Herpetic keratitis in zinc-deficient rabbits

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1 Herpetic keratitis in zinc-deficient rabbits. Scott Fetter, Gilbert Smolin, Masao Okumoto, and Deborah Condon In a study of rabbits, we found that zinc played a vital role in the growth of the rabbit and in its immunocompetence. Both humoral and cellular immunity were depressed in the zinc-deficient rabbit. Not only did zinc-deficient animals fail to gain weight, differing in that respect from rabbits on a regular diet, but they also had a high death rate, in contrast with no deaths among normal rabbits. Herpes simplex virus keratitis, both epithelial and stromal, was more severe in the zinc-deficient rabbit but was not improved by local zinc replacement (zinc sulphate ointment,.%); Zinc sulphate ointment (%), which is four times stronger than commercially available zinc ophthalmic preparations, was severely toxic for the rabbit cornea. (INVEST OPHTHAMO VIS SCI :-,.) Key words: herpes simplex, keratitis, zinc deficiency I n recent years the importance of zinc in the maintenance of a properly functioning immune system has become increasingly clear. " With this in mind, we decided to investigate the relationship of zinc deficiency to both humoral and cellular immunity in the rabbit. We next explored the natural course of herpes simplex virus (HSV) keratitis in the zinc-deficient animal and attempted to influence favorably, with local zinc replacement therapy, the course of the keratitis. Materials and methods In two experiments we used New Zealand white rabbits of both sexes, each weighing to kg. In the first experiment, animals were divided into lour groups. We fed the first two groups Purina abbit Chow, and the other two groups were fed an identical food lacking only zinc (pre- From the Francis I. Proctor Foundation for esearch in Ophthalmology and the Department of Ophthalmology, University of California, San Francisco. Supported in part by National Institutes of Health grant EY and by Fight for Sight, Inc. Submitted for publication Dec.,. eprint requests: Gilbert Smolin, M.D., Francis. Proctor Foundation, University of California, San Francisco, -S, San Francisco, Calif.. pared by the University of California Vivarium Service). The first two groups drank tap water, and the other two groups received only triple-distilled water. We used galvanized steel cages to house the first two groups and stainless steel cages to house the other two. On day after starting the rabbits on the two diets, we inoculated all of them with. ml of type HSV. We used the PH strain with atcid of (>. The corneas to be inoculated were anesthetized topically with proparacaine HC (Ophthaine) and were then sacrificed with a indner spatula horizontal and vertical scratches. In a double-blind fashion, two independent observers measured the resultant epithelial disease, assigning a percentage value to the area that stained with fluorescein (Figs. and ). The grading scale used in measuring the stromal disease ranged from (no disease) to (maximal disease with leukomatous corneal changes). The first and third groups of rabbits were treated with topical % zinc sulphate ointment in an ionic base. Three times daily on postinoculation days,, and, they were given Vz inch of ointment squeezed from a tube with a. mm opening. Three times daily on the same days, groups two and four (controls) were treated with petroleum alba in an ionic base. The animals were weighed on days,,, and after the two diets were started. Serum samples were obtained by venipuncture -//+$./ Assoc. for es. in Vis. and Ophthal., Inc. Downloaded From: on //

2 Volume Number Herpetic keratitis in zinc-deficient rabbits Table I. Serum zinc levels in rabbits fed a zinc-free diet for days and rabbits fed a regular diet for days* egular diet Zinc-free diet abbit Serum-zinc level (mglloo ml) abbit Died Serum-zinc level (mglloo ml) Of Of *p <. (Student's t test) when zinc-free and regular-diet groups were compared. t Not enough blood to do the test. Fig.. Irregular light reflex indirectly indicates the area of epithelial irregularity. Mild stromal edema, necrosis, and neovascularization is present on day in an untreated animal receiving a regular diet. Table II. Serum zinc levels in rabbits before and after day zinc-free diets* abbit Serum zinc level (mglloo ml) before zinc-free diet Serum zinc level (mgfloo ml) after zinc-free diet *p <. (Student's t test). with stainless steel needles on day after the special diets were started. Serum zinc levels were determined by subjecting the serum samples to atomic absorption. We evaluated antibody production by sensitizing the rabbits with an intravenous injection of ml of % (by volume) sheep red blood cells (SBC) prior to starting the special diets; days later we obtained serum specimens by venipuncture and measured the antibody titers by the hemagglutination technique. Skin tests were performed by sensitizing the animals with drops of a solution of % oxazolone in acetone dropped on the right ear daily for days, beginning on day. Ten days later the rabbits were challenged with. ml of four dilutions of the % oxazolone solution (%,.%,.%, and.%) dropped on their depilated backs. They were graded hr later according to the number of dilutions capable of inducing an Downloaded From: on // Fig.. Arrows indicate the area of epithelial defect overlying the leukomatous stroma. Marked stromal necrosis and neovascularization is present on day in an untreated animal receiving a zinc-deficient diet. erythematous reaction, from (anergy) to (reactive to all four oxazolone dilutions). In the second experiment, animals were divided into three groups. The first two groups were fed the zinc-free diet, and the third group received a regular diet. As in the first experiment, we inoculated all of the rabbits with HSV. This time we treated the first group with V inch of.% zinc sulphate ointment in an ionic base (from a tube with a. mm opening) and treated the second and third groups with the same amount of petroleum alba in an ionic base. The treatment schedule was the same as that in the first experiment.

3 Feiler et al. Invest. Ophthalmol. Vis. Sci. June Table III. Weight changes (gm) of rabbits on zinc-free diets and rabbits on regular diets in weeks Number of days on diet abbit Net change Zinc-free diet * egular diet * p value! < < <. NSD = no significant difference. * Died before day. tsignificant difierences between zinc-free diet group and regular-diet group by Student's t test Sacrificed Sacrificed + Sacrificed NSD <. esults First experiment. Serum specimens for zinc level determinations were taken at random from of the rabbits, seven from the zinc-free diet group and seven from the regular diet group. The average serum zinc level was mg/ ml in the zinc-free group and mg/ ml in the regular diet group (Table I). When the Student's t test was applied to the group averages, the difference between the two groups was statistically significant (p <.). In addition, the serum zinc levels declined significantly in each of three randomly selected animals in the zincdeficient diet group (Table II). We weighed the surviving animals of the (Nos. and died before day ) on days,,, and after starting their respective diets. Group averages on these days were determined and compared (Table III). Downloaded From: on //

4 Volume Number Herpetic keratitis in zinc-deficient rabbits Fig.. Marked cornea! stromal edema and infiltrate secondary to zinc toxicity. Table IV. Hemagglutination antibody response to SBC of rabbits fed regular or zinc-free diets for days* egular diet Zinc-free diet abbit Titer on day after SBC inoculation- Negative Undiluted Negative : t : : : Undiluted Undiluted : : Negative lit : abbit Titer on day after SBC inoculation t : : : : : : : * Difference between zinc-free diet group and regular-diet group by applying Student's t test on log scores: p <.. t Died before day. There was a statistically significant difference between the two dietary groups after each interval except on day. In the regular-diet group, of the animals had a net gain in weight, with an average increase of gm. In the zinc-free diet group, of showed a net loss, with an average loss of gm. The weight loss per week was greatest at the end of the first week. The antibody titers to SBC were sig- Downloaded From: on // Fig.. Histologic section of the limbus in a rabbit with zinc toxicity showing infiltration with polylnorphonuclear leukocytes and rnononuclear cells. Table V. Oxazolone skin tests performed on rabbits that survived zinc-free or regular diets for days* Zinc-free diet abbit Skin-test reaction egular diet abbit Skin-test reaction abbits and sacrificed for histologic examination. * Difference between zinc-free diet group and regular-diet group was determined by Mann-Whitney test: p <.. nificantly reduced in the zinc-free diet group (Table IV). Four of the zinc-deficient animals had negative titers, and none had a titer greater than. In the regular-diet group, only one animal had a titer of less than and seven had titers of :. The difference between the two groups, which was statistically significant, was. as determined by applying the Student's t test to the log of the antibody scores. Despite the death of of the zincdeficient animals by day, the surviving seven animals were significantly less reactive to oxazolone than were animals in the reg-

5 Feiler et al. Invest. Ophthalmol. Vis. Sci. June Table VI. Percentage of rabbit corneas taking fluorescein stain on days to after HS V inoculation (days to of special diets) abbit Day () Zinc-free diet (untreated) egular diet (untreated) p value* NSD Day () <. Day () <. Day () Sacrificed = right eye; = left eye; NSD = no significant difference. Mann-Whitney test when regular and zinc-free diet groups at above intervals were compared. NSD Day () Infectec NSD ular-diet group when the Mann-Whitney test for nonparametric data was used (Table V). All of the regular-diet group were reactive to oxazolone whereas five of the seven zinc-free animals were anergic. In this first experiment, % zinc sulphate ointment was used to treat two groups of rabbits. The entire corneas of these rabbits showed dense white infiltrates, some with rings, after days of thrice-daily applications of the ointment (Fig. ). Histopathologic examination of sections of these corneas was performed to determine the type of reaction that was occurring. These sections showed intense cellular infiltration at the limbus and in the corneal stroma, with polymorphonuclear leukocytes predominating (Fig. ). This led us to believe that a toxic reaction was occurring. The toxicity of the % zinc sulphate for the rabbits' corneas meant that only the untreated groups on zinc-free and regular diets could be compared. The untreated regulardiet group had significantly less epithelial keratitis than did the untreated zinc-free diet group on postinoculation days and (Table VI). The zinc-free diet group also had a % mortality in contrast to the regular-diet group's %. Second experiment. The application of % zinc sulphate to the corneas of a group of normally fed rabbits not infected with HSV confirmed its potential toxicity (Fig. ). A.% concentration was found to be almost free of toxicity, although it caused some transient edema; to ensure safety,.% was used in the rest of the experiment. There was no significant difference in the severity of the epithelial keratitis between the treated and untreated groups on zinc-free diets. The regular-diet group had less severe epithelial keratitis than that in either of these two zinc-deficient groups, treated or untreated (Table VII), not only on postinoculation days and but also on days and. The zinc-deficient group also had more stro- Downloaded From: on //

6 Volume Number Herpetic keratitis in zinc-deficient rabbits Table VII. Percentage of rabbit corneas taking fluorescein stain on days to after HSV inoculation (days to of special diets) /ihhit XUUUIV Day () Zinc-free diet (treated)* Zinc-free diet (untreated) egular diet (untreated) p valuet NSD Day () NSD Day (). Day () <. Day () <. = ight eye; = left eye; NSD = no significant difference. "Treatment with.% zinc sulfate. t Mann-Whitney test, comparing regular diet group (untreated) with zinc-free diet group (treated and untreated). Day () <. mal keratitis, and topical zinc sulphate therapy did not favorably influence either its incidence or its severity (Table VIII). Discussion Zinc is essential for to enzyme systems in the body, including several in the eye. One would expect to see a wide variety of symptoms in zinc-deficient animals. This fact is confirmed by the variety of symptoms present in acrodermatitis enteropathica, an inborn error of zinc metabolism. The morbidity and mortality in our zinc-deficient animals were expected. Several reports have documented the efficacy of zinc in the treatment of various diseases. It is of known benefit in acrodermatitis enteropathica, alcoholic cirrhosis, and various malabsorption syndromes, and there is speculation that it can have a beneficial role in rheumatoid arthritis, sickle-cell anemia, acne, wound healing, chronic debilitation, and chronic infection. ' Golden et al. showed that the local application of zinc sulphate could restore reactivity of the skin to Candida antigen in a group of proteinanergic children. They believed that the skin's lack of reactivity represented a defect in cell-mediated immunity, and concluded that the topical zinc could locally enhance this immunity. This fascinating report led us to initiate this Downloaded From: on //

7 Feiler et al. Invest. Ophthalmol. Vis. Sci. June Table VIII. Clinical scores of herpetic stromal lesions in rabbits fed zinc-free or regular diets days after herpetic inoculation* abbit Stromal lesion Zinc-free diet + zinc sulfate ointment + Zinc-free diet + control ointment egular diet control ointment = right eye; = left eye. *p =. Mann-Whitney test, comparing regular diet group with zinc-free diet groups, both treated and untreated. study, in which we immunosuppressed rabbits with a zinc-deficient diet and attempted to restore cell-mediated immunity topically with the use of topical zinc. The model we used was not the enhancement of a delayed hypersensitivity skin reaction but the amelioration of herpes keratitis. This model was employed because of our interest in this entity and because of the far-reaching ramifications of ameliorating a viral keratitis (e.g. measles) in a malnourished population. Golden and co-workers concluded that zinc deficiency was a major cause of the immunoincompetence seen in malnutrition. Their observations corroborated the results of earlier clinical and laboratory studies. For example, in, Williams and oeb showed that zinc was required for DNA replication in human lymphocytes. Shortly thereafter, Phillips and Azari found that zinc transferrin increased DNA synthesis in peripheral blood lymphocytes. Despite this increased DNA synthesis, however, uhl et al. could not find an associated increase in lymphocyte cytotoxicity. Cunningham et al. found that zinc stimulated human B lymphocytes as strongly as pokeweed antigen, purified protein derivative, and diptheria toxoid, which suggested that it influenced the humoral as well as the cellular immune response. On a more clinical level, Cavdar et al. studied a group of patients with Hodgkin's disease and found that the more impaired the patient's immune responses, the lower the serum zinc level. As noted above, we believed it would be appropriate to develop a rabbit model of zinc deficiency and to determine whether or not the rabbit showed concomitant immunosuppression and developed a more severe HSV keratitis than that in a normal rabbit. In our zinc-deficient rabbit model we documented both impaired cellular immunity (as shown by the reduced reactivity to oxazolone skin testing) and impaired humoral immunity (as shown by a reduction in the formation of antibodies to SBC). The fact that the more severe HSV keratitis developed in the zincdeficient animals and that their mortality was higher probably also signified an impaired immune status. We wanted to improve the immune status of the animals by the local application of zinc, as Golden et al. had done, but found that % zinc sulphate was toxic and that the results of its use were inconclusive. A.% sulphate ointment was not toxic but did not alter the HSV keratitis. The reason for the failure may be related to the lack and inability of lymphocytes to reach the cornea and the rapid dispersion of the ointment from the rabbit eye. Experiments are planned to confirm the work of Golden et al. l and to solve some of the problems related to this model. EFEENCES. Pekarek S, Hoagland AM, and Powanda MC: Humoral and cellular immune responses in zincdeficient rats. Nutr ep Int :,.. Williams O and oeb A: Zinc requirement for Downloaded From: on //

8 Volume Number Herpetic keratitis in zinc-deficient rabbits DNA replication in stimulated human lymphocytes. J Cell Biol :,.. Cunningham-undles S, Cunningham-undles C, Dupont B et al: Zinc-induced activation of human B lymphocytes. Clin Immunol Immunopathol :,.. Powanda MC, Cockerell G, and Pekarek S: Amino acid and zinc movement in relation to protein synthesis in early inflammation. Am J Physiol :,.. Hall JM and O'Connor G: Correlation between ocular inflammation and antibody production. J Immunol :,.. Neldner KH and Hambidge : Zinc therapy of acrodermatitis enteropathica. N Engl J Med :,.. Murray J, Murray A, and Murray N: Anorexia: sentinel of host defense? Prosp Biol Med :,.. Halsted JA, Smith J JC, and Irwin Ml: A conspectus of research on zinc requirements of man. J Nutr :,.. Cunliffe WJ, Burke B, Dodman B, and Gould DJ: A double-blind trial of zinc sulfate/citrate complex and tetracycline in the treatment of acne vulgaria. Br J Dermatol :,.. Golden MHN, Golden BE, Harland PSEG et al: Zinc and immunocompetence in protein-energy malnutrition. ancet :,.. Phillips J and Azari P: Zinc transferrin. Cell Immunol :,.. iihl H, Kirchner H, and Bochert G: Kinetics of the zinc stimulation of human peripheral lymphocytes. Proc Soc Exp Biol Med :,.. Cavdar AO, Babacan E, and Arcasoy A: elation of zinc to lymphocytes in pediatric Hodgkin's disease. Pediatr es :,. Downloaded From: on //