Paula K. Yu

Correlation of Histologic and Clinical Images to Determine the Diagnostic Value of Fluorescein Angiography for Studying Retinal Capillary Detail

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.

Quantitative confocal imaging of the retinal microvasculature in the human retina.

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.

Diabetic retinopathy: early functional changes.

1. The present review reports some of the earliest physiological changes that occur in the diabetic retina prior to any clinical or anatomical changes in an animal model of diabetes.

Quantitative morphometry of perifoveal capillary networks in the human retina.

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.

Microstructure and Network Organization of the Microvasculature in the Human Macula

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 Ringers, 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.

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.

The Structural Relationship between the Microvasculature, Neurons, and Glia in the Human Retina

PURPOSE To develop a new technique for detailed study of the spatial distribution of retinal and choroidal microvasculature and their relationship to neurons and glial cells at the cellular level in human cadaveric eyes. METHODS Twenty-six human donor eyes were used. Wherever possible, the central retinal artery and a branch of the posterior ciliary artery were individually cannulated and perfused with oxygenated Ringers solution with 0.5% bovine serum albumin. The perfusion pressure was continuously monitored. Once residual blood was washed out, the perfusate solutions were switched to fixative, membrane-permeabilizing solution and selected labeling solutions. The eyes were then immersion fixed and the retina and choroid flat-mounted for immunolabeling and confocal imaging before cryosectioning. The microstructures of vascular, glial, and neuronal cells in the retina and the stroma in the choroid were studied. RESULTS The retinal microvasculature was fully perfused and stained by cannulation of the central retinal artery. Regional distribution of choroidal vasculature perfusion was dependent on the specific feeder artery cannulated. The detailed spatial relationship between endothelial cells, glial cells, and neurons at the cellular and subcellular levels was identified with confocal microscopy and immunohistochemical labeling of retinal sections. In the choroid, endothelial cells were clearly identifiable down to the level of the intracellular cytoarchitecture of the choriocapillaris, along with their relationship to Bruchs membrane and the feeding and drainage vessels. CONCLUSIONS A microperfusion fixation and staining technique has been developed that allows studies of the structural relationships of vascular, glial, and neuronal elements at the cellular level in human donor eyes.

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.

Pathogenesis and intervention strategies in diabetic retinopathy

Diabetic retinopathy is the leading cause of new blindness in the working‐age population. If improved treatment regimens are to be developed it is crucial that the underlying pathophysiological mechanisms responsible for diabetic retinopathy are better understood. The multifactorial nature of the many pathways implicated in diabetic retinopathy requires a very detailed approach to elucidate the key mechanisms involved and their interactions in order to develop logical strategies aimed at therapeutic intervention. Fortunately, the streptozotocin rat model of diabetes displays many of the morphological and functional changes to the retinal vasculature that are evident in human diabetic retinopathy. This study reviews some of the recent experimental work by the authors in the streptozotocin rat, compares their findings to the human pathology, and outlines potential new avenues for therapeutic intervention. In particular, the improved understanding of which layers of the inner retina have the most stringent metabolic demands has helped identify which retinal layers are most susceptible to metabolic or hypoxic/ischaemic insult. It is concluded that improved treatment outcomes may ensue if the therapy is targeted at the appropriate tissue at specific stages of the disease.

Altered vasoactivity in the early diabetic eye : measured in the isolated perfused rat eye

The effect of 4 weeks streptozotocin-induced diabetes on ocular vascular resistance responses to noradrenalin (NA), adrenalin (A), phenylephrine (PHE), isoproterenol (ISOP), prostaglandin F2 alpha (PGF2 alpha). 5-hydroxytryptamine (5-HT) and angiotensin II (ANG II), was determined using a newly-developed, isolated, arterially-perfused rat eye preparation, by comparing responses from control and diabetic eyes. After extensive preliminary experiments to establish optimum parameters, the ophthalmic artery of enucleated control and diabetic rat eyes was cannulated and the retinal and uveal vasculature perfused at a constant flow with Na(+)-Krebs solution after streptozotocin-induced diabetes had been established for 4 weeks. The eyes were maintained in an environment-controlled organ bath. Perfusion pressure was monitored as increasing log M concentrations of agonists were added to the perfusate. Total ocular resistance could be calculated from knowledge of flow and pressure. In control eyes, NA, A, PHE, PGF2 alpha, and 5-HT all produced dose-dependent increases in total vascular resistance, with the following order of potency: NA = A > 5-HT > PHE = PGF2 alpha at 10(-4) M. The ocular circulation was not sensitive to isoproterenol and angiotensin II. In diabetic eyes responses to NA, A, PGF2 alpha and 5-HT were altered. Diabetic responses to NA and A had lower thresholds with larger resistance increases at low concentrations. However, the rate of increase in resistance with concentration was more gradual in diabetic eyes so that at 10(-4) M control responses were larger. Diabetic resistance responses to PGF2 alpha had the same threshold as in control eyes, but were greater in magnitude with an earlier peak at 10(-4) M. In contrast diabetic resistance responses to 5-HT were reduced, peaked at a lower resistance at 10(-4) M, but had the same threshold as those in the control eye. Basal vascular resistances in control: 3.14 +/- 0.32 mmHg min microliter-1 (n = 28), and diabetic eyes: 3.44 +/- 0.19, mmHg min microliter-1 (n = 36), were not significantly different. Vasoactivity in the early diabetic eye is disturbed with the effective balance between different agonists altered in favour of catecholamines at physiological concentrations. This may be related to the early changes in blood flow and oxygen distribution already reported in the rat eye, as well as changes to autonomic function. The isolated perfused rat eye is a valuable technique for investigating such vascular reactivity in animal models of retinal disease.