Corneal Sensitivity and Substance P in Experimental Herpes Simplex Keratitis in Mice

Andrew B. Tullo,* Perer Keeaf William A. Dlyrh.J Terry J. H1II4 and David L. Easry*

Experimental herpes simplex keratitis in the mouse produced a rapid fall in both corneal sensitivity and levels of corneal substance P (SP). This finding supports the association of SP with sensory neurones and shows that such levels can be used as an indication of damage to neurones resulting, for example, from infection with HSV. However, the delay in recovery of SP compared to the more rapid and complete recovery of sensitivity suggests that SP in the cornea is not directly involved in mediation of the blink reflex. Invest Ophthalmol Vis Sci 24:596-598, 1983

Neuropeptides are currently receiving much atten- 4 weeks previously,8 were inoculated by corneal scar- tion because of their widespread distribution within ification of the left eye7 through 5 n\ of growth me- the nervous system and because of their possible role dium containing 3.5 X 106 pfu of the same strain of in neurotransmission. One of these small peptides virus. As controls the corneas of uninfected mice were substance P (SP) is known to be particularly strongly scarified through medium without virus. Scarification associated with primary afferent neurones.1 It is to of the cornea in both groups consisted often parallel be found not only at the central terminals of such strokes with a 26-gauge needle followed by a further nerve fibers, but also in the peripheral endings,2 and ten perpendicular to the first. Before use all mice were recently its presence in the cornea was demonstrated examined with a Zeiss 10SL slit-lamp to exclude any by immunohistochemistry3'4 and by radioimmu- with ocular abnormalities. noassay.5 However, the function of substance P in such peripheral sites is far from clear. By surgical and Measurement of Corneal Sensitivity chemical denervation it has been shown that substance P levels in the cornea are reduced. However, Blink reflex was tested in unanesthetized mice by no correlation between changes in SP and corneal holding the animal by the scruff of the neck under sensitivity was found.5 In the human cornea infected a dissecting microscope (X10 magnification) and by herpes simplex virus (HSV), sensitivity is reduced,6 touching the center of the cornea with a sterile fila- and this has also been shown to be true of experi- ment of nylon (0.24 mm diameter). A 2 cm length mental ocular infection.7 In the experiments reported of nylon held in a loop holder was found to be con- here, SP levels were measured in the cornea of mice venient to avoid touching whiskers and eyelashes. A with HSV keratitis, first to use SP as an indicator of scoring system was used where 2.0 = normal brisk the integrity of sensory neurones, and second to ex- reflex, 1.0 = reflex reduced in speed or amplitude, amine the relationship of corneal SP to corneal sen- and 0 = absent reflex. sitivity. Assay of Substance P Materials and Methods Mice were killed by intraperitoneal injection of pentobarbitone immediately after examination. Both Infection of Mice eyes were enucleated, and each was transfixed with Eight-week-old outbred male mice, which had been a pin for removal of the cornea under a dissecting infected with HSV type 1 strain SCI6 in the right ear microscope. Care was taken to remove all conjunc- tival and limbal tissue. All corneas were treated sep- arately using the right (uninfected) eye as a control. From the Departments of Ophthalmology,* Pharmacology,f After placing in 200 y\ of 10 mM HC1 containing 1 and Microbiology,t University of Bristol, Bristol, United mM EDTA, 1 mM dithiothreitol, corneas were boiled Kingdom. and homogenized. The homogenates were freeze- Supported by the Wellcome Trust and the South Western Re- dried, and their SP content was determined by ra- gional Health Authority. dioimmunoassay using a C-terminal directed anti- Submitted for publication May 5, 1982. 9 125 8 Reprint requests: Andrew B. Tullo, MD, Bristol Eye Hospital, serum, and I Tyr SP as tracer. The test had a sen- Lower Maudlin Street, Bristol BS1 2LX, United Kingdom. sitivity of 5 pg per tube. 0146-0404/83/0500/596/S0.95 © Association for Research in Vision and Ophthalmology

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Results CORNEAl SENSITIVITY AND SP LEVELS DURING HERPES SIMPLEX KERATITIS

A group of 45 mice that had been infected as in 100 ft the Methods section was examined and tested for the blink reflex. At intervals, four to six mice chosen by clinical examination to be representative of the group were killed for SP assay of both corneas (Fig. 1). All mice showed signs of infection varying from transient epithelial lesions to geographic ulceration associated with stromal opacification. Signs were largely reversible, although in the more severe keratitis moderate limbal vascularization and early break-up of the tear film persisted for longer than 14 days. A marked fall in both corneal sensitivity and SP levels was apparent on the day after corneal inoculation. Minimum sensitivity was reached on day 2, after which it was regained quickly and reached normal values on day 7. The corneal sensitivity of mice assayed for SP corresponded well with that of the A 6 SENSITIVITY IN ALL REMAINING MICE 0 — 0 SENSITIVITY IN MICE ASSAYED FOR SP remainder of the group (Fig. 1). X----X SP LEVELS ( AND S.E.M.I Substance P levels fell at the same time as the fall in sensitivity; the minimum occurred on day 4, but

the level only reached 70% of normal values by day 5 6 7 II 12 11. On day 2 when sensitivity was minimal, three out DAYS AFTER INFECTION of six mice assayed for SP had no blink reflex in the Fig. 1. Corneal sensitivity and substance P levels during Herpes infected eye. The mean value of SP in these three simplex keratitis. Figures are expressed as a percentage of the value corneas was 47% of normal. In a further group of five of the contralateral (uninfected) eye. mice that had shown a complete recovery of corneal sensitivity, corneal SP levels were only 81% of normal disease there is a rapid and largely reversible fall in values 23 days after infection. corneal sensitivity. In control mice (four to five daily) whose cornea The most striking finding of the present study was had been scarified in the absence of virus the level that concurrent with this loss of sensitivity there was of corneal SP fell to a minimum of 67% of that in a marked fall in levels of corneal SP. In the uninfected the unscarified eye (Fig. 2). On day 7, the last day of cornea, sensitivity was not altered by scarification. observation, the level was 84% of normal. Corneal Although the minimum SP value was reached on day sensitivity remained normal throughout. 4 in both groups of mice, the fall was both more rapid and more pronounced in the infected group. Al- Discussion though slit-lamp examination, aided by Rose-Bengal stain in control mice, revealed no abnormality after The mouse has advantages for studies of ocular 36 hrs, damage to nerve endings as a result of scar- herpes simplex in that it is convenient and econom- ification was probably responsible for the fall in cor- ical to use relatively large numbers of animals, thus neal SP in these mice. The concomitant fall in corneal the reactions of a group of animals can be considered sensitivity and the levels of SP after infection with together. However, the size of the mouse eye is such HSV, suggest that the changes underlying both effects that no attempt was made to distinguish between ei- are as a result of the disease process. Such a process ther the center and periphery of the cornea, or be- might involve damage to nerve endings by virus, or tween ulcerated and nonulcerated areas when testing by factors released into the tissue during the inflam- for the blink reflex. Primary infection with HSV fre- matory response. Moreover, since virus is transported quently leads to destructive keratitis and extensive rapidly to the trigeminal ganglion10" it is possible periocular disease. Infection of the immune mouse, that even by one to two days after infection, damage however, results in largely reversible disease that is to the nerve cell body may have contributed to both 7 localized to the infected cornea and that more closely loss of sensitivity and fall in corneal SP. resembles human herpes simplex keratitis. Both den- The concurrent fall in sensitivity and SP levels sup- dritic and geographic ulceration occur associated with ports the association of SP with primary afferent neu- a variable degree of stromal infiltration and residual rones and shows that SP may be used as a marker of limbal vascularisation. Coincident with signs of acute neuronal integrity. However, on day 2 SP levels were

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CORNEAL SENSITIVITY AND SP LEVELS AFTER SCARIFICATION if any, of SP in the cornea is unknown. It remains 1 1 1 r— 1 1 1 a possibility that SP may be a mediator of the trophic 100 influence of corneal nerves. Key words: herpes simplex virus, corneal sensitivity, substance P, keratitis

Acknowledgment The skilled technical assistance of Carolyn Shimeld is gratefully acknowledged.

References 1. Jessell TM: The role of substance P in sensory transmission and pain perception. Adv Biochem Psychopharmacol 28:189, 1981. 2. Brodin E, Gazelius B, Lundberg JM, and Olgart L: Substance 50 P in trigeminal nerve endings: occurrence and release. Acta Physiol Scand 111:501, 1981. 3. Miller A, Costa M, Furness JB, and Chubb IW: Substance P immunoreactive sensory nerves supply the rat iris and cornea. Neurosci Lett 23:243, 1981. 4. Tervo K, Tervo T, Eranko L, and Eranko O: Substance P immunoreactive nerves in the rodent cornea. Neurosci Lett 25:95, 1981. 5. Keen P, Tullo AB, Blyth WA, and Hill TJ: Substance P in the mouse cornea: effects of chemical and surgical denervation. Neurosci Lett 29:231, 1982. 6. Duke-Elder S, editor. System of Ophthalmology. Vol. VIII: Diseases of the Outer Eye. Part 1, St. Louis, CV Mosby, 1965; A A SENSITIVITY IN MICE ASSAYED FOR SP p. 317. X----X SP LEVELS ( AND S.E.M. ) 7. Tullo AB, Shimeld C, Hill TJ, Blyth WA, and Easty DL: Ocular herpes simplex in non-immune and immune mice. Arch Ophthalmol, in press. 2 3 4 5 6 8. Hill TJ, Field HJ, and Blyth WA: Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying DAYS AFTER SCARIFICATION latency and recurrent disease. J Gen Virol 28:341, 1975. Fig. 2. Corneal sensitivity and substance P levels following scar- 9. Harmar A, Schofield JG, and Keen P: Cycloheximide-sensitive ification. Figures are expressed as a percentage of the value of the synthesis of substance P by isolated dorsal root ganglia. Nature contralateral (unscarified) eye. 284:267, 1980. 10. Knotts FB, Cook ML, and Stevens JG: Pathogenesis of her- petic encephalitis in mice after ophthalmic inoculation. J In- only reduced to 47% of normal in corneas with no fect Dis 130:16, 1974. 11. Tullo AB, Shimeld C, Blyth WA, Hill TJ, and Easty DL: blink reflex. On day 4 when SP levels were minimal, Spread of virus and distribution of latent infection following the reflex was reduced in some of the animals assayed ocular herpes simplex in non-immune and immune mice. J but normal in others. Moreover, in five mice with Gen Virol, 63:95, 1982. normal reflexes three weeks after infection corneal SP 12. Lembeck Fand Donnerer J: Time course of capsaicin-induced had not reached normal values. Thus, as suggested functional impairments in comparison with changes in neu- in a previous study5 SP in the cornea does not appear ronal substance P content. Naunyn Schmiedebergs Arch Phar- macol 316:240, 1981. to be involved directly in mediation of the blink re- 13. Mishima S: The effects of the denervation and the stimulation flex. Similarly changes in functional impairment and of the sympathetic and trigeminal nerve on the mitotic rate 12 SP levels do not correlate in capsaicin treated rats. of corneal epithelium in the rabbit. Jpn J Ophthalmol 1:65, The influence of corneal nerves on the epithelium 1957. is well documented.13"15 It has been suggested that 14. Mackie I A: Role of the corneal nerves in destructive disease of the cornea. Trans Ophthalmol Soc UK 98:343, 1978. the destruction of nerves that occurs in herpes zoster 15. Beuerman RW and Schimmelpfennig B: Sensory denervation ophthalmicus contributes to some of the corneal 16 of the rabbit cornea affects epithelial properties. Exp Neurol changes in this condition. Such changes as chronic 69:196, 1980. ulceration and thinning also occur in the cornea made 16. Marsh RJ: Herpes zoster keratitis. Trans Ophthalmol Soc UK anesthetic by surgery and infection with HSV, al- 93:181, 1973. though it seems unlikely that denervation is the only 17. Lewis RA, Keltner JL, and Cobb CA: Corneal anesthesia after 17 percutaneous radiofrequency trigeminal rhizotomy; a retro- factor involved in causing these changes. The role, spective study. Arch Ophthalmol 100:301, 1982.

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