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
layer extended from layer 2 just superficial to the basal layer upwards to the corneal surface. Care was taken that
The number of grids observed from limbus to limbus the section evaluated passed through the axial portion of
was 50 to 52, however, only data from 50 grids were the cornea such that the superior and inferior poles were
included for analysis. Thus, there were 25 segments of present. Mitotic indexes MI were computed as the
0.32 mm each evaluated across the corneal epithelium of number of mitotic basal or suprabasal cells divided by the
each rat. The orientation of mitotic figures relative to the total number of basal or suprabasal cells with nuclei 3100.
basement membrane was often difficult to distinguish, and All cells with nuclei in the entire extent of the corneal
analysis of this parameter was not conducted. In agreement epithelium were counted; the cornea was assessed by the
with Bertalanffy and Lau [2], no evidence of diurnal use of an ocular grid measuring 0.16 mm in length at
rhythm could be deciphered in specimens receiving col- 4003 magnification. The cornea was identified by the
chicine at different hours of the day or that were sacrificed presence of a subjacent stroma. Four 0.16 mm grid lengths
at different time points each day; the time of wounding of were counted at the junction of the cornea and limbus on
the cornea was kept constant i.e., 0800–1000 h. Mitotic the superior and inferior aspects of the eye and considered
figures were seldom noted above layer 2 in the cornea, as the limbus; stromal material was underlying the basal
limbus, or conjunctiva.
172 I
3.1. The number and distribution of mitotic epithelial or conjunctiva. The number of cells undergoing mitosis in
cells in the uninjured cornea an approximately 0.64 mm extent constituting the center of
the cornea had a mean MI of 0.8. 3.1.1. Basal cells
The range of the MI of basal cells in the unwounded 3.1.2. Suprabasal cells
cornea was 0 to 8.7, and for the four grids of limbus and The index of mitotic suprabasal cells in the corneal
conjunctiva the MI ranged from 0 to 5.5 and 0 to 3.7, epithelium of uninjured animals was 2.660.5, and for
respectively Figs. 1A,C, 2. It should be noted that in the limbus and conjunctiva the MI was less than 0.5
some specimens there were no mitotic cells in one or more Figs. 1B,D, 2. The region of 0.64 mm extent constituting
of the 0.32 mm segments of the peripheral cornea, limbus, the center of the cornea rarely contained mitotic figures. As
mentioned earlier, the mitotic cells in the suprabasal layer, were predominately located in layer 2.
Study of 1 mm plastic thick sections revealed that some of the mitotic cells in layer 2 were noted to have a ‘stalk’
of cytoplasm that connected to the basement membrane Fig. 1B,D. Evaluation of 31 cells with mitotic figures in
layer 2 from 20 thin sections showed that 16 had stalks compared to 84 of the cells without visible extensions to
the basement membrane. However, the number of cells with stalks may be an underestimation because the plane of
section may have obscured such connections.
3.1.3. Distribution of mitotic cells The number of mitotic cells in the basal and suprabasal
layers of the ocular surface epithelium of the uninjured eye was calculated. In the epithelium of the cornea, limbus,
and conjunctiva, each region contained mitotic figures in a ratio of approximately 30 in the basal layer to 70 in
layer 2 Fig. 3.
3.2. The number and distribution of mitotic epithelial cells after injury
3.2.1. Wounded region of the cornea Mitotic cells were not recorded in the originally abraded
area in the first 12 h following injury Fig. 4. From 18 h until the conclusion of the 5-day examination period, little
or no mitotic activity was observed in the basal epithelial cells populating the regenerating region of the cornea;
however, at 18 h some segments at the margins of the wound did have an MI that reached 6–8.
No mitotic activity was noted in the suprabasal layer for the first 12 h post-injury Fig. 4. From 18 h after abrasion
until termination of the experiment i.e., 5 days, mitosis was infrequent in these suprabasal cells. At the margins of
the wound, some segments observed at 18 h and 24 h had an MI that occasionally reached 5–8.
3.2.2. Cornea peripheral to the wounded region Within 6 h following injury, the MI of the basal cells of
Fig. 1. Photomicrographs of the corneal epithelium in uninjured animals
the corneal epithelium extending from the margins of the
demonstrating mitotic cells in the basal arrow A and suprabasal B
wound to the limbus was comparable to specimens ob-
layers of paraffin sections, and the basal C and suprabasal layers D of
tained from uninjured animals Fig. 4, although there was
1 mm plastic sections. All animals were injected with colchicine 6 h prior
a general impression of a subnormal MI in many segments.
to termination. Note the stalk-like cytoplasmic processes arrowheads
A significant elevation in the MI was noted at 18 h
extending from mitotic cells in layer 2 to the basal lamina B, D. ep5epithelium; st5stroma. Bar514 mm.
P,0.05 that persisted at the 24, 30, and 36 h time points
I .S. Zagon et al. Brain Research 882 2000 169 –179
173
Fig. 2. Histogram of the mean mitotic index of basal and suprabasal epithelial cells occupying the ocular surface of unwounded rats; standard errors are not included on the graphs because they were minimal. Epithelial cells located in the inferior and superior aspects of the cornea are denoted to the left and right
sides, respectively, of each figure. Animals received colchicine 6 h prior to termination at the designated times.
P,0.001, with up to 5-fold more mitotic figures ob- MI of approximately 4 at 36 h following denuding of the
served at 30 h than at 6 h following abrasion. The MI at 30 corneal epithelium; this value differed significantly P,
h and 36 h in the cornea peripheral to the wounded region 0.05 from those recorded in uninjured rats.
differed significantly P,0.001 from preparations of Summation of mitotic cells in both the basal and
uninjured animals. suprabasal layers revealed significant P,0.05 elevations
With regard to the suprabasal region of the corneal in mitotic activity at 18, 30, and 36 h relative to both
epithelium adjacent to the wounded region, the MIs were homeostatic levels and in abraded specimens at 6, 12, 24,
comparable to uninjured specimens for the first 36 h Fig. 48, 72, and 120 h Fig. 5.
4. At 36 h the MI was increased significantly P,0.001 by 7-fold from the 6 h level of injured corneas, and 2-fold
3.2.4. Conjunctiva over the unwounded preparations. The MIs at 48, 72, and
The MI of the basal epithelium of the conjunctiva was 120 h were similar to both the 6 h indexes of injured
not changed from homeostatic levels after injury to the corneas and unwounded specimens.
cornea except at 24 h when a 3-fold MI was noted; this Summation of mitotic cells in both the basal and
difference was statistically significant P,0.05 Fig. 4. suprabasal layers of the corneal epithelium adjacent to the
The difference in MI of the conjunctiva at 24 h in injured abraded area Fig. 5 showed that at 30 h and 36 h there
animals was statistically significant from the 12, 48, and 72 were 3-fold P,0.01 and 5-fold P,0.001 increases,
h time points P,0.05. respectively, from unwounded and wounded subjects.
In the suprabasal epithelial cells of the conjunctiva from Examination of the proportion of mitotic cells in the
animals with an abrasion of the corneal epithelium, mitotic basal and suprabasal layers in the corneal epithelium
activity was negligible at all time points studied Fig. 4 as adjacent to the abraded region indicated that from 12 h to
well as from homeostatic levels. 24 h post-injury approximately 81 of the cells undergo-
Summation of mitotic cells in both the basal and ing mitosis were in layer 1 whereas 19 of the mitotic
suprabasal layers in the conjunctiva revealed no significant figures were in layer 2 Fig. 3. Relative to the proportion
difference between wounded and unwounded specimens, of mitotic cells in layers 1 and 2 during homeostasis, there
or across the time points of wounded cornea preparations was a change of 2.5-fold within the first day following
Fig. 5. injury. During the second day following abrasion, layer 1
had 62 of the mitotic figures and layer 2 had 38. At 72 h, the proportion of mitotic cells in layers 1 and 2 of
4. Discussion
