Optical coherence tomography angiography (OCTA) is a volumetric, non-invasive, high-resolution vascular imaging modality capable of acquiring highly detailed visualizations of retinal microvasculature. It has become an important tool for diagnosis and prognosis in prevalent diseases and pathologies such as diabetic retinopathy, retinopathy of prematurity, and vein occlusions, as well as more rare conditions, including inherited retinal dystrophies. It is also useful for measuring treatment response and assessing which patients would benefit from treatment. Unlike dye-based angiography, OCTA eliminates risks such as anaphylaxis. It also often outperforms fundus photography in feature detection. However, conventional OCTA imaging has been limited by its small field of view, which restricts simultaneous visualization of the posterior pole and peripheral retina, causing single images to potentially miss widely spaced critical biomarkers and pathological features. Recent technological advances in widefield OCTA have addressed this limitation, extending the field of view to the mid-periphery and beyond. This breakthrough enhances the simultaneous detection of macular and peripheral retinal pathology and significantly broadens OCTA's diagnostic and research applications. This review explores the technical innovations enabling widefield OCTA and highlights its clinical utility across various conditions, emphasizing its growing importance as a powerful tool in ophthalmic practice and research.

Experimental elevation of intraocular pressure (IOP), a major glaucoma risk factor, has been a mainstay of research into mechanisms of glaucomatous optic nerve damage for decades. Methods that produce sustained IOP elevation can mimic the chronic nature of glaucoma and produce optic nerve damage. However, the pressure course for individual animals can be variable, unpredictably high at times, and difficult to monitor with current tonometry methods. All of this can complicate correlations of pressure history with axonal injury. An alternative is to control the extent and duration of IOP elevation over a period of several hour-long enough to produce axonal injury and gene expression changes within the optic nerve head that may indicate cellular mechanisms of glaucomatous optic nerve damage. The prolonged general anesthesia that this requires does have the potential to reduce systemic blood pressure, which may contribute to axonal injury in the face of elevated IOP. This chapter will describe our Controlled Elevation of IOP (CEI) model in laboratory rats. We will include methods for applying this to several animals at a time, as well as how to maintain blood pressure, oxygenation, and body temperature to ensure that the resulting injury and tissue events reflect the effects of elevated IOP on optic nerve tissues and not simply reduced ocular perfusion and ischemia.

Animal models that help us understand how elevated intraocular pressure (IOP) causes axonal injury will lead to new glaucoma therapies. Because reliable measurements are difficult to obtain in chronic models, we developed the controlled elevation of IOP (CEI) approach. Here, a cannula connected to an elevated balanced salt solution (BSS) reservoir is inserted into the anterior chamber of anesthetized Brown Norway rats. The extent and duration of IOP exposure is controlled by adjusting the reservoir height. We now describe a method for creating CEI in awake animals. A Pinport, which has a silicone plug that can be penetrated repeatedly, is modified, attached to the skull, and connected to a microcannula that is implanted in the posterior chamber. To elevate IOP, BSS from a reservoir is allowed to flow through a pressure transducer to a swivel-mounted tether and injector. The injector is placed on the Pinport, bypassing the need for anterior chamber cannulation and general anesthesia during CEI. The surgical technique and equipment required for implantation are described, as well as the equipment and methods for performing awake CEI in several animals at a time. The ability of this system to control the level of IOP is demonstrated by TonoLab measurement, and by comparing reservoir (Pinport) pressures to direct measurement using an independent anterior chamber cannula and transducer. We also demonstrate that IOP elevation can be maintained over several hours. Specific pitfalls during and after surgical implantation are highlighted to help other researchers adopt these techniques.

This study introduces an ultra-wide field (UWF) and high-resolution swept-source optical coherence tomographic angiography (OCTA) system for rat retinal imaging. Using an asymmetrical optics design, the system achieves unprecedented details of retinal structures and vascular plexuses over a large field of view (112°) in a single-shot acquisition. Views of single-nerve fiber bundles and single capillary vessels are consistently visible over a 112° field of view. The system has a long imaging range and high penetration and allows a full view of vitreous hyaloid vessels, retina, choroid, sclera, and posterior ciliary arteries, down to sub-sclera connective tissues. In a longitudinal study of oxygen-induced retinopathy (OIR) in rats, the system successfully revealed the progression and regression of OIR-related vascular pathologies, such as ischemia, neovascularization, and tortuosity. To our knowledge, this novel UWF-OCT/OCTA prototype designed for rat retinal imaging will be a vital tool for monitoring disease progression and evaluating therapeutic interventions in preclinical models.

Our purpose was to develop a protocol for prolonged anesthesia in mice and evaluate optic nerve axon injury in response to 4 h of controlled elevation of intraocular pressure (CEI). During CEI, C57BL/6 male mice (3-5 months old) were anesthetized with 1.5% isoflurane with 100% oxygen for 4 h and placed on a warm platform, with expired gas and anesthetic actively evacuated. Lactated ringers (0.5 ml) with 5% dextrose was administered subcutaneously at the start and end of CEI. Physiological parameters (oxygen saturation = O2, heart rate = HR, systolic blood pressure = SBP, and temperature) were monitored throughout the 4-h CEI. One eye was cannulated with polyurethane tubing connected to a balanced salt solution reservoir and IOP elevated to 20 (N = 18), 30 (N = 13), 50 (N = 14), and 60 mmHg (N = 16). An additional group of 22 female mice was exposed to CEI of 60 mmHg. Fourteen days after CEI, optic nerves were assessed for axonal injury by masked observers that assigned a grade on a scale from 1 (normal) to 5 (>50% of axons degenerating). CEI optic nerve injury was compared to injury assessed in contralateral optic nerves (N = 84) and naïve optic nerves (N = 18) using a one-way ANOVA followed by Kruskal-Wallis test for multiple comparisons. The relationship between optic nerve injury, physiological parameters, and IOP were assessed by linear regression analyses. Physiologic parameters remained stable throughout CEI (O2 = 95 ± 9%; HR = 450 ± 39; SBP = 102 ± 15 mmHg, and temperature = 38 ± 0.7 °C) and were not statistically different between groups (all comparisons had P > 0.5). Mean optic nerve injury grades (±SD) for naïve optic nerves (1.01 ± 0.02) were not significantly different from fellow/contralateral optic nerves (1.03 ± 0.07, P > 0.99), or from CEI of 20 mmHg (1.04 ± 0.08, P > 0.99) or 30 mmHg (1.05 ± 0.06, P = 0.6). However, animals exposed to CEI of 50 mmHg (2.09 ± 1.43, P = 0.0005) and 60 mmHg (male: 2.86 ± 1.30, P < 0.0001, female: 1.63 ± 1.00, P = 0.0006) developed significant optic nerve injury relative to their fellow/contralateral optic nerves. Axonal injury grades following a CEI of 60 mmHg were not significantly different between male and female mice (P = 0.19). Optic nerve injury positively correlated (P < 0.0001) with IOP and not with physiological parameters, indicating that the optic nerve injury is IOP-related. In conclusion, prolonged anesthesia in mice requires careful attention to animal physiology. With this, a 4-h exposure to elevated IOP can produce significant optic nerve injury with IOPs equal to or greater than 50 mmHg. We provide detailed descriptions of methods and materials for producing prolonged elevations of IOP in mice while maintaining and monitoring their physiology, as well as a unique, cost-effective transducer system for monitoring pressure delivery.

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 TGFβ 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 TGFβ pathway genes suggest that TM responses may prevent TGFβ-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.

Purpose: The rat controlled elevation of intraocular pressure (CEI) model allows study of in vivo responses to short-term exposure to defined intraocular pressures (IOP). In this study, we used 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 subjected to CEI for 8 hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5× IOP (CEI-50). Naïve animals that received no anesthesia or surgical interventions served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA was isolated, and samples were analyzed with a NanoString panel containing 770 genes. Postprocessing, 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 versus CEI-20 and CEI-50 versus naïve comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and transforming growth factor beta (TGFβ) pathways to be the most up- and downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. For ONH, 22 significantly differentially regulated genes were identified in the CEI-50 versus naïve comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. Conclusions: This study demonstrated the ability to assay short-term IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFβ pathway genes suggests that TM responses may reduce TGFβ-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

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.

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.

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.

Optical coherence tomography (OCT) is widely used in ophthalmic practice because it can visualize retinal structure and vasculature in vivo and 3-dimensionally (3D). Even though OCT procedures yield data volumes, clinicians typically interpret the 3D images using two-dimensional (2D) data subsets, such as cross-sectional scans or en face projections. Since a single OCT volume can contain hundreds of cross-sections (each of which must be processed with retinal layer segmentation to produce en face images), a thorough manual analysis of the complete OCT volume can be prohibitively time-consuming. Furthermore, 2D reductions of the full OCT volume may obscure relationships between disease progression and the (volumetric) location of pathology within the retina and can be prone to mis-segmentation artifacts. In this work, we propose a novel framework that can detect several retinal pathologies in three dimensions using structural and angiographic OCT. Our framework operates by detecting deviations in reflectance, angiography, and simulated perfusion from a percent depth normalized standard retina created by merging and averaging scans from healthy subjects. We show that these deviations from the standard retina can highlight multiple key features, while the depth normalization obviates the need to segment several retinal layers. We also construct a composite pathology index that measures average deviation from the standard retina in several categories (hypo- and hyper-reflectance, nonperfusion, presence of choroidal neovascularization, and thickness change) and show that this index correlates with DR severity. Requiring minimal retinal layer segmentation and being fully automated, this 3D framework has a strong potential to be integrated into commercial OCT systems and to benefit ophthalmology research and clinical care.

In this study, we present a sensorless adaptive optics swept-source optical coherence tomographic angiography (sAO-SS-OCTA) imaging system for mice. Real-time graphics processing unit (GPU)-based OCTA image acquisition and processing software were applied to guide wavefront correction using a deformable mirror based on signal strength index (SSI) from both OCT and OCTA images. High-resolution OCTA images with aberrations corrected and contrast enhanced were successfully acquired. Fifty-degree field of view high-resolution montaged OCTA images were also acquired.