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.

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.

Retinal capillary perfusion: Spatial and temporal heterogeneity.

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.

Intraretinal oxygen distribution and consumption during retinal artery occlusion and graded hyperoxic ventilation in the rat.

PURPOSE: To determine intraretinal oxygen distribution and consumption in a rat model of retinal artery occlusion during air breathing and stepwise systemic hyperoxia. METHODS: Laser occlusion of the pair of retinal arteries feeding the area of retina under investigation was performed. Oxygen-sensitive microelectrodes were then used to measure oxygen tension as a function of depth through the retina. Breathing mixtures were manipulated to produce stepwise increments in systemic oxygen levels, and the measurement of intraretinal oxygen distribution was repeated. Oxygen distribution in the retina was analyzed by an established eight-layer mathematical model of retinal oxygen consumption. RESULTS: Intraretinal oxygen distribution in the occluded area confirmed that the choroid was the only source of retinal oxygenation. Under air-breathing conditions, the oxygen supply from the choroid was sufficient to support the photoreceptor inner segments. Any remaining oxygen was consumed by the outer plexiform layer. Increases in inspired oxygen level reduced the extent of inner retinal anoxia. However, some degree of anoxia in the innermost retina was usually present. CONCLUSIONS: Occlusion of the retinal circulation renders most of the inner retina anoxic. Ventilation with 100% oxygen does not generally avoid some degree of intraretinal anoxia. With 100% oxygen ventilation, the oxygen consumption of the inner retina was more than four times that of the outer retina. A marked degree of heterogeneity in oxygen uptake of different retinal layers was evident. The dominant oxygen consumers were the inner segments of the photoreceptors, the outer plexiform layer, and the inner plexiform layer.

Vasoconstrictive effects of sodium fluorescein on retinal vessels is increased by light exposure.

PURPOSE: To determine whether clinically relevant doses of sodium fluorescein produce changes in vascular tone in retinal arterioles and veins and whether any such effects were light dependent. METHODS: Segments of porcine retinal arterioles and veins were dissected, cannulated, and perfused and their outer diameter monitored during intraluminal application of increasing doses (10(-10) to 10 (-3) g/ml) of sodium fluorescein under either brightly lit (350 lux) or dimly lit (4 lux) conditions. The significance of any induced change in vessel diameter was assessed in relation to the initial vessel diameter. RESULTS: At the higher light level, sodium fluorescein produced a significant dose-dependent contraction in porcine retinal arterioles and veins with a threshold of 10 (-5) g/ml and 10 (-9) g/ml sodium fluorescein, respectively. At the maximal dose tested (10(-3) g/ml), vessel diameter was reduced to approximately 85% of the initial vessel diameter in retinal arterioles and veins. Under dimly lit conditions, the vasoconstrictive effect of sodium fluorescein was still evident but the constriction was significantly smaller (p < 0.05) in both arteries and veins, reaching approximately 91% and 93%, respectively. CONCLUSIONS: Sodium fluorescein induced light-dependent vasoconstrictive effects on the retinal vasculature of the pig. Should a similar effect be present in human retinal vessels, then reduced illumination level may limit the vasoconstrictive effects of sodium fluorescein when used in routine ophthalmic examinations.

Oxygen distribution and consumption in the developing rat retina.

PURPOSE: To determine the intraretinal oxygen distribution and oxygen consumption in the rat before eye opening and maturation of the retina. METHODS: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized Sprague-Dawley rats at postnatal day (P)15. Measurements were made under air-breathing conditions and at increasing levels of systemic oxygenation (20%, 40%, 60%, 80%, and 100% oxygen) under light-adapted conditions. Oxygen consumption in the outer retina and in the predominantly avascular region of the inner retina was assessed by fitting the oxygen profiles to a mathematical model of oxygen consumption. The retinas were processed for histology and compared with retinas from mature animals. RESULTS: Under normal conditions, the intraretinal oxygen distribution at P15 was significantly different in some respects from that in mature animals. Oxygen consumption analysis indicated an average outer retinal oxygen consumption rate of 103 +/- 15 nL/min per cm2 and an inner retinal oxygen consumption rate of 42.4 +/- 11.7 nL/min per cm2. Inner retinal oxygen consumption was significantly (P < 0.001) lower than that previously measured in mature rats, but outer retinal oxygen consumption was similar. Systemic hyperoxia increased the oxygen level throughout the retina, but choroidal PO2 in particular remained significantly lower than in adult rats (P < 0.001). At P15 there were marked differences in the relative thickness of some retinal layers when compared with adult rats. In particular the inner and outer nuclear layers were much thicker at P15, the outer segments of the photoreceptors and the inner and outer plexiform layers were not fully developed. CONCLUSIONS: At P15, before eye opening, the oxygen consumption of the inner retina is lower than in mature retinas, presumably reflecting the immaturity of the retina in such young animals so soon after their first exposure to light stimuli.

Structure and function of myelinated nerve fibers in the rabbit eye following ischemia/reperfusion injury.

The rabbit eye presents a valuable model to study the effects of vascular occlusion on the function and structure of myelinated nerve fibers. The rabbit eye has a band of myelinated nerve fibers within the intraocular compartment that are supplied by a narrow band of retinal vasculature. These vessels were transiently occluded ( approximately 8 hours) using laser photocoagulation and the transmission of electrical signals along the nerve fibers was assessed by recording the visual evoked response (VER). Morphological damage was assessed by histological techniques. The ischemic insult produced no permanent change in retinal function as assessed by electroretinography, but the VER was suppressed, indicating failure of nerve fiber transmission. Histologically, the visible damage to the region supported by the retinal vasculature worsened following reperfusion, showing evidence of demyelination and necrosis followed by macrophage responses and gliosis. This rabbit model of ischemia/reperfusion of the retinal vasculature offers a rare opportunity to reliably study the response of myelinated nerve fibers to ischemia/reperfusion insults and has demonstrated the susceptibility of myelinated nerve fibers to such insults.

Local Modulation of Retinal Vein Tone.

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.

Intraretinal oxygen distribution in the monkey retina and the response to systemic hyperoxia.

PURPOSE: To measure the intraretinal oxygen distribution and consumption in the fovea, the parafovea, and the inferior retina in the monkey eye and determine the influence of graded systemic hyperoxia. METHODS: Oxygen sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized monkeys (n = 8) under normoxic and hyperoxic conditions. Oxygen consumption rates in the avascular regions of retina were determined by fitting the oxygen profiles to established oxygen consumption models. RESULTS: Under normoxic conditions, in the foveal area, the intraretinal oxygen distribution reflected the absence of retinal capillaries and the predominantly choroidal origin of retinal oxygenation. A similar shape of oxygen distribution was seen in the parafoveal retina with the addition of local perturbations in the inner retina attributed to the presence of retinal capillaries. In the inferior retina the same general shape was found. Oxygen consumption in the outer retina was higher in the parafoveal region, and the minimum oxygen tension was lower. During hyperoxia, choroidal oxygen levels in all areas increased dramatically, but the increase in oxygen tension in the inner retina was much less. The avascular nature of the foveal area allowed oxygen consumption analysis of both the inner and outer retina and showed that inner retinal oxygen consumption increased significantly during hyperoxic ventilation to a level equivalent to that of the outer retina. CONCLUSIONS: In the outer retina of the monkey the P(O2) minimum is lower, and the oxygen consumption rate is higher in the parafoveal region. During systemic hyperoxia, outer retinal oxygen consumption is unaffected, but in the foveal area, total oxygen consumption increases. This regulation of oxygen consumption in the monkey retina is comparable to that reported in lower mammals and may represent an important mechanism in retinal homeostasis.

Isolated preparations of ocular vasculature and their applications in ophthalmic research.

The purpose of this review is to outline the techniques and applications for isolated ocular vascular preparations and their significance to ophthalmic research. Various isolated ocular vascular preparations have been utilized in studies of ocular vascular biology, physiology and pharmacology, including work in both normal and diseased conditions. However, there is still significant potential for further studies to improve our understanding of the ocular circulation and its regulation. Experience has shown that there is no single preparation capable of addressing all of the questions that must be answered if a complete understanding of mechanisms of vascular regulation in the eye is to be achieved. Rather, it is necessary to select the appropriate preparation and techniques to address each individual question in the most appropriate manner. In this review, particular emphasis is placed on the applications for isolated ocular preparations and the relevance of such studies to our understanding of the pathogenesis of eye diseases involving the vasculature. Examples are given where therapeutic approaches in diabetes and glaucoma are assessed in terms of their impact on the vasoactive properties of the ocular vasculature.A significant heterogeneity is present in the different parts of the ocular vasculature, not only in the structural and functional properties of vessel itself, but also in terms of the tissue environment and innervation. A single vasoactive agent may also have different effects when applied to the inside or the outside of the same region of a vessel. The vasoactive response of the vascular system as a whole is what determines the rate of blood flow through the system, but this is regulated by a multitude of factors in different regions of the vascular network. Isolating individual components of the ocular vasculature is readily achievable for the extraocular vessels such as the ophthalmic or ophthalmocilliary arteries, which can be studied in myograph type systems measuring the mechanical vasoactive force generated by the vessel. Retinal vessels from very large animals can also be studied in this way, but the small diameter of the retinal vessels in most species requires a perfusion rather than myograph based technique. Perfusion based studies of vessel diameter in response to vasoactive stimuli can be applied to individual retinal arteries and their branches. Perfusion of more complex elements of the ocular vasculature such as isolated segments of the retina or ciliary body, or whole isolated perfused eyes may use the perfusate pressure as the determinant of vasoactive state. However, when several components of the ocular vasculature are being perfused simultaneously it may be difficult to separate out the contribution from the different vascular elements. The advantage of isolated preparations is that systemic influences can be eliminated, and vascular components can be studied that are inaccessible in vivo. The disadvantage is that no matter how well controlled the in vitro environment may be, it will always be a relatively poor mimic of the in vivo conditions. However, such in vitro work has certainly improved our understanding of the vasoactive properties of different regions of the ocular vasculature in both health and disease.

Endothelial F-actin cytoskeleton in the retinal vasculature of normal and diabetic rats.

PURPOSE: The purpose of this study was to characterize the endothelial (EC) F-actin cytoskeleton at different orders of the retinal microvasculature in the normal and diabetic rat and determine if changes in F-actin are associated with different stages of diabetes. METHODS: The EC F-actin cytoskeleton distribution, nuclei shape, and capillary diameter in the retinal vasculature of rats after 5 and 28 weeks of streptozotocin (STZ)-induced diabetes were compared to those in age-matched controls. The eyes were enucleated, arterially perfused, and labeled for F-actin cytoskeleton and nuclei (YO-PRO-1) or microvascular leakage (FITC-dextran). Retinal whole mounts were then examined by confocal microscopy. RESULTS: The EC F-actin distribution and nuclear size and shape were highly dependent on the location down the vascular tree. The retinal arterial system in the rat shows a high level of F-actin stress fibre (SF) staining. Peripheral border (PB) staining was present in the ECs of all vessels. Diffuse F-actin staining was observed in endothelial cytoplasm in capillaries, venules, and veins. EC nuclei became distinctly less elongated down the vascular tree. In diabetic rats at 5 weeks, at the capillary level the F-actin staining was more diffuse, and areas of F-actin loss were evident. Both dot-like and diffuse leakage was detected in retinal capillaries, and these leakage types were closely associated with the degree of F-actin changes. In diabetic rats at 28 weeks, there was an increased level of SF staining in the arterial system in addition to capillary F-actin changes. CONCLUSIONS: The EC F-actin cytoskeleton and nuclei shape retinal microvasculature of the normal rat change with location along the vascular tree. In the early stages of diabetes, there are changes to the F-actin cytoskeleton that are clearly associated with microvascular leakage. F-actin distribution could indicate important structural changes in the pathogenesis of diabetic retinopathy.

Sphincter activity in retinal arterioles feeding the deeper capillary layer in pig.

PURPOSE: To determine whether a sphincter-like control point is present in 90 degrees branches of the retinal vasculature. Such a control point could selectively control retinal blood flow to the deeper capillary layers. METHODS: A microperfusion system was used in which porcine retinal arteries with intact branches were cannulated and perfused intraluminally at physiological flow rates. The vasoactive response of the proximal and distal regions of either 90 degrees -or Y-type branches to intraluminal potassium (124 mM) were monitored simultaneously in real-time. RESULTS: The proximal region of the 90 degrees branches demonstrated a localized vasoconstriction when compared to the more distal region (p < 0.0001). In contrast, the Y branches contracted evenly along their length. CONCLUSIONS: The localized vasoactivity near the branch point of the 90 degrees -type branches may explain previous observations of so-called sphincter-like activity in these vessels. These sphincters may selectively regulate blood flow to the deeper retinal capillary layer.

Role of Endothelium in Abnormal Cannabidiol-Induced Vasoactivity in Retinal Arterioles.

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.

Intraretinal oxygen consumption in the rat in vivo.

PURPOSE: To make the first quantitative assessment of the rate of oxygen consumption in high oxygen-consuming layers of both the outer and inner retina of the rat in vivo. METHODS: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized rats. Individual PO2 profiles were fitted to a multilayer mathematical model of PO2 distribution that is able to determine the oxygen uptake in those retinal layers in which the oxygen supply is through diffusion from vascular layers of the retina. This includes the entire outer retina and the region of the inner retina containing the inner plexiform layer. Measurements were performed in the light (13 mW/cm2 at the cornea) and in the dark and the amplitude and time constant for light-induced changes in outer retinal oxygenation determined. RESULTS: Under light-adapted conditions, the oxygen consumption of the outer retina was 148 +/- 11 nL O2/min/cm2 (n = 20) [corrected] and that of the included portion of the inner retina was 184 +/- 17 nL O2/min/cm2 [corrected]. In the dark, outer retinal oxygen consumption increased by 47.8% (P < 0.001), and the time constant for the resultant PO2 decrease in the outer retina was 14.9 +/- 1.8 seconds (n = 16). There was no significant change in inner retinal oxygen consumption between light and dark conditions (P = 0.89). CONCLUSIONS: Under light-adapted conditions the oxygen uptake by the selected region of the inner retina (primarily the inner plexiform layer) is greater than that of the outer retina (P < 0.01). Dark adaptation rapidly and significantly increases outer retinal oxygen consumption, but the inner retina remains unaffected.

Vasoactive response of isolated pulpal arterioles to endothelin-1.

The vasoactive effect of endothelin-1 applied intraluminally or extraluminally was studied in vitro in isolated perfused porcine pulpal arterioles using a microperfusion system. Pulpal arterioles (outer diameter, 94.2 +/- 2.8 microm, n = 12) were cannulated and perfused at a constant flow rate in an environment-controlled bath on the stage of an inverted microscope. The vessel diameters were measured online. Both intraluminal and extraluminal application of endothelin-1 (10(-16) M to 10(-8) M) induced dose-dependent constrictions, reaching 82.3 +/- 1.7% (n = 12) and 70.5 +/- 1.3% (n = 12) at 10(-8) M, respectively. Nifedipine reversed endothelin-1-induced constriction dose-dependently at 10(-7) M and above. These data demonstrate that endothelin-1 induces calcium-dependent vasoconstriction in porcine pulpal arterioles, with extraluminal application more potent, which seems to reflect the possible modulation of vascular endothelium in the control of vascular tone.

Functional and morphological characteristics of the retinal and choroidal vasculature.

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.

Retinal ganglion cells: Energetics, compartmentation, axonal transport, cytoskeletons and vulnerability.

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.

The critical role of the conjunctiva in glaucoma filtration surgery.

This review considers the critical role of the conjunctiva in determining the success or failure of glaucoma filtration surgery. Glaucoma filtration surgery can be defined as an attempt to lower intraocular pressure (IOP) by the surgical formation of an artificial drainage pathway from the anterior chamber to the subconjunctival space. Many types of glaucoma filtration surgery have been developed since the first attempts almost 180 years ago. The wide range of new techniques and devices currently under investigation is testament to the limitations of current techniques and the need for improved therapeutic outcomes. Whilst great attention has been paid to surgical techniques and devices to create the drainage pathway, relatively little attention has been given to address the question of why drainage from such artificial pathways is often problematic. This is in contrast to normal drainage pathways which last a lifetime. Furthermore, the consequences of potential changes in aqueous humour properties induced by glaucoma filtration surgery have not been sufficiently addressed. The mechanisms by which aqueous fluid is drained from the subconjunctival space after filtration surgery have also received relatively little attention. We propose that factors such as the degree of tissue damage during surgery, the surrounding tissue reaction to any surgical implant, and the degree of disruption of normal aqueous properties, are all factors which influence the successful formation of long term drainage channels from the conjunctiva, and that these channels are the key to successful filtration surgery. In recent years it has been suggested that the rate of fluid drainage from the subconjunctival space is actually the determining factor in the resultant IOP reduction. Improved knowledge of aqueous humour induced changes in such drainage pathways has the potential to significantly improve the surgical management of glaucoma. We describe for the first time a novel type of drainage surgery which attempts to minimise surgical trauma to the overlying conjunctiva. The rationale is that a healthy conjunctiva allows drainage channels to form and less opportunity for inflammation and scar tissue formation which are a frequent cause of failure in glaucoma filtration surgery. Successful drainage over extended periods of time has been demonstrated in monkey and rabbit eyes. Long lasting drainage pathways were clearly associated with the presence of lymphatic drainage pathways. A new philosophy in glaucoma drainage surgery is proposed in which minimisation of surgical trauma to the conjunctiva and the encouragement of the development of conjunctival drainage pathways, particularly lymphatic pathways, are central pillars to a successful outcome in glaucoma filtration surgery.