Life Sciences

Yol. 5, pp. 2309-2314, 1966 . Pergagon Press Ltd.Printed in Great Britain.

ACTIVE CONTROL OF CORNEAL THICKNESS

Keith Green

Ophthalmological Research Unit, W.K . Kellogg FoundationLaboratories, The Wilmer Institute, Johns Hopkans University

School of Medicine, Baltimore, Maryland

(Received 5 August 1966 ; in final forte 5 October 1966)

There has been much speculation as to the mechanisms by which

the rabbit cornea maintains a constant thickness and hydration . A

large amount of evidence now indicates that the control is an

active process : conditions that inhibit cellular metabolism, such

as cooling below 10°C and various drugs, produce a reversible

corneal swelling . Many findings also support the concept that

corneal thickness Is controlled by an active ion transport system

(1) . Assuming the correctness of this view, the process would seem

to require an active movement of solute (and/or solvent) out of the

stroma into the bathing fluids . Recently, however, it has been

shown that there is an active inward transport of sodium and chlor-

ide across the epithelium from the tear film into the stroma (2,3) .

In view of these recent developments in corneal physiology, it has

been important to determine whether or not this active transport

system is the controlling factor in the maintenance of corneal

thickness .

Adult albino rabbits (2-3 kg) were killed with an intravenous

overdose of sodium pentobarbital (Nembutal) . The corneas were re-

moved and each was mounted on an open ended Lucite chamber ; a Lucite

plate, with a hole exactly the size of the chamber opening, was

placed over the open end of the chamber to hold the cornea firmly

in place, and was held tightly by four nylon screws . Both sides of

2309

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CORNEAL THICSNE88 CONTROL

Yol. 5, No . 24

the cornea were bathed initially in Krebs-bicarbonate Ringer's

solution (pH 7 .4) at 25 ° C . A 15cm head of Ringer was placed on the

aqueous humor side of the cornea to maintain a constant shape for

measuring thickness, which was performed using the instrument of

Maurice and Giardini (4) . Normal corneas were mounted as described

and one of two procedures followed . The first was that the thick-

ness was measured in normal corneas under three conditions : after

mounting, after 30 min in Ringer, and after 1 h of applied potential ;

all measurements were made at 25 ° C . An external voltage was applied

to the cornea in the same direction as the normal potential differ-

ence (P,D,) (endothelium positive with respect to the epithelium) .

The potential was supplied from a pHM4c Radiometer and connected

to the bathing solutions via calomel electrodes and agar/KC1 bridges .

The other procedure consisted of measuring corneal thickness under

these four conditions : after mounting, after 30 min in Ringer at

room temperature, after 2 h at 6°C and after 1 h of applied poten-

tial .

In this case the external voltage was applied in the opposite

direction to the normal corneal P,D, All the experiments were

performed to paired corneas, one of the pair in each case serving

as the control and the other receiving the applied voltage .

Zerahn (5) and Andersen (6) have shown that active ion trans-

port in biological membranes can readily be controlled by applying

an external voltage in one direction or the other across the

membrane . The effect is on the transport system alone and does not

produce large changes in rates of diffusion across the tissue .

Every pair of corneas were found to be of the same thickness

(± 0 .02 rtm) for the first two measurements . Application of a volt-

age such that the endothelial surface was made more positive caused

an increase in thickness of normal corneas (Fig . 1) . The control

Vol . 5, Ro . 24

COR'AEAL THICl~FSB CO1fTE0L

2311

O.SSI

É0.50'E

O.~.S'F-JWZ

Ou0.40

EMF

0 . 350 30 0 90

TIME ~1~ IIIÎ~

FIG . 1 .

Response of corneal thickness to an external voltage

applied opposite to the normal corneal P,D, External

voltage applied to experimental corneas atT EMF . C,

control corneas ; E, experimental corneas with applied

voltages in parenthesis . Values are the mean ± S,E .

of at least 8 corneas .

corneas usually increased by 0 .02 mm in the 90 min experimental

period (from 0 .38 ± 0.005 mm to 0.39 ± 0.005 mm : mean ± Standard

2312

coRAEAL Tszc>~>Jss cox~oL

vol . 5, xo. 24

FIG . 2,

Response of corneas swollen in the cold to an external

voltage applied in the same direction as the corneal P,D,

External voltage applied at ~ EMF,

C,

control corneas ;

E,

experimental corneas . Values are the mean ± S .E, of 8 corneas .

F,h-ror of 100 control corneas) . Application of a voltage in the

same direction as the corneal P,D� however, caused a greater

increase in corneal thickness . The increase in thickness was found

to be dependent upon the applied voltage, within the limits of

50 mV and 450 mV ; below 50 mV no change in thickness occurred as

compared to the control, and above 450 mV no further increases in

thickness were found (Fig . l) . The values seen after one hour of

applied voltage are steady state values, for maintenance of the

particular thickness continues for the duration of the period of

Qol . 5, No . 24

CORNEAL THICSNESS CONTROL

2313

voltage application .

Application of a voltage such that the positive electrode is

placed on the epithelial surface caused a decrease in thickness of

the swollen cornea (Fig . 2), although the application of such a

voltage caused no change in thickness of normal corneas . Cooling

of the cornea for 2 h caused an increase in thickness from 0,39 ±

0.005 mm to 0 .46 ± 0.01 mm (mean ± S,E, of 8 corneas) ; upon removal

from the refrigerator the Ringer was not t~eplaced with warm Ringer

but was allowed to regain heat from the room ; the temperature was

found to rise from 6°C to 18°C in 1 h . The control cornea continued

to swell after removal from the cold : 0 .46 + 0 .01 mm to 0.50 +

0.01 mm in .l h . However, following 1 h of an applied voltage of

500 mV (the minimum required to cause thinning was found to be

350 mV) the experimental cornea had thinned : 0 .46 + 0.01 mm to

0.43 ± 0 .01 mm (mean ± S.E, of 8 corneas.) .

On the basis that the applied poten~tal either accelerates or

nullifies the active transport system in the cornea, depending

upon the direction of the applied potential, the experiments show

that active ion transport may well be the factor by which corneal

thickness is maintained constant . This interpretation, however,

does not preclude the effects of the voltage on passive ion move-

ments under the influence of the applied current .

In the case of

the normal cornea, the applied voltage reduces the transport of ions

into the stroma . In the swollen cornea, where the transport

system is inhibited by cold, the applied voltage stimulates the

normal transport system or creates a greater driving force for the

ion transport, thereby increasing the ton concentration in the

stroma . The effects that are seen following application of the

applied voltage to the corneas in various states indicate that by

2~1~

COldiEAL TSIC)QPE88 CO1fTROL

Yol . 5, No. 24

controlling the active transport system one can control corneal

thickness . It is highly probable, therefore, that the active ion

transport system in the epithelium serves to maintain a constant

corneal hydration and thickness - a concept previously proposed

by Donn, Maurice and Mills (7) . The way In which corneal thickness

is controlled is at present unknown, but it may well be connected

to the binding of cations onto the acid mucopolysaccharide in the

stroma ; the transport pump may serve to control this process by

making ions available in greater or .lesser quantities and thereby

controlling the reversible imbibition of water .

Acknowledgements

This work was aided by U,S . Public Health Research Grant

N,B, 04854 from the National Institute of Neurological Diseases

and Blindness . The author wishes to thank Dr . T . Otorl for

performing the corneal thickness determinations .

References

1 .

Maurice,

D,M�

in The Eve , vol .

1,

(H .

Davson,

ed.) Academic

Press, London . (1962) .

2 . Green, K ., Am . J . Physiol . 20~, 1311 (1965) .

3 . Green, K., Exatl . Eve Res . ~, 110 (1966) .

4 . Maurice, D,M, and Glardini, A,A,, Brit . J . Ophthalmol . ~,,

169 (1958) .

5 . Zerahn, K ., Oxygen consumption and active sodium transport .

Universitetsforlaget . Aarhus . (1958) .

6 .

Andersen, B .,

reported by Ussing, H,H� J . Gen .

Physiol . 4~,,

135 (1960) .

7 .

Donn, A., Maurice,

D,M, and Mills, N,L� Arch . Ophthalmol . 6~2 ,

748 (1959) .