Download Corneal epithelial wound healing in partial limbal deficiency.

Investigative Ophthalmology & Visual Science, Vol. 31, No. 7, July 1990
Copyright © Association for Research in Vision and Ophthalmology
Corneal Epithelial Wound Healing
in Partial Limbal Deficiency
James J. Y. Chen and Scheffer C. G. Tseng
Previous studies have shown that the corneal epithelial stem cells are located at the limbal basal layer.
The limbal stem cells are regarded as the ultimate source for corneal epithelial cell proliferation and
differentiation. This paper examines epithelial wound healing in rabbit corneas with partial limbal
deficiency (PLD), which was created by the surgical removal of two-thirds of the limbal zone (superior
and inferior). Four to eight months after PLD creation, all corneas appeared normal, without vascularization. The residual stem cell capacity then was challenged by two sizes of corneal epithelial debridement created with combined n-heptanol and mechanical scraping. In the first group, two consecutive
6-mm defects were created 1 month apart. After the first wounding, three of eight PLD corneas had
delayed wound healing and two of the three had vascularization, as compared to controls (n = 7). After
the second wounding, both controls (n = 7) and the remaining PLD (n = 5) corneas showed similar
rapid healing. In the second group, a large defect of up to 1 mm within the limbus was created. Healing
was completed in 25-40 days in PLD (n = 6) corneas, a more marked delay compared to the 10-12
days for controls (n = 6) (P - 0.001). In addition, all PLD corneas showed increased vascularization
and had epithelium of the conjunctival phenotype, verified by the immunofluorescent staining positive
to AM-3 monoclonal antibody but negative to AE-5 monoclonal antibody. Thus, a deficiency of limbal
stem cells contributes to the triad of conjunctival epithelial ingrowth, corneal vascularization, and
delayed healing with recurrent erosion. In PLD, corneal epithelium is still compromised, particularly
when a large epithelial cell mass is removed. Invest Ophthalmol Vis Sci 31:1301-1314, 1990
The normal ocular surface is covered by corneal,
limbal, and conjunctival epithelia, each of which has
a distinct cellular phenotype. The cornea is covered
by a tightly adherent nonkeratinized stratified squamous epithelium, which ensures optical smoothness.
In contrast, conjunctival epithelium contains mucinsecreting goblet cells. The limbal epithelium is the
transitional zone between the corneal and conjunctival epithelia. It differs morphologically from the cornea in that it possesses Langerhans cells and melanocytf and from the conjunctiva in that it lacks goblet
cells.1 These three epithelial phenotypes maintain the
ocular surface integrity.
In postnatal development, stem cells are needed for
all self-renewing tissues that have a high degree of
From the Department of Ophthalmology, Bascom Palmer Eye
Institute, University of Miami School of Medicine, Miami, Florida.
Presented in part at the Association for Research in Vision and
Ophthalmology, May 1989, Sarasota, Florida.
Supported in part by United States Public Health Service grant
EY-06819 (SCGT) and in part by Core Grant EY-02180, Department of Health and Human Services, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
Submitted for publication: August 1, 1989; accepted December
6, 1989.
Reprint requests: SchefTer C. G. Tseng, MD, PhD, Bascom
Palmer Eye Institute, P.O. Box 016880, Miami, FL 33101.
cellular differentiation. Corneal epithelium is well
known for its rapid self-renewing capacity, which together with a coordinate cellular differentiation
maintains the entire corneal epithelial cell mass. In
exploring this self-renewing process, several studies
have shown that there is centripetal cellular movement responsible for the replacement of central corneal epithelial cellular attrition, resulting from either
normal desquamation2"4 or traumatic loss.5"9 The
exact anatomic location of the stem cells of the corneal epithelium remained unclear until Schermer et
al10 indicated their limbal location based on evidence
provided by the expression pattern of a major 64-kD
corneal epithelial keratin. Additional evidence supporting this finding was provided by Ebato et al," 12
who showed that limbal epithelial cells grow much
better than peripheral or central corneal epithelia in
an explant culture, and by Cotsarelis et al,13 who
showed that limbal basal cells can be selectively stimulated by topical ocular application of a tumor promoter, as well as by wounding of the central corneal
epithelium.
Based on these findings, we speculated that the
limbal stem cells play a crucial role as an ultimate
source of corneal epithelial cellular proliferation and
differentiation. Our laboratory previously studied
corneal epithelial wound healing in a rabbit model by
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1990
surgically removing the entire limbal zone, in a procedure called total limbal deficiency.14 In that model,
the limbal zone, including 2 mm of the peripheral
cornea and 3 mm of the adjacent conjunctiva, was
surgically removed. In response to two consecutive
central 7.5-mm epithelial debridements, the corneas
with total limbal deficiency exhibited abnormal
wound healing characterized by delayed healing, recurrent erosion, increased corneal vascularization,
and conjunctivalization of the resultant epithelial
phenotype.14 This finding demonstrated that the corneal epithelium loses its renewal capacity in the absence of the limbus. Clinically, Kenyon and Tseng15
recently reported encouraging results in the use of
limbal autograft transplantation for various ocular
surface disorders that cause destruction of the limbal
zone. In a rabbit model, Tsai, Sun, and Tseng16 also
showed that limbal transplantation is a better source
than conjunctival transplantation in restoring the
corneal epithelial phenotype for a combined chemically and mechanically damaged corneal surface.
These results indicate that the corneal epithelial phenotype can be recovered by the replacement of the
limbus.
The objective of the current study is to examine the
reserve stem cell capacity in corneas with partial limbal deficiency (PLD), a problem that can be encountered in some clinical situations as well as in donor
eyes for limbal autograft transplantation.
Materials and Methods
The use of animals in these investigations conformed to the ARVO Resolution on the Use of Animals in Research.
Rabbit Model of PLD
A total of 14 New Zealand albino rabbits of both
sexes, weighing 2-3 kg, were anesthetized with intramuscular xylazine HC1 (50 mg) and ketamine HC1
(50 mg). In one eye of each rabbit, two pieces of
limbal strips involving the limbal circumference of
10-2 and 4-8 o'clock were removed. To ensure thorough removal of limbal epithelium, a dissection of
the limbal zone was performed with a partial-thickness corneal incision 2 mm from the limbus with a
no. 69 Beaver blade, followed by lamellar dissection
toward the limbus with a no. 66 Beaver blade, and
completed by conjunctival peritomy 3 mm beyond
the corneolimbal junction with a pair of scissors at
the same locations along the limbal circumference.
No suture was used to secure the free conjunctival
edge. Topical gentamicin sulfate and dexamethasone
ointments were applied three or four times per day
for 1 week, followed by dexamethasone ointment
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alone once or twice per day for a second week. All of
the injured eyes healed without complication within
1-2 weeks. Four to eight months later, these 14 rabbits were subdivided into two groups, each receiving a
different size epithelial debridement.
Challenge of the Reserve Stem Cell Capacity of PLD
The reserve capacity of limbal stem cells in PLD
rabbits was challenged by two different sizes of epithelial debridement, as follows.
In the first group, eight PLD corneas and seven
normal control corneas received two consecutive
6-mm central corneal epithelial debridements created
at a 1-month interval. In brief, after adequate general
anesthesia was induced with intramuscular injection
of xylazine HC1 and ketamine HC1, one eye was
proptosed. After a trephine marking, the central
6-mm corneal surface was rubbed with a cotton tip
wet with n-heptanol17 for a period of 1-2 min. This
rubbing was performed in a repetitive circular motion, starting from the central cornea to the wound
margin and then moving backward from the wound
margin to the central cornea. The ocular surface then
was rinsed with sterile saline and the corneal epithelium removed by gentle scraping with a Bard-Parker
blade. Topical gentamicin ointment was given twice
per day for 3 days. One month later, the eyes were
examined with a Zeiss surgical microscope, and those
corneas without corneal vascularization received the
second debridement using the same procedure.
In the second group, six PLD corneas and six normal controls received one large corneal epithelial debridement up to 1 mm within the limbus. The wound
margin was outlined with a Bard-Parker blade. The
rest of the wounding procedure was the same as described above. Topical gentamicin ointment was also
applied twice per day for 3 days.
Study of the Reserve Proliferative Capacity
To study the reserve proliferative capacity of PLD
corneas, the healing process was evaluated by external photographs, with and without 1% fluorescein
staining. Photographs were taken under general anesthesia daily in the first week after wounding, and then
every 2-3 days until the wound was completely
healed. Those corneas healed with vascularization
were examined weekly until the rabbits were sacrificed. The longest follow-up was 6 months. The fluorescein-stained external photograph of each wound
was projected and traced onto a piece of paper. The
wound area was determined by a Zeiss Videoplan 2
image analyzer with the Mop-Videoplan program
(Kontron Elektronik GmbH, Eching, West Germany). The healing curve was plotted by tracing the
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mean and standard deviation of the wound area measurement of each given postoperative day. The curves
of the experimental PLD group and normal control
group were compared.
Study of Reserve Differentiative Capacity
During the entire follow-up period of 6 months,
one rabbit of each group was sacrificed with an intravenous overdose of pentobarbital. For normal controls, the small defect group was sacrificed at day 6
and 14, and the large defect group at day 1,6,18, and
60 after healing. For PLD, the small defect group was
sacrificed at day 3 and 21 after healing, and the large
defect group at 4, 7, 8, 12, and 24 weeks after wounding. The resulting epithelial phenotype, determined
by immunofluorescent study, was used to study the
reserve differentiative capacity of the PLD corneas.
The corneoscleral button was removed, sectioned
into two halves, and embedded for frozen sections.
The resulting epithelial phenotype over the corneal
surface healed from the two different sizes of epithelial debridement was evaluated by immunofluorescent staining with two monoclonal antibodies, AM-3
and AE-5, which recognize specifically the nonglycosylated portion of rabbit ocular mucin core protein1819 and a 64-kD keratin specific to corneal epithelium,10 respectively. AM-3 was developed and
characterized in our laboratory and used to verify
conjunctival epithelial phenotype. AE-5 (a kind gift
of Tung-Tien Sun), has been characterized previously
by his laboratory and used as a marker for corneal
epithelial phenotype.10
The conditioned supernatant of the hybridoma of
both of the above mentioned antibodies was used as a
primary antibody. The supernatant of the parental
secretor myeloma cells was used as primary antibody
control. A dilution of 1:200 fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody
was used as a secondary antibody. After immunofluorescent staining, the slide was examined with a Zeiss
Axiophot equipped with epifluorescence capacity
and photographed. A composite of serial photographs
taken under a 10X objective lens was made for each
specimen and included the epithelia covering corneal, limbal, and adjacent conjunctival surfaces and
is reported here for purposes of comparison.
Statistical Analysis
The paired and pooled data of both experimental
and control groups were analyzed by the student ttest and the Wilcoxon-Mann-Whitney test, with the
assistance of the Department of Biostatistics, Bascom
Palmer Eye Institute.
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Results
In all 14 PLD corneas, the limbal wounds healed
rapidly within 1-2 weeks, even though two thirds of
the limbal circumference had been surgically removed. The corneal epithelium of all of these corneas
remained intact and the corneal surface was smooth
and clear during the entire follow-up period of 4-8
months. To study the reserve proliferative capacity of
PLD corneas, we compared the healing curve of PLD
corneas to that of normal controls in response to two
different sizes of corneal epithelial debridement.
In the group of small epithelial defects, the first
6-ram corneal epithelial debridement was created in
eight PLD and seven normal control corneas. When
the healing curve was compared as a group, PLD
corneas appeared to heal slightly more slowly, but the
results were not statistically significant when compared to the normal controls (P > 0.5; Fig. 1A). Extensive variation indicated by a large standard deviation was noted in the PLD group and possibly was
due to the fact that three PLD corneas showed a
marked delay in wound healing (Fig. 1 A). The second
6-mm epithelial debridement was performed 1
month after thefirst.Three animals in the PLD group
were excluded: two developed corneal vascularization and one died. The remainingfivePLD and seven
control corneas showed rapid healing in 3-4 days and
4-9 days, respectively (Fig. IB). The difference in
healing rate between these two groups was not statistically significant (P > 0.5; Fig. IB). No additional
corneal vascularization was noted in either group
after the second wounding. Despite the lack of statistical significance, the healing rate in the second
wounding tended to be faster than the first for both
the control and PLD groups (Fig. 1A, B).
When individual eyes were examined after the first
wounding, all seven controls and five of eight PLD
corneas healed smoothly within 4-9 days without
corneal vascularization. An example (case 6) of each
group is shown in the left and central columns of
Figure 2. However, the remaining three PLD corneas
showed delayed wound healing, especially notable on
the superior and inferior corneal aspects, adjacent to
the site of limbus removal. One such example (case 8)
can be seen in the Figure 2 (right column). Indeed,
the extension of the wound to the peripheral cornea
also was noted in these three corneas on the first
postoperative day (Fig. 2, second row, right column).
Two of these three PLD corneas healed with corneal
vascularization coming from the limbal deficient
areas (data not shown).
In the group of animals with large epithelial defect,
both PLD (n = 6) and normal control (n = 6) corneas
received one large epithelial debridement that ex-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1990
A
90-i
OCONTROL
• PLD
80-
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O CONTROL
• PLD
70-
| 60E
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50LU
cc 4 0 30-
J
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8 9 10
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20-
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DAYS
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tended up to 1 mm within the limbus. All wounds in
control corneas without limbal removal healed rapidly, in 10-12 days, but none of the PLD wounds
healed until 25-40 days after wounding (Fig. 3). The
healing rate seemed to be comparable for both control and PLD corneas in the first 5 days after wounding, but the healing was significantly delayed from the
8th day onward after the debridement (P = 0.001;
Fig. 3). All control corneas healed smoothly; one such
example (case 2) is shown in Figure 4A and 4B. No
corneal vascularization was noted in any of the controls (Fig. 4A) except in one, which showed mild peripheral vascularization on the upper cornea during
the early healing course, which regressed immediately
after complete epithelialization (data not shown). In
contrast, all PLD corneas healed with irregular
wound edges, and most importantly, with progressive
corneal vascularization and inflammation. One example (case 3) is shown in Figure 4C-H. All six PLD
corneas became heavily vascularized, except case 5,
which had only mild vascularization during the follow-up period of 6 months (Fig. 5).
To study the potential reserve differentiative capacity of PLD corneas, we compared their resulting
epithelial phenotypes to those of normal controls
using cell-specific monoclonal antibodies in the immunofluorescent test. AM-3 monoclonal antibody
reacts specifically with ocular mucin,18'19 and recog-
Fig. 1. Comparison of the healing curves of the control and PLD
corneas in response to (A) the first central 6-mm epithelial debridement and (B) the second debridement. The healing curve was derived
by plotting the mean of the residual defect area with its standard
deviation. For (A), control n = 7 and PLD n = 8; for (B), control n
= 7, and PLD n = 5. There is no statistical significance (P> 0.5) for
either (A) or (B).
nizes conjunctival goblet cells of normal rabbits (Fig.
6A). AE-5, which recognizes a major 64-kD corneal
keratin,10 decorates the full-thickness corneal epithelium and suprabasal cell layers of limbal epithelium10
(Fig. 7A).
After two consecutive 6-mm epithelial debridements, all control corneas retained the corneal epithelial phenotype, as indicated by staining negative to
AM-3 (Fig. 6B) but positive to AE-5 (Fig. 7B). Five of
eight PLD corneas that healed without vascularization also retained a corneal epithelial phenotype
(Figs. 6C, 7C). However, the remaining two vascularized PLD corneas exhibited AM-3-positive goblet
cells extending to the midperiphery (Fig. 6D) as well
as absence of normal full-thickness corneal epithelial
AE-5 staining, leaving only punctate pattern (Fig.
7D). This result suggests the coexistence of isolated
AE-5 positive cells intermixed with AM-3 positive
goblet cells. The central cornea still retained the corneal epithelial phenotype, which was negative to
AM-3 but positive to AE-5 staining (Figs. 6D and
7D). This distribution was consistent with the presence of peripheral corneal vascularization.
In the study of one large epithelial debridement, all
normal control corneas healed without vascularization and maintained the corneal epithelial phenotype
as indicated by positive AE-5 staining and negative
AM-3 staining. One specimen obtained 18 days after
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LIMDUS AND CORNEAL EPITHELIAL WOUND HEALING / Chen ond Tseng
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Fig. 2. Serial external photographs of normal control (case 6, left) and PLD corneas (case 6, center; case 8, right) after the first 6-mm central
epithelial debridement. To illustrate the defect areas, 1% fluorescein staining was used. Photographs were taken immediately after debridement (OP, top row), and day I, 5, and 9 thereafter (Dl, 5, 9).
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200
o Control
• PLD
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mal control groups, there was no statistically significant difference, as a result of the two consecutive
small epithelial debridements (P > 0.4), even though
two of eight PLD corneas did develop corneal conjunctivalization. However, the difference between the
two groups was statistically significant in response to
one large epithelial debridement (P = 0.002). None of
the control corneas, but all six PLD corneas, exhibited corneal vascularization and conjunctivalization.
Discussion
10
DAYS
Fig. 3. Comparison of the healing curve between control and
PLD corneas in response to one large epithelial debridement. The
healing curves were derived in the same manner as described in the
legend of Figure 1 and in Materials and Methods. Statistically
significant delay of wound healing had been noted ever since day 8
after debridement (P = 0.001, n = 6 for each group).
healing is shown in Figures 6E and IE. The positive
expression of AE-5-specific antigen extended to the
perilimbal conjunctiva with concommitant loss of
AM-3 specific antigen expression at that region (Figs.
6E, 7E).
In contrast, all PLD corneas showed conjunctivalization of the resultant epithelial phenotype, which
stained positive with AM-3 but weak or negative with
AE-5 antibody. For example, 4 weeks after debridement, the cross section through the limbal deficient
area showed AM-3-stained goblet cells on most parts
of the cornea except the central area, which still retained the corneal epithelial phenotype (Figs. 6F, 7F).
However, a section adjacent to the residual limbus
showed extension of the goblet cells only to the perilimbal area, and the AE-5-positive epithelium on the
rest of the cornea (Figs. 6G, 7G). Twelve weeks after
debridement, one section of the corneal surface was
covered by conjunctival epithelium containing more
AM-3-positive goblet cells (Fig. 6H), and there was a
nearly total loss of AE-5 expression (Fig. 7H). Six
months after debridement, the corneal surface still
had an epithelium mixed with both AM-3-positive
and AE-5-positive characteristics (data not shown).
When we compared the relative risk of developing
corneal vascularization and conjunctivalization of
the resultant epithelial phenotype in PLD and nor-
By definition, stem cells are present in all self-renewing tissues that have a high degree of cellular differentiation.20'21 These cells are long-lived and relatively quiescent with respect to cell mitosis under the
state of steady growth, but have a great potential for
clonogenic cell division, and ultimately are responsible for cellular proliferation and differentiation. Most
of our knowledge about stem cells comes from studies
of blood cells and such epithelial tissues as intestinal
epithelium, seminiferous epithelium, and epidermal
keratinocytes.22'23 According to the information from
these studies, stem cells, after a round of mitosis, generate transient amplifying cells that are short-lived
and function primarily to amplify cell numbers
through active mitosis. The transient amplifying cells
finally differentiate into postmitotic cells that are
committed to cellular differentiation. The ultimate
expression of the functional aspect of the tissue is
achieved by the terminal differentiation of the postmitotic cells. Therefore, stem cells and transient amplifying cells compose the proliferative compartment
of the tissue and differ from each other in life span as
well as in mitotic activity.
Evidence has now been gathered to demonstrate
that stem cells of the corneal epithelium are located at
the limbal region. Previously, several investigations
had suggested that peripheral cornea and limbus play
a significant role in corneal epithelial renewal,24"29
but Schermer et al10 pointed out the limbal location
from their study of the expression pattern of a major
64-kD corneal keratin. They further suggested that
transient amplifying cells are located at the corneal
basal layer and postmitotic cells at the suprabasal cell
layers. Recently, other studies have indicated that the
mitotic activity in the peripheral cornea and limbus is
higher than that in the central cornea," 12 suggesting
that more transient amplifying cells are located in the
peripheral cornea. The current study together with
Fig. 4. Examples of the healing pattern after one large corneal epithelial debridement, of the normal control (case 2, A, B) and PLD corneas
(case 3, C-H). To illustrate the residual epithelial defect, the correspondingfluoresceinstaining was used and shown in the right column. The
control cornea healed in 10 days without corneal vascularization (A, B). The PLD cornea showed markedly delayed wound healing with
irregular margin (F, H) and recurrent erosion (H). Most importantly, increasing corneal vascularization was noted (E, G).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1990
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Fig. 5. External photographs of all six PLD corneas (cases 1-6) after one large epithelial debridement. In each case, the photograph was
chosen from the last available photographic examination, of which the duration of follow-up is provided in the upper right. All six corneas
except case 5 showed heavy vascularization. In addition, stromal opacity and granuloma pyogenicum developed on all of the control corneas.
several of our previous reports14"16 further substantiates this important concept. Other properties of
limbal stem cells are described in detail in a recent
review by Tseng.30 However, it remains unknown
whether stem cells and transient amplifying cells assume a different role in the healing of a central cor-
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LIMDUS AND CORNEAL EPITHELIAL WOUND HEALING / Chen and Tseng
neal epithelial wound. In this regard, the results of the
current study provide some useful information,
which deserves more discussion.
In this study, the limbal wounds of all PLD corneas
healed rapidly even when two thirds of the limbal
circumference was removed. In contrast to the characteristic AE-5 suprabasal staining of the normal
limbal zone (Fig. 7A), the limbal deficient area
showed full-thickness AE-5 positivity, immediately
contiguous to conjunctival epithelium containing
AM-3 positive goblet cells (data not shown). This result indicates that the limbal epithelium had been
surgically removed thoroughly at the wound area and
that the limbal wound was healed by the surrounding
transient amplifying cells, with possible contribution
by the adjacent conjunctival epithelium. Because the
resultant corneal epithelium did not show any epithelial breakdown during 4-8 months of follow-up, the
remaining stem cells and transient amplifying cells in
PLD corneas were still sufficient to maintain the ordinary corneal epithelial cell renewal. It is unknown
whether the corneal epithelial integrity can be maintained by these residual proliferative compartments
for an indefinite period of time. To challenge the
residual proliferative capacity, two consecutive central 6 mm epithelial debridements were created. The
defects of PLD corneas were healed without difficulty, as compared to the normal controls with intact
limbus (Fig. 1A and B). This result indicates that the
reserved proliferative capacity of the remaining stem
cells and transient amplifying cells in the peripheral
PLD corneas could still handle the challenge of at
least two small central epithelial defects. We do not
know, however, how many challenges these cells can
tolerate. One can speculate that if stem cells can
manage to generate sufficient numbers of transient
amplifying cells, then there should not be a problem
in toleration.
It should be noted that three of eight PLD corneas
did show delayed healing, although not statistically
significant when PLD as a group was compared to the
control. This can be explained by the further loss of
the remaining transient amplifying cells that occurred during the extension of the wound area to the
peripheral cornea, noted on the first postoperative
day (Fig. 2, right column). We cannot explain why
this wound extension happened only in these 3 PLD
corneas. Because transient amplifying cells on the peripheral cornea were damaged, the healing rate was
delayed to a similar extent to those of the PLD corneas receiving one large epithelial debridement.
When the remaining transient amplifying cells
were nearly totally removed by one large epithelial
debridement, up to 1 mm within the limbus, the
healing rate in PLD corneas was still comparable to
1309
the controls during the first 5 days after wounding
(Fig. 3). This early rapid healing in PLD corneas presumably was effected by the remaining transient amplifying cells in the suprabasal portion of the limbus
or in the peripheral 1 mm of cornea; however, because of their limited life span, from the 8th day onward, the healing in PLD corneas was significantly
slower than that of the controls (P = 0.001; Fig. 3).
These results indicate that transient amplifying cells
on the peripheral cornea and limbus seem to be responsible for immediate wound healing. We speculate that upon tissue demand for cellular regeneration, the limbal stem cells may need more time to be
activated to generate new transient amplifying cells.
Without sufficient stem cells, there would be no sustained supply of transient amplifying cells, and thus
the wound healing would eventually be retarded.
This concept can be supported further by the observation of markedly delayed healing when the limbus
was totally removed, as described in a recent experiment.14
If the limbus is intact, a normal corneal epithelial
phenotype could be maintained even after two consecutive small epithelial debridements (Figs. 6B, 7B)
or one large debridement (Figs. 6E, 7E). This result
supports the notion that the limbus indeed contains
the stem cells for corneal epithelium. The corneal
epithelial phenotype could also be maintained in the
PLD corneas when sufficient transient amplifying
cells were preserved in the peripheral cornea (Figs.
6C, 7C). Nevertheless, when the populations of these
transient amplifying cells were further depleted either
by one large epithelial debridement (Figs. 6G, H and
7G, H) or by subsequent wound extension after a
small epithelial debridement (Figs. 6D, 7D), the PLD
corneas manifested a mixed corneal and conjunctival
phenotype. These results suggest that the stem cell
population in the remaining one third of the limbus
cannot sustain the corneal epithelial phenotype when
most of the transient amplifying cells are removed.
But with additional surrounding transient amplifying
cells, the corneal epithelial phenotype could be maintained. The mixed expression of corneal and conjunctival epithelial phenotype indicates that the residual limbal stem cells either are nonfunctional or are
insufficient to generate enough transient amplifying
cells for reepithelialization and prevention of conjunctival epithelial ingrowth. This observation suggests that there might be dysfunction of stem cell
renewal or generation of transient amplifying cells
under this posttraumatic circumstance. Further investigations in the aspects of stem cell regulation will
help us clarify this question.
The ingrowth of conjunctival epithelium signifies
the loss of limbal epithelium as a barrier between
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AM-3
B
H
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Fig. 6. Composite immunofluorescent staining by the monoclonal antibody AM-3. The detailed procedures of specimen preparation and
immunofluorescent staining are provided in Materials and Methods. Arrows are placed underneath the limbal region with the arrow pointing
to the cornea and the arrowhead placed directly beneath the anatomic corneolimbal junction. AM-3 recognizes ocular mucin and only stains
the goblet cells in conjunctiva under normal circumstances (A). In the group receiving two small epithelial debridements, AM-3 staining was
negative on the control (B) or nonvascularized PLD corneas (C). However, in vascularized PLD corneas, AM-3 stained goblet cells covering
the paracentral corneal surface (D). In the group receiving one large epithelial debridement, AM-3 was negative in the entire cornea as well as
perilimbal conjunctiva even 18 days after reepithelialization in the control cornea (E). (The spot above the arrow is an artifact.) In PLD
corneas, 4 weeks after wounding, AM-3-positive goblet cells invaded the midperipheral cornea through the limbal-deficient zone (F) and only
the perilimbal area through the zone adjacent to the remaining intact limbus (G). However, 12 weeks after wounding, corneal surface was
covered by AM-3-positive goblet cells (H).
corneal and conjunctival epithelia. The reason that
the normal limbus has such an epithelial barrier can
be explained by the following observations. Activation of mitotic activity in perilimbal conjunctival epithelium has been observed by Danjo et al31 when a
small central corneal epithelial defect was created in
rabbits. As a response to a central wound, the stem
cells are activated, giving rise to transient amplifying
cells which spread centripetally as one can expect in
the classical "centripetal cell movement."2"9 During
the healing process of the normal controls, we observed the positive expression of AE-5 with concomitant negative expression of AM-3 at the perilimbal
conjunctiva (Fig. 6C, E, and 7C, E). Recently, Thoft
et al32 reported positive AE-5 expression on the perilimbal conjunctival area next to the superior limbus
in human corneas. We interpreted this result to be
caused by the activation of stem cells on the superior
limbus to generate transient amplifying cells from the
frequent mechanical stimulation of the upper lid
blinking. Based on these findings, we speculate that
through this kind of centripetal and centrifugal
growth pressure, the limbal epithelium can serve as a
barrier between corneal and conjunctival epithelium.
It is worth reiterating that the conjunctival epithelial ingrowth is accompanied by corneal vascularization. Such a constellation, conjunctivalization with
vascularization, is a hallmark of limbal stem cell deficiency, or limbal deficiency. This is confirmed by
the current report, and by our recent experiments
with total limbal deficiency in rabbits,14'33 in which
increasing vascularization always was associated with
conjunctival epithelial ingrowth. In earlier studies,
we34"36 and others37"38 also have observed an incidence ranging between 14 and 68% of conjunctival
epithelial ingrowth and vascularization when total
corneal epithelial defects are extended beyond the
limbus in rabbit corneas debrided with n-heptanol
(n = 287, combining various reports). Recently, we
noted this abnormal healing pattern also resulting
from the total removal of limbal basal epithelium.33
We cannot explain why corneal vascularization is
closely associated with the development of conjunctival epithelial ingrowth. We speculate that corneal
epithelium derived from limbal epithelium is intrinsically different from that of conjunctival origin in
producing antiangiogenic or anti-inflammatory factors (cytokines). The conjunctival epithelium may
produce factors that induce stromal inflammation
and vascularization, which may explain why conjunctival stroma is normally vascularized.
Clinically, by the use of impression cytology, conjunctival epithelial ingrowth has been recognized in
several ocular surface disorders, including chemical/
thermal injuries, Stevens-Johnson syndrome, aniridia, and some contact lens-induced keratopathies.39 These disorders also are associated with
corneal vascularization. Based on the above experimental data, we can speculate that these disorders
may have the common pathogenic basis of limbal
stem cell deficiency. Such tissue alterations are the
basis of compromised corneal functions and are
consistent with the experimental data reported
earlier.40"42 Patients usually suffer from recurrent erosions and decreased vision as a result of irregular
optical surface, weak tensile strength,40 decreased
glycogen content,41 and incompetent barrier function.42 The fact that such abnormal corneal surfaces
can be treated successfully with limbal autograft
transplantations1516 further supports the pathogenic
theory.
In summary, stem cells and transient amplifying
cells may have a different role in corneal epithelial
wound healing. During the healing process, the cellular migration and mitosis are engaged primarily by
the transient amplifying cells. But because of their
limited life span, the increasing demand for new
transient amplifying cells triggers limbal stem cell activation and thus generates more transient amplifying
cells. The growth pressure generated by stem cell activation and increasing transient amplifying cells
constitutes the epithelial barrier between corneal and
conjunctival epithelia.
Therefore, we can conclude that limbal stem cells
serve as an ultimate source for corneal cellular regeneration and differentiation. Deficiency of limbal stem
cells, or limbal deficiency, gives rise to the triad of
conjunctival epithelial ingrowth (or conjunctivaliza-
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AE-5
H
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No. 7
LIMDU5 AND CORNEAL EPITHELIAL WOUND HEALING / Chen and Tseng
1313
Fig. 7. Composite immunofluorescent staining by the monoclonal antibody AE-5. Arrows are placed underneath the limbal region with the
arrow pointing to the cornea and the arrowhead placed directly beneath the anatomic corneolimbal junction. AE-5 recognizes the cornea-specific 64-kD keratin and stains the full thickness of the entire corneal epithelium and suprabasal cell layers of the limbal epithelium (A). In the
group receiving two small epithelial debridements, the AE-5 positive staining was noted in the entire epithelium of the corneal surface in both
normal control (B) and nonvascularized PLD corneas (C). In the vascularized PLD corneas, the paracentral corneal surface was largely
negative to AE-5 except for a few scattered superficial cells, and the central corneal surface was still positive to AE-5 (D). In the control cornea
receiving one large epithelial debridement, AE-5-positive staining was noted in the entire corneal epithelium (E) extending to the perilimbal
conjunctiva (also seen in C). In PLD corneas, 4 weeks after wounding, the AE-5 was positive on the remaining part of the corneal surface (F,
G) where AM-3 was negative (see also Fig. 6F, G). Twelve weeks after wounding, the entire corneal surface was negative to AE-5 (H).
tion), corneal vascularization, and delayed healing
with recurrent erosion. In the study of PLD, corneal
epithelial integrity is compromized significantly
when sufficient transient amplifying cells are lost.
Therefore, any cornea with PLD, whether surgically
or nonsurgically induced, should be carefully monitored.
Key words: conjunctival transdifferentiation, cornea, epithelium, limbal deficiency, limbus, monoclonal antibodies,
stem cells, wound healing
14.
15.
16.
17.
18.
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