C. F. Burgoyne

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.

A comparison of optic nerve head morphology viewed by spectral domain optical coherence tomography and by serial histology.

PURPOSE To compare serial optic nerve head (ONH) histology with interpolated B-scans generated from a three-dimensional (3-D) spectral domain (SD)-OCT ONH volume acquired in vivo from the same normal monkey eye. METHODS A 15 degrees ONH SD-OCT volume was acquired in a normal monkey eye, with IOP manometrically controlled at 10 mm Hg. After perfusion fixation at 10 mm Hg, the ONH was trephined, the specimen embedded in a paraffin block, and serial sagittal sections cut at 4-mum intervals. The location of each histologic section was identified within the optic disc photograph by matching the position of the retinal vessels and of Bruchs membrane opening. By altering the angles of rotation and incidence, interpolated B-scans matching the location of the histologic sections were generated with custom software. Structures identified in the histologic sections were compared with signals identified in the matched B-scans. RESULTS Close matches between histologic sections and interpolated B-scans were identified throughout the extent of the ONH. SD-OCT identified the neural canal opening as the termination of the Bruchs membrane-retinal pigment complex and border tissue as the innermost termination of the choroidal signal. The anterior lamina cribrosa and its continuity with the prelaminar glial columns were also detected by SD-OCT. CONCLUSIONS Volumetric SD-OCT imaging of the ONH generates interpolated B-scans that accurately match serial histologic sections. SD-OCT captures the anterior laminar surface, which is likely to be a key structure in the detection of early ONH damage in ocular hypertension and glaucoma.

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.

Changes in the Biomechanical Response of the Optic Nerve Head in Early Experimental Glaucoma

PURPOSE To investigate the biomechanical response of the optic nerve head (ONH) connective tissues to IOP elevation in three pairs of monkey eyes in which one eye had early experimental glaucoma (EG). METHODS A serial imaging technique was used to reconstruct the ONH and peripapillary sclera of three pairs of unilateral EG eyes fixed at 10 mm Hg. Eye-specific finite element models of the posterior pole were constructed with inhomogeneous material properties defined for the lamina cribrosa (LC) based on local connective tissue volume fraction (CTVF) and predominant LC beam orientation. These models were used to simulate an IOP increase from 10 to 45 mm Hg. A laminar material constant was varied to produce a range of LC displacements and scleral canal expansions, and the associated LC stress and strain were characterized. RESULTS The models suggest that the LC of normal and EG eyes can deform posteriorly or anteriorly when the LC material stiffness is low or high, respectively. Scleral canal expansion was generally, but not always, reduced in EG eyes. Strains in the EG eye were similar to or lower than those in the contralateral eye for the same average LC displacement and increased when the LC was more plaint. Laminar stresses were consistently lower in the EG eye, regardless of LC stiffness. CONCLUSIONS Connective tissue remodeling in EG alters the biomechanical response of the LC to IOP elevation in an eye-specific manner. The models indicated that the LC tissues in EG eyes were more plaint than those in the contralateral normal eyes in two of three monkeys.

Relative course of retinal nerve fiber layer birefringence and thickness and retinal function changes after optic nerve transection.

PURPOSE To test the hypothesis that alterations of RNFL birefringence precede changes in RNFL thickness in an experimental model of RGC injury and, secondarily, to determine the time course of RGC functional abnormalities relative to RNFL birefringence and thickness changes. METHODS RNFL birefringence was measured by scanning laser polarimetry (GDx VCC; Carl Zeiss Meditec, Inc., Dublin, CA). RNFL thickness was measured by spectral domain optical coherence tomography (SD-OCT, Spectralis HRA+OCT; Heidelberg Engineering, GmbH, Heidelberg, Germany). Retinal function was assessed by three forms of electroretinography (ERG): slow-sequence multifocal (mf)ERG (VERIS; EDI, San Mateo, CA); pattern-reversal (P)ERG (Utas-E3000; LKC Technologies, Inc. Gaithersburg, MD); and photopic full-field flash (ff)ERG (Utas-E3000; LKC Technologies). All measurements were obtained in both eyes of four adult rhesus macaque monkeys (Macaca mulatta) during two baseline sessions, and again 1 week and 2 weeks after unilateral optic nerve transection (ONT). RESULTS ONT was successfully completed in three subjects. RNFL birefringence declined by 15% 1 week after ONT (P = 0.043), whereas there was no significant change in RNFL thickness (+1%, P = 0.42). Two weeks after ONT, RNFL retardance had declined by 39% (P = 0.018), whereas RNFL thickness had declined by only 15% (P = 0.025). RGC functional abnormalities were present 1 week after ONT, including decreased amplitudes relative to baseline of the mfERG high-frequency components (-65%, P = 0.018), the PERG N95 component (-70%, P = 0.007), and the photopic negative response of the ffERG (-44%, P = 0.005). CONCLUSIONS RNFL birefringence declined before and faster than RNFL thickness after ONT. RGC functional abnormalities were present 1 week after ONT, when RNFL thickness had not yet begun to change. RNFL birefringence changes after acute RGC injury are associated with RGC dysfunction. Together, they reflect RGC abnormalities that precede axonal caliber changes and loss.

Longitudinal Detection of Optic Nerve Head Changes by Spectral Domain Optical Coherence Tomography in Early Experimental Glaucoma

PURPOSE We determined if the detection of spectral-domain optical coherence tomography (SDOCT) optic nerve head (ONH) change precedes the detection of confocal scanning laser tomography (CSLT) ONH surface, SDOCT retinal nerve fiber layer (RNFL), scanning laser perimetry (SLP), and multifocal electroretinography (mfERG) change in eight experimental glaucoma (EG) eyes. METHODS Both eyes from eight monkeys were tested at least three times at baseline, and then every 2 weeks following laser-induced chronic unilateral IOP elevation. Event and trend-based definitions of onset in the control and EG eyes for 11 SDOCT neural and connective tissue, CSLT surface, SDOCT RNFL, SLP, and mfERG parameters were explored. The frequency and timing of onset for each parameter were compared using a logrank test. RESULTS Maximum post-laser IOP was 18 to 42 mm Hg in the EG eyes and 12 to 20 mm Hg in the control eyes. For event- and trend-based analyses, onsets were achieved earliest and most frequently within the ONH neural and connective tissues using SDOCT, and at the ONH surface using CSLT. SDOCT ONH neural and connective tissue parameter change preceded or coincided with CSLT ONH surface change in most EG eyes. The SDOCT and SLP measures of RNFL thickness, and mfERG measures of visual function demonstrated similar onset rates, but occurred later than SDOCT ONH and CSLT surface change, and in fewer eyes. CONCLUSIONS SDOCT ONH change detection commonly precedes or coincides with CSLT ONH surface change detection, and consistently precedes RNFLT, SLP, and mfERG change detection in monkey experimental glaucoma.

Comparison of clinical and three-dimensional histomorphometric optic disc margin anatomy.

PURPOSE To investigate the anatomic basis of the optic disc margin in the normal monkey eye by colocalizing optic disc photographs to three-dimensional (3D) histomorphometric reconstructions of the same optic nerve head. METHODS Optic disc photographs from 28 normal monkey eyes were overlaid onto 3D central retinal vessel reconstructions generated as part of postmortem optic nerve histomorphometric reconstructions for each eye. Within each reconstruction, the Bruchs membrane opening (BMO) was delineated. Alignment was achieved by matching the clinical vessel outline to the vessel reconstruction with parallel viewing software. An experienced observer viewed stereophotographs and marked the disc margin onto clinical photographs with custom software. Alignment of the delineated disc margin to the histomorphometrically defined BMO was qualitatively assessed within each image. RESULTS In 20 eyes, BMO aligned well to the disc margin delineation. In four eyes, alignment improved after repeated colocalization. Careful review of the histomorphometric reconstructions identified that in most cases Bruchs membrane extended beyond the termination of the border tissue of Elschnig, most substantially in the superior and nasal sectors. Misalignments could be explained by inaccurate BMO marking or where Bruchs membrane terminated externally to the inferior edge of the border tissue; this latter structure aligned to the disc margin. CONCLUSIONS BMO was a clinically detectable entity and represented the disc margin in most eyes in this study. The 3D architecture of the border tissue combined with the presence of an overhang of Bruchs membrane makes an important contribution to disc margin anatomy.

The effect of acute intraocular pressure elevation on peripapillary retinal thickness, retinal nerve fiber layer thickness, and retardance.

PURPOSE To determine whether acutely elevated intraocular pressure (IOP) alters peripapillary retinal thickness, retinal nerve fiber layer thickness (RNFLT), or retardance. METHODS Nine adult nonhuman primates were studied while under isoflurane anesthesia. Retinal and RNFLTs were measured by spectral domain optical coherence tomography 30 minutes after IOP was set to 10 mm Hg and 60 minutes after IOP was set to 45 mm Hg. RNFL retardance was measured by scanning laser polarimetry in 10-minute intervals for 30 minutes while IOP was 10 mm Hg, then for 60 minutes while IOP was 45 mm Hg, then for another 30 minutes after IOP was returned to 10 mm Hg. RESULTS RNFLT measured 1120 microm from the ONH center decreased from 118.1 +/- 9.3 microm at an IOP of 10 mm Hg to 116.5 +/- 8.4 microm at 45 mm Hg, or by 1.4% +/- 1.8% (P < 0.0001). There was a significant interaction between IOP and eccentricity (P = 0.0006). Within 800 microm of the ONH center, the RNFL was 4.9% +/- 3.4% thinner 60 minutes after IOP elevation to 45 mm Hg (P < 0.001), but unchanged for larger eccentricities. The same pattern was observed for retinal thickness, with 1.1% +/- 0.8% thinning overall at 45 mm Hg (P < 0.0001), and a significant effect of eccentricity (P < 0.0001) whereby the retina was 4.8% +/- 1.2% thinner (P < 0.001) within 800 microm, but unchanged beyond that. Retardance increased by a maximum of 2.2% +/- 1.1% 60 minutes after IOP was increased to 45 mm Hg (P = 0.0031). CONCLUSIONS The effects of acute IOP elevation on retinal thickness, RNFL thickness and retardance were minor, limited to the immediate ONH surround and unlikely to have meaningful clinical impact.

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.