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Vascularization of the rat cornea after prolonged zinc deficiency.

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THE ANATOMICAL RECORD 216:27-32 (1986)
Vascularization of the Rat Cornea After Prolonged
Zinc Deficiency
Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State
University, College of Medicine, Hershey, PA 17033
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,
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
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).
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
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.
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).
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).
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.
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
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).
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
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-
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
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
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
processes can be reversed by zinc supplementation. Cor- Hock, C.W., W.K. Hall, E.R. Pund, and V.P. Sydenstricker (1945) Vascularization of the cornea as a result of lysine deficiency. Fed. Proc.,
neal neovascularization is a multifactorial phenomenon.
The possibility of renewed neural invasion of the ante- Klevay,
L.M., H.G. Petering, and K.L. Stemmer (1971) A controlled
rior corneal stroma adds another dimension.
environment for trace metal experiments on animals. Environ. Sci.
I gratefully acknowledge Dr. John D. Connor for his
helpful suggestions during the course of this study and
Mrs. Doris Lineweaver for typing the manuscript.
This research was supported in part by U.S. Public
Health Service grant EY01438.
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