Peripheral retinal neovascularization in birdshot retinochoroidopathy.

A 69-year-old black woman had ocular findings consistent with birdshot retinochoroidopathy; these included cream-colored depigmented spots at the level of the retinal pigment epithelium and vitreitis. She also had peripheral retinal neovascularization with localized vitreous hemorrhage. There were no laboratory or systemic findings consistent with other disease entities.

Difference in effect of cultured fetal pancreas transplants on retinal and renal capillary basement membrane thickness in diabetic mice.

The goal of endocrine pancreas transplants should be the prevention of diabetic complications. The differential effect of grafts of organ-cultured fetal mouse pancreas on diabetic complications in the retina and kidney was tested by comparing capillary basement membrane thickness (BMT) in mice made diabetic with streptozotocin and transplanted either early or late, or treated with insulin. BALB/c female mice were grafted with a single organ-cultured syngeneic fetal pancreas at either 3 weeks or 7 months after induction of diabetes. Controls were sex- and age-matched nondiabetic; diabetic untreated; and diabetic insulin-treated mice. All mice were killed at 20 months of age and their eyes and kidneys fixed for electron microscopy. BMT was measured on coded micrographs. In all mice glomerular capillary BM were thicker than retinal capillary BM. Mice grafted early after the induction of diabetes had normal BMT in both sites, while those transplanted after 6 months of disease had normal retinal, but thickened glomerular, capillary BM. In each case the late-transplanted animals had BM thickness significantly less than the insulin-treated or untreated diabetics.

A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. Ticlopidine Aspirin Stroke Study Group.

We report the results of the Ticlopidine Aspirin Stroke Study, a blinded trial at 56 North American centers that compared the effects of ticlopidine hydrochloride (500 mg daily) with those of aspirin (1300 mg daily) on the risk of stroke or death. The medications were randomly assigned to 3069 patients with recent transient or mild persistent focal cerebral or retinal ischemia. Follow-up lasted for two to six years. The three-year event rate for nonfatal stroke or death from any cause was 17 percent for ticlopidine and 19 percent for aspirin--a 12 percent risk reduction (95 percent confidence interval, -2 to 26 percent) with ticlopidine (P = 0.048 for cumulative Kaplan-Meier estimates). The rates of fatal and nonfatal stroke at three years were 10 percent for ticlopidine and 13 percent for aspirin--a 21 percent risk reduction (95 percent confidence interval, 4 to 38 percent) with ticlopidine (P = 0.024 for cumulative Kaplan-Meier estimates). Ticlopidine was more effective than aspirin in both sexes. The adverse effects of aspirin included diarrhea (10 percent), rash (5.5 percent), peptic ulceration (3 percent), gastritis (2 percent), and gastrointestinal bleeding (1 percent). With ticlopidine, diarrhea (20 percent), skin rash (14 percent), and severe but reversible neutropenia (less than 1 percent) were noted. The mean increase in total cholesterol level was 9 percent with ticlopidine and 2 percent with aspirin (P less than 0.01). The ratios of high-density lipoprotein and low-density lipoprotein to total cholesterol were similar in both treatment groups. We conclude that ticlopidine was somewhat more effective than aspirin in preventing strokes in this population, although the risks of side effects were greater.

Cataract formation is prevented by administration of verapamil to diabetic rats.

The purpose of the present study was to examine the effects on cataractogenesis of daily sc administration of the Ca2+ antagonist drug verapamil to diabetic rats. Streptozotocin-induced diabetic rats were given verapamil half-way through the 8-week experimental period or during the full 8 weeks of diabetes. Verapamil administration had no effect on the high blood glucose values, low circulating insulin levels, or elevated triglyceride and cholesterol concentrations in the diabetic rats. Untreated diabetic rats had a 90% incidence of cataracts. Four weeks of verapamil administration reduced this incidence to 41%, and a full 8 weeks of drug treatment further lowered the incidence to 20%. Diltiazem, another Ca2+ antagonist, lowered the incidence of cataracts in the diabetic rats to a similar extent. Verapamil administration to the diabetic animals also partially protected against the presence of retinal microangiopathy in the diabetic animals. Lenticular hydration and lipid accumulation were only indirectly related to cataractogenesis in the diabetic rats and its protection by verapamil treatment. Lenticular electrolyte imbalance, particularly Ca2+, in the diabetic animals was closely correlated with cataract formation, and verapamil significantly reduced the alterations in these ion concentrations. The present results demonstrate the efficacy of verapamil as a protective agent against cataractogenesis and some retinal damage in diabetic animals. Most importantly, this occurs in the absence of any change in the glycemic status of the diabetic animals. The findings strongly support a role for lenticular Ca2+ imbalance in cataract development in diabetes and provide initial evidence to suggest its clinical use in the diabetic population at risk for blindness.

Histopathology of experimental preretinal neovascularization.

Despite the morbidity resulting from abnormal retinal neovascularization, morphological events associated with its development have not been fully described. We therefore studied sequential morphologic events during preretinal neovascularization in an experimental model induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of rabbits. Within 2 days following fibroblast injection, thickening of many venular and capillary endothelial cells resulted in partial obliteration of their lumina. 3H-thymidine incorporation occurred first in the nonvascular cells of the superficial medullary ray and thereafter in the preretinal vessels and extraretinal fibroblasts. Capillary budding was obvious within 3 days, with endothelial cells extending cytoplasmic processes into fragmented extracellular matrix (ECM). Endothelial cells, at the tips of budding vessels, and at more proximal sites in the parent vessel, incorporated 3H-thymidine and did not lose cell contact or migrate individually into the ECM. Lumina were present throughout the entire length of the buds and endothelial cells remained polarized. Neovascular events observed in this experimental model parallel those previously described in diabetic retinopathy and retinopathy of prematurity in humans.

Functional sorting of actin isoforms in microvascular pericytes.

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.

Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications.

Hyperglycemia is believed to be the major cause of diabetic vascular complications involving both microvessels and arteries as in the retina, renal glomeruli, and aorta. It is unclear by which mechanism hyperglycemia is altering the metabolism and functions of vascular cells, although changes in nonenzymatic protein glycosylation and increases in cellular sorbitol levels have been postulated to be involved. Previously, we have reported that the elevation of extracellular glucose levels with cultured bovine retinal capillary endothelial cells causes an increase in protein kinase C (PKC) activity of the membranous pool with a parallel decrease in the cytosol without alteration of its total activity. Now we demonstrate that the mechanism for the activation of PKC is due to an enhanced de novo synthesis of diacylglycerol as indicated by a 2-fold increase of [14C]diacylglycerol labeling from [14C]glucose. The elevated diacylglycerol de novo synthesis is secondarily due to increased formation of precursors derived from glucose metabolism; this formation is enhanced by hyperglycemia as substantiated by elevated [3H]glucose conversion into water. This effect of hyperglycemia on PKC is also observed in cultured aortic smooth muscle and endothelial cells and the retina and kidney of diabetic rats, but not in the brain. Since PKC in vascular cells has been shown to modulate hormone receptor turnover, neovascularization in vitro, and cell growth, we propose that this mechanism of enhancing the membranous PKC activities by hyperglycemia plays an important role in the development of diabetic vascular complications.

Decrease of diffusion of glycosylated albumin in retinal microcirculation by peptide fraction from bovine factor VIII.

The diffusion of the glycosylated albumin in the retinal vascular system has been studied in male New Zealand rabbits, using fluorangiographic techniques. A first group of animals was treated for 15 days with the peptide fraction from bovine Factor VIII (Vueffe); a second group, used as control group, was treated with physiological solution. At the end of treatment, glycosylated albumin was made fluorescent and then injected into the marginal vein of the rabbit. The direct observation and the photometric measurements performed on the digitized photograms with an image processing system showed a considerable reduction in retinic capillary diffusion of glycosylated albumin in the animals treated with the peptide fraction. The substance used in the study might therefore be of importance in the treatment of systemic disease with retinic vascular damage.

Macular subretinal neovascularization in choroidal tuberculosis.

A 54-year-old Chinese woman with miliary choroidal tuberculosis was followed for more than three years. She had had tuberculous meningitis for about one month before an ophthalmologic examination for blurred vision OU. There were 50 to 60 choroidal tubercles OU which were located mostly at the posterior poles including the macular areas. The meningitis and tubercular lesions resolved with antituberculous medications. In a series of fundus photographs and fluorescein angiograms, a macular subretinal neovascularization was noted in association with the tubercular lesions which resulted in disciform maculopathy. To the best of our knowledge, this case had the largest number of tubercles reported in this century, and the association of macular subretinal neovascularization with choroidal tuberculosis has never been reported.

Measurement of retinal blood velocity and flow rate in primates using a liposome-dye system.

A recently developed liposome-dye method was used to make quantitative measurements of blood velocity and volumetric flow rates in the retina of the rhesus monkey. After systemic injection of a liposome-encapsulated fluorescent dye (calcein), the release of a bolus of the dye in the retina was triggered by an ophthalmic argon laser. Initially, calcein was entrapped at a high concentration and the fluorescence of the dye in the lipid vesicle was quenched. After laser exposure, the liposomes released a bolus of dye at the laser exposure site in a specific vessel of the retina. The released dye formed a well-defined, fluorescent wavefront in the exposed artery or vein. By measuring the distance the dye traveled in the vein over a set time interval, it was possible to measure blood velocity in retinal vessels of various diameters, and to calculate volumetric flow rates in those vessels.

Intraretinal and preretinal PO2 response to acutely raised intraocular pressure in cats.

The effect of acutely raised intraocular pressure (IOP) on retinal oxygenation has been determined using oxygen-sensitive microelectrodes preretinally and intraretinally. Anesthetized cats were ventilated alternately with one of two oxygen concentrations, 21 or 100%, while the IOP was manipulated via a hydrostatic pressure head connected to the anterior chamber. Preretinal and inner retinal oxygen tension remained stable over a broad range of IOP for air breathing. In contrast, the outer retina was severely affected, with the PO2 reducing as IOP was raised, resulting in some areas becoming anoxic. Outer retinal oxygen consumption was reduced for perfusion pressures less than 50 mmHg. It is postulated that under air-breathing conditions the preretinal and inner retinal oxygen tension was stabilized by the autoregulatory capacity of the retinal circulation. The inability of the choroidal circulation to autoregulate was reflected in the reduction of outer retinal oxygen tension with increasing IOP. With 100% oxygen breathing, increases in IOP caused a significant reduction in PO2 throughout the entire retina and preretinal vitreous. It is proposed that the effect of the autoregulatory capacity of the retinal circulation on retinal oxygenation is reduced in hyperoxia.

Integrity of the blood-brain barrier in retinal xenografts is correlated with the immunological status of the host.

The purpose of this study was to determine the immunological correlates of blood-brain barrier breakdown in retinal xenografts in rats by utilizing skin grafting to initiate a timed immune response to the transplanted neural tissue. Embryonic day 13-14 CD-1 mouse retinae were grafted into the brainstem parenchyma of neonatal Sprague-Dawley rats. In one group of animals a 100 mm2 CD-1 skin graft was placed on the flank 21 days after the initial neural transplant in order to provoke an immune response to the neural graft. Control animals received no skin graft. Animals were injected with horseradish peroxidase (HRP) in the femoral vein 2-8 days after skin grafting. Brains were processed for Nissl, HRP-tetramethylbenzidine, and anti-M-6, -lymphocyte, -macrophage, and -astrocyte antibodies. Experimental and control animals injected 2-4 days after skin grafting showed no leakage of reaction product in the grafted tissue. A small percentage (one of eight) of 5-day animals showed isolated, patchy leakage, but no evidence of rejection of the neural graft. At 6 days all of the grafts showed evidence of leakage, and 71% of these grafts showed infiltration of lymphocytes. By 7-8 days extensive leakage of HRP and widespread infiltration of lymphocytes and macrophages were clearly evident. The present study demonstrates that blood-brain barrier breakdown is correlated closely with the sequence of immunological rejection of the graft. While these results confirm that a barrier exists in healthy neural transplants, they suggest that immunological factors should be considered in cases in which grafts are not protected by an intact barrier.

Idiopathic subretinal neovascularization.

Idiopathic subretinal neovascularization is a potentially blinding condition and often strikes patients in their prime of life. It appears to be seen in more females, at an earlier age than males. Treatment is possible by photoablation of the subretinal new vessel by Argon or Yag laser. There is a clinical impression that Argon laser is more effective than the Yag laser for this purpose.

The effect of arterial PCO2-variations on ocular and cerebral blood flow in the newborn piglet.

The response of ocular and cerebral blood flow to different arterial PCO2 levels was studied in ventilated paralyzed newborn piglets with the radionuclide-labeled microsphere method. The retina and the choroid have different blood flow responses to variations in arterial PCO2 levels. Retinal blood flow (ml/g/min) was increased during hypercarbia, from 0.26 +/- 0.03 at baseline to 0.51 +/- 0.07 (PaCO2 8.7 +/- 0.2 kPa) and 0.62 +/- 0.07 (PaCO2 11.0 +/- 0.2 kPa). However, no significant change was found in choroidal blood flow during hypercarbia. Cerebral blood flow was more responsive to PaCO2 than retinal blood flow, increasing from 0.71 +/- 0.03 at baseline to 2.25 +/- 0.25 (PaCO2 8.7 +/- 0.2) and 1.77 +/- 0.13 (PaCO2 11.0 +/- 0.2). Hypocarbia did not influence either retinal or choroidal blood flow.

Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells.

Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC). Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected. To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy. Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor. In contrast, no changes were observed in myoinositol or InsP levels. Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction. Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC. Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels. This decrease could be prevented by adding either PMA or sorbinil. Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.

Retinovitreal neovascularization in the Royal College of Surgeons rat.

Retinovitreal blood vessels, which we have previously reported in the dystrophic retinas of approximately 20% of older (greater than 15 months of age) spontaneously hypertensive (SHR) rats, also occur in about 20% of retinal dystrophic Royal College of Surgeons (RCS) rats greater than 1 year of age. We have demonstrated previously that these vessels, in the SHR rat, have the anatomic characteristics of retinovitreal neovascularization as it has been described in vasoproliferative retinopathies in humans (1). We now provide strong evidence that the endothelial cells and pericytes of the retinovitreal vessels that occur in RCS rats are proliferating, since they demonstrate by autoradiography significantly increased nuclear labeling with [3H]-thymidine over intra-retinal or choroidal vessels, or the abnormal vessels that grow within the retinal pigment epithelium in these dystrophic retinas. One important difference between the neovascularization that occurs in these rats and that which is observed in various human retinal vascular diseases is the frequent association of the new vessels in dystrophic rat retinas with surrounding cords of proliferating retinal pigment epithelium. The retinovitreal vessels in these strains of dystrophic rats represent a new animal model that may be useful for studying the fundamental processes underlying new blood vessel growth in the retina.

Abortive neovascular outgrowths discovered during vitrectomy for diabetic vitreous haemorrhage.

Abortive neovascular outgrowths from the retina were identified in 13 out of 34 eyes (38%) undergoing vitrectomy for diabetic vitreous haemorrhage. Postoperatively, fluorescein angiography of the lesions demonstrated a characteristic "smoke-stack" pattern of dye leakage. Each outgrowth appears to represent a focal variant of preretinal neovascularisation complicating retinal capillary non-perfusion and inner retinal ischaemia, their distinctive features being determined primarily by the absence of a cortical vitreous scaffold on the retinal surface at the time of vasoproliferation.

Experimental subretinal neovascularization in the rabbit.

Subretinal neovascularization (SRN) in the rabbit was induced by subretinal injection of vitreous without rupture of Bruch's membrane. Eight of 26 eyes developed SRN. The incidence of SRN rose from 33% to 57% in a period of 4-40 weeks. Because of the absence of any fluorescein angiographic indication of SRN, these occult new vessels were identified by light and transmission electron microscopy. Histological examination showed that these newly formed vessels are composed of continuous capillaries with the morphologic characteristics of choriocapillaris, including diaphragmed fenestrations, basement membranes, and junctional complexes. The new vessels originated from the choriocapillaris and penetrated through Bruch's membrane into the subretinal space, where they were associated with the degenerated sensory retina and proliferating glial and/or RPE cells. This experiment provides a model of SRN without breaks in Bruch's membrane.