Normal Monkey Eyes Research Articles

Endothelial Glycocalyx Morphology in Different Flow Regions of the Aqueous Outflow Pathway of Normal and Laser-Induced Glaucoma Monkey Eyes.

Glycocalyx morphology was examined in the trabecular outflow pathway of monkey eyes with and without experimental glaucoma. Laser burns were administered along ~270 degrees of the trabecular meshwork (TM) of one eye (n = 6) or both eyes (n = 2) of each monkey until intraocular pressure remained elevated. Portions of the TM were not laser-treated. Unlasered eyes (n = 6) served as controls. Enucleated eyes were perfused at 15 mmHg to measure the outflow facility, perfused with fluorescein to evaluate the outflow pattern, perfusion-fixed for glycocalyx labeling, and processed for electron microscopy. Coverage and thickness of the glycocalyx were measured in the TM, Schlemm’s canal (SC), collector channels (CCs), intrascleral veins (ISVs), and episcleral veins (ESVs) in non-lasered regions and high- and low-flow regions of controls. Compared to controls, laser-treated eyes had decreased outflow facility (p = 0.02). Glycocalyx thickness increased from the TM to ESVs in non-lasered regions and controls (p < 0.05). Glycocalyx coverage was generally greater distally in non-lasered regions (p < 0.05). In lasered regions, TM, SC, and CCs were partly to completely obliterated, and ISVs and ESVs displayed minimal glycocalyx. Whether the glycocalyx is decreased in the trabecular outflow pathway of human glaucomatous eyes warrants investigation.

Eye-specific 3D modeling of factors influencing oxygen concentration in the lamina cribrosa

Our goal was to identify the factors with the strongest influence on the minimum lamina cribrosa (LC) oxygen concentration as potentially indicative of conditions increasing hypoxia risk. Because direct measurement of LC hemodynamics and oxygenation is not yet possible, we developed 3D eye-specific LC vasculature models. The vasculature of a normal monkey eye was perfusion-labeled post-mortem. Serial cryosections through the optic nerve head were imaged using fluorescence and polarized light microscopy to visualize the vasculature and collagen, respectively. The vasculature within a 450 μm-thick region containing the LC – identified from the collagen, was segmented, skeletonized, and meshed for simulations. Using Monte Carlo sampling, 200 vascular network models were generated with varying vessel diameter, neural tissue oxygen consumption rate, inflow hematocrit, and blood pressures (arteriole, venule, anterior boundary, and posterior boundary). Factors were varied over ranges of baseline ±20% with uniform probability. For each model we first obtained the blood flow, and from this the neural tissue oxygen concentration. ANOVA was used to identify the factors with the strongest influence on the minimum (10th percentile) oxygen concentration in the LC. The three most influential factors were, in ranked order, vessel diameter, neural tissue oxygen consumption rate, and arteriole pressure. There was a strong interaction between vessel diameter and arteriole pressure whereby the impact of one factor was larger when the other factor was small. Our results show that, for the eye analyzed, conditions that reduce vessel diameter, such as vessel compression due to elevated intraocular pressure or gaze-induced tissue deformation, may particularly contribute to decreased LC oxygen concentration. More eyes must be analyzed before generalizing.

Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigmented epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of the small GTPases Rac and Rho, because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility, while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential. © 2022 The Pathological Society of Great Britain and Ireland.

Lamina cribrosa vessel and collagen beam networks are distinct

Our goal was to analyze the spatial interrelation between vascular and collagen networks in the lamina cribrosa (LC). Specifically, we quantified the percentages of collagen beams with/without vessels and of vessels inside/outside of collagen beams. To do this, the vasculature of six normal monkey eyes was labeled by perfusion post-mortem. After enucleation, coronal cryosections through the LC were imaged using fluorescence and polarized light microscopy to visualize the blood vessels and collagen beams, respectively. The images were registered to form 3D volumes. Beams and vessels were segmented, and their spatial interrelationship was quantified in 3D. We found that 22% of the beams contained a vessel (range 14%–32%), and 21% of vessels were outside beams (13%–36%). Stated differently, 78% of beams did not contain a vessel (68%–86%), and 79% of vessels were inside a beam (64%–87%). Individual monkeys differed significantly in the fraction of vessels outside beams (p < 0.01 by linear mixed effect analysis), but not in the fraction of beams with vessels (p > 0.05). There were no significant differences between contralateral eyes in the percent of beams with vessels and of vessels outside beams (p > 0.05). Our results show that the vascular and collagenous networks of the LC in monkey are clearly distinct, and the historical notions that each LC beam contains a vessel and all vessels are within beams are inaccurate. We postulate that vessels outside beams may be relatively more vulnerable to mechanical compression by elevated IOP than are vessels shielded inside of beams.

Adenosine receptor distribution in Rhesus monkey ocular tissue

Adenosine receptor (ADOR) antagonists, such as 7-methylxanthine (7-MX), have been shown to slow myopia progression in humans and animal models. Adenosine receptors are found throughout the body, and regulate the release of neurotransmitters such as dopamine and glutamate. However, the role of adenosine in eye growth is unclear. Evidence suggests that 7-MX increases scleral collagen fibril diameter, hence preventing axial elongation. This study used immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to examine the distribution of the four ADORs in the normal monkey eye to help elucidate potential mechanisms of action. Eyes were enucleated from six Rhesus monkeys. Anterior segments and eyecups were separated into components and flash-frozen for RNA extraction or fixed in 4% paraformaldehyde and processed for immunohistochemistry against ADORA1, ADORA2a, ADORA2b, and ADORA3. RNA was reverse-transcribed, and qPCR was performed using custom primers. Relative gene expression was calculated using the ΔΔCt method normalizing to liver expression, and statistical analysis was performed using Relative Expression Software Tool. ADORA1 immunostaining was highest in the iris sphincter muscle, trabecular meshwork, ciliary epithelium, and retinal nerve fiber layer. ADORA2a immunostaining was highest in the corneal epithelium, trabecular meshwork, ciliary epithelium, retinal nerve fiber layer, and scleral fibroblasts. ADORA2b immunostaining was highest in corneal basal epithelium, limbal stem cells, iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells and scattered scleral fibroblasts. ADORA3 immunostaining was highest in the iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells, and scleral fibroblasts. Compared to liver mRNA, ADORA1 mRNA was significantly higher in the brain, retina and choroid, and significantly lower in the iris/ciliary body. ADORA2a expression was higher in brain and retina, ADORA2b expression was higher in retina, and ADORA3 was higher in the choroid. In conclusion, immunohistochemistry and RT-qPCR indicated differential patterns of expression of the four adenosine receptors in the ocular tissues of the normal non-human primate. The presence of ADORs in scleral fibroblasts and the choroid may support mechanisms by which ADOR antagonists prevent myopia. The potential effects of ADOR inhibition on both anterior and posterior ocular structures warrant investigation.

Reproducibility of Perfusion Parameters of Optic Disc and Macula in Rhesus Monkeys by Optical Coherence Tomography Angiography.

Background:Optical coherence tomography (OCT) angiography is a novel technique by which we can detect the local perfusion of fundus directly. The aim of this study was to evaluate the reproducibility of optic disc and macular flow perfusion parameters in rhesus monkeys using OCT angiography.Methods:Eighteen healthy monkeys (18 eyes) were subjected to optic disc and macula flow index measurements via a high-speed and high-resolution spectral-domain OCT XR Avanti with a split-spectrum amplitude de-correlation angiography algorithm. Right eye was imaged 3 times during the first examination and once during each of the two following examinations. The intra-visit and inter-visit intraclass correlation coefficients (ICCs) were both determined.Results:The average flow indices of the four optic disc area layers were 0.171 ± 0.009 (optic nerve head), 0.015 ± 0.004 (vitreous), 0.052 ± 0.009 (radial peripapillary capillary), and 0.167 ± 0.011 (choroid). Average flow indices of the four macula area layers were 0.044 ± 0.011 (superficial retina), 0.036 ± 0.011 (deep retina), 0.016 ± 0.009 (outer retina), and 0.155 ± 0.013 (choroid). Intra-visit (ICC value: 0.821–0.954) and inter-visit (ICC value: 0.844–0.899) repeatability were both high.Conclusions:The study is about the reproducibility of optic disc and macular perfusion parameters as measured by OCT angiography in healthy rhesus monkeys. Flow index measurement reproducibility is high for both the optic disc and macula of normal monkey eyes. OCT angiography might be a useful technique to assess changes when examining monkeys with experimental ocular diseases.

Normative Values of Retinal Oxygen Saturation in Rhesus Monkeys: The Beijing Intracranial and Intraocular Pressure (iCOP) Study.

ObjectiveTo study the normal values of the retinal oxygen saturation in Rhesus monkeys and to evaluated repeatability and reproducibility of retinal oxygen saturation measurements.MethodsEighteen adult Rhesus macaque monkeys were included in this experimental study. An Oxymap T1 retinal oximeter (Oxymap, Reykjavik, Iceland) was used to perform oximetry on all subjects. Global arterial (SaO2) and venous oxygen saturation (SvO2), arteriovenous difference in SO2 were measured. In the first examination, each eye was imaged three times. At the following two examinations, each eye was imaged once. All examinations were finished in one month. P values were calculated to evaluate the difference between the measurements during three visits by performing an ANOVA. Intra-visit and inter-visit intraclass correlation coefficient (ICC) was determined.ResultsAt baseline, the average SaO2 and SvO2 were 89.48 ± 2.64% and 54.85 ± 2.18%, respectively. The global A-V difference was 34.63 ± 1.91%. The difference between the three visits was not significant (p>0.05). The highest A-V difference in SO2 and lowest saturations were found in the inferotemporal quadrant. Intra-session and inter-visit repeatability were both high. For all oxygen saturation parameters, the ICC values of the intra-session repeatability ranged between 0.92 and 0.96. As found previously, a relatively high ICC value for inter-visit repeatability also was found for all oxygen saturation measurements, ranging between 0.86 and 0.94, with the lowest values in the infero-nasal quadrant.ConclusionsOur study is the first to describe retinal SO2 in healthy Rhesus monkeys. In normal monkey eyes, the reproducibility and repeatability of retinal oximetry oxygen saturation measurements were high in the retinal arterioles and venules. Our results support that Oxymap T1 retinal oximetry is a suitable and reliable technique in monkey studies.

Lamina cribrosa microarchitecture in normal monkey eyes part 1: methods and initial results.

To introduce quantitative postmortem lamina cribrosa (LC) microarchitecture (LMA) assessment and characterize beam diameter (BD), pore diameter (PD), and connective tissue volume fraction (CTVF) in 21 normal monkey eyes. Optic nerve heads (ONHs) underwent digital three-dimensional (3D) reconstruction and LC beam segmentation. Each beam and pore voxel was assigned a diameter based on the largest sphere that contained it before transformation to one of twelve 30° sectors in a common cylinder. Mean BD, PD, and CTVF within 12 central and 12 peripheral subsectors and within inner, middle, and outer LC depths were assessed for sector, subsector, and depth effects by analysis of variance using general estimating equations. Eye-specific LMA discordance (the pattern of lowest connective tissue density) was plotted for each parameter. The ranges of mean BD, PD, and CTVF were 14.0 to 23.1 μm, 20.0 to 35.6 μm, and 0.247 to 0.638, respectively. Sector, subsector, and depth effects were significant (P < 0.01) for all parameters except subsector on CTVF. Beam diameter and CTVF were smaller and PD was larger within the superior-temporal (ST) and inferior-temporal (IT) sectors (P < 0.05). These differences were enhanced within the central versus peripheral subsectors. Beam diameter and CTVF were larger and PD was smaller (P < 0.05) within the middle LC layer. Lamina cribrosa microarchitecture discordance most commonly occurred within the ST and IT sectors, varied by eye, and generally diminished as CTVF increased. Our data support previous characterizations of diminished connective tissue density within the ST and IT ONH regions. The clinical importance of eye-specific LMA discordance warrants further study.

Effect of 0.04% AR-13324, a ROCK, and norepinephrine transporter inhibitor, on aqueous humor dynamics in normotensive monkey eyes.

To determine the mechanism by which topically applied AR-13324, a rho kinase inhibitor, and an inhibitor of the norepinephrine transporter, reduces intraocular pressure (IOP) in normotensive monkey eyes. Seven normotensive monkeys were used. Tonographic outflow facility (C) was measured before drug administration and repeated 6 hours after administration of 50 µL (25 µL×2) of 0.04% AR-13324 to 1 eye and an equal volume of vehicle to the contralateral control eye. Baseline aqueous humor flow rates (F) were measured hourly for 6 hours beginning at 10:00 AM on day 1. On day 2, 50 µL (25 µL×2) of 0.04% AR-13324 was applied to 1 eye of each animal and vehicle to the fellow eye at 8:00 AM. Aqueous humor flow rates were measured at the same times as on the baseline day beginning 2 hours after dosing. Six hours after a single dose of 0.04% AR-13324 to 7 normal monkey eyes, C was increased (P<0.05) by 53% in drug-treated eyes compared with either contralateral vehicle-treated control eyes or baseline measurements. The IOP measured by pneumatonometer in treated eyes was reduced (P<0.005) by 25% when compared with baseline measurements and by 24% when compared with contralateral vehicle-treated eyes. For 6 hours after a single dose of 0.04% AR-13324, F was reduced (P<0.05) by 20% and 23% when compared with contralateral vehicle-treated eyes and baseline values, respectively. AR-13324 reduces IOP in normotensive monkey eyes. A dual mechanism of action, increase in tonographic outflow facility, and decrease of aqueous humor flow rates, accounts for the IOP reduction in normotensive monkey eyes.

Reproducibility of Macular, Retinal Nerve Fiber Layer, and ONH Measurements by OCT in Rhesus Monkeys: The Beijing Intracranial and Intraocular Pressure (iCOP) Study

We evaluated repeatability and reproducibility of measurements of the retinal nerve fiber layer thickness (RNFLT), optic nerve head (ONH), and macular inner retinal layer (MIRL) by RTVue spectral-domain optical coherence tomography (OCT) in normal Rhesus monkeys. The experimental study included 15 adult Rhesus macaque monkeys. RNFLT, ONH parameters (area of disc, cup, and rim; volume of cup and rim; and cup-to-disc ratios), and MIRL thickness were imaged at three separate examinations within one month. Each eye was imaged three times at the first examination, and once at each of the two following examinations. We determined the intra-session and inter-session (inter-visit) within-subject SD (Sw), precision (1.96 × Sw), coefficient of variation (CVw, 100 × Sw/overall mean), and intraclass correlation coefficient (ICC). For the intra-session repeatability, ICC values ranged between 0.97 and 1.00 for all ONH parameters, and between 0.80 and 0.98 for all regional RNFLT parameters, with a value of 0.97 for the average RNFLT. For the inter-session (inter-visit) reproducibility, ICC values ranged from 0.93-1.00 for the ONH measurements and from 0.63-0.97 for regional RNFLT parameters, with a value of 0.96 for the average RNFLT. Again, the ICC values were lowest in the temporal inferior and temporal superior regions. The ICC value for the average MIRL thickness was 0.95. Repeatability and reproducibility of spectral-domain OCT measurements of the RNFL, ONH, and MIRL were high in normal monkey eyes. Spectral-domain OCT may be suitable to assess changes in follow-up examinations of monkeys with experimental glaucoma.

Reproducibility of Measuring Lamina Cribrosa Pore Geometry in Human and Nonhuman Primates with In Vivo Adaptive Optics Imaging

The ability to consistently resolve lamina cribrosa pores in vivo has applications in the study of optic nerve head and retinal disease mechanisms. Repeatability was assessed in imaging laminar pores in normal living eyes with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO). Reflectance images (840 nm) of the anterior lamina cribrosa were acquired using the AOSLO in four or more different sessions in two normal rhesus monkey eyes and three normal human eyes. Laminar pore areas, elongations (ratio of major to minor axes of the best-fit ellipse) and nearest neighbor distances were calculated for each session. Measurement repeatability was assessed across sessions. Pore areas ranged from 90 to 4365 μm(2) in monkeys and 154 to 6637 μm(2) in humans. Mean variabilities in measuring pore area and elongation (i.e., mean of the standard deviation of measurements made across sessions for the same pores) were 50 μm(2) (6.1%) and 0.13 (6.7%), respectively, in monkeys and 113 μm(2) (8.3%) and 0.17 (7.7%), respectively, in humans. Mean variabilities in measuring nearest neighbor distances were 1.93 μm (5.2%) in monkeys and 2.79 μm (4.1%) in humans. There were no statistically significant differences in any pore parameters across sessions (ANOVA, P > 0.05). The anterior lamina cribrosa was consistently imaged in vivo in normal monkey and human eyes. The small intersession variability in normal pore geometry suggests that AOSLO imaging could be used to measure and track changes in laminar pores in vivo during glaucomatous progression.

Comparative study of the retinal vessel anatomy of rhesus monkeys and humans

This study aims to compare retinal vessel anatomy of normal rhesus monkeys and humans and to provide a basis from a structural perspective for the use of rhesus monkey as an experimental model in future studies of retinal vessels. The retinas of six normal rhesus monkey eyes and eight human eyecups following corneal transplantation were obtained and stained using adenosine diphosphatase methods. The distributions, orders, layers of the retinal vessels and the perifoveal vascular ring were compared. With adenosine diphosphatase staining, distinct retinal vessels were fully discernable from the first order surrounding the optic disc to the fifth order. There were no statistically significant differences between rhesus monkeys and humans in the percentages of vessels surrounding the optic disc at the equator and the peripheral region. Vascular networks in both species were arranged in several layers around the optic disc, two anastomotic layers at the equator and one sparse layer peripherally. Capillaries at the macular area were quite dense and an intact perifoveal vascular ring was observed. No differences were observed between rhesus monkeys and humans in the percentage area of the vessels and the area, perimeter and diameter of the perifoveal vascular ring. The distributions, orders, layers and the perifoveal vascular ring of the retinal vessels of rhesus monkey are quite similar to those of humans. The data suggest that from an anatomical perspective, the rhesus monkey is a good animal model for the study of human retinal vessels, particularly the macular capillaries.

Effects of Topical Phenylephrine and Tafluprost on Optic Nerve Head Circulation in Monkeys with Unilateral Experimental Glaucoma

To compare the effects of a topical alpha agonist (vasoconstrictor) and a prostaglandin receptor (FP) agonist (vasodilator) on circulation in the optic nerve head (ONH) in experimental glaucomatous and normal eyes of monkeys. Tissue blood velocity in the ONH (NB(ONH)) was determined using the laser speckle method in both eyes of eight normal cynomolgus monkeys under systemic anesthesia for 180 minutes after bilateral instillation of 5% phenylephrine. The effect of 0.0015% tafluprost, a potent FP agonist, was also studied after single and once-daily 7-day instillations. Measurements were repeated in both eyes of the eight monkeys after establishment of unilateral laser-induced glaucoma. NB(ONH) decreased significantly in both eyes of normal monkeys 30 to 120 minutes after phenylephrine instillation by a maximum of 9% to 11% (P < 0.05) without significant change in intraocular pressure (IOP). A similar decrease in NB(ONH) was found in non-laser-treated eyes in glaucomatous monkeys despite the absence of significant changes in contralateral experimental glaucomatous eyes. NB(ONH) increased by 16% (P < 0.05) 60 minutes after a single instillation and also after 7-day repeated instillations of tafluprost in both eyes of normal monkeys. A similar increase in NB(ONH) occurred in both eyes after the establishment of unilateral glaucoma but was completely abolished by 5 mg/kg indomethacin injected intravenously 15 minutes after tafluprost instillation. Tafluprost significantly reduced IOP only in experimental glaucomatous eyes by 34%. The ONH vasculature in glaucomatous and normal eyes reacts differently to an exogenous alpha agonist, whereas it reacts similarly to an FP agonist.

A Comparison of Optic Nerve Head Morphology Viewed by Spectral Domain Optical Coherence Tomography and by Serial Histology

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. 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 Bruch's 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. 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 Bruch's 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. 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.

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

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.

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

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. 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. 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. The termination of Bruch's 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.

Comparison of Clinical and Three-Dimensional Histomorphometric Optic Disc Margin Anatomy

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. 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 Bruch's 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. 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 Bruch's 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 Bruch's membrane terminated externally to the inferior edge of the border tissue; this latter structure aligned to the disc margin. 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 Bruch's membrane makes an important contribution to disc margin anatomy.

Effect of Flunarizine, a Calcium Channel Blocker, on Intraocular Pressure and Aqueous Humor Dynamics in Monkeys

To evaluate the effects of flunarizine, a nonselective calcium channel blocker, on intraocular pressure (IOP) in monkeys with laser-induced unilateral glaucoma and on aqueous humor dynamics in normal monkeys. The IOP was measured before and hourly for 6 hours after single-dose administration of 0.5%, 1%, or 2% flunarizine to the glaucomatous eye of 8 monkeys with unilateral laser-induced glaucoma. In a separate multiple-dose study, 0.5% flunarizine was applied twice daily for 5 consecutive days to the glaucomatous eye of the same 8 monkeys. IOP was measured at untreated baseline, after treatment with vehicle only, and on treatment days 1, 3, and 5. Tonographic outflow facility and fluorophotometric flow rates of aqueous humor were measured in 7 normal monkeys before and after the fifth dose of twice-daily treatment with 0.5% flunarizine. Unilateral application of 50 microL of 0.5%, 1%, or 2% flunarizine reduced IOP bilaterally. In the treated glaucomatous eyes, flunarizine reduced the IOP for 2, 3, or 5 hours, with a maximum reduction of 2.5+/-0.5 (mean+/-SEM) mm Hg (9%), 3.0+/-0.4 mm Hg (10%), and 5.0+/-0.8 mm Hg (18%) following the 0.5%, 1%, and 2% concentrations, respectively (P<0.01). The maximum reductions in IOP in the contralateral untreated eyes were 1.3+/-0.5 mm Hg, 1.5+/-0.3 mm Hg, and 2.9+/-0.7 mm Hg following the 0.5%, 1%, and 2% concentrations, respectively (P<0.05). Both the magnitude and duration of the ocular hypotensive effect of 0.5% flunarizine were enhanced with twice-daily administration for 5 days. Outflow facility in normal monkey eyes was increased (P<0.05) by 39% in the treated eyes compared with vehicle-treated contralateral eyes and by 41% compared with baseline values, and aqueous humor flow rates were unchanged (P>0.30). Flunarizine reduces IOP in a dose-dependent manner when administered to glaucomatous monkey eyes, but also has an ocular hypotensive effect on the contralateral untreated eyes. An increase in tonographic outflow facility seems to account for the IOP reduction in normal monkey eyes.

Effects of Spectral Characteristics of Ganzfeld Stimuli on the Photopic Negative Response (PhNR) of the ERG

To determine flash and background colors that best isolate the photopic negative response (PhNR) and maximize its amplitude in the primate ERG. Photopic full-field flash ERGs were recorded from anesthetized macaque monkeys before and after pharmacologic blockade of Na(+)-dependent spiking activity with tetrodotoxin (TTX, 1 to 2 muM, n = 3), blockade of ionotropic glutamatergic transmission with cis-2,3 piperidine dicarboxylic acid (PDA, 3.3-3.8 mM, n = 3) or laser-induced monocular experimental glaucoma (n = 6), and from six normal human subjects. Photopically matched colored flashes of increasing stimulus strengths were presented on scotopically matched blue, white, or yellow backgrounds of 100 scot cd/m(2) using an LED-based stimulator. PhNRs that could be eliminated by TTX or severe experimental glaucoma were present in responses to brief (<5 ms) and long-duration (200 ms) stimuli of all color combinations. In normal monkey and human eyes for brief low-energy flashes, PhNR amplitudes were highest for red flashes on blue backgrounds and blue flashes on yellow backgrounds. For high-energy flashes, amplitudes were more similar for all color combinations. For long-duration stimuli, the PhNR(on) at light onset in monkeys was larger for red and blue stimuli, regardless of background color, than for spectrally broader flashes, except for stimuli >17.7 cd/m(2) when PhNR(on)s were all of similar amplitude. For red flashes, eliminating the PhNR(on) pharmacologically or by glaucoma removed the slowly recovering negative wave that normally followed the transient b-wave and elevated the whole ON response close to the level of the b-wave peak. However, for white, blue, and green flashes, a lower-amplitude plateau that could be removed by PDA remained. For weak to moderate flash strengths, the best stimulus for maximizing PhNR amplitude is one that primarily stimulates one cone type, on a background with minimal adaptive effect on cones. For stronger stimuli, differences in amplitude are smaller. For long-duration stimuli, red best isolates the PhNR(on) because it minimizes the overlapping lower-level plateau that originates from the activity of second-order hyperpolarizing retinal neurons.

Effect of 15-Keto Latanoprost on Intraocular Pressure and Aqueous Humor Dynamics in Monkey Eyes

To compare the ocular hypotensive effects of 15-keto latanoprost (KL) with the commercial preparation of latanoprost (Xalatan; Pfizer, New York, NY) in monkey eyes with laser-induced unilateral glaucoma and to evaluate the effects of topical 0.005% KL on aqueous humor dynamics in normal monkey eyes. Intraocular pressure (IOP) was measured hourly for 6 hours beginning at 9:30 AM on day 1 (untreated baseline); day 2 (vehicle only); and treatment days 1, 3, and 5 (topical, 30 microL of study drug) in the glaucomatous eyes of four to eight monkeys with unilateral laser-induced glaucoma. KL concentrations of 0.0001%, 0.001%, and 0.01% and latanoprost at 0.005% were studied separately, with a minimum washout period of 2 weeks between studies. Tonographic outflow facility (C) and fluorophotometric aqueous humor flow rates (F) were measured in nine normal monkeys before and after a single topical dose of 0.005% KL in one eye, with a vehicle-only control in the fellow eye. When applied once daily to glaucomatous monkey eyes, all three concentrations of KL and a 0.005% concentration of latanoprost produced significant (P < 0.05) reductions in IOP, with the maximum reduction on treatment day 5, regardless of the drug or concentration studied. The maximum reduction (P < 0.001) from vehicle-only baseline IOP was (mean +/- SEM) 3.0 +/- 0.3 mm Hg (9%) for 0.0001% KL, 7.6 +/- 0.6 mm Hg (23%) for 0.001% KL, 6.3 +/- 0.4 mm Hg (18%) for 0.01% KL, and 6.6 +/- 0.6 mm Hg (20%) for 0.005% latanoprost. After application of a single dose of 0.005% KL in nine normal monkey eyes, neither C nor F was altered (P > 0.80) when compared with untreated baseline values or vehicle-treated control eyes. The reduction in IOP produced by 0.001% KL was equivalent to, and at some measured time points, greater than the effect produced by 0.005% latanoprost. The IOP reduction by KL in normal monkeys appeared to have no effect on aqueous humor production or tonographic outflow facility and may thus indicate a drug-induced increase in uveoscleral outflow.