THE ANATOMICAL RECORD 216:27-32 (1986) Vascularization of the Rat Cornea After Prolonged Zinc Deficiency ALPHONSE E. LEURE-DUPREE Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, PA 17033 ABSTRACT Neovascularization of the anterior stroma of the rat cornea was associated with prolonged zinc deficiency (in this model). There was also a n increase in the myelinated nerves of the cornea. Blood vessels were not observed in the corneas of the pair-fed and ad-libitum-fed control animals. The invading blood vessels were frequently associated with Schwann cells and neurites. Unmyelinated nerves were observed in the corneal stroma of all three experimental groups. The cornea in man and most animals is normally avascular except at the extreme periphery. Numerous methods for producing ocular vascularization have been reviewed by Henkind (1978), Patz (1982), and Klintworth and Burger (1983). In most instances, vascularization of the cornea is associated with some degree of inflammation and implicates a variety of leukocytes (Klintworth and Burger, 1983) and activated macrophages (Polverini et al., 1977). Of special interest here is the role of diet. It has been suggested that the degree of corneal vascularization is a reliable index of nutritional status. Blood vessels in the cornea have been observed in animals maintained on diets deficient in tryptophan and/or lysine (Albanese, 1945; Hock et al., 19451, methionine (Hock et al., 1945; Sydenstricker et al., 1946), riboflavin (Bessey and Wolbach, 1939), and vitamin A (Wolbach and Howe, 1925). The possibility of multifactorial interactions was revealed by the observation that vitamin-A-deficient rats, housed in a germ-free environment and fed a diet supplemented with retinoic acid, did not develop corneal vascularization (Carter-Dawson et al., 1980). References to the influence of trace metals are sparse. Follis and co-workers (1941) reported that two of seven rats maintained on a diet low in zinc had vascularized corneas. A clinical report on acrodermatitis enteropathica, a n autosomal recessive defect that results in diminished absorption cf intestinal zinc, mentioned a n ingrowth of blood vessels into the cornea (Wirsching, 1962). In this regard, it also may be relevant that corneal vascularization has been reported as a consequence of intoxication with thallium, a heavy metal (Busche, 1943). The present study deals with morphological observations of neovascularization of the corneal stroma in zincdeficient rats. Histological examination of all corneas in this study did not reveal the presence of inflammatory cells. ually in stainless-steel cages were kept in a temperature-controlled room (23 f 2°C) with 45-55% humidity and with a 12-hour light/dark cycle. Rats used in this study were isolated from animals used in other investigations. Water bottles were presoaked in EDTA solution, acid washed, and rinsed several times with deionized, double-distilled water. The bottles were equipped with silicone stoppers. Rats were randomly assigned to one of three experimental groups. All were fed a commercially prepared zinc-deficient, pelleted diet (Teklad, Madison, Wisconsin) that contained 0.7 ppm zinc as determined in our laboratory by atomic absorption spectroscopy. Groups called “zinc-deficient” and “ad libitum” controls received the diet ad libitum. The “pair-fed” group received the diet in an amount equivalent in weight to that consumed by zinc-deficient rats. Distilled deionized water with 30 ppm zinc (as zinc acetate) was freely available to the pair-fed and ad libitum groups, whereas there was no zinc in the water of the zinc-deficient rats. Food intake and animal weight changes were measured daily between 0800 and 0900 hours. Unconsumed food was weighed at the same time. After being placed on the diet, seven animals in each group were killed at intervals from 5 to 7 weeks. Electron microscopy Corneas from animals after 5, 6 , and 7 weeks of controlled zinc intake were fixed with 3% glutaraldehyde (Ladd) in 0.1 M cacodylate buffer (pH 7.3) containing 0.02% calcium chloride. The tissues were rinsed in buffer and postfixed in 1%osmium tetroxide in 0.1 M cacodylate buffer. The corneas were processed by a procedure previously described (Leure-duPree, 1974). RESULTS Zinc assessment As was reported in previous studies from this laboratory (Leure-duPree and McClain, 1982; Leure-duPree et al., 1982), the earliest manifestation of zinc deficiency, a MATERIALS AND METHODS measurable decrease in food consumption, was observed Seventy weanling male Sprague-Dawley rats, weigh- within 7 days. Subsequent evidence of zinc deficiency ing 40 to 50 gm (Charles River Breeding Laboratories), included impairment of growth and development of skin were maintained in a n environment designed for trace _ _ _ ~ Received September 19,1985;accepted March 17,1986. metal studies (Klevay et al., 1971).Rats housed individ0 1986 ALAN R. LISS, INC. 28 A.E. LEURE-DUPREE lesions. Zinc deficiency was ultimately confirmed by measuring zinc concentration in bone by atomic absorption spectroscopy. In agreement with previously reported findings with this diet, femur zinc concentration in the zinc-deficient rats was approximately 35% of the control values (Leure-duPree et al., 1982). on a zinc-deficient diet without zinc supplementation (Fig. 3). These myelinated fibers contained neurotubules, neurofilaments, and channels of endoplasmic reticulum. Some neurites contained clear or dense-cored vesicles. Mesaxons were also observed (Fig. 3). Morphology The general structural organization of the rat cornea was basically similar to that of other mammalian corneas. The morphological characteristics of the ad libitum and pair-fed control rats were similar. A photomicrograph of a cornea of a n ad-libitum-fed control rat on the diet for 7 days with zinc supplementation is seen in Figure 1. The thickness of the cornea of the control rats was about 200 pm, of which 60 pm constituted the epithelial layer and approximately 130 pm the stroma. Corneal innervation was organized essentially as described for the mouse (Whitear, 1960). Nerve fibers entered the cornea either from the region of the limbus or from the sclera. Unmyelinated nerve fibers were commonly observed in the anterior stroma of the corneas of all three diet groups. Axons were either completely or partially surrounded by Schwann cell cytoplasm. Typically, a distinct basal lamina surrounded a group of nerve fibers (Fig. 2). Myelinated nerves, although occasionally present in the anterior Of the controls, were more apparent in the stroma maintained Fig. 1. Cornea of ad libitum control rat maintained on the diet for 7 weeks. Basal cells (arrows)of the epithelium appear columnar (x1,500). Fig. 2. Cornea of zinc-deficient rat maintained on the diet for 7 weeks. Fibroblast (F); unmyelinated neurites surrounded by basal lamina (arrows) located in the anterior stroma ( x 69,000). VASCULARIZATIONOF CORNEA 29 Fig. 3. Myelinated and unmyelinated axons ensheathed by fibroblast (F) processes in the anterior stroma of a zinc-deficient rat maintained on the diet for 7 weeks. The neurite contains dense-cored vesicles (dvc). Mesaxon (arrow) of myelinated nerve; junctional complex (hollow arrowhead) ( x 43,000). One of the more novel observations was the apparent ensheathing of the Schwann cells and the myelinated nerves by long branching processes of the fibroblasts (Fig. 3). This relationship is similar to that seen in association with the autonomic innervation of blood vessels. This juxtaposition was not evident in the corneas of the ad-libitum- or pair-fed animals. An unexpected finding was the presence of blood vessels in the anterior corneal stroma of the zinc-deficient rats. Sixteen of the 21 rats maintained on a zinc-deficient diet for 5-7 weeks without zinc supplementation showed neovascularization of the cornea. The invading vessels often contained erythrocytes (Fig. 4).Although the vessels varied in size, no muscular coat was observed. The endothelial cells were not fenestrated (Fig. 5). Nerve fibers were observed in close association with ingrowing blood vessels. For example, in Figure 6, a Schwann cell, a bouton, and a neurite are in close association with the invading blood vessel located in the anterior stroma of the cornea. The Schwann cell, unlike the bouton and neurite, was not covered with basal lamina. The bouton contained a complement of mitochondria and neurotubules; some neurites contained clear vesicles. There was also a n alteration in the corneal epithelial cells (Fig. 4),particularly flattening of the basal cells (Fig. 5). The basal cells, which were characteristically columnar, were broader than those of Of the ad-1ibitum- and pair-fed No the changes were observed in the corneal epithelium of the control animals. Fig. 4. Cornea of a zinc-deficient rat maintained on the diet for 6 weeks, Blood vessels (arrows) in the anterior stroma of the cornea contain erythrocytes. Note the flattened basal cells of the corneal epithelium ( X 1,800). 30 A.E. LEURE-DUPREE Fig. 5. Cornea of zinc-deficient rat maintained on the diet for 6 weeks. The basal cells of corneal epithelium (BC) are flattened. Fibroblast (F);blood vessel (BV) in anterior stroma of cornea. Nucleus (Nu) of endothelial cell of blood vessel. Note the usual proximity of the fibroblasts (F) to the epithelium (~20,535). DISCUSSION served in the ad-libitum- and pair-fed controls. At the The morphological observations in this study demon- time selected for microscopic examination (5-7 weeks) strate that a significant number of rats maintained on a polymorphonuclear leukocytes were not observed in the zinc-deficient diet without supplementation have a n in- corneas. In a light microscopic study, Follis and co-workgrowth of blood vessels in the anterior stroma of the ers (1941) observed leukocytes in the corneas of rats on cornea. Neovascularization of the cornea was not ob- low levels of dietary zinc. A considerable body of evi- VASCULARIZATION OF CORNEA 31 Fig. 6. Anterior corneal stroma of zinc-deficient rat maintained on the diet for 7 weeks. Schwann cell (S);bouton (b) and processes of the neurites are in close association with blood vessel (bv). Neurite with dense-cored vesicles (arrow). The nucleus of a pericyte (P)and a stromal cell (ST)are present (X21,OOO). dence points to leukocytes as being a n important stimulus for corneal neovascularization (Klintworth and Burger, 1983). Further studies are presently underway to determine whether at earlier times leukocytes are present. The intimate association of Schwann cells and neurites with corneal vessels suggests a concurrent neurovascular invasion. This, of course, begs the question of whether the invading blood vessels carry their neural element in with them, invade along pathways already occupied by neurites, follow newly invading neurites into the stroma, or are followed by such neurites. An additional question is whether the Schwann cells myelinate nerve processes already resident in the cornea or myelinate newly arrived neurites. Whether Schwann cells precede, accompany, or follow the invading blood vessels is under investigation. However, this seems to be the first report that Schwann cells and neurites are in close association with vessels invading the corneal stroma. Generally, the Schwann cells associated with blood vessels lacked a defined basal lamina, although a distinct basal lamina was present on boutons and neurites in intimate contact with vessels. Absence of a basal lamina in Schwann cells is generally a characteristic of early embryonic development. Bressler and Munger (1983) reported that Schwann cells in young monkeys had minimal basal laminae compared to Schwann cells around axon bundles. Therefore, the Schwann cells associated with blood vessels in the anterior stroma of zinc-deficient rats may be embryonic (or newly divided) in nature. An alternative explanation for the lack of basal lamina is that Schwann cells have migrated into the corneal stroma. It has been reported that migrating Schwann cells generally do not have a basal lamina (Billings-Galiardi et al., 1974). Unmyelinated nerves not associated with blood vessels were commonly observed in the corneas of all three treatment groups and are probably comparable to the polymodal C fibers (Beuerman and Tanelian, 1979). The association of fibroblasts with Schwann and myelinated nerves was a frequent observation, and it raises questions about the interaction between fibroblasts and Schwann cells. Fibroblasts may serve as a tunnel or framework for the invading neurites that may be stimulated as a result of some neurogenic factor yet unknown. Morphological alteration in the epithelium consisted primarily of a flattening of the basal cells, and some loss of cell-to-cell functions. Issues raised in these experiments include the identification of the precise time that blood vessels enter the corneal stroma during zinc deficiency, and whether these 32 A.E. LEURE-DUPREE processes can be reversed by zinc supplementation. Cor- Hock, C.W., W.K. Hall, E.R. Pund, and V.P. 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