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Purpose: Optic nerve head (ONH) atrophy is frequently associated with hydrocephalic conditions. Cerebrospinal fluid (CSF)-containing meninges form a subarachnoid space that terminates at the ONH, which physically impacts it. This study aims to characterize optic neuropathy in congenital hydrocephalic mice with genetic disruption of the Ccdc13 gene., Methods: The ccdc13 germline knockout mice were generated. The hydrocephalus phenotype and subarachnoid space surrounding the optic nerve were evaluated using routine histology and Evans blue stain. Optic neuropathy was examined with immunohistochemistry and transmission electron microscopy (TEM). Axon transport was indicated by cholera toxin subunit B (CTB) fluorescence conjugate. Retinal function was evaluated by electroretinography (ERG), and Ccdc13 expression was revealed by a knock-in Gfp reporter., Results: Ccdc13 mutant mice manifested hydrocephalus at birth. ONH displacement, or negative cupping, and enlarged subarachnoid space at the optic terminus occurred as early as 1 month after birth. Intraocular pressure (IOP) was normal. Optic neuropathy was first observed at the ONH, followed by a distal-to-proximal progression of optic nerve pathology indicated by alteration of axonal ultrastructure and deposition of unphosphorylated neurofilament heavy chain. Anterograde axonal transport was also hampered. Retinal ganglion cell (RGC) function was compromised as early as postnatal day 21 (P21), along with reduced neurofilament heavy chain expression. Optic neuropathy caused by disruption of Ccd13 was non-cell autonomous, stemming from hydrocephalus with presumed high intracranial pressure (ICP), which physically impacts the ONH by increasing the translaminar pressure gradient., Conclusions: We provided knowledge of optic neuropathy from a congenital mouse model for hydrocephalus. The hydrocephalus in mice could damage the ONH by increasing the translaminar pressure gradient and negative cupping, leading to impairment in axoplasmic transport and RGC pathology. Our findings highlight the importance of the interplay between IOP and ICP in the development of glaucoma.

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC) and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Purpose: To determine alteration of dendritic spines and associated changes in the primary visual cortex (V1 region) related to unilateral optic nerve crush (ONC) in adult mice., Methods: Adult unilateral ONC mice were established. Retinal nerve fiber layer (RNFL) thickness was measured by spectral-domain optical coherence tomography. Visual function was estimated by flash visual evoked potentials (FVEPs). Dendritic spines were observed in the V1 region contralateral to the ONC eye by two-photon imaging in vivo. The neurons, reactive astrocytes, oligodendrocytes, and activated microglia were assessed by NeuN, glial fibrillary acidic protein, CNPase, and CD68 in immunohistochemistry, respectively. Tropomyosin receptor kinase B (TrkB) and the markers in TrkB trafficking were estimated using western blotting and co-immunoprecipitation. Transmission electron microscopy and western blotting were used to evaluate autophagy., Results: The amplitude and latency of FVEPs were decreased and delayed at 3 days, 1 week, 2 weeks, and 4 weeks after ONC, and RNFL thickness was decreased at 2 and 4 weeks after ONC. Dendritic spines were reduced in the V1 region contralateral to the ONC eye at 2, 3, and 4 weeks after ONC, with an unchanged number of neurons. Reactive astrocyte staining was increased at 2 and 4 weeks after ONC, but oligodendrocyte and activated microglia staining remained unchanged. TrkB was reduced with changes in the major trafficking proteins, and enhanced autophagy was observed in the V1 region contralateral to the ONC eye., Conclusions: Dendritic spines were reduced in the V1 region contralateral to the ONC eye in adult mice. Reactive astrocytes and decreased TrkB may be associated with the reduced dendritic spines.

Purpose: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head., Methods: Twenty-five Wistar rats were killed by CO 2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 μm meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research., Results: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with β3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001)., Conclusions: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach., (© 2019 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

The purpose of this study was to investigate the effects of latanoprost, an ocular hypotensive prostaglandin analog, on scleral collagen fibers and laminar pores in myopic guinea pigs. Young guinea pigs underwent monocular form deprivation (FD; white plastic diffusers) from 14-days of age for 10-weeks. After the first week, FD eyes also received daily topical A) latanoprost (Lat, 0.005%, n = 5) or B) artificial tears (AT; n = 5). At the end of the treatment period, animals were sacrificed, eyes enucleated and optic nerve heads (ONH) excised to include a 4 mm diameter ring of surrounding sclera for scanning electron microscopy (SEM), and an additional 6 mm ring of sclera surrounding the ONH was excised for transmission electron microscopy (TEM). For SEM, ONH samples were first immersed in 0.2M NaOH for 30 h to isolate the collagenous structures. All samples were stained with osmium tetroxide, dried through an ethanol series and finally subjected to critical point drying before imaging. Image J was used to analyze the dimensions of laminar pores (SEM images) and scleral collagen fibers (TEM images). As previously reported in a related study, latanoprost was effective in inhibiting myopia progression in FD eyes of the guinea pigs. The scleral fibers of FD myopic eyes treated with AT were smaller and more variable in cross-sectional areas compared to untreated (fellow) eyes (mean areas: 0.0059 ± 0.0013 vs. 0.0085 ± 0.002 μm 2 ; p < 0.001), consistent with scleral changes reported for human myopia. In contrast, the scleral fibers of the Lat-treated FD eyes were similar to those of fellow eyes (0.0083 ± 0.002 vs. 0.0078 ± 0.0014 μm 2 ). However, laminar pore size appeared unaffected by either the FD or drug treatments, with no significant difference found between FD eyes and their fellows, for either treatment group. That daily topical latanoprost appeared to protect against myopia-related changes in scleral collagen, rather than exaggerating them, as might be predicted from its known action on the uveoscleral extracellular matrix, lends further support its use for myopia control. In this guinea pig myopia model, the lamina cribrosa appeared unaffected., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Photoreceptor (PR) axons project from the retina to the optic lobe in brain and form a precise retinotopic map in the Drosophila visual system. Yet the role of retinal basal glia in the retinotopic map formation is not previously known. We examined the formation of the retinotopic map by marking single PR pairs and following their axonal projections. In addition to confirming previous studies that the spatial information is preserved from the retina to the optic stalk and then to the optic lamina, we found that the young PR R3/4 axons transiently overshoot and then retract to their final destination, the lamina plexus. We then examined the process of wrapping glia (WG) membrane extension in the eye disc and showed that the WG membrane extensions also follow the retinotopic map. We show that the WG is important for the proper spatial distribution of PR axons in the optic stalk and lamina, suggesting an active role of wrapping glia in the retinotopic map formation.

Purpose: To study age- and intraocular pressure-induced changes in the glial lamina of the murine optic nerve on the ultrastructural level., Methods: Naïve C57bl/6 mice at various ages spanning the time between early adulthood (3 months) and senescence (30 months) were used in this study. In addition, the intraocular pressure (IOP) was increased in a group of young mice by injection of microbeads into the anterior chamber. The unmyelinated segments of the optic nerve containing the glial lamina were prepared for transmission electron microscopy and imaged at high resolution., Results: Axon packing density decreased slightly with age. Aging nerves contained higher numbers of enlarged and degenerating axons. Mean axonal diameter and in particular the variance of axonal diameter correlated well with age. Axonal mitochondria also showed age-dependent signs of pathology. The mean diameter of axonal mitochondria increased, and aged axons often contained profiles of mitochondria with very few or no cristae. Astrocytic mitochondria remained normal even in very old nerves. Changes to axons and axonal mitochondria in young glaucomatous nerves were comparable with those of 18- to 30-month-old naïve mice. In addition to axons and mitochondria, aged and glaucomatous nerves showed thickening of the blood vessel basement membranes and increased deposition of basement membrane collagen., Conclusions: On the ultrastructural level, the effects of age and elevated IOP are quite similar. One month of elevated IOP seems to have as strongly detrimental effects on the nerve as at least 18 months of normal aging.

Purpose: To examine the early glial reactivity and neuron damage in response to short-term cerebrospinal fluid pressure (CSFp) reduction, as compared with intraocular pressure (IOP) elevation., Methods: The experiment included 54 male Sprague-Dawley rats with elevated translaminar cribrosa pressure difference (TLPD), defined as IOP minus CSFp. These rats underwent either continuous CSF drainage for 6 hours (n = 18), or unilateral IOP elevation to 40 mm Hg for 6 hours (n = 18). For control, 18 normal rats were anesthetized for 6 hours. Orthograde axonal transport was examined by intravitreal injection of 3% rhodamine-β-isothiocyanate. We also used transmission electron microscopy to display the ultrastructural features of retinal ganglion cell axons in the optic nerve head. Early glial reactivity in the retina, lateral geniculate nucleus (LGN), and superior colliculus (SC) was detected by immunostaining and Western blot for the glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). We also observed the glial reactivity in the inferior colliculus and hippocampus to rule out possible influences of CSF dynamics and composition., Results: Anterograde staining with 3% rhodamine-β-isothiocyanate revealed decreased fluorescence intensity of the SC and LGN projected from both lower CSFp and higher IOP eyes. Transmission electron microscopy showed loss of axons from the optic nerve head in the high-IOP group, but not in the low-CSFp group. Compared with the anesthesia control group, GFAP expression was significantly increased in the retina, LGN, and SC, whereas GS expression was only increased in the retina following CSFp reduction. However, their expressions were not significantly elevated in the inferior colliculus and hippocampus. In the high-IOP group, expressions of GFAP and GS were significantly increased in the retina, LGN, and SC., Conclusions: Visual system neurons may be much more sensitive than other nervous tissues. Following short-term CSFp reduction, early glial reactivity may precede axonal loss. Changes of translaminar cribrosa pressure difference in both experimental low-CSFp and high-IOP groups induce selective early glial reactivity. The neuron damage from abnormally low CSFp may be pathogenetically similar to high IOP.

There is increasing clinical evidence that the eye is not only affected by intraocular pressure (IOP), but also by intracranial pressure (ICP). Both pressures meet at the optic nerve head of the eye, specifically the lamina cribrosa (LC). The LC is a collagenous meshwork through which all retinal ganglion cell axons pass on their way to the brain. Distortion of the LC causes a biological cascade leading to neuropathy and impaired vision in situations such as glaucoma and idiopathic intracranial hypertension. While the effect of IOP on the LC has been studied extensively, the coupled effects of IOP and ICP on the LC remain poorly understood. We investigated in-vivo the effects of IOP and ICP, controlled via cannulation of the eye and lateral ventricle in the brain, on the LC microstructure of anesthetized rhesus monkeys eyes using the Bioptigen spectral-domain optical coherence tomography (OCT) device (Research Triangle, NC). The animals were imaged with their head upright and the rest of their body lying prone on a surgical table. The LC was imaged at a variety of IOP/ICP combinations, and microstructural parameters, such as the thickness of the LC collagenous beams and diameter of the pores were analyzed. LC microstructure was confirmed by histology. We determined that LC microstructure deformed in response to both IOP and ICP changes, with significant interaction between the two. These findings emphasize the importance of considering both IOP and ICP when assessing optic nerve health.

Purpose: Optic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage., Methods: Mice expressing green fluorescent protein in individual astrocytes were used to evaluate the early response of astrocytes in the glial lamina of the optic nerve head after increasing the IOP using the microbead occlusion method. Tissue sections from the glial lamina were imaged consecutively by confocal and electron microscopy., Results: Confocal and electron microscope images show that astrocytes close to the myelination transition zone in the hypertensive nerve heads extend new processes that follow the longitudinal axis of the optic nerve and invade axon bundles in the nerve head. Ultrastructurally, the longitudinal processes were largely devoid of subcellular organelles except for degenerating mitochondria., Conclusions: The longitudinal processes are a common feature of glaucomatous optic nerve astrocytes, whereas they are not observed after traumatic nerve injury. Thus, astrocytes appear to fine-tune their responses to the nature and/or timing of the injury to the neurons that they surround.

Purpose: The purpose of this study was to leverage polarized light microscopy (PLM) to visualize the collagen fiber architecture of posterior pole and optic nerve head with micrometer-scale resolution and to identify and quantify major organizational components., Methods: Eight sheep posterior poles were cryosectioned and imaged using PLM. Collagen fiber orientation was determined by using custom scripts, and the resulting orientation maps were inspected and quantified to identify major structural elements and tested for differences in mean fiber orientation and anisotropy, using linear mixed effect models., Results: Images revealed an intricate organization of collagen fibers in the posterior pole. In the lamina cribrosa, interweaving fibers formed large knots and wrapped around nerve fiber pores, with beam insertions into the scleral canal wall that were either narrow and straight or wide. In the peripapillary sclera, three significantly different (P < 0.0001) components were identified: fibers oriented circumferentially proximal to the canal, radially in the innermost sclera, and unaligned with interweaving fibers. The radial fibers were between 60 and 180 μm thick, extending at least 3 mm from the canal., Conclusions: PLM revealed structural aspects of the lamina cribrosa and sclera that may have important biomechanical roles but that were previously unreported or not characterized quantitatively. In the lamina cribrosa, these roles included wide and narrow beam insertions and details of collagen fibers interweaving and wrapping around the pores. In the sclera, we described regions of circumferential, radial, and unaligned "random" fibers. Although there is consensus that circumferential fibers protect neural tissues by resisting canal expansion, the role of the radial fibers remains unclear.

Purpose: To investigate how the lamina cribrosa (LC) microstructure changes with distance from the central retinal vessel trunk (CRVT), and to determine how this change differs in glaucoma., Methods: One hundred nineteen eyes (40 healthy, 29 glaucoma suspect, and 50 glaucoma) of 105 subjects were imaged using swept-source optical coherence tomography (OCT). The CRVT was manually delineated at the level of the anterior LC surface. A line was fit to the distribution of LC microstructural parameters and distance from CRVT to measure the gradient (change in LC microstructure per distance from the CRVT) and intercept (LC microstructure near the CRVT). A linear mixed-effects model was used to determine the effect of diagnosis on the gradient and intercept of the LC microstructure with distance from the CRVT. A Kolmogorov-Smirnov test was applied to determine the difference in distribution between the diagnostic categories., Results: The percent of visible LC in all scans was 26 ± 7%. Beam thickness and pore diameter decreased with distance from the CRVT. Glaucoma eyes had a larger decrease in beam thickness (-1.132 ± 0.503 μm, P = 0.028) and pore diameter (-0.913 ± 0.259 μm, P = 0.001) compared with healthy controls per 100 μm from the CRVT. Glaucoma eyes showed increased variability in both beam thickness and pore diameter relative to the distance from the CRVT compared with healthy eyes (P < 0.05)., Conclusions: These findings results demonstrate the importance of considering the anatomical location of CRVT in the assessment of the LC, as there is a relationship between the distance from the CRVT and the LC microstructure, which differs between healthy and glaucoma eyes.

The guinea pig is becoming an increasingly popular model for studying human myopia, which carries an increased risk of glaucoma. As a step towards understanding this association, this study sought to characterize the normal, developmental intraocular pressure (IOP) profiles, as well as the anatomy of the optic nerve head (ONH) and adjacent sclera of young guinea pigs. IOP was tracked in pigmented guinea pigs up to 3 months of age. One guinea pig was imaged in vivo with OCT and one with a fundus camera. The eyes of pigmented and albino guinea pigs (ages 2 months) were enucleated and sections from the posterior segment, including the ONH and surrounding sclera, processed for histological analyses - either hematoxylin and eosin (H&E) staining of paraffin embedded, sectioned tissue (n = 1), or cryostat sectioned tissue, processed for immunohistochemistry (n = 3), using primary antibodies against collagen types I-V, elastin, fibronectin and glial fibrillary acidic protein (GFAP). Transmission and scanning electron microscopy (TEM, SEM) studies of ONHs were also undertaken (n = 2 & 5 respectively). Mean IOPs ranged from 17.33 to 22.7 mmHg, increasing slightly across the age range studied, and the IOPs of individual animals also exhibited diurnal variations, peaking in the early morning (mean of 25.8, mmHg, ∼9 am), and decreasing across the day. H&E-stained sections showed retinal ganglion cell axons organized into fascicles in the prelaminar and laminar region of the ONHs, with immunostained sections revealing collagen types I, III, IV and V, as well as elastin, GFAP and fibronectin in the ONHs. SEM revealed a well-defined lamina cribrosa (LC), with radially-oriented collagen beams. TEM revealed collagen fibrils surrounding non-myelinated nerve fiber bundles in the LC region, with myelination and decreased collagen posterior to the LC. The adjacent sclera comprised mainly crimped collagen fibers in a crisscross arrangement. Both the sclera and LC were qualitatively similar in structure in pigmented and albino guinea pigs. The well-organized, collagen-based LC of the guinea pig ONH is similar to that described for tree shrews and more similar to the human LC than that of other rodents that lack collagen. Based on these latter structural similarities the guinea pig would seem a promising model for investigating the relationship between myopia and glaucoma., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The purpose of this study is to three-dimensionally (3D) characterize the principal macroscopic and microscopic relationships within the rat optic nerve head (ONH) and quantify them in normal control eyes. Perfusion-fixed, trephinated ONH from 8 normal control eyes of 8 Brown Norway Rats were 3D histomorphometrically reconstructed, visualized, delineated and parameterized. The rat ONH consists of 2 scleral openings, (a superior neurovascular and inferior arterial) separated by a thin connective tissue strip we have termed the "scleral sling". Within the superior opening, the nerve abuts a prominent extension of Bruch's Membrane (BM) superiorly and is surrounded by a vascular plexus, as it passes through the sclera, that is a continuous from the choroid into and through the dural sheath and contains the central retinal vein (CRV), (inferiorly). The inferior scleral opening contains the central retinal artery and three long posterior ciliary arteries which obliquely pass through the sclera to obtain the choroid. Bruch's Membrane Opening (BMO) is irregular and vertically elongated, enclosing the nerve (superiorly) and CRV and CRA (inferiorly). Overall mean BMO Depth, BMO Area, Choroidal Thickness and peripapillary Scleral Thickness were 29 μm, 56.5 × 10(3) μm(2), 57 μm and 104 μm respectively. Mean anterior scleral canal opening (ASCO) and posterior scleral canal opening (PSCO) radii were 201 ± 15 μm and 204 ± 16 μm, respectively. Mean optic nerve area at the ASCO and PSCO were 46.3 × 10(3)±4.4 × 10(3) μm(2) and 44.1 × 10(3)±4.5 × 10(3) μm(2) respectively. In conclusion, the 3D complexity of the rat ONH and the extent to which it differs from the primate have been under-appreciated within previous 2D studies. Properly understood, these anatomic differences may provide new insights into the relative susceptibilities of the rat and primate ONH to elevated intraocular pressure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cases Report: Two patients presented with headache and bilateral papillary edema. Patient 1 was found to have a papilledema (P) with intracranial pressure of 32cmH2O. Patient 2 was found to have a migraine with a pseudopapilledema (PP) (optic nerve head drusen). SD-OCT was used to image the optic disc, subretinal hyporeflective space (SHS), and alpha-angle (Aα)., Discussion: Optic disc SD-OCT may be useful for differentiating disc morphology in P and PP. The area of the SHS and the Aα were higher in the P patient than in the patient with PP., (Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.)

The aim of this study was to quantify connective tissue fibre orientation and alignment in young, old and glaucomatous human optic nerve heads (ONH) to understand ONH microstructure and predisposition to glaucomatous optic neuropathy. Transverse (seven healthy, three glaucomatous) and longitudinal (14 healthy) human ONH cryosections were imaged by both second harmonic generation microscopy and small angle light scattering (SALS) in order to quantify preferred fibre orientation (PFO) and degree of fibre alignment (DOFA). DOFA was highest within the peripapillary sclera (ppsclera), with relatively low values in the lamina cribrosa (LC). Elderly ppsclera DOFA was higher than that in young ppsclera (p < 0.00007), and generally higher than in glaucoma ppsclera. In all LCs, a majority of fibres had preferential orientation horizontally across the nasal-temporal axis. In all glaucomatous LCs, PFO was significantly different from controls in a minimum of seven out of 12 LC regions (p < 0.05). Additionally, higher fibre alignment was observed in the glaucomatous inferior-temporal LC (p < 0.017). The differences between young and elderly ONH fibre alignment within regions suggest that age-related microstructural changes occur within the structure. The additional differences in fibre alignment observed within the glaucomatous LC may reflect an inherent susceptibility to glaucomatous optic neuropathy, or may be a consequence of ONH remodelling and/or collapse.

This work intends to assess circumpapillary retinal vessel density (RVD) at a 3.46 mm diameter circle and correlate it with circumpapillary retinal nerve fiber layer (RNFL) thickness measured with Fourier-Domain Optical Coherence Tomography. Furthermore, it aims to evaluate the reduction of intersubject variability of RNFL when considering RVD as a source of information for RNFL distribution. For that, 106 healthy subjects underwent circumpapillary RNFL measurement. Using the scanning laser ophthalmoscope fundus image, thickness and position of retinal vessels were assessed and integrated in a 256-sector RVD profile. The relationship between local RVD value and local RNFL thickness was modeled by linear regression. RNFL was then compensated for RVD variation by regression formulas. A strong statistically significant intrasubject correlation was found for all subjects between RVD and RNFL profiles (mean R = 0.769). In the intersubject regression analysis, 247 of 256 RNFL sectors showed a statistically significant positive correlation with RVD (mean R = 0.423). RVD compensation of RNFL resulted in a relative reduction of up to 20% of the intersubject variance. In conclusion, RVD in a 3.46 mm circle has a clinically relevant influence on the RNFL distribution. RVD may be used to develop more individualized normative values for RNFL measurement, which might improve early diagnosis of glaucoma.

Introduction: Clinical determination of the outer limits of the optic disk (OD) doesn't always correspond to the true anatomic limits of the optic nerve head (ONH) defined by the Bruch's membrane opening (BMO). A new index analyzing the OD with optical coherence tomography (OCT), "minimal rim width" (BMO-MRW), evaluates the smallest thickness of the neuroretinal rim between the BMO and the internal limiting membrane. The purpose of this study was to evaluate new software for automatic measurement of the BMO-MRW., Materials and Methods: This study investigated 95 eyes: 40 control eyes and 55 eyes followed and treated for primary open angle glaucoma (42 early glaucoma, 7 moderate glaucoma and 6 advanced glaucoma). After a precise localization of the OD center, 24 radial scans of the ONH are taken with the Spectralis OCT (Heidelberg Engineering, Germany). From the 48 measurements of BMO-MRW, the mean thickness as well as that in each of the 6 papillary sectors of this new index are calculated. ROC curves analysis (receiver operating characteristic) was used to assess the diagnostic capabilities of the various parameters., Results: Thicknesses of all parameters were statistically lower in glaucoma than in controls. The mean value and inferotemporal sector (IT) had the best diagnostic capabilities without significant difference between them (BMO-MRW-average = 0.890 ± 0.062, BMO-MRW-IT = 0.881 ± 0.066, P = 0.59). The area under the curve was lowest in the temporal sector (0.820 ± 086 statistically lower than the average value, P = 0.04)., Conclusions: This preliminary study of a new automated analysis of the neuroretinal rim highlights the diagnostic value of the BMO-MRW index. This evaluation appears to be best correlated with the anatomy of the ONH with good diagnostic sensitivity., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes., Methods: The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed., Results: A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors., Conclusions: Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

Retinal ganglion cells (RGCs), the output neurons of the retina, have axons that project via the optic nerve to diverse targets in the brain. Typically, RGC axons do not branch before exiting the retina and thus do not provide it with synaptic feedback. Although a small subset of RGCs with intraretinal axon collaterals has been previously observed in human, monkey, cat, and turtle, their function remains unknown. A small, more recently identified population of RGCs expresses the photopigment melanopsin. These intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit an irradiance-coding signal to visual nuclei in the brain, contributing both to image-forming vision and to several nonimage-forming functions, including circadian photoentrainment and the pupillary light reflex. In this study, using melanopsin immunolabeling in monkey and a genetic method to sparsely label the melanopsin cells in mouse, we show that a subgroup of ipRGCs have axons that branch en route to the optic disc, forming intraretinal axon collaterals that terminate in the inner plexiform layer of the retina. The previously described collateral-bearing population identified by intracellular dye injection is anatomically indistinguishable from the collateral-bearing melanopsin cells identified here, suggesting they are a subset of the melanopsin-expressing RGC type and may therefore share its functional properties. Identification of an anatomically distinct subpopulation in mouse, monkey, and human suggests this pathway may be conserved in these and other species (turtle and cat) with intraretinal axon collaterals. We speculate that ipRGC axon collaterals constitute a likely synaptic pathway for feedback of an irradiance signal to modulate retinal light responses.

Purpose: morphobiometric evaluation of macula and peripapillary retinal nerve fiber layer thickness with the use of high-resolution optical coherence tomography in children with a history of regressed retinopathy or prematurity., Materials and Methods: 18 patients at the age of 8 to 14 years with a history of spontaneously regressed retinopathy of prematurity were studied prospectively. For statistical purposes a control group of 21 matched subjects at the age of 8 to 15 years was used. Ophthalmic examination and optical coherence tomography were performed in each patient. Peripapillary nerve fibre layer thickness, foveal and parafoveal thickness ratio, total macular volume and subfoveal choroidal thickness were measured in both groups., Results: in the optical coherence tomography, the foveal thickness in children with retinopathy of prematurity was significantly higher [269.5 μm (232-321)] compared to the controls [224.5 μm (207-267)]. The macular volume in the study group was also higher (8.68 mm³). The subfoveal choroidal thickness was reduced in study group [321 μm (112-365)] compared to the control group [337 μm (294-358)]. There was no statistical significant difference in total peripapillary nerve fibre layer thickness between the two groups., Conclusion: The morphobiometric macular changes in eyes with a history of regressed retinopathy of prematurity are possibly related to the developmental abnormalities, which retinopathy of prematurity is due to the presence of the abnormal foveal structure across all retinal layers.

Although clinical and experimental observations indicate that the optic nerve head (ONH) is a major site of axon degeneration in glaucoma, the mechanisms by which local retinal ganglion cell (RGC) axons are injured and damage spreads among axons remain poorly defined. Using a laser-induced ocular hypertension (LIOH) mouse model of glaucoma, we found that within 48 h of intraocular pressure elevation, RGC axon segments within the ONH exhibited ectopic accumulation and colocalization of multiple components of the glutamatergic presynaptic machinery including the vesicular glutamate transporter VGLUT2, several synaptic vesicle marker proteins, glutamate, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and active zone cytomatrix components, as well as ultrastructurally identified, synaptophysin-containing vesicles. Ectopic vesicle exocytosis and glutamate release were detected in acute preparations of the LIOH ONH. Immunolocalization and analysis using the ionotropic receptor channel-permeant cation agmatine indicated that ONH axon segments and glia expressed glutamate receptors, and these receptors were more active after LIOH compared with controls. Pharmacological antagonism of glutamate receptors and neuronal activity resulted in increased RGC axon sparing in vivo. Furthermore, in vivo RGC-specific genetic disruption of the vesicular glutamate transporter VGLUT2 or the obligatory NMDA receptor subunit NR1 promoted axon survival in experimental glaucoma. As the inhibition of ectopic glutamate vesicular release or glutamate receptivity can independently modify the severity of RGC axon loss, synaptic release mechanisms may provide useful therapeutic entry points into glaucomatous axon degeneration.

Purpose: To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression., Design: Observational, consecutive case series., Participants: Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG)., Methods: Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro., Main Outcome Measures: Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa., Results: Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro., Conclusions: The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Pseudoexfoliation (PEX) syndrome is a systemic disorder of the elastic fiber system that can lead to PEX glaucoma. Elastotic alterations in the lamina cribrosa (LC) of PEX eyes suggested biomechanical implications predisposing to pressure-induced optic nerve damage. In this pilot study, the stiffness of LC and peripapillary sclera (ppSC) in eyes with and without PEX syndrome were analyzed by atomic force microscopy (AFM) nanoindentation., Methods: Unfixed cryosections (5-μm thick) were prepared from the optic nerve heads (ONH) of three donor eyes with PEX syndrome and three age-matched control eyes. AFM force mapping was performed in selected regions of the central, midperipheral, and peripheral LC and the ppSC using a spherical cantilever tip. To determine the local Young's modulus of elasticity (YME) as a measure of tissue stiffness, force curves were acquired and analyzed using the spherical Hertz model., Results: For the LC, the median YME values calculated from single stiffness maps averaged 17.2 (±2.7) kPa in normal eyes and 10.1 (±1.4) kPa in PEX eyes, indicating a significant PEX-related decrease in stiffness by over 40% (P < 0.01). The corresponding YME values for the ppSC, which revealed a 9-fold higher tissue stiffness than in the LC, averaged 158.3 (±59.8) kPa for control and 85.8 (±16.9) kPa for PEX samples., Conclusions: AFM was proven suitable for determining the stiffness of ONH tissues, encouraging further large-scale analyses. The marked decrease in stiffness, implying an increased deformability of the ONH in PEX eyes, may reflect an inherent tissue weakness rendering these eyes more vulnerable to glaucomatous damage.

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.

Purpose: Sigma receptor 1 (σR1) is expressed abundantly in the eye, and several reports suggest that this putative molecular chaperone plays a role in lens cell survival, control of intraocular pressure (IOP), and retinal neuroprotection. The present study examined the consequence of the absence of σR1 on ocular development, structure, and function., Methods: Wild-type (σR1⁺/⁺), heterozygous (σR1⁺/⁻), and homozygous (σR1⁻/⁻, knockout) mice aged 5 to 59 weeks were subjected to comprehensive electrophysiological testing and IOP measurement. The eyes were examined by light and electron microscopy and subjected to morphometric examination and detection of apoptosis., Results: Cornea and lens of σR1⁻/⁻ mice were similar to wild-type mice in morphologic appearance at all ages examined, and IOP was within normal limits. Comprehensive ERG and morphometric analyses initially yielded normal findings in the σR1⁻/⁻ mice compared with those in the wild-type. By 12 months, however, significantly decreased ERG b-wave amplitudes and diminished negative scotopic threshold responses, consistent with inner retinal dysfunction, were detected in σR1⁻/⁻ mice. Concomitant with these late-onset changes were increased TUNEL- and active caspase 3-positive cells in the inner retina and significant loss of cells in the ganglion cell layer, particularly in the central retina. Before these functional and structural abnormalities, there was ultrastructural evidence of axonal disruption in the optic nerve head of σR1⁻/⁻ mice as early as 6 months of age, although there were no alterations observed in retinal vascularization in σR1⁻/⁻ mice., Conclusions: These data suggest that lack of σR1 leads to development of late-onset retinal dysfunction with similarities to optic neuropathy.

Purpose: This article describes the first retinal histopathologic findings in a patient with Susac's syndrome (SS)., Design: Observational case report., Participant: A 51-year-old white woman diagnosed with SS., Methods: Eyes from a 51-year-old white woman diagnosed with SS were obtained at autopsy. One retina was dissected and processed for adenosine diphosphatase (ADPase) flat embedding. Selected areas were processed further for transmission electron microscopy., Main Outcome Measures: Histopathologic examination using ADPase flat-embedding technique., Results: There were vaso-occlusive changes in the retinal periphery resulting in small areas of capillary dropout. Cross-sections demonstrated serous filled spaces between the retinal blood vessels and the internal limiting membrane. Lumens adjacent to these spaces appeared compressed and sometimes closed, but without thrombosis. Decreased ADPase activity in some peripheral blood vessels suggested endothelial cell dysfunction and vaso-occlusion. In the optic nerve head, numerous corpora amylacea were observed in the vicinity of capillaries with thickened walls and narrow lumens. Transmission electron microscopy demonstrated thickened and amorphous vascular basal lamina and open endothelial cell junctions in some retinal blood vessels., Conclusions: The serous deposits with compression of retinal vessel lumens observed histologically probably represent the so-called string of pearls described clinically in SS. Chronic extension of these serous deposits along the vessel wall possibly are the cause of retinal arterial wall plaques as described by Gass and other investigators. In the optic nerve head, corpora amylacea are probably a result of microinfarcts resulting from optic nerve head capillary angiopathy. Accumulation of amorphous material in the basal lamina, loss of viable endothelial cells, and capillary dropout suggest that SS may be an endotheliopathy., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease., Competing Interests: The authors have declared that no competing interests exist.

Purpose: A new experimental glaucoma model was developed by using an ex vivo rat retinal preparation to examine the effects of elevated hydrostatic pressure on retinal morphology and glutamine synthetase (GS) activity., Methods: Ex vivo rat retinas were exposed to elevated hydrostatic pressure for 24 hours in the presence of glutamate or glutamate receptor antagonists and examined histologically. GS activity was assessed by colorimetric assay., Results: Pressure elevation induced axonal swelling in the nerve fiber layer. Axonal swelling was prevented by a combination of non-N-methyl-d-aspartate (non-NMDA) receptor antagonist and an NMDA receptor antagonist, indicating that the damage results from activation of both types of glutamate receptor. When glial function was preserved, the typical changes induced by glutamate consisted of reversible Müller cell swelling resulting from excessive glial glutamate uptake. The irreversible Müller cell swelling in hyperbaric conditions may indicate that pressure disrupts glutamate metabolism. Indeed, elevated pressure inhibited GS activity. In addition, glutamate exposure after termination of pressure exposure exhibited apparent Müller cell swelling., Conclusions: These results suggest that the neural degeneration observed during pressure elevation is caused by impaired glial glutamate metabolism after uptake.

Purpose: We study the vitreous interface of the optic disc to delimit the passages for the flow of fluids through the prelaminar tissue of porcine eyes., Methods: Wet scanning electron microscope (SEM), conventional SEM and transmission electron microscope (TEM) were used to explore the surface of the optic nerve of the pig. The vitreous cavity was perfused with a fluorescent marker and colloidal gold at controlled pressure. Samples of perfused optic nerve head were cryosectioned and observed with the confocal laser microscope (lectin) or resin embedded and observed under TEM (gold)., Results: Fenestrations were present under the SEM in all three regions of the vitreous interface. SEM results were confirmed at the TEM level and under the wet-SEM. Perfusion experiments traced the flow of a fluorescent molecule delineating routes of preferential flow with origin in the fenestrations. Colloidal gold marked the site of entrance in the prelaminar tissue identifying major fenestrations in the basal membrane., Conclusions: Interchange of fluid between the optic nerve and the vitreous cavity in the pig is facilitated by fenestrations of varied sizes in the basal membrane and preferential flow routes through the prelaminar tissue. Preferential flow routes exist in the extracellular spaces of Elschnig and Kuhn' astrocytes and give a sharply distinct image when compared with flow through zones in which astrocytes envelope axons. Escape routes may be instrumental in preventing oedemas of the optic nerve head, but they could also serve as entrance doors for fluids from the vitreous and aqueous and play a pathogenic role in ageing and glaucoma., (© 2010 The Authors. Journal compilation © 2010 Royal Australian and New Zealand College of Ophthalmologists.)

Detection of the optic nerve head (ONH) is a key preprocessing component in algorithms for the automatic extraction of the anatomical structures of the retina. We propose a method to automatically locate the ONH in fundus images of the retina. The method includes edge detection using the Sobel operators and detection of circles using the Hough transform. The Hough transform assists in the detection of the center and radius of a circle that approximates the margin of the ONH. Forty images of the retina from the Digital Retinal Images for Vessel Extraction (DRIVE) dataset were used to test the performance of the proposed method. The center and boundary of the ONH were independently marked by an ophthalmologist for evaluation. Free-response receiver operating characteristics (FROC) analysis as well as measures of distance and overlap were used to evaluate the performance of the proposed method. The centers of the ONH were detected with an average distance of 0.36 mm to the corresponding centers marked by the ophthalmologist; the detected circles had an average overlap of 0.73 with the boundaries of the ONH drawn by the ophthalmologist. FROC analysis indicated a sensitivity of detection of 92.5% at 8.9 false-positives per image. With an intensity-based criterion for the selection of the circle and a limit of 40 pixels (0.8 mm) on the distance between the center of the detected circle and the manually identified center of the ONH, a successful detection rate of 90% was obtained with the DRIVE dataset.

Background: The vitreous interface of the optic nerve has been studied to delimit the covering of Elschnig's astrocytes and interstitial pathways of flow through the prelaminar region., Methods: Perfusion of the prelaminar tissue under controlled pressure with a fluorescent marker injected into the vitreous cavity in pig eyes. The prelaminar region of the optic nerve and adjacent retina was fixed and flatmounted or frozen and cryosectioned and examined with the confocal laser microscope. Samples were also prepared for conventional transmission electron microscopy., Results: The surface of the vitreous surface of the optic nerve is covered by a cobblestone-like pavement made of astrocytic projections. Intensely stained passages of different thickness indicate the presence of wide interconnected intercellular spaces in the covering of Elschnig's astrocytes. Those passages are absent in the intervascular areas occupied by axons and axon-linked astrocytes., Conclusions: Delineation of the astrocytic pavement and the preferred flow routes formed by wide extracellular spaces are conspicuous features of the prelaminar region when examined with the confocal laser microscope and the help of sticky fluorescent tracer. This suggests that excess extracellular fluid can be interchanged with the vitreous by a network of interconnected extracellular spaces or preferred flow routes. Some pathogenic mechanisms can be related to fluid interchange in the optic nerve head.

Purpose: To determine the degree of correlation between spatial characteristics of the retinal nerve fiber layer (RNFL) birefringence (Delta n(RNFL)) surrounding the optic nerve head (ONH) with the corresponding anatomy of retinal ganglion cell (RGC) axons and their respective organelles., Methods: RNFL phase retardation per unit depth (PR/UD, proportional to Delta n(RNFL)) was measured in two cynomolgus monkeys by enhanced polarization-sensitive optical coherence tomography (EPS-OCT). The monkeys were perfused with glutaraldehyde and the eyes were enucleated and prepared for transmission electron microscopy (TEM) histologic analysis. Morphologic measurements from TEM images were used to estimate neurotubule density (rho(RNFL)), axoplasmic area (A(x)) mode, axon area (A(a)) mode, slope (u) of the number of neurotubules versus axoplasmic area (neurotubule packing density), fractional area of axoplasm in the nerve fiber bundle (f), mitochondrial fractional area in the nerve fiber bundle (x(m)), mitochondria-containing axon profile fraction (m(p)), and length of axonal membrane profiles per unit of nerve fiber bundle area (L(am)/A(b)). Registered PR/UD and morphologic parameters from corresponding angular sections were then correlated by using Pearson's correlation and multilevel models., Results: In one eye there was a statistically significant correlation between PR/UD and rho(RNFL) (r = 0.67, P = 0.005) and between PR/UD and neurotubule packing density (r = 0.70, P = 0.002). Correlation coefficients of r = 0.81 (P = 0.01) and r = 0.50 (P = 0.05) were observed between the PR/UD and A(x) modes for each respective subject., Conclusions: Neurotubules are the primary source of birefringence in the RNFL of the primate retina.

Radial optic neurotomy (RON) has been proposed for alleviation of the "scleral outlet compartment syndrome" at the level of the lamina cribrosa, which is thought to play a pathoetiologic role in central retinal vein occlusion (CRVO). The aim of this study has been to analyze the ultrastructural alterations of the lamina cribrosa after RON to gain new insights in the underlying pathomechanical factors. Fifteen donor eyes underwent a standardized open-sky-vitrectomy and RON after removal of the anterior eye segment for keratoplasty. Using a microvitreoretinal blade, a radial incision was performed on the nasal hemisphere of the optic nerve head radial to the optic disc and parallel to the nerve fibre layer. The lamina cribrosa and the surrounding scleral ring were then prepared for light microscopy, scanning (SEM) and transmission electron microscopy (TEM). Ultrastructural analysis demonstrated that in 60% (n=9) of the evaluated cases the scleral ring was dissected completely and in 40% (n=6) only partially. The adjacent neuronal tissue to the dissection area showed only minimal injury. The central retinal vessels were not injured in all cases. Only complete incision of the circular ring of collagen fibrils surrounding the lamina cribrosa via RON resulted in effective relaxation of the scleral outlet and was achieved in 60% of all eyes under standardized conditions. In all cases the adjacent tissue showed only minimal injury. The high rate of incomplete dissection of the scleral outlet may be an explanation for the variable outcome seen in different studies on RON.

Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, alpha and beta. In the rod cells, the Cngb1 locus encodes the channel beta-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the beta-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5'-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel alpha-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the beta-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric alpha-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the beta-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the beta-subunit is required for normal light sensitivity of the rods.

Purpose: This paper aims to study the anterior surface of the optic nerve in relation to its ability to support a source of stress acting from the vitreous cavity. The intercellular junctions of the lining astrocytes mediated by cellular adhesion molecules (CAMs) may be the main targets for ionic stress., Methods: The optic nerve of the domestic pig was prepared for light, confocal laser and transmission electron microscopy. Immunostaining was performed for antibodies against glial fibrillary acidic protein, neural cadherin (N-cadherin) and neural CAM (N-CAM)., Results: Only 1 type of intercellular junction was found among the bordering astrocytes, which was characterized as a zonula adherens. Unions between lining cells showed a positive immunogold effect and immunofluorescence against N-cadherin in the zonula adherens and membrane apposition. N-CAM was also present in areas of nonjunctional cellular adhesion., Conclusion: The stability of intercellular junctions of the nerve-vitreous boundary is sensitive to altered concentrations of Ca(2+). Since aqueous humor has half the Ca(2+) concentration of plasma, any contact of aqueous humor with the optic nerve head can interfere with the ionic concentration of calcium in the extracellular spaces. This mechanism may contribute to age-related changes and some types of glaucoma., (Copyright 2009 S. Karger AG, Basel.)

Purpose: To compare the optic nerve parameters measured by confocal scanning laser in normal, ocular hypertensive and glaucomatous eyes; and in groups of ocular hypertensive eyes, classification of these according to the optic nerve appearance and to short-wavelength automated perimetry (SWAP) results., Methods: 101 eyes of 101 normal subjects, 247 eyes of 247 ocular hypertensive subjects and 102 eyes of 102 glaucomatous subjects were studied. Subjects were classified based on intraocular pressure and standard automated perimetry (AP) performance. Hypertensive subjects were classified into ocular hypertensive and preperimetric glaucoma, and into ocular hypertensive with normal SWAP and hypertensive with pathological SWAP findings. Every patient underwent evaluation of the optic nerve, AP, SWAP and confocal scanning laser ophthalmoscopy (HRT II), resulting in the acquisition of topographic parameters of the optic nerve, which were then compared between the different groups., Results: Significant differences (p<0.05) were found between normal subjects and glaucomatous eyes in all optic nerve parameters except disc area (2.23/2.1), height variation contour (0.41/0.39) and average variability (0.05/0.04). Ocular hypertensive eyes showed an overlap when compared with normal and glaucoma groups. No differences were found between normal subjects and those with ocular hypertension in mean retinal nerve fiber layer thickness (0.24/0.24) or between those with ocular hypertension and glaucoma in mean cup depth (0.28/0.3). Nevertheless, when those with ocular hypertension were segregated based on clinical evaluation of optic nerve or SWAP performance, the ability to define the presence of structural glaucoma damage improved significantly., Conclusions: HRT enables moderate discrimination between normal, ocular hypertensive and glaucoma subjects. The use of diagnostic tests to detect early glaucomatous damage (such as short-wavelength automated perimetry) in ocular hypertensive eyes improves the ability of HRT to discriminate glaucoma.

Aim: To compare Stratus-OCT measurements in controls, ocular hypertensive (OHT) patients with (FDT+) and without (FDT-) frequency-doubling technology (FDT) abnormalities, and in patients affected with early primary open-angle glaucoma (POAG)., Methods: Thirty-two controls, 78 OHT patients (38 FDT- and 40 FDT+), and 45 early POAG patients (six FDT- and 39 FDT+) underwent the following tests within 3 months: standard automated perimetry (SAP) HFA 30-2; FDT N-30-F; and, Stratus-OCT imaging with retinal nerve fibre layer (RNFL) and optic nerve head (ONH) scans. One eye per patient was considered. Differences among groups were evaluated using the Kruskal-Wallis, analysis of variance, and Duncan's tests., Results: There were no significant differences in all Stratus-OCT parameters between POAG and OHT FDT+ patients. Statistically significant differences were found between the control group and both the POAG and OHT FDT+ groups for 15 of the 21 Stratus-OCT parameters. Control eyes compared to OHT FDT- showed significant differences in 13 of the 21 parameters. The comparison between the OHT FDT- group, and both the POAG and OHT FDT+ group resulted in 13 of the 21 parameters to be significantly different., Conclusions: Stratus-OCT seems to show a higher ability in detecting significant differences between healthy, OHT, and early POAG eyes when compared to SAP and FDT. This suggests that the Stratus-OCT could show structural abnormalities before SAP or FDT visual field defects appear in patients at risk of developing glaucoma, which may be beneficial in making therapeutic decisions, especially in OHT patients.

Rats are becoming an increasingly popular model system for understanding mechanisms of optic nerve injury in primary open-angle glaucoma (POAG). Although the anatomy of the rat optic nerve head (ONH) is different from the human, the ultrastructural relationships between astrocytes and axons are quite similar, making it likely that cellular processes of axonal damage in these models will be relevant to human glaucoma. All of these models rely on elevating intraocular pressure (IOP), a major risk factor for glaucoma. Methods that produce increased resistance to aqueous humor outflow at the anterior chamber angle, specifically hypertonic saline injection of aqueous outflow pathways and laser treatment of the limbal tissues, appear to produce a specific regional pattern of injury that may have a particular relevance to understanding regional injury in human glaucoma. Because increased pressure fluctuations are a characteristic of such models and the rodent ONH appears to have high susceptibility to elevated IOP, special instrumentation and measurement techniques are required to document pressure exposure in these eyes and understand the pressure levels that the eyes and the optic nerve are exposed to. With these techniques, it is possible to obtain an excellent correlation between pressure and the extent of nerve damage. Continued use of these models will lead to a better understanding of cellular mechanisms of pressure-induced optic nerve damage and POAG.

Glaucoma, the second most prevalent cause of blindness worldwide, is a degenerative disease characterized by loss of vision due to loss of retinal ganglion cells. There is no cure for glaucoma, but early intervention with drugs and/or surgery may slow or halt loss of vision. Increased intraocular pressure (IOP), age, and genetic background are the leading risk factors for glaucoma. Our laboratory and other investigators have provided evidence that astrocytes are the cells responsible for many pathological changes in the glaucomatous optic nerve head (ONH). Over several years, in vivo and in vitro techniques characterized the changes in quiescent astrocytes that lead to the reactive phenotype in glaucoma. Reactive astrocytes alter the homeostasis and integrity of the neural and connective tissues in the ONH of human and experimental glaucoma in monkeys. During the transition of quiescent astrocytes to the reactive phenotype altered astrocyte homeostatic functions such as cell-cell communication, migration, growth factor pathway activation, and responses to oxidative stress may impact pathological changes in POAG. Our data also suggests that the creation of a non-supportive environment for the survival of RGC axons through remodeling of the ONH by reactive astrocytes leads to progression of glaucomatous optic neuropathy.

Purpose: To correlate the optic nerve head topographic parameters measured by the Heidelberg Retina Tomograph II (HRT) with the perimetric indices of standard automated perimetry (SAP)., Methods: This study included 101 normal subjects, 247 ocular hypertensive eyes (increased intraocular pressure with normal SAP) and 102 glaucomatous subjects (IOP above 21 mm Hg and abnormal standard automated perimetry). Only one eye was randomly chosen from each subject for the study. The visual field was evaluated by means of Humphrey Field Analyzer (24-2 full threshold strategy). The HRT II (Heidelberg Engineering) was used to acquire and measure the optic disc topographic parameters. Pearson correlations between topographic data and perimetric indices were performed for the total sample and each group of patients. The distribution of values obtained in the samples was normal., Results: A significant correlation was found between several optic disc parameters and the global indices of SAP. Rim area, rim volume, cup/disc area ratio, rim/disc area ratio, cup shape measurement, RNFL cross-sectional area, and discriminant functions FSM and RB, showed the strongest correlation with the visual field indices in the total and glaucoma groups (RIM AREA: total group: r=0.32; p=4.14x10(-11)/glaucoma group: r=0.28; p=0.004. RIM VOLUME: total group: r=0.26; p=1.55x10(-7)/glaucoma group: r=0.26; p=0.006). The ocular hypertensive group showed few significant correlations., Conclusions: The correlations found between standard automated perimetry and HRT defined topographic parameters allow a better understanding of glaucomatous damage and make decision-making easier.

Purpose: To determine the diagnostic capability of PULSAR-T30W, FDT-Threshold-N30 and HRT-II in glaucoma suspects., Methods: Forty-seven eyes from 47 referred glaucoma suspects (GS) were examined twice with each technique. Cases with TOP-WW-MD>6dB were excluded. Results were compared with those of 70 eyes from 70 normal controls (C)., Results: Mean MD value using TOP-WW in the GS group (0.96dB. sd=1.7) was not significantly different from C (0.8dB. sd=1.77) (p>0.05). Disc area in GS group (2.12 mm(2). sd=0.34) was significantly greater than in C (1.97 mm2. sd=0.45) (p<0.01). For 95.7% specificity, PULSAR-sLV showed the highest sensitivity of 30.9% in individual examinations. The highest reproducible sensitivity in the two examinations was obtained using HRT-II maximum contour elevation (23.4%) and reference height (23.4%), and was 14.9% for various indices after correcting for the influence of disc area (cup area, cup/disc area ratio, maximum contour depression and mean RNFL thickness). Reproducible sensitivity of the perimetric indices was: PULSAR-MD=8.5%, PULSAR-sLV=17%, FDT-MD=6.4%, FDT-PSD=4.3%. The association of perimetric and HRT-II indices achieved high sensitivity but low diagnostic reproducibility., Conclusions: The most effective indices were maximum contour elevation, reference height and PULSAR-sLV, although the inclusion of the optic nerve head assessment in the selection of the GS sample may have favored the HRT-II results.

Purpose: To identify the structure and composition of the tree shrew optic nerve to determine its potential as a model for glaucoma., Methods: Tree shrew optic nerves, aged 4 weeks to 5 years, were wax or cryoembedded for analysis of overall morphology and cellular (glial fibrillary acidic protein [GFAP]) and extracellular matrix (collagen types I, III, IV, V, VI; fibronectin; and elastin) immunolocalization studies. In addition, transmission and scanning electron microscopy were performed. In vivo optic disc imaging was performed by HRT2 and fundus camera photography., Results: The optic nerve of the tree shrew comprised regions comparable to the human prelaminar and lamina cribrosa (LC) in the optic nerve head and the retrolaminar region, immediately posterior. The multilayered connective tissue plates of tree shrew LC stretched across the optic nerve canal at the level of the sclera and consisted of collagen types I, III, IV, V, and VI; elastin; and fibronectin. Significant age-related alterations in connective tissue components were indicated. Connective tissue was present in the central retinal vessel sheaths and was identified as longitudinally oriented collagen fibrils in the retrolaminar optic nerve. GFAP immunofluorescence indicated a high concentration of astrocytic processes in the LC. Myelination of axons was evident in the retrolaminar optic nerve. Ultrastructural studies supported the structural organization and spatial distribution of connective tissue., Conclusions: In contrast to many rodent models of glaucoma, since the tree shrew optic nerve resembles that in humans, especially at the LC, the tree shrew offers an ideal opportunity to investigate glaucoma pathophysiology in a subprimate model.

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.

The arrangement of connective tissue components (i.e., collagen, reticular, and elastic fibers) and glial elements in the optic nerve head of the human eye was investigated by the combined use of light microscopy and scanning electron microscopy (SEM). Light-microscopically, the optic nerve head could be subdivided into four parts from the different arrangements of the connective tissue framework: a surface nerve fiber layer, and prelaminar, laminar, and postlaminar regions. The surface nerve fiber layer only possessed connective tissue elements around blood vessels. In the prelaminar region, collagen fibrils, together with delicate elastic fibers, formed thin interrupted sheaths for accommodating small nerve bundles. Immunohistochemistry for the glial fibrillary acidic protein (GFAP) showed that GFAP-positive cells formed columnar structures (i.e., glial columns), with round cell bodies piled up into layers. These glial columns were located in the fibrous sheaths of collagen fibrils and elastic fibers. In the laminar region, collagen fibrils and elastic fibers ran transversely to the optic nerve axis to form a thick membranous layer - the lamina cribrosa - which had numerous round openings for accommodating optic nerve fiber bundles. GFAP-positive cellular processes also ran transversely in association with collagen and elastin components. The postlaminar region had connective tissues which linked the lamina cribrosa with fibrous sheaths for accommodating nerve bundles in the extraocular optic nerve, where GFAP-positive cells acquired characteristics typical of fibrous astrocytes. These findings indicate that collagen fibrils, as a whole, form a continuous network which serves as a skeletal framework of the optic nerve head for protecting optic nerve fibers from mechanical stress as well as for sustaining blood vessels in the optic nerve. The lamina cribrosa containing elastic fibers are considered to be plastic against the mechanical force affected by elevation of the intraocular pressure. The present study has also indicated that glial cells with an astrocytic character play an important role in constructing the connective tissue framework characteristic of the optic nerve head.

Purpose: To evaluate the diagnostic ability of three digital imaging technologies (HRT-II, OCT and GDx VCC) in detecting glaucomatous damage., Methods: A total of 427 eyes from 427 subjects were included in this cross-sectional study. Each subject was classified as healthy, ocular hypertensive or glaucomatous, according to intraocular pressure levels, standard automated perimetry and optic disc morphology. All of them underwent examination by means of HRT, OCT and GDx VCC. Receiver operating characteristic (ROC) curves were plotted and sensitivities at fixed specificities (85% and 95%) were calculated for each parameter assessed., Results: The parameters with the higher diagnostic ability were: FSM discriminant function from HRT-II (AUC=0.899), retinal nerve fiber layer average thickness from OCT (AUC=0.929) and NFI from GDx VCC (AUC=0.879), with no statistically significant differences between them., Conclusion: The HRT-II, OCT and GDx VCC provide measurements of the retinal nerve fiber layer and the optic nerve head with high discriminating ability in open-angle glaucoma.

Purpose: To investigate the induction of heat shock protein (Hsp)70 in the optic nerve head by localized laser application in transpupillary thermotherapy (TTT)., Methods: TTT was performed on the right eye of Norwegian brown rats with an 810-nm diode laser installed on a slit lamp biomicroscope. The laser was aimed at the center of the optic nerve head with a 50-microm spot size. Various exposures (range, 60-200 mW) were used with an exposure duration of 60 seconds, and the various exposure durations (range, 1-5 minutes) were used with a power of 100 mW. Twenty hours after laser irradiation, immunohistochemical staining and Western blot analyses were performed. For morphologic analysis of the optic nerve head, confocal scanning laser ophthalmoscopy and scanning electron microscopy were performed., Results: In the control eyes, Hsp70 was detected minimally in the optic nerve tissues by immunohistochemistry. After TTT, Hsp70 in the optic nerve tissue was induced more than in the control eyes. By Western blot, Hsp70 expression was found to increase progressively after TTT as the power was increased, but it also decreased slightly at powers >140 mW. The optimal setting of TTT without tissue damage was determined to be 100 mW for 60 seconds., Conclusions: Transpupillary laser irradiation of the optic nerve head induces Hsp70 expression. This result can be applied to the neuroprotective experiments in glaucoma by enhancement of a natural cytoprotective stress response.

Purpose: To determine the safety and efficacy of transsclerally placed intrapapillary wire microelectrodes implanted chronically into the optic nerve head of rabbit eyes., Methods: Four platinum wire microelectrodes were passed through the sclera and implanted into the optic nerve head of five rabbit eyes for 4-6 months. Color fundus photography, fluorescein angiography, electroretinograms (ERGs), and visually evoked potentials (VEPs) were used to monitor the retina. Electrically evoked potentials (EEPs) were elicited by bipolar electrical stimulation of the optic nerve axons by different combinations of the four electrodes immediately after the implantation and at 1-month intervals thereafter. The effects of the chronic implantation of the electrodes on the morphology of the optic nerve were evaluated by histological and immunohistochemical examinations at 4 and 6 months after the implantation., Results: All of the electrodes remained stable in the implanted sites throughout the post-implantation period, except for one electrode that had pulled out of the optic nerve head at 1 month after implantation. No intraocular infection, inflammation, or vitreoretinal proliferation was observed in any eye. EEPs could be elicited from each pair of electrodes at all testing times. The mean threshold currents (charge densities) to evoke EEPs increased from 19.3 +/- 9.2 microA (6.0 +/- 2.9 microC/cm2) on the implantation day to 78.8 +/- 31.9 microA (24.6 +/- 10.0 microC/cm2) at 1 month after implantation, but did not change significantly thereafter. The implicit time and amplitude of the a- and b-waves of the ERGs and of P1 of the VEPs did not change significantly throughout the post-implantation period. Histological evaluation of the optic nerve head revealed slight tissue encapsulations surrounding the electrode and increased expression of glial fibrillary acidic protein near the surface of the optic nerve., Conclusions: Implantation of transscleral intrapapillary microelectrodes appears to be safe and effective. These findings indicate that the implantation of microelectrodes in the optic nerve head should be considered for an optic nerve-based prosthesis.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head (ONH) include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. Transforming growth factor-beta (TGF-beta) is an important pro-fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non-glaucomatous controls. We hypothesize that in POAG, lamina cribrosa (LC) glial cells respond to elevated TGF-beta, producing a remodeled ONH ECM. Using Affymetrix microarrays, we report the first study examining the effect of TGF-beta1 on global gene expression profiles in glial fibrillary acidic acid (GFAP)-negative LC glial cells in vitro. Prominent among the differentially expressed genes were those with established fibrogenic potential, including CTGF, collagen I, elastin, thrombospondin, decorin, biglycan, and fibromodulin. Independent TaqMan and Sybr Green quantitative PCR analysis significantly validated genes involved in regulation of cell proliferation (platelet-derived growth factor [PDGF-alpha]), angiogenesis (vascular endothelial growth factor [VEGF]), ECM accumulation and degradation (CTGF, IL-11, and ADAMT-S5), and growth factor binding (ESM-1). Bioinformatic analysis of the ESM-1 promoter identified putative Smad and Runx transcription factor binding sites, and luciferase assays confirmed that TGF-beta1 drives transcription of the ESM-1 gene. TGF-beta1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro-fibrotic cell type and an attractive target for novel therapeutic strategies., (Copyright 2005 Wiley-Liss, Inc.)