c<orneal dystrophies and degenerations

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1 Oval corneal opacities in beagles III. Histochemical demonstration of stromal lipids without hyperlipidemia Alan M. Roth, Marilyn B. Ekins, George O. Waring III, Lata M. Gupta, and Leon S. Rosenblatt We found oval stromal avascular corneal opacities in 128 eyes of 75 beagles from 497 studied. There were three morphologic types that progressed in severity with time: nebular, racetrack, and white arc. Histochemical sti'^n r "the earliest morphologic type (nebular) revealed neutral fats, cholesterol, phospholipids, and sometimes fatty acids both intracellularly and extracellularly. We found no elevation of serum cholesterol or triglycerides except in dogs with the most advanced inorphologic type (white arc) and no alteration in thyrometabolic function. We think that oval stromal opacities in beagle corneas are a primary disorder ofcorneal lipid metabolism closely resembling the central crystalline dystrophy of Schnyder and may be an animal model for this human disease. Key words: corneal metabolism, primary lipid dystrophy, Schnyder's crystalline dystrophy, lipid histochemistry, animal model c<orneal dystrophies and degenerations have been described rarely in dogs. 1 Although sporadic cases have been reported, none has resembled the oval stromal corneal opacities we previously described in 128 eyes of 75 purebred beagles from a colony of 497 dogs. 2 These anterior stromal opacities were oval, about 3 by 5 mm, located at the junction of the middle and inferior thirds of the avascular cornea, and were usually bilaterally From the Departments of Ophthalmology (Drs.. Roth, Ekins, Waring, and Ms. Gupta) and Pathology (Dr. Roth), School of Medicine, and the Radiobiology Laboratory, University of California, Davis, and Genticon, Walnut Creek, Calif. (Dr. Rosenblatt). Supported by National Eye Institute Grant EY03302 and grants from the National Society for the Prevention of Blindness, the U.S. Department of Energy, and the Medical Research Council of Canada. Submitted for publication Nov. 2, Reprint requests: George O. Waring, M.D., Emory University Clinic, Department of Ophthalmology, 1365 Clifton Road N.E., Atlanta, Ga symmetrical. We observed three morphologic types: nebular, with a homogenous gray appearance (Fig. 1); racetrack, with a gray oval surrounding a slightly depressed, brownish center (Fig. 2); and white arc, with subepithelial, dense, white plaques of granular or spicule-shaped material (Fig. 3). These three patterns represent different stages in the natural history of the disorder, progressing from the nebular (least severe) to the white arc (most advanced). 3 Because the colony was outbred, 4 we could not establish a hereditary pattern. We have also seen similar corneal opacities in beagles that were not part of this colony (Fig. 4). When we initially described the clinical appearance of oval stromal corneal opacities in beagles, 2 we did not know what material composed these lesions. Negative findings with standard histochemical stains and the presence of vacuoles and crystals in transmission electron micrographs led us to search for lipids. Although we recognized that lipid /81/ $01.20/ Assoc. for Res. in Vis. and Ophthal., Inc. 95

2 96 Roth et at. Invest. Ophthalmol. Vis. Sci, July 1981 Fig. 1. Nebular oval corneal opacity has homogeneous gray appearance with indistinct margins. Fig. 2. Racetrack oval opacity has slightly depressed, brownish center. histochemistry is an imprecise art and that only a few techniques define lipids with certainty, we decided to perform a large battery of techniques to give the most comprehensive data. These may then be interpreted in light of the limitations of such testing and correlated with more precise biochemical analysis. We report here the histopathologic findings and histochemical demonstration of lipids in these opacities and show that serum lipid levels and thyroid hormone assays are similar in both affected beagles and age- and sex-matched controls. Materials and methods All beagles came from a colony involved in lifelong radionuclide toxicity studies at the Radiobiology Laboratory, University of California, Davis. 1 ' 8 As each dog was scheduled for sacrifice, we reviewed our previous records to determine

3 " 'f--'" Volume 21 Number 1, Part I Oval corneal opacities in beagles 97 Fig. 3. White arc oval opacity is densely white, granular subepithelial deposit. Fig. 4. Oval opacity in beagle from outside the colony studied shows double row of gray crystalloid deposits (left), which stand out prominently in retroillumination (right). the type of corneal opacity present. After the dog was anesthetized with intravenous sodium pentobarbital, we verified the type of corneal opacity by inspection with a penlight and enucleated the eyes. We immediately removed a corneosclerat shell by cutting into the epichoroidal space 4 mm posterior to the limbus, excising the shell with scissors, and disinserting the ciliary body from the scleral spur. The cornea was placed epithelial side clown on a silicone block and trisected through the oval opacity; one third was fixed in cold 2% glutaraldehyde in Millonig's buffer for electron microscopy, one third was frozen in liquid nitrogen for biochemical determinations, and one third was fixed in Baker's fonnol-calcium solution 7 for light microscopy. We recorded the nutritional and medical history of each dog. An experienced veterinary pathologist performed a complete autopsy. Histochetnistry. We studied 29 corneas from 16 affected beagles that had the nebular type opacity and two corneas from two unaffected beagles. Three specimens from two dogs were blocked in paraffin only. Because routine histochemical stains

4 98 Roth et al. Invest. Ophthalmol. Vis. Sri. July 1981 Table I. Histochemical stains done for lipid Oil red 0 Suden black B Baker Pearse Menschik Landing et al. Fischler Holczinger Schultz Adams Okamoto Bromine-Sudan black B Mukherji Norton Osmium tetroxide Seligman-Ashbel Lipid-soluble dye Lipid-soluble dye Type of reaction Acid hematein with pyridine extraction Copper phthalocyanin Nile blue sulfate Phosphomolybdic acid Cupric soap-lithium hematoxylin Cupric soap-rubeanic acid Acetic-sulfuric acid Perchloric acid-naphthoquinone Sulfuric-iodide Bromination unmasking Bromine-silver Bromine-silver Lipid-soluble reagent Naphthoic hydrazide Lipid demonstrated Neutral fat Neutral fat especially phospholipid Phospholipid Phospholipid Phospholipid Choline-containing phospholipid Fatty acid Fatty acid Cholesterol Cholesterol Cholesterol Unmasked cholesterol Unsaturated lipid Unsaturated lipid Unsaturated lipid Active carbonyl lipids Reference Specificity High Moderate Moderate High Moderate High Moderate Moderate Moderate of sections cut from these blocks revealed no abnormal substances, because the ultrastructural studies on these specimens showed intracellular vacuoles and crystals suggestive of lipids (Spangler, Waring and Morrin, unpublished data), and because the clinical appearance was compatible with lipid deposits, we divided the formol-calcium -fixed tissue from each of the remaining 26 corneas into two pieces. We embedded one of these in paraffin and stained sections with hematoxylin and eosin, Masson's trichrome method, the periodic acid-schiff reaction, and Alcian blue at ph 2.5. The other piece was frozen, and sections were cut 10 to 15 /xm thick and stained by 16 histochemical methods for demonstrating lipids (Table I). We stained sections of brain known to contain the different lipids with each corneal specimen as a control. Sections being analyzed for cholesterol were examined within 15 min of staining because of rapid oxidation of the reaction and because the sections were destroyed by the technique. Paraffin and frozen sections were also examined under plane-polarized light for birefringence. Serum lipid measurements. We measured serum cholesterol and triglycerides in affected beagles and in age- and sex-matched control beagles on two occasions, 1 year apart. In December 1977, we paired each of 48 affected beagles (28 males, 20 females) with an unaffected beagle of the same age and sex ( males, 21 females) in the same radiation group ( 90 Sr, 226 Ra, or not radiated). Some of the affected and control dogs were littermates. In January 1979, we re-examined the 29 surviving affected beagles (18 male, 11 female) and the 40 surviving controls (24 male, 16 female). The mean ages of the control and affected dogs were comparable for both sexes (male 12.5 years, female 12.6 years). After the clogs had fasted 6 hr, we collected 10 ml of clotted blood with a 20-gauge needle from the left jugular vein. The samples were immediately centrifuged at 3000 rpm at 4 C; the serum was removed and stored at 4 C. We measured total cholesterol by the Technicon AutoAnalyzer II (Clinical Method No. 24/preliminary March 1972) for the first determination and by the enzymatic method of Patsch 8 for the second. The high-density-lipoprotein cholesterol (CHDL) <X cholesterol) was determined by NIH protocol, 9 and triglycerides by Fletchers technique. l0 The combined low-density-, and very-low-density-lipoprotein cholesterol (C LD L and C V LDL> j3 and pre-/3 cholesterol) was then calculated by subtracting the CHDL from the total cholesterol. 9 All solvents used were reagent grade. To confirm the accuracy of our serum lipid measurements, we employed standards of known lipid concentration and also sent random beagle serum samples to an independent laboratory that used the same methods. We employed analysis of variance and regression analysis to compare serum lipid measurements from the total number of affected and control dogs, to compare each of the three affected subgroups (nebular, racetrack, and white arc) to each other and to the controls, and to compare sexes and ages. We used data only from beagles whose serum lipids were measured twice. Each of

5 Volume 21 Number 1, Part 1 Oval cornea! opacities in beagles 99 the affected dogs was classed according to the lesion of its more severely affected eye. Between the first and second lipid determination, the opacities in some of the affected dogs progressed; thus the same dog may be in one affected subgroup in 1977 and in another in Thyroid hormone assays. We tested thyrometabolic function in 24 affected beagles and in 39 age- and sex-matched control beagles. Blood was collected from the jugular vein and the serum was immediately separated as described above. The samples were stored at 4 C for no longer than 48 hr before analysis. Thyroxine (T 4 ) levels were measured by the Quantimune T-4 Radioimmunoassay technique (Bio-Rad Laboratories Bulletin 4202, October 1978), and the binding capacity of thyroid hormone carrier proteins was assayed by the Quanta-Count T 3 resin uptake method (Bio- Rad Bulletin 4212, March 1977). The free thyroid index (T 7 ) was calculated by multiplying the T 4 level and the T 3 percent resin uptake (%RU). We employed chi-square analysis to compare the results between control and affected dogs, between control dogs and those in each affected subgroup (nebular, racetrack and white arc), and between dogs in each subgroup. Statistical review was performed by Dr. Rosenblatt. Results Complete autopsies on all dogs revealed no changes suggestive of a systemic lipid metabolic disorder. Histology and general histochemistry. Paraffin sections stained with hematoxylin and eosin and with Masson's trichrome method demonstrated that the epithelium and its basal lamina generally were intact with occasional focal acanthosis (facet formation). Although Bowman's layer is not normally present in beagle corneas (Morrin, Waring, Spangler, unpublished data), the normal anterior stroma is hypocellular, with interlacing collagen bundles. The major abnormality in the affected corneas was in this area, with disruption of collagen architecture, focal areas of collagen fibril fragmentation, stromal vacuole formation, and scattered enlarged vacuolated keratocytes. No blood vessels were seen. Periodic acid-schiff and Alcian blue reactions revealed no gross abnormalities in glycosaminoglycans. Plane-polarized light showed no birefringence in sections from paraffin blocks. Frozen sections, however, contained many doubly refractile crystals, primarily within keratocytes, but occasionally in the extracellular stroma. The posterior stroma, Descemet's membrane, and endothelium were normal. Lipid histochemistry. Table II shows the results of the histochemical reactions for lipids in 26 affected corneas. The right and left corneas from each dog usually stained similarly. All stains were positive on the known control tissue and showed no reaction in the two unaffected control corneas. The majority of positive stains were intracellular with many having extracellular staining as well. No case showed only extracellular stain, although a few had such dense overall reaction that we could not ascertain its location. Stains for neutral fats (oil red O and Sudan black B) were positive in all corneas, primarily in the anterior half. In general, the intracellular stain was in large globules, whereas the stromal stain was in finer granules (Fig. 5). At least one of the methods for demonstrating cholesterol (Schultz, Okamoto, and Adams) was positive in each cornea (Fig. 6). The majority were positive by all three techniques. All corneas demonstrated phospholipids with at least one of four reactions for phospholipid (Baker, Pearse, Landing, and Menschik), and 12 of the 26 corneas demonstrated fatty acids by at least one of the tests (Fischler, Holczinger). The remainder of the histochemical reactions were negative, although sporadic intracellular staining was sometimes present. Serum lipid measurements. There was no significant difference between affected and control dogs in either 1977 or 1979 for total cholesterol, C H DL, C LDL, and C V LDL, or triglycerides (Table III). The influence of sex on serum lipids was also negligible except that, in 1979, affected females showed higher triglycerides than affected males. The influence of age on serum lipids was less clear-cut (Table III). In 1977, there was a statistically significant trend for serum cholesterol to rise

6 100 Roth et al. Invest. Ophthalmol. Vis. Sci. July 1981 Fig. 5. Neutral fat appears as large globules in keratocytes, whereas smaller extracellular granules accumulate in anterior stroma. (Frozen section, oil red O; X200.) Table II. Results of histochemical stains foi lipid A EI C D I F Dog and cornea No Oil red O Suden black B Bromine-Sudan black B Schultz Okamoto Adams Baker* Pearse Landing Menschik Fischler Holczinger OsO 4 Norton Mukherji Seligman-Ashbel = Negative; + = mildly positive; ++ = moderately positive; + = strongly positive. Dogs D and M supplied only one cornea each. *With pyridine extraction control

7 Volume 21 Number 1, Part 1 Oval corneal opacities in beagles 101 Fig. 6. Cholesterol accumulates predominantly in keratocytes of anterior stroma. (Frozen section, Okamoto's method; x200.) G H I J K L M N f + -f

8 102 Roth et al. Invest. Ophthalmol. Vis. Sci. July 1981 Table III. Serum lipid measurements (mg/dl) in beagles with oval opacities and unaffected controls Total cholesterol c HDL First sample Second sample First sample Second sample Control Opacity Control Opacity Control Opacity Control Opacity Male: Mean S.D. N Female: Mean S.D. N Analysis of variance*: By sex By group By sex and group Linear regression against age: Male r 0.40 P(r) 0.04 Female r 0.57 P(r) First sample = 1977; second sample = 1979; = not significant = p >0.05. * Normal vs. abnormal; male vs. female. with age, but this was not confirmed in When we analyzed total serum cholesterol, CHDL> C LDL, and C V LDL measurements in the three different clinical patterns in 1977, dogs with white arc opacities consistently had higher absolute values, but we could not confirm these differences in 1979 (Table IV). Thyroid hormone assays. The 39 control dogs had T 3 %RU of 35.2% ± 4.6, T 4 level of 1.37/xg/100 ml ± 0.68, and T 7 of Eight beagles with the nebular opacity had T 3 %RU of 34.6% ± 5.4, T 4 level of 1.45 jug/loo ml ± 5.4, and T 7 of Eight dogs with the race track lesion showed T 3 %RU of 36.6% ± 5.1, T 4 levels of 1.46 ^g/100 ml ± 0.32 and T 7 of Six animals with the white arc corneal opacity had T 3 %RU of 35.3% ± 4.2, T 4 level of 1.70 /Ag/100 ml ± 0.70 and T 7 of Chi-square analysis showed no significant difference in T 3 %RU, T 4, or T 7 between control and affected dogs, between control dogs and those in each subgroup, or between beagles in each subgroup. Comment Relatively little is known about the composition, source, and metabolism of normal corneal lipids. There is no evidence for lipid storage in the normal cornea in vivo and lipids are probably limited to corneal cell membranes in the form of neutral fats (predominantly cholesterol and its esters) and phospholipids (primarily sphingomyelin). 11 These are relatively stable and there appears to be a low lipid turnover rate. Normal corneal stromal cells can synthesize lipids 12 ' 13 and can incorporate labeled phosphorus into phospholipids. 14 Lipids appear abnormally in the human cornea in a variety of patterns (Table V). Three factors may contribute to these lipid deposits: (1) stromal blood vessels such as those in old scars or in limbal masses, (2) abnormal systemic lipid metabolism such as type II hyperlipoproteinemia or Tangier disease, and (3) primary corneal abnormalities such as central crystalline corneal dystrophy.

9 Volume 21 Number 1, Part 1 Oval corneal opacities in beagles 103 C L0L ana First sample Control Opacity CvLDL Second sample Control Opacity Triglycerides First sample Second sample Control Opacity Control Opacity P = We searched for these three contributing factors in beagles with oval lipid corneal opacities. The corneas were avascular both clinically and histopathologically. In a few cases, corneal ulceration had occurred in conjunction with the oval opacities, and the corneas vascularized secondarily. Serum lipid measurements in affected dogs were normal. The environment, nutrition, and medical care of this colony were carefully monitored. 15 ' 16 A complete autopsy on each dog revealed neither atherosclerosis nor excess lipid deposits in parenchymal organs. Our initial analysis of serum cholesterol suggested elevated C H DL i n dogs with the racetrack and white arc patterns. 17 However, further statistical analysis and a second sampling of affected and control dogs failed to reveal statistically significant differences (Table IV). Moreover corneal opacities have not been described in reports of hyperlipemic beagles. 18> 19 Therefore we concluded that a systemic disorder of lipid metabolism did not contribute to the oval lipid corneal opacities. However, we did observe consistently higher levels of serum cholesterol in dogs with the white arc type opacity, although the level was not always statistically significant and the number of these dogs was small (Table IV). It is possible that these subepithelial white deposits, which we presume are lipids but have not studied histochemically, are related to the elevated serum lipids. On the other hand, although we found clinical progression of these opacities from nebular to racetrack to white arc types, we have not been able to document either a concurrent rise in serum lipids or increased serum lipids with age (Tables III and IV). In hypothyroidism, serum cholesterol levels may be elevated because of altered metabolism; this metabolic effect has been reported in beagles. 20 Because our pilot study suggested that affected beagles had elevated serum cholesterol levels, we measured thyroxin and thyroglobin binding capacity, cal-

10 104 Roth et al. Invest. Ophthalmol. Vis. Sci. Julij 1981 Table IV. Total serum cholesterol measurements in beagles with nebular, race track, and white arc oval opacities First sample Second sample Control Nebular Racetrack White arc Control Nebular Racetrack White arc Male: Mean S.D. N Female: Mean S.D. N Analysis of variance* By sex By type By sex and type P = First sample = 1977; second sample = 1979; = not significant = p > * Normal vs. abnormal; male vs. female. Table V. Corneal stromal lipid deposits in man Type Corneal arcus Normal aging >40 years Hyperlipoproteinemia II and III, <40 years Secondary (pre-existing stromal vessels) Vascularized scar Limbal mass (e.g., dermoid, nevus) Primary (no known pre-existing corneal pathology) Limbal Central White ring (of Coats) Central crystalline dystrophy (of Sch'nyder) Systemic disorders Tangier disease (decreased C H DL in serum) Lecithin cholesterol acyltranferase deficiency Juvenile xanthogranuloma Reference 37, , Clinical appearance Gray paralimbal arc with lucid interval Gray-white, random pattern, spiculated margin Gray arc, lucid interval D-shaped, yellow, solid, deep stromal, initially vascular Round, yellow, initially avascular Small, white, round with gray dots Central anterior ring of fine, white crystals, hazy gray background, corneal arcus Diffuse fine gray spots Diffuse fine gray spots Flat, yellow, vascularized limbal mass dilated T 7) and found no significant differences between affected and control dogs or dogs with different subgroups of opacities. Thus thyroid dysfunction does not play a part in the pathogenesis of the oval opacities. We think that oval stromal corneal opacities in beagles represent a primary disorder of corneal lipid metabolism. Of the human disorders in which lipid is deposited in the cornea, these opacities most closely resemble the central crystalline corneal dystrophy of Schnyder. 21 ' 22 Both occur in avascular, noninflamed corneas, are bilateral, and appear relatively symmetrical. The clinical appearance of both includes a ring-shaped configuration in which a central gray stromal opacity that is most dense superficially is studded with subepithelial white crystals. In ad-

11 Volume 21 Number 1, Part 1 Oval corneal opacities in beagles 105 vanced cases of both disorders, the opacity may occupy full-thickness stroma. Histochemically, both contain cholesterol and neutral fats, 15 18< ' 19 whereas the beagle corneas also have histochemical evidence of phospholipids and sometimes fatty acids. Both have similar ultrastructure with elongated crystals, extracellular vacuoles and debris, disruption of collagen lamellae, hyperplasia of keratocyte organelles, and stromal cellular degeneration. 26 Hyperlipoproteinemia has been found in some patients with central crystalline dystrophy and in family members of affected individuals. 21 ' 22 Although we did not find hyperlipidemia in affected beagles, the dogs with white arc opacities did have serum cholesterol levels higher than the control population, which may contribute to their clinically more advanced opacity. Bron and colleagues 22 speculated that the stromal keratocytes in central crystalline corneal dystrophy are unable to properly metabolize lipids, so that local lipid deposits occur. Elevated serum lipids then further overload the cells and increase the lipid deposition. In man, the disorder is autosomal dominant; it is unfortunate that we cannot determine the hereditary influences in our beagle colony because of the carefully planned outbreeding. 4 Oval corneal lipid opacities in beagles may be an animal model of the human corneal dystrophy of Schnyder. The demonstration of lipids by histochemical techniques is a tedious process. The methods are not well documented, many reagents are difficult to obtain, and only after diligent search by laboratory personnel can a meaningful battery of lipid stains be performed (Table I). Even when histochemical methods demonstrate the presence and location of lipids, distinguishing among the classes of lipids is difficult. Many of the reactions are nonspecific for the different types of lipid and can give false-positive and/or false-negative results. The presence of other types of compounds in the tissue may cause capricious staining. Diffusion from the sections can occur and subcellular fractionation has been found. 28 Biochemical analysis of lipids 28 ' 29 is necessary to accurately identify and quantitate the varieties in the oval opacities; we are presently performing these studies on affected beagle corneas. REFERENCES 1. Vai Nisi SJ and Goldberg MF: Animal models of inherited human eye disease. In Genetics and Metabolic Eye Disease, Goldberg MF, editor. Boston, 1974, Little Brown & Co., pp Waring GO, Muggli FM, and MacMillan A: Oval corneal opacities in beagles. J Am Anim Hosp Assoc 13:204, Ekins MB, Waring GO, and Harris RR: Oval lipid corneal opacities in beagles. II. Natural history over four years and study of tear function. J Am Anim Hosp Assoc 16:601, McKelvie DH, Shultz FT, Parcher JW, et al: Random selection of beagles to maintain heterogeneity and minimize bias in a life-span experiment. Lab Anim Care 16:337, Goldman M, Delia Rosa RJ, and McKelvie DH: Metabolic, dosimetricand pathological consequences in skeletons of beagles fed 90 Sr. In Delayed Effects of Bone-Seeking Radionuclides, Mays CW, editor. Salt Lake City, 1969, University of Utah Press, pp Goldman M, Dungworth DL, Bulgin MS, et al: Radiation-induced neoplasms in beagles after administration of 90 Sr and 226 Ra. In Radiation-Induced Cancer. Vienna, 1969, International Atomic Energy Agency, pp Luna LG, editor: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, ed. 3. New York, 1968, McGraw-Hill Book Co., Inc., pp. 4, Patsch W, Sartor A, and Braunsteiner H: An enzymatic method for the determination of the initial rate of cholesterol esterification in human plasma. J Lipid Res 17:182, Lipid and lipoprotein analysis. In Manual of Laboratory Operations. Lipid Research Clinics Program, Vol. 1. DHEW Publication (NIH) No , Fletcher M: A colorimetric method for estimating serum triglycerides. Clin Chim Acta 22:393, Feldman GL: Human ocular lipids: their analysis and distribution. Surv Ophthalmol 12:207, Andrews JS: Corneal lipids. I. Sterol and fatty acid synthesis in intact calf cornea. INVEST OPHTHALMOL 5:367, Culp TW, Cunningham RD, Tucker PW, et al: In vivo synthesis of lipids in rabbit iris, cornea and lens tissue. Exp Eye Res 9:98, Broekhuyse RM and Daumen FGM: The eye. In Monographs in Lipid Research: Lipid Metabolism in Mammals, ed. 2, Snyder F, editor. New York, 1977, Plenum Press, pp Andersen AC and Goldman M: Outdoor kennel for dogs. J Am Vet Med Assoc 137:129, Wolf HG, Delia Rosa RJ, and Andersen AC: Nutri-

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