Download Hydrostatic pressure effects on deswelling of de

Investigative Ophthalmology
July 1976
546 Reports
7.
8.
9.
10.
of gluconeogenesis in the perfused livers of
rats, J. Biol. Chem. 242:2622, 1967.
Thoft, R. A., and Friend, J.: Biochemical
aspects of contact lens wear, Am. J. Ophthalmol. 80: 139, 1975.
Riley, M. V.: Aerobic glycolysis in the ox
cornea, Exp. Eye Res. 8: 201, 1969.
Reddy, V. N.: Distribution of free amino
acids and related compounds in rabbit cornea, Ophthalmol. Res. 1: 46, 1970.
Schonheyder, F., Ehlers, N., and Hust, B.:
Amino acids in aqueous humor and plasma
in chronic ocular disorders, Acta Ophthalmol.
53: 627, 1975.
Hydrostatic pressure effects on deswelling
of de-epithelialized and de-endothelialized corneas.
KAREN
A.
BOWMAN AND
KEITH GREEN.
The effect of varying hydrostatic pressure on
the thinning rate of preswollen de-epithelialized
or de-endothelialized corneas has been determined
in the specular microscope. The appropriate membrane was removed, the cornea given access to
Ringer to swell, and then fluid exchange at that
surface blocked with oil. De-epithelialized corneas
thin more slowly as hydrostatic pressure on the
posterior surface is increased, until fluid movement
ceases at 60 to 70 mm. Hg. Fluid movement can
occur, therefore, against a considerable hydrostatic
pressure. De-endothelialized corneas thin at a
higher rate as hydrostatic pressure is increased;
this effect is probably a mechanical one with increasing pressure forcing fluid out across the
epithelium.
Since its introduction, the specular microscope
has been used extensively in the study of corneal
physiology.1"3 With this system, the cornea can
be excised atraumatically from the eye and
mounted in appropriate chambers for determination of thickness to an accuracy of ± 2 fim. With
little variation most investigators have elected
to use hydrostatic pressure of about 15 mm. Hg1-3
on the endothelial surface, although lower pressures have been used, e.g., 8 mm. Hg.4 One
study has reported no effect of hydrostatic pressure variation between 6 and 37 mm. Hg on the
deswelling rate of the de-endothelialized cornea.5
The present work was a study of the effects
of varying the applied hydrostatic pressure between 5 and 50 mm. Hg on the deswelling rate
of both de-epithelialized and de-endothelialized
corneas.
Materials and methods. Adult albino rabbits,
2 to 3 kilograms, were killed with an overdose
of sodium pentobarbital and the eyes enucleated
with the lids as described previously.1 Corneal excision and mounting in the appropriate chambers
also followed the standard procedure.1"3
De-epithelialized corneas. For convenience the
epithelium was removed from the cornea with
a Gill corneal knife prior to enucleation and
mounting. The stroma, therefore, was allowed
access to fluid over a time period of about 10
minutes prior to placement in the chamber. Krebsbicarbonate-Ringer's solution,1 with added adenosine (5.0 mM.) and reduced glutathione (0.3
mM.), 6 was placed on the exposed stromal surface and the stroma allowed to swell for 15
minutes after mounting; the fluid was removed
and the stroma covered with silicone oil (No.
20 CSKS Dow Corning, Midland, Mich.) to prevent further fluid exchange across the exposed
stromal surface. Thickness was measured by alternately focusing on the anterior stromal surface
and the endothelium. The endothelial surface was
perfused with modified Krebs-bicarbonate-Ringer's
solution at 37° C. at a rate of 33 /tl per minute.
A control series consisted of thickness measurements at 15 mm. Hg for 5 hours. The experimental series consisted of an initial 1 hour of
15 mm. Hg, for comparison with the controls, followed by one of three other subsequent pressures
5, 30, or 50 mm. Hg applied for the second
hour, a different pressure from this series applied for the third hour, and the remaining unused pressure from this series applied for the
fourth hour, which allowed at least four corneas
to be subjected to each possible sequence of these
three pressures. A final period of 15 mm. Hg
followed between hours 4 and 5. Each pressure
was applied for 1 hour, and the deswelling rate
determined over the final 30 minutes of each
hour, since initial experiments showed that the
thickness adjustment became regular within 30
minutes.
De-endothelialized corneas. The endothelium
was removed by gentle scraping with the cornea
attached to the mounting rod. The posterior surface of the cornea was given free access to Ringer's
solution and allowed to swell for 20 minutes.
The normal mounting procedure was then followed except that silicone oil was perfused across
the exposed posterior stromal surface and the
epithelial surface was perfused with modified
Ringer's solution. The perfusion system delivered
Ringer's at 1 ml. per minute and 32° C. to the
epithelial surface and the fluid level was maintained with a vacuum overflow. The normal volume needed to fill the anterior space bordering
the epithelium was 1 ml. without immersion of
the objective, thus the turnover was 1 volume
per minute, sufficient to prevent stagnation of
the epithelial fluid bath which, by evaporation,
could become more hypertonic. The objective was
immersed into the perfusing solution every 30
minutes for thickness determinations.
A similar experimental protocol to that outlined above for the de-epithelialized corneas was
followed; that is, at least two corneas were used
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017
Reports
Volume 15
Number 7
547
560
i 15 mmHg
54
5 mmHg
50 mmHg
i
°i-^<
520-
••30///hr*)
500H
V
480-
"•>
\
460H
440H
4 20
20
40
80
60
100
120
140
160
180
TIME (min.)
Fig. 1. Deswelling response of de-epithelialized cornea to adjustment in applied hydrostatic
pressure as a function of time. A representative of six corneas is shown to illustrate stabilization of deswelling rate within 20 minutes.
Table I. Deswelling rates of de-epithelialized and de-endothelialized corneas at different
pressures when compared at different time intervals"
Time(hr)
Pressure (mm. Hg.)
De-epithelialized:
5
15
30
50
De-endo th elialized:
5
'
15
30
50
0-1
1-2
—
28.7
36.5
14.4
8.5
18.3 ± 3.5
—
+0.3
15.0
16.5
—
32.3 ± 6.8
2-3
3-4
± 3.9
± 1.2
± 7.2
± 3.1
31.3 ±
19.0 ±
20.7 ±
9.5 ±
5.7
6.9
12.1
3.5
± 5.8
± 2.9
± 5.6
50.3 ± 4.7
3.8 ±
7.5 ±
12.0 ±
24.5 ±
9.9
4.8
2.5
9.7
25.0
24.8
16.0
11.3
±
±
±
±
4-5
7.2
2.0
2.1
2.2
± 11.4
± 4.0
± 3.0
27.8 ± 7.5
2.5
9.0
4.5
1.0 ± 1.9
1.4 ±2.9
0
De-epithelialized corneas: Values (/im/hr.) are the mean ± S.E. of four determinations except for 0-1 hr. and 4-5 hr.
15 mm. Hg, which represent the mean ± S.E. of 12 determinations.
De-endothelialized corneas: Values are the mean ± S.E. of eight determinations except for 0-1 hr. and 4-5 hr. for 15
mm. Hg, which represents the mean ± S.E. of 24 determinations. The positive sign for 5 mm. Hg, 1-2 hr., indicates a
gain in thickness.
The data in the table combine both the data from the constant pressure series at 15 mm. Hg and the variable pressure
data from the experimental series.
at each pressure which was applied in a pattern
as described above.
Results. Initial experiments using six deepithelialized and 2 de-endothelialized corneas
provided a time course for stabilization of deswelling after a pressure change. A typical result
for a de-epithelialized cornea is shown in Fig. 1,
where the effect of the pressure change per se
was absorbed within 15 to 20 minutes after that
change; the final 30 minutes of each hour was
used, therefore, for deswelling rate determinations.
A similar rapid change in response to pressure
was found for de-endothelialized corneas.
De-epithelialized corneas. The initial thickness
of corneas after swelling was 549.3 ± 7.3 jum
(16 corneas) (mean ± S.E.). The deswelling rate
over the first 4 hours was found to be consistent
(see Table I for 15 mm. Hg); between 4 and 5
hours, however, the rate slows from 28.1 ± 2.8
/*m per hour4 over the previous four hours to
1.0 ± 1.9 ^m per hour. At 15 mm. Hg between
4 and 5 hours, after prior exposure to the various
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017
Investigative Ophthalmology
July 1976
548 Reports
n
i
O
i
15-
i
5
u>
20-
THINNING ,
s^
>
10
20
30
40
50
60
PRESSURE (mmHg)
Fig. 2. Deswelling rate as a function of hydrostatic
pressure applied to the posterior surface of deepithelialized corneas. X indicates the mean
deswelling rate found for corneas subjected to
constant 15 mm. Hg for 4 hours. Each point is
the mean ± S.E. of 12 measurements.
other pressures, the rate was 10.3 ± 5.6 fim per
hour, due to the difference between this value and
that obtained at 15 mm. Hg during the preceding 4 hours the final time period data were not
utilized in the analysis. The good agreement between deswelling rates at each pressure at any
time interval (Table I) allowed the data to be
pooled and the result is shown in Fig. 2. The
deswelling rate at 50 mm. Hg. is significantly
less than that found at either 5 (0.01 > p >
0.005) or 15 mm. Hg (0.05 > p > 0.025),
but not significantly different from the rate at
30 mm. Hg.
De-endolhelialized corneas. This initial thickness of corneas after swelling was 505.2 ± 4.0
/mi (28 corneas). The deswelling rate at 15 mm.
Hg over 4 hours was consistent (Table I) at
12.5 ± 2.1 /fin per hour. In other experiments,
where another protocol was followed after either 1
or 2 hours, the deswelling rate was found to be
13.3 ± 1.6 (n = 24) pm per hour. The consistency of deswelling at each pressure at any
time allowed the experimental data to be pooled.
At 15 mm. Hg, after being subjected to other
pressures during the preceding 4 hours, the thickness reduction was 1.4 ± 2.9 /mi per hour. Only
the data obtained during the first 4 hours were
used since there was such a large decrease in
thinning rate after this time. The results are
shown in Fig. 3, where the slope indicates that
as the applied pressure is increased so the deswelling rate increases. The deswelling rate at
50 mm. Hg is significantly greater than that
found at either 5, 30, or 15 mm. Hg (p > 0.05).
The value at 5 mm. Hg (Fig. 3) is significantly different (p < 0.001) from that at 30
and 50 mm. Hg and the 15 and 30 mm. Hg
values are significantly different from the 50
mm. Hg value (p < 0.01); the 15 and 30 mm.
Hg values are not significantly different from
each other. The deswelling rate and total quantity
of thinning are far in excess of any significant
contribution of the epithelial thickness, and must
reflect stromal thickness changes.
Discussion.
De-epithelialized corneas. The rate of deturgescence of de-epithelialized corneas, at 15 mm.
Hg, is similar to that seen by previous workers,1"1 thus the endothelia were functioning normally. The initial 15 mm. Hg deswelling rate
in the series of corneas subjected to alternating
pressures was the same as that determined in
the corneas at constant 15 mm. Hg for 4 hours
(see Table 1 and Fig. 2). The constant-pressure
experiments reveal that the first 4 hours could
be directly compared (Table 1, 15 mm. Hg)
and that changes caused by altering the applied
pressure were complete within 20 minutes (Fig.
1).
The data shown in Fig. 2 indicate that at
pressures greater than 15 mm. Hg there is an
influence of pressure on the deswelling rate. Between 5 and 15 mm. Hg there is no significant
difference in deswelling rate, thus a curve is
shown with a pressure-independent component
between 5 and 15 mm. Hg and a linear relationship indicated from 15 to 50 mm. Hg. Hodson'1
reported that a pressure change from 6 to 37 mm.
Hg had no efFect on the deturgescence rate, although others' have reported that at about 60
to 80 mm. Hg the fluid movement across the
endothelium from the stroma to aqueous humor
surface is no longer able to offset the fluid leak
into the cornea. The difference between these
findings is perhaps related to the higher pressures used by others" and ourselves which caused
a significant difference to be found in the thinning rate. By extrapolating the linear curve in
Fig. 2, values for the pressure where no net
flow occurs across the endothelium would be
obtained of between 65 and 75 mm. Hg. The
net fluid movement out of the stroma across the
endothelium, therefore, can occur against a considerable hydrostratic pressure gradient. Whatever the driving force, this movement implies a
considerable physiologic force exists for fluid
movement across the endothelium.
De-endolhelialized corneas. The deswelling rate
of de-endothelialized corneas is somewhat larger
than that reported in one instance^ +0.9 tun per
hour, and about the same as that reported by
Zadunaisky." Klyce* mentioned that de-endothe-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017
Reports 549
Volume 15
Number 7
35-
"^
30
"
J. M
!S . O H
§
15-
kj
io-
I
M
0-
b
+ 5-
—r—
20
—r—
30
—r—
40
—I
50
PRESSURE (mmHg)
Fig. 3. Deswelling rate as a function of hydrostatic pressure applied to the posterior surface
of de-endothelialized corneas. X indicates the mean deswelling rate found for corneas subjected to constant is 5 mm. Hg for 4 hours. Each point is the mean ± S.E. of 12 measurements.
lialized corneas deswell at about 15 nm per hour
if the epithelial bathing solution is not replenished, but in our experiments the perfusion rate
was 1 ml. per minute, giving a complete replacement of fluid each minute. Evaporation, with a
consequent increase in osmotic pressure of the
epithelial bathing solution, was not a factor,
therefore, in the present experiments and must
reflect activity of the epithelium.
Contrary to the effect of increasing pressure on
the de-epithelialized cornea, the de-endothelialized
cornea deswells more rapidly as the hydrostatic pressure on the posterior surface is increased. This finding may be a consequence of
tissue compression; since the cornea is held
tightly between the two chambers raising the
pressure on the posterior surface will effectively
squeeze more fluid out of the cornea at a faster
rate, especially since the relatively impermeable
epithelium10 is present. It appears, therefore, that
the increased water loss across the epithelium
in response to elevated hydrostatic pressures is
the result of a mechanical phenomenon rather than
a reflection of a physiologic process. In the deepithelialized cornea, however, compression of
the stroma is not a relevant explanation for the
steady-state results, since the deswelling rate
falls rather than increases. The endothelium is
relatively leaky to water10 and as such will allow
more water to pass as the hydrostatic pressure is
increased and thus more fluid will be forced into
the stroma against the effect of the endothelial
fluid movement out of the stroma and under these
conditions, the cornea will not deswell as fast.
These results emphasize that, under nonstimulated conditions, both membranes remove fluid
from the stroma at zero hydrostatic pressure. The
fluid movement, however, occurs at a rate dependent on the stromal hydration since the rate
falls markedly once a near-normal thickness is
achieved.
We thank Ms. Debbie Hancock for her secretarial assistance.
From the Departments of Ophthalmology and
Physiology, Medical College of Georgia, Augusta,
Ga. Supported in part by Public Health Service
Research Grant EY 01413 from the National Eye
Institute. Lions Eye Bank-Augusta made the
specular microscopes available. Submitted for publication Feb. 10, 1976. Reprint requests: K.
Green, Ph.D., 3 D 11, R & E Building, Department of Ophthalmology, Medical College of
Georgia, Augusta, Ga. 30902.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017
550
Key words: rabbit, cornea, epithelium, endothelium, hydrostatic pressure, deswelling rate,
specular microscope.
REFERENCES
1. Maurice, D. M.: The location of the fluid
pump in the cornea, J. Physiol. 221: 43,
1972.
2. McCarey, B. E., Edelhauser, H. F., and Van
Horn, D. L.: Functional and structural
changes in the corneal endothelium during in
vitro perfusion, INVEST. OPHTHALMOL. 12:
3.
4.
5.
6.
7.
8.
9.
10.
Investigative Ophthalmology
July 1976
Reports
410, 1973.
Fischbarg, J.: Active and passive properties
of the rabbit corneal endothelium, Exp. Eye
Res. 15: 615, 1973.
Coles, W. H.: Pilocarpine toxicity. Effects
on the rabbit corneal endothelium, Arch.
Ophthalmol. 93: 36, 1975.
Hodson, S.: The regulation of corneal hydration by a salt pump requiring the presence
of sodium and bicarbonate ions, J. Physiol.
236: 271, 1974 .
Dikstein, S., and Maurice, D. M.: The
metabolic basis to the fluid pump in the
cornea, J. Physiol. 221: 29, 1972.
Kaye, G. I., Sibley, R. C , and Hoefle, F. B.:
Recent studies on the nature and function
of the corneal endothelial barrier, Exp. Eye
Res. 15: 585, 1973.
Klyce, S. D.: Transport of Na, Cl and
water by the rabbit corneal epithelium at
resting potential, Am. J. Physiol. 228: 1446,
1975.
Zadunaisky, J. A.: The control of comeal
thickness by activation of epithelial transport.
Paper presented at 1st International Congress
of Eye Research, June 2-7, 1974, Capri, Italy.
Green, K., and Green, M. A.: Permeability
to water of rabbit corneal membranes, Am.
J. Physiol. 217: 635, 1969.
Automatic recording of corneal thickness
in vitro. STEPHEN D. KLYCE AND DAVID
M. MAURICE.
An addition to the specular microscope is described which allows it to record the thickness
of the excised cornea automatically as a function
of time. The focus of the instrument is scanned
mechanically through the tissue, and the position
of the reflecting surfaces is detected by a photoelectric system and marked on a chart recorder.
The system is able to follow thickness changes
over periods of many liours and with an accuracy greater than obtainable by manual operation. This system has been helpful in the evaluation of a new medium which considerably extends
the useful lifetime of the corneal endothelial fluid
pump.
The specular microscope1' 2 has been used extensively to measure the thickness of the excised
and perfused cornea. Generally, readings are
taken at intervals by a trained observer, which
is an acceptable procedure when the preparation
survives for only about 7 hours, as is commonly
the case. However, several workers are attempting to find which are the missing factors which
would extend its lifetime, and any degree of success results in inconveniently long working periods.
It was evident that an automated system was
called for, and this report describes an attachment to the specular microscope that has been
developed for the continuous measurement of
cornealthickness.
Materials and methods. Under the specular
microscope, readings of corneal thickness are obtained by difference on manually focusing either
up or down through the cornea and recording the
calibrations on the fine-focus dial when a slit
of light projected through the objective is focused
on its surfaces. A reflecting surface forms an
image of the slit at the eyepiece, and this image
shifts across the field when the microscope is
moved through focus. The position of the slit
on an eyepiece scale proved to be a more reproducible indicator of the position of the surface than a subjective estimate of sharpness of
its image.
Description of apparatus. In the automated
version (Fig. 1) the focus of the microscope is
scanned mechanically through the cornea while
the positions of the surface reflections are sensed
and recorded electronically. The rotary motion
of the fine-focus dial is sensed by a ten-turn
potentiometer coupled to the dial to provide a
10 mV. increment in signal for each 3.60° (equivalent to 1 /mi) of rotation. This signal is fed
through a sample-and-hold module to drive the
Y axis of a 10" chart recorder (Model SR-255B,
Heath Co.) while the chart is driven at slow
speed (0.01" per minute). Motorized vertical
scan (100 /mi per minute) is accomplished by
driving the fine-focus dial with a reversible 4
r.p.m. synchronous motor. The motor alternately
raises and lowers the microscope body through
an adjustable scan range of 50 to 950 /mi. As
the microscope is scanned through the cornea,
the focused images are transmitted by fiber optics
to a photometric system whose output triggers
the chart recorder to make a dot (by lowering
the pen) corresponding to the position of the
fine-focus dial each time a peak of a relatively
intense reflection is sensed.
As mechanical hysteresis is inherent in the
microscope, a double dotted line is traced for
each reflecting surface when both directions of
scan are recorded. To check for accuracy and
stability, the thickness of a coverglass slip was
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017