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The Effect of Corticosteroid Hormones on Experimental Herpes Simplex Keratitis
PINE POLLEN GRANULOMATA suits in granulomata of the iris after 14-21 days. 640 South Kingshighway Boulevard (10).
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ACKNOWLEDGMENT
I should like to thank Edward G. Locke, Chief, Division of Wood Chemistry, U.S. Dept. of Agri culture for supplying the pine pollen necessary for this study.
REFERENCES
1. Cummings, M. M., Dunner, E., Schmidt, R. H., Jr., and Bamwell, J. B.: Concepts of epidemiology of sarcoidosis; preliminary report of 1,194 cases reviewed with special reference to geographic ecology Postgrad. Med., 19:437, 1956. 2. Cummings, M. M., and Hudgins, P. C : Chemical constituents of pine pollen and their possible relationship to sarcoidosis, Am. J. M. Sc, 236:31, 1958. 3. Utz, J. P., Moderator. Sarcoidosis. Clinical Staff Conference at the Nat. Inst. of Health Ann. of Int. Med., 51:1356, 1959. 4. Bullington, S. J., and Waksman, B. H.: A study of iridocyclitis occurring in rats with arthritis induced by the injection of Freund's adjuvant, Abstract, Am. J. Oph., 49:1424, 1960.
T H E E F F E C T O F CORTICOSTEROID H O R M O N E S ON E X P E R I M E N T A L H E R P E S SIMPLEX K E R A T I T I S A CLINICAL, HISTOPATHOLOGIC, AND HISTOCHEMICAL STUDY S A M U E L J. K I M U R A , M.D., V I C T O R D I A Z - B O N N E T , M.D., M A S A O O K U M O T O , M.A. A N D M I C H A E L J. H O G A N ,
M.D.*
San Francisco
It is generally agreed that the topical use of corticosteroid hormones has a deleterious effect on herpes simplex. We have shown this effect in previous studies.1 The experi mental disease in rabbits treated with topical corticosteroids was a much more severe dis ease and had a more prolonged course. This effect was thought by us to be due to an in creased multiplication of the virus, because cortiscosteroids have been shown to have this effect on other viruses. 2 ' 3 Kilbourne and Horsfall showed that cortisone caused an in creased influenza virus multiplication in chicken eggs, and Teodoru and Shwartzman showed the same effect in experimental polio myelitis in hamsters. Jawetz, Okumoto and Sonne,4 however, were not able to show that * From the Department of Ophthalmology and the Francis I. Proctor Foundation for Research in Ophthalmology, University of California School of Medicine. This investigation was supported in part by the National Council to Combat Blindness Grantin-aid No. G-238. Read at Midwinter National Meet ing of the Association for Research in Ophthalmol ogy, New Orleans, December 5, 1960.
corticosteroids increase the amount of herpes simplex virus in rabbit corneas. This sug gests that the effect is not due to suppression of antibody formation by the steroid. The only other possibility is that the drugs prob ably alter the cornea so as to decrease its re sistance to the herpes simplex virus. This in vestigation is designed to study the cellular effects of this virus on corneal tissues as de termined by clinical and histologic changes. MATERIALS AND METHODS
Animals. Black Dutch rabbits weighing approximately two kilograms were used. All of the injections were performed with the eyes anesthetized with one drop of Ophthaine. Virus. The P H ("O") strain of herpes simplex virus was used. The stock virus was prepared by injecting mouse brain and mak ing a 20 percent mouse brain suspension in skim milk. The preparation was stored at —40°C. The twenty-third mouse.brain pas sage having an LD50 of 10~5 was used. Steroids. Prednisolone acetate (Meticor-
946/74
S. J. KIMURA, V. DIAZ-BONNET, M. OKUMOTO AND M. J. HOGAN
telone acetateR) 25 mg/ml was used. Two days prior to the virus injection 0.2 ml (5 mg.) of the suspension was injected subconjunctivally and it was repeated every other day for the length of the experiment. Steroid vehicle control. The vehicle solu tion of Meticortelone acetate was prepared according to the formula issued with each multidose vial (phenylethyl alcohol 5 mg/ml, benzalkonium chloride 0.1 mg/ml, H 2 0 C.P.). This vehicle solution was injected subconjunctivally (0.2 ml) into eyes of the control series. Methods. Thirty rabbits were divided into three groups. Group I) 12 rabbits were given subconjunctival injections of corticosteroids to each eye and two days later were injected with 0.03 ml of the virus suspen sion P H M.23 beneath Tenon's capsule of each eye; Group II) 12 rabbits were treated in the same manner as Group I, except that the steroid vehicle solution was substituted for the steroid suspension; Group I I I ) con trol series, six rabbits were injected with the virus and treated with injections of saline regularly every other day until the animals were sacrificed. Clinical examination of the rabbits were made every other day and the cornea and anterior chamber of the rabbits were studied with a biomicroscope. Rabbits from each group were sacrified daily from the fourth to nineteenth postvirus inoculation day. The eyes were immediately fixed in 20 percent formalin solution. After fixation for 24 hours the eyes were cut and corneal specimens were taken for sectioning. The following histologic and histochemical stains were used: Hematoxylin and eosin ( H & E ) , colloidal iron ( A M P ) , Alcian Blue, Giemsa, toluidine blue, periodic acid-Schiff ( P A S ) , and Verhoeff's elastic tissue stain. The stained sections were studied with a light microscope. RESULTS
Clinical. Clinically the corticosteroid treated animal developed a slight discharge E Meticortelone acetate furnished by Schering Corporation.
and redness of the eyes as early as the sec ond day after the virus was inoculated. By the fourth day all these animals developed a moderately severe keratoconjunctivitis. The control animals, on the other hand, developed keratitis about the fourth day but clinically only a few corneal lesions were seen with the biomicroscope and often only in one eye. There was no discharge until the fifth day or later. The course of the clinical disease was much longer in the corticosteroid treated series. The corneal lesions progressed until the cornea was often completely denuded of the epithelium and the stroma was edematous and hazy. Of the six treated rabbits ob served beyond the eleventh postvirus inocu lation day, all developed a diffuse epithelial lesion with edema. Three developed shallow ulcers (disciform keratitis). Five of the six rabbits developed uveitis on or about the twelfth day. The control series observed be yond the eleventh postinoculation day devel oped corneal scarring but all of the corneas were healed by the fourteenth day. Two rabbits developed bilateral uveitis in this group. The clinical picture is summarized in Table I. Pathological. Hematoxylin and eosin stained sections of the treated and control eyes showed the typical epithelial lesion in the early stages. The epithelial cells border ing the dendritic ulcer showed intranuclear inclusions and viral-type giant cells. The corticosteroid treated eyes showed the pres ence of inflammatory cells in the epithelial ulcer at an earlier period. This is in keeping with the clinical picture which showed slight discharge in the treated eyes from the sec ond day after virus inoculation. The control eyes showed the presence of inflammatory cells in the epithelial lesion after the seventh day. The stroma of the cornea also appeared different in the corticosteroid treated eyes. There was edema from the fourth day as contrasted with controls which showed edema on the seventh day. This edema per sisted longer in the steroid treated eyes. The
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EXPERIMENTAL HERPES SIMPLEX TABLE I Days Postinoculation
Steroid
Steroid Vehicle Control
Saline Control
4
Keratoconjunctivitis
Keratitis—slight
Keratitis—slight
5
Keratoconjunctivitis—Moder ately severe
Keratoconjunctivitis—Mild
Keratoconjunctivitis—Mild
7- 8
Keratoconjunctivitis—Severe
Keratocon j unctivitis—M ild
Keratocon j uncti vi tis—M ild
10-11
Keratocon j u nctivi tis—Severe
Keratocon j unctivitis—M ild, healing
Keratoconjunctivitis—-al most healed
12-13
Keratoconjunctivitis—Severe Disciform keratitis Uveitis (5/6 eyes)
Cornea healed Uveitis (1/3 eyes)
Eyes healed
14-19
Disciform keratitis Uveitis Severe corneal scarring
Most cases healed 2 cases uveitis
control eyes showed no edema after the twelfth day. Along with edema there were increased numbers of corneal fibrocytes in both the treated and control series after the twelfth day. The nuclei of these cells often were larger and rounder than normal. The steroid-treated eyes also showed an earlier stromal infiltration of inflammatory cells. There were neutrophils from the fourth day in the treated rabbits but not until the eighth day in the control eyes. Necrosis of the superficial stroma of the cornea was present in the corticosteroid treated eyes from around the twelfth day. This was not seen in the control eyes. The necrosis is in keeping with the clinical find ing of disciform keratitis in the treated rab bits after the eighth day. Inclusion bodies persisted in diminishing number up to the nineteenth day in treated eyes. In the control eyes none were seen after the twelfth day. Most of the inclusion bodies in the late stages were of the eosinophilic type. Giantcells were found through the twelfth day after virus inoculation in both series. Histochemical studies. Corticosteroid treated and control corneas were subjected to histochemical procedures. Under the con ditions of the experiment we were not able to determine any significant alteration in the mucopolysaccharide or collagen content
of the cornea. Edema of the stroma caused all of the stains to be lighter in color in both the treated and control series. DISCUSSION
Experimental herpes simplex keratitis in the rabbit is more severe following treat ment with corticosteroid hormone (Prednisolone acetate). The treated eyes develop a more severe keratoconjunctivitis with hyperemia and discharge which has an earlier onset and lasts longer. Histopathological ex amination of the cornea shows that treated cases develop more extensive epithelial lesions with stromal edema and infiltration of inflammatory cells. Inclusion bodies per sist longer in the treated corneas but the viral type giant cells were not seen after the twelfth postinoculation day in both series. The giant cells are formed by the condensa tion of chromatin into smaller locules, each containing inclusion material. The loculated nuclei continue to grow as the inclusions in crease in size. Each compartment assumes the size and shape of an individual nucleus. Therefore, giant cells are found only during the stage that virus is multiplying and their presence probably indicates viral activity in the cornea. The presence of edema and inflammatory cells in the corneal stroma indicates a more severe keratitis. Necrosis of the superficial
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S. J. KIMURA, V. DIAZ-BONNET, M. OKUMOTO AND M. J. HOGAN
corneal stroma also indicates a more severe reaction. Clinically this is seen in the late stages of disciform keratitis. The corneal fibrocytes were more numer ous and were larger, but no intranuclear in clusions were seen in these cells. If the deleterious effect of the corticosteroid hormone on herpetic keratitis is due to changes in the corneal ground substance, and acts by decreasing its resistance to spread of the infection, one might expect changes in permeability of the ground substance of the cornea. Two of the functions of the ground substance are thought to be: 1) to act as a mechanical barrier against spread of foreign substance; and 2) to provide a substrate for repair of connective tissue. It is known that corticosteroids decrease connective tissue elements such as fibroblasts and thus prevent normal wound healing. Therefore, histochemical stains and reactions were applied but no change was found. The difference in amount of mucopolysaccharide present may be too small to be detected by these stains. It is possible that the resistance of the cornea is decreased by an epithelial disease of such severity that the epithelium is lost. Smelser5 felt that the synthesis of the ground substance is accomplished by the epi thelium of the cornea and the connective tis sue. He interpreted the role of the epithelium as a source of energy or as a precursor necessary to the synthesis of chondroitin sulfate and/or keratosulfate.
FURTHER RESEARCH
It is certainly evident that the mechanism by which the corticosteroids cause more severe corneal disease in herpetic keratitis remains unanswered. Perhaps electron micro scopic examination of tissue sections will reveal whether there is prolongation of sur vival of the virus. Kilbourne6 showed this to be true of influenza B virus in chick embryo cultures. Electron microscopic studies are now under way in our laboratories. Perhaps another approach to the solution of this problem is to carry out enzyme histochemistry, for it is conceivable that the decreased resistance of the cornea is a function of enzymatic action.
SUMMARY
1. Clinical herpes simplex infection of the cornea runs a prolonged course and many of the animals develop a picture of disciform keratitis on prolonged treatment with Prednisolone acetate. 2. Histopathologically the steroid treated rabbits show more extensive epithelial and stromal involvement. 3. Histochemical studies under the condi tions of the experiment failed to show any change in the mucopolysaccharide or the collagen of the cornea. San Francisco (22).
REFERENCES
1. Kimura, S. J., and Okumoto, M.: The effect of corticosteroids on experimental herpes simplex keratoconjunctivitis in the rabbit. Am. J. Ophth., 43:131-134, Part II, Apr. 1957. 2. Kilbourne, E. D., and Horsfall, F. L., Jr.: Increased virus in eggs injected with cortisone. Proc. Soc. Exper. Biol. & Med., 76:116-118, 1951. 3. Teodoru, C. V., and Shwartzman, G.: Endocrine factors in pathogenesis of experimental poliomyeli tis in hamsters. Role of inoculatory and environmental stress. Proc. Soc. Exper. Biol. & Med., 91:181-187, 1956. 4. Jawetz, E., Okumoto, M., and Sonne, M.: Studies on herpes simplex. X. The effect of corticosteroids on herpetic keratitis in the rabbit. J. Immunology, 83:486-490, Nov. 1959. 5. Smelser, George K.: The importance of the epithelium in the synthesis of the sulfated ground substances in corneal connective tissue. Tr. N.Y. Acad. Sciences, 21:575-577, May 1959. 6. Kilbourne, E. D.: The influence of cortisone on experimental viral infection. I. Prolongation of survival time and suppression of inflammation in chick embryos infected with influenza B virus. J Im munology, 74:57-62, 1955.
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ACKNOWLEDGMENT
I should like to thank Edward G. Locke, Chief, Division of Wood Chemistry, U.S. Dept. of Agri culture for supplying the pine pollen necessary for this study.
REFERENCES
1. Cummings, M. M., Dunner, E., Schmidt, R. H., Jr., and Bamwell, J. B.: Concepts of epidemiology of sarcoidosis; preliminary report of 1,194 cases reviewed with special reference to geographic ecology Postgrad. Med., 19:437, 1956. 2. Cummings, M. M., and Hudgins, P. C : Chemical constituents of pine pollen and their possible relationship to sarcoidosis, Am. J. M. Sc, 236:31, 1958. 3. Utz, J. P., Moderator. Sarcoidosis. Clinical Staff Conference at the Nat. Inst. of Health Ann. of Int. Med., 51:1356, 1959. 4. Bullington, S. J., and Waksman, B. H.: A study of iridocyclitis occurring in rats with arthritis induced by the injection of Freund's adjuvant, Abstract, Am. J. Oph., 49:1424, 1960.
T H E E F F E C T O F CORTICOSTEROID H O R M O N E S ON E X P E R I M E N T A L H E R P E S SIMPLEX K E R A T I T I S A CLINICAL, HISTOPATHOLOGIC, AND HISTOCHEMICAL STUDY S A M U E L J. K I M U R A , M.D., V I C T O R D I A Z - B O N N E T , M.D., M A S A O O K U M O T O , M.A. A N D M I C H A E L J. H O G A N ,
M.D.*
San Francisco
It is generally agreed that the topical use of corticosteroid hormones has a deleterious effect on herpes simplex. We have shown this effect in previous studies.1 The experi mental disease in rabbits treated with topical corticosteroids was a much more severe dis ease and had a more prolonged course. This effect was thought by us to be due to an in creased multiplication of the virus, because cortiscosteroids have been shown to have this effect on other viruses. 2 ' 3 Kilbourne and Horsfall showed that cortisone caused an in creased influenza virus multiplication in chicken eggs, and Teodoru and Shwartzman showed the same effect in experimental polio myelitis in hamsters. Jawetz, Okumoto and Sonne,4 however, were not able to show that * From the Department of Ophthalmology and the Francis I. Proctor Foundation for Research in Ophthalmology, University of California School of Medicine. This investigation was supported in part by the National Council to Combat Blindness Grantin-aid No. G-238. Read at Midwinter National Meet ing of the Association for Research in Ophthalmol ogy, New Orleans, December 5, 1960.
corticosteroids increase the amount of herpes simplex virus in rabbit corneas. This sug gests that the effect is not due to suppression of antibody formation by the steroid. The only other possibility is that the drugs prob ably alter the cornea so as to decrease its re sistance to the herpes simplex virus. This in vestigation is designed to study the cellular effects of this virus on corneal tissues as de termined by clinical and histologic changes. MATERIALS AND METHODS
Animals. Black Dutch rabbits weighing approximately two kilograms were used. All of the injections were performed with the eyes anesthetized with one drop of Ophthaine. Virus. The P H ("O") strain of herpes simplex virus was used. The stock virus was prepared by injecting mouse brain and mak ing a 20 percent mouse brain suspension in skim milk. The preparation was stored at —40°C. The twenty-third mouse.brain pas sage having an LD50 of 10~5 was used. Steroids. Prednisolone acetate (Meticor-
946/74
S. J. KIMURA, V. DIAZ-BONNET, M. OKUMOTO AND M. J. HOGAN
telone acetateR) 25 mg/ml was used. Two days prior to the virus injection 0.2 ml (5 mg.) of the suspension was injected subconjunctivally and it was repeated every other day for the length of the experiment. Steroid vehicle control. The vehicle solu tion of Meticortelone acetate was prepared according to the formula issued with each multidose vial (phenylethyl alcohol 5 mg/ml, benzalkonium chloride 0.1 mg/ml, H 2 0 C.P.). This vehicle solution was injected subconjunctivally (0.2 ml) into eyes of the control series. Methods. Thirty rabbits were divided into three groups. Group I) 12 rabbits were given subconjunctival injections of corticosteroids to each eye and two days later were injected with 0.03 ml of the virus suspen sion P H M.23 beneath Tenon's capsule of each eye; Group II) 12 rabbits were treated in the same manner as Group I, except that the steroid vehicle solution was substituted for the steroid suspension; Group I I I ) con trol series, six rabbits were injected with the virus and treated with injections of saline regularly every other day until the animals were sacrificed. Clinical examination of the rabbits were made every other day and the cornea and anterior chamber of the rabbits were studied with a biomicroscope. Rabbits from each group were sacrified daily from the fourth to nineteenth postvirus inoculation day. The eyes were immediately fixed in 20 percent formalin solution. After fixation for 24 hours the eyes were cut and corneal specimens were taken for sectioning. The following histologic and histochemical stains were used: Hematoxylin and eosin ( H & E ) , colloidal iron ( A M P ) , Alcian Blue, Giemsa, toluidine blue, periodic acid-Schiff ( P A S ) , and Verhoeff's elastic tissue stain. The stained sections were studied with a light microscope. RESULTS
Clinical. Clinically the corticosteroid treated animal developed a slight discharge E Meticortelone acetate furnished by Schering Corporation.
and redness of the eyes as early as the sec ond day after the virus was inoculated. By the fourth day all these animals developed a moderately severe keratoconjunctivitis. The control animals, on the other hand, developed keratitis about the fourth day but clinically only a few corneal lesions were seen with the biomicroscope and often only in one eye. There was no discharge until the fifth day or later. The course of the clinical disease was much longer in the corticosteroid treated series. The corneal lesions progressed until the cornea was often completely denuded of the epithelium and the stroma was edematous and hazy. Of the six treated rabbits ob served beyond the eleventh postvirus inocu lation day, all developed a diffuse epithelial lesion with edema. Three developed shallow ulcers (disciform keratitis). Five of the six rabbits developed uveitis on or about the twelfth day. The control series observed be yond the eleventh postinoculation day devel oped corneal scarring but all of the corneas were healed by the fourteenth day. Two rabbits developed bilateral uveitis in this group. The clinical picture is summarized in Table I. Pathological. Hematoxylin and eosin stained sections of the treated and control eyes showed the typical epithelial lesion in the early stages. The epithelial cells border ing the dendritic ulcer showed intranuclear inclusions and viral-type giant cells. The corticosteroid treated eyes showed the pres ence of inflammatory cells in the epithelial ulcer at an earlier period. This is in keeping with the clinical picture which showed slight discharge in the treated eyes from the sec ond day after virus inoculation. The control eyes showed the presence of inflammatory cells in the epithelial lesion after the seventh day. The stroma of the cornea also appeared different in the corticosteroid treated eyes. There was edema from the fourth day as contrasted with controls which showed edema on the seventh day. This edema per sisted longer in the steroid treated eyes. The
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EXPERIMENTAL HERPES SIMPLEX TABLE I Days Postinoculation
Steroid
Steroid Vehicle Control
Saline Control
4
Keratoconjunctivitis
Keratitis—slight
Keratitis—slight
5
Keratoconjunctivitis—Moder ately severe
Keratoconjunctivitis—Mild
Keratoconjunctivitis—Mild
7- 8
Keratoconjunctivitis—Severe
Keratocon j unctivitis—M ild
Keratocon j uncti vi tis—M ild
10-11
Keratocon j u nctivi tis—Severe
Keratocon j unctivitis—M ild, healing
Keratoconjunctivitis—-al most healed
12-13
Keratoconjunctivitis—Severe Disciform keratitis Uveitis (5/6 eyes)
Cornea healed Uveitis (1/3 eyes)
Eyes healed
14-19
Disciform keratitis Uveitis Severe corneal scarring
Most cases healed 2 cases uveitis
control eyes showed no edema after the twelfth day. Along with edema there were increased numbers of corneal fibrocytes in both the treated and control series after the twelfth day. The nuclei of these cells often were larger and rounder than normal. The steroid-treated eyes also showed an earlier stromal infiltration of inflammatory cells. There were neutrophils from the fourth day in the treated rabbits but not until the eighth day in the control eyes. Necrosis of the superficial stroma of the cornea was present in the corticosteroid treated eyes from around the twelfth day. This was not seen in the control eyes. The necrosis is in keeping with the clinical find ing of disciform keratitis in the treated rab bits after the eighth day. Inclusion bodies persisted in diminishing number up to the nineteenth day in treated eyes. In the control eyes none were seen after the twelfth day. Most of the inclusion bodies in the late stages were of the eosinophilic type. Giantcells were found through the twelfth day after virus inoculation in both series. Histochemical studies. Corticosteroid treated and control corneas were subjected to histochemical procedures. Under the con ditions of the experiment we were not able to determine any significant alteration in the mucopolysaccharide or collagen content
of the cornea. Edema of the stroma caused all of the stains to be lighter in color in both the treated and control series. DISCUSSION
Experimental herpes simplex keratitis in the rabbit is more severe following treat ment with corticosteroid hormone (Prednisolone acetate). The treated eyes develop a more severe keratoconjunctivitis with hyperemia and discharge which has an earlier onset and lasts longer. Histopathological ex amination of the cornea shows that treated cases develop more extensive epithelial lesions with stromal edema and infiltration of inflammatory cells. Inclusion bodies per sist longer in the treated corneas but the viral type giant cells were not seen after the twelfth postinoculation day in both series. The giant cells are formed by the condensa tion of chromatin into smaller locules, each containing inclusion material. The loculated nuclei continue to grow as the inclusions in crease in size. Each compartment assumes the size and shape of an individual nucleus. Therefore, giant cells are found only during the stage that virus is multiplying and their presence probably indicates viral activity in the cornea. The presence of edema and inflammatory cells in the corneal stroma indicates a more severe keratitis. Necrosis of the superficial
948/76
S. J. KIMURA, V. DIAZ-BONNET, M. OKUMOTO AND M. J. HOGAN
corneal stroma also indicates a more severe reaction. Clinically this is seen in the late stages of disciform keratitis. The corneal fibrocytes were more numer ous and were larger, but no intranuclear in clusions were seen in these cells. If the deleterious effect of the corticosteroid hormone on herpetic keratitis is due to changes in the corneal ground substance, and acts by decreasing its resistance to spread of the infection, one might expect changes in permeability of the ground substance of the cornea. Two of the functions of the ground substance are thought to be: 1) to act as a mechanical barrier against spread of foreign substance; and 2) to provide a substrate for repair of connective tissue. It is known that corticosteroids decrease connective tissue elements such as fibroblasts and thus prevent normal wound healing. Therefore, histochemical stains and reactions were applied but no change was found. The difference in amount of mucopolysaccharide present may be too small to be detected by these stains. It is possible that the resistance of the cornea is decreased by an epithelial disease of such severity that the epithelium is lost. Smelser5 felt that the synthesis of the ground substance is accomplished by the epi thelium of the cornea and the connective tis sue. He interpreted the role of the epithelium as a source of energy or as a precursor necessary to the synthesis of chondroitin sulfate and/or keratosulfate.
FURTHER RESEARCH
It is certainly evident that the mechanism by which the corticosteroids cause more severe corneal disease in herpetic keratitis remains unanswered. Perhaps electron micro scopic examination of tissue sections will reveal whether there is prolongation of sur vival of the virus. Kilbourne6 showed this to be true of influenza B virus in chick embryo cultures. Electron microscopic studies are now under way in our laboratories. Perhaps another approach to the solution of this problem is to carry out enzyme histochemistry, for it is conceivable that the decreased resistance of the cornea is a function of enzymatic action.
SUMMARY
1. Clinical herpes simplex infection of the cornea runs a prolonged course and many of the animals develop a picture of disciform keratitis on prolonged treatment with Prednisolone acetate. 2. Histopathologically the steroid treated rabbits show more extensive epithelial and stromal involvement. 3. Histochemical studies under the condi tions of the experiment failed to show any change in the mucopolysaccharide or the collagen of the cornea. San Francisco (22).
REFERENCES
1. Kimura, S. J., and Okumoto, M.: The effect of corticosteroids on experimental herpes simplex keratoconjunctivitis in the rabbit. Am. J. Ophth., 43:131-134, Part II, Apr. 1957. 2. Kilbourne, E. D., and Horsfall, F. L., Jr.: Increased virus in eggs injected with cortisone. Proc. Soc. Exper. Biol. & Med., 76:116-118, 1951. 3. Teodoru, C. V., and Shwartzman, G.: Endocrine factors in pathogenesis of experimental poliomyeli tis in hamsters. Role of inoculatory and environmental stress. Proc. Soc. Exper. Biol. & Med., 91:181-187, 1956. 4. Jawetz, E., Okumoto, M., and Sonne, M.: Studies on herpes simplex. X. The effect of corticosteroids on herpetic keratitis in the rabbit. J. Immunology, 83:486-490, Nov. 1959. 5. Smelser, George K.: The importance of the epithelium in the synthesis of the sulfated ground substances in corneal connective tissue. Tr. N.Y. Acad. Sciences, 21:575-577, May 1959. 6. Kilbourne, E. D.: The influence of cortisone on experimental viral infection. I. Prolongation of survival time and suppression of inflammation in chick embryos infected with influenza B virus. J Im munology, 74:57-62, 1955.