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.

Superior cervical ganglionectomy in monkeys: light and electron microscopy of the anterior eye segment.

Morphological changes in the anterior eye segment of eight cynomolgus monkeys were investigated 2 days to 2.2 years after unilateral surgical superior cervical ganglionectomy (SCGx). SCGx was confirmed by histologic examination of the excised surgical specimen and persistent ipsilateral miosis. In four short-term monkeys (2, 4, 7 and 11 days), iris, ciliary muscle and trabecular meshwork were studied by electron microscopy. In the other four longer-term monkeys (3 week, 4 week, 5 week, 2.2 year) the anterior eye segment was investigated with tyrosine hydroxylase immunohistochemistry (TH-IR) and catecholamine fluorescence (CF). Electron microscopy of experimental eyes showed characteristic signs of Wallerian degeneration in numerous nerve fibers and terminals in the iris, but to a lesser extent in the ciliary muscle and the trabecular meshwork. TH-IR and CF showed marked interindividual differences. In all experimental eyes, there was a marked reduction, but never a complete absence of adrenergic nerves in the iris. In two animals (4 week and 2.2 years), the adrenergic innervation of the ciliary body and the chamber angle was similarly reduced. In contrast, in the experimental eyes of the other two animals (3 and 5 weeks), changes in adrenergic innervation to the ciliary body and chamber angle were minimal or absent. The results indicate that following apparently complete SCGx in the cynomolgus monkey, reduction of adrenergic innervation to the iris as evidenced by pupillary physiology, electron microscopy, TH-IR and CF does not guarantee reduction in adrenergic innervation to the ciliary body and trabecular meshwork. SCGx may not extirpate all third order sympathetic neurons in the distal stump, or there may be a significant contribution of accessory ganglion cells to the adrenergic innervation of the anterior eye segment.

The color of the human eye: a review of morphologic correlates and of some conditions that affect iridial pigmentation.

Iris color can be affected by a variety of ocular disorders. It is suspected that iris color may not remain constant throughout life. These observations have drawn attention to the morphologic correlates of iris color and its regulation. Differences in the iris color of normal eyes are the result of variable amounts of melanin pigment granules within a constant number of melanocytes in the superficial stroma of the iris. These melanocytes seem to reach their genetically determined amount of melanin in early childhood, and their melanin content usually remains constant in adulthood. Diseases such as Horner's syndrome and Fuchs' heterochromic iridocyclitis affect iris color, resulting in a decrease of iris pigmentation. Evidence suggests that melanin content of some melanocytes is subject to adrenergic regulation even past childhood. Application of the prostaglandin analogue latanoprost, on the other hand, leads to an increase in iris pigmentation in some patients. Studies with cultured dermal and uveal melanocytes, as well as with uveal melanoma cells, however, show no increase in cell proliferation when treated with latanoprost in vitro. The mechanisms by which latanoprost affects regulation of iris pigmentation requires further investigation.

Clinicopathologic correlation of intraretinal microvascular abnormalities.

PURPOSE: To define the cross-sectional morphology of intraretinal microvascular abnormalities, which previously have been described only in terms of trypsin digestion. MATERIAL/METHODS: Fourteen vascular lesions of five patients with diabetic retinopathy were identified on fundus photographs and/or fluorescein angiograms and classified as intraretinal microvascular abnormalities. Eyes of these patients were obtained after the patients' deaths. The period between the time at which the photographs were taken and that at which enucleation was performed was 3-20 months. The duration of autolysis before fixation was 5 hours or less. The embedded tissue was evaluated by light and electron microscopy, and these findings were correlated with the clinical appearance. RESULTS: The lesions consisted of multiple, closely spaced, thin-walled vascular lumina with a caliber of 20-70 microns. They were located in the inner retina and surrounded by a wide cuff containing randomly oriented collagen fibers. Endothelial cell nuclei were numerous. Pericyte degeneration and multiplication of the endothelial and pericyte basement membrane had occurred. Endothelial junctions were short, and gaping of junctions was not seen. However, occasional fenestrations were present. CONCLUSION: The cross-sectional morphology of intraretinal microvascular abnormalities is consistent with vascular pathology typical for intraretinal diabetic microangiopathy, but also includes features usually seen in new vessels. This supports the concept that intraretinal microvascular abnormalities have the particular potential for neovascularization.

Melanocytes and iris color. Electron microscopic findings.

OBJECTIVE: To quantitatively associate iris color with melanocyte pigment content. METHODS: Autopsy eyes were classified as uniform-blue, uniform-hazel, or uniform-brown or showing a darker peripupillary ring. Using electron microscopic images and computerized image analysis, area, number, and size of mature melanosomes within the perinuclear cytoplasmic area only or within perinuclear and peripheral cytoplasmic areas of the superficial stromal melanocytes combined were measured. RESULTS: Average melanosomal area per perinuclear cytoplasmic area (AMAC) and average number of melanosomes per perinuclear area (AMNC) significantly differed across iris color groups (overall P<.001). This result reflects the large difference between blue-uniform and all other color groups. A marginally significant (nominal) trend from blue-ring through brown-ring was also detected (P=.06 for AMAC and P=.07 for AMNC). The average perinuclear cytoplasmic area was larger in the central iris zone (within 1 mm around the pupillary margin) than in the intermediate iris zone (between 1 and 2 mm around the pupillary margin) (P=.002), but AMAC and AMNC did not significantly differ between zones. The average melanosome size did not differ significantly across color groups (P=.11). CONCLUSION: Differences in iris colors are at least partially attributed to variable AMNC and AMAC within superficial melanocytes.

Melanocytes and iris color. Light microscopic findings.

OBJECTIVE: To systematically evaluate morphologic differences in iris stroma that contribute to clinically perceptible differences in iris color, using immunohistochemical identification of stromal melanocytes and fluorescence microscopy. METHODS: Paraffin-embedded sections from 51 human irides were stained with S100a and fluorescein isothiocyanate. Cells were counted and scored as melanocytes or other. Melanocyte number, proportion, and density were determined for light-colored (blue), medium-colored (hazel) and dark-colored (brown) irides and compared. RESULTS: No statistically significant difference was observed for mean total cellularity or mean melanocyte number among the three color groups. Mean total stromal cell count was 1177 +/- 259 (mean +/- SEM), and mean melanocyte number was 778 +/- 196 per 5-micrometer section. In human irides, 65.9% of the iris stroma is composed of melanocytes. Melanocyte density (number of cells per square millimeter) is not related to iris color. CONCLUSION: The number of melanocytes, the proportion of melanocytes, and iris stromal cellularity are not major contributors to iris color.

Renin mRNA is synthesized locally in rat ocular tissues.

Components of the Renin Angiotensin System (RAS) have been detected in ocular tissues and fluids. The source of the ocular RAS proteins is unknown but possibilities include diffusion or leakage from the systemic circulation, specific uptake from the blood, or local synthesis. We have used RT-PCR and in situ hybridization (ISH) to show that renin mRNA is present in ocular tissues from 3 strains of rats. By RT-PCR, we found 10 of 15 ciliary body samples, 13 of 16 iris samples, and 1 of 3 retina samples were positive for renin mRNA. Also, 6 of 6 brain and 7 of 8 kidney samples were positive. Using ISH, we found renin mRNA in the ciliary muscle adjacent to the sclera extending into the choroid. Tissue near the outflow channels of the anterior chamber angle also labeled. Retinal labeling was weak but present in the nerve fiber layer. Clusters of grains, possibly representing blood vessels, were also seen in the ciliary body, iris, and retina using ISH. These results suggest the presence of a local ocular RAS.

Ocular renin angiotensin: EM immunocytochemical localization of prorenin.

Prorenin (PR) was localized by electron microscopic (EM) immunostaining of cryo-ultramicrotomy sections of human ciliary body and correlated with light microscopic immunostaining. Both layers of the ciliary epithelium contained the prohormone. However, density was much higher in the adjacent extracellular spaces, particularly in the vitreous cortex. This observation adds further evidence to a role of the ciliary epithelium in the transfer, storage or synthesis of components of a putative ocular renin angiotensin system.

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.

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.

Levator aponeurosis elastic fiber network.

This light and electron microscopic study demonstrates an elastic fiber network (EFN) for the levator palpebrae superioris muscle complex, which forms an intricate insertion into the upper eyelid. The EFN is examined in the monkey, in a fresh exenteration specimen, and in fresh frozen cadaver specimens from both sexes of different age groups. Multiple elastic insertions of the levator aponeurosis and Muller's muscle attachment with well-organized elastic fibers are demonstrated using special staining techniques and serial microscopic sectioning. Transmission electron microscopy (TEM) confirms the ultrastructure of "mature" elastin fibers in Muller's muscle tendon and their close relationship with the elastin-related fiber, oxytalan. Current thinking concerning the nature of elastic fibers and their possible implications in acquired involutional blepharoptosis is discussed. This microscopic study of the EFN of the upper eyelid focuses attention on the multiple elastic fiber insertions of the levator muscle complex that includes the levator aponeurosis, the conjoined fascia, the lid crease area, and Muller's muscle tendon, which have not been previously described.

Identification of orbital lymphatics: enzyme histochemical light microscopic and electron microscopic studies.

The presence of orbital lymphatics in the primate model is demonstrated using light and electron microscopic enzyme histochemistry. In addition, strictly morphological definitions of lymphatics, such as discontinuous basal lamina, thin and irregular walls, anchoring filaments, and attenuated endothelial cell cytoplasm, were applied. This study confirmed the presence of conjunctival lymphatics reported by others. It also clearly demonstrated the presence of orbital arachnoid and lacrimal gland lymphatics that have not been previously described. A few areas of the extraocular muscles and connective tissue at the orbital apex also showed evidence of the presence of lymphatic vessels. Additional work is needed to define the nature and extent of orbital lymphatics as well as their connection to the extraorbital lymphatic system.

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.

Sympathetic nerve anatomy in the cavernous sinus and retrobulbar orbit of the cynomolgus monkey.

We present new information regarding the sympathetic nerve anatomy in the cavernous sinus and retrobulbar orbit of the cynomolgus monkey. Postganglionic sympathetic nerves were identified using an immunoperoxidase technique in which the primary antiserum was directed against tyrosine hydroxylase, the rate-limiting enzyme in norepinephrine synthesis. Our work is unique in adapting this staining method to paraffin-embedded tissue. This technique allows sympathetic nerve fibers to be distinguished from other autonomic, sensory, and motor nerves. A large sympathetic nerve bundle lateral to the internal carotid artery in the cavernous sinus gave off one or more branches that leave the artery to encircle the abducens nerve. Further division occurs within the cavernous sinus, but all sympathetic nerve fibers destined for the orbit entered it through the superior orbital fissure. None pass through the optic canal. In the orbit, sympathetics were associated with the ophthalmic artery and some of its branches and with the sensory root to the ciliary ganglion. After entering the ganglion, the sympathetic fibers were lost to detection in most specimens, but they were again seen in a single short ciliary nerve in one instance. Sympathetic nerve fibers were not detected adjacent to several structures identified in the human anatomy literature, such as the intracranial and intracanalicular segments of the ophthalmic artery, the nasociliary nerve, the long ciliary nerves, the nerve to the inferior oblique muscle, or the lacrimal artery and nerve.

Parasympathetic denervation of the ciliary muscle following panretinal photocoagulation.

Cynomolgus monkeys underwent unilateral panretinal scatter photocoagulation (PRP) and/or nasal and temporal horizontal retinal meridional photocoagulation (HRMP) with xenon arc or argon or krypton laser light. Shortly thereafter, in the PRP-treated eyes, accommodative responsiveness to topical eserine and electrical stimulation of the Edinger-Westphal nucleus (EWN) was diminished, accommodative responsiveness to intramuscular (i.m.) pilocarpine was enhanced, and the number of muscarinic receptors in the ciliary muscle was reduced compared to the contralateral controls. In most instances, these parameters returned to normal over 6-12 wks and the abnormalities could be induced again by another round of PRP. However, in some PRP-treated eyes, accommodative responsiveness to EWN stimulation and topical eserine remained subnormal permanently (greater than 1 yr). Shortly after HRMP alone, accommodative responses to i.m. pilocarpine, topical eserine, and central stimulation did not differ markedly in the treated and control eyes. Morphologic studies 1 to 78 wk following PRP revealed that myelinated and unmyelinated nerves within the entire circumference of the choroid and ciliary muscle were severely damaged early on. The number of unmyelinated nerves between the individual ciliary muscle fibers was drastically reduced, those which remained were swollen or deteriorated, and agranular synaptic vesicles were rarely seen. Thereafter, the nerves in the choroid and ciliary muscle gradually regenerated. Following HRMP, only the choroidal nerves which passed through the photocoagulated areas and the ciliary muscle nerves in the corresponding meridians showed signs of deterioration, and there was minimal effect on the physiologic responses examined. These findings collectively indicate that intraocular parasympathetic denervation of the ciliary muscle is produced by PRP, although all nerve types are likely damaged.

Clinicopathologic correlation of diode laser burns in monkeys.

One hundred twenty-five retinochoroidal photocoagulation burns, produced by a transpupillary diode laser (810 nm) in six eyes of three cynomolgus monkeys, were evaluated by clinicopathologic correlation for up to 9 weeks after laser treatment. Diode burns of clinical grade 2 strength were comparable to those described for argon laser. However, diode burns of clinical grade 3 strength produced choroidal changes more intense than those described for argon laser. Where present underneath photocoagulation sites, ciliary nerves in choroid or sclera consistently showed scarring. Prospective randomized controlled clinical trials to document possible clinical equivalence or superiority of diode treatment have not yet been performed. Potential clinical advantages of the diode laser include its weight, size, durability, price, absence of visible flash, and its ability to produce burns that profoundly affect the choroid.

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.

Immunocytochemical study of an eye with proliferative vitreoretinopathy and retinal tacks.

After an eye-wall resection for a choroidal melanoma, a 32-year-old woman had subsequent retinal detachment with proliferative vitreoretinopathy (PVR), and an unsuccessful attempt at repair with retinal tacks. Gross and light-microscopic examination of the globe revealed a total retinal detachment with extensive preretinal and subretinal membranes. The membranes surrounded the tack heads and extended in taut bands to form a tractional detachment of the pars plana. The membranes contained glial and nonglial cells. The glial cells immunolabeled for glial fibrillary acidic protein (GFAP), carbonic anhydrase-C (CA-C), vimentin, and glutamine synthetase (GS), thus suggesting that they were Müller's cells. While the tacks did not seem to cause PVR, in this case they may have provided an anchoring point from which membranes were able to exert traction on the retina and pars plana.

Immunostaining of preretinal membranes for actin, fibronectin, and glial fibrillary acidic protein.

The frequency and extent of immunostaining for actin, fibronectin (FN), and glial fibrillary acidic protein (GFAP) were determined in 37 preretinal membranes (PRMs) obtained at vitrectomy from 35 patients with proliferative diabetic retinopathy (PDR) (n = 16), proliferative vitreoretinopathy (PVR) (n = 18), or idiopathic macular pucker (MP) (n = 3). All three proteins were detected in the vast majority of specimens (actin, 86%; FN, 95%; GFAP, 96%), although the extent of staining varied for each. Actin-FN co-localization was observed in all diagnostic groups on comparison of adjacent sections and in double-labeled sections. The extent of actin staining did not correlate with clinical grading of PRM contraction. In PDR membranes, FN staining was low overall, but proportional to the vascular content of the PRM. Fibronectin staining of PVR membranes was greater, and extensive even in avascular specimens. In MP membranes, most cells were GFAP-positive, whereas in PDR and PVR specimens, GFAP staining was variable. The lack of correlation of clinical contractility and membrane composition, as studied in this article by immunostaining, indicates that other factors must play significant roles in determining membrane behavior.