Posterior and anterior permeability defects? Morphologic observations on streptozotocin-treated rats.

Structural abnormalities of the blood-ocular barrier were examined in streptozotocin (STZ)-treated hyperglycemic rats, after 9 days, 6 months, and 10 months' duration of "diabetes," and in normoglycemic control animals using the horseradish peroxidase tracer technique combined with light and electron microscopy. The most frequent abnormalities consisted of small areas of diffuse dense staining by the tracer of (1) the retinal pigment epithelium and (2) the nonpigmented ciliary epithelium. Pigment epithelium abnormalities occurred occasionally in both groups of animals with approximately equal frequency and extent. Ciliary body abnormalities occurred also in both groups, but were frequent; statistically, the probability of these changes was not significantly different between the two groups. At the ora serrata, tracer escape was present through the retinal pigment epithelium into subretinal space and retina. Retinal vascular leakage occurred rarely and may be related to tracer toxicity rather than hyperglycemia. Thus, using the HRP method, we cannot confirm the claim that sustained STZ-induced hyperglycemia causes breakdown of the blood-retinal barrier in the rat.

HRP/trypsin technique for studies of the retinal vasculature.

The HRP/trypsin technique is a new histologic method for the light microscopic study of the retinal blood vessels. A two-stage procedure, the first step results in a retinal whole amount preparation which permits visualization of the three-dimensional architecture of perfused vessels and their relationship within the retina. This allows analysis of gross vessel morphology and differentiation of deep and superficial vascular beds. The second step involves digesting the whole mount with trypsin and staining with hematoxylin. This permits detailed evaluation of the density of retinal capillary endothelial cells and pericytes, recognition of basement membrane ghosts, microaneurysms, and other intraretinal microvascular abnormalities, with intravascular horseradish peroxidase as a perfusion marker. This technique has been employed with success in studies of the retinal vasculature in both normal and RCS rat retinas, and in a monkey model of branch retinal vein occlusion.

Endothelial fenestrae in proliferative diabetic retinopathy.

The ultrastructure of old neovascular preretinal membranes was examined in both eyes of a patient with proliferative diabetic retinopathy treated in one eye with photocoagulation. Membranes in both eyes consisted of a matrix rich in mature collagen surrounding viable new vessels and "ghost vessels." Viable vessels of different calibers frequently showed endothelial fenestrae bridged by diaphragms. Occasionally tight junctions between endothelial cells appeared altered. Fenestrae and incompetent junctions may account for the characteristic "leakiness" of newly formed vessels.

Actin filaments in retinal pericytes and endothelial cells.

The contractile protein actin was identified within retinal capillary pericytes and endothelial cells. In capillary cross-sections, circumferential pericyte processes showed numerous parallel bundles of actin filaments forming a cap over the adjacent endothelial cells. The latter contained few actin filaments but a considerable number of intermediate filaments. Pericytes and endothelial cells communicated via gaps in the adluminal basal lamina. These special anatomic relationships are interpreted as evidence for a contractile role of retinal capillary pericytes and against a contractile role of retinal capillary endothelial cells

Permeability and patency of retinal blood vessels in experimental diabetes.

Increased permeability of retinal blood vessels in human diabetic retinopathy is well known clinically. Its morphologic equivalent is unknown. In dogs with 5 years of poorly controlled alloxan diabetes and nonproliferative diabetic retinopathy comparable to that of man, permeability and patency of retinal blood vessels were tested with the protein tracer horseradish peroxidase and evaluated by electron microscopy. A breakdown of the blood-retinal barrier was found associated with extensive tracer leakage around retinal blood vessels. Tracer had seemingly permeated endothial junctions, and was not transported through the endothelial cytoplasm. Blood vessels which had lost their endothelial cells and were partially occluded by glial cells retained some patency to tracer. These findings suggest the following. (1) Endothelial tight junctions are not a static cell specialization but one that can open due to chronic metabolic or osmotic factors prevailing in diabetes. Opened tight junctions may account for plasma leakage seen clinically in human diabetic retinopathy. (2) In the absence of endothelial cells perfusion does not necessarily end abruptly. The tracer method and electron microscopy may show details of vascular obstruction that are not readily demonstrated clinically.

Pericyte changes in branch retinal vein occlusion.

The model of experimental branch vein occlusion (BVO) in the monkey offers the opportunity to examine retinal capillaries under stress. Electron microscopic morphometry was done on 812 capillaries of 13 eyes of cynomolgus monkeys, comparing 579 capillary collaterals of 9 BVO eyes with 233 normal capillaries of 4 control eyes. The tissue underwent the myosin subfragment-1 technique to decorate and quantify bundles of actin filaments in capillary pericytes. The duration of BVO was 2-48 months. Capillary collaterals of BVO eyes had an enlarged caliber, endothelial hyperplasia, and pericyte hypertrophy, but no proportional increase in basement membrane area. Collaterals near the inner plexiform layer (IPL) had a greater wall thickness, pericyte coverage, and actin coverage than collaterals near the outer plexiform layer (OPL). Pericyte hypertrophy was proportionate to caliber increase in OPL vessels and exceeded caliber increase only in IPL vessels. Actin coverage was proportional with the vessel dilation and size of pericyte cytoplasm in all vessels. These findings indicate that capillary collaterals in BVO are not equipped morphologically for an increased regulatory role in microvascular flow beyond their normal function.

Fibronectin distribution in the rat eye. An immunohistochemical study.

Previous studies of fibronectin (FN) distribution in eye tissue have relied on immunofluorescence (IF) techniques on frozen sections, and have not included the rat. Using rat eyes, a technique was developed for immunoperoxidase (IP) staining of formalin fixed paraffin embedded material, and the results were compared to those obtained by IF. IP was technically more difficult, and required pepsinization of tissue after formalin fixation to obtain consistent results. The optimum pepsin time varied for different structures in the eye. IP offers better tissue preservation and stain resolution. Results with IF were consistent with those observed with the newly applied IP. The distribution of FN in rat eyes was similar though not identical to that reported in other species. Prominent stain was observed in the conjunctiva, basement membrane of the corneal epithelium, corneal stroma, anterior aspect of Descemet's membrane, trabecular meshwork, perivascular stroma of the ciliary body, choroid and retinal blood vessels. Lens structures, vitreous, and internal limiting membrane of the retina were negative.

An ocular renin-angiotensin system. Immunohistochemistry of angiotensinogen.

The circulating renin-angiotensin system (RAS) is an important determinant in maintaining adequate systemic blood pressure, and it also may modify organ-specific blood flow. All recognized RAS components have been identified in the eye. In this study, angiotensinogen (ANG) was localized using an affinity-purified antibody and paraffin sections of seven human eyes. An antibody for human serum albumin was used for comparison. The ANG was present selectively in the cytoplasm of the nonpigmented ciliary epithelium (NPCE), more prominently in the pars plana than in the pars plicata. Both ANG and albumin were present in the blood vessel lumina of the uvea and retina. Both antibodies also stained perivascular tissue in the uvea, but not in the retina, reflecting the relative tightness of blood-tissue barriers. The detection of ANG in the NPCE may be significant in view of previous descriptions localizing prorenin and angiotensin-converting enzyme in the same cell layer.

Systemic hypertension produces pericyte changes in retinal capillaries.

PURPOSE: The purpose of this study was to examine retinal capillaries and their pericytes that previous research suggests to be contractile. A contractile role regulating capillary blood flow may be more apparent when the vasculature is subjected to the stress of systemic hypertension. METHODS: Using ultrastructural morphometry and the myosin subfragment-1 technique, retinal capillaries of normal and hypertensive rats were measured at three different time points, early, intermediate, and late (24, 44, and 68 wk). RESULTS: Hypertensive capillaries seemed to dilate at the early time point (P = 0.002), were constricted at the intermediate time point (P < 0.001), and did not redilate later. Wall thickness was enlarged at all times, pericyte coverage (the ratio of plasma membrane length in contact with the vascular circumference to the outer circumference of the endothelial tube) was greater at early and intermediate time points, and the total area of viable cytoplasm relative to the vessel wall area was increased at the intermediate time (all P < 0.001). Also, at the intermediate time, the circumferential coverage of the endothelial tube by actin filament bundles within pericytes and the actin area relative to the vessel wall area had increased (P < 0.001). CONCLUSIONS: These data indicate that the effects of systemic hypertension extend into the retinal capillary bed, causing pericyte change with actin increase and capillary constriction. They represent the first in vivo indirect evidence by morphologic criteria for pericyte contractility in retinal vascular disease.

Ocular renin-angiotensin: immunohistochemical evidence for the presence of prorenin in eye tissue.

Angiotensin II (A2) is a vasoconstrictor generated by the renin-angiotensin system. A2 appears to act also as an angiogenic factor. Recent evidence suggests that renin is synthesized at many tissue sites and may generate A2 locally. Local A2 may have important functions in the normal and diseased eye. We examined eight human eyes by immunostaining with an antibody to prorenin, the biosynthetic precursor of renin. In all eyes, prorenin staining was extensive in the pars plicata of the ciliary body suggesting that the ciliary body synthesizes renin and this renin may be part of an ocular A2 generating system.

Septic choroiditis with serous detachment of the retina in dogs.

The present study describes a model of multifocal septic choroiditis with serous retinal detachment after intracarotid injection of Staphylococcus aureus or Streptococcus faecalis. The fundus lesions occurred mainly in the tapetal area and, on ophthalmoscopic examination, were more extensive after S. aureus than after S. faecalis injection. On histopathologic examination there were microabscesses in the inner choroid and subretinal space, disrupting the outer retina but sparing the inner retina.

Transgenic mice with pigmented intraocular tumors: tissue of origin and treatment.

PURPOSE: To describe the cell of origin, tumor progression, light and electron microscopic appearance, immunohistochemical properties, and response to frequently used anticancer therapies in two transgenic models of intraocular melanoma. METHODS: Two lines of transgenic mice that develop pigmented intraocular tumors were produced with the SV40 T and t antigens under the control of the mouse tyrosinase gene. Tumors were sequentially studied and characterized by light microscopy, electron microscopy, and immunohistochemistry stains. Tumor response to two cycles of dacarbazine was assessed on the basis of tumor size in one group of animals. Response to external beam irradiation was measured by survival time in other animals. RESULTS: Two lines of transgenic mice developed bilateral intraocular tumors with complete penetrance and without primary cutaneous melanomas. Tumors developed first in the retinal pigment epithelial layer, with subsequent retinal and choroidal invasion, extraocular extension, and metastasis. Tumors stained positive for S-100, HMB-45, and Fas-ligand. Electron microscopy revealed polarization of tumor cells with basement membrane formation, microvilli, immature melanosomes, and abundant endoplasmic reticulum. Dacarbazine significantly reduced tumor size in these mice, and a trend toward dose-dependent decrease in survival was found with external beam irradiation. CONCLUSIONS: Tumors developed from the retinal pigment epithelium. Their histology and growth, however, closely resembled that of human choroidal melanoma. This model may be a useful tool for future studies of endogenous primary pigmented tumors limited to the eye. Response to standard therapies suggests it can serve as a model with which to evaluate therapeutic modalities.

Repair of the pigment epithelial barrier following photocoagulation.

Mild argon laser burns produced in the retina of cynomolgus monkeys underwent differentiated retinal repair with frequent restoration of the pigment epithelial barrier. Permeability of the restored barrier was tested by two tracers injected intravenously--horseradish peroxidase followed by electron microscopic evaluation and fluorescein sodium followed by fluorescence microscopic examination of freeze-dried tissue. Tightness of the barrier was associated with regularly arranged pigment epithelial cells joined by zonulae occludentes. Leakage of the barrier was associated with discontinuities in the line of regularly proliferated pigment epithelial cells. At times, the tracers stained the pigment epithelial cells diffusely but they did not accumulate in the subretinal space. Variable changes in the choriocapillaris were noted. Mild photocoagulation may be effective in treating central serous choroidopathy in man when restoration of the pigment epithelial barrier is stimulated.

Clinicopathologic correlation of xenon arc and argon laser photocoagulation. Procedure in human diabetic eyes.

Xenon arc and argon laser lesions applied as scatter treatment to two diabetic patients were studied clinically and histopathologically. Seventy-one burns were grouped as very severe, severe, and moderately severe, according to pigmentation and diameter of the retinal scar. All lesions except half of the moderately severe ones showed full-thickness retinal involvement. In very severe xenon burns, whether xenon or argon, only proliferated retinal pigment epithelium provided a thin retinal scar, corresponding to an ophthalmoscopic appearance of "bare sclera". In the center of severe and moderately severe lesions, both glial cells and pigment epithelium bridged the retinal defect, forming a thick pigmented scar that contained pigment-laden macrophages. Rephotocoagulation over such thick scars may be less hazardous than is generally assumed. Nonvascular preretinal membranes were frequently present over very severe xenon lesions but rarely over very severe argon burns.

Histopathology of focally photocoagulated preretinal new vessels.

The histopathologic effects of focal xenon arc and argon laser photocoagulation on three patches of preretinal new vessels were studied in a patient with proliferative diabetic retinopathy. Both treatment modalities achieved total obliteration of the vessels. However, the adjacent retina and choroid were severely damaged, and preretinal membranes were seen over all three patches. Local tissue destruction is minimized when preretinal neovascularization is treated indirectly by scatter photocoagulation. Currently, scatter photocoagulation is our treatment of choice.

Squamous cell carcinoma of the conjunctiva presenting with intraocular extension.

A 73-year-old man, when first seen clinically, had extensive intraocular invasion from a limbal squamous cell carcinoma after only a six-week history of symptoms. This mode of initial presentation is rare for this tumor type.

Recessive dystrophic epidermolysis bullosa.

Recessive epidermolysis bullosa dystrophica (REBD), a bullous disorder of the skin and mucous membranes, is frequently associated with ocular complications. A 40-year-old woman presented with lid ulcerations, chronic conjunctivitis, diffuse subepithelial corneal scarring, corneal ulceration, and cataracts. Management with intensive lubricant therapy, soft-bandage contact lenses, and cataract extraction successfully restored her sight. Histologic examination via light and electron microscopy revealed blister formation and scarring beneath the epithelial basement membrane of both the skin and cornea, confirming the diagnosis of REBD. The ultrastructure of the skin and cornea and the ocular complications of the major forms of epidermolysis are described herein. To our knowledge, successful ocular management of a confirmed case of REBD with complete ocular histopathologic features has not been documented.

Experimental photocoagulation of the human retina. I. Correlation of physical, clinical, and pathologic data.

Thirty-eight retinal burns were inflicted by a modified xenon-arc photocoagulator in the eyes of eight human volunteers. The ophthalmoscopic characteristics of these lesions were correlated with the degree of histopathologic damage. Definitive clinical criteria that helped to predict the severity of the retinal burns were established. The dose of radiant energy received was further correlated with the histopathologic changes in determining the threshold of safety standards of bright flashes of white light to the human eye. Lesions from patients with blue irises were produced by considerably higher dose rates than comparable lesions from patients with brown irises and the fovea was more susceptible to damage than the rest of the retina.

Experimental photocoagulation of the human retina. II. Electron microscopic study.

Minimal retinal damage in xenon-arc photocoagulation lesions in man 14 hours to three days after exposure was confined to the retinal pigment epithelium (RPE) and consisted of accumulation of lysosome-related bodies in the cytoplasm. Slightly more intense damage consisted of dilation, vacuolization, and breakdown of the lamellae of the smooth-surfaced endoplasmic reticulum, preferentially around melanin-rich cell portions. Underneath more heavily vacuolated cells of the RPE, already in the periphery of ophthalmoscopically just-visible lesions, damage extended into the endothelial cells of the choriocapillaris and into the photoreceptor elements. Macrophages escaped from the choroidal circulation to appear on both sides of the Bruch membrane and within the subretinal space. More intense lesions showed features identical to those described in monkeys and are expected to show the same morphologic pattern of healing.

Sub-pigment epithelial membranes after photocoagulation for diabetic macular edema.

OBJECTIVE: The chronic histopathologic effects of focal and grid argon laser photocoagulation were examined in eyes obtained at autopsy that had previously been treated for diabetic macular edema. The focus was on further characterizing fibrous sub-pigment epithelial membranes that previously had been shown to extend beyond burn edges. DESIGN: A total of 131 argon laser burns were evaluated in five eyes. Tissue was embedded in paraffin or glycol methacrylate, serially sectioned, and examined by light microscopy. MAIN OUTCOME MEASURE: Outer and inner nuclear layer defects were measured, and the frequency and extent of sub-pigment epithelial membranes was estimated. The presence of Müller cell processes among membranes was evaluated by immunostaining for glial fibrillary acidic protein and enzyme histochemical staining for carbonic anhydrase. RESULTS: Burns consistently produced defects in the outer nuclear layer that were larger than the spot size of the laser beam. Inner nuclear layer defects were present in only seven of 131 burns. Glycol methacrylate--embedded tissue sections from 73 burns showed sub-pigment epithelial membranes in all five eyes. In one eye, membranes were confluent between burns. In the remaining four eyes, 37 individual membranes were found among 53 burns, and 47% of membranes contained Müller cell processes. The membranes in paraffin-embedded tissue could not be adequately evaluated. CONCLUSIONS: After focal laser treatment for diabetic macular edema, the inner retina was usually spared. Fibrous sub-pigment epithelial membranes were frequent among burns in all five eyes, and they showed a conspicuous contribution by Müller cell processes. We speculate that by impairing the overlying pigment epithelium, these membranes may contribute to a progressive enlargement of laser scars.