Juan Reynaud

Remodeling of the Connective Tissue Microarchitecture of the Lamina Cribrosa in Early Experimental Glaucoma

PURPOSE To characterize the trabeculated connective tissue microarchitecture of the lamina cribrosa (LC) in terms of total connective tissue volume (CTV), connective tissue volume fraction (CTVF), predominant beam orientation, and material anisotropy in monkeys with early experimental glaucoma (EG). METHODS The optic nerve heads from three monkeys with unilateral EG and four bilaterally normal monkeys were three dimensionally reconstructed from tissues perfusion fixed at an intraocular pressure of 10 mm Hg. A three-dimensional segmentation algorithm was used to extract a binary, voxel-based representation of the porous LC connective tissue microstructure that was regionalized into 45 subvolumes, and the following quantities were calculated: total CTV within the LC, mean and regional CTVF, regional predominant beam orientation, and mean and regional material anisotropy. RESULTS Regional variation within the laminar microstructure was considerable within the normal eyes of all monkeys. The laminar connective tissue was generally most dense in the central and superior regions for the paired normal eyes, and laminar beams were radially oriented at the periphery for all eyes considered. CTV increased substantially in EG eyes compared with contralateral normal eyes (82%, 44%, 45% increases; P<0.05), but average CTVF changed little (-7%, 1%, and -2% in the EG eyes). There were more laminar beams through the thickness of the LC in the EG eyes than in the normal controls (46%, 18%, 17% increases). CONCLUSIONS The substantial increase in laminar CTV with little change in CTVF suggests that significant alterations in connective and nonconnective tissue components in the laminar region occur in the early stages of glaucomatous damage.

Comparison of Clinical and Spectral Domain Optical Coherence Tomography Optic Disc Margin Anatomy

PURPOSE To investigate spectral domain optical coherence tomography (SD-OCT)-detected optic disc margin anatomy in the monkey eye by colocalizing disc photographs to SD-OCT scans acquired from the same eyes. METHODS The neural canal opening (NCO) was delineated within 40 digital radial sections generated from SD-OCT volumes acquired from 33 normal monkey eyes (15 degrees, 290 x 768 horizontal grid pattern). Each volume was colocalized to its disc photograph by matching the retinal vessels within each photograph to vessel outlines visible within en face SD-OCT images. Border tissue was delineated where it extended internally to the NCO. A clinician (masked to delineated points) marked the disc margin onto each photograph while viewing the relevant stereophotograph pair. Alignment of the clinician-ascribed disc margin to the NCO and border tissue delineation was assessed. The process was repeated in a single myopic human eye. RESULTS In 23 eyes, the NCO aligned to the disc margin. In 10 eyes, externally oblique border tissue was detectable in the temporal disc. In these regions of the disc, the termination of border tissue was the disc margin. An exaggerated form of this phenotype was observed in the myopic human eye. In this case, temporal border tissue terminated at the anterior scleral canal opening, which was detected as the disc margin. CONCLUSIONS The termination of Bruchs membrane, border tissue, and the anterior scleral canal opening may constitute the disc margin within the same eye, depending on the border tissue architecture; this anatomy is consistently visualized by SD-OCT.

Correlation between Local Stress and Strain and Lamina Cribrosa Connective Tissue Volume Fraction in Normal Monkey Eyes

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

24-Hour IOP Telemetry in the Nonhuman Primate: Implant System Performance and Initial Characterization of IOP at Multiple Timescales

PURPOSE IOP is the most common independent risk factor for development and progression of glaucoma, but very little is known about IOP dynamics. Continuous IOP telemetry was used in three nonhuman primates to characterize IOP dynamics at multiple time scales for multiple 24-hour periods. METHODS An existing implantable telemetric pressure transducer system was adapted to monitoring anterior chamber IOP. The system records 500 IOP, ECG, and body temperature measurements per second and compensates for barometric pressure in real time. The continuous IOP signal was digitally filtered for noise and dropout and reported using time-window averaging for 19, 18, and 4 24-hour periods in three animals, respectively. Those data were analyzed for a nycthemeral pattern within each animal. RESULTS Ten-minute time-window averaging for multiple 24-hour periods showed that IOP fluctuated from 7 to 14 mm Hg during the day, and those changes occurred frequently and quickly. Two-hour time-window averages of IOP for multiple 24-hour periods in three animals showed a weak nycthemeral trend, but IOP was not repeatable from day-to-day within animals. CONCLUSIONS The measured IOP was successfully measured continuously by using a new, fully implantable IOP telemetry system. IOP fluctuates as much as 10 mm Hg from day to day and hour to hour in unrestrained nonhuman primates, which indicates that snapshot IOP measurements may be inadequate to capture the true dynamic character of IOP. The distributions, magnitudes, and patterns of IOP are not reproducible from day to day within animals, but IOP tends to be slightly higher at night when IOP data are averaged across multiple 24-hour periods within animals.

Deformation of the rodent optic nerve head and peripapillary structures during acute intraocular pressure elevation.

PURPOSE. To evaluate the effect of acutely elevated intraocular pressure (IOP) on retinal thickness and optic nerve head (ONH) structure in the rat eye by spectral domain-optical coherence tomography (SD-OCT). METHODS. Fourteen adult male Brown-Norway rats were studied under anesthesia (ketamine/xylazine/acepromazine, 55:5:1 mg/kg intramuscularly). Both eyes were imaged by SD-OCT on two baseline occasions several weeks before and again 2 and 4 weeks after the acute IOP imaging session. During the acute IOP session, SD-OCT imaging was performed 10 minutes after IOP was manometrically set at 15 mm Hg and then at 10, 30, and 60 minutes after IOP had been elevated to 50 mm Hg (n = 8) and again 10 and 30 minutes after IOP had been lowered back to 15 mm Hg (recovery). In two additional groups, IOP elevation was set to 70 mm Hg (n = 4) or 40 mm Hg (n = 2). Acute IOP results are reported for a pattern of 49 horizontal B-scans spanning a 20° square and follow-up results for peripapillary circular B-scans. Retinal and retinal nerve fiber layer (RNFL) thicknesses were measured with custom software by manual image segmentation. Friedman and Dunns tests were used to assess acute and longer-term effects of acute IOP elevation. RESULTS. Acute IOP elevation to 50 mm Hg caused rapid (within seconds) deformation of the ONH and peripapillary structures, including posterior displacement of the ONH surface and outward bowing of peripapillary tissue; retinal thickness decreased progressively from 10 to 30 to 60 minutes by 16%, 18%, and 20% within the area of Bruchs membrane opening (BMO; P < 0.0001) by 8%, 9%, and 11% within the central 10° (excluding the BMO; P < 0.0001) but only by 1%, 2%, and 2.4% beyond the central 10° (P < 0.0001). Recovery was progressive and nearly complete by 30 minutes. Acute IOP elevation to 40 and 70 mm Hg produced similar structural changes, but 70 mm Hg also interfered with retinal blood flow. There were no changes in peripapillary retinal or RNFL thickness (P = 0.08 and P = 0.16, respectively) measured 2 and 4 weeks after acute elevation to 50 mm Hg. CONCLUSIONS. Acute IOP elevation in the rodent eye causes rapid, reversible posterior deformation of the ONH and thinning of the peripapillary retina, with only minimal retinal thinning beyond 5° of the ONH. No permanent changes in peripapillary retinal or RNFL thickness (for up to 1 month of follow-up) were caused by 60 minutes of IOP elevation to 50 mm Hg.

Onset and Progression of Peripapillary Retinal Nerve Fiber Layer (RNFL) Retardance Changes Occur Earlier Than RNFL Thickness Changes in Experimental Glaucoma

PURPOSE Longitudinal measurements of peripapillary RNFL thickness and retardance were compared in terms of time to reach onset of damage and time to reach a specific progression endpoint. METHODS A total of 41 rhesus macaques with unilateral experimental glaucoma (EG) each had three or more weekly baseline measurements in both eyes of peripapillary RNFL thickness (RNFLT) and retardance. Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic elevation of intraocular pressure and weekly imaging continued. Pairwise differences between baseline observations were sampled by bootstrapping to determine the 95% confidence limits of each measurements repeatability. The first two sequential measurements below the lower confidence limit defined the endpoint for each parameter. Segmented linear and exponential decay functions were fit to each RNFL-versus-time series to determine the time to damage onset. RESULTS In all, 29 (71%) of the EG eyes reached endpoint by RNFL retardance and 25 (61%) reached endpoint by RNFLT. In total, 33 (80%) reached endpoint by at least one of the RNFL parameters and 21 (51%) reached endpoint by both RNFL parameters. Of the 33 EG eyes reaching any endpoint, a larger proportion reached endpoint first by retardance (n = 26, 79%) than did by RNFLT (n = 7, 21%; P = 0.002). Survival analysis indicated a shorter time to reach endpoint by retardance than by RNFLT (P < 0.001). Of the 21 EG eyes that reached endpoint by both measures, the median duration to endpoint was 120 days for retardance and 223 days for RNFLT (P = 0.003, Wilcoxon test). The time to onset was faster for retardance than that for RNFLT based on either segmented fits (by 31 days; P = 0.008, average R(2) = 0.89) or exponential fits (by 102 days; P = 0.01, average R(2) = 0.89). CONCLUSIONS The onset of progressive loss of RNFL retardance occurs earlier than the onset of RNFL thinning. Endpoints of progressive loss from baseline also occurred more frequently and earlier for RNFL retardance as compared with RNFLT.

Anatomic vs. acquired image frame discordance in spectral domain optical coherence tomography minimum rim measurements.

Purpose To quantify the effects of using the fovea to Bruchs membrane opening (FoBMO) axis as the nasal-temporal midline for 30° sectoral (clock-hour) spectral domain optical coherence tomography (SDOCT) optic nerve head (ONH) minimum rim width (MRW) and area (MRA) calculations. Methods The internal limiting membrane and BMO were delineated within 24 radial ONH B-scans in 222 eyes of 222 participants with ocular hypertension and glaucoma. For each eye the fovea was marked within the infrared reflectance image, the FoBMO angle (θ) relative to the acquired image frame (AIF) horizontal was calculated, the ONH was divided into 30°sectors using a FoBMO or AIF nasal/temporal axis, and SDOCT MRW and MRA were quantified within each FoBMO vs. AIF sector. For each sector, focal rim loss was calculated as the MRW and MRA gradients (i.e. the difference between the value for that sector and the one clockwise to it divided by 30°). Sectoral FoBMO vs. AIF discordance was calculated as the difference between the FoBMO and AIF values for each sector. Generalized estimating equations were used to predict the eyes and sectors of maximum FoBMO vs. AIF discordance. Results The mean FoBMO angle was −6.6±4.2° (range: −17° to +7°). FoBMO vs. AIF discordance in sectoral mean MRW and MRA was significant for 7 of 12 and 6 of 12 sectors, respectively (p<0.05, Wilcoxon test, Bonferroni correction). Eye-specific, FoBMO vs. AIF sectoral discordance was predicted by sectoral rim gradient (p<0.001) and FoBMO angle (p<0.001) and achieved maximum values of 83% for MRW and 101% for MRA. Conclusions Using the FoBMO axis as the nasal-temporal axis to regionalize the ONH rather than a line parallel to the AIF horizontal axis significantly influences clock-hour SDOCT rim values. This effect is greatest in eyes with large FoBMO angles and sectors with focal rim loss.

Details of Glaucomatous Damage Are Better Seen on OCT En Face Images Than on OCT Retinal Nerve Fiber Layer Thickness Maps.

PURPOSE High-resolution images of glaucomatous damage to the retinal nerve fiber layer (RNFL) were obtained with an adaptive optics-scanning light ophthalmoscope (AO-SLO) and used as a basis for comparisons between en face slab images and thickness maps derived from optical coherence tomography (OCT) scans. METHODS Wide-field (9 × 12 mm) cube scans were obtained with swept-source OCT (DRI-OCT) from six eyes of six patients. All eyes had a deep defect near fixation as seen on a 10-2 visual field test. Optical coherence tomography en face images, based on the average reflectance intensity, were generated (ATL 3D-Suite) from 52-μm slabs just below the vitreal border of the inner limiting membrane. The RNFL thickness maps were generated from the same OCT data. Both were compared with the AO-SLO peripapillary images that were previously obtained. RESULTS On AO-SLO images, three eyes showed small regions of preserved and/or missing RNFL bundles within the affected region. Details in these regions were seen on the OCT en face images but not on the RNFL thickness maps. In addition, in the healthier hemi-retinas of two eyes, there were darker, arcuate-shaped regions on en face images that corresponded to abnormalities seen on AO-SLO. These were not seen on RNFL thickness maps. CONCLUSIONS Details of local glaucomatous damage, missing or easily overlooked on traditional OCT RNFL thickness analysis used in clinical OCT reports, were seen on OCT en face images based on the average reflectance intensity. While more work is needed, it is likely that en face slab imaging has a role in the clinical management of glaucoma.

Relationship between Orbital Optic Nerve Axon Counts and Retinal Nerve Fiber Layer Thickness Measured by Spectral Domain Optical Coherence Tomography

PURPOSE We determined the relationship between total optic nerve axon counts and peripapillary retinal nerve fiber layer thickness (RNFLT) measured in vivo by spectral domain optical coherence tomography (SDOCT). METHODS A total of 22 rhesus macaques had three or more baseline measurements in both eyes of peripapillary RNFLT made by SDOCT. Laser photocoagulation then was applied to the trabecular meshwork of one eye to induce chronic unilateral IOP elevation. SDOCT measurements of RNFLT continued approximately every two weeks until the predefined study endpoint was reached in each animal. At endpoint, animals were sacrificed and the optic nerve was sampled approximately 2 mm behind the globe to obtain thin sections for histologic processing and automated axon counting across 100% of the optic nerve cross-sectional area. RESULTS At the final imaging session, the average loss of RNFLT was 20 ± 21%, ranging from essentially no loss to nearly 65% loss. Total optic nerve axon count in control eyes ranged from 812,478 to 1,280,474. The absolute number of optic nerve axons was related linearly to RNFLT (axon count = 12,336 × RNFLT((μm)) - 257,050, R(2) = 0.65, P < 0.0001), with a Pearson correlation coefficient of 0.81. There also was a strong linear relationship between relative optic nerve axon loss (glaucomatous-to-control eye) and relative RNFLT at the final imaging session, with a slope close to unity but a significantly negative intercept (relative axon loss((%)) = 1.05 × relative RNFLT loss((%)) - 14.4%, R(2) = 0.75, P < 0.0001). The negative intercept was robust to variations of fitted model because relative axon loss was -14% on average for all experimental glaucoma (EG) eyes within 6% (measurement noise) of zero relative loss. CONCLUSIONS There is a strong linear relationship between total optic nerve axon count and RNFLT measured in vivo by SDOCT. However, substantial loss of optic nerve axons (∼10%-15%) exists before any loss of RNFLT manifests and this discrepancy persists systematically throughout a wide range of damage.

Automated Quantification of Optic Nerve Axons in Primate Glaucomatous and Normal Eyes—Method and Comparison to Semi-Automated Manual Quantification

PURPOSE To describe an algorithm and software application (APP) for 100% optic nerve axon counting and to compare its performance with a semi-automated manual (SAM) method in optic nerve cross-section images (images) from normal and experimental glaucoma (EG) nonhuman primate (NHP) eyes. METHODS ON cross sections from eight EG eyes from eight NHPs, five EG and five normal eyes from five NHPs, and 12 normal eyes from 12 NHPs were imaged at 100×. Calibration (n = 500) and validation (n = 50) image sets ranging from normal to end-stage damage were assembled. Correlation between APP and SAM axon counts was assessed by Deming regression within the calibration set and a compensation formula was generated to account for the subtle, systematic differences. Then, compensated APP counts for each validation image were compared with the mean and 95% confidence interval of five SAM counts of the validation set performed by a single observer. RESULTS Calibration set APP counts linearly correlated to SAM counts (APP = 10.77 + 1.03 [SAM]; R(2) = 0.94, P < 0.0001) in normal to end-stage damage images. In the validation set, compensated APP counts fell within the 95% confidence interval of the SAM counts in 42 of the 50 images and were within 12 axons of the confidence intervals in six of the eight remaining images. Uncompensated axon density maps for the normal and EG eyes of a representative NHP were generated. CONCLUSIONS An APP for 100% ON axon counts has been calibrated and validated relative to SAM counts in normal and EG NHP eyes.