Introduction Thoft and Friend [26] proposed the X, Y, Z hypothesis as
Brain Research 882 2000 169–179 www.elsevier.com locate bres
Research report
Cellular dynamics of corneal wound re-epithelialization in the rat III. Mitotic activity
a , b,c
a a
Ian S. Zagon , Joseph W. Sassani
, Torre B. Ruth , Patricia J. McLaughlin
a
Department of Neuroscience and Anatomy , The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine,
500 University Drive, Hershey, PA 17033, USA
b
Department of Ophthalmology and Pathology , The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine,
500 University Drive, Hershey, PA 17033, USA
c
The Pennsylvania Lions Vision and Research Center , The Milton S. Hershey Medical Center, The Pennsylvania State University,
College of Medicine , 500 University Drive, Hershey, PA 17033, USA
Accepted 15 August 2000
Abstract
The number and distribution of mitotic epithelial cells in the ocular surface during homeostasis and in response to abrasion of the mammalian cornea were determined. Normal rats and those receiving a 3 mm diameter centrally located epithelial defect, received an
intraperitoneal injection of colchicine 6 h prior to sacrifice. Mitosis in the basal epithelium during homeostasis was comparable in magnitude across the ocular surface epithelium, with the exception of a few mitotic figures in the midline. Thirty percent of the mitotic
figures were in the basal layer layer 1, and 70 were in layer 2; the cells in layer 2 were often noted to retain connection to the basal lamina by cytoplasmic stalks. Mitosis was rarely noted in the regenerating epithelium. However, summation of M phase cells in both the
basal and suprabasal epithelium adjacent to the wound showed increases of 3- and 5-fold at 30 and 36 h after abrasion, respectively, from levels at homeostasis and the time of injury. In striking contrast to homeostatic epithelium, 80 of the mitotic cells were located in layer
1 of the corneal epithelium, with normal distribution observed by 72 h. Mitosis in the limbus and conjunctiva was increased 3-fold at 30 h and 24 h, respectively, from values at homeostasis and the time of debridement. These results, using rigorous statistical analysis and
precise topographic assessment, showed that mitosis is not impeded — but rather often accelerated — following denuding of the corneal epithelium and that the spatial distribution of mitotic cells is correlated with wounding. The data revealed that re-epithelialization of the
corneal epithelium is not dependent on mitosis in the regenerating epithelium, but rather in the adjacent unwounded epithelium of the cornea, with most cells being located in the basal layer until re-epithelialization is completed. Mitotic cells in the limbus and conjunctiva
may be related to replenishment of ocular surface epithelial cells used in the repair process rather than directly supplying the abraded surface.
2000 Elsevier Science B.V. All rights reserved.
Theme : Development and regeneration
Topic : Regeneration
Keywords : Cornea; Mitosis; Colchicine; Epithelium; Wound healing; Limbus; Conjunctiva
1. Introduction Thoft and Friend [26] proposed the X, Y, Z hypothesis as
the organizing principle of corneal epithelial maintenance, The ocular surface epithelium is in a constant state of
with X representing the proliferation of basal epithelial physiological renewal, with a dependence upon a balance
cells, Y the contribution to the cell mass by centripetal between the generation of cells and the loss of cells. In
movement of peripheral cells, and Z the epithelial cell loss humans and rats, for example, the corneal epithelium has
from the surface. Thus, the kinetics of the corneal epi- been estimated to become renewed every 7 days [2,12].
thelium can be expressed as X1Y5Z, with cell loss balancing cell replacement. This steady state of cell
renewal is crucial to the functions of the ocular surface
Corresponding author. Tel.: 11-717-531-6409; fax: 11-717-531-
epithelium, which serves as a protective barrier between
5003. E-mail address
: isz1psu.edu I.S. Zagon.
the external and intraocular environments, and for modula-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 6 4 - X
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-