Topographic distribution and phenotypic heterogeneity of Schlemm's canal endothelium in human donor eyes.

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.

Endothelial contraction of retinal veins.

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.

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system.

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.

Regulation of Oxygen Tension in the Mammalian Retina During Systemic Hyperoxia Is Species Dependent.

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.

Structural characteristics of the optic nerve head influencing human retinal venous pulsations.

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.

Intracellular cytoskeleton and junction proteins of endothelial cells in the porcine iris microvasculature.

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.

Quantitative study of the microvasculature and its endothelial cells in the porcine iris.

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.

Quantitative study of age-related endothelial phenotype change in the human vortex vein system.

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.

Correlation between the radial peripapillary capillaries and the retinal nerve fibre layer in the normal human retina.

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.

Comparative quantitative study of astrocytes and capillary distribution in optic nerve laminar regions.

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.

Regional heterogeneity of endothelial cells in the porcine vortex vein system.

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.

Phenotypic heterogeneity in the endothelium of the human vortex vein system.

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.

Alterations to vascular endothelium in the optic nerve head in patients with vascular comorbidities.

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.

Optimal Calculation of Mean Pressure From Pulse Pressure.

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.

An assessment of microvascular hemodynamics in human macula.

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.

Posture-Induced Changes in Intraocular, Orbital, Cranial, Jugular Vein, and Arterial Pressures in a Porcine Model.

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.

Quantitative study of the topographic distribution of conjunctival lymphatic vessels in the monkey.

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.

An experimental study of VEGF induced changes in vasoactivity in pig retinal arterioles and the influence of an anti-VEGF agent.

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.

Damping of intraocular pressure fluctuations.

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.