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The purpose of this experiment is to assess the morphology
of the corneal endothelial cells in the central paracentral and
peripheral regions of both the rat (CD-IGS) and the mouse (CD-1
& C-57). Once obtained this data will be compared with previous
research conducted with rabbits in order to arrive at a model that
will most closely resemble the human corneal endothelium for the
betterment of future human corneal research. It is hypothesized
that due to the fact that the rabbit rat and mouse are all rodents
the results obtained from the present study will be similar to those
obtained from the rabbit studies or at least will exhibit a similar
trend. The central corneal endothelium is hypothesized to have higher
ECD than the peripheral region. 10 Rat corneas (average age 50 weeks
average diameter 6.28 mm) and 10 mice corneas 5 C-57 (average age
8.10 weeks average diameter 2.96) and 5 CD-1 (average age 7.10 average
diameter 2.84 mm) were enucleated. Following excision the corneas
were stained with 2% alizarin red solution for 7-10 minutes in order
to better visualize the morphology of the constituent cells. A flat
mount of the corneas was then made and the slide was viewed under
light microscopy. It was then possible to photograph the excised
corneas. The photography printout was analyzed via tracing and digitization
in order to attain the cellular density in the central paracentral
and peripheral regions of the corneal endothelium. From this information
it was possible to view which areas of the endothelium had the highest
cellular density. RESULTS: The rats had a mean central ECD (in cells/mm2)
of 2601+280 paracentral ECD was 2506+231 and peripheral ECD was
2196+390. A two tailed t test revealed a significant difference
between the central and peripheral ECD’s (p=.0219). Pigmented
mouse data revealed a mean central ECD of 2715+84 paracentral ECD
was 2592+158 and peripheral ECD was 2426+199. A two tailed t test
revealed a significant difference between the central and peripheral
ECD’s (p=.03). Statistical tests did not reveal any statistical
significance in the different regions of the albino corneal endothelium
nor in the combined data of the pigmented-albino mouse corneas although
the ECD’s were higher in the central cornea as compared to
the periphery. Also data obtained from the albino mice showed a
statistically significant increase in cellular density in the central
(p=.04) paracentral (p=.01) and peripheral (p=.03) regions of the
corneal endothelium as compared to the pigmented mice. CONCLUSION:
The results of this study show that the rat and mouse may not be
an applicable model on which to base future human corneal experiments
as the density trend is opposite to that obtained via the human
studies.
The corneal endothelium has been shown to be important
in stromal hydration corneal thickness and hence corneal clarity.
The corneal endothelium’s ability to absorb stains most notably
2% Alizarin Red stain has enabled researchers to view the corneal
endothelial cell morphology. The resulting cell sizes and shapes
as determined via the staining and morphological appearance correlate
with the functional capacity of the endothelium. Functional capacity
is defined as the corneal endothelium’s ability to respond
to damage inflammation and maintenance of corneal transparency.
Prior research evaluating human corneal endothelial cells has determined
the ECD in the central paracentral and peripheral regions of the
cornea. Research has been conducted and ECD’s have been deteremined
by a variety of methods most commonly via the use of the noncontact
specular microscope histologic cross sections and alizarin red staining.
These human studies have shown that there is a significant increase
in the ECD of the peripheral corneal endothelium as compared with
the central corneal endothelium. See Figure 1. It has been postulated
that perhaps this is due to the fact that there may be stem cell
activity in the periphery. The peripheral corneal endothelium does
not have the ability to regenerate however it may serve as a reserve
or storage area for the nearly continuous central endothelial cells;
the peripheral corneal endothelium may function as a migration zone.
Research as also been conducted with rabbits. Research has shown
that the corneal endothelium of the rabbit and human are not as
similar as previously thought with different morphological properties.
The coefficient of variation of cell area (CV) of the rabbit and
human corneal endothelium were compared and the results show that
there is a decrease in CV in rabbits following staining that is
not correlated with staining technique. The CV in human corneal
endothelial cells remained relatively constant and shows no significant
change. Furthermore recent research has found that the central corneal
endothelium in the rabbit has a higher ECD than does the periphery
a finding that is in opposition to that observed in the human corneal
endothelium. See Table 1. The results of these two studies make
it obvious that perhaps the rabbit is not the ideal candidate to
be used as a model of the human corneal endothelium. Perhaps the
mouse or rat endothelium will provide researchers with a more accurate
model.
- The rats (CD-IGS) average age 50 weeks received an overdose
of sodium pentobarbital via an intracardiac injection; the mice
(C-57 & CD-1) average age 7.6 weeks were sacrificed via an
overdose of carbon dioxide.
- The eyes of both the rat and mouse were enucleated the corneas
were excised leaving a small (mm) ring of sclera.
- At this time small radial incisions were made along the diameter
of the cornea so that a flat mount of the cornea could be made.
- The endothelial surface was stained with the intercellular
alizarin red S using the method described by Taylor and Hunt.5
- A staining time of between 7-10 minutes was found to be optimal
in allowing for stain demarcation of intercellular borders.
- After this time the alizarin red S was rinsed from the endothelial
surface in a solution of sterile isotonic Balanced Salt Solution
(BSS).
- The flat mount was photographed using light microscopy.
- For each photograph 100 contiguous cells were traced and digitized.
The computer assisted analysis provided the parameters of cell
area coefficient of variation and the percentage of cells which
were hexagonal.
- Rat data exhibits an increased ECD in the central corneal endothelium
which decreases paracentrally with the lowest ECD in the peripheral
region.
- Statistical significance (p=.0219) was demonstrated between
the central and peripheral ECD in the rats.
- Pigmented mouse ECD similarly decreases from the corneal center
to the periphery.
- Statistical significance (p=.03) was demonstrated between the
central and peripheral ECD in the pigmented mouse.
- No statistical significance was demonstrated in either the albino
mouse or the combined pigmented-albino mice data between any of
the corneal regions although peripheral ECD’s were lower
than those in the central corneal endothelium.
- ECD’s obtained from the albino mice were higher than those
obtained from the pigmented mice with statistical significance
demonstrated in the central (p=.04) paracentral (p=.01) and peripheral
(p=.03) regions.
- This data exhibits a trend similar to that obtained from the
rabbit studies and opposite to the trend obtained from human studies.3
- Hence neither the rat nor the mouse is an appropriate model
on which to base human corneal endothelial studies.
Supported in part by: NEI grants R0-1-EY00933, P30-EY06360, and
RPB HHMI Grant No. 52003727.
Basically my laboratory is aiming to determine whether or not the
human cornea has stem cells cells that have the ability to regenerate
following trauma speeding up the healing process. It is known that
stem cells occur in areas of high endothelial cell density (many
cells per square millimeter) and the periphery of the human corneal
endothelium has a high ECD more so than does either the central
or paracentral corneal endothelium. It was my responsibility to
determine the ECD trend in both rats and mice in order to determine
if the trend was the same as or different from that observed in
the humans. After I removed the eyes from the rodents I carefully
extracted the cornea stained it so that I would be able to view
individual cells under the light microscope and then rinsed the
stain. At that time I was able to view the cells of the three different
areas of the cornea under the microscope and take pictures of them.
With the pictures I used a machine called a digitizer to trace out
100 continuous cells from each area and then the computer generated
the ECD of each area. The results allowed me to determine that in
the rat and the mouse (which yields the same results as previously
published rabbit data) the ECD was highest in the central corneal
endothelium as opposed to the peripheral corneal endothelium. These
results lead us to believe that perhaps the rabbit rat and mouse
are not accptable models on which to base future corneal endothelial
studies as the trends exhibited are opposite to those observed in
humans.
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