Evidence for ceramide induced cytotoxicity in retinal ganglion cells.

Ceramides are bioactive compounds that play important roles in regulating cellular responses to extracellular stimuli and stress. Previous studies have shown that ceramides contribute to retinal degeneration associated with ischemic and ocular hypertensive stress. Acid sphingomyelinase (ASMase) is one of the major enzymes responsible for the stress-induced generation of ceramides. The goals of this study are to investigate the effects of ceramides on retinal ganglion cells (RGCs) and of ASMase inhibition in ocular hypertensive mice. Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human optic nerve head astrocytes were used to characterize the response to C2-ceramide. Microbead-induced ocular hypertension in the ASMase heterozygote mouse model was used to confirm the physiological relevance of in vitro studies. In mice, RGC function and morphology were assessed with pattern ERG (pERG) and immunofluorescence. The addition of C2-ceramide to iPSC-derived RGCs produced a significant concentration- and time-dependent reduction in cell numbers when compared to control cultures. While the addition of C2-ceramide to astrocytes did not affect viability, it resulted in a 2.6-fold increase in TNF-α secretion. The addition of TNF-α or conditioned media from C2-ceramide-treated astrocytes to RGC cultures significantly reduced cell numbers by 56.1 ± 8.4% and 24.7 ± 4.8%, respectively. This cytotoxic response to astrocyte-conditioned media was blocked by TNF-α antibody. In ASMase heterozygote mice, functional and morphological analyses of ocular hypertensive eyes reveal significantly less RGC degeneration when compared with hypertensive eyes from wild-type mice. These results provide evidence that ceramides can induce RGC cell death by acting directly, as well as indirectly via the secretion of TNF-α from optic nerve head astrocytes. In vivo studies in mice provide evidence that ceramides derived through the activity of ASMase contribute to ocular hypertensive injury. Together these results support the importance of ceramides in the pathogenesis of ocular hypertensive injury to the retina.

Multiple roles for Pax2 in the embryonic mouse eye.

The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2GFP/+ knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.

Microstructure and resident cell-types of the feline optic nerve head resemble that of humans.

The lamina cribrosa (LC) region of the optic nerve head (ONH) is considered a primary site for glaucomatous damage. In humans, biology of this region reflects complex interactions between retinal ganglion cell (RGC) axons and other resident ONH cell-types including astrocytes, lamina cribrosa cells, microglia and oligodendrocytes, as well as ONH microvasculature and collagenous LC beams. However, species differences in the microanatomy of this region could profoundly impact efforts to model glaucoma pathobiology in a research setting. In this study, we characterized resident cell-types, ECM composition and ultrastructure in relation to microanatomy of the ONH in adult domestic cats (Felis catus). Longitudinal and transverse cryosections of ONH tissues were immunolabeled with astrocyte, microglia/macrophage, oligodendrocyte, LC cell and vascular endothelial cell markers. Collagen fiber structure of the LC was visualized by second harmonic generation (SHG) with multiphoton microscopy. Fibrous astrocytes form glial fibrillary acidic protein (GFAP)-positive glial columns in the pre-laminar region, and cover the collagenous plates of the LC region in lamellae oriented perpendicular to the axons. GFAP-negative and alpha-smooth muscle actin-positive LC cells were identified in the feline ONH. IBA-1 positive immune cells and von Willebrand factor-positive blood vessel endothelial cells are also identifiable throughout the feline ONH. As in humans, myelination commences with a population of oligodendrocytes in the retro-laminar region of the feline ONH. Transmission electron microscopy confirmed the presence of capillaries and LC cells that extend thin processes in the core of the collagenous LC beams. In conclusion, the feline ONH closely recapitulates the complexity of the ONH of humans and non-human primates, with diverse ONH cell-types and a robust collagenous LC, within the beams of which, LC cells and capillaries reside. Thus, studies in a feline inherited glaucoma model have the potential to play a key role in enhancing our understanding of ONH cellular and molecular processes in glaucomatous optic neuropathy.

Isolation and characterization of human optic nerve head astrocytes and lamina cribrosa cells.

The lamina cribrosa is the initial site of glaucomatous injury. Pathological changes to the lamina cribrosa include posterior displacement of the lamina cribrosa, loss of trophic support, and remodeling of the extracellular matrix. Optic nerve head (ONH) astrocytes and lamina cribrosa cells synthesize extracellular matrix proteins to support and maintain the lamina cribrosa under physiological conditions. During glaucoma, these cells respond to mechanical strain and other stimuli, which leads to pathological remodeling of the ONH. Although ONH astrocytes and lamina cribrosa cells have been previously cultured, there is no well-accepted, straightforward technique to isolate both cell types from a single dissected human ONH. To better understand the pathophysiology of glaucoma, we obtained and cultured lamina cribrosa explants from human donor eyes. Initially, cells that grew out from the explant were ONH astrocytes and lamina cribrosa cells. Using a specialized medium, we isolated pure populations of lamina cribrosa cells and ONH astrocytes. ONH astrocytes expressed glial fibrillary acidic protein (GFAP). Lamina cribrosa cells expressed alpha-smooth muscle actin (α-SMA), but were negative for GFAP. This method of ONH cell isolation and cell-culture will provide a technique to better understand the molecular and cell-specific changes in glaucomatous damage to the ONH.

Statins Suppress TGF-ß2-Mediated MMP-2 and MMP-9 Expression and Activation Through RhoA/ROCK Inhibition in Astrocytes of the Human Optic Nerve Head.

Purpose: Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-ß2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation. Methods: Primary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-ß2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry. Results: MMP-2 and MMP-9 levels were upregulated by TGF-ß2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-ß2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-ß2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs. Conclusions: Statins inhibited the TGF-ß2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.

Relationship between peripapillary choroidal thickness and degree of tessellation in young healthy eyes.

PURPOSE: To determine the relationship between the peripapillary choroidal thickness (ppCT) and the degree and distribution of the tessellation in the fundus of normal eyes. METHODS: This was a prospective, observational cross-sectional study of 118 right eyes of young healthy volunteers. The ppCT was measured from the optical coherence tomography (OCT) circle scans manually at eight sectors: the nasal, supranasal superior, supratemporal, temporal, infratemporal, inferior, and infranasal sectors. The subjective degree of the tessellation in the color fundus photographs (CFPs) was classified into three categories: non-tessellated (NT), weakly tessellated (WT), and strongly tessellated (ST) in same sectors. The objective degree of tessellation designated by the tessellation fundus index (TFI) which was calculated as TFI = (R - G)/(R + G + B) using the mean value of the red-green-blue intensities of the CFPs. The differences in the ppCT and TFI for the three tessellation groups were analyzed. The correlations between the TFI and the ppCT were also determined. RESULTS: The mean age of the subjects was 25.8 years and the mean axial length of the eye was 25.5 mm. The inter-rater agreement of the subjective classifications was high with a Fleiss kappa of 0.71. The ppCT was significantly thinner in eyes with higher degrees of tessellation (P < 0.05) in all sectors. The TFIs were significantly and negatively correlated with the ppCTs in all sectors (r = - 0.44 to - 0.24, P < 0.05) except the nasal and the supranasal sectors. CONCLUSION: The degree of peripapillary tessellation is significantly correlated with the ppCT in young healthy eyes, and it has large individual and geographic variations.

Rodent retinal microcirculation and visual electrophysiology following simulated microgravity.

How the absence of gravity affects the physiology of human beings is generating global research interest as space exploration, including missions aboard the International Space Station, continues to push boundaries. Here, we examined changes in retinal microcirculation and visual electrophysiology in mice suspended by their tails to simulate the cephalad movement of blood that occurs under microgravity conditions. Tail suspension was performed with a head-down tilt with a recommended angle of 30°. Mice in the control groups were similarly attached to a tether but could maintain a normal position. Morphologically, the 15-day tail-suspended mice showed retinal microvascular dilation, tortuosity, and a relatively long fluorescence retention; however, the average diameter of the major retinal vessels was not notably changed. In addition, optical coherence tomography showed their optic nerve head had an increased diameter. However, the mice could adapt to the change, with microcirculation and the optic nerve head recovering following 30-day tail suspension. Expression of rhodopsin and cone-opsins was not notably changed, and no retinal apoptotic-positive cells were detected between 15- and 30-day tail suspensions. Moreover, the three experimental groups of suspended mice showed normal retinal layers and thickness. Functionally, following 15-day tail suspension, scotopic electroretinograms showed a decline in the oscillatory potentials (OPs), but not in the b wave; simultaneously, the peak time of flash visual evoked potential component N1 was delayed compared to its baseline and the time-matched control. Following 30-day tail suspension, the OPs (O2) amplitude recovered to approximately 97% of its baseline or 86% of the time-matched control level. By simulating cephalad shifting of blood, short-term tail suspension can affect rodent retinal microcirculation, the optic nerve head, and disturb visual electrophysiology. However, the change is reversible with no permanent injury observed in the retina. The mice could adapt to the short-term change of retinal microcirculation, indicating new conditions that could be combined with, or could enhance, simulated microgravity for further studying the impact of short- or long-term outer space conditions on the retina.

Effects of fasting on peripapillary capillary density, peripapillary nerve fiber layer, intraocular pressure and central corneal thickness.

PURPOSE: To investigate the effects of fasting on intraocular pressure (IOP), central corneal thickness (CCT), radial peripapillary capillary (RPC) density and retinal nerve fiber layer (RNFL) thickness during Islamic fasting month of Ramadan. METHODS: Twenty-seven healthy fasting volunteers were enrolled. All subjects underwent full ophthalmic examination and optical coherence tomography angiography (OCTA) of both eyes. All measurements were recorded first in the morning (8:00-10:00 a.m.) and then in the evening (4:00-6:00 p.m.). The first visit was performed during the second and third week of Ramadan and then two months later in a nonfasting routine day. RESULTS: Mean age of participants was 40.07 ± 9.29 years. A significant decrease was found for evening IOP (11.17 ± 2.29 mmHg) in comparison with morning IOP (12.00 ± 2.28) (p = 0.00) only on fasting days. A decrease was observed for CCT both on fasting (6 µm) and nonfasting days (3 µm) (p = 0.00 and p = 0.02, respectively) in the evening. There was a significant increase in whole and peripapillary RPC density (%) on fasting days (48.79 ± 3.08 morning, 49.72 ± 2.85 evening for whole and 50.57 ± 4.06 morning, 51.64 ± 3.71 evening for peripapillary) (p = 0.00). Average RNFL thickness was decreased from morning to evening both on fasting days (0.80 µm) and nonfasting days (1.25 µm) (p = 0.00). Optic nerve head (ONH) vertical cup/disc (C/D) ratio was greater on fasting days (0.30 ± 0.25 morning, 0.31 ± 0.24 evening) in comparison with nonfasting days (0.27 ± 0.25 morning, 0.28 ± 0.25 evening) (p = 0.02). CONCLUSION: Fasting decreases the IOP and CCT in healthy subjects. OCTA revealed significant difference in RPC vessel density, RNFL thickness and ONH vertical C/D ratio during fasting hours in comparison with nonfasting days.

The relationship between glutathione levels in leukocytes and ocular clinical parameters in glaucoma.

PURPOSE: To investigate the effect of mitochondrial dysfunction on the autoregulation of blood flow, by measuring levels of glutathione, an indicator of mitochondrial dysfunction, in glaucoma patients. METHODS: Fifty-six OAG patients and 21 age-matched controls underwent a blood assay. Mitochondrial function was measured according to the levels of total glutathione (t-GSH), reduced GSH (GSH), and oxidized GSH (GSSG, glutathione disulfide) in peripheral blood mononuclear cells. Ocular blood flow in the optic nerve head was assessed with laser speckle flowgraphy parameters, including acceleration time index (ATI). We determined correlations between these measurements and other clinical parameters. Furthermore, we investigated the association between glutathione levels and glaucoma with a logistic regression analysis. Finally, we calculated the area under the receiver operating characteristic (ROC) curve in order to determine the power of redox index (the log GSH/GSSG ratio) to distinguish the groups. RESULTS: OAG patients demonstrated significantly higher GSSG levels and a lower redox index than the controls (p = 0.01, p = 0.01, respectively), but total GSH and reduced GSH levels were similar in the OAG subjects and controls (p = 0.80, p = 0.94, respectively). Additionally, redox index was significantly correlated with mean deviation (MD) of the visual field (r = 0.29, p = 0.03) and ATI (r = -0.30, p = 0.03). Multiple linear regression analysis showed that redox index contributed to MD (p = 0.02) and ATI (p = 0.04). The receiver operating characteristic curve (AUC) analysis suggested that redox index could differentiate between control eyes and eyes with glaucoma (AUC; 0.70: 95% interval; 0.57-0.84). The cutoff point for redox index to maximize its sensitivity and specificity was 2.0 (sensitivity: 91.1%, specificity: 42.9%). CONCLUSIONS: These results suggest that redox index is lower in OAG patients than in controls. Thus, it is possible that mitochondrial dysfunction contributes to glaucoma pathogenesis by causing vascular alterations.

Quantification of collagen fiber structure using second harmonic generation imaging and two-dimensional discrete Fourier transform analysis: Application to the human optic nerve head.

Second harmonic generation (SHG) microscopy is widely used to image collagen fiber microarchitecture due to its high spatial resolution, optical sectioning capabilities and relatively nondestructive sample preparation. Quantification of SHG images requires sensitive methods to capture fiber alignment. This article presents a two-dimensional discrete Fourier transform (DFT)-based method for collagen fiber structure analysis from SHG images. The method includes integrated periodicity plus smooth image decomposition for correction of DFT edge discontinuity artefact, avoiding the loss of peripheral image data encountered with more commonly used windowing methods. Outputted parameters are as follows: the collagen fiber orientation distribution, aligned collagen content and the degree of collagen fiber dispersion along the principal orientation. We demonstrate its application to determine collagen microstructure in the human optic nerve head, showing its capability to accurately capture characteristic structural features including radial fiber alignment in the innermost layers of the bounding sclera and a circumferential collagen ring in the mid-stromal tissue. Higher spatial resolution rendering of individual lamina cribrosa beams within the nerve head is also demonstrated. Validation of the method is provided in the form of correlative results from wide-angle X-ray scattering and application of the presented method to other fibrous tissues.

Bruch's membrane opening minimum rim width and retinal nerve fiber layer thickness in a Brazilian population of healthy subjects.

OBJECTIVE: To determine Bruch's membrane opening (BMO) minimum rim width (MRW) and peripapillary retinal nerve fiber layer thickness (RNFLT) measurements, acquired with optical coherence tomography (OCT) in healthy Brazilian individuals self-reported as African Descent (AD), European Descent (ED) and Mixed Descent (MD). METHODS: 260 healthy individuals (78 AD, 103 ED and 79 MD) were included in this cross-sectional study conducted at the Clinics Hospital of the University of Campinas. We obtained optic nerve head (24 radial B scans) and peripapillary retinal nerve fiber layer (3.5-mm circle scan) images in one randomly selected eye of each subject. RESULTS: After adjustment for BMO area and age, there were no significant differences in mean global MRW (P = 0.63) or RNFLT (P = 0.07) among the three groups. Regionally, there were no significant differences in either MRW or RNFLT in most sectors, except in the superonasal sector, in which both MRW and RNFLT were thinner among ED (P = 0.04, P<0.001, respectively). RNFLT was also thinner in ED in the inferonasal sector (P = 0.009). In all races, global MRW decreased and global RNFLT increased with BMO area. AD subjects had higher rates of global RNFLT decay with age (-0.32 µm/year) compared to ED and MD subjects (-0.10 µm/year and -0.08 µm/year, respectively; P = 0.01 and P = 0.02, respectively). CONCLUSIONS AND RELEVANCE: While we found no significant differences in global MRW and RNFLT among the three races, age-related thinning of the RNFLT was significantly higher in the AD subgroup, which warrants further study.

Rapamycin mediates mTOR signaling in reactive astrocytes and reduces retinal ganglion cell loss.

Damage and loss of retinal ganglion cells (RGCs) can cause visual impairment. The underlying molecular mechanisms that mediate RGC death in ischemic retinal diseases are still unclear. In this study, we sought to understand the neuroprotective effect of rapamycin, the selective inhibitor of mTORC1, on RGC survival and the cellular mechanics that mediate this effect. Recent studies have reported that the epidermal growth factor (EGF) receptor shows an increase in expression in astrocytes after injury, and this receptor can promote their transformation into reactive astrocytes. Our results, along with previous works from others, show the colocalization of phosphor-EGF receptors with the astrocyte marker glial fibrillary acidic proteins in reactive astrocytes in the injured retina. In our in vitro studies, using primary astrocyte cultures of the optic nerve head of rats, showed that rapamycin significantly blocked EGF-induced mTOR signaling mainly through the PI3K/Akt pathway in primary astrocytes, but not through the MAPK/Erk pathway. Additionally, rapamycin dramatically inhibited the activation of mTOR signaling in our ratinal ischemia-reperfusion (I/R) injury model in vivo. Astrocyte activation was assessed by immunostaining retinal flat mounts or cross sections with antibody against GFAP, and we also used western blots to detect the expression of GFAP. Taken together, these results revealed that rapamycin decreases the activation of astrocytes after retinal ischemia-reperfusion injury. Furthermore, rapamycin can improve retinal RGC survival in rats during I/R, as detected by FluoroGold labeling. Our data reveals the neuroprotective effects of rapamycin in an experimental retina injury model, possibly through decreasing glial-dependent intracellular signaling mechanisms for suppressing apoptosis of RGCs. Our study also presents an approach to targeting reactive astrocytes for the treatment of optic neurodegenerations.

Precision of Optic Nerve Head and Retinal Nerve Fiber Layer Parameter Measurements by Spectral-domain Optical Coherence Tomography.

PURPOSE: The aim of this study was to assess the repeatability and reproducibility (R&R) of Bruch membrane opening based on minimum rim width (BMO-MRW), minimum rim area (BMO-MRA) and peripapillary retinal nerve fiber layer thickness (RNFLT) with the Spectralis optical coherence tomography (Heidelberg Engineering) for normal and glaucoma subjects. Precise measurement of these parameters can support detection of structural glaucomatous damage and progression. METHODS: This cross-sectional study included 16 healthy controls and 16 patients with glaucoma. One eye was randomly selected and included in this study. Subjects underwent 1 baseline and 3 follow-up measurements, using 3 different Spectralis optical coherence tomography devices in randomized order, each operated by a single operator. Outcome measures were global and sectorial averages of BMO-MRW and BMO-MRA, and of peripapillary RNFLT obtained from 12/14/16-degree circle scans. Coefficients of variation (COV) were calculated and a mixed-effects analysis of variance was performed to compare R&R between devices. RESULTS: COVs of global and sectorial BMO-MRW measurement under repeatability conditions ranged from 0.51% to 1.7% (normal, 0.62% to 1.3%; glaucoma, 0.64% to 2.3%). Respective COVs under reproducibility conditions ranged from 0.89% to 1.9% (normal, 0.77% to 2.8%; glaucoma, 1.1% to 2.6%). COVs of global and sectorial RNFLT measurements under repeatability conditions ranged from 0.5% to 2.8%. Respective COVs under reproducibility conditions ranged from 1.6% to 3.5%. CONCLUSIONS: For R&R, the COVs of measured parameters were by trend higher for glaucoma eyes compared with normal controls. The BMO-MRW measurement system has an excellent precision taking into account that major and minor corrections of segmentation have to be done by the examiner before evaluation.

Relationship between Sigma-1 receptor and BDNF in the visual system.

Glaucoma is an incurable optic neuropathy characterized by dysfunction and death of retinal ganglion cells (RGCs). Brain derived neurotrophic factor (BDNF) is an essential neurotrophin that supports RGC function and survival. Despite BDNF's importance, our knowledge of molecular mechanisms that modulate BDNF processing and secretion is incomplete. Sigma-1 receptor (S1R) is associated with increased BDNF in hippocampus and with BDNF secretion by brain-derived astrocytes and neuronal cell lines. Much less is known about the relationship between S1R and BDNF in the visual system. Here, we examine how S1R activation and deletion alter expression of mature BDNF (mBDNF) and proBDNF in retina and cultured optic nerve head (ONH) astrocytes. For S1R activation, the S1R agonist (+)-pentazocine (PTZ, 0.5 mg/kg) was administered by intraperitoneal injection to C57BL/6J mice, 3 times per week, for 5 weeks. Expression of proBDNF and mBDNF was also examined in S1R knockout and age-matched C57BL/6J mice. In vitro, cultured ONH astrocytes were treated with 3 µM PTZ for 24 h followed by collection of media and ONH astrocyte lysates. Results showed that treatment with (+)-PTZ increased mBDNF protein in both retina and hippocampus. In contrast, S1R deletion was associated with retinal mBDNF deficits. In ONH astrocytes S1R agonist (+)-PTZ significantly increased levels of secreted BDNF and proBDNF in cell lysates. These findings support a role for S1R in the modulation of BDNF levels within the retina and optic nerve head. Treatment with S1R agonists might provide benefit in diseases such as glaucoma by increasing BDNF levels from endogenous sources.

Effects of Selective Serotonin Reuptake Inhibitors on Macular Ganglion Cell Complex Thickness and Peripapillary Retinal Nerve Fiber Layer Thickness.

PURPOSE: To evaluate macular ganglion cell complex (GCC) thickness and peripapillary retinal nerve fiber layer (RNFL) thickness in patients treated with SSRIs. METHODS: The present study included 62 eyes of 31 patients who were using SSRIs and 60 eyes of 30 healthy, age- and gender-matched control subjects. All patients underwent a full ophthalmological examination in which macular thickness, GCC thickness, and peripapillary RNFL thickness were measured using optical coherence tomography (OCT). The Mann-Whitney U test was used to compare the patients' group with the age- and gender-matched control group. Pearson correlation analyses were also performed to assess the relationships between macular thickness, GCC thickness, RNFL thickness, and the duration of SSRI usage. RESULTS: The mean duration of SSRI usage was 29.96 ± 27.19 (range 6-120) months. The foveal thickness was 253.48 ± 22.77µm in the patients' group and 266.60 ± 20.64 µm in the control group; the difference between the groups was statistically significant. In addition, the perifoveal GCC thickness in the inferonasal and inferotemporal quadrant were significantly smaller thinner in the patient group (Mann-Whitney U test, p = 0.021and p = 0.013, respectively). CONCLUSIONS: Our results suggest a relation between SSRIs and decreased retinal GCC thickness and RNFL thickness. Future long-term prospective studies should elucidate the actual effect of SSRIs on GCC and RNFL thickness.

Interocular symmetry of retinal nerve fiber layer and optic nerve head parameters measured by Cirrus high-definition optical coherence tomography in a normal pediatric population.

PURPOSE: To determine interocular differences in the retinal nerve fiber layer (RNFL) and optic nerve head (ONH) parameters in a pediatric population using Cirrus high-definition optical coherence tomography (HD-OCT). METHODS: Seventy normal Indian children aged 5-17 years presenting to the Pediatric Clinic were included in this observational cross-sectional study. All subjects underwent a comprehensive ophthalmologic examination and an evaluation of the RNFL and ONH by Cirrus HD-OCT. Differences between the right and left eyes were calculated and values were compared by means of a paired t-test. Subjects were also divided into two groups based on age (under or over 10 years of age). Interocular differences in RNFL and ONH parameters together with sex and age variations for these differences were determined. RESULTS: The mean age of studied pediatric population was 11.83 ± 3.3 years (range 5-17). Average RNFL thickness was 94.46 ± 8.7 µm (± SD) (range 77-111). Differences in the average RNFL between right and left eyes were not statistically significant (P = 0.060). Superior quadrant RNFL was thicker in the left eye and temporal quadrant was thicker in the right eye. Among ONH parameters, there were no statistically significant differences in any parameters, except vertical cup-disc (CD) ratio which was significant (P = 0.007). The 2.5%-97.5% limits of asymmetry were 9 µm for average RNFL, 0.14 for average CD ratio, and 0.22 for vertical CD ratio. Mean interocular RNFL thickness differences in superior, superior nasal, and temporal superior quadrants were 10.61 (P < 0.001), 12.57 (P < 0.001), and 4.46 (P = 0.002) µm, respectively. Interocular nerve fiber layer thickness differences were not significantly correlated with sex, while only significant differences with age were observed in 12 clock hour sector analysis, mainly in nasal inferior and inferior quadrant. CONCLUSIONS: We report the degree of interocular symmetry of RNFL and ONH parameters measured by Cirrus HD-OCT in a healthy pediatric population. The normal interocular RNFL asymmetry should not exceed 9 µm and vertical CD ratio beyond 0.22 should be considered for further investigations. The physiologic asymmetry provided by this study may assist in identifying changes in RNFL thickness and ONH parameters in pediatric glaucoma and ONH disorders.

Determinants and Characteristics of Bruch's Membrane Opening and Bruch's Membrane Opening-Minimum Rim Width in a Normal Japanese Population.

Purpose: To identify determinants of Bruch's membrane opening (BMO), and BMO-minimum rim width (BMO-MRW) and circumpapillary retinal nerve fiber layer thickness (RNFLT) centered on BMO center and characterize these parameters in a normal Japanese population. Methods: Spectral-domain optical coherence tomography images of optic nerve head and circumpapillary and macular retina were obtained in 258 eyes of 258 normal Japanese with mean (standard deviation) age of 51.7 (18.2) years. BMO area, BMO-MRW, RNFLT (measured with a 3.5-mm-diameter circle scan) were all acquired and analyzed relative to the eye-specific fovea to BMO (FoBMO) axis. One randomly selected eye of each subject was analyzed. Multiple regression analysis was used to identify determinants to the parameters. Results: BMO area, global BMO-MRW, RNFLT, and FoBMO angle averaged 2.06 (0.45) mm2, 305.5 (50.0) µm, 101.8 (9.6) µm, and -7.8° (3.8°), respectively. There was a modest correlation between global BMO-MRW and RNFLT (r = 0.337; P < 0.001), while the sectorwise correlations were highest in the superior-temporal sector (r = 0.500; P < 0.001) and lowest in the nasal sector (r = 0.117; P = 0.063). Global BMO-MRW and RNFLT declined with age at -1.04 µm/y (P < 0.001) and -0.12 µm/y (P = 0.001), and the former correlated negatively (P = 0.001) and the latter positively (P < 0.001) with BMO area after adjustment for other factors (R2 = 0.191 and 0.272, respectively). BMO area correlated positively with axial length (P = 0.023) and negatively with age (P < 0.001) (R2 = 0.157). Conclusions: BMO-MRW and RNFLT declined with age with a difference between them in their relationship to BMO area. BMO area positively correlated with axial length and negatively with age.

Statins reduce TGF-beta2-modulation of the extracellular matrix in cultured astrocytes of the human optic nerve head.

Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-ß2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-ß2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-ß2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-ß2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-ß2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling.

Measuring Deformation in the Mouse Optic Nerve Head and Peripapillary Sclera.

Purpose: To develop an ex vivo explant system using multiphoton microscopy and digital volume correlation to measure the full-field deformation response to intraocular pressure (IOP) change in the peripapillary sclera (PPS) and in the optic nerve head (ONH) astrocytic structure. Methods: Green fluorescent protein (GFP)-glutamate transporter-GLT1 (GLT1/GFP) mouse eyes were explanted and imaged with a laser-scanning microscope under controlled inflation. Images were analyzed for regional strains and changes in astrocytic lamina and PPS shape. Astrocyte volume fraction in seven control GLT1/GFP mice was measured. The level of fluorescence of GFP fluorescent astrocytes was compared with glial fibrillary acidic protein (GFAP) labeled astrocytes using immunohistochemistry. Results: The ONH astrocytic structure remained stable during 3 hours in explants. Control strain-globally, in the central one-half or two-thirds of the astrocytic lamina-was significantly greater in the nasal-temporal direction than in the inferior-superior or anterior-posterior directions (each P≤ 0.03, mixed models). The PPS opening (perimeter) in normal eye explants also became wider nasal-temporally than superior-inferiorly during inflation from 10 to 30 mm Hg (P = 0.0005). After 1 to 3 days of chronic IOP elevation, PPS area was larger than in control eyes (P = 0.035), perimeter elongation was 37% less than controls, and global nasal-temporal strain was significantly less than controls (P = 0.007). Astrocyte orientation was altered by chronic IOP elevation, with processes redirected toward the longitudinal axis of the optic nerve. Conclusions: The explant inflation test measures the strain response of the mouse ONH to applied IOP. Initial studies indicate regional differences in response to both acute and chronic IOP elevation within the ONH region.

Peripapillary Nerve Fiber Elevation in Young Healthy Eyes.

PURPOSE: Eyes with a peripapillary nerve fiber elevation (pNFE) are those with a discrepancy between the optic disc margin in a color fundus photograph and the Bruch's membrane opening in an optical coherent tomographic (OCT) cross-sectional image. The purpose of this study was to determine the prevalence of pNFE in young healthy eyes, and to compare the axial length and optic disc tilt between pNFE and non-pNFE groups. METHODS: This was a prospective, observational, cross-sectional study of 117 right eyes. All participants (mean age 25.8 ± 4.0 years) underwent a comprehensive ophthalmologic examination. The pNFE was determined from the color fundus photographs, scanning laser ophthalmoscopic images, and optic disc cross-sectional OCT images. The degree of optic disc tilt was determined by the sine curve technique. Mann-Whitney U tests were used to determine the significance of the differences in the axial length and optic disc tilt between the pNFE and non-pNFE groups. RESULTS: Fifty-nine eyes were placed in the pNFE group and 58 eyes in the non-pNFE group. The axial length of the pNFE group (26.0 ± 1.4 mm) was significantly longer than that of the non-pNFE group (24.9 ± 1.2 mm; P < 0.001). The optic disc tilt of the pNFE group (42.1 ± 16.5 pixels) was significantly greater than that of the non-pNFE group (33.2 ± 16.8 pixels; P = 0.003). CONCLUSIONS: The presence of pNFE is not rare in young healthy eyes. The eyes with pNFE have longer axial lengths and greater optic disc tilt. The pNFE should be considered when the disc margin is assessed.