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We have examined the development of microglia in the rat retina, using a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Retinas were studied from animals aged from E(embryonic day)12, just after the invagination of the optic cup and prior to the closure of the optic fissure, to adulthood. The lectin also proved a sensitive label for the endothelial cells of the developing retina. Our results provide some support for the view that microglia are derived from the monocyte-macrophage series of blood cells. At E12, most labeled cells were found at the vitreal surface, suggesting that they had come from the hyaloid circulation, while some had entered the retina and appeared to be migrating towards its ventricular surface. From E14 to early postnatal ages, most labeled cells had processes and resembled the amoeboid microglial cells described in silver carbonate staining studies (Ling, 1982). The number of labeled cells rose from about 700 to E14 to a peak of about 27,000 at P(postnatal day)7, and fell to about 19,600 by P12. As early as E16, a regularity was apparent in the distribution of microglial cells over the surface of the retina, the cells tending to avoid each other. Microglial cells are found throughout the thickness of the very young retina, but as the layers of the retina differentiate, they are increasingly restricted to the inner half of the retina. Our findings indicate that microglia enter the retina well before the period of neuronal death, making it unlikely that they invade the retina solely in response to cell death. Our results confirm however that, once in the retina, microglia become associated with, and appear to phagocytose, the pyknotic debris which appears during the period of neuronal death. They also become closely associated with the retinal vasculature. In the adult, the intensity of the labeling of microglia was much reduced. Those cells which were labeled appeared more differentiated, resembling the "resting microglia" described in earlier studies.

The results of many investigations on the morphology of the eyes of Vertebrates are proposed, comparing our personal data with the literature available on this argument. It is firstly taken into account the retinal nourishment: it takes place according to direct and indirect mechanisms (Scheme 1). The former (Scheme 2) consist of intraretinal blood vessels and are particularly rare among the Vertebrates; the latter are more numerous and can be classified as constant (choriocapillaris) (Schemes 3A, B), if they are present in all the species up today investigated, or unconstant [Müller cells increased in their number and size (Scheme 5A), papillary cone (Schemes 5B, 8B), membrana vascularis retinae (Schemes 6A, C), falciform process (Schemes 6B, 7A, B, 8B), pecten oculi (Schemes 8A, B)], if they can be demonstrated only in some species, even if belonging to different classes. Their structural and ultrastructural organization, likewise their embryological processes, are examined in detail. The effects of the cyclic changes of light and darkness during a 24 hrs period on the eyeball are then examined; quantitative and qualitative modifications of the mitochondria (Schemes 9A, B) and of the synaptic ribbons (Schemes 10A, B) in the outer plexiform layer, of the photosensitive disks of rods and cones, and of the secretory cells and of the excretory ducts of the Meibomian tarsal glands (Schemes 11A, B, 12A, B) are described. If the animals are exposed to prolonged darkness, no structural changes can be demonstrated; nevertheless, a dark environment can induce the differentiation of peculiar structural specializations, such as the retinal or choroidal tapetum lucidum (Schemes 13, 14, 15). On the contrary, the light, if prolonged or of high intensity, is able to induce irreversible lesions on the photoreceptors and on the pigment epithelium, according to similar mechanisms in all the animals up to now investigated (Scheme 16). In conclusion, it is confirmed the peculiar structural complexity of the eyeball in all the classes of Vertebrates, due to adaptation to the various environmental requests and developed either during the evolutionary processes or during the different steps of the life of a single animal.

Cytogenesis in the cat retina was studied using tritiated (3H) thymidine autoradiography and nuclear stains. Three zones of cell division were recognized. In the first zone cell cleavage occurs at the outer limiting membrane. The distribution of these mitotic figures is uniform as early as an embryonic age of 29 days (E29) until E50. At about E50 a "cold spot", made apparent by the absence of mitotic figures, is evident at the site of the developing area centralis. This spreads to encompass the whole retina by postnatal day 10 (P10). A second zone of cell division was recognized by the presence in the developing inner nuclear layer of 3H-thymidine labelled nuclei which do not migrate to the outer limiting membrane (ventricular surface) to divide. Some of these labelled nuclei are located in regions of the retina where cytogenesis at the ventricular surface has ceased. A third zone was observed in the optic nerve fibre/ganglion cell layers from about E54 until beyond the first postnatal month. This activity gives rise to vascular endothelial cells in the nerve fibre/ganglion cell layers. Once established, the developing vascular cells invade the inner plexiform and inner nuclear layers to form the deeper capillary net.

We have observed that the lectin Ricinus communis agglutinin I (RCA), which binds to terminal galactose moieties, serves as a marker for vasculature in the mouse retina. The binding of fluorescein-isothiocyanate-conjugated RCA was used to study the development of retinal vasculature in normal mice and in rd (retinal degeneration) mutant mice, which exhibit a massive loss of photoreceptor cells during the first month of life. In the normal mouse, retinal capillaries develop in an ordered manner and are concentrated in three major zones between the inner limiting membrane and the outer plexiform layer. In the rd mouse, the vessels appear to form normally but begin to degenerate by the end of the second postnatal week. By the end of the fourth postnatal week there is approximately a 35% reduction in the total number of vascular profiles in the rd retina compared to normal littermate controls. This reduction in vascularity is temporally associated with the photoreceptor degeneration.

A pathologic analysis of 40 autopsy cases of chronic retinopathy of prematurity is presented. The type of retinal detachment in chronic retinopathy is unique, resulting from progressive changes in the peripheral retina: folding at the site of extraretinal vasoproliferation; scroll-like rolling anteriorly towards the lens; foreshortening of entire retina with detachment; and closure of the retinal "funnel." The biomechanical intraretinal and extraretinal factors operating during this process are discussed. Also presented are some interesting atypical features found in this series: arrested retinopathy; "sea fan" extraretinal vascular fronds; retinopathy in anencephaly; cribriform vanguard; and extramedullary erythropoiesis.

Cultured mural cells (intramural pericytes) from adult rhesus retinal capillaries were examined for the presence of the sorbitol pathway. A radioimmunoassay for human aldose reductase, cross-reactive with rhesus lens aldose reductase, showed the presence of this enzyme in our cultured cells. Mural cells grown in culture media containing normal (10 mM) and high (40 mM) levels of glucose were examined for polyol accumulation by gas-liquid chromatography. Cells incubated in high glucose medium showed a threefold increase in sorbitol concentration over cells grown at low glucose levels. After 30 days in high glucose medium, mural cells formed dense multilayered areas with extensive cellular debris. These findings suggest the presence of the sorbitol pathway in cultured retinal mural cells and cellular degeneration in high glucose medium; this may have possible implications in the pathogenesis of diabetic retinopathy.

By cannulating the stroma of rabbit corneae, and introducing extracts of retina and vitreous from newborn kittens subjected to vaso-obliterative doses of oxygen, an attempt was made to test the hypothesis that ischaemic retinal tissue releases a factor with vasoformative properties. It was found that, although retino-vitreal extracts from the test animals elicited a positive response in just over half the experiments, the degree of corneal vascularisation provoked by extracts from intact control animals was only marginally less. Consequently our findings do not permit any definite answer regarding the existence of a specific factor responsible for the proliferation of new vessels on the retinal surface in states associated with retinal hypoxia.

Maintenance of newborn kittens in an oxygen rich atmosphere followed by a recovery period in a normal atmosphere mimicked the effects of the human disease retrolental fibroplasia. The retinas of such kittens contained significantly raised levels of low molecular weight angiogenic material (as measured by procollagenase activation) when compared with those of a control group of kittens.

Monosodium glutamate (MSG) administered to neonatal rats on postnatal days 1 to 10 caused a generalized degeneration of the inner retinal layers. MSG administered only on postnatal days 8, 9, 10, and/or 11 caused a retinopathy limited to more peripheral retinal areas corresponding to currently existing regions of immature retinal vessels. Ink-injected retinal vessel studies showed a delay in development of the retinal vessel network but no alterations in vessel patency. Fluorescence microscopic examination of freeze-dried tissues revealed to abnormalities of the blood-retinal barriers to sodium fluorescein. We conclude, as demonstrated by these methods, that MSG retards development of the retinal vessels but does not affect development of the blood-retinal barriers. The retinotoxic effect of MSG apparently results from a mechanism(s) other than a breakdown of the blood-retinal barrier.

As a rule, in long-lasting diabetes type 1 there appear vascular complications in a form of proliferative retinopathy. While looking for the causes of fast vascular changes, the authors considered the possibility of immunological disorders. They started prospective examinations of patients with diabetes type 1 and proliferative retinopathy changes in the fundus of the eye. The authors evaluated the activity of angiogenic factors released by mononuclear cells isolated from peripheral blood of the patients examined. The authors found a significantly higher activity of these factors in patients with proliferative retinopathy in comparison with a control group of healthy persons and a group of patients with diabetes type 1 and simple retinopathy. The results obtained may indicate to immunological changes in the pathogenesis of proliferative retinopathy.

The pattern of retinal vasculative is described and the position at which cell proliferation at the ventral retinal margin is maximal was shown to be at the point of entry of the ventral blood vessels. To test whether there is a causal relation between retinal blood supply and retinal cell production, surgical inversion of the eye, transplantations and excisions of retina were done to change the pattern of retinal vasculature. The growth pattern of inverted eyes was normal with respect to the internal axes of the eyes. After excision of part of the retina or after fusion of retinal fragments to form compound eyes, the pattern of retinal cell proliferation was not correlated with the distribution of retinal blood vessels, but was correlated with the position(s) of the choroidal fissure(s).

Forty-two eyes in 30 patients with disc neovascularization were treated by the method of extensive photocoagulation of the retinal periphery (ablation). Total obliteration of disc neovascularization was accomplished in 76% of eyes treated. Partial regression was noted in 21% of eyes treated. There were only 2 major complications--traction retinal detachment and one major hemorrhage--in this series. Feeder-frond treatment was necessary to obliterate recurrent disc neovascularization in 2 eyes. It is concluded that therapy is efficacious in the treatment of papillary neovascularization.

A 32-year-old obese woman with hypertension and a three-year history of pseudotumor cerebri developed bilateral juxtapapillary subretinal neovascular membranes. To our knowledge, this is the first reported case of bilateral subretinal neovascular membranes complicating the course of this disease. The subretinal neovascular membrane in the left eye spontaneously involuted, but because the membrane in the right eye threatened the foveola, the patient underwent argon-laser photocoagulation. The subretinal fluid and hemorrhage progressively resolved, the membrane was replaced by fibrous tissue, and visual acuity improved. The pathogenesis of the subretinal neovascular membranes was presumably secondary to pressure deformity of the border of Bruch's membrane at the optic disk, creating a discontinuity of normal anatomic apposition of the chorioretinal layers. This anatomic dehiscence, coupled with hypoxia created by axonal tissue swelling and resultant impaired vascular perfusion of the tissues, led to the development of subretinal neovascular membranes.

Capillaries from bovine, monkey, and human retinas maintained in tissue culture produced a monolayer of cells. Autoradiographic and electron microscopic evidence indicated that the mural cells (intramural pericytes) were the cells that proliferated. Since intramural pericytes are damaged selectively in diabetes mellitus, their availability in culture will be useful in seeking means to control diabetic retinopathy.

The murine retina provides an ideal model for the study of vascular development. In this investigation we have examined the development of blood vessels in flat-mounted whole retinas from C57B6 mice ranging from birth to 4 months of age. Basement membrane components of blood vessels were visualized by indirect immunofluorescence with antibodies against type IV collagen and laminin. Endothelial cells (EC) were labeled with a plant lectin, Ricinus communis agglutinin I (RCA), and antibodies against angiotensin converting enzyme. Results show three stages of vascular differentiation. During the first stage (postnatal days P0-P10), vessels develop radially from optic disc to ora serrata within the presumptive nerve fiber layer. In the second stage beginning P4, vessels form within deeper retinal layers. In the third stage beginning P7, a capillary network develops as branches of radial vessels in the nerve fiber layer. The entire vascular system begins as a polygonal network of capillary-like vessels. Selective regression of various segments of these polygons leads to the ultimate arborous pattern of arteries, arterioles, veins, venules, and capillaries seen in the adult. Some individual EC appear to be left behind during this retraction process and pericytes may have a role in determining which vessel segments regress. This combination of flat-mounted whole retinas and probes specific for vascular elements provides an ideal system for the study of retinal vascularization and the characterization of vasculogenesis in general.

Metabolic demands of the ocular tissues are determinants for the presence, morphology, and permeability of the ocular blood vessels. Changes in those metabolic requirements, such as those which occur during tissue maturation or a disease process, can lead to an alteration of the unique permeability and morphology of each nutrient vessel system. Those alterations during the course of a disease are the basis for the well-known visually destructive complications associated with vascular retinopathies. In order to prevent or circumvent those complications, we must come to understand both normal and abnormal tissue environmental factors which are determinants of vessel morphology and permeability. Investigations of those determinants in animals having fully, partially, or non-vascularized retinae and in animals with experimental retinopathies are necessary to the understanding of tissue/blood vessel inter-relationships. It is also necessary to evaluate those same relationships as fully as possible in human eyes with and without vascular abnormalities. Then we can begin to understand and to control blood vessel development.

Increasing numbers of immature infants are surviving with incompletely developed retinal vessels at risk for ROP. Since adverse perinatal events seem closely associated with the more severe forms of the disorder, neonatologists may be able to contribute most to the reduction of vision loss if they can optimize the neonatal course of these infants. However, to date, no particular intervention has been proven to be efficacious in preventing ROP. On the other hand, cryotherapy has been found effective in stopping the progress of severe ROP to retinal detachments about half the time. This places a responsibility on the neonatologist and ophthalmologist to assure that each at-risk infant is examined by 4-6 weeks of age to enable the prompt application of cryotherapy if severe ROP develops. This is presently the infant's best chance of retaining useful vision, and appropriate nursery policies should be developed to ensure its availability. Additional research is needed to understand the control of regression vs progression in ROP and to explore potential means of preventing the disease altogether.

We studied the ultrastructural characteristics of the mitoses of the retinal vascular endothelium during normal vasculogenesis in the immature retina of 45 newborn albino rats, from birth to the end of the 1st month of postnatal development. For the first time mitotic phenomena in the endothelial cells to the adjacent endothelium during all mitosis stages. Direct budding from the pre-existing vessels seems to be the actual mechanism of growth of the retinal vessels in the holangiotic immature retina.