Reactivation of Latent Herpes Simplex Virus by Excimer Laser Photokeratectomy Jay S. Pepose, M.D., Keith A. Laycock, Ph.D., Judith Kelvin Miller, Ph.D., Ekktet Chansue, M.D., Eric J. Lenze, Larry A. Gans, M.D., and Morton E. Smith, M.D.
We tested whether excimer laser photorefractive and phototherapeutic keratectomy may reactivate latent herpes simplex and cause recurrent keratitis in mice. Two of ten latently infected mice that were treated with ten excimer laser pulses to the corneal epithelium shed herpes simplex virus type 1, as did four of ten mice that were treated with 50 excimer laser pulses. Ocular shedding of herpes simplex virus was detected in four of ten mice that were treated with ethylenediaminetetraacetic acid (EDTA) scraping of the corneal epithelium without laser keratectomy, and in six of ten mice on which combined EDTAfacilitated epithelial removal was performed followed by the application of ten excimer laser pulses. In both EDTA-treated groups, viral shedding was prolonged and 18 of 20 mice developed marked corneal opacification or neovascularization, or both. Corneal photoablation with the excimer laser may induce reactivation of latent herpes simplex virus, even in mice with clear and smooth-appearing corneas, and should be considered in the differential diagnosis of humans with persistent corneal epithelial defects after refractive or therapeutic excimer procedures.
T H E HERPES SIMPLEX VIRUS, after primary infec tion of a host, becomes latent and establishes a
Accepted for publication April 8, 1992. From the Departments of Ophthalmology and Visual Sciences (Drs. Pepose, Laycock, Miller, Chansue, Gans, and Smith; and Mr. Lenze) and Pathology (Drs. Pepose and Smith), Washington University School of Medicine, St. Louis, Missouri. This study was supported in part by a grant from Research to Prevent Blindness, Inc., New York, New York. Reprint requests to Jay S. Pepose, M.D., Department of Ophthalmology and Visual Sciences, Box 8096, Wash ington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
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finely balanced and unique host-pathogen rela tionship. 1 Humans are the only natural hosts for herpes simplex virus. Periodic reactivation of the dormant virus in neuronal tissue and subsequent axonal transport to the ocular sur face, mucous membranes and skin, allows repli cating virus to spread and infect other members of the community. Only in unusual circum stances that result in systemic viral dissemina tion or encephalitis, does herpetic reactivation result in death, which simultaneously ends its own life cycle. See also p. 96 Many factors and stimuli can reactivate latent herpes simplex. These include mechani cal,2,3 chemical,4"6 or photodynamic 710 injuries to the skin or ocular surface; systemic immunosuppression11"13; hyperthermia 14 ; and mechani cal,15 surgical, 16 electrical, 17 or chemical stimula tion 1821 of nerves of latently infected sensory or autonomie ganglia. Penetrating keratoplasty, 22 radial keratotomy, 23 anterior superficial kera tectomy, cryogenic injury of the cornea, transection of corneal nerves at the corneoscleral limbus, 24 and intrastromal injections 25 have effi ciently induced herpes simplex recurrences in animals. Persistent epithelial defects have been culture-positive for herpes simplex after pene trating keratoplasty in patients in whom surgi cal procedures were performed because of pre vious herpetic scarring, 2632 as well as in patients with nonherpetic indications. 3335 Considerable attention has been recently fo cused on the argon-fluoride excimer laser, a device that has entered phase III trials in the United States for photorefractive and phototherapeutic keratectomy. The laser emits 193nm ultraviolet light at a high fluence, delivering sufficient energy to break carbon-carbon bonds within the superficial corneal cells that absorb the laser energy. 8687 We used a well-character1992
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ized animal model of herpetic latency 10 to deter mine whether excimer laser photokeratectomy is capable of reactivating latent herpes simplex, resulting in viral replication at the ocular sur face.
Material and Methods Virus and cells—Herpes simplex virus type 1 McKrae strain was used for all experiments. A plaque-purified virus stock was grown and as sayed on Vero cells in minimum essential medi um containing Earle's balanced saline solution and 5% fetal bovine serum. Material from eye swabs was cultured similarly on Vero cells. Cells were cultured at 36 C in a humidified incubator that contained 5% carbon dioxide. Mice and inoculation procedures—Female 4to 6-week-old National Institutes of Health inbred strain mice were obtained from Harlan Olac Limited (Bicester, Oxford, England). The mice were examined before inoculation to en sure they had no ocular abnormalities. All in vestigations conformed to the Association for Research in Vision and Ophthalmology Resolu tion on the Use of Animals in Research. Mice were anesthetized by intraperitoneal injection of xylazine (10 m g / k g of body weight) followed by ketamine (70 m g / k g of body weight). The surface of the right cornea was scarified in a grid pattern with a No. 15 scalpel blade. A drop of viral inoculum that contained 106 plaque-forming units of herpes simplex virus type 1 McKrae strain in 5 μΐ of minimum essential medium was placed on the scarified area and a drop of Hanks' balanced saline solution was placed on the uninfected eye to prevent drying of the cornea while the mouse was unconscious. Concomitant with viral inoculation, 1.0 ml of pooled human serum (Chemicon International, Temecula, California) containing antibodies to herpes simplex virus type 1 (effective dose for 50% viral neutralization, 1:640) was injected intraperitoneally to protect the ocular tissues from damage during the acute phase of infec tion. Three days after inoculation, the infected eyes of the mice were swabbed with Dacron surgical spears (Xomed-Treace, Jacksonville, Florida) to detect infectious virus. The mice were housed for five weeks to permit the virus to establish latency. Treatment of mice before exposure to laser— Shortly before laser treatment, the epithelial
layer of the cornea was removed from selected groups of mice. To facilitate this procedure, a 15-μ1 drop of an ethylenediaminetetraacetic acid (EDTA) solution (6.83 g of NaCl, 0.2 g of KC1, 1.14 g of Na 2 HP0 4 / 0.2 g of KH 2 P0 4 ; 0.12 ml of 1 % phenol red, 7.6 g of Na 4 EDTA per one liter; pH 7.2 to 7.438,39) was placed on the eye of the anesthetized mouse, where it remained for 45 to 60 minutes. The corneal epithelium was then scraped away with a dry Dacron swab. Shortly after EDTA application and epithelial scraping, ten of the mice with deepithelialized corneas and 20 additional anesthetized mice were subjected to excimer corneal ablation. Excimer laser photokeratectomy—Anesthe tized mice in the photokeratectomy groups were treated with a Summit Excimed UV200LA argon fluoride excimer laser (Summit Technolo gy, Waltham, Massachusetts) with laser emis sions at wavelength 193 nm, optical zone 1 mm 2 , and estimated fluence at the eye of ap proximately 180 mj/cm 2 . Using a 10-nanosecond pulse width and a 10-Hz repetition rate, ten or 50 laser pulses were applied to the central cornea of each mouse. Selected mice from all four treatment groups were killed. The treated eye was enucleated and processed for standard light microscopy to assess the corneal histopathologic characteristics and depth of ex cimer photoablation. Detection of viral shedding—To improve the detection rate of viral replication at the ocular surface, 1% prednisolone acetate drops were instilled once daily starting 48 hours after laser treatment or removal of the corneal epithelium, or both. Our previous studies showed that topi cal corticosteroid administration alone is not sufficient to induce herpetic reactivation in mice.10 To detect infectious virus at the ocular sur face, the cornea was swabbed with a Weck-Cel Dacron surgical spear (Xomed-Treace, Jackson ville, Florida) previously soaked in 0.5 ml of minimum essential medium. The swab material was transferred to 12 x 75-mm culture tubes, and 0.2-ml aliquots were placed on confluent monolayers of Vero cells in 24-well titer plates. If infectious virus was present, cytopathic ef fects were visible after two to five days.
Results Excimer laser photokeratectomy was per formed directly on the corneal epithelium of
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two groups of ten latently infected mice. The mice in group 1 were treated with ten pulses of the laser, resulting in an ablation depth in the midcorneal epithelium. The second group of mice was treated with 50 laser pulses to the corneal epithelium, with a resultant ablation depth in the anterior stroma as assessed by light microscopy. In a third and fourth group (ten mice each), the corneal epithelium was first removed by soaking with EDTA. Whereas me chanical means are sufficient to remove the epithelium in humans, incubation with EDTA is necessary in mice, in which the tightly adher ent corneal epithelium is difficult to remove with a blade alone without risking corneal laceration. In one of these groups, the deepithelialized cornea was treated with ten pulses of the excimer laser, resulting in a deeper stromal ablation depth, and the other deepithelialized group was not laser treated. The eyes of the mice in all four groups were swabbed one day before the described treatment, immediately afterward, and on each successive day for the next ten days. Infectious virus was recovered from the ocular surface in two of ten mice treated with ten laser pulses, in four of ten mice treated with 50 excimer laser pulses, in four of ten mice treated with EDTA without photokeratectomy, and in six of ten mice subjected to combined EDTA and laser treatment. Previous ly, swabbing has been insufficient to induce reactivation of latent herpes simplex virus, 10 and, as confirmed by our negative cultures of swab material at the time of treatment, there is minimal spontaneous viral shedding in mice. The detection of viral shedding at the ocular surface is therefore in response to the specific treatment administered to each group of mice. The duration of viral shedding in the mice treated with EDTA was generally longer than in those that were only exposed to the laser with out previous EDTA treatment. There were 12 days of viral shedding in the mice treated only with EDTA/scraping, seven days of viral shed ding in the mice treated with ten excimer laser pulses, six days of viral shedding in the mice treated with 50 excimer laser pulses, and 16 days of viral shedding in the mice treated with EDTA/scraping plus ten excimer laser pulses. Of the mice that did shed virus, three of the four mice treated with EDTA/scraping alone shed virus for three days or longer; one of two of the mice treated with ten excimer laser pulses shed virus for three days or longer; none of four of the mice treated with 50 excimer laser pulses produced detectable virus on three or more
47
days; four of six of the mice that were treated with combined EDTA/scraping plus ten ex cimer laser pulses shed virus for three days or longer. In addition to swabbing, the eyes of the mice were observed with a biomicroscope one month after treatment. The eyes of mice undergoing photokeratectomy in the absence of EDTA treat ment were either clinically normal or showed focal stromal opacification and no characteris tic epithelial herpetic lesions. In contrast, nine of ten of the eyes of mice subjected to combined EDTA and laser treatment, or EDTA treatment alone, had marked disease at this time. Seven of the ten mice treated with EDTA alone had some degree of stromal opacity and nine of the ten mice had developed corneal neovascularization. In the combined treatment group, three of ten mice had stromal opacity and eight of ten mice had neovascularization. Only one mouse in each group was clinically normal after treat ment.
Discussion We have demonstrated that excimer laser photokeratectomy can reactivate herpes sim plex virus in latently infected mice. Thus, ex cimer laser corneal surgical procedures must be added to the list of other corneal surgical proce dures22"25 that can induce herpetic reactivation in animals. The mechanism by which laser photokeratectomy triggers reactivation is un known, but a common feature of many of the inducing factors is damage or irritation of the dense corneal nerve plexus.22,24 This is evi denced clinically by the degree of patient dis comfort on the first night after excimer laser photorefractive or phototherapeutic keratectomy. Factors that lead to neuronal death, such as ganglionic explantation and maintenance in organ culture 1 or the depletion of nerve growth factor trophic support to neurons, 40 are power ful stimuli that most efficiently elicit the reacti vation of latent herpes simplex virus in vitro. It is not surprising, from the perspective of viral survival, that reactivation of herpes simplex is linked to several stimuli that also portend neu ronal cell death. In the mice studied, excimer laser treatment to the corneal epithelium and anterior stroma elicited viral shedding of generally short dura tion with little resultant disease. Previous re moval of corneal epithelium with EDTA fol-
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lowed by excimer laser application resulted in more prolonged shedding with subsequent stromal opacification and corneal neovascularization. Kaufman, Brown, and Ellison 41 demon strated ocular herpes simplex virus shedding with few or no associated clinical signs, which was similar to the minimal disease seen in our mice that shed virus after treatment of the cornea with the excimer laser alone. As predicted by our model, recurrent ocular herpes simplex has been recently described in the clinical setting after excimer laser photorefractive keratectomy for posttransplantation astigmatism in a patient with a history of her pes keratitis 42 and in two patients after excimer laser phototherapeutic keratectomy for herpes simplex scars, described by Vrabec and associ ates elsewhere in this issue. This latter condi tion thereby appears to represent a relative contraindication for excimer laser therapeutic keratectomy, although selected cases of quies cent herpetic scars have been successfully treat ed by excimer laser ablation without incident. 43 At a minimum, prophylactic antiviral treatment and frequent follow-up examination appear in dicated in such cases. Similar reactivations of herpes simplex virus after other types of corne al surgical procedures have been described in which epithelial defects were culture-positive for herpes simplex after corneal transplantation in patients with26"33 and without 34 · 35 a history of recurrent ocular herpetic disease. An epithelial dendrite developed in a patient using trifluorothymidine and topical corticosteroids after la ser photocoagulation of corneal neovascularization with a 577-nm yellow dye laser. This patient with a history of herpetic stromal kera titis had not had recurrent dendritic disease in the preceding ten years before corneal laser treatment. 44 The latently infected mice undergoing ex cimer laser corneal ablation in our study had clear and smooth corneas without lesions, simi lar to patients undergoing excimer laser photorefractive keratectomy. The pooled human ser um containing neutralizing antibodies to her pes simplex virus administered intraperitoneally at the time of viral inoculation prevented the development of corneal disease during acute infection. Epidemiologie studies indicat ed that most cases of primary ocular herpes simplex infection do not result in permanent corneal disease. 45 However, herpes simplex DNA has been demonstrated in corneal tissue of patients with a history of herpes kerati tis, 4648 and in clear corneas without clinical
signs of disease. 49 The biological state of herpes simplex nucleic acid in such corneas is un clear.50·51 These factors must be considered in performing excimer laser photorefractive kera tectomy on myopic patients with clear corneas. Selected candidates for this procedure may have had a previous subclinical herpetic ocular infection and may even harbor some form of herpes simplex virus DNA in corneal tissue. A larger percentage of these patients will have latent infections of the trigeminal ganglia with herpes simplex. The delayed time course of viral recovery of herpes simplex at the ocular surface in our experiments seemed most consistent with reac tivation at the level of the trigeminal ganglia with axonal transport to the ocular surface (estimated in in vitro models to be 3 to 5 mm per hour 52 ), although replication of persistent herpes simplex nucleic acid in corneal tissue cannot be strictly excluded. The corneal surface reepithelializes within three days of excimer photorefractive keratectomy in most patients. Our experimental results suggested that herpes simplex keratitis be considered in the differen tial diagnosis of patients with persistent epithe lial defects after corneal excimer laser proce dures.
References 1. Cook, M. L., Bastone, V. B., and Stevens, J. G.: Evidence that neurons harbor latent herpes simplex virus. Infect. Immun. 9:946, 1974. 2. Hill, T. J., Field, H. J., and Blyth, W. A.: Acute and recurrent infection with herpes simplex virus in the mouse. A model for studying latency and recur rent disease. J. Gen. Virol. 28:341, 1975. 3. Hill, T. J., Blyth, W. A., and Harbour, D. A.: Trauma to the skin causes recurrence of herpes sim plex in the mouse. J. Gen. Virol. 39:21, 1978. 4. Harbour, D. A., Hill, T. J., and Blyth, W. A.: Recurrent herpes simplex in the mouse. Inflamma tion in the skin and activation of virus in the ganglia following peripheral stimulation. J. Gen. Virol. 64:1491, 1983. 5. Schmidt, J. R., and Rasmussen, A. F.: Activation of latent herpes simplex encephalitis by chemical means. J. Infect. Dis. 106:154, 1960. 6. Laibson, P. R., and Kibrick, S.: Reactivation of herpetic keratitis by epinephrine in rabbits. Arch. Ophthalmol. 75:254, 1966. 7. Perna, J. J., Mannix, M. L., Rooney, J. F., Notkins, A. L., and Straus, S. E.: Reactivation of latent herpes simplex virus infection by ultraviolet light. A human model. J. Am. Acad. Dermatol. 17:473, 1987.
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8. Spruance, S. L., Freeman, D. J„ and Stewart, J. C. B.: The natural history of ultraviolet radiationinduced herpes simplex labialis and response to ther apy with peroral and topical formulations of acyclovir. J. Infect. Dis. 163:728, 1991. 9. Shimeld, C , Hill, T. J., Blyth, W. A., and Easty, D. L.: Reactivation of latent infection and induction of recurrent herpetic eye disease in mice. J. Gen. Virol. 71:397, 1990. 10. Laycock, K. A., Lee, S. F., Brady, R. H., and Pepose, J. S.: Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation. Invest. Ophthalmol. Vis. Sci. 32:2741, 1991. 11. Blyth, W. A., Harbour, D. A., and Hill, T. J.: Effect of immunosuppression on recurrent herpes simplex in mice. Infect. Immun. 29:902, 1980. 12. Underwood, G. E., and Weed, S. D.: Recurrent cutaneous herpes simplex in hairless mice. Infect. Immun. 10:471, 1974. 13. Kurata, T., Kurata, K., and Aoyama, Y.: Reacti vation of herpes simplex virus (type 2) infection in trigeminal ganglia and oral lips with cyclophosphamide treatment. Jpn. J. Exp. Med. 48:427, 1978. 14. Sawtell, N. M., and Thompson, R. L.: Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia. J. Virol. 66:2150, 1992. 15. Walz, M. A, Price, R. W., and Notkins, A. L.: Latent infec tion with herpes simplex virus type 1 and 2. Viral reactivation in vivo after neurectomy. Science 184:1185, 1974. 16. Nesburn, A. B., Dickinson, R., and Radnoti, M.: The effect of trigeminal nerve and ganglion manipulation on recurrences of ocular herpes sim plex in rabbits. Invest. Ophthalmol. 15:726, 1976. 17. Green, M. T„ Rosborough, J. P., and Dunkel, E. C : In vivo reactivation of herpes simplex virus in rabbit trigeminal ganglia. Electrode model. Infect. Immun. 34:69, 1981. 18. Se Kwon, B., Gangarosa, L. P., Burch, K. D., DeBack, J., and Hill, J. M.: Induction of ocular herpes simplex virus shedding by iontophoresis of epinephrine into rabbit cornea. Invest. Ophthalmol. Vis. Sci. 21:442, 1981. 19. Shimomura, Y., Gangarosa, L. P., Katoaka, M., and Hill, J. M.: HSV-1 shedding by iontophoresis of 6-hydroxydopamine followed by topical epinephrine. Invest. Ophthalmol. Vis. Sci. 24:1588, 1983. 20. Willey, D. E., Trousdale, M. D., and Nesburn, A. B.: Reactivation of murine latent HSV infection by epinephrine iontophoresis. Invest. Ophthalmol. Vis. Sci. 25:945, 1984. 21. Gordon, Y. J., Araullo-Cruz, T. P., Romanow ski, E., Ruziczka, L., Balouris, C , Oren, J., Cheng, K. P., and Kim, S.: The development of an improved murine iontophoresis reactivation model for the study of HSV-1 latency. Invest. Ophthalmol. Vis. Sci. 27:1230, 1986. 22. Beyer, C. F., Arens, M. Q., Hill, J. M., Rose, B. T., Hill, G. A., and Lin, D. T. C : Penetrating kera-
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toplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used. Curr. Eye Res. 8:1323, 1989. 23. Haruta, Y., Maguire, L. ]., Rootman, D. S., and Hill, J. M.: Recurrent herpes simplex virus type 1 corneal epithelial lesions after radial keratotomy in the rabbit. Arch. Ophthalmol. 105:692, 1987. 24. Beyer, C. F., Hill, J. M., Reidy, J. J., and Beuerman, R. W.: Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1. Invest. Ophthalmol. Vis. Sci. 31:925, 1990. 25. Gordon, Y. J., Romanowski, E., and AraulloCruz, T.: A fast, simple reactivation method for the study of HSV-1 latency in the rabbit ocular model. Invest. Ophthalmol. Vis. Sci. 31:921, 1990. 26. Pfister, R. F., Richards, ]. S. F., and Dohlman, C. H.: Recurrence of herpetic keratitis in corneal grafts. Am. J. Ophthalmol. 73:192, 1972. 27. Polack, F. M., and Kaufman, H. E.: Penetrating keratoplasty in herpetic keratitis. Am. J. Ophthalmol. 73:908, 1972. 28. Langston, R. H. S., Pavan-Langston, D., and Dohlman, C : Penetrating keratoplasty for herpetic keratitis. Prognostic and therapeutic determinants. Trans. Am. Acad. Ophthalmol. Otolaryngol. 79:77, 1975. 29. Foster, C. S., and Duncan, J.: Penetrating kera toplasty for herpes simplex keratitis. Am. J. Ophthal mol. 92:336, 1981. 30. Cohen, E. J., Laibson, P. R., and Arentsen, J. J.: Corneal transplantation for herpes simplex keratitis. Am. J. Ophthalmol. 95:645, 1983. 31. Cobo, L. M., Coster, D. J., Rice, N. S. C , and Jones, B. R.: Prognosis and management of corneal transplantation for herpetic keratitis. Arch. Ophthal mol. 98:1755, 1980. 32. Epstein, R. J„ Seedor, J. A., Dreizen, N. G., Stulting, R. D., Waring, G. O., Wilson, L. A., and Cavanagh, H. D.: Penetrating keratoplasty for herpes simplex keratitis and keratoconus. Ophthalmology 94:935, 1987. 33. Salisbury, J. D„ Berkowitz, R. A., Gebhardt, B. M., and Kaufman, H. E.: Herpesvirus infection of cornea allografts. Ophthalmic Surg. 15:406, 1984. 34. Beyer, C. F., Byrd, T. J., Hill, J. M , and Kauf man, H. E.: Herpes simplex virus and persistent epithelial defects after penetrating keratoplasty. Am. J. Ophthalmol. 109:95, 1990. 35. Mannis, M. J., Plotnik, R. D., Schwab, I. R., and Newton, R. D.: Herpes simplex dendritic kerati tis after keratoplasty. Am. J. Ophthalmol. 111:480, 1991. 36. L'Espérance, F., Warner, J., Telfair, W., Yoder, P., and Martin, C : Excimer laser instrumentation and technique for human corneal surgery. Arch. Ophthalmol. 107:131, 1989. 37. Marshall, J., Trokel, S., Rothery, S., and Schu bert, H.: An ultrastructural study of corneal incisions induced by an excimer laser at 193 nm. Ophthalmol ogy 92:749, 1985. 38. Mackenzie, I. C , and Squier, C. A.: Cytochem-
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ical identification of ATPase-positive Langerhans cells in EDTA-separated sheets of mouse epidermis. Br. J. Dermatol. 92:523, 1975. 39. Gillette, T. E., and Chandler, J. W.: Immunofluorescence and histochemistry of corneal epithelial flat mounts. UseofEDTA. Curr. Eye Res. 1:249, 1981. 40. Wilcox, C. L., and Johnson, E. M., Jr.: Nerve growth factor deprivation results in the reactivation of latent herpes simplex in vitro. J. Virol. 61:2311, 1987. 41. Kaufman, H. E., Brown, D. C , and Ellison, E. M.: Recurrent herpes simplex in rabbit and man. Science 156:1628, 1967. 42. McDonnell, P. J., Moreira, H., Clapham, T. N., D'Arcy, J., and Munnerlyn, C. R.: Photorefractive keratectomy for astigmatism. Initial clinical results. Arch. Ophthalmol. 109:1370, 1991. 43. Sher, N. A., Bowers, R. A., Zabel, R. W., Frantz, J. M., Eiferman, R. A., Brown, D. C , Rowsey, J. J., Parker, P., Chen, V., and Lindstrom, R. L.: Clinical use of the 193 nm excimer laser in the treatment of corneal scars. Arch. Ophthalmol. 109:491, 1991. 44. Baer, J. C , and Foster, C. S.: Corneal laser photocoagulation for treatment of neovascularization. Efficacy of 577 nm yellow dye laser. Ophthal mology 99:173,1992. 45. Liesegang, T. J.: Epidemiology of ocular her pes simplex. Natural history in Rochester, Minn, 1950 through 1982. Arch. Ophthalmol. 107:1160, 1989.
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46. Rong, B. L., Pavan-Langston, D., Weng, Q. P., Martinez, R., Cherry, J. M., and Dunkel, E. C : Detec tion of herpes virus thymidine kinase and latencyassociated transcript gene sequences in human herpetic corneas by polymerase chain reaction am plification. Invest. Ophthalmol. Vis. Sci. 32:1808, 1991. 47. Kaye, S. B., Lynas, C , Patterson, A., Risk, J. M., McCarthy, K., and Hart, C. A.: Evidence for herpes simplex viral latency in the human cornea. Br. J. Ophthalmol. 75:195, 1991. 48. Pavan-Langston, D., Rong, B. L., and Dunkel, E. C : Extraneuronal herpetic latency: animal and human corneal studies. Acta Ophthalmol. 67:135, 1989. 49. Crouse, C. A., Pflugfelder, S. C , Pereira, I., Cleary, T., Rabinowitz, S., and Atherton, S. S.: De tection of herpes viral genomes in normal and dis eased corneal epithelium. Curr. Eye Res. 9:569,1990. 50. Pepose, J. S.: Herpes simplex keratitis. Role of viral infection versus immune response. Surv. Oph thalmol. 35:345, 1991. 51. Cook, S. D., and Hill, J. M.: Herpes simplex virus. Molecular biology and the possibility of corne al latency. Surv. Ophthalmol. 36:140, 1991. 52. Lynche, E., Kristensson, K., Svennerholm, B., Vohlne, A., and Ziegler, R.: Uptake and transport of herpes simplex virus in neuntes of rat dorsal root ganglia cells in culture. J. Gen. Virol. 65:55, 1984.
We tested whether excimer laser photorefractive and phototherapeutic keratectomy may reactivate latent herpes simplex and cause recurrent keratitis in mice. Two of ten latently infected mice that were treated with ten excimer laser pulses to the corneal epithelium shed herpes simplex virus type 1, as did four of ten mice that were treated with 50 excimer laser pulses. Ocular shedding of herpes simplex virus was detected in four of ten mice that were treated with ethylenediaminetetraacetic acid (EDTA) scraping of the corneal epithelium without laser keratectomy, and in six of ten mice on which combined EDTAfacilitated epithelial removal was performed followed by the application of ten excimer laser pulses. In both EDTA-treated groups, viral shedding was prolonged and 18 of 20 mice developed marked corneal opacification or neovascularization, or both. Corneal photoablation with the excimer laser may induce reactivation of latent herpes simplex virus, even in mice with clear and smooth-appearing corneas, and should be considered in the differential diagnosis of humans with persistent corneal epithelial defects after refractive or therapeutic excimer procedures.
T H E HERPES SIMPLEX VIRUS, after primary infec tion of a host, becomes latent and establishes a
Accepted for publication April 8, 1992. From the Departments of Ophthalmology and Visual Sciences (Drs. Pepose, Laycock, Miller, Chansue, Gans, and Smith; and Mr. Lenze) and Pathology (Drs. Pepose and Smith), Washington University School of Medicine, St. Louis, Missouri. This study was supported in part by a grant from Research to Prevent Blindness, Inc., New York, New York. Reprint requests to Jay S. Pepose, M.D., Department of Ophthalmology and Visual Sciences, Box 8096, Wash ington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
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finely balanced and unique host-pathogen rela tionship. 1 Humans are the only natural hosts for herpes simplex virus. Periodic reactivation of the dormant virus in neuronal tissue and subsequent axonal transport to the ocular sur face, mucous membranes and skin, allows repli cating virus to spread and infect other members of the community. Only in unusual circum stances that result in systemic viral dissemina tion or encephalitis, does herpetic reactivation result in death, which simultaneously ends its own life cycle. See also p. 96 Many factors and stimuli can reactivate latent herpes simplex. These include mechani cal,2,3 chemical,4"6 or photodynamic 710 injuries to the skin or ocular surface; systemic immunosuppression11"13; hyperthermia 14 ; and mechani cal,15 surgical, 16 electrical, 17 or chemical stimula tion 1821 of nerves of latently infected sensory or autonomie ganglia. Penetrating keratoplasty, 22 radial keratotomy, 23 anterior superficial kera tectomy, cryogenic injury of the cornea, transection of corneal nerves at the corneoscleral limbus, 24 and intrastromal injections 25 have effi ciently induced herpes simplex recurrences in animals. Persistent epithelial defects have been culture-positive for herpes simplex after pene trating keratoplasty in patients in whom surgi cal procedures were performed because of pre vious herpetic scarring, 2632 as well as in patients with nonherpetic indications. 3335 Considerable attention has been recently fo cused on the argon-fluoride excimer laser, a device that has entered phase III trials in the United States for photorefractive and phototherapeutic keratectomy. The laser emits 193nm ultraviolet light at a high fluence, delivering sufficient energy to break carbon-carbon bonds within the superficial corneal cells that absorb the laser energy. 8687 We used a well-character1992
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ized animal model of herpetic latency 10 to deter mine whether excimer laser photokeratectomy is capable of reactivating latent herpes simplex, resulting in viral replication at the ocular sur face.
Material and Methods Virus and cells—Herpes simplex virus type 1 McKrae strain was used for all experiments. A plaque-purified virus stock was grown and as sayed on Vero cells in minimum essential medi um containing Earle's balanced saline solution and 5% fetal bovine serum. Material from eye swabs was cultured similarly on Vero cells. Cells were cultured at 36 C in a humidified incubator that contained 5% carbon dioxide. Mice and inoculation procedures—Female 4to 6-week-old National Institutes of Health inbred strain mice were obtained from Harlan Olac Limited (Bicester, Oxford, England). The mice were examined before inoculation to en sure they had no ocular abnormalities. All in vestigations conformed to the Association for Research in Vision and Ophthalmology Resolu tion on the Use of Animals in Research. Mice were anesthetized by intraperitoneal injection of xylazine (10 m g / k g of body weight) followed by ketamine (70 m g / k g of body weight). The surface of the right cornea was scarified in a grid pattern with a No. 15 scalpel blade. A drop of viral inoculum that contained 106 plaque-forming units of herpes simplex virus type 1 McKrae strain in 5 μΐ of minimum essential medium was placed on the scarified area and a drop of Hanks' balanced saline solution was placed on the uninfected eye to prevent drying of the cornea while the mouse was unconscious. Concomitant with viral inoculation, 1.0 ml of pooled human serum (Chemicon International, Temecula, California) containing antibodies to herpes simplex virus type 1 (effective dose for 50% viral neutralization, 1:640) was injected intraperitoneally to protect the ocular tissues from damage during the acute phase of infec tion. Three days after inoculation, the infected eyes of the mice were swabbed with Dacron surgical spears (Xomed-Treace, Jacksonville, Florida) to detect infectious virus. The mice were housed for five weeks to permit the virus to establish latency. Treatment of mice before exposure to laser— Shortly before laser treatment, the epithelial
layer of the cornea was removed from selected groups of mice. To facilitate this procedure, a 15-μ1 drop of an ethylenediaminetetraacetic acid (EDTA) solution (6.83 g of NaCl, 0.2 g of KC1, 1.14 g of Na 2 HP0 4 / 0.2 g of KH 2 P0 4 ; 0.12 ml of 1 % phenol red, 7.6 g of Na 4 EDTA per one liter; pH 7.2 to 7.438,39) was placed on the eye of the anesthetized mouse, where it remained for 45 to 60 minutes. The corneal epithelium was then scraped away with a dry Dacron swab. Shortly after EDTA application and epithelial scraping, ten of the mice with deepithelialized corneas and 20 additional anesthetized mice were subjected to excimer corneal ablation. Excimer laser photokeratectomy—Anesthe tized mice in the photokeratectomy groups were treated with a Summit Excimed UV200LA argon fluoride excimer laser (Summit Technolo gy, Waltham, Massachusetts) with laser emis sions at wavelength 193 nm, optical zone 1 mm 2 , and estimated fluence at the eye of ap proximately 180 mj/cm 2 . Using a 10-nanosecond pulse width and a 10-Hz repetition rate, ten or 50 laser pulses were applied to the central cornea of each mouse. Selected mice from all four treatment groups were killed. The treated eye was enucleated and processed for standard light microscopy to assess the corneal histopathologic characteristics and depth of ex cimer photoablation. Detection of viral shedding—To improve the detection rate of viral replication at the ocular surface, 1% prednisolone acetate drops were instilled once daily starting 48 hours after laser treatment or removal of the corneal epithelium, or both. Our previous studies showed that topi cal corticosteroid administration alone is not sufficient to induce herpetic reactivation in mice.10 To detect infectious virus at the ocular sur face, the cornea was swabbed with a Weck-Cel Dacron surgical spear (Xomed-Treace, Jackson ville, Florida) previously soaked in 0.5 ml of minimum essential medium. The swab material was transferred to 12 x 75-mm culture tubes, and 0.2-ml aliquots were placed on confluent monolayers of Vero cells in 24-well titer plates. If infectious virus was present, cytopathic ef fects were visible after two to five days.
Results Excimer laser photokeratectomy was per formed directly on the corneal epithelium of
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two groups of ten latently infected mice. The mice in group 1 were treated with ten pulses of the laser, resulting in an ablation depth in the midcorneal epithelium. The second group of mice was treated with 50 laser pulses to the corneal epithelium, with a resultant ablation depth in the anterior stroma as assessed by light microscopy. In a third and fourth group (ten mice each), the corneal epithelium was first removed by soaking with EDTA. Whereas me chanical means are sufficient to remove the epithelium in humans, incubation with EDTA is necessary in mice, in which the tightly adher ent corneal epithelium is difficult to remove with a blade alone without risking corneal laceration. In one of these groups, the deepithelialized cornea was treated with ten pulses of the excimer laser, resulting in a deeper stromal ablation depth, and the other deepithelialized group was not laser treated. The eyes of the mice in all four groups were swabbed one day before the described treatment, immediately afterward, and on each successive day for the next ten days. Infectious virus was recovered from the ocular surface in two of ten mice treated with ten laser pulses, in four of ten mice treated with 50 excimer laser pulses, in four of ten mice treated with EDTA without photokeratectomy, and in six of ten mice subjected to combined EDTA and laser treatment. Previous ly, swabbing has been insufficient to induce reactivation of latent herpes simplex virus, 10 and, as confirmed by our negative cultures of swab material at the time of treatment, there is minimal spontaneous viral shedding in mice. The detection of viral shedding at the ocular surface is therefore in response to the specific treatment administered to each group of mice. The duration of viral shedding in the mice treated with EDTA was generally longer than in those that were only exposed to the laser with out previous EDTA treatment. There were 12 days of viral shedding in the mice treated only with EDTA/scraping, seven days of viral shed ding in the mice treated with ten excimer laser pulses, six days of viral shedding in the mice treated with 50 excimer laser pulses, and 16 days of viral shedding in the mice treated with EDTA/scraping plus ten excimer laser pulses. Of the mice that did shed virus, three of the four mice treated with EDTA/scraping alone shed virus for three days or longer; one of two of the mice treated with ten excimer laser pulses shed virus for three days or longer; none of four of the mice treated with 50 excimer laser pulses produced detectable virus on three or more
47
days; four of six of the mice that were treated with combined EDTA/scraping plus ten ex cimer laser pulses shed virus for three days or longer. In addition to swabbing, the eyes of the mice were observed with a biomicroscope one month after treatment. The eyes of mice undergoing photokeratectomy in the absence of EDTA treat ment were either clinically normal or showed focal stromal opacification and no characteris tic epithelial herpetic lesions. In contrast, nine of ten of the eyes of mice subjected to combined EDTA and laser treatment, or EDTA treatment alone, had marked disease at this time. Seven of the ten mice treated with EDTA alone had some degree of stromal opacity and nine of the ten mice had developed corneal neovascularization. In the combined treatment group, three of ten mice had stromal opacity and eight of ten mice had neovascularization. Only one mouse in each group was clinically normal after treat ment.
Discussion We have demonstrated that excimer laser photokeratectomy can reactivate herpes sim plex virus in latently infected mice. Thus, ex cimer laser corneal surgical procedures must be added to the list of other corneal surgical proce dures22"25 that can induce herpetic reactivation in animals. The mechanism by which laser photokeratectomy triggers reactivation is un known, but a common feature of many of the inducing factors is damage or irritation of the dense corneal nerve plexus.22,24 This is evi denced clinically by the degree of patient dis comfort on the first night after excimer laser photorefractive or phototherapeutic keratectomy. Factors that lead to neuronal death, such as ganglionic explantation and maintenance in organ culture 1 or the depletion of nerve growth factor trophic support to neurons, 40 are power ful stimuli that most efficiently elicit the reacti vation of latent herpes simplex virus in vitro. It is not surprising, from the perspective of viral survival, that reactivation of herpes simplex is linked to several stimuli that also portend neu ronal cell death. In the mice studied, excimer laser treatment to the corneal epithelium and anterior stroma elicited viral shedding of generally short dura tion with little resultant disease. Previous re moval of corneal epithelium with EDTA fol-
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lowed by excimer laser application resulted in more prolonged shedding with subsequent stromal opacification and corneal neovascularization. Kaufman, Brown, and Ellison 41 demon strated ocular herpes simplex virus shedding with few or no associated clinical signs, which was similar to the minimal disease seen in our mice that shed virus after treatment of the cornea with the excimer laser alone. As predicted by our model, recurrent ocular herpes simplex has been recently described in the clinical setting after excimer laser photorefractive keratectomy for posttransplantation astigmatism in a patient with a history of her pes keratitis 42 and in two patients after excimer laser phototherapeutic keratectomy for herpes simplex scars, described by Vrabec and associ ates elsewhere in this issue. This latter condi tion thereby appears to represent a relative contraindication for excimer laser therapeutic keratectomy, although selected cases of quies cent herpetic scars have been successfully treat ed by excimer laser ablation without incident. 43 At a minimum, prophylactic antiviral treatment and frequent follow-up examination appear in dicated in such cases. Similar reactivations of herpes simplex virus after other types of corne al surgical procedures have been described in which epithelial defects were culture-positive for herpes simplex after corneal transplantation in patients with26"33 and without 34 · 35 a history of recurrent ocular herpetic disease. An epithelial dendrite developed in a patient using trifluorothymidine and topical corticosteroids after la ser photocoagulation of corneal neovascularization with a 577-nm yellow dye laser. This patient with a history of herpetic stromal kera titis had not had recurrent dendritic disease in the preceding ten years before corneal laser treatment. 44 The latently infected mice undergoing ex cimer laser corneal ablation in our study had clear and smooth corneas without lesions, simi lar to patients undergoing excimer laser photorefractive keratectomy. The pooled human ser um containing neutralizing antibodies to her pes simplex virus administered intraperitoneally at the time of viral inoculation prevented the development of corneal disease during acute infection. Epidemiologie studies indicat ed that most cases of primary ocular herpes simplex infection do not result in permanent corneal disease. 45 However, herpes simplex DNA has been demonstrated in corneal tissue of patients with a history of herpes kerati tis, 4648 and in clear corneas without clinical
signs of disease. 49 The biological state of herpes simplex nucleic acid in such corneas is un clear.50·51 These factors must be considered in performing excimer laser photorefractive kera tectomy on myopic patients with clear corneas. Selected candidates for this procedure may have had a previous subclinical herpetic ocular infection and may even harbor some form of herpes simplex virus DNA in corneal tissue. A larger percentage of these patients will have latent infections of the trigeminal ganglia with herpes simplex. The delayed time course of viral recovery of herpes simplex at the ocular surface in our experiments seemed most consistent with reac tivation at the level of the trigeminal ganglia with axonal transport to the ocular surface (estimated in in vitro models to be 3 to 5 mm per hour 52 ), although replication of persistent herpes simplex nucleic acid in corneal tissue cannot be strictly excluded. The corneal surface reepithelializes within three days of excimer photorefractive keratectomy in most patients. Our experimental results suggested that herpes simplex keratitis be considered in the differen tial diagnosis of patients with persistent epithe lial defects after corneal excimer laser proce dures.
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46. Rong, B. L., Pavan-Langston, D., Weng, Q. P., Martinez, R., Cherry, J. M., and Dunkel, E. C : Detec tion of herpes virus thymidine kinase and latencyassociated transcript gene sequences in human herpetic corneas by polymerase chain reaction am plification. Invest. Ophthalmol. Vis. Sci. 32:1808, 1991. 47. Kaye, S. B., Lynas, C , Patterson, A., Risk, J. M., McCarthy, K., and Hart, C. A.: Evidence for herpes simplex viral latency in the human cornea. Br. J. Ophthalmol. 75:195, 1991. 48. Pavan-Langston, D., Rong, B. L., and Dunkel, E. C : Extraneuronal herpetic latency: animal and human corneal studies. Acta Ophthalmol. 67:135, 1989. 49. Crouse, C. A., Pflugfelder, S. C , Pereira, I., Cleary, T., Rabinowitz, S., and Atherton, S. S.: De tection of herpes viral genomes in normal and dis eased corneal epithelium. Curr. Eye Res. 9:569,1990. 50. Pepose, J. S.: Herpes simplex keratitis. Role of viral infection versus immune response. Surv. Oph thalmol. 35:345, 1991. 51. Cook, S. D., and Hill, J. M.: Herpes simplex virus. Molecular biology and the possibility of corne al latency. Surv. Ophthalmol. 36:140, 1991. 52. Lynche, E., Kristensson, K., Svennerholm, B., Vohlne, A., and Ziegler, R.: Uptake and transport of herpes simplex virus in neuntes of rat dorsal root ganglia cells in culture. J. Gen. Virol. 65:55, 1984.
