Download A new in vitro corneal preparation to study epithelial wound

Investigative Ophthalmology & Visual Science, Vol. 33, No. 11, October 1992
Copyright © Association for Research in Vision and Ophthalmology
A New In Vitro Corneal Preparation to
Study Epithelial Wound Healing
Darrell L. Tanelian and Kammi Bisla
Corneal epithelial wound healing is an important process necessary for maintenance of visual integrity.
Corneal epithelial wound healing occurs by cellular migration and proliferation. However, the molecular basis of reepithelialization is not known. To investigate individual molecular contributions to the
wound healing process, an in vitro corneal preparation comparable to the in vivo condition is needed.
This investigation developed a new whole mount in vitro rabbit cornea preparation and studied epithelial wound healing rates for epithelial and subepithelial wounds. The wound closure rates obtained in
this study for epithelial and subepithelial wound healing (52 ± 14 jum/hr and 38 ± 7 Atm/hr, respectively) are comparable to in vivo rates of wound healing determined by other laboratories for rabbits.
This preparation, achieved by functionally separating the epithelial and endothelial sides of the cornea,
allows application of agents to the cornea in a manner that approximates the in vivo condition. This in
vitro system is promising for future studies designed to investigate corneal wound healing while reducing potential ocular discomfort associated with in vivo corneal wounding. Invest Ophthalmol Vis Sci
33:3024-3028,1992
Corneal epithelium forms a protective barrier1 and
modulates fluid transport to maintain normal stromal hydration.2 Because of the epithelium's functional importance, reepithelialization after injury
from trauma or surgery has been studied extensively.3"8 Reepithelialization occurs by epithelial cell
migration and proliferation to cover the denuded
area,9"13 and healing rates for this process have been
determined.14"17 Effects of therapeutic agents or
growth factors on corneal wound healing also have
been examined. However, the effects of these agents
on wound healing has produced inconsistent results
that may result from differences in the wound healing
models used.18"24 In vivo studies have difficulty establishing steady-state delivery of agents, such that applied concentrations are equivalent to effector site
concentrations. These studies also have difficulty manipulating environmental conditions. In vitro experiments, while controlling experimental variables, have
not maintained normal corneal shape or tissue pressure, and in the case of epithelial tissue culture, other
corneal layers are not present. Also, fetal calf serum
(FCS), containing an unknown composition of
growth factors and other products, has been added to
the tissue culture medium of some in vitro studies, '7>25"30 potentially altering the effect of applying further therapeutic agents.
The present study introduces a new in vitro whole
mount corneal preparation to study the effects of therapeutic agents on epithelial wound healing. This organ culture preparation combines attributes of
current in vivo and in vitro systems, allowing reepithelialization to occur without the addition of supplemental growth factors or FCS to the culture medium.
Furthermore, normal corneal shape, tissue pressure,
and tissue clarity are maintained. Wound healing
rates comparable to in vivo preparations are achieved.
Materials and Methods
From the Pain Research Center, Department of Anesthesiology,
University of Texas, Southwestern Medical Center at Dallas.
Supported by NIH grant 1RO1 NS28646-01A1.
Submitted for publication: January 10, 1992; accepted April 15,
1992.
Reprint requests: Darrell L. Tanelian, University of Texas, Southwestern Medical Center, Department of Anesthesiology, 5323
Harry Hines Blvd., Dallas, TX 75235-9068.
Animal care and treatment in this study complied
with the Institutional Animal Care Review Committee at Stanford University School of Medicine and the
ARVO resolution on the use of laboratory animals.
New Zealand white rabbits, weighing 2-3 kg, were
killed with an intravenous injection of Beuthanasia
(pentobarbital/phenytoin; Schering Corp., Kenilworth, NJ) after anesthesia with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/
kg). The eye was proptosed and rinsed with corneal
Ringer's solution equilibrated with 95% O2/5% CO2
by bubbling with this gas mixture at 21 °C. The cornea
and a 2 mm rim of sclera were excised, and the iris
and lens were removed. The cornea then was
mounted onto an in vitro perfusion chamber made
3024
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No. 11
VITRO CORNEAL EPITHELIAL WOUND HEALING / Tonelion ond Dislo
WOUNDED AREA
TEMPERATURE
PROBE
3025
wound, the epithelium within the marked area was
scraped away and the basement membrane was left
intact (Fig. 2A). In the subepithelial wound, the epithelium, basement membrane, and part of the anterior stroma were scraped away (Fig. 2B). Wounding
was confirmed by staining with 0.5% fluorescein and
by histologic studies.
Analysis of Wound Area
Fig. 1. Schematic of in vitro whole mount corneal chamber with
mounted, wounded cornea. The corneal epithelium is kept in a
humidified air environment, while the endothelium is perfused
with tissue culture medium entering through the inlet valve. Temperature and intraocular pressure may be constantly monitored as
indicated. There is constant flow through the system at 5 ml/hr.
out of Plexiglas, which was designed and built by the
authors (Fig. 1). The epithelial side of the cornea was
kept in a warmed (35°C), humidified air environment
to prevent drying. This was accomplished by placing
the corneal mounting chamber (Fig. 1) inside an electronically heated and temperature-controlled covered
Plexiglas box partially filled with normal saline. The
endothelial side was perfused at 5 ml/hr with Gibco's
(Grand Island, NY) Medium 199 and 50 tig/ml gentamicin equilibrated with 95% O2/5% CO2 and brought
to a pH of 7.4. Corneas were maintained at 35°C, and
the intraocular pressure was 15 mmHg.
Rates of wound closure were monitored by staining
with fluorescein every 5 hr and recording the average
radius of the remaining wound. Corneas were allowed
to heal until fluorescein failed to stain the cornea,
which was reported as the time of wound closure. The
quality and extent of reepithelialization was evaluated
by stereo microscopic (M3Z; Wild Leitz, Heerbrugg,
Switzerland) visualization after fluorescein staining.
The wound radius was measured with a calibrated
grid. The wound area and radius were calculated using the methods of Crosson, Klyce, and Beuerman.14
Wound area was determined from wound radius by
considering corneal curvature and comparing wound
radius to the entire corneal radius. The equation is: As
= 2 R[R - (R2 - r2)*], where As is the remaining
wound area, R is corneal radius, and r is the radius of
the remaining wound area. Data were statistically analyzed using repeated measures analysis of variance,
and the mean ± standard error of the mean was plotted on the graphs.
Histology
Corneal Wounding
A Bard-Parker No. 15 blade was used to create one
of two types of corneal wounds: a superficial epithelial
or an anterior keratectomy subepithelial wound. A 5
mm circular trephine mark was made in the center of
the cornea to mark the wound area. In the epithelial
Fig. 2. (A) Cross-section
of corneal tissue immediately after epithelial wounding confirms that the epithelium has been removed from
the wounded area. Arrow indicates wound margin. (B)
Subepithelia! wounding of
cornea extends through the
epithelium and basal lamina
and into the anterior
stroma, as this photograph
confirms. The arrow specifies the wound margin. (All
bars= 100 Mm.)
After wound closure, corneas were fixed in 10%
buffered formalin fixative for histologic studies. The
corneas then were stained with hematoxylin-eosin/
phloxine counterstain as well as periodic acid-Schiff
stain to highlight the basal lamina. Corneas then were
sectioned (6 /xm) and mounted with Permount
B
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3026
Vol. 33
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1992
Fig. 3. Photographs of
corneal epithelial wound
healing from initial subepithelial wounding through 70
hours. Time 0 is the time of
initial wounding. Wounds
have been stained with 2%
fluorescein for visualization, and wound edges are
marked with arrows. Complete wound closure for this
type of wound occurred on
average in 69 ± 4.5 hr (mean
± standard deviation).
O hr
10 hr
20 hr
30 hr
40 hr
50 hr
6 0 hr
70 hr
(Fisher Chemical, Fair Lawn, NJ). Sections were
viewed with a Nikon Diaphot inverted microscope.
Results
Wound healing occurred in a circularly symmetrical manner in both types of corneal wounds, and corneas remained transparent throughout the healing
process (Fig. 3). Graphs summarizing the mean decrease in epithelial wound area with time (n = 5) for a
5 mm diameter epithelial wound are shown in Figure
4. The subepithelial wound healing experiments (n
= 10) have been plotted on the same graph (Fig. 4).
Examination of this graph reveals the epithelial
wound healed more rapidly than the subepithelial
wound. Complete wound closure occurred in 51 ± 5.5
hr for the epithelial wound and in 69 ± 4.5 hr for the
subepithelial wound. These curves reveal a nonlinear
decline in wound area with time. In the early phase of
healing (0-25 hr), the epithelial wound healing rate
was 0.57 ± 0.07 mm2/hr, and later (30-55 hr) the rate
decreases to 0.21 ± 0.06 mm2/hr. The subepithelial
curve shows a similar pattern with a fast rate in the
first part of the healing curve (0-25 hr) of 0.42 ± 0.07
mm2/hr, followed by a decrease to 0.25 ± 0.05 mm2/
hr (30-55 hr).
Another way to quantitate wound healing is to
measure the velocity of epithelial cell migration. This
method, initially described by Crossen, Klyce, and
Beuerman,14 measures the actual epithelial migration
rate independent of wound size and allows for comparison between different wound healing models. The
results of the epithelial and subepithelial wound healing experiments are plotted as a function of wound
radius in Fig. 5. This plot approximates a linear function from which linear regression analysis can be used
to calculate the epithelial migration rates. The rate of
epithelial migration is 52 ± 14 /im/hr for epithelial
wounds and 38 ± 7 j^m/hr for subepithelial wounds.
A brief latency period was noted for the epithelial and
subepithelial wounds. However, the initial measurement intervals used in these experiments were not
short enough to accurately evaluate this early phase of
healing.
IN VITRO WOUND HEALING
SUBEPITHELIAL WOUND
EPITHELIAL WOUND
30
TIME
45
(hr)
Fig. 4. The average epithelial (n = 5; circles) and subepithelial (n
= 10; squares) wound area at specific times after wounding. This
curve appears to show two separate healing phases (0-25 hr and
30-55 hr). The average rate of decrease in wound area for the first
phase is 0.57 ± 0.07 mm 2 /hr and 0.42 ± 0.07 mm 2 /hr (mean
± standard error of the mean) for the epithelial and subepithelial
wounds, respectively. Both rates decrease in the second half of the
healing process. The epithelial wound heals at a rate of 0.21 ± 0.06
mm 2 /hr (30-55 hr), and the subepithelial wound healing rate decreases to 0.25 ± 0.05 mm 2 /hr (mean ± SEM). Time to complete
wound closure is 51 ± 5.5 and 69 ± 4.5 hr(mean ± standard deviation) for the epithelial and subepithelial wounds, respectively. Error
bars indicate SEM.
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No. 11
VITRO CORNEAL EPITHELIAL WOUND HEALING / Tonelion and Dislo
IN VITRO WOUND HEALING
•
•
SUBEPITHELIAL WOUND
EPITHELIAL WOUND
Fig. 5. Average decrease in wound radius for the epithelial wound
(n = 5; circles) and for the subepithelial wound (n = 10; squares).
The average rate of epithelial wound closure was 52 ± 14 jum/hr
(mean ± standard error of the mean), and the average rate of subepithelial wound closure was 38 ± 7 (mean ± SEM). Error bars indicate SEM.
Histology of healed epithelial and subepithelial corneas revealed a continuous layer of epithelial cells covering the wounded area. The epithelial lining the original wound and the area immediately surrounding was
thinned after wound closure. Normal corneal thickness was maintained throughout the entire wound
healing process.
Discussion
Epithelial cells at the wound margin were shown to
migrate toward the center of the corneal wound to
cover the denuded region, consistent with other investigations of corneal epithelial healing.910'31 During
this migration phase, DNA synthesis ceases in the migrating basal epithelial cells, and cell mitosis does not
occur until after wound closure.32 A temporary increase in mitotic rate is seen several days after initial
wounding.9 The molecular mechanisms underlying
corneal wound healing are not known, except that
functional actin filaments are required for wound closure.
The in vitro whole mount preparation was used in
this study to monitor wound healing for epithelial and
subepithelial wounds. Corneal transparency, normal
shape, and thickness were maintained for up to 94 hr,
although a maximum limit of viability was not established. The present study indicates that cellular components vital to wound healing are present in the intact in vitro cornea. Also, with the addition of a minimal medium, wound healing was able to proceed in a
normal fashion. The wound closure rates obtained in
this study for epithelial and subepithelial wound heal-
3027
ing (52 ± 14 /um/hr and 38 ± 7 /xm/hr, respectively)
are comparable to in vivo rates of wound healing for
rabbits, which are 64 ± 2 ^tm/hr for epithelial
wounds14 and approximately 50 jim/hr for subepithelial wounds.15 The in vitro condition does not exactly
replicate the in vivo state because factors such as plasminogen and plasmin in the tear film, which aid corneal epithelial migration,33 are not present. This may
explain the slightly decreased rates with this in vitro
model compared to in vivo studies. Other in vitro
organ culture assays have measured epithelial wound
healing rates ranging from 7.4 mm2/day (0.38 mm2/
hr) 34 to0.83mm 2 /hr. 16
This new in vitro corneal preparation is promising
as a model for investigating the epithelial wound healing process. The ability to maintain steady-state drug
concentrations is a great advantage for evaluation of
therapeutic agents and growth factors. Manipulation
of environmental conditions also may reveal valuable
insights into the mechanisms of corneal epithelial
wound healing.
Key words: cornea, epithelial, in vitro, organ culture, wound
healing
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