Profiling IOP-responsive genes in anterior and posterior ocular tissues in the rat CEI glaucoma model.

Purpose: The rat Controlled Elevation of Intraocular pressure (CEI) model allows study of in vivo responses to defined intraocular pressures (IOP). In this study, we use Nanostring technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N=35) were subject to CEI for 8-hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5x IOP (CEI-50). Naïve animals, receiving no anesthesia or surgical interventions, served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA isolated, and samples were analyzed with a Nanostring panel containing 770 genes. Post-processing, raw count data were uploaded to Rosalind® for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the "CEI-50 vs. CEI-20" and "CEI-50 vs. naïve" comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and TGFß pathways to be the most up- and down-regulated KEGG pathways, respectively. For ONH, 22 significantly regulated genes were identified in the "CEI-50 vs. naïve" comparison. Pathway analysis identified 'defense response' and 'immune response' as two significantly upregulated biological process pathways. Conclusions: This study demonstrates the ability to assay IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggest that TM responses may prevent TGFß-induced extracellular matrix synthesis. For ONH, the initial response to elevated IOP may be protective, with astrocytes playing a key role in these gene responses.

Optic Nerve Head Gene Transcription Sequelae to a Single Elevated IOP Exposure Provides Insights Into Known Responses to Chronically Elevated IOP.

Purpose: To clarify the optic nerve head (ONH) gene expression responses associated with a single, axon-damaging exposure to elevated IOP in relation to the composite cellular events previously identified in models of chronically elevated IOP. Methods: Anesthetized rats were exposed unilaterally to an 8-hour pulse-train controlled elevation of IOP (PT-CEI) at 60 mm Hg, while others received normotensive CEI at 20 mm Hg. ONH RNA was harvested at 0 hours and 1, 2, 3, 7, and 10 days after either CEI and from naïve animals. RNA sequencing was performed to analyze ONH gene expression. DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Gene function was compared between PT-CEI and two models of chronic ocular hypertension from the literature. Results: The number of significantly changed genes peaked immediately (n = 1354) after PT-CEI (0 hours). This was followed by a lull (<4 genes per time point) at 1 and 2 days after PT-CEI. Gene activity increased again at 3 days (136 genes) and persisted at 7 (78 genes) and 10 (339 genes) days. Significant gene functional categories included an immediate upregulation of Defense Response at 0 hours, followed by upregulation in Cell Cycle, a reduction in Axonal-related genes at 3 to 10 days, and upregulation of Immune Response-related genes at 10 days following PT-CEI. The most commonly upregulated gene expression across our PT-CEI study and two chronic models of ocular hypertension were cell cycle related. Conclusions: The PT-CEI model places in sequence ONH gene expression responses previously reported in models with chronically elevated IOP and may provide insights into their role in optic nerve damage.

Longitudinal Observation of Retinal Response to Optic Nerve Transection in Rats Using Visible Light Optical Coherence Tomography.

Purpose: To characterize rat retinal responses after optic nerve transection (ONT) by visible-light optical coherence tomography (vis-OCT). Methods: Unilateral ONT was performed in Brown Norway rats (n = 8). In vivo, vis-OCT retinal imaging was performed on the experimental eyes before ONT (baseline), and two days, one week, two weeks, and four weeks (endpoint) after ONT, as well as on fellow eyes at the endpoint. The system was operated at a 70 kHz A-line sampling rate with both raster scans (512 × 2 × 512 A-lines), and circular scans (2048 × 100 A-lines) acquired around the optic disc. Retinal layers were segmented to calculate layer thicknesses and project en face images for visualization and quantifications. Vessel densities and oxygen saturation were used to evaluate the morphologic and functional impact on the retinal vasculature. Results: After ONT, retinal nerve fiber bundles demonstrated significant degeneration, starting at two weeks, with a reduction of thicknesses quantified on the nerve fiber layer, ganglion cell complex, and total retina. Along with that, the activation of macrophage-like cells in the vitreoretinal interface was also observed. Vessel densities for all three retinal plexuses were unaffected over the period of observation. However, oxygen saturation in retinal arteries and veins was significantly reduced at four weeks after ONT. Conclusions: Vis-OCT can provide high-definition, in vivo characterization of retinal responses to ONT in rats. Despite a significant reduction in retinal layer thickness, this was not accompanied by alterations in vascular density. Despite this, oximetry indicates reduced retinal oxygen saturation, suggesting that altered vascular physiology is not reflected in the anatomic appearance of retinal blood vessel density alone.

A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes.

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Early Optic Nerve Head Glial Proliferation and Jak-Stat Pathway Activation in Chronic Experimental Glaucoma.

Purpose: We previously reported increased expression of cell proliferation and Jak-Stat pathway-related genes in chronic experimental glaucoma model optic nerve heads (ONH) with early, mild injury. Here, we confirm these observations by localizing, identifying, and quantifying ONH cellular proliferation and Jak-Stat pathway activation in this model. Methods: Chronic intraocular pressure (IOP) elevation was achieved via outflow pathway sclerosis. After 5 weeks, ONH longitudinal sections were immunolabeled with proliferation and cell-type markers to determine nuclear densities in the anterior (unmyelinated) and transition (partially myelinated) ONH. Nuclear pStat3 labeling was used to detect Jak-Stat pathway activation. Nuclear density differences between control ONH (uninjected) and ONH with either early or advanced injury (determined by optic nerve injury grading) were identified by ANOVA. Results: Advanced injury ONH had twice the nuclear density (P < 0.0001) of controls and significantly greater astrocyte density in anterior (P = 0.0001) and transition (P = 0.006) ONH regions. An increased optic nerve injury grade positively correlated with increased microglia/macrophage density in anterior and transition ONH (P < 0.0001, both). Oligodendroglial density was unaffected. In glaucoma model ONH, 80% of anterior and 66% of transition region proliferating cells were astrocytes. Nuclear pStat3 labeling significantly increased in early injury anterior ONH, and 95% colocalized with astrocytes. Conclusions: Astrocytes account for the majority of proliferating cells, contributing to a doubled nuclear density in advanced injury ONH. Jak-Stat pathway activation is apparent in the early injury glaucoma model ONH. These data confirm dramatic astrocyte cell proliferation and early Jak-Stat pathway activation in ONH injured by elevated IOP.

Optic Nerve Head Astrocytes Display Axon-Dependent and -Independent Reactivity in Response to Acutely Elevated Intraocular Pressure.

Purpose: Optic nerve head (ONH) astrocytes provide support for axons, but exhibit structural and functional changes (termed reactivity) in a number of glaucoma models. The purpose of this study was to determine if ONH astrocyte structural reactivity is axon-dependent. Methods: Using rats, we combine retrobulbar optic nerve transection (ONT) with acute controlled elevation of intraocular pressure (CEI), to induce total optic nerve axon loss and ONH astrocyte reactivity, respectively. Animals were euthanized immediately or 1 day post CEI, in the presence or absence of ONT. ONH sections were labeled with fluorescent-tagged phalloidin and antibodies against ß3 tubulin, phosphorylated cortactin, phosphorylated paxillin, or complement C3. ONH label intensities were quantified after confocal microscopy. Retrobulbar nerves were assessed for axon injury by light microscopy. Results: While ONT alone had no effect on ONH astrocyte structural orientation, astrocytes demonstrated significant reorganization of cellular extensions within hours after CEI, even when combined with ONT. However, ONH astrocytes displayed differential intensities of actin (phosphorylated cortactin) and focal adhesion (phosphorylated paxillin) mediators in response to CEI alone, ONT alone, or the combination of CEI and ONT. Lastly, label intensities of complement C3 within the ONH were unchanged in eyes subjected to CEI alone, ONT alone, or the combination of CEI and ONT, relative to controls. Conclusions: Early ONH astrocyte structural reactivity to elevated IOP is multifaceted, displaying both axon dependent and independent responses. These findings have important implications for pursuing astrocytes as diagnostic and therapeutic targets in neurodegenerative disorders with fluctuating levels of axon injury.

Investigation of MicroRNA Expression in Experimental Glaucoma.

MicroRNAs are small, endogenous noncoding RNAs that modulate post-transcriptional gene expression. Recent evidence suggests that they may have a potential role in the regulation of the complex biological responses that develop in response to elevated intraocular pressure. However, contemporary microRNA assay techniques (e.g., microarrays and next-generation sequencing) typically require large amounts of RNA template that are often times difficult to obtain from glaucomatous tissue. We describe in detail an experimental protocol utilizing targeted pre-amplification and low-density polymerase chain reaction arrays to circumvent this hurdle. This approach optimizes the simultaneous high-throughput screening of small tissue samples, such as the rodent optic nerve head, for up to 754 microRNA probes while also providing an opportunity for subsequent confirmatory reactions of technical or biological replicates.

Utilizing RNA-Seq to Identify Differentially Expressed Genes in Glaucoma Model Tissues, Such as the Rodent Optic Nerve Head.

Understanding the cellular pathways activated by elevated intraocular pressure (IOP) is crucial for the development of more effective glaucoma treatments. Microarray studies have previously been used to identify several key gene expression changes in early and extensively injured ONH, as well as in the retina. Limitations of microarrays include that they can only be used to detect transcripts that correspond to existing genomic sequencing information and their narrower dynamic range. However, RNA sequencing (RNA-seq) is a powerful tool for investigating known transcripts, as well as for exploring new ones (including noncoding RNAs and small RNAs), is more quantitative, and has the added benefit that the data can be re-analyzed as new sequencing information becomes available. Here, we describe an RNA-seq method specifically developed for identifying differentially expressed genes in optic nerve heads of eyes exposed to elevated intraocular pressure. The methods described here could also be applied to small tissue samples (less than 100 ng in total RNA yield) from retina, optic nerve, or other regions of the central nervous system.

Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: A Pilot Study.

Purpose: MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that have been detected in human aqueous humor (AH). Prior studies have pooled samples to obtain sufficient quantities for analysis or used next-generation sequencing. Here, we used PCR arrays with preamplification to identify and compare miRNAs from individual AH samples between patients with primary open-angle glaucoma (POAG) and normal controls. Methods: AH was collected before cataract surgery from six stable, medically treated POAG patients and eight age-matched controls. Following reverse transcription and preamplification, individual patient samples were profiled on Taqman Low Density MicroRNA Array Cards. Differentially expressed miRNAs were stratified for fold changes larger than ±2 and for significance of P < 0.05. Significant Kyoto Encyclopedia of Genes and Genomes pathways influenced by the differentially expressed miRNAs were identified using the predicted target module of the miRWalk 2.0 database. Results: This approach detected 181 discrete miRNAs, which were consistently expressed across all samples of both experimental groups. Significant up-regulation of miR-518d and miR-143, and significant down-regulation of miR-660, was observed in the AH of POAG patients compared with controls. These miRNAs were predicted to reduce cell proliferation and extracellular matrix remodeling, endocytosis, Wnt signaling, ubiquitin-mediated proteolysis, and adherens junction function. Conclusions: This pilot study demonstrates that miRNA expression within the AH of POAG patients differs from age-matched controls. AH miRNAs exhibit potential as biomarkers of POAG, which merits further investigation in a larger case-controlled study. This technique provides a cost-effective and sensitive approach to assay miRNAs in individual patient samples without the need for pooling.

A Period of Controlled Elevation of IOP (CEI) Produces the Specific Gene Expression Responses and Focal Injury Pattern of Experimental Rat Glaucoma.

Purpose: We determine if several hours of controlled elevation of IOP (CEI) will produce the optic nerve head (ONH) gene expression changes and optic nerve (ON) damage pattern associated with early experimental glaucoma in rats. Methods: The anterior chambers of anesthetized rats were cannulated and connected to a reservoir to elevate IOP. Physiologic parameters were monitored. Following CEI at various recovery times, ON cross-sections were graded for axonal injury. Anterior ONHs were collected at 0 hours to 10 days following CEI and RNA extracted for quantitative PCR measurement of selected messages. The functional impact of CEI was assessed by electroretinography (ERG). Results: During CEI, mean arterial pressure (99 ± 6 mm Hg) and other physiologic parameters remained stable. An 8-hour CEI at 60 mm Hg produced significant focal axonal degeneration 10 days after exposure, with superior lesions in 83% of ON. Message analysis in CEI ONH demonstrated expression responses previously identified in minimally injured ONH following chronic IOP elevation, as well as their sequential patterns. Anesthesia with cannulation at 20 mm Hg did not alter these message levels. Electroretinographic A- and B-waves, following a significant reduction at 2 days after CEI, were fully recovered at 2 weeks, while peak scotopic threshold response (pSTR) remained mildly but significantly depressed. Conclusions: A single CEI reproduces ONH message changes and patterns of ON injury previously observed with chronic IOP elevation. Controlled elevation of IOP can allow detailed determination of ONH cellular and functional responses to an injurious IOP insult and provide a platform for developing future therapeutic interventions.

Astrocyte Structural and Molecular Response to Elevated Intraocular Pressure Occurs Rapidly and Precedes Axonal Tubulin Rearrangement within the Optic Nerve Head in a Rat Model.

Glaucomatous axon injury occurs at the level of the optic nerve head (ONH) in response to uncontrolled intraocular pressure (IOP). The temporal response of ONH astrocytes (glial cells responsible for axonal support) to elevated IOP remains unknown. Here, we evaluate the response of actin-based astrocyte extensions and integrin-based signaling within the ONH to 8 hours of IOP elevation in a rat model. IOP elevation of 60 mm Hg was achieved under isoflurane anesthesia using anterior chamber cannulation connected to a saline reservoir. ONH astrocytic extension orientation was significantly and regionally rearranged immediately after IOP elevation (inferior ONH, 43.2° ± 13.3° with respect to the anterior-posterior axis versus 84.1° ± 1.3° in controls, p<0.05), and re-orientated back to baseline orientation 1 day post IOP normalization. ONH axonal microtubule filament label intensity was significantly reduced 1 and 3 days post IOP normalization, and returned to control levels on day 5. Phosphorylated focal adhesion kinase (FAK) levels steadily decreased after IOP normalization, while levels of phosphorylated paxillin (a downstream target of FAK involved in focal adhesion dynamics) were significantly elevated 5 days post IOP normalization. The levels of phosphorylated cortactin (a downstream target of Src kinase involved in actin polymerization) were significantly elevated 1 and 3 days post IOP normalization and returned to control levels by day 5. No significant axon degeneration was noted by morphologic assessment up to 5 days post IOP normalization. Actin-based astrocyte structure and signaling within the ONH are significantly altered within hours after IOP elevation and prior to axonal cytoskeletal rearrangement, producing some responses that recover rapidly and others that persist for days despite IOP normalization.

Circadian rhythm of intraocular pressure in the adult rat.

Ocular hypertension is a risk factor for developing glaucoma, which consists of a group of optic neuropathies characterized by progressive degeneration of retinal ganglion cells and subsequent irreversible vision loss. Our understanding of how intraocular pressure damages the optic nerve is based on clinical measures of intraocular pressure that only gives a partial view of the dynamic pressure load inside the eye. Intraocular pressure varies over the course of the day and the oscillator regulating these daily changes has not yet been conclusively identified. The purpose of this study was to compare and contrast the circadian rhythms of intraocular pressure and body temperature in Brown Norway rats when these animals are housed in standard light-dark and continuous dim light (40-90 lux) conditions. The results from this study show that the temperature rhythm measured in continuous dim light drifted forward relative to external time, indicating that the rhythm was free running and being regulated by an internal biological clock. Also, the results show that there is a persistent, but dampened, circadian rhythm of intraocular pressure in continuous dim light and that the circadian rhythms of temperature and intraocular pressure are not synchronized by the same central oscillator. We conclude that once- or twice-daily clinical measures of intraocular pressure are insufficient to describe intraocular pressure dynamics. Similarly, our results indicate that, in experimental animal models of glaucoma, the common practice of housing animals in constant light does not necessarily eliminate the potential influence of intraocular pressure rhythms on the progression of nerve damage. Future studies should aim to determine whether an oscillator within the eye regulates the rhythm of intraocular pressure and to better characterize the impact of glaucoma on this rhythm.

Development of a rat schematic eye from in vivo biometry and the correction of lateral magnification in SD-OCT imaging.

PURPOSE: Optical magnification in optical coherence tomography (OCT) depends on ocular biometric parameters (e.g., axial length). Biometric differences between eyes will influence scan location. A schematic model eye was developed to compensate for lateral magnification in OCT images of the healthy rat. METHODS: Spectral-domain optical coherence tomography images were acquired in 19 eyes of 19 brown Norway rats. Images were scaled using the OCT instrument's built-in scaling function and by calculating the micron per degree from schematic model eyes developed from in vivo biometry (immersion A-scan and videokeratometry). Mean total retinal thickness was measured 500 µm away from the optic nerve head and optic nerve head diameter was measured. Corneal curvature, lens thickness, and axial length were modified to calculate their effects on OCT scan location and total retinal thickness. RESULTS: Mean total retinal thickness increased by 21 µm and the SD doubles when images were scaled with the Built-in scaling (222 ± 13 µm) compared with scaling with individual biometric parameters (201 ± 6 µm). Optic nerve head diameter was three times larger when images were scaled with the Built-in scaling (925 ± 97 µm) than the individual biometric parameters (300 ± 27 µm). Assuming no other change in biometric parameters, total retinal thickness would decrease by 37 µm for every millimeter increase in anterior chamber depth due to changes in ocular lateral magnification and associated change in scan location. CONCLUSIONS: Scaling SD-OCT images with schematic model eyes derived from individual biometric data is important. This approach produces estimates of retinal thickness and optic nerve head size that are in good agreement with previously reported measurements.

Quantitative evaluation of factors influencing the repeatability of SD-OCT thickness measurements in the rat.

PURPOSE: To quantify repeatability and reproducibility of thickness measurements and the effects of realignment and image quality on measurements of retinal thickness from optical coherence tomographic (OCT) imaging in the rat eye. METHODS: Retinal imaging was performed in 16 brown norway rats (N = 16 EYES; X = 372 G). Precision metrics: 95% limits of agreement (LoA), intraclass correlation coefficient (ICC), and the coefficient of variation (CV), were calculated using manual and combined manual + automated realignment procedures for nerve fiber and retinal ganglion cell layer (NFL/GCL), NFL/GCL and inner plexiform layer (NFL/GCL + IPL), and total retina thicknesses (excluding blood vessels). The influence of image quality on NFL thickness measurement was assessed by comparing high- and low-quality image data (real and simulated) from the rat as well as clinical data. RESULTS: Mean NFL/GCL thickness was 26 3 M, NFL/GCL + IPL thickness was 70 3 M, AND total retinal thickness was 192 7 M. Thickness difference between imaging sessions for NFL/GCL was 1 M (95% LOA: -4 to 3 µm; ICC = 0.82; CV = 4.7%), for NFL/GCL + IPL was 0 µm (95% LoA: -4 to 4 µm; ICC = 0.88; CV = 1.4%), and total retinal thickness was 1 µm (95% LoA: -3 to 4 µm; ICC = 0.97; CV = 0.7%). Thickness differences were similar between realignment procedures (NFL/GCL: P = 0.43; NFL/GCL + IPL: P = 0.33; total retina: P = 0.62). Although NFL thickness measurements increased slightly in low-quality rat images (4 µm; P = 0.04), this was not true with clinical images (1.4 µm; P = 0.36). CONCLUSIONS: Precision of retinal layer thickness estimation from OCT imaging is excellent when manual and automated realignment procedures are combined, but may still be influenced by image quality and segmentation methods.