Stanislava Fialová

Melanin Pigmentation in Rat Eyes: In Vivo Imaging by Polarization-Sensitive Optical Coherence Tomography and Comparison to Histology.

PURPOSE The purpose of this study was to demonstrate polarization-sensitive optical coherence tomography (PS-OCT) for imaging pigmented structures in the posterior eye segments of albino and pigmented rats and to correlate depolarization contrast of the retinal pigment epithelium (RPE) and choroid in in vivo PS-OCT to melanin pigmentation detected in postmortem histologic serial sections. METHODS In vivo three-dimensional PS-OCT imaging was performed in adult albino and pigmented rat eyes at 70-kHz A-line rate. Degree of polarization uniformity (DOPU) fundus maps and radial DOPU profiles were generated. Postmortem histomorphologic analysis was performed in order to investigate melanin pigmentation of the RPE and choroid. Fundus pigmentation maps were extracted from histologic serial sections. Pigmentation profiles were correlated to DOPU profiles of the same eyes. RESULTS Strong depolarization was found in the RPE/choroid complex of pigmented rats, whereas the same structures exhibited uniform polarization in albino rats. The difference between the depolarization characteristics between albino and pigmented animals was statistically significant. In the fundus pigmentation maps, optical pigment density was zero in albino rat eyes. In pigmented rat eyes, a strong negative correlation between optical pigment density and DOPU was observed. CONCLUSIONS This in vivo and ex vivo investigation of posterior rat eyes indicates that melanin is the cause of depolarization in retinal PS-OCT images. It further demonstrates that melanin pigmentation in the RPE and choroid can be quantified via depolarization imaging and therefore suggests that PS-OCT is a useful tool for the noninvasive quantitative assessment of pigmentary changes in vision-threatening diseases such as age-related macular degeneration.

Peripapillary rat sclera investigated in vivo with polarization-sensitive optical coherence tomography.

PURPOSE To demonstrate polarization-sensitive (PS) optical coherence tomography (OCT) for noninvasive, volumetric, and quantitative imaging of the birefringent properties of the peripapillary rat sclera; to compare the findings from PS-OCT images to state-of-the-art histomorphometric analysis of the same tissues. METHODS A high-speed PS-OCT prototype operating at 840 nm was modified for imaging the rat eye. Densely sampled PS-OCT raster scans covering an area of ~1.5 × 1.5 mm centered at the papilla were acquired in the eyes of anesthetized male Sprague-Dawley rats. Cross-sectional PS-OCT images were computed, and fundus maps displaying the birefringent properties of the sclera were analyzed. Postmortem histomorphologic analysis was performed. RESULTS Polarization-sensitive OCT enables visualization of the polarization properties of ocular tissues in vivo. The birefringent characteristics of the rat sclera were quantitatively assessed. Scleral birefringence formed a donut-shaped pattern around the papilla with significantly increased values of 0.703 ± 0.089°/μm (i.e., 1.64 × 10(-3) ± 0.2 × 10(-3); mean ± standard deviation) and 0.721 ± 0.084°/μm (i.e., 1.68 × 10(-3) ± 0.2 × 10(-3)) at an eccentricity of 0.4 mm for the left and right eyes, respectively. Birefringent axis orientation maps revealed a ring-shaped distribution around the optic nerve. Postmortem PS-OCT micrographs provided access to retinal and scleral microstructure and were compared to standard histomorphologic analysis. CONCLUSIONS Polarization-sensitive OCT enables quantitative imaging of tissue polarization properties in addition to conventional OCT imaging based on reflectivity. In the rat sclera, in vivo PS-OCT provides access to volumetric mapping of birefringence. Scleral birefringence is associated with microstructural tissue organization. Therefore, PS-OCT should prove a valuable tool for the in vivo investigation of peripapillary sclera in glaucoma.

Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model

We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.

Polarization properties of single layers in the posterior eyes of mice and rats investigated using high resolution polarization sensitive optical coherence tomography

We present a high resolution polarization sensitive optical coherence tomography (PS-OCT) system for ocular imaging in rodents. The system operates at 840 nm and uses a broadband superluminescent diode providing an axial resolution of 5.1 µm in air. PS-OCT data was acquired at 83 kHz A-scan rate by two identical custom-made spectrometers for orthogonal polarization states. Pigmented (Brown Norway, Long Evans) and non-pigmented (Sprague Dawley) rats as well as pigmented mice (C57BL/6) were imaged. Melanin pigment related depolarization was analyzed in the retinal pigment epithelium (RPE) and choroid of these animals using the degree of polarization uniformity (DOPU). For all rat strains, significant differences between RPE and choroidal depolarization were observed. In contrast, DOPU characteristics of RPE and choroid were similar for C57BL/6 mice. Moreover, the depolarization within the same tissue type varied significantly between different rodent strains. Retinal nerve fiber layer thickness, phase retardation, and birefringence were mapped and quantitatively measured in Long Evans rats in vivo for the first time. In a circumpapillary annulus, retinal nerve fiber layer birefringence amounted to 0.16°/µm ± 0.02°/µm and 0.17°/µm ± 0.01°/µm for the left and right eyes, respectively.

Posterior rat eye during acute intraocular pressure elevation studied using polarization sensitive optical coherence tomography.

Polarization sensitive optical coherence tomography (PS-OCT) operating at 840 nm with axial resolution of 3.8 µm in tissue was used for investigating the posterior rat eye during an acute intraocular pressure (IOP) increase experiment. IOP was elevated in the eyes of anesthetized Sprague Dawley rats by cannulation of the anterior chamber. Three dimensional PS-OCT data sets were acquired at IOP levels between 14 mmHg and 105 mmHg. Maps of scleral birefringence, retinal nerve fiber layer (RNFL) retardation and relative RNFL/retina reflectivity were generated in the peripapillary area and quantitatively analyzed. All investigated parameters showed a substantial correlation with IOP. In the low IOP range of 14-45 mmHg only scleral birefringence showed statistically significant correlation. The polarization changes observed in the PS-OCT imaging study presented in this work suggest that birefringence of the sclera may be a promising IOP-related parameter to investigate.

Visible light spectral domain optical coherence microscopy system for ex vivo imaging

A visible light spectral domain optical coherence microscopy system operating in the wavelength range of 450-680 nm was developed. The resulting large wavelength range of 230 nm enabled an ultrahigh axial resolution of 0.88μm in tissue. The setup consisted of a Michelson interferometer combined with a homemade spectrometer with a spectral resolution of 0.03 nm. Scanning of 1 x 1 mm2 and 0.5 x 0.5 mm2 areas was performed by an integrated microelectromechanical mirror. After scanning the light beam is focused onto the tissue by a commercial objective with a 10 x magnification, resulting in a transverse resolution of 2 μm . Specification measurements showed that a -89 dB sensitivity with a 24 dB/mm roll-off could be achieved with the system. First of all the capabilities of the system were tested by investigating millimeter paper, tape and the USAF (US Air Force) 1951 resolution test target. Finally cerebral tissues from non-pathological and Alzheimers disease affected brains were investigated. The results showed that structures, such as white and gray matter, could be distinguished. Furthermore a first effort was made to differentiate Alzheimers disease from healthy brain tissue.

In Vivo Characterization of Spontaneous Retinal Neovascularization in the Mouse Eye by Multifunctional Optical Coherence Tomography

Purpose To investigate the early development of spontaneous retinal neovascularization in the murine retina by a multifunctional optical coherence tomography approach. To characterize involved tissue changes in vivo and describe structural and functional changes over time. Methods A multifunctional optical coherence tomography (OCT) system providing 3-fold contrast comprising reflectivity, polarization sensitivity, and OCT angiography (OCTA) was utilized to image very-low-density lipoprotein receptor (VLDLR) knockout mice. Baseline measurements were acquired as early as postnatal day 14 and a follow-up of neovascularization development was performed until the age of 3 months. Control mice were imaged accordingly and a multiparametric image analysis was performed to characterize different stages of pathologic vascular growth. Histology was conducted at the endpoint of the experiment. An interventional pilot experiment was conducted to investigate the effect of the anti-vascular endothelial growth factor (VEGF) agent aflibercept on the development of retinal neovascularization. Results Onset of neovascularization was imaged at baseline, and significant changes were encountered in the retina over time, including reduced retinal thickness, increase of lesion volume, migration of pigmented structures, and presence of abnormal blood flow in the outer retina. Multifunctional image contrast was correlated to ex vivo histology. Microscopic analysis of retinal flat mounts and cross-sectional samples confirmed the changes observed in in vivo structural and functional OCT images. Administration of an anti-VEGF agent resulted in a significantly reduced lesion volume. Conclusions Longitudinal, multifunctional OCT imaging of infant VLDLR-/- mouse retinas enabled a multiparametric, in vivo staging of neovascularization formation from before lesion onset until their manifestation.

High-resolution polarization sensitive OCT for ocular imaging in rodents

A new high-resolution polarization sensitive optical coherence tomography system was developed for imaging rodent retina. Various light-tissue interactions such as birefringence and depolarization can change the polarization state of light. In the eye, there are several tissues that have these properties, for example retinal pigment epithelium (depolarization) and sclera (birefringence). These layers play key roles in diseases like age-related macular degeneration or glaucoma. Animal models are an important component for understanding disease pathogenesis. The gold standard for the evaluation of preclinical experiments is histology, which is an invasive and terminal procedure. Since OCT is non-invasive, it has the potential to be an alternative to histology with the benefit of long-term study of the disease progression in the same animal. In this study, a superluminescent diode with spectrum width 100 nm and mean wavelength 840 nm is used as a light source in order to enable high axial resolution. Spectrometers are custom built to enable high imaging speed that allows acquiring 3D data sets with 1024x200x1536 voxels in 3.44 s. From the acquired data, images displaying phase retardation induced by birefringence and orientation of birefringent axis were calculated. In first measurements, we were able to identify the RPE-choroid complex (depolarization effect) and the sclera (strong birefringence) in the retina of Long-Evans and Sprague-Dawley rats. Our preliminary results demonstrate the feasibility of the system for high speed/resolution imaging of the rodent retina. This is useful for longitudinal studies of disease models of retinal disease in rats and mice

Ocular fundus pulsations within the posterior rat eye : chorioscleral motion and response to elevated intraocular pressure

A multi-functional optical coherence tomography (OCT) approach is presented to determine ocular fundus pulsations as an axial displacement between the retina and the chorioscleral complex in the albino rat eye. By combining optical coherence elastography and OCT angiography (OCTA), we measure subtle deformations in the nanometer range within the eye and simultaneously map retinal and choroidal perfusion. The conventional OCT reflectivity contrast serves as a backbone to segment the retina and to define several slabs which are subsequently used for quantitative ocular pulsation measurements as well as for a qualitative exploration of the multi-functional OCT image data. The proposed concept is applied in healthy albino rats as well as in rats under acute elevation of the intraocular pressure (IOP). The evaluation of this experiment revealed an increased pulsatility and deformation between the retinal and chorioscleral complex while increasing the IOP level from 15 mmHg to 65 mmHg. At IOP levels exceeding 65 mmHg, the pulsatility decreased significantly and retinal as well as choroidal perfusion vanished in OCTA. Furthermore, the evaluation of the multi-parametric experiment revealed a spatial correlation between fundus pulsatility and choroidal blood flow. This indicates that the assessed pulsatility may be a valuable parameter describing the choroidal perfusion.

Multi-functional optical coherence tomography imaging of spontaneous neovascularization in the mouse retina

A multi-functional OCT approach is used to identify different tissue types during the early development of spontaneous neovascularizations in the mouse retina based on their intrinsic optical properties.