Evaluation and Correlation of Morphological, Blood Flow and Physiological Retinal Changes in a Rat Model of Glaucoma with a Combined Optical Coherence Tomography and Electroretinography System

Glaucoma is a chronic disease associated with progressive dysfunction of the retinal ganglion cells (RGC), reduction of the retinal blood flow, thinning of the retinal nerve fiber layer (RNFL) and deformation of the optical nerve head (ONH). It is the second leading cause of blindness worldwide, with an estimate of 64.3 million people between the ages of 40 to 80 years affected in 2013, 76.7 million by 2020, and 111.8 million by 2040. Currently, there is no cure for glaucoma and any clinically available pharmaceutical or surgical approaches to treating the disease can only slow its progression. Therefore, early detection and treatment are essential for managing the glaucoma progression. Elevated intraocular pressure (IOP) is one of the most well studied and documented pathogenic risk factors for open-angle glaucoma (OAG), and as such, numerous animal models have been developed to study the acute and chronic IOP elevation effect on the ONH structure, retinal blood perfusion and RGC function. However, most of these studies utilized static chronic IOP elevation, while the relation between the IOP dynamics and the progression of glaucoma is still poorly understood. Joos et al proposed a rat model of glaucoma that utilized a dynamic approach to IOP elevation by use of a vascular loop that consists of short duration (~1h), intermittent IOP elevation. This model resembles closely the daily IOP spiking observed in glaucomatous patients, especially during the early stages of the disease. Better understanding of how the retina (human and animal) responds to such intermittent spikes of the IOP can provide ophthalmologists with valuable information on the origins and early stages of glaucoma development when treatment would be most efficient, as well as insights into developing new therapeutic approaches for glaucoma. Over the past few decades, a number of ex-vivo and in-vivo optical imaging modalities ranging from histopathology to confocal microscopy and optical coherence tomography (OCT) have been used to image changes in the morphology of the retina and the optic nerve head (ONH) in human subjects and animal models of OAG. Laser Doppler Flowmetry, Doppler OCT (DOCT) and Optical Coherence Angiography (OCTA) have been utilized to image and quantify changes in the total retinal blood flow and the blood perfusion in retinal capillaries during IOP elevation. Furthermore, electroretinography (ERG) has been used to assess changes in the retinal function (response to visual stimulation) during elevated IOP. However, all previous studies collected information about the morphological, functional and blood flow / perfusion changes in the retina during elevated IOP separately, at different time points, which prevented the researchers from correlating those changes and uncovering the relationship between them, typically referred to as neurovascular coupling. Since OCT provides both intensity and phase information in a single acquisition, this imaging technology is able to assess changes in the retinal morphology, function and blood flow/perfusion in-vivo and simultaneously. Therefore, the main goals of this PhD project were to: • Develop a combined OCT+ERG imaging system that can image in-vivo and record simultaneously, changes in the retinal morphology, retinal response to visual stimulation and retinal blood flow / perfusion at normal and elevated IOP. • Test the performance of the OCT+ERG system in a rat model of glaucoma. • Utilize the OCT+ERG technology and the dynamic IOP rat model of glaucoma based on the vascular loop, to investigate the effects of acute and chronic IOP elevation to ischemic and non-ischemic IOP levels on the rat retina. • Utilize the OCT+ERG technology to investigate neurovascular coupling in the rat retina at normal and abnormal IOP levels. Results from this PhD research have been published or summarized in manuscripts that are currently under review. Therefore, this PhD thesis was prepared in such a way that individual manuscripts represent separate thesis chapters

Comparison of phase-resolved Doppler optical coherence tomography and optical coherence tomography angiography for measuring retinal blood vessels size

The goal of this study was to compare two OCT-based methods for measuring retinal blood vessels size: Phase-resolved Doppler OCT (DOCT) and OCT angiography (OCTA). The study was conducted in rats (n= 6) using a SD-OCT system operating at 1060 nm with 92 kHz image acquisition rate. Arteries and veins were separated by the phase polarity. Results from this study showed that the venal diameters are significantly larger than the arterial diameters, and there is no significant difference in the vessel diameters measured by both methods

Multimodal imaging of the mouse eye using visible light photoacoustic ophthalmoscopy and near-infrared-II optical coherence tomography

Non-invasive imaging plays a crucial role in diagnosing and studying eye diseases. However, existing photoacoustic ophthalmoscopy (PAOM) techniques in mice have limitations due to handling restrictions, suboptimal optical properties, limited availability of light sources and permissible light fluence at the retina. This study introduces an innovative approach that utilizes Rose Bengal, a contrast agent, to enhance PAOM contrast. This enables visualization of deeper structures like the choroidal microvasculature and sclera in the mouse eye using visible light. The integration of near-infrared-II optical coherence tomography (NIR-II OCT) provides additional tissue contrast and insights into potential NIR-II PAOM capabilities. To optimize imaging, we developed a cost-effective 3D printable mouse eye phantom and a fully 3D printable tip/tilt mouse platform. This solution elevates PAOM to a user-friendly technology, which can be used to address pressing research questions concerning several ocular diseases such as myopia, glaucoma and/or age-related macular degeneration in the future.Comment: 14 pages, 4 figure

Additive manufacturing for multimodal photoacoustic ophthalmoscopy

Photoacoustic ophthalmoscopy in rodents is gaining research momentum, due to advancement in transducer shape and technology. Needle transducers emerged as most valuable tool for photoacoustic retinal imaging and have proven to be sensitive enough to resolve retinal vasculature in-vivo. Nevertheless, placement of the eye and screening of the retina remains challenging, since needle transducers must remain static during image acquisition, while the optical field of view is limited. Such restriction mandates movement of the mouse to rotate the eye and therefore the imaging area on the retina. The needle transducer needs to be temporarily detached during this process to avoid damage to the eye or the transducer. Re-attachment involves additional application of ultrasound gel and doesn’t guarantee ideal placement for optimized imaging performance. Additive manufacturing can help to tackle those challenges and allows to design novel rotational rodent holders for imaging. Hence, we present a fully 3D printable rotatable tip/tilt mouse platform with the eye in the center of rotation, combined with a printable needle transducer holder. Such system guarantees optimal placement of the needle transducer during imaging and rotation of the mouse eye, avoiding detachment of the transducer and effortless screening of the retina. The capabilities for retinal screening are demonstrated by a multimodal optical coherence photoacoustic ophthalmoscopy system employing two separated wavelengths, 1310 nm for optical coherence and 570 nm for photoacoustic ophthalmoscopy.This proceeding is published as Haindl, Richard, Valentina Bellemo, Praveenbalaji Rajendran, Bingyao Tan, Mengyang Liu, Rainer Leitgeb, Wolfgang Drexler, Leopold Schmetterer, and Manojit Pramanik. "Additive manufacturing for multimodal photoacoustic ophthalmoscopy." In Photons Plus Ultrasound: Imaging and Sensing 2023, vol. 12379, pp. 27-33. SPIE, 2023. doi: https://doi.org/10.1117/12.2648234. Copyright 2023 Society of Photo-Optical Instrumentation Engineers (SPIE). Posted with permission

Localized transverse flow measurement with dynamic light scattering line-scan OCT

A novel decorrelation-based approach for measuring localized transverse flow velocity using line-scan (LS) optical coherence tomography (OCT) is proposed. The new approach allows for separation of the flow velocity component along the line-illumination direction of the imaging beam from other orthogonal velocity components, from particle diffusion motion, and from noise-induced distortion in the OCT signal's temporal autocorrelation. The new method was verified by imaging flow in a glass capillary and a microfluidic device and mapping the spatial distribution of the flow velocity within the beam's illumination plane. This method can be extended in the future to map the three-dimensional flow velocity fields for both ex-vivo and in-vivo applications.National Medical Research Council (NMRC)National Research Foundation (NRF)Published versionCanada First Research Excellence Fund; Canadian Institutes of Health Research (446387); Natural Sciences and Engineering Research Council of Canada (312037); National Research Foundation Singapore (NRF2019-THE002-0006); National Medical Research Council (CG/C010A/2017_SERI, MOH-001015-00)

Correlation of Visually Evoked Functional and Blood Flow Changes in the Rat Retina Measured With a Combined OCTþERG System

PURPOSE. To correlate visually evoked functional and blood flow changes in the rat retina measured simultaneously with a combined optical coherence tomography and electroretinography system (OCTþERG). METHODS. Male Brown Norway (n ¼ 6) rats were dark adapted and anesthetized with ketamine/xylazine. Visually evoked changes in the retinal blood flow (RBF) and functional response were measured simultaneously with an OCTþERG system with 3-lm axial resolution in retinal tissue and 47-kHz image acquisition rate. Both single flash (10 and 200 ms) and flicker (10 Hz, 20% duty cycle, 1-and 2-second duration) stimuli were projected onto the retina with a custom visual stimulator, integrated into the OCT imaging probe. Total axial RBF was calculated from circular Doppler OCT scans by integrating over the arterial and venal flow. RESULTS. Temporary increase in the RBF was observed with the 10-and 200-ms continuous stimuli (~1% and~4% maximum RBF change, respectively) and the 10-Hz flicker stimuli (~8% for 1-second duration and~10% for 2-second duration). Doubling the flicker stimulus duration resulted in~25% increase in the RBF peak magnitude with no significant change in the peak latency. Single flash (200 ms) and flicker (10 Hz, 1 second) stimuli of the same illumination intensity and photon flux resulted in~23 larger peak RBF magnitude and~25% larger RBF peak latency for the flicker stimulus. CONCLUSIONS. Short, single flash and flicker stimuli evoked measureable RBF changes with larger RBF magnitude and peak latency observed for the flicker stimuli

The new era of retinal imaging in hypertensive patients

Structural and functional alterations in the microcirculation by systemic hypertension can cause significant organ damage at the eye, heart, brain, and kidneys. As the retina is the only tissue in the body that allows direct imaging of small vessels, the relationship of hypertensive retinopathy signs with development of disease states in other organs have been extensively studied; large-scale epidemiological studies using fundus photography and advanced semi-automated analysis software have reported the association of retinopathy signs with hypertensive end-organ damage includes the following: stroke, dementia, and coronary heart disease. Although yielding much useful information, the vessels assessed from fundus photographs remain limited to the larger retinal arterioles and venules, and abnormalities observed may not be that of the earliest changes. Newer imaging modalities such as optical coherence tomography angiography and adaptive optics technology, which allow a greater precision in the structural quantification of retinal vessels, including capillaries, may facilitate the assessment and management of these patients. The advent of deep learning technology has also augmented the utility of fundus photographs to help create diagnostic and risk stratification systems. Particularly, deep learning systems have been shown in several large studies to be able to predict multiple cardiovascular risk factors, major adverse cardiovascular events within 5 years, and presence of coronary artery calcium, from fundus photographs alone. In the future, combining deep learning systems with the imaging precision offered by optical coherence tomography angiography and adaptive optics could pave way for systems that are able to predict adverse clinical outcomes even more accurately.Agency for Science, Technology and Research (A*STAR)Nanyang Technological UniversityNational Medical Research Council (NMRC)National Research Foundation (NRF)Published versionSupported by the grants from the National Medical Research Council (CG/C010A/ 2017; OFIRG/0048/2017; OFLCG/004c/2018; TA/MOH-000249-00/2018; and MOH-OFIRG20nov-001), National Research Foundation Singapore (NRF-CRP24-2020-0001 and NRF2019-THE002-0006), A*STAR (A20H4b0141), the Singapore Eye Research Institute & Nanyang Technological University (SERI-NTU Advanced Ocular Engineering (STANCE) Program) the Duke-NUS Medical School (Duke-NUS-KP(Coll)/2018/0009A), the SERI-Lee Foundation (LF1019-1) Singapore

Novel approaches for imaging-based diagnosis of ocular surface disease

Imaging has become indispensable in the diagnosis and management of diseases in the posterior part of the eye. In recent years, imaging techniques for the anterior segment are also gaining importance and are nowadays routinely used in clinical practice. Ocular surface disease is often synonymous with dry eye disease, but also refers to other conditions of the ocular surface, such as Meibomian gland dysfunction or keratitis and conjunctivitis with different underlying causes, i.e., allergies or infections. Therefore, correct differential diagnosis and treatment of ocular surface diseases is crucial, for which imaging can be a helpful tool. A variety of imaging techniques have been introduced to study the ocular surface, such as anterior segment optical coherence tomography, in vivo confocal microscopy, or non-contact meibography. The present review provides an overview on how these techniques can be used in the diagnosis and management of ocular surface disease and compares them to clinical standard methods such as slit lamp examination or staining of the cornea or conjunctiva. Although being more cost-intensive in the short term, in the long term, the use of ocular imaging can lead to more individualized diagnoses and treatment decisions, which in turn are beneficial for affected patients as well as for the healthcare system. In addition, imaging is more objective and provides good documentation, leading to an improvement in patient follow-up and education.Published versionThis research was funded by the Medical Scientific Fund of the Mayor of the City of Vienna, Project No. 17059

Line-scanning SD-OCT for in-vivo, non-contact, volumetric, cellular resolution imaging of the human cornea and limbus

In-vivo, non-contact, volumetric imaging of the cellular and sub-cellular structure of the human cornea and limbus with optical coherence tomography (OCT) is challenging due to involuntary eye motion that introduces both motion artifacts and blur in the OCT images. Here we present the design of a line-scanning (LS) spectral-domain (SD) optical coherence tomography system that combines 2 × 3 × 1.7 µm (x, y, z) resolution in biological tissue with an image acquisition rate of ∼2,500 fps, and demonstrate its ability to image in-vivo and without contact with the tissue surface, the cellular structure of the human anterior segment tissues. Volumetric LS-SD-OCT images acquired over a field-of-view (FOV) of 0.7 mm × 1.4 mm reveal fine morphological details in the healthy human cornea, such as epithelial and endothelial cells, sub-basal nerves, as well as the cellular structure of the limbal crypts, the palisades of Vogt (POVs) and the blood microvasculature of the human limbus. LS-SD-OCT is a promising technology that can assist ophthalmologists with the early diagnostics and optimal treatment planning of ocular diseases affecting the human anterior eye.Published versionCanada First Research Excellence Fund; Canadian Institutes of Health Research (446387); Natural Sciences and Engineering Research Council of Canada (312037)