Search

Purpose: The purpose of this study was to determine posture-induced changes in arterial blood pressure (ABP), intraocular pressure (IOP), orbital pressure (Porb), intracranial pressure (ICP), and jugular vein pressure (JVP) at various tilt angles in an in vivo pig., Methods: Anesthetized and ventilated pigs (n = 8) were placed prone on a tiltable operating table. ABP, IOP, Porb, ICP, and JVP were monitored while the table was tilted at various angles between 15 degrees head up tilt (HUT) and 25 degrees head down tilt (HDT) either in stepwise changes (5 degrees per step) or continuously. The mean pressure was calculated from digitized pressure waveforms from each compartment. For stepwise changes in tilt angle the pressures were plotted as a function of tilt angle. For continuous tilt changes, the pressures were plotted as a function of time., Results: In the case of stepwise changes, ABP remained relatively stable whilst IOP, Porb, ICP, and JVP demonstrated significant differences between most angles (typically P < 0.0001). The difference was greatest for IOP (P < 0.0001) where the average IOP increased from 13.1 ± 1.23 mm Hg at 15 degrees HUT to 46.3 ± 2.03 mm Hg at 25 degrees HDT. The relationship between pressure and tilt angle was almost linear for ICP and JVP, and sigmoidal for IOP and Porb. Interestingly, the effect of changes in tilt angle occurred very rapidly, within a few seconds., Conclusions: Our results in a pig model demonstrate that changes in posture (tilt angle) induce rapid changes in IOP, Porb, ICP, and JVP, with IOP affected most severely.

Background: There are six different formulae for estimating mean arterial pressure (MAP) from systolic and diastolic pressure readings. This study is to determine the optimum formula for calculating MAP when compared to the gold standard approach, which is the area under the curve of an invasively measured pulse waveform divided by the cardiac cycle duration., Methods: Eight live pigs were used as the experimental model for the invasive measurement of femoral artery pressure (AP) by a fluid filled catheter connected with a pressure transducer. In addition, intraocular pressure (IOP) and jugular vein pressure (JVP) were also recorded. The mean pressure (MP) was calculated from digital waveforms sampled at 1,000 points per second with the six formulae and area method for AP, IOP and JVP., Results: The absolute mean difference between the area MAP and each formula's MAP ranged from 0.98 to 3.23 mm Hg. Our study also found that even under physiological conditions, area MAP can vary between successive pulses by up to 5 mm Hg. For mean IOP and JVP, the mean difference between a formula's MP and the area method's was less than 1 mm Hg for most formulae. With the pooled data, there was excellent agreement amongst all formulae for MAP with the intra-class correlation coefficient (ICC) ranging from 0.97 to 0.99, while the ICC of most formulae for IOP and JVP was 1.0., Conclusions: Our study suggests that all current formulae are adequate for estimating MAP, though some formulae are not suitable for mean IOP and JVP., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd.)

An adequate blood supply to meet the energy demands is essential for any tissue, particularly for high energy demand tissues such as the retina. A critical question is: How is the dynamic match between neuronal demands and blood supply achieved? We present a quantitative assessment of temporal and spatial variations in perfusion in the macular capillary network in 10 healthy human subjects using a non-invasive and label-free imaging technique. The assessment is based on the calculation of the coefficient of variation (CoV) of the perfusion signal from arterioles, venules and capillaries from a sequence of optical coherence tomography angiography images centred on the fovea. Significant heterogeneity of the spatial and temporal variation was found within arterioles, venules and capillary networks. The CoV values of the capillaries and smallest vessels were significantly higher than that in the larger vessels. Our results demonstrate the presence of significant heterogeneity of spatial and temporal variation within each element of the macular microvasculature, particularly in the capillaries and finer vessels. Our findings suggest that the dynamic match between neuronal demands and blood supply is achieved by frequent alteration of local blood flow evidenced by capillary perfusion variations both spatially and temporally in the macular region., (© 2023. The Author(s).)

We have previously reported that porcine retinal veins can be contracted by vasoactive factors such as endothelin-1, but it is still unknown which cells play the major role in such contraction responses. This study seeks to confirm whether retinal vein endothelial cells play a significant role in the endothelin-1 induced contraction of porcine retinal veins. This is a novel study which provides confirmation of the endothelial cells' ability to contract retinal veins using a live vessel preparation. Retinal veins were isolated from porcine retina and cannulated for perfusion. The vessels were exposed to extraluminal delivery of endothelin-1 (10 -8  M) and change in vessel diameter recorded automatically every 2 s. A phase contrast objective lens was also used to capture images of the endothelial cell morphometries. The length, width, area, and perimeter were assessed. In addition, vein histology and immuno-labeling for contractile proteins was performed. With 10 -8  M endothelin-1 contractions to 63.6% of baseline were seen. The polygonal shape of the endothelial cells under normal tone became spindle-like after contraction. The area, width, perimeter and length were significantly reduced by 54.8%, 48.1%, 28.5% and 10.5% respectively. Three contractile proteins, myosin, calponin and alpha-SMA were found in retinal vein endothelial cells. Retinal vein endothelial cells contain contractile proteins and can be contracted by endothelin-1 administration. Such contractile capability may be important in regulating retinal perfusion but could also be a factor in the pathogenesis of retinal vascular diseases such as retinal vein occlusion. As far as we are aware, this is the first study on living isolated veins to confirm that endothelial cells contribute to the endothelin-1 induced contraction., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Endothelium phenotype is known to be closely associated with flow shear stress. This study is to determine the topographic distribution of endothelial cells and the phenotype of different quadrants and regions of Schlemm's canal using human donor eyes. This study infers differences in flow dynamics based on cell shape and intracellular structure. The Schlemm's canal from 15 human donor eyes were either perfusion labelled using silver stain or dissected for float labeling with Phalloidin to enable visualization of endothelial cell border and intracellular structure. Data were acquired for endothelial cells from the outer and inner wall of Schlemm's canal and grouped according to quadrant of origin. Measurements included endothelial cell length, width, area, and aspect ratio and compared between quadrants. Endothelial cells are mostly spindle-shape and the cell size on the outer wall are larger and longer than those from the inner wall. Significant differences in endothelial cell size and shape were seen in different quadrants. The endothelial cells have varied shapes and orientations close to large ostia in the outer wall and remarkably long endothelial cells were found in the walls of collector channels. F-actin aggregation was found at all endothelial cell borders, and inside some of the endothelial cytoplasm. The presence of various spindle shapes, significant phenotype heterogeneity and F-actin aggregation of endothelial cells indicates aqueous humor flow likely creates variations in shear stress within Schlemm's canal. Further investigation of the relationship between the phenotype heterogeneity and hydrodynamics of aqueous flow may help us understand the mechanisms of outflow resistance changes in glaucoma., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: We studied the topographic distribution of contractile protein in different orders of the human macular microvasculature to further understanding of the sites for capillary blood flow regulation., Methods: Nine donor eyes from eight donors were cannulated at the central retinal artery and perfusion labeled for alpha smooth muscle actin (αSMA) and filamentous actin (F-actin). Confocal images were collected from the macula region, viewed, projected, and converted to a 255 grayscale for measurements. The mean intensity was measured for macular arterioles, venules, and capillary segments. The diameter of each vessel segment measured was recorded. The normalized mean intensity values from all images were ranked according to vessel types and size with a total of nine categories., Results: F-actin was present throughout the macular microvasculature whereas αSMA labeling showed variations. Overall, αSMA has a more prominent presence in the macular arterioles than in the macular capillaries and venules, and αSMA strongly labeled the smaller macular arterioles. Some capillaries also labeled positive for αSMA, including some of the capillaries in the innermost capillary ring surrounding the foveola. It was weakly present in the capillaries on the venous side and larger venules. In the larger macular arterioles closer to 100 μm in diameter, αSMA labeling was weakly present and not as ubiquitous as in the smaller arterioles., Conclusions: Nonuniform distribution of contractile proteins in the different types, orders, and sizes of macular microvasculature indicates that these vessels may have different contractile capability and roles in macular flow regulation.

The central role of the cardiovascular system is to maintain adequate capillary perfusion. The spatially and temporally heterogeneous nature of capillary perfusion has been reported in some organs. However, such heterogeneous perfusion properties have not been sufficiently explored in the retina. Arguably, spatial and temporal heterogeneity of capillary perfusion could be more predominant in the retina than that in other organs. This is because the retina is one of the highest metabolic demand neural tissues yet it has a limited blood supply due to optical requirements. In addition, the unique heterogeneous distribution of retinal neural cells within different layers and regions, and the significant heterogeneity of intraretinal oxygen distribution and consumption add to the complexity. Retinal blood flow distribution must match consumption of nutrients such as oxygen and glucose within the retina at the cellular level in order to effectively maintain cell survival and function. Sophisticated local blood flow control in the microcirculation is likely required to control the retinal capillary perfusion to supply local retinal tissue and accommodate temporal and spatial variations in metabolic supply and demand. The authors would like to update the knowledge of the retinal microvessel and capillary network and retinal oxidative metabolism from their own studies and the work of others. The coupling between blood supply and energy demands in the retina is particularly interesting. We will mostly describe information regarding the retinal microvessel network and retinal oxidative metabolism relevant to the spatial and temporal heterogeneity of capillary perfusion. We believe that there is significant and necessary spatial and temporal heterogeneity and active regulation of retinal blood flow in the retina, particularly in the macular region. Recently, retinal optical coherence tomography angiography (OCTA) has been widely used in ophthalmology, both experimentally and clinically. OCTA could be a valuable tool for examining retinal microvessel and capillary network structurally and has potential for determining retinal capillary perfusion and its control. We have demonstrated spatial and temporal heterogeneity of capillary perfusion in the retina both experimentally and clinically. We have also found close relationships between the smallest arterioles and capillaries within paired arterioles and venules and determined the distribution of smooth muscle cell contraction proteins in these vessels. Spatial and temporal heterogeneity of retinal capillary perfusion could be a useful parameter to determine retinal microvessel regulatory capability as an early assay for retinal vascular diseases. This topic will be of great interest, not only for the eye but also other organs. The retina could be the best model for such investigations. Unlike cerebral vessels, retinal vessels can be seen even at the capillary level. The purpose of this manuscript is to share our current understanding with the readers and encourage more researchers and clinicians to investigate this field. We begin by reviewing the general principles of microcirculation properties and the spatial and temporal heterogeneity of the capillary perfusion in other organs, before considering the special requirements of the retina. The local heterogeneity of oxygen supply and demand in the retina and the need to have a limited and well-regulated retinal circulation to preserve the transparency of the retina is discussed. We then consider how such a delicate balance of metabolic supply and consumption is achieved. Finally we discuss how new imaging methodologies such as optical coherence tomography angiography may be able to detect the presence of spatial and temporal heterogeneity of capillary perfusion in a clinical setting. We also provide some new information of the control role of very small arterioles in the modulation of retinal capillary perfusion which could be an interesting topic for further investigation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system. The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy. There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions. Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: The iris allows effective delivery of nutrients into the aqueous humor supplying the surrounded avascular tissues. However, possible underlying mechanisms of the iris vasculature have not been well established. This study aims to quantitatively assess the human iris vascular network, endothelial cell morphometries, and characterize endothelial junctions to better understand the properties of the iris vasculature., Materials and Methods: The irises from human donor eyes were dissected and short fixed before float staining for VE-cadherin and claudin-5, f-actin and nuclei and flat-mounted for confocal imaging. The iris microvasculature was studied for its distribution and branch orders. The endothelial and nuclear morphometrics were measured for each vessel order. Characteristics of cellular junction staining and intracellular cytoskeleton were investigated., Results: The human iris vasculature was found to comprise of six orders of arteries, three orders of veins, and capillaries. The endothelial cell shape was long and narrow in all arteries, suggesting a high hemodynamic shear stress. Relatively large vessels ran radially in the superficial two-thirds of the iris, while smaller and denser vessels ran in the deepest third. Significant heterogeneity in vascular diameter, shape of the endothelia and nuclei, and the nuclear position was evident between artery, capillary and vein. Staining of junction proteins VE-cadherin and claudin-5 appeared non-uniform at the cell borders, especially in large veins., Conclusions: High rates of blood flow and special barrier properties are indicated by the morphological properties of the human iris vasculature. Detailed information of the iris vasculature combined with the inter- and intra-endothelial structure may help us further understand the physiological and pathogenic roles of the iris.

The oxygen supply to the retina in man and most mammals is derived from both the retinal and choroidal circulations. However, some mammals have only a partially vascularized retina, and some have a completely avascular retina. Here we contrast the retinal oxygen levels during systemic hyperoxia in a fully vascularized retina (rat), a partially vascularized retina (rabbit), and an avascular retina (guinea pig). Oxygen sensitive microelectrodes were used to measure the intraretinal oxygen distribution in anaesthetized rats, rabbits and guinea pigs during air breathing and 100% oxygen ventilation. In the vascularized rat retina the increase in oxygen tension in the choroid, reflected the increase in systemic oxygen levels during hyperoxic ventilation. However, the rise in oxygen levels in the inner retina was muted. In the avascular region of the partially vascularized rabbit retina, the increase in choroidal oxygen tension resulted in a large increase in oxygen tension across the full thickness of the retina. In the avascular retina of the guinea pig, very little change in choroidal or retinal oxygen tension was seen during systemic hyperoxia. Remarkably different responses to systemic hyperoxia are evident in the rat, rabbit, and guinea pig, three conventional laboratory animals that are commonly used in ophthalmic research. Neither the regulatory mechanisms responsible for the increase in oxygen consumption in the rat retina, or the stability of the choroidal oxygen tension in the guinea pig during systemic hyperoxia are currently understood. A better understanding of oxygen regulation in the mammalian retina could open up new avenues for improving the oxygen environment in the human retina in a range of ischaemic retinal diseases that account for the majority of blindness in the developed world.

Purpose: The prevailing view is that the human retina is supplied by the central retinal artery (CRA), the short posterior ciliary arteries (SPCAs) support the choroid, and both the CRA and the SPCAs are so-called "end artery" systems. In this study, we investigate whether vascular connections among the retina, choroid, and the optic nerve head (ONH) exist, using selective cannulation and microperfusion-labeling techniques., Methods: The CRA and/or one or more of the SPCAs were selected for cannulation in 18 human donor eyes. Fluorescent probes with different excitation wavelengths were perfused through different arteries on the same eye to distinguish the supply sources of different vascular beds. After labeling and fixation, the ONH region was dissected either longitudinally or transversely as thick sections for confocal microscopy. Retina, choroid, and ONH were imaged from whole-mount specimens., Results: Probes perfused through the CRA or the SPCA alone labeled the microvessels in the retina, choroid, and ONH regions, as well as the optic nerve trunk. The vessels of the lamina cribrosa and the optic nerve trunk were labeled when probes were perfused through the SPCA. Perfusion through both the CRA and SPCA produced double labeling of vessels in the retina, the choroid, and the ONH., Conclusions: The results indicate an inter-relationship of arterial supply to the retina, choroid, and ONH in the human eye. This has important implications in understanding clinical observations and disease mechanisms such as that of glaucoma and ischemic optic nerve disease.

Primary angle-closure glaucoma (PACG) is a common cause of blindness. Angle closure is a fundamental pathologic process in PAGC. With the development of imaging devices for the anterior segment of the eye, a better understanding of the pathogenesis of angle closure has been reached. Aside from pupillary block and plateau iris, multiple-mechanisms are more common contributors for closure of the angle such as choroidal thickness and uveal expansion, which may be responsible for the presenting features of PACG. Recent Genome Wide Association Studies identified several new PACG loci and genes, which may shed light on the molecular mechanisms of PACG. The current classification systems of PACG remain controversial. Focusing the anterior chamber angle is a principal management strategy for PACG. Treatments to open the angle or halt the angle closure process such as laser peripheral iridotomy and/or iridoplasty, as well as cataract extraction, are proving their effectiveness. PACG may be preventable in the early stages if future research can identify which kind of angles and/or persons are more likely to benefit from prophylactic treatment. New treatment strategies like adjusting the psychological status and balancing the sympathetic-parasympathetic nerve activity, and innovative medicines are needed to improve the prognosis of PACG. In this review, we intend to describe current understanding and unknown aspects of PACG, and to share the clinical experience and viewpoints of the authors., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The relationship between structural characteristics of the optic nerve head and venous pulsations in the human eye remain unknown. Using photoplethysmographic techniques we investigated whether properties of the human retinal veins and their surrounding structures influence venous pulsation. 448 locations of venous pulsation were analysed from 26 normal human eyes. Green channel densitometry derived from video recordings of venous pulsations were used to generate a map of venous pulsation amplitudes along retinal veins. Optical coherence tomography was used to perform quantitative measurements of tissue characteristics at sites of high and low amplitude points as well as in a second analysis, at maximal amplitude pulsation sites from superior and inferior halves of the eyes. Structural characteristics measured included venous diameter, distance from pulsation point to cup margin, vessel length from pulsation point to vein exit, tissue thickness overlying vein, optic disc diameter and presence of a proximal arteriovenous crossing. Increasing venous pulsation amplitudes were associated with larger applied ophthalmodynamometry force, increasing venous diameter, and decreasing absolute cup margin distance (all p < 0.001). Increasing distance of maximal amplitude pulsation point to cup margin was associated with the presence of a proximal arteriovenous crossing, increasing venous diameter, and decreasing tissue depth (all p ≤ 0.001). Venous diameter and tissue depth alter venous compliance, which is likely to be a major factor determining sites of venous pulsation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: To determine whether vascular tone of isolated porcine retinal veins can be modulated by tissue-generated vasoactive factors such as endothelin-1 and adenosine. Such information may be useful in understanding the role of the retinal veins in regulating blood flow, and also provide a model for investigating new hypotheses suggesting a role for vasoactive factors in retinal vascular diseases such as retinal vein occlusion., Methods: An isolated perfused retinal vein preparation was used for this study. Segments of porcine retinal veins were dissected, cannulated, and perfused, and their diameter was monitored during vasoactive agent application of increasing doses of endothelin-1 (10(-12)-10(-8) M) or adenosine (10(-10)-10(-4) M). Adenosine (10(-6) M) was also applied on veins during preconstriction with endothelin-1 (10(-8) M). The significance of any induced change in vessel diameter was assessed in relation to the baseline vessel diameter prior to any drug delivery., Results: Dose-dependent vasocontractile responses were induced by endothelin-1 administration. Endothelin-1 produced a significant contraction at doses of 10-11 M and above. At 10(-8) M the maximal endothelin-1-induced contractions were to 70.2 ± 2.1% of baseline. Adenosine produced a dose-dependent dilation reaching 113.0 ± 2.4% at 10(-4) M. Adenosine (10(-6) M) induced a significant dilation in endothelin-1 (10(-8) M)-contracted vessels., Conclusions: Porcine retinal veins can be modulated by both vasocontraction and vasodilation agents, suggesting that the retinal veins may play a regulatory role in the retinal circulation, particularly in regard to the capillary pressure upstream from the draining retinal veins. To our knowledge, this is the first study of vasoactivity in isolated perfused retinal veins, providing an opportunity to study the direct vasoactive effects of specific vasoactive agents.

Recently we reported studies of the iris microvasculature and its endothelial cells using intra-luminal micro-perfusion, fixation, and silver staining, suggesting that the iris vascular endothelium may be crucial for maintaining homeostasis in the ocular anterior segment. Here we present information regarding the intracellular structure and cell junctions of the iris endothelium. Thirty-seven porcine eyes were used for this study. The temporal long posterior ciliary artery was cannulated to assess the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining with phalloidin for intracellular cytoskeleton f-actin, and with antibodies against claudin-5 and VE-cadherin for junction proteins. Nuclei were counterstained with Hoechst. The iris was flat-mounted for confocal imaging. The iris microvasculature was studied for its distribution, branch orders and endothelial morphometrics with endothelial cell length measured for each vessel order. Our results showed that morphometrics of the iris microvasculature was comparable with our previous silver staining. Abundant stress fibres and peripheral border staining were seen within the endothelial cells in larger arteries. An obvious decrease in cytoplasmic stress fibres was evident further downstream in the smaller arterioles, and they tended to be absent from capillaries and veins. Endothelial intercellular junctions throughout the iris vasculature were VE-cadherin and claudin-5 immuno-positive, indicating the presence of both adherent junctions and tight junctions between vascular endothelial cells throughout the iris microvasculature. Unevenness of claudin-5 staining was noted along the endothelial cell borders in almost every order of vessels, especially in veins and small arterioles. Our results suggest that significant heterogeneity of intracellular structure and junction proteins is present in different orders of the iris vasculature in addition to vascular diameter and shape of the endothelia. Detailed information of the topography and intracellular structure and junction proteins of the endothelium of the iris microvasculature combined with unique structural features of the iris may help us to further understand the physiological and pathogenic roles of the iris vasculature in relevant ocular diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Cannabinoids have been reported to mediate changes in vascular resistance through endothelial receptor targets. We examined involvement of the endothelium in cannabinoid-mediated vasoactive responses in resistance arterioles of the retina., Methods: Vascular responses to both intraluminal (IL) and extraluminal (EL) administration of the atypical cannabinoid, abnormal cannabidiol (abn-CBD), a prototypical agonist at the non-CB1/CB2 endothelial cannabinoid receptor (CBeR), were studied in endothelial intact and endothelial denuded, isolated perfused porcine retinal arterioles with and without endothelin-1 (ET-1) precontraction. The effects of AM251, a CB1 receptor antagonist, and O-1918, an analog of CBD reported to antagonize CBeR, were also studied., Results: Dose-dependent vasocontractile responses were induced by both IL and EL administration of abn-CBD in the absence of precontraction. Significantly greater vasoconstriction was induced by IL administration of abn-CBD than with EL administration. In contrast, only vasodilation to abn-CBD was observed in ET-1 precontracted retinal arterioles. Endothelium removal significantly reduced abn-CBD-induced vasoactivity when abn-CBD was used IL but not when applied EL. IL abn-CBD-induced vasoactivity was antagonized by O-1918 and AM251., Conclusions: Cannabinoids show complex vasoactive actions in isolated perfused retinal arterioles. The fact that abn-CBD-mediated vasorelaxation was seen only in precontracted retinal vessels indicates that the abn-CBD-induced vasoactive response is highly dependent on vascular tone. Furthermore, IL and EL administration produced differential responses, and removal of endothelium blunted abn-CBD vasoactivity, highlighting the critical role of endothelium in abn-CBD vasoactivity. AM251 and O-1918 inhibition of abn-CBD-induced vasoactivity suggests the possibility of modulating abn-CBD-induced vasoactivity.

The roles of the iris microvasculature have been increasingly recognised in the pathogenesis of glaucoma and cataract; however limited information exists regarding the iris microvasculature and its endothelium. This study quantitatively assessed the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining of the porcine iris. The temporal long posterior ciliary artery of 11 isolated porcine eyes was cannulated, perfusion-fixed and labelled using silver nitrate. The iris microvasculature was studied for its distribution, orders and endothelial morphometrics. The density of three layers of microvasculature was measured. Endothelial cell length and width were measured for each vessel order. The iris has an unusual vascular distribution which consisted of abundant large vessels in the middle of the iris stroma, branching over a relatively short distance to the microvasculature located in the superficial and deep stroma as well as the pupil edge. The average vascular density of the middle, superficial, and deep layers were 38.9 ± 1.93%, 10.9 ± 1.61% and 8.0 ± 0.79% respectively. Multiple orders of iris vessels (capillary, 6 orders of arteries, and 4 orders of veins) with relatively large capillary and input arteries (319.5 ± 25.6 μm) were found. Significant heterogeneity of vascular diameter and shape of the endothelia was revealed in different orders of the iris vasculature. Detailed information of topography and endothelium of the iris microvasculature combined with unique structural features of the iris may help us to further understand the physiological and pathogenic roles of the iris in relevant ocular diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study aims to provide evidence of the importance of radial peripapillary capillaries (RPCs) by quantitative study of the relationship between the RPCs and retinal nerve fibre layer (RNFL) in normal human donor eyes. The retinal microvasculature in eleven normal human donor eyes was perfused, fixed and labelled after cannulation of the central retinal artery. The retinas were dissected and whole-mounted for confocal microscopy. Six study regions were taken radially from the edge of the optic disc. RPCs from the optic disc edge to a radial distance up to 2.5 mm were imaged and their diameters, inter-capillary distance and volume occupation measured. These were correlated with the study region as well as thickness of the RNFL. It was found that the pooled average diameter of the RPCs in the first 2.5 mm from the optic disk was 8.9 μm. Significant differences in capillary diameter were present in the six regions, with larger diameter RPCs in the superior, inferior and nasal regions, and significantly smaller diameter in the temporal region. RPCs in the arcuate fibre regions extend the furthest from the optic disc, maintained a close inter-capillary distance for a longer distance than other regions, and have the highest RPCs volume occupancy. The RPCs volume was generally correlated with RNFL thickness. In conclusion, a close correlation between RNFL and RPCs presence has been demonstrated which is supportive of their functional reliance/co-dependence. The significantly smaller temporal RPCs may be a result of the greater presence of RPCs in the two bordering arcuate fibre regions and therefore a richer availability of nutrients diffusing from these two regions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To determine whether morbidity and mortality in patients with cardiovascular comorbidities, but no known ocular disease, is related to demonstrable quantitative changes in the retinal microvasculature., Methods: Eleven eyes from 8 donors with cardiovascular comorbidities as a diseased group were compared with 16 eyes from 14 donors free from vascular disease as a control group. All eyes had no known ocular disease. The retina was perfusion-fixed and labeled for endothelial f-actin using micro-cannulation techniques. The retinal microvasculature 3 mm superior to the optic disc was imaged with confocal scanning laser microscopy. Quantitative measurements of capillary diameter and density were obtained using two-dimensional image reconstructions. Pathological vascular changes in other regions of the retinal vasculature found in the diseased group were identified and reconstructed in two or three dimensions., Results: Capillary densities were significantly different between each capillary network in the diseased group. There was a significant decrease in density between both the nerve fiber layer and retinal ganglion cell layer of the diseased group when compared with those layers in the control eyes. There were pathological vascular changes including microaneurysms and tortuous, dilated venules identified in the diseased group., Conclusions: Cardiovascular comorbidities may be associated with changes to the capillary density within the human retinal microvasculature, before the manifestation of known ocular diseases. These differences in capillary density may have important correlations with neuronal function and facilitates the basis of understanding pathogenic mechanisms in retinal vascular disease., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: We have previously reported significant phenotype heterogeneity in the vortex vein system. This study is to quantify the age-related change of such endothelial phenotype heterogeneity., Method: The inferior temporal vortex vein system of 10 eyes from 7 young donors (30±4.1 years) and 9 eyes from 6 aged (72±4.7 years) donors were dissected after perfusion fixation and labeled for f-actin and nucleic acid. Confocal images of endothelial cells were obtained from nine anatomic regions and measurements made of the cell and nucleus sizes. The results were compared between the two age groups., Results: Similar regional endothelial heterogeneity was observed in both age groups through the different regions of the vortex vein system. Age-related increase in endothelial cell area was observed in all the study regions. Age-associated regional differences were also observed in the endothelial length, width, and nucleus parameters. Endothelial nuclei were also found to be located further downstream within the cell in aged donor eyes., Conclusion: Age related enlarged endothelial cells have been identified in this venous system, a likely indicator of senescence. The relationship between the endothelial senescence, regional endothelial phenotype change and endothelial dysfunction in possible pathological changes needs to be further defined., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This review is about vascular endothelial phenotype heterogeneity in the retinal and choroidal circulations. It is becoming increasingly clear that the functional and structural heterogeneity is present in the retinal and choroidal circulations. Differential responses of the vessels to vasoactive substances have been shown with intraluminal and extraluminal delivery and in different regions of the same vascular bed. Vascular endothelial phenotype is highly heterogenic and site-specific, particularly in the retinal and choroidal veins. Updated information of such heterogeneity may help us to further understand the control mechanisms of the retinal and choroidal circulations which are important in compensating for the physiological and pathological challenges faced by these vascular beds. The site-specific changes of vascular endothelial phenotype may be linked with endothelium dysfunction, and site-specific diseases such as central and branch retinal vein occlusion. Endothelial dysfunction has been recognized as an initial step for many vascular diseases. Endothelial cells are a strategic and valid target for therapeutic intervention. Fundamentally important questions regarding the role of vascular endothelial cell function in the eye are discussed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Retinal ganglion cell (RGC) axonal structure and function in the optic nerve head (ONH) is predominantly supported by astrocytes and capillaries. There is good experimental evidence to demonstrate that RGC axons are perturbed in a non-uniform manner following ONH injury and it is likely that the pattern of RGC axonal modification bears some correlation with the quantitative properties of astrocytes and capillaries within laminar compartments. Although there have been some excellent topographic studies concerning glial and microvascular networks in the ONH our knowledge regarding the quantitative properties of these structures are limited. This report is an in-depth quantitative, structural analysis of astrocytes and capillaries in the pre laminar, lamina cribrosa and post laminar compartments of the ONH. 49 optic nerves from human (n = 10), pig (n = 12), horse (n = 6), rat (n = 11) and rabbit (n = 10) eyes are studied. Immunohistochemical and high-magnification confocal microscopy techniques are used to co-localise astrocytes, capillaries and nuclei in the mid-portion of the optic nerve. Quantitative methodology is used to determine the area occupied by astrocyte processes, microglia processes, nuclei density and the area occupied by capillaries in each laminar compartment. Comparisons are made within and between species. Relationships between ONH histomorphometry and astrocyte-capillary constitution are also explored. This study demonstrates that there are significant differences in the quantitative properties of capillaries and astrocytes between the laminar compartments of the human ONH. Astrocyte processes occupied the greatest area in the lamina cribrosa compartment of the human ONH implicating it as an area of great metabolic demands. Microglia were found to occupy only a small proportion of tissue in the rat, rabbit and pig optic nerve suggesting that the astrocyte is the predominant glia cell type in the optic nerve. This study also demonstrates that there is significant uniformity, with respect to astrocyte and capillary constitution, in the post laminar region of species with an unmyelinated anterior optic nerve. This implicates an important role served by oligodendrocytes and myelin in governing the structural characteristics of the post laminar optic nerve. Finally, this study demonstrates that eyes with similar lamina cribrosa structure do not necessarily share an identical cellular constitution with respect to astrocytes. The quantitative properties of astrocytes in the pre laminar and lamina cribrosa regions of the rat, which has a rudimentary lamina cribrosa with only a few collagenous beams, shared more similarities to the human eye than the pig or horse. The quantitative properties of astrocytes and capillaries in the laminar compartments of the ONH provide a basis for understanding the pathogenic mechanisms that are involved in diseases such as glaucoma and ischemic optic neuropathy. The findings in this study also provide valuable information about the distinct advantages of different animal models for studying human optic nerve diseases. Utilisation of structural data provided in this report together with emerging in vivo technology may potentially permit the early identification of RGC axonal injury by quantifying changes in ONH capillaries and astrocytes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The vortex vein system is the drainage pathway for the choroidal circulation and serves an important function in the effective drainage of the exceptionally high blood flow from the choroidal circulation. As there are only 4-6 vortex veins, a large volume of blood must be drained from many choroidal veins into each individual vortex vein. The vortex vein system must also cope with passing through tissues of different rigidity and significant pressure gradient as it transverses from the intrao-cular to the extra-ocular compartments. However, little is known about how the vortex vein system works under such complex situations in both physiological and pathological condition. Endothelial cells play a vital role in other vascular systems, but they have not been studied in detail in the vortex vein system. The purpose of this study is to characterise the intracellular structures and morphology in both the intra-and extra-ocular regions of the human vortex vein system. We hypothesise the presence of endothelial phenotypic heterogeneity through the vortex vein system. The inferior temporal vortex vein system from human donor eyes were obtained and studied histologically using confocal microscopy. The f-actin cytoskeleton and nuclei were labelled using Alexa Fluor conjugated Phalloidin and YO-PRO-1. Eight regions of the vortex vein system were examined with the venous endothelium studied in detail with quantitative data obtained for endothelial cell and nuclei size and shape. Significant endothelial phenotypic heterogeneity was found throughout the vortex vein system with the most obvious differences observed between the ampulla and its downstream regions. Variation in the distribution pattern of smooth muscle cells, in particular the absence of smooth muscle cells around the ampulla, was noted. Our results suggest the presence of significantly different haemodynamic forces in different regions of the vortex vein system and indicate that the vortex vein system may play important roles in regulation of the choroidal circulation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to investigate whether region-dependent endothelial heterogeneity is present within the porcine vortex vein system., Methods: The superior temporal vortex vein in young adult pig eyes were dissected out and cannulated. The intact vortex vein system down to the choroidal veins was then perfused with labels for f-actin and nucleic acid. The endothelial cells within the choroidal veins, pre-ampulla, anterior portion of the ampulla, mid-ampulla, posterior portion of the ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein regions were studied in detail using a confocal microscopy technique. The endothelial cell and nuclei length, width, area and perimeter were measured and compared between the different regions., Results: Significant regional differences in the endothelial cell and nuclei length, width, area and perimeter were observed throughout the porcine vortex vein system. Most notably, very narrow and elongated endothelia were found in the post-ampulla region. A lack of smooth muscle cells was noted in the ampulla region compared to other regions., Conclusions: Heterogeneity in endothelial cell morphology is present throughout the porcine vortex vein system and there is a lack of smooth muscle cells in the ampulla region. This likely reflects the highly varied haemodynamic conditions and potential blood flow control mechanisms in different regions of the vortex vein system., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Retinal ganglion cells (RGCs) are specialized projection neurons that relay an immense amount of visual information from the retina to the brain. RGC signal inputs are collected by dendrites and output is distributed from the cell body via very thin (0.5-1 μm) and long (∼50 mm) axons. The RGC cell body is larger than other retinal neurons, but is still only a very small fraction (one ten thousandths) of the length and total surface area of the axon. The total distance traversed by RGCs extends from the retina, starting from synapses with bipolar and amacrine cells, to the brain, to synapses with neurons in the lateral geniculate nucleus. This review will focus on the energy demands of RGCs and the relevant tissues that surround them. RGC survival and function unexceptionally depends upon free energy, predominantly adenosine triphosphate (ATP). RGC energy metabolism is vastly different when compared to that of the photoreceptors. Each subcellular component of the RGC is remarkably different in terms of structure, function and extracellular environment. The energy demands and distribution of each component are also distinct as evidenced by the uneven distribution of mitochondria and ATP within the RGC - signifying the presence of intracellular energy gradients. In this review we will describe RGCs as having four subcellular components, (1) Dendrites, (2) Cell body, (3) Non-myelinated axon, including intraocular and optic nerve head portions, and (4) Myelinated axon, including the intra-orbital and intracranial portions. We will also describe how RGCs integrate information from each subcellular component in order achieve intracellular homeostatic stability as well as respond to perturbations in the extracellular environment. The possible cellular mechanisms such as axonal transport and axonal cytoskeleton proteins that are involved in maintaining RGC energy homeostasis during normal and disease conditions will also be discussed in depth. The emphasis of this review will be on energetic mechanisms within RGC components that have the most relevance to clinical ophthalmology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To determine if patients with cardiovascular comorbidities but no clinically detectable retinal disease demonstrate quantitative alterations to perifoveal capillary networks., Methods: Comparisons were made between 10 eyes from patients with vascular comorbidities and 17 control eyes. All eyes were absent of clinically evident ocular disease. Microcannulation techniques were used to label the retinal microvasculature. Retinae were flat mounted, and the peripapillary region 2 mm nasal to the fovea was imaged using confocal laser scanning microscopy. Two- and three-dimensional image reconstructions were used to perform quantitative measurements of individual capillary networks within the perifovea. Parameters measured included capillary diameter, capillary loop area, capillary loop length, capillary density, and capillary surface area., Results: Capillary diameter was increased in the retinal ganglion cell and superficial inner plexiform layer capillary network in patients with vascular comorbidities. Capillary loop area and capillary loop length were reduced in all capillary networks in patients with vascular comorbidities except the deep capillary network of the inner nuclear layer. Capillary density was reduced in the nerve fiber layer capillary network in patients with vascular comorbidities. There was no difference in the relative occupied capillary surface area between control and diseased eyes., Conclusions: The results in this study suggest that the quantitative characteristics of perifoveal capillary networks are nonuniformly altered in patients with vascular comorbidities, before the onset of clinically identifiable eye diseases. These findings may be important for understanding pathophysiological mechanisms involved in retinal vascular diseases.

Vascular comorbidities are inherently linked to the pathogenesis of central retinal artery occlusion (CRAO) and central retinal vein occlusion (CRVO). However, the endothelial-mediated pathogenic mechanisms that precede, and therefore modulate, luminal occlusion have not been clarified. The aim of this study was to delineate the pattern of endothelial morphometric alteration in the central retinal artery and vein in patients with vascular comorbidities. Eyes with a previous history of vascular occlusion were not included in this study in order to avoid the confounding effects of post-occlusion endothelial changes. This study also sought to determine if vascular comorbidities had a disparate effect on arterial and venous endothelium in the optic nerve head. Comparisons were made between 13 human eyes from patients with vascular comorbidities and 22 control eyes from patients with no known systemic disease. Novel micro-cannulation techniques developed in our laboratory were used to label the cytoskeleton and nuclei of endothelial cells in the central retinal artery and vein following which images were captured using confocal microscopy. Endothelial and nuclear morphometric parameters were quantified in different laminar regions of the optic nerve head. F-actin stress fibre expression was also quantified. Analysis of covariance was used to determine statistical differences between the two groups. Interestingly, age did not influence endothelial morphometry, nuclear morphometry or f-actin expression in central retinal vessels. There were also no arterial endothelial differences between control and disease groups in any laminar region. Endothelial f-actin stress fibre expression increased significantly in the central retinal vein in patients with vascular comorbidities. The greatest change in these eyes was found to occur at the posterior lamina cribrosa. Increased venous endothelial f-actin stress fibre expression may reflect vascular comorbid disease-induced alterations to hemodynamic properties and coagulation cascades in the central retinal vein. The posterior lamina may be an important site for thrombus formation in CRVO as venous endothelia in this region are most influenced by the presence of vascular comorbidities. The findings of this study suggest that the role of endothelial dysfunction in CRVO and CRAO pathogenesis could be different., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: There is increasing evidence that relatively rapid spikes in intraocular pressure may contribute to axonal damage in glaucoma. The present study seeks to quantify the ability of a compressible damping element (a simple air bubble) to reduce intraocular pressure fluctuations induced by a known change in intraocular fluid volume., Methods: A mathematical model describing the damping of intraocular pressure increases for a given infusion volume was developed and compared with experimental data obtained from isolated pig eyes. A damping element (100 µL to 2 mL of air) was added to the system, and the effect on the induced intraocular pressure change for a given infusion volume was assessed., Results: The introduction of the damping element reduced the intraocular pressure change in a volume-dependent manner consistent with the mathematical modelling. The maximum bubble size tested (2 mL) dampened the intraocular pressure change by an average of 63.5 ± 8.7% at a baseline pressure close to 20 mmHg. Close agreement was seen between the mathematical model and the experimental data., Conclusion: Mathematical modelling and experiments in isolated pig eyes demonstrated that the addition of a damping element in the form of a compressible air bubble is capable of significantly reducing induced intraocular pressure spikes., (© 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina., Methods: A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks., Results: Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks., Conclusions: The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.

Purpose: To quantify the distribution and morphometric characteristics of capillary networks in the human perifovea. To determine correlations between the location of neuronal subcellular compartments and the morphometric features of regional capillary networks in the layered retina., Methods: The perifoveal region, located 2 mm nasal to the fovea, was studied in 17 human donor eyes. Novel micropipette technology was used to cannulate the central retinal artery and label the retinal microcirculation using a phalloidin perfusate. γ-synuclein, Goα, and parvalbumin antibodies were also used to co-localize the nerve fiber layer (NFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), and inner nuclear layer (INL). Confocal scanning laser microscopy was used for capillary imaging. Capillary diameter, capillary density, and capillary loop area measurements were compared between networks., Results: Four capillary networks were identified in the following retinal layers: (1) NFL, (2) RGCL and superficial portion of IPL, (3) deep portion of IPL and superficial portion of INL, and (4) deep portion of INL. Laminar configurations were present in NFL and deep INL networks. Remaining networks demonstrated three-dimensional configurations. Capillary density was greatest in the networks serving the IPL. Capillary loop area was smallest in the two innermost networks. There was no difference in capillary diameter between networks., Conclusions: Capillary networks in the human perifovea are morphometrically heterogeneous. Morphometric features of regional capillary networks in the layered retina may serve a critical role in supporting neuronal homeostasis. Improved knowledge of these features may be important for understanding pathogenic mechanisms underlying retinal vascular diseases.

Background: Vascular endothelial growth factor (VEGF) plays an important role in ocular physiology. Anti-VEGF agents are now used for treatment of common retinal diseases. This study characterises the vasoactive properties of VEGF in isolated perfused pig retinal arterioles under normal tone or endothelin-1 (ET-1) pre-contracted conditions and determines the influence of an anti VEGF agent on VEGF induced vasoactivity., Methods: An isolated perfused retinal arteriole preparation was used. The outer diameter of retinal vessels was monitored at 2 second intervals in response to VEGF and the anti VEGF agent, bevacizumab. The effect of intraluminal delivery of VEGF was determined over a wide concentration range (10(-16) to 10(-7) M) both with and without pre-contraction with ET-1 (3 x 10(-9) M). Bevacizumab (0.35 mg mL(-1)) was applied extraluminally to determine the influence of bevacizumab on VEGF induced vasoactive changes on ET-1 pre-contracted vessels., Results: In retinal arterioles with normal tone, VEGF induced a concentration dependent contraction at low concentrations, reaching 93.5% at 10(-11) M and then contraction was reduced at higher concentrations, recovering to 98.1% at 10-7 M. VEGF produced a potent concentration dependent vasodilatation in arterioles pre-contracted with ET-1. VEGF induced vasodilatation in arterioles pre-contracted with ET-1 was significantly inhibited by bevacizumab., Conclusions: VEGF induced vasoactive changes in pig retinal arterioles are dependent on concentration and vascular tone. Bevacizumab inhibits VEGF-induced vasodilatation in pre-contracted arterioles.

Purpose: To investigate the venous endothelial phenotype at retinal artery-vein (AV) crossings and age-related changes in human cadaveric eyes., Methods: Eighteen human donor eyes free of known ocular diseases were divided into two groups according to age (≤30 and >50 years). The central retinal artery was cannulated and perfused with oxygenated Ringer's solution with 1% bovine serum albumin. The perfusate solutions were switched to fixative, membrane permeabilizing solution, and selected labeling solutions for microfilament F-actin and nucleic acid in vascular endothelial cells and smooth muscle cells. The eyes were then immersion fixed and the retinas flat mounted. The venous endothelial cells were examined by confocal microscopy at the AV, pre-AV, and post-AV crossing regions., Results: There was no significant difference between the younger and older groups in endothelial cell length upstream or downstream from an AV crossing. At the AV crossing, the venous endothelial cells were shorter in the younger group and longer in the older group compared with those upstream or downstream from the AV crossing. Stress fibers were not frequently observed in the endothelial cells of younger donors. However, the older group had numerous stress fibers in their endothelia at AV crossing points., Conclusions: Age-related phenotype changes in venous endothelial cells have been identified in the region of AV crossings providing supportive evidence for the hypothesis of age-related and site-specific changes in the vascular endothelial cells as an important factor contributing to the pathogenesis of branch retinal vein occlusion.

The purpose of this study was to quantify the topographic distribution of bulbar conjunctival microlymphatic vessels in the monkey. Sixteen eyes from 8 rhesus monkeys were used. Full thickness pieces of globe wall were excised from each quadrant. Cryosections were stained for 5'-nucleotidase, an enzyme histochemical staining for lymphatic vessels, or vascular endothelial growth factor receptor-3, an immunohistochemical marker for the identification of lymphatic endothelial cells, and then counterstained by hematoxylin. The remaining bulbar conjunctiva was dissected and flat mounted. The tissue was then processed with 5'-nucleotidase and alkaline phosphatase, an enzyme histochemical stain with higher activity in blood vessels. Microscope images were further analysed by image processing. The density of lymphatics, diameter of lymphatic vessels, and the size of the drainage zone of each blind end of the initial lymphatics were studied. Conjunctival lymphatics consisted of initial lymphatics and pre-collectors. The initial lymphatics with blind ends were predominately distributed just under the epithelium. The density of these lymphatics (∼50%) and the drainage zone area (∼0.81 mm(2)) was similar in each quadrant, with no difference in the limbus and fornix regions. The average diameter of lymphatic vessels in each quadrant ranged from 82 to 111 μm, and was greater in the superior and nasal regions. Larger calibre pre-collectors with valve-like structures were mostly located sub Tenon's membrane and predominantly located in the region mid-way between the limbus and fornix. There was a marked depth difference in initial lymphatic distribution, with the initial lymphatics mostly confined to the region between Tenon's membrane and the conjunctival epithelium. Detailed knowledge of the topographic distribution of conjunctival lymphatics have significant relevance to a better understanding of immunology, drug delivery, glaucoma filtration surgery, and tumour metastasis in the conjunctiva., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pressure changes within the central nervous system (CNS) have the capacity to provoke neurodegeneration by perturbing axonal homeostatic processes. The pathogenic role of axonal transport dysfunction in diseases characterised by sustained pressure elevation have been clearly delineated, however the patho-physiogical mechanisms underlying neurological disorders typified by measurable fluctuations in CNS pressure remains unclarified. This study utilises the rabbit optic nerve, a myelinated, mammalian neuronal tract to compare the effects of sustained pressure elevation and fluctuating pressure change on axonal transport processes. In 5 rabbits, neural pressure was sustained at 10mm Hg and 40mm Hg in the right and left optic nerves, respectively, for 6 hours. In another 5 rabbits, neural pressure was modulated between 7.5mm Hg and 57.5mm Hg at 30minute intervals in the right optic nerve, and sustained at 40mm Hg in the left optic nerve for 6hours. Rhodamine-β-isothiocyanate, an axonal transport tracer, was used to quantify axonal transport differences between normal-, high- and fluctuating-pressure nerves. Axonal transport rates in high-pressure nerves were significantly lower than normal-pressure nerves. The effects of fluctuating-pressure and sustained high-pressure on axonal transport processes were not significantly different. The findings of this study suggest that the magnitude of pressure elevation and the profile of pressure change over time is important in modulating axonal function. It also implicates the importance of axonal transport dysfunction in the process of neurodegeneration. These results may have relevance for understanding patho-physiological mechanisms involved in pseudotumor cerebri, syringomyelia, hydrocephalus and glaucoma-diseases characterised by fluctuating pressure changes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: To investigate the effects of acute elevations in intraocular pressure (IOP) on the cup, prelaminar, and lamina cribrosa regions of the porcine optic nerve head (ONH)., Methods: Ex vivo imaging of 10 porcine ONHs was performed using spectral-domain optical coherence tomography (OCT). The IOP was manipulated with a pressure head and measured with a pressure transducer. Reference scans were taken at 0 mm Hg, before further scanning was performed at 7-mm Hg steps, up to 49 mm Hg. Morphometric parameters were measured across centrally located OCT B-scans at different IOPs, and the relationship between IOP and changes in these parameters was analyzed., Results: As IOP increased from 0 to 49 mm Hg, mean cross-sectional cup area increased (28% ± 3%, P < 0.001), lamina cribrosa area decreased (18% ± 2%, P < 0.001), and prelaminar tissue area decreased (5.5% ± 0.5%, P < 0.001). Multivariate regression demonstrated that most of the change in cup area is associated with changes in both lamina cribrosa position and thickness (r = 0.89, P < 0.001)., Conclusions: Acute elevations in IOP were shown to result in posterior displacement of ONH, as well as lamina cribrosa and prelaminar tissue deformation in the porcine ONH.

Aim To correlate morphometric parameters of the porcine optic nerve head (ONH) in spectral domain optical coherence tomography (SD-OCT) B-scans with matched histological sections. Methods Ten porcine eyes were scanned using the Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany) at 7 mm Hg and subsequently fixed, cryosectioned and stained with Van Giessen stain. B-scans were matched with histological sections and morphometric parameters measured on both. Results SD-OCT measurements of the Bruch membrane opening (BMO) cross-sectional diameter and the mean distance from the BMO plane to the anterior surface of the collagenous lamina cribrosa correlated strongly with matched histological measurements (r=0.80 (p=0.005) and 0.83 (p=0.003), respectively) while measurements of the mean prelaminar tissue thickness, cup cross-sectional area and neuroretinal rim cross-sectional area had lower correlation coefficients (r=0.62 (p=0.055), 0.57 (p=0.08) and 0.33 (p=0.354), respectively). SD-OCT measurement of lamina cribrosa mean cross-sectional thickness also showed a lower correlation with matched histological measurements (r=0.64, p=0.048). Conclusion A high correlation exists between SD-OCT B-scans and matched histological sections for anterior collagenous structures of the ONH, while a lower correlation exists for morphometric parameters involving the prelaminar region and lamina cribrosa thickness.

Oligodendrocyte-derived myelin retards the ability of CNS axons to regenerate following transection. The intrinsic response of CNS axons to an axotomy insult may be vastly different in the absence of myelin. However, the paucity of adequate experimental models has limited detailed investigation of cellular behaviour following axon transection in an unmyelinated CNS environment. In this study we perform laser-induced axotomy of the porcine retinal ganglion cell axon, a physiologically unmyelinated, mature CNS axon that is structurally similar to humans to infer knowledge about axonal behaviour in the absence of myelin. Axotomy-induced changes to the neuronal cytoskeleton and supporting astrocytes during the early stages after transection are delineated by examining the sequence of neurofilament subunit, microtubule (TUB), microtubule associated protein (MAP), glial fibrillary acidic protein (GFAP) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) modification. Axonal transection induced an increase in the expression of neurofilament light at regions within, and immediately adjacent to, sites of axotomy. Other neurofilament subunits were not altered at sites of transection. Unlike myelinated axons where an increase in GFAP staining within hypertrophic glial scars have been shown to inhibit axonal repair we demonstrate a decrease in GFAP staining within regions of increased or preserved neurofilament expression. The behaviour of TUB and MAP proteins following transection of unmyelinated CNS axons are similar to what has previously been described in myelinated CNS axons. This study provides fundamental insights into astrocyte and axonal behaviour acutely after axotomy and demonstrates a series of degenerative events in unmyelinated CNS axons, which in comparison to prior reports are different to myelinated CNS axons. The findings of this report have relevance to understanding pathogenic mechanisms underlying neuro-degeneration in the CNS., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study documents the morphometric features of arterial and venous endothelia in the different laminar regions of the normal human optic nerve head and speculates on the hemodynamic characteristics of the central retinal artery (CRA) and central retinal vein (CRV)., Methods: Twenty normal human eyes were used. Microcannulation techniques were used to label the cytoskeleton and nuclei of endothelial cells in the CRA and CRV, after which images were captured using confocal microscopy. Length, width, length-to-width ratio, and area measurements were obtained from endothelium and its nuclei. Nucleus position with respect to cell apex and direction of blood flow was also quantified. Comparisons were made between prelaminar, anterior lamina cribrosa, posterior lamina cribrosa, and retrolaminar regions. Venous and arterial endothelial cell morphology was also compared., Results: There was significant variation in venous endothelial morphology across the different laminar regions; however, no differences were found in arterial endothelial characteristics (all P > 0.1065). Significant differences were found between arterial and venous endothelium in all laminar regions apart from the posterior lamina cribrosa, where only nuclear area (P = 0.0001) and nucleus position (P = 0.0088) were found to be different., Conclusions: Arterial-like appearance of venous endothelium in the posterior lamina cribrosa, where pressure gradient forces are predicted to be greatest and CRV luminal diameter is known to be narrowest, implicates this as a site of altered hemodynamic stress. Heterogeneity of venous endothelium may have relevance for understanding ocular vascular diseases such as central retinal vein occlusion.

Ultraviolet (UV) lasers have the capability to precisely remove tissue via ablation; however, due to strong absorption of the applicable portion the UV spectrum, their surgical use is currently limited to extraocular applications at the air/tissue boundary. Here we report the development and characterization of a fiber-optic laser delivery system capable of outputting high-fluence UV laser pulses to internal tissue surfaces. The system has been developed with a view to intraocular surgical applications and has been demonstrated to ablate ocular tissue at the fluid/tissue boundary. The fifth (213 nm) and fourth(266 nm) harmonics of a Nd:YAG laser were launched into optical fibers using a hollow glass taper to concentrate the beam. Standard and modified silica/silica optical fibers were used, all commercially available. The available energy and fluence as a function of optical fiber length was evaluated and maximized. The maximum fluence available to ablate tissue was affected by the wavelength dependence of the fiber transmission; this maximum fluence was greater for 266 nm pulses (8.4 J/cm2) than for 213 nm pulses (1.4 J/cm2). The type of silica/silica optical fiber used did not affect the transmission efficiency of 266 nm pulses, but transmission of 213 nm pulses was significantly greater through modified silica/silica optical fiber. The optical fiber transmission efficiency of 213 nm pulses decreased as a function of number of pulses transmitted, whereas the transmission efficiency of 266 nm radiation was unchanged. Single pulses have been used to ablate fresh porcine ocular tissue. In summary, we report a method for delivering the fifth (213 nm) and fourth (266 nm) harmonics of a Nd:YAG laser to the surface of immersed tissue, the reliability and stability of the system has been characterized, and proof of concept via tissue ablation of porcine ocular tissue demonstrates the potential for the intraocular surgical application of this technique.

Background: Triamcinolone acetonide (TA) has applications for the treatment of a large range of intraocular vascular diseases. The present study in pigs was performed to investigate histopathological and histochemical changes in the levels of myocilin deposition in the anterior segment in a model of branch retinal vein occlusion (BRVO) after vitreal administration of TA., Methods: After ophthalmoscopic examination, intraocular pressure (IOP) measurement and fundus photography, a BRVO was created photothrombotically in each eye of six pigs, using argon green photocoagulation. The left eye was then injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined, animals sacrificed, and eyes enucleated and processed in paraffin and epoxy resin. Immunofluorescence cytochemistry on paraffin sections was performed to localise the distribution of myocilin in the anterior segment and histology by light and transmission electron microscopy on epoxy resin sections on TA-treated and untreated eyes., Results: Histology revealed pathological changes in the TA-treated eye, including swollen mitochondria, layered long endoplasmic reticulum, pleomorphic nuclei, dense fibrillar extracelluar deposits and aggregates of unusual cell inclusions. Myocilin levels were significantly higher in the TA-treated eyes in the trabecular meshwork (p = 0.001), ciliary process (p = 0.011) and iris (p = 0.030) than in the untreated eyes., Conclusions: This study suggests that increased myocilin synthesis and related ultrastructural changes in the anterior segment after treatment with intravitreal TA in a porcine model of retinal oedema in BRVO may contribute to IOP elevation.

Purpose: To characterize the topography and cellular structure of the macular microvasculature using a recently developed technique of arterial cannulation, perfusion, fixation, and staining of human donor eyes., Methods: Sixteen human donor eyes were used. The central retinal artery was cannulated and perfused with Ringer's, then fixative, membrane permeabilizing, and selected labeling solutions. The eyes were immersion fixed, and the retina was flat mounted for confocal microscopy. The macular area, including the foveola, fovea, and parafovea, was sampled. The intracellular cytoskeleton of vascular endothelial and smooth muscle cells was studied in different orders of arterioles and venules and in the capillaries. To evaluate the degree of asymmetry within vascular networks, the distribution of generation numbers and the Horton-Strahler approach to vessel naming were compared., Results: The distribution of the microvascular network in the macular region was complex but followed a general theme. The parafoveal region was supplied by dense vasculature with approximately nine closely arranged pairs of arterioles and venules. Each arteriole had abundant branches and a high degree of asymmetry (∼10 generations and 3.5 orders within 1.2-mm length). Only a few arterioles (average ∼2.9) supplied the terminal capillary ring. Very long spindle endothelial cells were seen in the superficial and deep capillaries. Significant heterogeneity of distribution and shape of the endothelial and smooth muscle cells was evident in different orders of the macular vasculature., Conclusions: The authors have demonstrated for the first time the cellular structure and topographic features of the macular microvasculature in human donor eyes.

Purpose: To delineate morphometric and quantitative features of the capillary image derived from high-resolution fundus fluorescein angiography (FFA) and consequently determine the diagnostic value of FFA for studying the retinal capillary circulation., Methods: Retinal capillary images obtained from healthy young subjects using high-resolution FFA were compared with confocal scanning laser microscopic capillary images derived from the retinas of age-matched human donors. Confocal microscopic images were acquired from retinal flatmount tissue after central retinal artery cannulation, perfusion fixation, and antibody labeling. Capillary images from equivalent retinal regions were morphologically and quantitatively analyzed in both groups., Results: Ten human subjects (mean age, 27.4 years) were used for FFA studies, and five cadaveric eyes (mean donor age, 26.5 years) were used for histologic studies. In histologic specimens the density of the superficial capillary network was significantly greater than that of the deep capillary network. Despite use of a healthy young population, only 30% of high-resolution FFA studies provided clear capillary images. The configuration of the capillary network in FFA images was comparable to the superficial capillary network in confocal microscope images; however, the density of the capillary network in FFA images was consistently lower than that of histologic images., Conclusions: FFA provides incomplete morphologic information about the superficial capillary network and even less information about the deep capillary network. Caution should, therefore, be exercised when using FFA data to extrapolate information about microvascular histopathologic processes. The usefulness of newer technology for studying retinal capillary detail should be investigated.

New and more precise subretinal surgical techniques would be useful in a range of retinal diseases. The purpose of this study is to determine the feasibility of using fiberoptically delivered ultraviolet laser energy to transect or ablate subretinal tissues. Choroid segments dissected from fresh porcine eyes, with or without the retinal pigment epithelium (RPE), were clamped in a fluid bath. Pulsed fourth harmonic (266 nm) of a Nd:YAG laser radiation was delivered via an optical fiber probe at fluence levels between 0.08 and 0.40 J/cm(2). The tissue was then fixed and sectioned for histological examination. Radiation induced damage was categorized by the degree of tissue disruption and ablation depth. Tissue ablation and the severity of the tissue injury varied with both tissue properties and applied laser parameters. Disruption of Bruch's membrane was typically induced by 10 pulses of 0.30 J/cm(2) or 2 pulses of 0.40 J/cm(2). Lower radiation doses did not disrupt Bruch's membrane, but did damage the choroidal tissue and produce vacuoles in the underlying choroid. The full thickness of the choroid was ablated by 200 pulses of 0.40 J/cm(2). The presence of the RPE produced a shielding effect which was greater than would be expected for an equivalent thickness of choroidal tissue. Ablation characteristics of subretinal tissue are highly dependent on the laser parameters used and the type of tissue involved. To perform well controlled laser surgery on subretinal tissues both laser parameters and the properties of the target cells and tissues have to be considered., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Purpose: To examine the time-dependent effects of focal axonal ischemia on the retinal ganglion cell (RGC) cytoskeleton., Methods: Eight pigs were used. Small retinal arteriolar branches were occluded by argon laser to induce focal ischemic insults that were maintained for a period of 6 hours or 1 hour. Treated and untreated retinal segments were dissected from the eye after euthanatization. Each retinal segment followed the longitudinal projection of RGC axons from peripheral retina to the optic disc. Antibodies to phosphorylated neurofilament heavy, phosphorylation-independent neurofilament heavy (NFH), neurofilament light, neurofilament medium, microtubule, and microtubule-associated proteins were used to study the axonal cytoskeleton. Glial fibrillary acidic protein and TUNEL staining were also used to examine astrocyte and apoptotic changes, respectively. Comparisons were made between treated and untreated retinal segments., Results: Cytoskeleton protein changes occurred within ischemic regions and also within retinal tissue on the disc side and peripheral side of the ischemic regions. NFH and microtubule proteins were the earliest cytoskeleton subunits that underwent change. Changes to all cytoskeleton proteins, apart from NFH, occurred in a time-dependent manner within regions of ischemia. In the time points studied, cytoskeleton changes occurred in the absence of detectable astrocyte changes and RGC apoptosis., Conclusions: An ischemic insult induces RGC cytoskeleton protein change, implying that the local environment plays an important role in modulating axonal structure and function. Cytoskeleton proteins are likely to be important pathogenic mediators of neuronal dysfunction in diseases such as glaucoma and retinal vascular disease.