Apolipoprotein E allele-dependent pathogenesis: a model for age-related retinal degeneration.

Age-related macular degeneration (AMD) is a late-onset, multifactorial, neurodegenerative disease of the retina and the leading cause of irreversible vision loss in the elderly in the Western world. We describe here a murine model that combines three known AMD risk factors: advanced age, high fat cholesterol-rich (HF-C) diet, and apolipoprotein E (apoE) genotype. Eyes of aged, targeted replacement mice expressing human apoE2, apoE3, or apoE4 and maintained on a HF-C diet show apoE isoform-dependent pathologies of differential severity. ApoE4 mice are the most severely affected. They develop a constellation of changes that mimic the pathology associated with human AMD. These alterations include diffuse sub-retinal pigment epithelial deposits, drusenoid deposits, thickened Bruch's membrane, and atrophy, hypopigmentation, and hyperpigmentation of the retinal pigment epithelium. In extreme cases, apoE4 mice also develop marked choroidal neovascularization, a hallmark of exudative AMD. Neither age nor HF-C diet alone is sufficient to elicit these changes. We document choroidal neovascularization and other AMD-like ocular pathologies in an animal model that exploits known AMD risk factors. The model is additionally attractive because it is not complicated by invasive experimental intervention. Our findings in this model implicate the human apoE E4 allele as a susceptibility gene for AMD and support the hypothesis that common pathogenic mechanisms may underlie AMD and Alzheimer's disease.

Role of neutrophils in breakdown of the blood-retinal barrier following intravitreal injection of platelet-activating factor.

Breakdown of the blood-retinal barrier occurs in inflammatory conditions and in ischemic retinal diseases such as diabetic retinopathy. Platelet-activating factor (PAF) is a potent inflammatory mediator which increases vascular permeability. The purpose of this study was to determine if intravitreally-injected PAF would cause breakdown of the blood-retinal barrier and, if so, by what mechanism. Fluorescein angiography was performed before and at 0.5, 1, 2, 3 and 4 hr after PAF injection into the vitreous cavity of rabbit eyes and the eyes were enucleated immediately for light and electron microscopy. Slow flowing thrombi were observed in all PAF-injected eyes. Complete vascular occlusion was observed in 10 of 16 eyes after 3 and 4 hr. There was no fluorescein leakage in any of eyes before or at 0.5 or 1 hr after PAF injection. Fourteen of 20 eyes had fluorescein leakage at 2, 3 and 4 hr after PAF injection. The extent of fluorescein leakage correlated with the degree of polymorphonuclear leukocyte (PMN) margination, disruption of the endothelial cell layer, infiltration into vascular walls and migration into the vitreous cavity. PMNs appeared to migrate by both intercellular and transcellular routes across the endothelium. Pretreatment of rabbits with a PAF inhibitor, BN52021, prevented most of the abnormal findings.

Neutrophils plug capillaries in acute experimental retinal ischemia.

Capillary plugging by neutrophils appears to be the mechanism responsible for the no reflow phenomenon following experimental ischemia in many tissues. The purpose of this study was to determine if neutrophils plug capillaries in experimental retinal ischemia. Unilateral retinal ischemia was produced in albino rats by focally exposing three adjacent retinal arterioles to argon blue-green laser light at 100 mW. Total occlusion was achieved in at least two vessels in each eye. The animals were euthanized at 3, 6, 8, or 24 hr following laser treatment. Nonlasered eyes of 17 animals served as controls. Trypsin digests were prepared of the retinal vasculature following formalin fixation. The total number of neutrophils present in the capillaries of each retina was counted using a light microscope and expressed as the number of cells per retina. Other eyes were fixed in glutaraldehyde:paraformaldehyde and the retinal tissue prepared for light and electron microscopy. The mean number of neutrophils in capillaries of retinas subjected to laser treatment was significantly higher than that in untreated control retinas at all time periods studied and increased with time from 3 to 6 hr. Intact as well as degranulated PMNs were present in the capillaries. The presence of large numbers of neutrophils plugging capillaries and their increased number with duration of ischemia supports the hypothesis that they contribute to capillary nonperfusion following acute retinal ischemia.

Ultrastructure of fiber cells and multilamellar inclusions in experimental diabetes.

PURPOSE: The goal of this ultrastructural study was to examine fiber cell shape and intercellular junctions during the early stages of fiber cell breakdown and edema in diabetic rabbit lenses. METHODS: Lens abnormalities were recorded with a slit lamp. Between 6-10 mo after drug treatment, diabetic lenses and untreated control lenses were freshly enucleated and sectioned with a vibrating knife microtome. The thick tissue sections were chemically fixed and processed for thin-section electron microscopy. RESULTS: Alloxan-induced diabetes in albino rabbits produced clinically apparent cataracts as soon as 1 mo after the animals became hyperglycemic. The cataracts displayed cortical fluid-filled vacuoles in the equatorial region and at the cortex-nucleus interface, white specks scattered throughout the cortex, and posterior subcapsular cataracts. Fiber cells just deeper than the large cortical vacuoles had oval or spindle-shaped cross sections. Multilamellar inclusions, not reported previously for diabetic lenses, were observed at or near the fiber cell interfaces and were composed of concentric or spiral rings of plasma membrane-bound cytoplasmic processes. Undulating membranes were present throughout most of the multilamellar inclusions. Transparent lenses from untreated controls did not have such multilamellar bodies or extensive membrane undulations in cells at the same distance from the lens surface. CONCLUSIONS: Fiber cells respond to the diabetic insult differently depending on their stage of differentiation and age. The observed changes are consistent with the hypothesis that hyperglycemia accelerates the formation of age-related changes in fiber cells.

Aclacinomycin A in the treatment of experimental proliferative vitreoretinopathy. Efficacy and toxicity in the rabbit eye.

PURPOSE: Aclacinomycin A is an oligosaccharide anthracycline that, by contrast with daunomycin, lacks carcinogenicity. The authors evaluated the efficacy of aclacinomycin A in prevention of experimental proliferative vitreoretinopathy (PVR) and its toxicity on the rabbit retina. METHODS: Dutch-belted rabbit were used to create a model for traction retinal detachment. Seven to 10 days after vitreous gas compression, 25,000 homologous fibroblasts were injected into the vitreous cavity. Subsequently, the eyes received either sham injections or doses of 6, 30, or 60 nmol of aclacinomycin A, respectively. The fundus findings were documented on days 7, 14, and 28 after the fibroblast injection. The toxicity studies were conducted according to the same protocol as was used for the efficacy evaluation but without the fibroblast injection. Simultaneous electroretinograms were recorded on days 0, 3, 7, and 14 from the right eyes that were injected with 30 or 60 nmol of aclacinomycin A and the left eyes that were sham injected. Morphologic studies were conducted on the eyes enucleated on days 3, 7, and 14 after drug exposure. RESULTS: Intraocular administration of 30 nmol of aclacinomycin A on day 2 after fibroblast injection resulted in a detachment rate of 37.5% (controls, 100%; P < 0.01, by Fisher's exact test). Administration of 60 nmol of aclacinomycin A 3 days after fibroblast injection resulted in a detachment rate of 26.7% (controls, 100%; P < 0.0001). Six nanomoles of aclacinomycin A 3 days after fibroblast injection had no effect. No electroretinogram changes were present in eyes treated with 30 nmol of aclacinomycin A. Such recordings from eyes exposed to 60 nmol of aclacinomycin A demonstrated decreased a- and b-waves on day 3; these completely recovered by day 7. Morphologic studies of these eyes revealed no damage to the retina. CONCLUSIONS: These results suggest that aclacinomycin A should be considered an alternative to daunomycin for treatment of human PVR because, in addition to its lack of carcinogenicity, it shows good efficacy and causes less retinal toxicity.

The effect of combined daunorubicin and triamcinolone acetonide treatment on a refined experimental model of proliferative vitreoretinopathy.

Prior studies have shown that intravitreal daunorubicin (9-15 nmol) and triamcinolone acetonide (2 mg) are effective individually in preventing retinal detachment in experimental proliferative vitreoretinopathy. This report compares the efficacy of the combination of daunorubicin (15 nmol) and triamcinolone acetonide (2 mg) with that of daunorubicin alone in a refined experimental model of proliferative vitreoretinopathy. The degree of retinal detachment in each treatment group was graded, with the unequivocal absence or presence of retinal detachment used as an indicator of treatment success or failure. Both treatments (daunorubicin alone and in combination with triamcinolone acetonide) effectively prevented retinal detachment. However, there was no significant difference in the rate of retinal detachment between the two treatment groups. These results indicate that combination therapy with daunorubicin/triamcinolone is no more effective at preventing retinal detachment than daunorubicin alone.

Treatment of experimental preretinal neovascularization using photodynamic thrombosis.

Retinal or preretinal neovascularization (NV) is the result of many ischemic conditions of the retina and is an important factor leading to severe visual loss in diabetic retinopathy. Panretinal photocoagulation does not always control its growth or bleeding sequelae. A potential new treatment modality, photodynamic therapy (PDT), was evaluated for limiting the progression of experimental NV in the rabbit eye. The NV was produced by injecting cultured dermal fibroblasts into the preretinal vitreous space after combined enzymatic and mechanical vitreolysis. This method results in traction retinal detachment with a rapid and consistent growth of NV. After administration of the photosensitizing dye rose bengal (20 mg/kg intravenously), PDT was done using a slit-lamp light source focused through a fundus contact lens (45 J/cm2). The NV was treated on two separate occasions during the active phase of growth (on days 13 and 21 after fibroblast injection). Control animals were exposed to light before injection of rose bengal. Eight randomly assigned animals in each group were followed between treatments and for 28 days after the second treatment. The appearance of NV was documented by frequent photography and fluorescein angiography. The PDT resulted in thrombosis of NV for at least 3 days. Reperfusion, however, was consistently noted at 7 days. Thrombosis was associated with a delay in the growth and maturation of NV fronds, which resumed after reperfusion. Twenty-eight days after the second treatment, NV in both experimental and control eyes had undergone atrophy. At that time (the conclusion of follow-up), however, the size of treated NV fronds (estimated from computerized image analysis of fluorescein angiograms) was significantly less than that of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Daunorubicin treatment in a refined experimental model of proliferative vitreoretinopathy.

A condition similar to proliferative vitreoretinopathy (PVR) in man can be produced by injecting 25,000 homologous dermal fibroblasts into rabbit eyes following gas compression of the vitreous. Daunorubicin (15 nmol) was effective in preventing retinal detachment in this model when injected simultaneously with the fibroblasts or in two doses (10 nmol followed by 5 nmol 4 h later) on the 3rd day after fibroblast injection. A single dose of 15 nmol on the 3rd day was not effective in preventing retinal detachment. These results suggest that daunorubicin may be clinically useful in preventing PVR when given by injection both at the time of vitrectomy as well as later, when protein exudation and pigment clumps in the vitreous cavity herald the onset of PVR.

The safety of intravitreal hyaluronidase. A clinical and histologic study.

The authors previously developed a new model of preretinal neovascularization in the rabbit eye using hyaluronidase for enzymatic vitreolysis. The purpose of this study was to evaluate the safety of intravitreal injections of hyaluronidase. Concentrations of 1, 15, 30, 50, and 150 IU of hyaluronidase in 0.1 ml of 0.9% saline were injected intravitreally and aspirated repetitively until the vitreous was partially liquified. The animals were examined with indirect ophthalmoscopy, fundus photography, and fluorescein angiography before injection and on days 1 and 7 after injection. Light and electron microscopic retinal sections were prepared from enucleated eyes at days 1 and 7. All concentrations of hyaluronidase were effective in producing partial vitreolysis. Eyes treated with 1 IU showed no abnormalities on days 1 or 7. Eyes treated with 15 IU showed no retinal abnormalities on day 1, but on day 7 histologic abnormalities were present in two of four eyes. At higher concentrations, clinical and histologic changes were seen in proportion to the concentration and included focal whitening, edema, vitreous haze, vascular abnormalities, and retinal necrosis at the highest doses. Histologic evaluation of the retina revealed marked destruction in all layers at the higher concentrations. The authors conclude that 1 IU of intravitreal hyaluronidase is sufficient for partial vitreolysis and nontoxic to the rabbit retina.

Reduction of basement membrane thickening in diabetic cat retina by sulindac.

Capillary basement membrane thickening is one of the earliest histologic lesions in diabetic retinopathy. Its pathogenesis is not understood; however, recent evidence suggests that aldose reductase may play a role. In this study, a new animal model, the diabetic cat, was used to determine whether retinal capillary basement membrane thickening occurred early in the course of hyperglycemia, and if so, whether it could be inhibited by sulindac, an aldose reductase inhibitor. Retinal capillary basement membrane thickness was significantly increased in diabetic cats compared to normal cats (114 +/- 15 vs 72 +/- 12 nm, mean +/- SD) [corrected]. Basement membrane thickness was significantly less in sulindac-treated diabetic cats (93 +/- 9 nm) compared to the untreated diabetic cats (114 +/- 15 nm). In addition, quantitation of endothelial cells, pericytes, and contacts between endothelial cells and pericytes in electron micrographs revealed that they were not reduced in number in untreated diabetic cats compared to normal or sulindac-treated diabetic animals.