Download Corneal sensitivity and substance P in experimental herpes

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 attention because of their widespread distribution within
the nervous system and because of their possible role
in neurotransmission. One of these small peptides
substance P (SP) is known to be particularly strongly
associated with primary afferent neurones.1 It is to
be found not only at the central terminals of such
nerve fibers, but also in the peripheral endings,2 and
recently its presence in the cornea was demonstrated
by immunohistochemistry3'4 and by radioimmunoassay.5 However, the function of substance P in
such peripheral sites is far from clear. By surgical and
chemical denervation it has been shown that substance P levels in the cornea are reduced. However,
no correlation between changes in SP and corneal
sensitivity was found.5 In the human cornea infected
by herpes simplex virus (HSV), sensitivity is reduced,6
and this has also been shown to be true of experimental ocular infection.7 In the experiments reported
here, SP levels were measured in the cornea of mice
with HSV keratitis, first to use SP as an indicator of
the integrity of sensory neurones, and second to examine the relationship of corneal SP to corneal sensitivity.
4 weeks previously,8 were inoculated by corneal scarification of the left eye7 through 5 n\ of growth medium containing 3.5 X 106 pfu of the same strain of
virus. As controls the corneas of uninfected mice were
scarified through medium without virus. Scarification
of the cornea in both groups consisted often parallel
strokes with a 26-gauge needle followed by a further
ten perpendicular to thefirst.Before use all mice were
examined with a Zeiss 10SL slit-lamp to exclude any
with ocular abnormalities.
Materials and Methods
Mice were killed by intraperitoneal injection of
pentobarbitone immediately after examination. Both
eyes were enucleated, and each was transfixed with
a pin for removal of the cornea under a dissecting
microscope. Care was taken to remove all conjunctival and limbal tissue. All corneas were treated separately using the right (uninfected) eye as a control.
After placing in 200 y\ of 10 mM HC1 containing 1
mM EDTA, 1 mM dithiothreitol, corneas were boiled
and homogenized. The homogenates were freezedried, and their SP content was determined by radioimmunoassay using a C-terminal directed antiserum,9 and 125I Tyr8SP as tracer. The test had a sensitivity of 5 pg per tube.
Measurement of Corneal Sensitivity
Blink reflex was tested in unanesthetized mice by
holding the animal by the scruff of the neck under
a dissecting microscope (X10 magnification) and
touching the center of the cornea with a sterile filament of nylon (0.24 mm diameter). A 2 cm length
of nylon held in a loop holder was found to be convenient to avoid touching whiskers and eyelashes. A
scoring system was used where 2.0 = normal brisk
reflex, 1.0 = reflex reduced in speed or amplitude,
and 0 = absent reflex.
Assay of Substance P
Infection of Mice
Eight-week-old outbred male mice, which had been
infected with HSV type 1 strain SCI6 in the right ear
From the Departments of Ophthalmology,* Pharmacology,f
and Microbiology,t University of Bristol, Bristol, United
Kingdom.
Supported by the Wellcome Trust and the South Western Regional Health Authority.
Submitted for publication May 5, 1982.
Reprint requests: Andrew B. Tullo, MD, Bristol Eye Hospital,
Lower Maudlin Street, Bristol BS1 2LX, United Kingdom.
0146-0404/83/0500/596/S0.95 © Association for Research in Vision and Ophthalmology
596
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No.
5
597
CORNEAL SENSITIVITY AND SUBSTANCE P IN HERPES KERATITIS / Tullo er ol.
CORNEAl SENSITIVITY AND SP LEVELS DURING HERPES SIMPLEX KERATITIS
Results
A group of 45 mice that had been infected as in
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
remainder of the group (Fig. 1).
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
11. On day 2 when sensitivity was minimal, three out
of six mice assayed for SP had no blink reflex in the
infected eye. The mean value of SP in these three
corneas was 47% of normal. In a further group of five
mice that had shown a complete recovery of corneal
sensitivity, corneal SP levels were only 81% of normal
values 23 days after infection.
In control mice (four to five daily) whose cornea
had been scarified in the absence of virus the level
of corneal SP fell to a minimum of 67% of that in
the unscarified eye (Fig. 2). On day 7, the last day of
observation, the level was 84% of normal. Corneal
sensitivity remained normal throughout.
Discussion
The mouse has advantages for studies of ocular
herpes simplex in that it is convenient and economical to use relatively large numbers of animals, thus
the reactions of a group of animals can be considered
together. However, the size of the mouse eye is such
that no attempt was made to distinguish between either the center and periphery of the cornea, or between ulcerated and nonulcerated areas when testing
for the blink reflex. Primary infection with HSV frequently leads to destructive keratitis and extensive
periocular disease. Infection of the immune mouse,
however, results in largely reversible disease that is
localized to the infected cornea7 and that more closely
resembles human herpes simplex keratitis. Both dendritic and geographic ulceration occur associated with
a variable degree of stromal infiltration and residual
limbal vascularisation. Coincident with signs of acute
100 ft
A 6 SENSITIVITY IN ALL REMAINING MICE
0 — 0 SENSITIVITY IN MICE ASSAYED FOR SP
X----X SP LEVELS ( AND S.E.M.I
5
6
7
II
12
DAYS AFTER INFECTION
Fig. 1. Corneal sensitivity and substance P levels during Herpes
simplex keratitis. Figures are expressed as a percentage of the value
of the contralateral (uninfected) eye.
disease there is a rapid and largely reversible fall in
corneal sensitivity.
The most striking finding of the present study was
that concurrent with this loss of sensitivity there was
a marked fall in levels of corneal SP. In the uninfected
cornea, sensitivity was not altered by scarification.
Although the minimum SP value was reached on day
4 in both groups of mice, the fall was both more rapid
and more pronounced in the infected group. Although slit-lamp examination, aided by Rose-Bengal
stain in control mice, revealed no abnormality after
36 hrs, damage to nerve endings as a result of scarification was probably responsible for the fall in corneal SP in these mice. The concomitant fall in corneal
sensitivity and the levels of SP after infection with
HSV, suggest that the changes underlying both effects
are as a result of the disease process. Such a process
might involve damage to nerve endings by virus, or
by factors released into the tissue during the inflammatory response. Moreover, since virus is transported
rapidly to the trigeminal ganglion10" it is possible
that even by one to two days after infection, damage
to the nerve cell body may have contributed to both
loss of sensitivity and fall in corneal SP.
The concurrent fall in sensitivity and SP levels supports the association of SP with primary afferent neurones and shows that SP may be used as a marker of
neuronal integrity. However, on day 2 SP levels were
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598
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / May 1983
CORNEAL SENSITIVITY AND SP LEVELS AFTER SCARIFICATION
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if any, of SP in the cornea is unknown. It remains
a possibility that SP may be a mediator of the trophic
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
50
A
A SENSITIVITY IN MICE ASSAYED FOR SP
X----X SP LEVELS ( AND S.E.M. )
2
3
4
5
6
DAYS AFTER SCARIFICATION
Fig. 2. Corneal sensitivity and substance P levels following scarification. Figures are expressed as a percentage of the value of the
contralateral (unscarified) eye.
only reduced to 47% of normal in corneas with no
blink reflex. On day 4 when SP levels were minimal,
the reflex was reduced in some of the animals assayed
but normal in others. Moreover, in five mice with
normal reflexes three weeks after infection corneal SP
had not reached normal values. Thus, as suggested
in a previous study5 SP in the cornea does not appear
to be involved directly in mediation of the blink reflex. Similarly changes in functional impairment and
SP levels do not correlate in capsaicin treated rats.12
The influence of corneal nerves on the epithelium
is well documented.13"15 It has been suggested that
the destruction of nerves that occurs in herpes zoster
ophthalmicus contributes to some of the corneal
changes in this condition.16 Such changes as chronic
ulceration and thinning also occur in the cornea made
anesthetic by surgery and infection with HSV, although it seems unlikely that denervation is the only
factor involved in causing these changes.17 The role,
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Ocular herpes simplex in non-immune and immune mice.
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ocular herpes simplex in non-immune and immune mice. J
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12. Lembeck Fand Donnerer J: Time course of capsaicin-induced
functional impairments in comparison with changes in neuronal substance P content. Naunyn Schmiedebergs Arch Pharmacol 316:240, 1981.
13. Mishima S: The effects of the denervation and the stimulation
of the sympathetic and trigeminal nerve on the mitotic rate
of corneal epithelium in the rabbit. Jpn J Ophthalmol 1:65,
1957.
14. Mackie I A: Role of the corneal nerves in destructive disease
of the cornea. Trans Ophthalmol Soc UK 98:343, 1978.
15. Beuerman RW and Schimmelpfennig B: Sensory denervation
of the rabbit cornea affects epithelial properties. Exp Neurol
69:196, 1980.
16. Marsh RJ: Herpes zoster keratitis. Trans Ophthalmol Soc UK
93:181, 1973.
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percutaneous radiofrequency trigeminal rhizotomy; a retrospective study. Arch Ophthalmol 100:301, 1982.
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