increase in IOP and identify the main factors and interactions that determine ...these responses in the monkey.
Eye-specific baseline models of the LC and sclera of both eyes of three normal monkeys were constructed. Morphing techniques were used to generate 888 models with controlled variations in LC thickness, position and modulus (stiffness), scleral thickness and modulus, and scleral canal size and eccentricity. Finite element modeling was used to simulate an increase in IOP from 10 to 15 mm Hg. A two-level, full-factorial experimental design was used to select factor combinations and to determine the sensitivity of LCD and SCE to the eight factors, independently and in interaction.
LCD was between 53.6 μm (posteriorly) and -12.9 μm (anteriorly), whereas SCE was between 0.5 and 15.2 μm (all expansions). LCD was most sensitive to laminar modulus and position (24% and 21% of the variance in LCD, respectively), whereas SCE was most sensitive to scleral modulus and thickness (46% and 36% of the variance in SCE, respectively). There were also strong interactions between factors (35% and 7% of the variance in LCD and SCE, respectively).
IOP-related LCD and SCE result from a complex combination of factors, including geometry and material properties of the LC and sclera. This work lays the foundation for interpreting the range of individual sensitivities to IOP and illustrates that predicting individual ONH response to IOP will require the measurement of multiple factors.
We sought to visualize the aqueous outflow system in 3 dimensions (3D) in living human eyes, and to investigate the use of commercially available spectral-domain optical coherence tomographic ...(SD-OCT) systems for this purpose.
Prospective, observational study.
One randomly determined eye in each of 6 normal healthy subjects was included.
We performed 3D SD-OCT imaging of the aqueous humor outflow structures with 2 devices: The Cirrus HD-OCT and the Bioptigen SDOIS.
We created 3D virtual castings of Schlemm's canal (SC) and more distal outflow structures from scan data from each device.
Virtual casting of the SC provided visualization of more aqueous vessels branching from SC than could be located by interrogating the 2-dimensional (2D) image stack. Similarly, virtual casting of distal structures allowed visualization of large and small aqueous outflow channel networks that could not be appreciated with conventional 2D visualization.
The outflow pathways from SC to the superficial vasculature can be identified and tracked in living human eyes using commercially available SD-OCT.
Advances in imaging have made it increasingly common to study soft tissues without first embedding them in plastic or paraffin and without using labels or stains. The process, however, usually still ...involves fixation and cryosectioning, which could deform the tissues. Our goal was to quantify the morphological changes of ocular tissues caused by formalin fixation and cryosectioning. From each of 6 porcine eyes, 4 regions were obtained: cornea, equatorial and posterior sclera, and posterior pole containing the optic nerve head. Samples were imaged using visible light microscopy fresh, 1-minute and 24-hours post-fixation, and post-cryosectioning. Effects were assessed by 14 parameters representing sample size and shape. Overall, formalin fixation and sectioning caused only minimal changes to the ocular tissues, with average percentage parameter differences of 0.1%, 1%, and 1.2% between fresh and post-fixing by 1 minute, 24 hours, and post-cryosectioning, respectively. Parameter changes were not directional, and were only weakly dependent on the duration of fixation and the region of the eye. These results demonstrate that formalin fixation and cryosectioning are good choices for studying ocular tissue morphology and structure, as they do not cause the large tissue shrinkage or distortions typically associated with other, more complicated, techniques.
The lamina cribrosa (LC) is a prime location of glaucomatous damage. The purpose of this study was to compare LC 3-dimensional micro-architecture between healthy and glaucomatous eyes in vivo by ...using optical coherence tomography (OCT).
Sixty-eight eyes (19 healthy and 49 glaucomatous) from 47 subjects were scanned in a 3.5 × 3.5 × 3.64-mm volume (400 × 400 × 896 pixels) at the optic nerve head by using swept-source OCT. The LC micro-architecture parameters were measured on the visible LC by an automated segmentation algorithm. The LC parameters were compared to diagnosis and visual field mean deviation (VF MD) by using a linear mixed effects model accounting for age.
The average VF MD for the healthy and glaucomatous eyes was -0.50 ± 0.80 dB and -7.84 ± 8.75 dB, respectively. Beam thickness to pore diameter ratio (P = 0.04) and pore diameter standard deviation (P < 0.01) were increased in glaucomatous eyes. With worse MD, beam thickness to pore diameter ratio (P < 0.01), pore diameter standard deviation (P = 0.05), and beam thickness (P < 0.01) showed a statistically significant increase while pore diameter (P = 0.02) showed a significant decrease. There were no significant interactions between any of the parameters and age (all P > 0.05).
Glaucomatous micro-architecture changes in the LC, detected by OCT analysis, reflect beams remodeling and axonal loss leading to reduction in pore size and increased pore size variability.
Characterizing the collagen fiber orientation and organization in the eye is necessary for a complete understanding of ocular biomechanics. In this study, we assess the performance of polarized light ...microscopy to determine collagen fiber orientation of ocular tissues. Our results demonstrate that the method provides objective, accurate, repeatable and robust data on fiber orientation with µm-scale resolution over a broad, cm-scale, field of view, unaffected by formalin fixation, without requiring tissue dehydration, labeling or staining. Together, this shows that polarized light microscopy is a powerful method for studying collagen architecture in the eye, with applications ranging from normal physiology and aging, to pathology and transplantation.
To investigate the biomechanical response to IOP elevation of normal monkey eyes using eye-specific, three-dimensional (3-D) finite element (FE) models of the ONH that incorporate lamina cribrosa ...(LC) microarchitectural information.
A serial sectioning and episcopic imaging technique was used to reconstruct the ONH and peripapillary sclera of four pairs of eyes fixed at 10 mm Hg. FE models were generated with local LC material properties representing the connective tissue volume fraction (CTVF) and predominant LC beam orientation and used to simulate an increase in IOP from 10 to 45 mm Hg. An LC material stiffness constant was varied to assess its influence on biomechanical response.
Strains and stresses within contralateral eyes were remarkably similar in both magnitude and distribution. Strain correlated inversely, and nonlinearly, with CTVF (median, r (2) = 0.73), with tensile strains largest in the temporal region. Stress correlated linearly with CTVF (median r(2) = 0.63), with the central and superior regions bearing the highest stresses. Net average LC displacement was either posterior or anterior, depending on whether the laminar material properties were compliant or stiff.
The results show that contralateral eyes exhibit similar mechanical behavior and suggest that local mechanical stress and strain within the LC are correlate highly with local laminar CTVF. These simulations emphasize the importance of developing both high-resolution imaging of the LC microarchitecture and next-generation, deep-scanning OCT techniques to clarify the relationships between IOP-related LC displacement and CTVF-related stress and strain in the LC. Such imaging may predict sites of IOP-related damage in glaucoma.
To characterize optic nerve head (ONH) connective tissue deformation after acute (15 or 30 minutes) intraocular pressure (IOP) elevation in six adult normal monkeys using three-dimensional (3-D) ...histomorphometry.
Trephined ONH and peripapillary sclera from both eyes of six monkeys, each perfusion fixed with one eye at IOP 10 mm Hg (IOP-10) and the other at IOP 30 or 45 mm Hg (IOP-30 or IOP-45, by anterior chamber manometer), were serially sectioned, 3-D reconstructed, 3-D delineated, and quantified according to standard parameters. For each monkey, intereye differences (high-IOP eye minus IOP-10) for each parameter were calculated and compared by ANOVA and EPIDmax both overall and regionally. EPIDmax deformations for each parameter were defined to be those statistically significant differences that exceeded the maximum physiologic intereye difference within six bilaterally normal monkeys in a previous report.
Regional EPIDmax laminar thinning, posterior bowing of the peripapillary sclera, and thinning and expansion of the scleral canal were present in most high-IOP eyes and were colocalized in those demonstrating the most deformation. Laminar deformation was minimal, not only posteriorly but in some cases anteriorly in the high-IOP eyes. No increase in deformation was seen in the IOP-45 versus the IOP-30 eyes.
ONH connective tissue alterations after acute IOP elevation involve regional thinning, stretching, and deformation of the lamina cribrosa and peripapillary sclera that are minimal to modest in magnitude. The time-dependent character of these alterations and their compressive, expansile, and shear effects on the axons, the astrocytes, and the laminar and posterior ciliary circulations remain to be determined.
The lamina cribrosa is a primary site of damage in glaucoma. While mechanical distortion is hypothesized to cause reduction of axoplasmic flow, little is known about how the pores, which contains the ...retinal ganglion cell axons, traverse the lamina cribrosa. We investigated lamina cribrosa pore paths in vivo to quantify differences in tortuosity of pore paths between healthy and glaucomatous eyes. We imaged 16 healthy, 23 glaucoma suspect and 48 glaucomatous eyes from 70 subjects using a swept source optical coherence tomography system. The lamina cribrosa pores were automatically segmented using a previously described segmentation algorithm. Individual pore paths were automatically tracked through the depth of the lamina cribrosa using custom software. Pore path convergence to the optic nerve center and tortuosity was quantified for each eye. We found that lamina cribrosa pore pathways traverse the lamina cribrosa closer to the optic nerve center along the depth of the lamina cribrosa regardless of disease severity or diagnostic category. In addition, pores of glaucoma eyes take a more tortuous path through the lamina cribrosa compared to those of healthy eyes, suggesting a potential mechanism for reduction of axoplasmic flow in glaucoma.
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