In vivo volumetric imaging of human retinal circulation with phase-variance optical coherence tomography

We present in vivo volumetric images of human retinal micro-circulation using Fourier-domain optical coherence tomography (Fd-OCT) with the phase-variance based motion contrast method. Currently fundus fluorescein angiography (FA) is the standard technique in clinical settings for visualizing blood circulation of the retina. High contrast imaging of retinal vasculature is achieved by injection of a fluorescein dye into the systemic circulation. We previously reported phase-variance optical coherence tomography (pvOCT) as an alternative and non-invasive technique to image human retinal capillaries. In contrast to FA, pvOCT allows not only noninvasive visualization of a two-dimensional retinal perfusion map but also volumetric morphology of retinal microvasculature with high sensitivity. In this paper we report high-speed acquisition at 125 kHz A-scans with pvOCT to reduce motion artifacts and increase the scanning area when compared with previous reports. Two scanning schemes with different sampling densities and scanning areas are evaluated to find optimal parameters for high acquisition speed in vivo imaging. In order to evaluate this technique, we compare pvOCT capillary imaging at 3x3 mm^2 and 1.5x1.5 mm^2 with fundus FA for a normal human subject. Additionally, a volumetric view of retinal capillaries and a stitched image acquired with ten 3x3 mm^2 pvOCT sub-volumes are presented. Visualization of retinal vasculature with pvOCT has potential for diagnosis of retinal vascular diseases

Vascular changes in diabetic retinopathy-a longitudinal study in the Nile rat.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies

Depth-resolved rhodopsin molecular contrast imaging for functional assessment of photoreceptors

Rhodopsin, the light-sensing molecule in the outer segments of rod photoreceptors, is responsible for converting light into neuronal signals in a process known as phototransduction. Rhodopsin is thus a functional biomarker for rod photoreceptors. Here we report a novel technology based on visible-light optical coherence tomography (VIS-OCT) for in vivo molecular imaging of rhodopsin. The depth resolution of OCT allows the visualization of the location where the change of optical absorption occurs and provides a potentially accurate assessment of rhodopsin content by segmentation of the image at the location. Rhodopsin OCT can be used to quantitatively image rhodopsin distribution and thus assess the distribution of functional rod photoreceptors in the retina. Rhodopsin OCT can bring significant impact into ophthalmic clinics by providing a tool for the diagnosis and severity assessment of a variety of retinal conditions

Detailed Visual Cortical Responses Generated by Retinal Sheet Transplants in Rats with Severe Retinal Degeneration.

To combat retinal degeneration, healthy fetal retinal sheets have been successfully transplanted into both rodent models and humans, with synaptic connectivity between transplant and degenerated host retina having been confirmed. In rodent studies, transplants have been shown to restore responses to flashes of light in a region of the superior colliculus corresponding to the location of the transplant in the host retina. To determine the quality and detail of visual information provided by the transplant, visual responsivity was studied here at the level of visual cortex where higher visual perception is processed. For our model, we used the transgenic Rho-S334ter line-3 rat (both sexes), which loses photoreceptors at an early age and is effectively blind at postnatal day 30. These rats received fetal retinal sheet transplants in one eye between 24 and 40 d of age. Three to 10 months following surgery, visually responsive neurons were found in regions of primary visual cortex matching the transplanted region of the retina that were as highly selective as normal rat to stimulus orientation, size, contrast, and spatial and temporal frequencies. Conversely, we found that selective response properties were largely absent in nontransplanted line-3 rats. Our data show that fetal retinal sheet transplants can result in remarkably normal visual function in visual cortex of rats with a degenerated host retina and represents a critical step toward developing an effective remedy for the visually impaired human population.SIGNIFICANCE STATEMENT Age-related macular degeneration and retinitis pigmentosa lead to profound vision loss in millions of people worldwide. Many patients lose both retinal pigment epithelium and photoreceptors. Hence, there is a great demand for the development of efficient techniques that allow for long-term vision restoration. In this study, we transplanted dissected fetal retinal sheets, which can differentiate into photoreceptors and integrate with the host retina of rats with severe retinal degeneration. Remarkably, we show that transplants generated visual responses in cortex similar in quality to normal rats. Furthermore, transplants preserved connectivity within visual cortex and the retinal relay from the lateral geniculate nucleus to visual cortex, supporting their potential application in curing vision loss associated with retinal degeneration

Optical Coherence Photoacoustic Microscopy (OC-PAM) for Multimodal Imaging

Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM) are two noninvasive, high-resolution, three-dimensional, biomedical imaging modalities based on different contrast mechanisms. OCT detects the light backscattered from a biological sample either in the time or spectral domain using an interferometer to form an image. PAM is sensitive to optical absorption by detecting the light-induced acoustic waves to form an image. Due to their complementary contrast mechanisms, OCT and PAM are suitable for being combined to achieve multimodal imaging. In this dissertation, an optical coherence photoacoustic microscopy (OC-PAM) system was developed for in vivo multimodal retinal imaging with a pulsed broadband NIR light source. To test the capabilities of the system on multimodal ophthalmic imaging, the retina of pigmented rats was imaged. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of melanin in the retina since the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye. By using the pulsed broadband light source, the OCT image quality highly depends on the pulse-to-pulse stability of the light source without averaging. In addition, laser safety is always a concern for in vivo applications, especially for eye imaging with a pulsed light source. Therefore, a continuous wave (CW) light source is desired for OC-PAM applications. An OC-PAM system using an intensity-modulated CW superluminescent diode was then developed. The system was tested for multimodal imaging the vasculature of a mouse ear in vivo by using Gold Nanorods (GNRs) as contrast agent for PAM, as well as excised porcine eyes ex vivo. Since the quantitative information of the optical properties extracted from the proposed NIR OC-PAM system is potentially able to provide a unique technique to evaluate the existence of melanin and lipofuscin specifically, a phantom study has been conducted and the relationship between image intensity of OCT and PAM was interpreted to represent the relationship between the optical scattering property and optical absorption property. It will be strong evidence for practical application of the proposed NIR OC-PAM system

Doppler holography for ophthalmology

A comprehensive assessment of retinal health demands reliable and precise methods to measure localized blood perfusion. Despite considerable advancements in imaging techniques, such as indocyanine green and fluorescein angiography, along with optical coherence tomography angiography, their capacity to monitor blood flow dynamics across the cardiac cycle faces significant limitations. For more effective care of those with ocular conditions, innovating new approaches is paramount. Doppler holography, an emerging non-invasive optical imaging technique, meets this challenge by offering high temporal resolution imaging of retinal and choroidal blood flow. Now a burgeoning interdisciplinary research field, Doppler holography intertwines functional optical imaging system design, high-performance computing, and clinical investigation. Through collaborative efforts among universities, industry partners, and ophthalmic clinics, a network for its advancement and application is taking shape. This endeavor promises to propel the discovery of novel functional biomarkers, transforming the diagnosis and treatment of retinal diseases, refining disease severity categorization, and enhancing therapeutic monitoring-ultimately leading to improved healthcare outcomes.Comment: 60 pages. Accreditation to Supervise Research (Habilitation), CNRS - ESPCI Paris - PSL (Paris Sciences & Lettres University

Novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis

We present a novel non-invasive and non-contact system for reflex-free retinal imaging and dynamic retinal vessel analysis in the rat. Theoretical analysis was performed prior to development of the new optical design, taking into account the optical properties of the rat eye and its specific illumination and imaging requirements. A novel optical model of the rat eye was developed for use with standard optical design software, facilitating both sequential and non-sequential modes. A retinal camera for the rat was constructed using standard optical and mechanical components. The addition of a customized illumination unit and existing standard software enabled dynamic vessel analysis. Seven-minute in-vivo vessel diameter recordings performed on 9 Brown-Norway rats showed stable readings. On average, the coefficient of variation was (1.1 ± 0.19) % for the arteries and (0.6 ± 0.08) % for the veins. The slope of the linear regression analysis was (0.56 ± 0.26) % for the arteries and (0.15 ± 0.27) % for the veins. In conclusion, the device can be used in basic studies of retinal vessel behavior

The Use of Preclinical Models to Improve the Treatment of Retinoblastoma

Rodent models play an essential role in the development of new chemotherapeutics and dosing regimes. It is often difficult to carryout a clinical study for pediatric cancers due to the small patient population. Retinoblastoma, a pediatric cancer of the eye, is one example of a pediatric cancer that can benefit from preclinical studies. Over the years various retinoblastoma rodent models have been developed used to test various combination of broad-spectrum systemic chemotherapy. It was found form these studies that the combination of topotecan and carboplatin was effective. However both drugs cause myelosuppression and therefore administrating both of these drugs systemically is not possible. An alternative effective therapy in the clinic was the use of a subconjunctival administration. We thought if we could administer both drugs, one by systemic and one by a subconjunctival injection, perhaps we could decrease the systemic exposure with good tumor response. Detailed pharmacokinetic studies were conducted to understand the subconjunctival injections of topotecan and carboplatin. It was found that both drugs could successfully penetrate the eye and increase drug exposure. In addition, in the presence of a tumor, drug exposure to the vitreous was greater. Additionally comparative pharmacodynamic studies combining topotecan subconjunctival injection with carboplatin intraperitoneal or carboplatin subconjunctival injection with topotecan intraperitoneal were conducted. The tumor response, systemic toxicity and local toxicity were studied. There was tumor response in both combinations and no ocular toxicity was seen with a single eye subconjunctival injection for either drug. However, rats that received the combination with topotecan subconjunctival injection and carboplatin intraperitoneal experienced great toxicity and morbidity. The data and observations suggest the death is due to dehydration. Therefore it was concluded that the alternative combination was better. The above data suggested an appropriate drug combination and schedule for a preclinical study. However, the noninvasive methods to follow tumor progression and choosing the correct genetic model needed to be determined. This was essential to ensure the preclinical study could be easily translated for future clinical studies. A characterization study of five modalities, retina camera, optomotry, tonometer, ultrasound and MRI, was done with retinoblastoma mice. We determined the feasibility of each technique. It was found that the retina camera could detect the tumor the earliest in a high throughput manner. Additionally, the tonometer and optomotry machines could assess ocular health. While the ultrasound and MRI could image the eye and tumor in one field of view, MRI could capture the posterior chamber in more detail along with the extraocular space. With different software programs, the tumor to eye ratio volume measurement were determined and compared to the gold standard of enucleation, embedding, serial sectioning and hand tracing. It was found that there was a better correlation between the ultrasound and hand tracing histological sections. Concurrently, the tumor progression of six different genotypes was assessed. The tumor progression depended on the number and different genes deleted. Additionally, based on genotypes, it was determined there was not a strong genotypic trend in the increase in IOP or the loss of vision. From the studies of tumor progression we have learn more about the influence of genes on tumor progression, which will benefit additional genetic studies in mouse model systems and human tumors