Investigative Ophthalmology & Visual Science, Vol. 32, No. 2, February 1991
Copyright © Association for Research in Vision and Ophthalmology
Reports
Flow Cytometry Measurements of the DNA Content
of Corneal Epithelial Cells during Wound Healing
Hilary W. Thompson,* James S. Malrer,t Thomas L. Steinemann,* and Roger W. Beuerman*
an increase in mitotic activity in the corneal epithelium during the period of cell migration before
wound closure.
Materials and Methods. These procedures were
consistent with the ARVO Resolution on the Use of
Animals in Research. Albino rabbits, 5 weeks of age
and 2-3 kg in weight, were anesthetized by intramuscular injection of a ketamine/xylazine mixture (1.5
mg/kg body weight). Each rabbit's eyes were
wounded in an identical manner. As in a previous
study,5 the eye was proptosed through a sterile rubber
dam, and two drops of a topical anesthetic (proparacaine, 0.5%) were applied to the cornea. A 6-mm-diameter central keratectomy 150-200-jum deep was
created, and topical antibiotic ointment (gentamicin
sulfate 3 mg/g) was applied.
For tissue collection, animals were killed by lethal
pentobarbital injection and the globes were enucleated. Immediately after enucleation, eyes were transported to the flow cytometer in moist chambers on
crushed ice. The cornea was dabbed with a sterile
Dacron-tipped swab to remove surface debris.
Three types of specimens were collected: 1) Epithelial cells were obtained from the area that surrounded
the wound at seven intervals after wounding: immediately (time zero), and at 12, 24, 36, 48, 60, and 72
hr (Fig. 1, legend for numbers of eyes at each time
point). The cornea was marked with a 10-mm diameter trephine centered on the original 6-mm wound.
Tissue was harvested from the annulus between the
10-mm and 6-mm marks by mechanical debridement with a #15 scalpel blade (Storz Ophthalmic Instruments, St. Louis, MO). Electron microscopic
studies have shown that scraping the corneal surface
removes the epithelial cell layer completely; little or
no cellular debris is left on the cornea and the basal
lamina remains intact (Steinemann, Thompson,
Malter, Beuerman, 1990, unpublished data).
2) Epithelium was collected from the surface of
the wound area at 78 hr after wound closure in eight
eyes of four rabbits. Slit-lamp examination without
fluorescein before tissue removal showed complete
healing of the wound. A 6-mm-diameter trephine
This study presents the first application of flow cytometry
(FCM) techniques to the assessment of cell cycle dynamics
in the corneal epithelium after experimental wounding. Anterior keratectomies 6 mm in diameter were created in the
central corneas of albino rabbits. The authors sampled the
epithelial tissue obtained outside the wound at 12-hr intervals until wound closure at 72 hr. Regenerated epithelium
from the surface of the wounded area was collected at
78 hr. The percentages of nuclei in the G0/G1 (growth), S
(DNA synthesis), and G2/M (tetraploid/mitosis) phases
were determined by FCM. An increase in the percentage of
nuclei in the G2/M phase at 36 hr was seen, compared with
cell populations in samples from unwounded control corneas. The authors found an increase in mitotic activity in
the corneal epithelium during the period of cell migration
before wound closure. Invest Ophthalmol Vis Sci 32:433436, 1991
Flow cytometry (FCM) measures the DNA content
of individual cells, which provides an accurate indication of cell cycle stage. During the S (synthetic) phase
of the cell cycle, the amount of DNA increases and is
doubled in the G2 (tetraploid) and M (mitotic) phases
of the cell cycle.' Recently, cell nuclei, rather than
whole cells, have been used in FCM studies because
DNA staining in isolated nuclei is more reliable.2-3
FCM provides a more accurate assessment of cell
cycle stage than has been available with thymidine
uptake methods.3 Burns et al4 used pepsin to liberate
nuclei from acetic acid-treated tissue to determine the
cell cycle kinetics in the normal mouse cornea by
FCM. We used FCM to evaluate the mitotic status of
cell nuclei from the rabbit corneal epithelium after
experimental wounding. Our goal was to determine
whether mitotic activity increased after wounding.
We examined the changes in the percentages of nuclei
at various stages of the cell cycle in the epithelial cells
that surrounded the wound. We also studied the distribution of DNA content in the regenerated epithelium that covered the wound surface after wound
closure.
We found a significant increase in the number of
cells in G2/M phase at 36 hr. This finding indicated
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434
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / February 1991
24-
EZ2 G2/M
2016-
I
o
CL
84-
CONTROL
0
12
24
36
48
72
TIME (HRS)
Fig. 1. Mean percentages and standard errors derived from flow
cytometry of corneal epithelial tissue at 12-hr intervals after
wounding. Control (unwounded), n = 32; 0, n = 10; 12, 24, 36, and
48 hr, n = 9; 60 hr, n = 4; 72 hr, n = 6. S (synthetic) phase, solid bar;
G2/M (tetraploid) phase, striped bar.
with the obturator retracted was used to make an
incision over the previous wound circumference and
the central, 6-mm-diameter disk of loosely adherent,
regenerated epithelium was removed intact. Tissue
from these eight eyes was pooled for FCM.
3) Epithelium was collected from 32 unwounded
eyes of 16 rabbits after application of antibiotic ointment. A 10-mm-diameter trephine was centered on
the cornea, and epithelial tissue was scraped from
inside the trephine mark, as described above.
Tissue specimens were immediately placed in
Hank's balanced saline solution (HBSS). The epithelial cells were dissociated by gentle aspiration through
a 25-gauge needle after filtration through nylon mesh
to remove undissociated cells. After low-speed centrifugation (1500 rpm for 5-8 min) of the cell suspension, excess HBSS was removed and the specimens
were stained for DNA using 100 /xl of a 50-jug/ml
propidium iodide stain mix (50 mg propidium iodide, 0.2 mg RNAse, 1.016 g MgCl2, 0.6% NP40, 1
mg sodium azide, 0.01 M Tris, buffered to pH 7.0)
immediately before FCM. This procedure, which imparted a hypoosmotic shock to the cells, causing lysis
and the release of nuclei, improved propidium iodide
staining of the nuclear DNA.2
FCM was performed on an Epics Profile (Coulter
Electronics, Hialeah, FL) set to acquire a maximum
of 25,000 events, which included cellular debris.
After the data were downloaded to floppy disks, 256
channel histograms of cell number vs fluorescence
intensity were generated. Cell debris and unlysed cells
were excluded from the analysis by selective gating as
well as the application of criteria described below.
The 2N and 4N peaks were established by analysis of
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Vol. 32
nuclei from rabbit white blood cells stained under the
same conditions as the epithelial cell nuclei. The
numbers and relative percentages of nuclei in the
G0/G1, S, and G2/M phases were calculated using
the quadratic method with cytologic software
(Coulter Electronics, version 2.01).
Counts of nuclei from each specimen were evaluated for inclusion in the study on the basis of two
criteria. First, the total number of events recorded for
analysis had to be greater than 6000 (maximum:
25,000). This criterion excluded runs in which the
numbers of epithelial cells in the specimens were inadequate. Second, the number of events made up by
the sum of nuclei in the G0/G1, S, and G2/M phases
of the cell cycle had to exceed 80% of the total number of events. This criterion excluded samples that
had too high a percentage of noncellular debris or
unlysed cells.
Significant differences between the percentages of
nuclei in the S and the G2/M (tetraploid) phases were
detected using analysis of variance (ANOVA). Specific F tests were conducted to compare mean percentages of nuclei in each phase in unwounded control specimens with the percentages at each time
point after wounding.6
Results. FCM was performed on specimens from
102 corneas; 96 specimens met the criteria for inclusion in the study, and these data were used for further
analysis.
The percentages of S and G2/M phase nuclei in the
unwounded controls and the specimens from the
wounded corneas are shown in Figure 1. The basal
rate of mitosis seen in the 32 unwounded control
specimens was characterized by 13.5% ± 0.70%
(mean ± SEM) of the nuclei in S phase and 8.7%
± 0.42% in G2/M phase. In the specimens collected
immediately after wounding (time zero), the percentage of nuclei in S phase was significantly larger than
the percentage of S phase nuclei in the unwounded
control (P < 0.01); however, there was no significant
difference in percentages for G2/M phase nuclei.
Twelve hours after wounding, the percentage of S
phase nuclei was not significantly different from that
of unwounded controls. There were, however, significantly more nuclei in G2/M phase (P < 0.015), compared with the controls. As seen in Figure 1, the mean
percentage of nuclei in S phase was greater than the
mean percentage in G2/M phase, whereas at later
times (24-48 hr), the percentage of nuclei in G2/M
phase was greater than the percentage in S phase. This
phenomenon is an indication of a burst of mitotic
activity.
At 24 hr, the percentage of S phase nuclei was significantly smaller than the value in unwounded control specimens (ANOVA, unwounded vs 24 hr, P
No. 2
Reports
< 0.05). From 24-48 hr, the percentages of nuclei in
the G2/M phase were larger than the percentage in
unwounded controls, with a peak at 36 hr (unwounded vs 24 hr, P < 0.051; unwounded vs 36 hr, P
< 0.0005; unwounded vs 48, P < 0.001). At 60 and
72 hr, the percentages of 4N nuclei were statistically
similar to control values.
Overall, the results in Figure 1 showed a decline
in the percentage of nuclei in S phase after wounding and an increase in the percentage of nuclei
in G2/M (tetraploid) phase, which peaked 36 hr after
wounding.
Temporal relationships among the stages of the cell
cycle are shown in a three-dimensional plot (Fig 2) in
which the numbers of S phase and tetraploid nuclei
can be compared with the numbers of G0/G1 nuclei.
The increase in tetraploid nuclei was mirrored in a
decline in the numbers of diploid nuclei, whereas the
numbers of nuclei in S phase remained relatively
constant after an initial decline.
The distribution of DNA in the regenerated epithelium collected from the wound area showed 71.3% of
the nuclei in G0/G1 phase, 19.8% in S phase, and
8.9% in the G2/M phase. These percentages were
nearly identical to the percentages seen in the unwounded control specimens (72.9%, G0/G1; 13.5%,
S; 8.7%, G2/M).
Discussion. This is, to our knowledge, the first report of flow cytometry analysis of cell cycle kinetics
in corneal epithelium after wounding. Our results do
not confirm the previously seen suppression of mitotic activity after a wound.7 Rather, a burst of mitotic activity was seen before wound closure.
In the unwounded tissue, there were more nuclei in
the G0/G1 phase than in the other phases of the cell
cycle, and more S phase nuclei than G2/M phase
nuclei. Thus, in the cell cycle in the corneal epithe-
(O
Fig. 2. Numbers of nuclei in the G0/G1, S, and G2/M phases at
each time after wounding. Mean cell numbers are displayed on the
ordinate versus cell-cycle stage (ploidy) and time after wounding.
Control = unwounded.
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435
lium, the percentages of nuclei in each phase indicated that Gl was the longest phase, S was the next
longest, and G2 and M were the shortest phases.8
During healing, the relative durations of the cell cycle
phases did not change, and nuclei did not remain in
G2 but progressed to mitosis.8 The peak percentage
of nuclei in G2/M phase at 36 hr most likely represents an increase in cells undergoing mitosis. On the
cell cycle surface in Figure 2, more nuclei become
tetraploid, whereas there was no increase in S phase
nuclei. The percentage of cells in S phase showed
little change after wounding. This finding suggests
that the numbers of cells that progressed to S phase
remained constant, although increased numbers of
cells were in transition from S phase to G2 and from
G2 to mitosis. In contrast, a study by Friedenwald
and Buschke7 of rat corneal epithelium wounded
with a series of 30-/Km wounds showed complete suppression of mitosis until after the epithelial defect
closed. In that study, however, the small wound
radius may have resulted in healing before the change
in cell cycle kinetics. In the FCM study of normal
corneas by Burns et al,4 the percentage of cells in the
G2/M phase made up less than 1% of the total. However, their methods included acid digestion and prolonged storage.
Friedenwald and Buschke7 showed suppression of
mitosis by counting mitotic figures in stained tissue
sections. This technique could not indicate the total
number of cells obtained, the percentage of the cell
sample in the synthetic or S phase, or the points at
which G2 to M phase transitions occurred. In a previous study from our laboratories,9 colchicine was
used to cause mitotic figures to accumulate around
the wound margin for counting by means of light
microscopy. However, this method did not permit a
true determination of the natural peak of mitosis.
The increased percentage of cells in S phase in the
time zero specimens, compared with unwounded
controls, suggests the possibility of a rough regionalization in DNA synthesis between the central and
peripheral epithelium. Additional studies must be
done to investigate this theory.
FCM is an excellent means to study the cell cycle of
an accessible cell population such as the corneal epithelium. The distribution of DNA content in cell
populations can be accurately and rapidly determined, offering advantages over autoradiography or
scintillation counting. In contrast to older methods,
flow cytometry provides more precise information
about the cell cycle of corneal epithelial cells. This
method will also permit us to answer questions about
the nature of the trophic influences of corneal innervation on corneal wound healing, and to provide a
better background for our understanding of the con-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Februory 1991
trol of cell proliferation and the stimuli that evoke it
in the cornea.
Key words: cell cycle, cornea, epithelium, flow cytometry,
wound healing
Acknowledgments. The authors thank Joan Avant and
Connie Palmer for assistance with flow cytometry.
From the *LSU Eye Center, Louisiana State University Medical
Center School of Medicine, New Orleans and the fDepartment of
Pathology, Tulane University School of Medicine, New Orleans,
Louisiana. Supported in part by Clinical Investigator Award
CA-01427-02 from the National Cancer Institute (JSM) and U.S.
Public Health Service grants EY04074 (RWB) and EY02377 (departmental) from the National Eye Institute, National Institutes of
Health, Bethesda, Maryland. Submitted for publication: November 21, 1989; accepted September 17, 1990. Reprint requests:
Roger W. Beuerman, PhD, LSU Eye Center, 2020 Gravier Street,
Suite B, New Orleans, LA 70112.
References
1. Alberts B, Bray D, Lewis J, RaffM, Roberts K, and Watson
JD: Molecular Biology of the Cell. New York, Garland, 1989.
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2. Vindelov LL, Christensen J, and Nissen NI: A detergent-trypsin method for the preparation of nuclei for flow cytometric
DNA analysis. Cytometry 3:323, 1982.
3. Shapiro HM: Practical Flow Cytometry. New York, Alan R.
Liss, 1988, pp. 132-175.
4. Burns ER, Bagwell CB, Hinson WG, Pipkin JL, and Hudson
JL: Preparation and stability of sixteen murine tissues and
organs forflowcytometric cell cycle analysis. Cytometry 4:150,
1983.
5. Frantz JM, Dupuy BM, Kaufman HE, and Beuerman RW:
The effect of collagen shields on epithelial wound healing in
rabbits. Am J Ophthalmol 108:524, 1989.
6. Freund RJ and Littel RC: SAS for Linear Models. SAS Institute, Cary, North Carolina, 1981.
7. Friedenwald JS and Buschke W: The influence of some experimental variables on the epithelial movements in the healing of
corneal wounds. Journal of Cellular and Comparative Physiology 23:95, 1944.
8. Marcus M, Fainsod A, and Diamond G: The genetic analysis
of mammalian cell-cycle mutants. Annual Review of Genetics
19:389, 1985.
9. Beuerman RW, Tanelian DL, and Schimmelpfennig B: Nerve
tissue interactions in the cornea. In The Cornea: Transactions
of the World Congress on the Cornea III, Cavanagh HD, editor. New York, Raven Press, 1988, pp. 59-62.
Investigative Ophthalmology & Visual Science, Vol. 32, No. 2, February 1991
Copyright © Association for Research in Vision and Ophthalmology
Anomolous Motion VEPs in Infonts ond in Infontile Esotropio
Anthony M. Norcia,* Horacio Garcia,f Roger Humphry,^ Alexander Holmes,* Russell D. Hamer,* and Deborah Orel-Dixler*
Visual evoked potentials (VEPs) were recorded monocularly in response to vertical gratings that underwent oscillatory apparent motion at a temporal frequency of 10 Hz. In
normal infants 6 months or younger and in patients with a
history of constant strabismus onset before 6 months of age,
the oscillatory motion VEP contains a prominent first harmonic component that is temporally 180° out of phase in
the two eyes. This pattern is not seen in normal adults and is
consistent with the presence of a nasalward/temporalward
asymmetry of cortical responsiveness in infants and in patients with early onset strabismus. Invest Ophthalmol Vis
Sci 32:436-439,1991
The oculomotor behavior of patients with a history
of infantile esotropia bears a striking resemblance to
that of normal neonates. Monocular optokinetic nystagmus is asymmetric in both esotropic patients1 and
neonates2; slow-phase gain is higher for nasalward vs
temporalward motion. Esotropic patients are known
to show asymmetric smooth pursuit of small targets3
and to have sluggish open-loop pursuit acceleration
for temporally directed motion in the step-ramp
task.4 These patients also perceive nasally directed
motion to be faster than temporally directed motion
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of the same velocity.4 Since visual cortex provides the
dominant afferent input to the pursuit system5 and
since there is a perceptual correlate of the nasalward
vs temporalward motion asymmetry, the visual cortex may be involved in the genesis of the oculomotor
asymmetries found in infantile esotropia.4 In this report, VEP evidence for cortical involvement in the
production of asymmetric eye movements is prevented.
Materials and Methods. Observers: Thirteen
members of the laboratory staff participated in the
study. None had a history of abnormal binocular vision, and each had acuity correctable to 6/6 or better
in each eye. Eleven normal infants between 6-26
weeks of age, who had no strabismus or significant
refractive errors, were recruited from parent education classes. Fifteen patients who had constant esotropia onset before 6 months of age were also tested.
Onset of esotropia was documented either by history
or from medical records. Each patient had 20/30 or
better acuity in their worst eye, and no patient had
more than an octave acuity difference between eyes.
Twelve of the fifteen esotropic patients had strabismus surgery after 2 yr of age. The three patients