Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure.

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 (TGFß) 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 TGFß pathway genes suggests that TM responses may reduce TGFß-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

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.

Retinal capillary oximetry with visible light optical coherence tomography.

Assessing oxygen saturation (sO2) remains challenging but is nonetheless necessary for understanding retinal metabolism. We and others previously achieved oximetry on major retinal vessels and measured the total retinal oxygen metabolic rate in rats using visible-light optical coherence tomography. Here we extend oximetry measurements to capillaries and investigate all three retinal vascular plexuses by amplifying and extracting the spectroscopic signal from each capillary segment under the guidance of optical coherence tomography (OCT) angiography. Using this approach, we measured capillary sO2 in the retinal circulation in rats, demonstrated reproducibility of the results, validated the measurements in superficial capillaries with known perfusion pathways, and determined sO2 responses to hypoxia and hyperoxia in the different retinal capillary beds. OCT capillary oximetry has the potential to provide new insights into the retinal circulation in the normal eye as well as in retinal vascular diseases.

Imaging retinal structures at cellular-level resolution by visible-light optical coherence tomography.

In vivo high-resolution images are the most direct way to understand retinal function and diseases. Here we report the use of visible-light optical coherence tomography with volumetric registration and averaging to achieve cellular-level retinal structural imaging in a rat eye, covering the entire depth of the retina. Vitreous fibers, nerve fiber bundles, and vasculature were clearly revealed, as well as at least three laminar sublayers in the inner plexiform layer. We also successfully visualized ganglion cell somas in the ganglion cell layer, cells in the inner nuclear layer, and photoreceptors in the outer nuclear layer and ellipsoid zone. This technique provides, to the best of our knowledge, a new means to visualize the retina in vivo at a cellular resolution and may enable detection or discovery of cellular neuronal biomarkers to help better diagnose ocular disease.

Monitoring retinal responses to acute intraocular pressure elevation in rats with visible light optical coherence tomography.

Elevated intraocular pressure (IOP) is an important risk factor for glaucoma. However, the role of IOP in glaucoma progression, as well as retinal physiology in general, remains incompletely understood. We demonstrate the use of visible light optical coherence tomography to measure retinal responses to acute IOP elevation in Brown Norway rats. We monitored retinal responses in reflectivity, angiography, blood flow, oxygen saturation ( sO 2 ), and oxygen metabolism over a range of IOP from 10 to 100 mmHg. As IOP was elevated, nerve fiber layer reflectivity was found to decrease. Vascular perfusion in the three retinal capillary plexuses remained steady until IOP exceeded 70 mmHg and arterial flow was noted to reverse periodically at high IOPs. However, a significant drop in total retinal blood flow was observed first at 40 mmHg. As IOP increased, the venous sO 2 demonstrated a gradual decrease despite steady arterial sO 2 , which is consistent with increased arterial-venous oxygen extraction across the retinal capillary beds. Calculated total retinal oxygen metabolism was steady, reflecting balanced responses of blood flow and oxygen extraction, until IOP exceeded 40 mmHg, and fell to 0 at 70 and 80 mmHg. Above this, measurements were unattainable. All measurements reverted to baseline when the IOP was returned to 10 mmHg, indicating good recovery following acute pressure challenge. These results demonstrate the ability of this system to monitor retinal oxygen metabolism noninvasively and how it can help us understand retinal responses to elevated IOP.

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.

Real-time cross-sectional and en face OCT angiography guiding high-quality scan acquisition.

Defocusing, vignetting, and bulk motion degrade the image quality of optical coherence tomography angiography (OCTA) more significantly than structural OCT. The assessment of focus, alignment conditions, and stability of imaging subjects in commercially available OCTA systems are currently based on OCT signal quality alone, without knowledge of OCTA signal quality. This results in low yield rates for further quantification. In this Letter, we developed a novel OCTA platform based on a graphics processing unit (GPU) for a real-time, high refresh rate, B-san-by-B-scan split-spectrum amplitude-decorrelation angiography. The GPU provides a real-time display of both cross-sectional and en face images to assist operators during scan acquisition and ensure OCTA scan quality.

Automated phase unwrapping in Doppler optical coherence tomography.

Phase wrapping is a crucial issue in Doppler optical coherence tomography (OCT) and restricts its automatic implementation for clinical applications that quantify total retinal blood flow. We propose an automated phase-unwrapping technique that takes advantage of the parabolic profile of blood flow velocity in vessels. Instead of inspecting the phase shift manually, the algorithm calculates the gradient magnitude of the phase shift on the cross-sectional image and automatically detects the presence of phase wrapping. The voxels affected by phase wrapping are corrected according to the determined flow direction adjacent to the vessel walls. We validated this technique in the rodent retina using a prototype visible-light OCT and in the human retina with a commercial infrared OCT system. We believe this signal processing method may well accelerate clinical applications of Doppler OCT in ophthalmology.

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.

Evaluating changes of blood flow in retina, choroid, and outer choroid in rats in response to elevated intraocular pressure by 1300 nm swept-source OCT.

We report the development of a 1300 nm swept-source optical coherence tomography (SS-OCT) system specifically designed to perform OCT imaging and optical microangiography (OMAG) in rat eyes in vivo and its use in evaluating the effects of intraocular pressure (IOP) elevation on ocular circulation. The swept laser is operated in single longitude mode with a 90 nm bandwidth centered at 1300 nm and 200 kHz A-line rate, providing remarkable sensitivity fall-off performance along the imaging depth, a larger field of view of 2.5 × 2.5 mm2 (approximately 35°), and more time-efficient imaging acquisition. The advantage of the SS-OCT/OMAG is highlighted by an increased imaging depth of the entire posterior thickness of optic nerve head (ONH) and its surrounding vascular anatomy, to include, for the first time in vivo, the vasculature at the scleral opening, allowing visualization of the circle of Zinn-Haller and posterior ciliary arteries (PCAs). Furthermore, the capillary-level resolution angiograms achieved at the retinal and choroidal layers over a larger field of view enable a significantly improved quantification of the response of vascular area density (VAD) to elevated IOP. The results indicate that reduction in perfusion of the choroid in response to elevated IOP is delayed compared to that seen in the retina; while choroidal VAD doesn't reach 50% of baseline until ~70 mmHg, the same effect is seen for the retinal VAD at ~60 mmHg. The superior image quality offered by SS-OCT may allow more comprehensive investigation of IOP-related ocular perfusion changes and their pathological roles in glaucomatous optic nerve damage.

Rodent retinal circulation organization and oxygen metabolism revealed by visible-light optical coherence tomography.

Visible light optical coherence tomography (vis-OCT) is an emerging label-free and high-resolution 3-dimensional imaging technique that can provide retinal oximetry, angiography, and flowmetry in one modality. In this paper, we studied the organization of the arterial and venous retinal circulation in rats using vis-OCT. Arterioles were found predominantly in the superficial vascular plexus whereas veins tended to drain capillaries from the deep capillary plexus. After that, we determined the oxygen metabolic rate supported by retinal microcirculation by combining retinal vessel oxygen saturation and blood flow measurements. The ability to visualize and monitor retinal circulation organization and oxygen metabolism by vis-OCT may provide new opportunities for understanding the pathology of ocular diseases.

Automated spectroscopic retinal oximetry with visible-light optical coherence tomography.

Accurate, quantitative assessment of retinal blood oxygen saturation (sO2 ) may provide a useful early indicator of pathophysiology in several ocular diseases. Here, with visible-light optical coherence tomography (OCT), we demonstrate an automated spectroscopic retinal oximetry algorithm to measure the sO2 within the retinal arteries (A-sO2 ) and veins (V-sO2 ) in rats by automatically detecting the vascular posterior boundary on cross-sectional structural OCT. The algorithm was validated in vitro with flow phantoms and in vivo in rats by comparing the sO2 results, respectively, to those obtained using a blood gas analyzer and pulse oximetry. We also investigated the response of oxygen extraction (A-V sO2 ), including inter-session reproducibility, at different inhaled oxygen concentrations.

Fast and robust standard-deviation-based method for bulk motion compensation in phase-based functional OCT.

Phase-based optical coherence tomography (OCT), such as OCT angiography (OCTA) and Doppler OCT, is sensitive to the confounding phase shift introduced by subject bulk motion. Traditional bulk motion compensation methods are limited by their accuracy and computing cost-effectiveness. In this Letter, to the best of our knowledge, we present a novel bulk motion compensation method for phase-based functional OCT. Bulk motion associated phase shift can be directly derived by solving its equation using a standard deviation of phase-based OCTA and Doppler OCT flow signals. This method was evaluated on rodent retinal images acquired by a prototype visible light OCT and human retinal images acquired by a commercial system. The image quality and computational speed were significantly improved, compared to two conventional phase compensation methods.

In Vivo Small Molecule Delivery to the Optic Nerve in a Rodent Model.

Small molecule delivery to the optic nerve would allow for exploration of molecular and cellular pathways involved in normal physiology and optic neuropathies such as glaucoma, and provide a tool for screening therapeutics in animal models. We report a novel surgical method for small molecule drug delivery to the optic nerve head (ONH) in a rodent model. In proof-of-principle experiments, we delivered cytochalasin D (Cyt D; a filamentous actin inhibitor) to the junction of the superior optic nerve and globe in rats to target the actin-rich astrocytic cytoskeleton of the ONH. Cyt D delivery was quantified by liquid chromatography and mass spectrometry of isolated optic nerve tissue. One day after Cyt D delivery, anterior ONH filamentous actin bundle content was significantly reduced as assessed by fluorescent-tagged phalloidin labeling, relative to sham delivery. Anterior ONH nuclear counts and axon-specific beta-3 tubulin levels, as well as peripapillary retinal ganglion cell layer nuclear counts were not significantly altered after Cyt D delivery relative to sham delivery. Lastly, the surgical delivery technique caused minimal observable axon degeneration up to 10 days post-surgery. This small molecule delivery technique provides a new approach to studying optic neuropathies in in vivo rodent models.

The effect of age on the response of retinal capillary filling to changes in intraocular pressure measured by optical coherence tomography angiography.

PURPOSE: To compare the effect of elevated intraocular pressure (IOP) on retinal capillary filling in elderly vs adult rats using optical coherence tomography angiography (OCTA). METHODS: The IOP of elderly (24-month-old, N=12) and adult (6-8month-old, N=10) Brown Norway rats was elevated in 10mmHg increments from 10 to 100mmHg. At each IOP level, 3D OCT data were captured using an optical microangiography (OMAG) scanning protocol and then post-processed to obtain both structural and vascular images. Mean arterial blood pressure (MAP), respiratory rate, pulse and blood oxygen saturation were monitored non-invasively throughout each experiment. Ocular perfusion pressure (OPP) was calculated as the difference between MAP for each animal and IOP at each level. The capillary filling index (CFI), defined as the ratio of area occupied by functional capillary vessels to the total scan area but excluding relatively large vessels of >30µm, was calculated at each IOP level and analyzed using the OCTA angiograms. Relative CFI vs IOP was plotted for the group means. CFI vs OPP was plotted for every animal in each group and data from all animals were combined in a CFI vs OPP scatter plot comparing the two groups. RESULTS: The MAP in adult animals was 108±5mmHg (mean±SD), whereas this value in the elderly was 99±5mmHg. All other physiologic parameters for both age groups were uniform and stable. In elderly animals, significant reduction of the CFI was first noted at IOP 40mmHg, as opposed to 60mmHg in adult animals. Individual assessment of CFI as a function of OPP for adult animals revealed a consistent plateau until OPP reached between 40 and 60mmHg. Elderly individuals demonstrated greater variability, with many showing a beginning of gradual deterioration of CFI at an OPP as high as 80mmHg. Overall comparison of CFI vs OPP between the two groups was not statistically significant. CONCLUSIONS: Compared to adults, some, but not all, elderly animals demonstrate a more rapid deterioration of CFI vs OPP. This suggests a reduced autoregulatory capacity that may contribute to increased glaucoma susceptibility in some older individuals. This variability must be considered when studying the relationship between IOP, ocular perfusion and glaucoma in elderly animal models.

Hypertonic Saline Injection Model of Experimental Glaucoma in Rats.

A reliable method of creating chronic elevation of intraocular pressure (IOP) in rodents is an important tool in reproducing and studying the mechanisms of optic nerve injury that occur in glaucoma. In addition, such a model could provide a valuable method for testing potential neuroprotective treatments. This paper outlines the basic methods for producing obstruction of aqueous humor outflow and IOP elevation by injecting hypertonic saline (a sclerosant) into the aqueous outflow pathway. This is one of several rodent glaucoma models in use today. In this method, a plastic ring is placed around the equator of the eye to restrict injected saline to the limbus. By inserting a small glass microneedle in an aqueous outflow vein in the episclera and injecting hypertonic saline toward the limbus, the saline is forced into Schlemm's canal and across the trabecular meshwork. The resultant inflammation and scarring of the anterior chamber angle occurs gradually, resulting in a rise in IOP after approximately 1 week. This article will describe the equipment necessary for producing this model and the steps of the technique itself.

Angiographic and structural imaging using high axial resolution fiber-based visible-light OCT.

Optical coherence tomography using visible-light sources can increase the axial resolution without the need for broader spectral bandwidth. Here, a high-resolution, fiber-based, visible-light optical coherence tomography system is built and used to image normal retina in rats and blood vessels in chicken embryo. In the rat retina, accurate segmentation of retinal layer boundaries and quantification of layer thicknesses are accomplished. Furthermore, three distinct capillary plexuses in the retina and the choriocapillaris are identified and the characteristic pattern of the nerve fiber layer thickness in rats is revealed. In the chicken embryo model, the microvascular network and a venous bifurcation are examined and the ability to identify and segment large vessel walls is demonstrated.