Ultrastructural Observations on Experimentally ... - Europe PMC

Ultrastructural Observations on Experimentally Produced Melanin Pigmentation of the Corneal Epithelium J. Stuart McCracken, MD, and Gordon K. Klintworth, MD, PhD

Mielanin pigmentation of the corneal epithelium was induced in pigmented guinea pigs by the topical application of colchicine to their eyes or bv corneal cauterization with silver nitrate. With colchicine the pigmentation was preceded by the development of an abnonnal corneal epithelium in which numerous cells became arrested in cell division. The corneal melanosis resulted largely from the migration of melanocytes into the corneal epithelium from the normally pigmented contiguous conjunctiva and to a lesser extent from the presence of melanin granules within corneal epithelial cells. In both models a leukocvtic and vascular invasion of the cornea preceded and accompanied the migration of melanocytes into the corneal epithelium. Electron microscopy disclosed cells with the same morphology as conjunctival melanocytes between the epithelial cells of the cornea. Miature melanin granules were also present within some squamous epithelial cells as individual granules or as clusters. The ultrastructural findings are viewed in relation to how melanin granules are transferred from melanocytes to epithelial cells. Evidence is presented which suggests that melanin granule transfer mav follow the fusion of the membranes of the melanocytes and epithelial cells. (Am J Pathol 85:167-182, 1976)

THE EY ES of many heavily pigmented animals and of certain human races possess a pigmented ring around the cornea. In some species this is due to melanocy-tes wvithin the sclera, -hile in others the melanocvtes are mainly wvithin the conjunctiva.l In many other species, this pericorneal pigmented ring consists predominantly of conjunctival melanocytes and melanin granules within conjunctival epithelial cells. Occasionall1 some melanin pigmentation may be present in the peripheral cornea in the absence of overt disease. Under normal circumstances most of the cornea is nonpigmented, with the possible exception of the manatee and a femv other species.`4 For a long time, ophthalmologists have recognized that melanin pigmentation of the cornea can occur in certain pathologic states in man and other animals.2 -22 W\'hen this occurs the pigmentation results mainly from melanin granules within epithelial cells and intraepithelial melanocy-tes. The entity has been designated epithelial melanosis and may accompany conjunctival nevi, melanomas, From the Department of Pathology. Duke U niversitx Nledical Center. Durham. North Carolina Supported in part bh Grants EY-00146 and \IH-11947 from the National Institutes of Health; Dr Khlintmsorth is the recipient of Research Career Des elopment AXward EY-4479.5 and an R. P. B. Iouis B. \laver Scholarship. Accepted for publication Jtune S. 1976. .ddress reprint re(quests to Dr. Gordon K. Klintsorth. Department of Patholog!. Duke U niversity \Medical Center. Durham. SC 27710. 167

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and other congenital, inflammatory, and neoplastic lesions. 5-7,9-17.19-22 These corneal melanocvtes mav account for the rare corneal melanomas.23-26 Experimentally, corneal epithelial melanosis has been produced in rabbits by corneal abrasion,"6,'729 arsenicals,3' sulfur and nitrogen mustard,32'33 and electrocauter.^34 Henkind 8 produced the phenomenon in pigmented guinea pigs by the topical application of colchicine to their eves. This study confirms and extends his light microscopic observations. Materials and Methods Fifty-tw-o healthy pigmented guinea pigs Nvere used (Camm Research Laboratories. Wayne. N.J.). Four of these ser'ed as normal controls; 0.2 ml colchicine injection (Lilly. Indianapolis. Ind.) was applied topically in one dose to the eves of 27 animals at a concentration of 0.5 mg ml. Tswenty of the remaining animals received the same dose of colchicine twice per day. To test the specificity of colchicine for melanalocy te migration. a silver nitrate applicator tip (Graham Field Surgical Company, New Hyde Park, N\.Y.) %vas applied to the comea of another guinea pig, under ether anesthesia, for 1 to 2 seconds. The eves of all animals were examined dail-v with the aid of a Zeiss operation microscope (Carl Zeiss, West Germans). Guinea pigs were killed at variable time intervals (I to 14 days) after colchicine treatment. The eves from all treated and normal animals w-ere prepared in the follow-ing manner: for light microscopy, specimens were fixed in buffered 10% formalin (pH 7). For electron microscopy, globes were fixed by immersion in 4% cacodylate-buffered glutaraldehy de (pH 7.2 to 7.4), osmolaritv = 640 to 740 mOsmol/liter. After fixation the comeas and adjacent conjunctival and scleral tissues were excised, and the specimens were washed in sodium cacodylate buffer and postfixed with 1 % osmic acid buffered with sym-collidine (pH 7.2) for 1 hour, then dehydrated by passage through increasing concentrations of ethanol. To insure proper orientation of the tissue, the specimens were placed in plastic block molding cups s%ith a 12 X 16 X 5 mm specimen chamber (Sorvall, New-ton. Conn.) and embedded in Epon. Sections 40 M thick were cut w-ith a sliding microtome (American Optical, Buffalo, N.Y.) and mounted on a slide for light microscopic observation as unstained preparations. Selected areas of the comea were cut away and mounted on a prepolvrmerized Epon block. Sections of the originally examined material w-ere cut at 0.3 A. and stained with toluidine blue for the purpose of selecting the area for electron microscopic examination. Thin sections cut at 30 to 60 mp were mounted on copper grids and stained with 4% uranyl acetate and lead citrate according to the method of Reynolds.-5

Results Normal Guinea Pigs

Pigmentation was prominent in the conjunctival epithelium immediately adjacent to the corneoscleral limbus in normal pigmented untreated guinea pigs (Figures 1 and 2). The conjunctival melanin w,as present within epithelial cells and melanocytes. Occasional melanocvtes existed in the subepithelial tissue, especially adjacent to blood vessels. The more peripheral conjunctival epithelium wvas not pigmented. The nonpigmented stratified squamous epithelium of the normal

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guinea pig consisted of an average of five to six layers (Figure 3). When viewed in the transmission electron microscope, the corneal epithelial cells were characterized by extensive arrays of intracytoplasmic tonofibrils, as well as multiple desmosomal connections with surrounding cells of the same type, as described in the epidermis of the guinea pig by Snell." Intracellular and extracellular melanin granules were conspicuously absent. The basal epithelial cells rarely displayed mitotic figures (1.8 mitoses/100 cells); others had none. The posterior surface of the corneal epithelium with its regularly spaced hemidesmosomes rested on a delicate basement membrane. Cddine-Treatd Gue P*s

By the second day of colchicine application more basal epithelial cells contained mitotic figures (3%) than the controls (1.8%). The more superficial layers still lacked mitoses. By 3 days the comeal epithelium consisted of one to five layers of cells, and many cells particularly in the basal layer displayed mitotic figures at 4 days (Figure 4). Some areas of the comea lacked an epithelium. From 7 days until the end of the experiment, two to three layers of epithelium usually remained and the mitotic figures in the basal epithelial cells remained high (3 to 4%). Aside from the aforementioned epithelial alterations, colchicine induced an acute inflammatory response. Within 2 days of colchicine application, the pericomeal conjunctival vascular arcades were usually hyperemic. These vascular loops remained prominent, and by the fourth day, blood vessels first extended into the clear comeal periphery. Between the sixth and seventh days of colchicine application, some comeas became opaque after being as crystal clear as normal comeas. This coincided with a progressive thickening of the corneal stroma and a separation of the collagen lamellae. At this time the advancing border of radially oriented, growing blood vessels involved 90% of some comeas, sparing only a small opaque central area. Light microscopy disclosed capillaries of varying sizes predominantly in the outer third of the comeal stroma. A few neutrophilic leukocytes appeared in the superficial corneal stroma by 2 days. Mononuclear leukocytes appeared within 7 days, and both cell types gradually became more numerous and extensively infiltrated the entire comeal stroma by the 14th day. Ulceration of some comeas was observed after 4 days, but in most instances it did not become pronounced until 10 or more days after colchicine application. Melanin made its first appearance in the peripheral comea, both in epithelial cells and in melanocytes interspersed among them, 4 days after colchicine administration. From then onward, stellate melanocytes with

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elongated cytoplasmic processes were evident between basal corneal epithelial cells (Figures 5-7). The melanocytic processes contained mitochondria and rough endoplasmic reticulum and were packed with mature melanin granules as well as granules containing the matrix typical of premelanosomes (Figures 6, 7, 11, and 13). Morphologic evidence of melanin degradation was not observed in these cells. Unlike epithelial cells, melanocytes lacked desmosomes and tonofibrils. The cytoplasm of some epithelial cells contained melanosomes as individual granules with and without encircling membranes, either peripherally or alongside the nucleus (Figures 6-10). The melanin granules were present in all epithelial layers but were most numerous in the cells of the basal epithelium, some of which contained as many as 20 in clusters within a single electron micrograph of a cell. Cellular junctions between the melanocytes and epithelial cells were not observed, but in some sites where melanocytes and squamous epithelial cells were closely approximated, the cytoplasmic membrane of adjacent cells could not always be clearly identified around the entire cells (Figures 12 and 13). Cytoplasmic processes of some epithelial cells enveloped melanocytes, (Figure 14) but phagosomes containing melanin granules and other cytoplasmic organelles in various stages of degradation were not observed in epithelial cells. The cell membranes of an occasional melanocytic process were disrupted (Figure 15), but extracellular melanin granules were not observed in the numerous tissue sections examined. In the superficial corneal stroma, very few cells containing melanin were observed beneath the epithelium. These cells with morphologic features of macrophages contained lysosomes and melanin granules within phagosomes, but conspicuously lacked premelanosomes, tonofibrils, and desmosomes (Figures 16 and 17). The epithelial melanosis reached a sufficient intensity by 6 to 7 days to become evident as areas of superficial black corneal pigmentation in the living animal. This was most apparent in the vascularized area and, especially, peripheral to the vascular front. The colchicine did not arrest corneal endothelial cells or melanocytes in cell division. The lesions produced in guinea pigs with both dosages of colchicine differed only in severity. Sbw Nitrate-Treate Guina

Pigs Silver nitrate cauterization caused a dark brown circular opacity of reduced silver at the site of corneal injury. An acute inflammatory response characterized by hyperemia of the conjunctival blood vessels, opacification of the cornea, and a neutrophilic infiltration of the cornea



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ensued. This was followed within 4 days by corneal vascularization and a melanocytic migration into the cornea. Marked intercellular epithelial edema surrounded the region of corneal cauterization. As in the colchicine-treated animals, melanocytes infiltrated the basal epithelium of the cornea, and melanosomes appeared within the cytoplasm of epithelial cells. Di

In theory the lack of melanin within the normal corneal epithelium can result from either a lack of melanocytes or the failure of corneal melanocytes to synthesize melanin. Regarding the latter possibility, it is of interest that dendriticly shaped cells have been recognized in the cornea of several species since as early as 1881.4.183` It is now established that at least some of these cells are Langerhans cells with their characteristic cytoplasmic "Birbeck granules." 1'240 " However, some dendritic cells may represent melanocytes that have lost their ability to synthesize melanin. In an electron microscopic study of the human cornea, Segawa 42 recognized three types of dendritic cells in the cornea: one containing mature melanin granules, another with immature and mature melanin granules, and yet another which lacked melanin granules. Because of this, he concluded that the corneal dendritic cells were amelanotic melanocytes. This study reinforces the observation of others that corneal epithelial melanosis is preceded and accompanied by corneal vascularization.6,7l 1,U This close association suggests common mechanisms in the stimulation of the melanocytic and vascular invasion of the cornea. With regard to corneal vascularization, the phenomenon is part of the inflammatory response, and evidence has been presented to implicate a crucial role of leukocytes-particularly polymorphonuclear leukocytes-in its pathogenesis.'5 It remains to be determined whether this holds true for corneal epithelial melanosis or not. Colchicine is known to inhibit cell division, and in corneas exposed to this compound some of the epithelium usually becomes denuded within a few days, probably because of its failure to regenerate. Eventually, neutrophilic leukocytes, capillaries, and melanocytes enter the peripheral cornea. The epithelial melanosis that follows corneal injury due to colchicine and silver nitrate is invariably associated with melanocytes within the contiguous peripheral cornea, and probably results from the migration of conjunctival melanocytes into the cornea. The same mechanism has been suggested for other causes of corneal epithelial melanosis.8 In pigmented rabbits, several investigators have observed an apparent migration of the limbal band of pigment towards the corneal lesion, leaving a non-

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pigmented zone at the comeoscleral limbus after corneal injury.27 29, Indeed, this observation, and the presence of goblet cells within the corneal epithelium after some corneal injuries, has given rise to the concept that at least some corneal epithelial defects heal by conjunctival epithelial cells spreading over the defect.2Ti29'-" It remains an open question whether the melanocytes enter the cornea because of an active migration, or whether they are passively carried into the cornea because of a sliding epithelial movement from the corneoscleral limbus. The present findings argue against a concomitant melanocytic proliferation as suggested by some investigators in other experimental models.32" As melanocytes are not arrested in mitoses by colchicine treatment, it is unlikely that these cells undergo a significant degree of cell division during the formation of corneal epithelial melanosis. As only an occasional melaninbearing cell was observed beneath the epithelium, it is most unlikely that melanocytes reach the corneal epitheliu.n by way of subepithelial migration. It is well established that only melanin-bearing cells containing deoxyphenylalanine (DOPA) oxidase synthesize melanin pigment from DOPA.47 As this enzyme has never been demonstrated in epithelial cells, they should not produce melanin, and indeed, premelanosomes were not observed within corneal epithelial cells. Although the nature of the present study does not definitively establish how corneal epithelial cells obtained melanin granules, in all likelihood the process is identical to that in the skin, which has intrigued investigators for years.'' In 1948 Masson51 suggested that melanocytes injected melanin granules into epithelial cells, but how such a process occurs has not been convincingly demonstrated. Swift " proposed that melanocytes secrete melanin granules- into the intercellular space to be taken up by the epithelial cells. If this is true, one would expect to observe occasional extracellular melanin granules between epithelial cells. We did not, and to the best of our knowledge this has not been the case in other ultrastructural studies as well.12.,052 Nevertheless, the theory cannot be discredited, as the phenomenon could be a transient one. Another hypothesis for pigment transfer was proposed by Mottaz and Zelickson.52 They observed cytoplasmic projections of epithelial cells around the dendritic processes of melanocytes and clusters of melanin granules bounded by two concentric membranes inside the epithelial cells, and they postulated that epithelial cells engulfed the tips of the processes of melanocytes. With time, these two concentric membranes were thought to fuse and release the melanosomes into the cytoplasm of the epithehal cell. Although such a mechanism could have been operable in the present models as well, it is of interest that morpho-



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logic evidence of cytoplasmic degradation within the membrane-bounded phagosomes, such as that which occurs when these structures fuse with lysosomes, was not observed within epithelial cells in neither their study nor ours. Our finding of an apparent continuity of the cytoplasm of epithelial cells and adjacent melanocytes supports Charles and Ingram's proposal'" of yet another mechanism, namely a fusion of the membranes of the melanocyte and the epithelial cells that receive the melanin granules. Although we could not convincingly establish that cell fusion did take place because of the vagaries of electron microscopy, cell fusion is a recognized biologic phenomenon. It occurs in foreign body and Langhans multinucleate giant cell formation."-" Cell fusion is also characteristic of cells infected with a variety of viruses including measles, visna, and Sendai (HVJ) viruses.59 Refeences 1. Klintworth GK: Unpublished observations 2. Bellhorn RW, Henkind P: Superficial pigmentary keratitis in the dog. J Am Vet Med Assoc 149:173-175, 1966 3. Duke-Eider S: The eye in evolution. System of Ophthalmology, Vol 1. St. Louis, C.V. Mosby Co., 1958, pp 453 4. Sugiura S, Wakui K, Kondo E: Comparative anatomical and embryological studies on the polygonal cell system in the basal cell layer of the cornea. Acta Soc Ophthalmol Jap 66:1010-1033, 1962 5. Berliner ML: Biomicroscopy of the Eye: Slit Lamp Microscopy of the Living Eye, Vol 1. New York, Hafner Publishing Co., Inc., 1966, pp 243-248, 270 6. Cowan TH: Striate melanokeratosis in negroes. Am J Ophthalmol 57:443 449, 1964 7. Dallos J: Zwei Falle von Melanosis bulbi. Arch Augenheilkd 105:542-546, 1932

8. Henkind P: Migration of limbal melanocytes into the corneal epithelium of guineapigs. Exp Eye Res 4:42-47, 1965 9. Herschendorfer A: Pigment changes in the conjunctiva of the lids in trachoma. Am J Ophthalmol 9:812-822, 1926 10. Heusser H: Ueber Flecken und Vaskularisation der Hornhaut des Pferdes. Arch Ophthalmol (Berl) 106:10-62, 1921 11. Hoffert JR, Fromm PO: Biomicroscopic, gross, and microscopic observations of corneal lesions in the lake-trout Salvelnus namaycush. J Fish Res Board Can 22:761-766, 1965 12. Jauregui HO, Klintworth GK: Pigmented squamous cell carcinoma of cornea and conjunctiva: A light miomscopic, histochemical and ultrastructural study. Cancer (In press) 1976 13. Kreibich C: Hornhautpigmentation. Arch Dermatol Syph (Berd) 135:277-282, 1921 14. Matsuoka H: Histologische Studien uiber das Melaninpigment in der Konjonktiva. Acta Soc Ophthalmol Jap 36:1683-1701, 1932 15. Pillat A: Ueber numcieben Melanosen und Pigentnrvi der Hornhautoberflache. Klin Monatsbl Augenheilkd 104:3945, 1940 16. Redslob E: Etude sur le pigment de l'epithelium conjonctival et corn&en. Ann Ocul (Paris) 159:523437, 1922 17. Reese AB: Tumors of the Eye, Second edition. New York, Harper and Row, Publishers, 1963, pp 330-341

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18. Segawa K: Electron microscopy of dendritic cells in the human corneal epithelium: A modified Masson's ammoniated silver nitrate stain. Invest Ophthalmol 4:264-269, 1965 19. Steiner R: La pigmentation de l'epithelium conjunctival et corneen. Ann Ocul (Paris) 160:137,1923 20. Stocker FW, Prindle RE: A new type of pigment line in the cornea. Am J Ophthalmol 27:341-45, 1944 21. Vogt A: Slit Lamp Microscopy of the Living Eye, Vol 3. Zurich, Schweizer Druck, and Verlagshaus, 1941, pp 1033-1048 22. Yamaguchi H: Beitrag zur Kenntnis der Melanosis corneae. Klin Monatsbl Augenheilkd 42:117-125, 1904 23. Darabos G: Primnres Melanom der HornhauthinterflIche. Klin Monatsbl Augenheilkd 143:529-534, 1963 24. Duke-Elder S: Pigmented tuinours. System of Ophthalmology, Vol 8, Pt 2. St. Louis, C.V. Mosby Co, 1965, pp 1227-1229 25. Lamers WPMA: Malignant melanoma of the cornea. Ophthalmologica 146:353-354, 1963 26. Stallard HB: Primary malignant melanoma of the cornea. Br J Ophthalmol 46:40-44, 1962 27. Lohlein W: Versuche uber die Pigmentwanderung in der Epithelschicht der Hornhaut. Arch Augenheilkd 100:385-401, 1929 28. LZhlein W: Versuche uber die Pigmentwanderung in der Epithelschict der Hornhaut und ihre Bedeutung fUar die Erkenntnis der Epithelregeneration. II. Histologische Befunde. Arch Augenheilkd 102:497-522, 1930 29. Mann I: A study of epithelial regeneration in the living eye. Br J Ophthalmol 28:26-40, 1944 30. Mann I, Pirie A, Pullinger BD: The treatment of Lewisite and other arsenical vesicant lesions of the eyes of rabbits with British anti-lewisite (BAL). Am J Ophthalmol 30:421-435, 1947 31. Mann I, Pirie A, Pullinger BD: An experimental and clinical study of the reaction of the anterior segment of the eye to chemical injury, with special reference to chemical warfare agents. Br J Ophthalmol Suppl 13:5-171, 1948 32. Maumenee AE, Scholz RO: The histopathology of the ocular lesions produced by the sulfur and nitrogen mustards. Bull Johns Hopkins Hosp 82:121-147, 1948 33. Mann I, Pullinger BD: A study of mustard-gas lesions of the eyes of rabbits and men. Am J Ophthalmol 26:1253-1277, 1943 34. Michaelson IC: Proliferation of limbal melanoblasts into the cornea in response to a corneal lesion: An experimental study. Br J Ophthalmol 36:657-665, 1952 35. Reynolds ES: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 36. Snell RS: An electron microscope study of the dendritic cells in the basal layer of guinea-pig epidermis. Z Zellforsch Mikrosk Anat 66:457-470, 1965 37. Ranvier LA: Cornee. Lecons d'Anatomie General faites au college de France. Paris, JB Bailliere, 1881, pp 1878-1879 38. Egorow I: Nervenelemente der Cornea in Meerschweinchenauge. Arch Ophthalmol 131:531-563, 1934 39. Scharenberg K: Cells and nerves of the human cornea: A study with silver carbonate. Am J Ophthalmol 40:368-379, 1955 40. Segawa K, Nakaizumi Y: Fine structure of corneal epithelium: So-called Langerhans cells and nerve endings. Acta Soc Ophthalmol Jap 66:53, 1962 41. Sugiura S, Wakui K: Polygonal cell at the basal cell layer of the corneal epithelium of man, with special reference to its role in movement of the ocular fluid. Acta Soc Ophthal Jap 65:2434-2438, 1961



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42. Segawa K: Electron microscopic studies on the human corneal epithelium: dendritic cells. Arch Ophthalmol 72:650-659, 1964 43. Teng CC: The fine structure of the corneal epithelium and basement membrane of the rabbit. Am J Ophthalmol 51:278-297, 1961 44. Riley PA: The Langerhans cell. The Physiology and Pathophysiology of the Skin. Edited A Jarrett. London, Academic Press, 1974, pp 1199-1218 45. Fromer CH, Klintworth GK: An evaluation of the role of leukocytes in the pathogenesis of experimentally induced corneal vascularization. III. Studies related to the vasoproliferative capability of polymorphonuclear leukocytes and lymphocytes. Am J Pathol 82:157-170, 1976 46. Duke-Elder S: The healing of the corneal tissues.2' pp 604-614 47. Riley PA: The biochemistry of pigment formation." pp 1149-1165 48. Charles A, Ingram JT: Electron microscope observations of the melanocyte of the human epidermis. J Biophys Cytol 6:41-44, 1959 49. Cohen J, Szabo G: Study of pigment donation in vitro. Exp Cell Res 50:418-434, 1968 50. Gazzolo L, Prunieras M: Melanin granules in keratinocytes in vitro. J Invest Dermatol 51:186-189, 1968 51. Masson P: Pigment cells in man. Spec Publ New York Acad Sci 4:15-51, 1948 52. Mottaz JH, Zelickson AS: Melanin transfer: A possible phagocytic process. J Invest Dermatol 49:605-610, 1967 53. Riley PA: Melanin and melanocytes." pp 1104-1130 54. Swift JA: Transfer of melanin granules from melanocytes to the cortical cells of human hair. Nature 203:976-977, 1964 55. Sapp JP: An ultrastructural study of nuclear and centriolar configurations in multinucleated giant cells. Lab Invest 34:109-114, 1976 56. Davis, JMG: The ultrastructural changes that occur during the transformation of lung macrophages to giant cells and fibroblasts in experimental asbestosis. Br J Exp Pathol 44:568-575, 1963 57. Mariano M, Spector WG: The formation and properties of macrophage polykaryons (inflammatory giant cells). J Pathol 113:1-19, 1974 58. Sutton JS, Weiss L: Transformation of monocytes in tissue culture into macrophages, epithelioid cells, and multinucleated giant cells: An electron microscope study. J Cell Biol 28:303-332, 1966 59. Cascardo MR, Karzon DT: Measles virus giant cell inducing factor (fusion factor). Virology 26:311-325, 1965 60. Harris H: The formation and characteristics of hybrid cells. Cell Fusion (Dunham Lecture Series). Oxford, Clarendon Press, 1970, pp 1-31 61. Harter DH, Choppin PW: Cell-fusing activity of visna virus particles. Virology 31:279-288, 1967 62. Giles RE, Ruddle FH: Production and characterization of proliferating somatic cell hybrids. Tissue Culture: Methods and Applications. Edited by PF Kruse, MK Patterson. New York Academic Press, Inc., 1973, pp 475-478 63. Okada Y: Factors in fusion of cells by HVJ. Curr Top Microbiol Immunol 48:102-128, 1969

Acknomwledgmets The authors gratefully acknowledge the valuable assistance of Jay Benbow, Carl M. Bishop, Jessie Calder, Henrv Estrada, Bernard Lloyd, and Allan Summers.

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Figure 1-The conjunctival epithelium that surrounds the cornea in the pigmented Figure 2guinea pig normally contains much melanin pigment (H&E, x 400). Pigmentation ceases in the normal pigmented guinea pig where the epithelium of Fgure 3-The the conjunctiva merges with that of the cornea (H&E, x 400). epithelium of the central cornea of the normal guinea pig lacks melanin pigmentation (H&E, x 400). Fgure 4-After topical colchicine application, numerous corneal epithelial cells become arrested in mitosis. This is the central corneal epithelium of a guinea pig 4 days after colchicine administration. (H&E, x 400) Figure 5-Numerous melanocytes are present in this thin corneal epithelium 7 days after colchicine administration. (Fontana stain, x 400)

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Fur 8-Epithelial cells with clusters of melanin granules are separated by intercellular edema and Fire 9connected to each other by thin cytoplasmic extensions containing desmosomes (x 10,000). Fiure 10This A membrane encircles a mature melanin granule within an epithelial cells (x 53,500). Fgure 11melanin granule inside an epithelial cell lacks a distinct surrounding membrane (x 53,500). Part of a melanocyte adjacent to a cornea] epithelial cell. Mature melanin granules and premelanosomes are present in the cytoplasm of the melanocyte. (x 37,900)

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in the coichicine-treated guinea pig corneas. The presence of lysosomes and melanin granules within phagosomes in these cells, together with the lack of premelanosomes argue that such cells are macrophages. (x 8300) Fgwue 17-Melanin granules enclosed in phagosomes are present in a subepithelial macrophage (x 36,100).