Shear rate and hematocrit dependence of fluorescence from retinal vessels in fluorescein angiography

The purpose of this work was to obtain more quantitative knowledge about the yield of fluorescence from retinal vessels during fluorescein angiography. The influence of shear rate, concentration of sodium fluorescein, hematocrit, and layer thickness on the yield of fluorescence from blood were investigated. Measurements were performedin vitro on samples of human blood in a cone-plate shear chamber using frontal illumination. Application of physiologically relevant levels of shear (>88/sec) decreased the yield of fluorescence from the blood sample considerably as compared with stasis. The yield of fluorescence was proportionally related to the logarithm of the sodium fluorescein concentration in blood up to a sodium fluorescein concentration of 1.2 mg/ml. Above that concentration quenching occurred. An increase in layer thickness at a hematocrit of 45% resulted only in an increase of the yield of fluorescence up to a layer thickness of 25 μm. In conclusion, the sodium fluorescein concentration in blood is the only important factor that determines the yield of fluorescence from the larger retinal vessels in the successive phases of the fluorescein angiogram in a subject with a given hematocrit and hemoglobin concentration. The yield of fluorescence from retinal vessels (>25 μm) is proportionally related to the logarithm of the sodium fluorescein concentration over a broad range of concentrations.     

The preprocessing of retinal images for the detection of fluorescein leakage

Images of the human retina are routinely used in clinical practice for the diagnosis and management of eye disease. Increased permeability of retinal blood vessels, which is a clinically significant feature, can be visualized with a process known as fluorescein angiography as leakage of fluorescence dye into the surrounding tissues. Analyses of such images can be quantified but significant degradation of images due to uneven illumination or occluded optical pathways is often incurred during image capture. We describe a procedure to restore fluorescein angiographic retinal images so that quantitative computation can be reliably performed. Analysis of the image acquisition system reveals that captured images are composed of two functions, one describing the true underlying image and the other the degradation incurred. These two functions are independent of one another and it is possible to estimate the degradation from an isolated captured image and restore it appropriately. Any leakage of fluorescein dye is then detected by analysing the restored angiographic sequence over time and finding areas of the image that do not have the usual decrease in fluorescence intensity.     

The development of astrocytes and blood vessels in the postnatal rabbit retina

Summary Antibodies to glial fibrillary acidic protein (GFAP) have been used to study the shape and location of astrocytes in whole mount preparations of developing postnatal rabbit retina. At all developmental stages GFAP-positive astrocytes were detectable. At birth, they were few in number and only weakly labelled. With further development, their number as well as their labelling intensity increased. Following Nissl counterstaining it was observed that GFAP-positive astrocytes, always situated in the nerve fibre layer, are capable of cell division during about the first 4 postnatal weeks.GFAP-positive astrocytes were always confined to a wing-shaped area extending horizontally from both sides of the optic nerve head. It is suggested that astrocytes are not generated in the entire rabbit retina, which is in clear contrast to the second glial cell type of the rabbit retina, the Müller cell; and it has been concluded that the confinement of astrocytes to the medullary rays region in the adult rabbit is established during ontogenesis, and is not due to a secondary restriction of astrocytes to this region.Horizontal sections cut through entire rabbit retinae at various postnatal stages revealed that the first intraretinal blood vessels are not found before postnatal day 9. This is more than 1 week later than the first astrocytes are detectable. It is suggested that, at least in the rabbit, retinal astrocytes do not co-migrate with blood vessel endothelial cells from the optic disc into the retina, a hypothesis considered recently for the cat retina.It was, however, not possible to decide unequivocally if, in this material, astroglial progenitors are derived from retinal neuroepithelial cells or invade the retina from the optic nerve head.     

The projection of the temporal retina in rats,studied by retrograde transport of horseradish peroxidase

Summary Horseradish peroxidase (HRP) was injected unilaterally into the lateral geniculate nucleus or tectum, or both, in 26 hooded rats in order to mark the exact extent of the retina from which uncrossed optic axons arise. This region occupied about a quarter of the retina, in the temporal periphery, following thalamic injections, but a much smaller region following tectal injections. By comparing the proportions of HRP positive neurones in nasal and temporal retinae of both eyes it was shown that: (1) within the region supplying uncrossed axons the majority of the ganglion cells nevertheless project contralaterally, (2) a large proportion of the ganglion cells from the temporal crescent project bilaterally, which does not occur from the remainder of the retina, (3) ganglion cells of all sizes contribute to both ipsilateral and contralateral projections. The results also support earlier suggestions that the smallest neurones in the ganglion cell layer do not send an axon into the brain, and are therefore not ganglion cells     

Optical model of the blood in large retinal vessels

Several optical techniques that investigate blood contained within the retinal vessels are available or under development. We present a mechanical model that simulates the optical properties of the eye, the retinal vessels, and the ocular fundus. A micropipette is chosen as the retinal vessel model, and a mechanical housing is constructed to simulate the eyeball. Spectralon is used to simulate the retinal layers. Filling the eye with fluid index matched to the glass pipette eliminates reflection and refraction effects from the pipette. An apparatus is constructed and used to set the oxygen, nitrogen, and carbon dioxide concentrations in whole human blood. These whole blood samples are pumped through the pipette at 34 microL/min. Measurements made in the model eye closely resemble measurements made in the human eye. This apparatus is useful for developing the science and testing the systems that optically investigate blood and blood flow in the large retinal vessels.     

The morphological correlates of X- and Y-like retinal ganglion cells in the retina of monkeys

Summary The morphology of the ganglion cells of the monkey's retina was revealed by filling the cells with horseradish peroxidase from their cut axons in the optic nerve. This procedure gave much more consistent results than the Golgi method, was much quicker and filled dendrites just as extensively. Quantitative measures of the dendritic tree of two types of ganglion cell, P and P, suggest that they correspond to the physiologically defined Y- and X-cells, respectively.Supported by MRC grant G971/397/B     

The transcapillary exchange of sodium fluorescein in ischaemic limbs measured by fluorescein flowmetry

The transcapillary exchange of sodium fluorescein in the skin of 36 extremities (19 patients) with arterial occlusive disease was measured using two different ways of analysing the tissue fluorescence, fluorescein flowmetry (FF) and dynamic fluorescein angiography (FA). 7 mg sodium fluorescein/kg body weight was given intravenously. With FF, the transcapillary exchange of sodium fluorescein is expressed in arbitrary units as a fluorescence index. With FA, the time interval from the bolus injection to the first appearance of the fluorescence in the tissue is measured. There was an inverse correlation between pain and fluorescence index. All eight extremities with a fluorescence index of 0.001 density units/s or lower needed amputation, whereas all extremities with higher indices were viable. All extremities requiring amputation had an appearance time of 150 s or more, which contrasted with those remaining viable, having an appearance time of 120 s or less. The results indicate that FF as well as FA are appropriate methods to measure a 'nutritive' transport of solutes in the skin. Theoretically, however, if quantitative values for blood flow are required, we recommend FF, this method being more adequate, since it is influenced both by the fraction of cardiac output distributed to the tissue and by cardiac output itself. If, however, only a prognosis of a limb's viability is needed, FA is sufficient, being easier to perform, only requiring the measurement of the appearance time.     

The development of astrocytes in the albino rabbit retina and their relationship to retinal vasculature

We have examined the development of astrocytes in the albino rabbit retina, using antibodies to glial fibrillary acidic protein (GFAP) and vimentin. Vimentin immunoreactive (vimentin⁺) astrocyte-like cells first appear at the 24th postconceptional day (24 PCD), in a pattern similar to that of the adult. GFAP immunoreactivity was first detected in astrocytes at the 29 PCD, in a similar pattern. Vessels enter the retina from 29 PCD. The presence of astrocytes in a mature distribution prior to the ingrowth of vessels indicates that astrocytes are not dependent on the vessels for their early positioning and differentiation. In contrast with the rat and cat, we found no evidence of migration of astrocytes into the rabbit retina from the optic nerve.     

Evaluation system for absolute brightness from the retinal vessels

The paper describes a method for the absolute evaluation of the luminance of a photographic image of the fundus, based on a comparison between this image and a referred grey scale. The absolute measurements of the luminance of the particular on the film are performed by a microprocessor-controlled microfilm reader. This new method can be applied in general studies regarding the fundus of the eye in particular for studying retinal diseases caused by hypertension and diabetes.     

Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location

Summary Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used forin situ andin vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable byin situ immunocytochemistry. However, B-type cells could be seen to express all three subunitsin vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation. The results also show that determination of the neurofilament protein content of a population of nerve cells can be critically dependent on examination of single isolated cells. Isolated rabbit horizontal cells provide a promising system for studies of the mechanical, molecular and biochemical properties of neurofilaments.     

Analysis of posterior retinal layers in spectral optical coherence tomography images of the normal retina and retinal pathologies

We present a computationally efficient, semiautomated method for analysis of posterior retinal layers in three-dimensional (3-D) images obtained by spectral optical coherence tomography (SOCT). The method consists of two steps: segmentation of posterior retinal layers and analysis of their thickness and distance from an outer retinal contour (ORC), which is introduced to approximate the normal position of external interface of the healthy retinal pigment epithelium (RPE). The algorithm is shown to effectively segment posterior retina by classifying every pixel in the SOCT tomogram using the similarity of its surroundings to a reference set of model pixels from user-selected area(s). Operator intervention is required to assess the quality of segmentation. Thickness and distance maps from the segmented layers and their analysis are presented for healthy and pathological retinas.     

Changes in retinal neurons in the guinea pig retina stimulated by strobe lights during development

The modern-day population is overexposed to visual stimuli accompanied by contrast and strength changes, such as the television or videogames, beginning early in life. These light stimuli may have an influence on the development of the visual system. The purpose of this study was to examine the effects of light stimuli on retinal development. We reared guinea pigs under a daily 12-h strobe light (2 Hz)/dark cycle from birth, while control animals were reared under a 12-h light/dark cycle. The animals were sacrificed 1, 2, and 4 weeks after birth. The thickness of the outer nuclear layer (ONL) thickness decreased by 14.8% in the strobe-reared animals compared to the control group at 4 weeks, but not at 1 and 2 weeks. The Müller cells of the strobe-reared animals showed a stouter branch compared to that of the control animals at 2 and 4 weeks. In the strobe-reared model, axon-like processes emerging from the rod bipolar cell bodies were observed in the outer plexiform layer (OPL). These findings show that strobe-light stimuli induce morphological changes in retinal neurons, which may lead to the disturbance of normal visual processing during development.