170 I
tion of fluid transport for normal stromal hydration and ocular surface. Colchicine was used to induce a mitotic
corneal transparency [9,19,35]. arrest in order to identify cells in the M phase of the cell
It is understandable that injury to the cornea must be cycle. We expected to resolve the following queries: a
repaired rapidly in order to re-establish function. Three Where does mitosis take place in ocular surface epithelium
phases of epithelial healing have been described: a during homeostasis? b Is there a relationship between the
epithelial cell migration and wound closure, b re-estab- location of mitosis and DNA synthesis during homeosta-
lishment of cell number by cellular replication and dif- sis? c Does the covering of the wounded surface depend
ferentiation, and c reassembly of adhesion structures upon mitosis of the cells populating the re-epithelialized
[1,4–8,10,11,14,15,21,23,25,27,28,35]. Unfortunately, dis- area? d Is wound closure contingent upon mitosis of
agreement exists regarding the time course of these events adjacent cells in the undamaged corneal epithelium? e Do
following epithelial injury. Some studies have reported that cells of the limbus, cited as progenitors of the corneal
the early stages of epithelial wound closure predominately epithelial cells [20,22,24] respond to injury of the central
rely on cell migration rather than cell replication, with region of the cornea by alterations in mitosis? f How do
mitosis inhibited up to 3 to 4 days after injury depending cells of the conjunctiva, which have the capability of
upon wound size [1,6,7,21]. Nevertheless, other workers covering the corneal epithelium [29–31], react to an insult
have suggested that DNA synthesis and mitosis do occur in in the central region of the cornea with respect to mitotic
the regenerating epithelium and or the adjacent corneal activity? g Which cell layers, basal and or suprabasal,
epithelium within 24 h following injury [5,10,13,14,23, undergo mitosis? h What is the relationship of mitotic
27,28]. Finally, it has been reported that little change in behavior and DNA synthesis during repair of abrasions of
cell division from homeostatic levels in the corneal the corneal epithelium? The information obtained in these
epithelium is observed after wounding [25]. There are experiments concerned with mitosis was integrated with
many factors that contribute to this lack of consensus about that from previous studies on DNA synthesis in order to
the process of wound healing of the corneal surface. Some reveal, in a more comprehensive fashion, the role of cell
of these include: differences in species used e.g., rat, proliferation in wound healing.
mouse, rabbit, lack of specification of the regions ex- amined e.g., corneal epithelium, limbus, conjunctiva or
layers i.e., basal, suprabasal or plane of section investi-
2. Materials and methods
gated, subjective impressions rather than statistically valid statements, methodology that may not be optimal and
2.1. Animals specific e.g., scintillation counting of the corneal surface,
differences in wounding techniques e.g., n-heptanol, Adult 250–300 g male Sprague–Dawley rats Charles
mechanical, type of wound superficial, full thickness, River Labs, Wilmington, MA were utilized in this study.
and or damage to the integrity of the basal lamina with Animals were housed in an environment of 2160.58C with
respect to injury. a relative humidity of 50610. The room had a complete
To address these concerns, we have designed a series of exchange of air 15–18 times per hour and a 12 h light–
experiments that incorporate statistical analysis in a rigor- dark cycle with no twilight. Water and Purina 5010 Rodent
ously defined model that examined a mechanical dis- Chow were continuously available. The rats were accli-
turbance of the epithelial surface that left the basal lamina mated to the animal facilities for at least 1 week prior to
intact. In two earlier reports [33,34] we tagged cells with study.
radioactive thymidine prior to abrasion [33] and at discrete All investigations conformed to the Association for
points after debridement [34] and inquired about DNA Research in Vision and Ophthalmology Resolution on the
synthesis during repair. Some of the principles formulated Use of Animals in Research, the regulations of the
from these earlier studies include: a DNA synthesis and National Institutes of Health, and the guidelines of the
centripetal migration of cells observed in homeostasis is Department of Comparative Medicine of The Pennsylvania
retained after injury to the corneal epithelium, b cell State University College of Medicine.
replication occurs simultaneously with centripetal migra- Animals were examined pre-operatively to exclude
tion during re-epithelialization, c vertical migration is not subjects with ocular surface disease.
repressed by injury, d the regenerating surface of epi- thelium is not dependent on DNA synthesis for repopula-
2.2. Wound healing tion, e re-epithelialization relies on the activity of cells in
the adjacent unwounded area, and f the repair of injuries The procedures for wounding followed Zagon and
to the central corneal epithelium does not rely upon the colleagues [33,34]. In brief, rats were anesthetized by
limbus or conjunctiva. intraperitoneal injections of ketamine 10 mg kg and
The present report extends these observations about xylazine 5 mg kg. Using an Olympus SZ-ET dissecting
DNA synthesis and cell migration by addressing questions microscope Tokyo, Japan and a Highlight 2000 cold light
related to mitosis in the homeostatic and re-epithelializing source Olympus, a centrally placed 3 mm diameter circle
I .S. Zagon et al. Brain Research 882 2000 169 –179
171
was demarcated only in the right cornea with a disposable epithelial cells of this area. The limbus was approximately
dermatology skin punch Acu-Punch, Acuderm, Inc., Ft. 2.3 mm from the edge of the wound. The conjunctiva was
Lauderdale, FL. The encircled corneal epithelium was analyzed over four 0.16 mm grid lengths beginning 1 mm
removed with a [15 Bard-Parker scalpel blade. To from the limbo-corneal junction on each of the superior
facilitate accurate measurements of the wounded areas, and inferior aspects of the eye; goblet cells distinguished
special efforts were made to produce abrasions with round this bulbar region of the conjunctiva.
and smooth perimeters. Following surgery, 50 ml of Polytrim Allergan Pharmaceuticals, Irvine, CA was ap-
2.5. Thin section analysis plied to the injured eye [33,34].
Animals were examined post-operatively to exclude In addition to evaluating paraffin embedded sections as
ocular surface disease, and removed from the study if to the location of mitotic figures in colchicine preparations,
inflammation or infection occurred. some unwounded animals were injected with colchicine
and 6 h later, their eyes were fixed in 2 glutaraldehyde, 2.3. Colchicine treatment
2.5 paraformaldehyde, 3 sucrose, and 0.025 CaCl in
2
0.1 M sodium cacodylate buffer at 48C for 18 h, postfixed Based on preliminary findings in which colchicine 10
in OsO for 2 h, and embedded in Epon 812. One-micron
4
mg kg; Sigma, Indianapolis, IN was injected intraperito- sections of the corneal epithelium were placed on glass
neally into 2 animals time point at 1, 2, 4, 6, and 24 h and slides and stained with toluidine blue. Images were cap-
examined for mitotic activity, 6 h and 24 h were found to tured with a three-chip Sony CCD camera model DKC-
be optimal for recording a mitotic index; the 6 h time point 5000 mounted on an Olympus BH-2 light microscope.
following colchicine injection was chosen for subsequent studies. At each time point, 4 animals were anesthetized
2.6. Data presentation and decapitated, and the eyes proptosed and enucleated.
Eyes were fixed in 10 neutral buffered formalin for 24 h, All studies were conducted in a masked fashion, and the
and embedded in paraffin. Six-micron sections that in- same individual performed the surgery and the cell count-
cluded the entire corneal surface, limbus, and conjunctiva ing. Data are presented as the mean mitotic index percent
were collected and stained with hematoxylin and eosin. for every two grid areas 5segment. In some cases, the
In some cases, surgery was performed and rats given average MI for a region e.g., regenerating surface was
colchicine for 6 h. All surgeries were performed at 0800– provided. Thus, there are 2 segments 54 grids each for
1000 h. Animals also were collected at 12, 18, 24, 30, 36, the inferior and superior aspects of the limbus and conjun-
48, 72, and 120 h following surgery. ctiva, and 25 segments 550 grids across the corneal
surface. No differences in the MIs of the superior and 2.4. Mitotic indexes
inferior poles in any region of the ocular surface epi- thelium at any time point could be detected, and data were
The number of cells with mitotic figures i.e., prophase, combined. All data within a region across time were
metaphase, anaphase or telophase in the basal and sup- subjected to ANOVA, with subsequent comparisons made
rabasal epithelial layers of the superior and inferior cornea, using the Newman–Keuls test.
limbus, and conjunctiva were counted from 2 non-serial sections per eye; 4 corneas were assessed at each time
point. Only cells in the deepest aspect of the basal epithelium were considered basal cells. The suprabasal
3. Results
