Download Measurement of centripetal migration of normal corneal

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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1985
account for the appearance and disappearance of
corneal LC. While LC are felt to have an immunologic
role in processing antigen,6 and in corneal transplantation,2 we believe these cells also play some role in
tissue damage and/or repair. The appearance of LC
in the cornea may be important after certain types
of corneal injury and infection. Roussel et al5 suggested
that the perpetuation of corneal inflammation may
be mediated by LC, and Rubsamen et al" proposed
a role for LC in corneal transplant rejection. Methods
for controlling the number of LC in the cornea may
prove useful in these situations.
Corneal LC are seen in large numbers after chronic
inflammation and trauma.4 In our studies, corneal
tissue was also damaged by suture placement and by
UV irradiation. LC appear to be active after a variety
of corneal perturbations. The exact role for LC in
nonimmunologic disturbances of the cornea will require further investigation.
Key words: Langerhans cell, guinea pig, cornea, nylon
suture, UV-irradiation
Acknowledgment.
T h e authors thank Ramsey H e m a d y
for his excellent technical assistance.
From the Francis I. Proctor Foundation for Research in Ophthalmology, San Francisco, California. Supported in part by NIH
grant EY-02502. Submitted for publication: July 6, 1984. Reprint
requests: Mitchell H. Friedlaender, MD, S-315, Proctor Foundation,
University of California, San Francisco, CA 94143.
Vol. 26
References
1. Lang R, Friedlaender M, and Schoenrock B: Langerhans cell
induction and migration in the guinea pig cornea. ARVO
Abstracts. Invest Ophthalmol Vis Sci 21(Suppl):l, 1981.
2. Lang RM, Friedlaender MH, and Schoenrock BJ: A new
morphologic manifestation of Langerhans cells in guinea pig
corneal transplants. Curr Eye Res 1:161, 1981.
3. Juhlin L and Shelley WB: New staining techniques for the
Langerhans cell. Acta Dermatovener (Stockholm) 57:289, 1977.
4. Gillette TC, Chandler JW, and Greiner JV: Langerhans cells
of the ocular surface. Ophthalmology 89:700, 1982.
5. Roussel TF, Osato MS, and Wilhelmus KR: Corneal Langerhans
cell migration following ocular contact hypersensitivity. Cornea
2:27, 1983.
6. Wolff K and Stingl G: The Langerhans cell. J Invest Dermatol
80:017s, 1983.
7. Aberer W, Schuler G, Stingl G, Honigsmann H, and Wolff K:
Ultraviolet light depletes surface markers of Langerhans cells.
J Invest Dermatol 76:202, 1981.
8. Jacobelli D, Takahashi S, and Hashimoto K: Cell membrane
damage of Langerhans cells by ultraviolet light. Clin Res 31:
574A, 1983.
9. Norlund J, Ackles A, and Lerner A: The effects of ultraviolet
light and certain drugs on la-bearing Langerhans cells in
murine epidermis. Cell Immunol 60:50, 1981.
10. Miyakazi H, Kawada A, Takaki Y, Sato T, and Masutani M:
Effects of ultraviolet light on epidermal dendritic cells of
hairless mice. In Sunlight and Man, Pathak M, Harber L, Seiji
M, and Kukita A, editors. Tokyo, University of Tokyo Press,
1972, pp. 217-229.
11. Rubsamen PE, McCulley J, Bergstresser PR, and Streilein JW:
On the la immunogenicity of mouse corneal allografts infiltrated
with Langerhans cells. Invest Ophthalmol Vis Sci 25:513, 1984.
Measurement of Centripetal Migration of Normal Corneal
Epithelial Cells in the Mouse
Robert C. Duck
Fine punctate marks were made in normal corneas of mice
using a needle rotating in a mixture of India ink and thorium
dioxide. After 7 days, the marker was visible in the stroma
and also in epithelial cells which had moved away from the
stromal marks and towards the center of the cornea. The
mean distance between these labels at the end of 7 days
was 94 urn ± 1 4 (SEM). The median distance migrated
was about 17 /im per day. This figure represents the
distance through which superficial and wing cells had
migrated; the distance migrated by basal cells was not
determined. Invest Ophthalmol Vis Sci 26:1296-1299, 1985
Recently Thoft and Friend1 have proposed an
"XYZ hypothesis" of corneal epithelial cell maintenance. They review the evidence, which is indirect,
that normal corneal epithelial cells are in a state of
constant centripetal migration, as well as proliferation
and desquamation. However, no experimental work
has been carried out on the specific question of the
possible migration of the epithelial sheet in the normal
cornea.
In order to test this hypothesis, certain of the
methods used to study morphogenetic movements of
cells and cell sheets during embryonic development
could be applied to the cornea. Markers have been
developed for this purpose and include carmine or
Nile blue dyes,2 carbon or India ink implantation,3
and H3-thymidine, the labeled cells being traced by
autoradiography.4 Fortunately, the corneal epithelial
cells are highly phagocytic towards the colloidal carbon
of India ink, and this simple method can be used to
trace their movement.
Materials and Methods. Swiss mice weighing 2025 g were anesthetized by an intraperitoneal injection
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of sodium pentobarbitol. Into each eye was then
instilled a drop of a mixture of Pelican India ink
(Gunther Wagner) and equal parts of a colloidal
solution of thorium dioxide (Thorotrast; Testagar,
Detroit), and the excess was blotted away. A short,
1-mm diameter steel needle with a very sharp point
mounted in the chuck of a high speed drill was
pressed lightly against the cornea, and power was
applied for about 10 sec. A tiny black spot marked
the position of the "tatoo" in epithelium and underlying stroma.
Preliminary experiments showed that an interval
of 7 days was satisfactory for checking the result
because the label was still seen in the epithelial cells
as well as in the stroma. Animals allowed to live for
2 wk showed ink marks only in the stroma; the
labeled epithelial cells had largely desquamated.
The animals were killed by cervical dislocation 7
days after marking the cells. The eyes were enucleated
and immersed for 4 hr in half-strength Karnovsky
fixative. The corneas were excised and cut into four
sections which were flattened between a weighted
coverslip and a miscroscope slide so that they could
be examined even by the oil immersion lens.
The ink marks were identified, the specimen was
rotated to give a consistent position of the limbus,
and a photograph was taken. The distance between
the leading edge of the migrating epithelial cells and
the closest edge of the stromal tatoo was measured.
Humane treatment of the mice was provided in
accordance with the ARVO Resolution on the Use
of Animals in Research.
Results. Most of the tatoos were not satisfactory
because ink had not marked the stroma. In order to
obtain a measure of migration, it was necessary to
have ink in the stroma to indicate the starting point,
and also ink in a cluster of the migrating cells.
In almost all cases, when a stromal tatoo was
observed in the cornea after 7 days, a group of cells
having particles in their cytoplasm extended from the
stromal tatoo towards the center of the cornea (Fig.
1). Epithelial cells containing the ink in phagocytic
vacuoles were easily distinguished from stromal cells
by their shape and by the level in the cornea, determined by their plane of focus using the oil immersion
lens (NA 1.0). Prior to the onset of migration the
spot was small and dense (Fig. 2).
Observations were made on 21 tatoo marks in 16
mice. The mean distance migrated in 7 days was 94
/im ± 14 (SEM). This figure is significantly different
from 0 (the null hypothesis), having a P value of
<0.01. This mean includes two specimens in which
no ink could be found in the epithelium and one
specimen in which migration appeared to be 30 jam
in the opposite direction from the rest of the group.
Fig. 1. Typical migration of epithelial cells away from mark in
stroma 7 days after impaling India ink. The cluster of labeled
epithelial cells is approaching the edge of the iris, which is the outof-focus line near the top of the picture. Distance between leading
margin of epithelial cells and nearest edge of stromal mark is 240
^m. Stromal cells show migration away from the implant point.
They are not in the plane of focus of epithelial cells (X135).
If these three are excluded, the mean distance migrated
becomes 111 ± 13 pm (SEM). The median distance
was 123 fjm. It seems that the best estimate for
distance migrated is about 17 ^m/day.
Although it was not the purpose of this research
to study the movement of conjunctival epithelium,
ink marks were made in conjunctiva close to the
limbus in several animals. Only six satisfactory tatoos
were observed in conjunctiva, and none of these
showed any movement towards or away from the
limbus after 7 days. The question of conjunctival
epithelium crossing the limbus in the normal eye
requires further study.
In order to determine which layers of the epithelium
were involved in the migration, some specimens,
after being studied by light microscopy, were prepared
in the usual way for transmission electron microscopy.
They were oriented so that sections would be cut
through the marked cells of both stroma and epithelium. The thorium dioxide, being electron dense, had
been added to the ink for this purpose. Phagocytic
inclusions were observed in all layers of the epithelium,
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INVESTIGATIVE OPHTHALMOLOGY G VI5UAL SCIENCE / September 1985
Vol. 26
The present results confirm migration in the normal
cornea, although the rate of migration is only about
V,o of that seen earlier in the peripheral part of the
corneas repairing a central defect.
Previous studies suggesting centripetal cell movement have generally been based on observations in
which some insult was made to an appreciable area
of corneal epithelium, which might therefore be said
to be reacting to injury. Mann 7 studied the migration
in living rabbits of pigmented epithelial cells as they
responded to lesions of the cornea made close to the
limbus, and by repeatedly scraping the cornea, she
was able to draw these pigmented cells far from the
limbus. Other examples given by Thoft and Friend1
suggest centripetal cell migration, such as observations
on sex chromatin of donor grafts and the movement
of epithelial dots after grafting. It is difficult to
evaluate how much effect, if any, such invasive
procedures might have on migration, and to decide
Fig. 2. Appearance of an ink spot 18 hr after impaling the ink
into the cornea. Migration has not yet begun, and the spot is very
dense since it contains the ink in stroma and also epithelium. The
spot lies in the peripheral cornea, just inside the edge of the very
dilated iris (lower denser area) (X135).
including basal cells (Fig. 3), although they appeared
to be larger and more numerous in wing and superficial cells. However, this tedious method does not
lend itself to the measurement of migration. Obviously, the probability of finding thorium inclusions
in thin sections is not high, and it is not possible to
determine whether the distance migrated by cells
observed to have inclusions is representative of all
migrating basal cells.
Discussion. I earlier demonstrated5 by the use of
ink labeling that during the resurfacing of a small
central epithelial defect of the cornea, mass movement
of epithelium in the cornea peripheral to the lesion
resulted. Subsequently, I observed that the hemidesmosomes of these peripheral cells were arranged in
rows.6 An analysis comparing the angle of these rows
to the radial axis of the cornea showed a highly
significant correlation, and I concluded that the pattern in rows was an expression of migration in a
radial direction. However, a similar pattern of hemidesmosome rows was also seen in the normal cornea
suggesting that normal epithelium too might be constantly undergoing centripetal migration.
Fig. 3. Electron micrograph of corneal epithelium containing
inclusion of impaled thorium dioxide at a distance of about 200
ym from the mark in the stroma. Inclusions are seen in wing and
basal cells'.'(X3,600).
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whether the migration represents a normal phenomenon in these cases.
Although a quantitative evaluation of the movement of basal cells could not be carried out by the
method used here, basal cells containing thorium
inclusions were observed to lie in centripetal positions
relative to the stromal marks. Whether they moved
as far as the more superficial cells remains to be
determined.
That they do move centripetally, as anticipated
from the study of the orientation of the hemidesmosomes, opens up the question of the participation of
hemidesmosomes in their migration. They cannot be
static. During the repair of suction blisters of skin,
hemidesmosomes apparently undergo a rapid dislocation and relocation, which takes place in less time
than is required for the epidermal cell to migrate
through its own length.8 Other investigators estimated
that less than an hour is required for their reformation
after being torn from the basal lamina by suction.9
Thus, the relatively slow rate of migration of the
normal corneal epithelium would pose no problem
for the repeated dislocation and re-attachment of its
hemidesmosomes to the basal lamina.
The significance of centripetal epithelial migration
for the maintenance of corneal epithelial integrity is,
of course, a matter for speculation. The convergence
of cells towards the center, by which central cells are
replaced with those derived from peripheral cornea,
could possibly reflect the gradient from peripheral
cornea to central cornea of certain substances derived
from the limbal capillaries. According to Maurice,10
substances spread through the cornea by diffusion
from the perilimbal capillaries. The gradient for large
molecules is steep, serum albumin being several times
more concentrated at the periphery than at the center.
Whether these molecules confer any advantage on
the peripheral epithelial cells is unknown, but possibly
such substances as growth factors, known to have
pronounced effects on corneal epithelial cells in vivo
as well as in vitro," could reach the epithelial cells
via these capillaries.
Key words: corneal epithelium, cell migration, mouse
From the Department of Anatomy, The University of Western
Ontario, London, Ontario, Canada. Supported by Grant MT-1011
from the Medical Research Council of Canada. Submitted for
publication: December 6, 1984. Reprint requests: Dr. Robert C.
Buck, Department of Anatomy, Health Sciences Centre, The
University of Western Ontario, London, Ontario, Canada
N6A 5C1.
References
1. Thoft RA and Friend J: The X, Y, Z hypothesis of corneal
epithelial maintenance. Invest Ophthalmol Vis Sci 24:442,
1983.
2. Keller RE: Vital dye mapping of the gastrula and neurula of
Xenopus laevis. II. Prospective areas and morphogenetic movements in the deep layer. Dev Biol 51:118, 1976.
3. Spratt NT: Localization of the prospective neural plate in the
early chick blastoderm. J Exp Zool 120:109, 1952.
4. Nicolet G: Avian gastrulation. Advances in Morphogenesis 9:
231, 1971.
5. Buck RC: Cell migration in repair of mouse corneal epithelium.
Invest Ophthalmol Vis Sci 18:767, 1979.
6. Buck RC: Hemidesmosomes of normal and regenerating mouse
corneal epithelium. Virchows Arch (Cell Pathol) 41:1, 1982.
7. Mann I: A study of epithelial regeneration in the living eye.
Br J Ophthalmol 28:26, 1944.
8. Krawczyk WS and Wilgram G: Hemidesmosome and desmosome morphogenesis during epidermal wound healing. J Ultrastruct Res 45:93, 1973.
9. Beerens EGJ, Slot JW, and van der Leun JC: Rapid regeneration
of the dermal-epidermal junction after partial separation by
vacuum: an electron microscopic study. J Invest Dermatol 65:
513, 1975.
10. Maurice DM: The cornea and sclera. In The Eye, Davson H,
editor. New York, Academic Press, 1962, pp. 289-386.
11. Gospodarowicz D and Giguere L: Growth factors, effect on
corneal tissue. In Cell Biology of the Eye, McDevitt DS, editor.
New York, Academic Press, 1982, pp. 97-142.
Iontophoresis of Epinephrine Isomers to Robbit Eyes
Induced HSV-1 Oculor Shedding
James M. Hill,* Yoshikazu Shimomura,t Byoung Se Kwon,:J: and Louis P. Gangarosa, Sr.§
Iontophoresis of 0.01% levo(—) epinephrine for 8 min at
0.8 mAmp once daily for 3 consecutive days induces ocular
shedding of herpes simplex virus type 1 (HSV-1) in latently
infected rabbits. In the present experiment, we tested dextro(+) and levo(—) epinephrine for their comparative effects
on induced HSV-1 ocular shedding. One hundred percent
of the eyes shed virus after either 0.01% dextro(+) or
levo(—) epinephrine iontophoresis (8 min, 0.8 mAmp).
However, the shedding frequency caused by 0.005% levo(—)
epinephrine was significantly higher (P < 0.05) than that
by 0.005% dextro(+) epinephrine when the iontophoresis
was conducted at 0.4 mAmp for 4 min. Iontophoresis of
0.001% levo(-) epinephrine for 8 min at 0.8 mAmp once
daily for 3 consecutive days and iontophoresis of 0.001%
dextro(+) epinephrine for 8 min at 0.8 mAmp once daily
for 3 consecutive days did not induce HSV-1 ocular shedding
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