Exptl Eye Res. (1967) 6, 93-96

O b s e r v a t i o n s o n C o r n e a l P o t e n t i a l *

KEITH GREEN

Ophtludmological Research Unit, W. K. Kellogg Fou~idation Laboratory, T~e IVilm~ Institute, The Johns Hopkins Univ~z~rsity School of Medicine, Baltimore, Maryland.,

U.S.A.

(Received 2.9 April 1966, and in ret.*ised form 29 September 1966)

T h e co rnea l p o t e n t i a l h a s b e e n s t u d i e d in t h e e x c i s e d co rne~ u n d e r c o n d i t i o n s in ~-hich i t w a s d e e m e d t o b e f r ee o f s t r ess . ~No d i f f e r ences w e r e obser~-ed b e t w e e n t h e p o t e n t i a l "difference m e a s u r e d w h e n no s t r e s s w a s a p p l i e d t o t h e c o r n e a a n d w h e n t h e c o r n e a w a s ~ u b j e c t e d to streaq. I t is s u g g e s t e d t h a t t h e c o r n e a l p o t e n t i a l o r i g i n a t e s e n t i r e l y f r o m ~c t ivo t r a n s p o r t a c r o ~ t h e m e m b r a n e , p a r t i c u l a r l y s ince t h e s h o r t - c i r c u i t c u r r e n t ( a n i n d e x o f a c t i v e i o n t r a n s p o r t ) r e m a i n s u n c h a n g e d a f t e r 4 i s * o r t i o n o f t h e m e m b r a n e .

1. Introduct ion

During the last few years, studies have been under taken on the origin of the t rans- corneal potent ia l in the rabbi t . In r i v e studies of the t ranscorneal potent ial have always been l imited by the lack of kaaowledge of the ionic composi t i 'm of tim solutions ba th ing the cornea. The in ~dvo conjunct iva, for example, has a potent ia l of 10 mV between the anter ior chamber and the normal external ' a th ing film of liquid; in vi t ro , however, no potent ia l can be detected (Green, unpubLished data) . An in vi t ro method of invest igat ion is a necessity, therefore, to unders tand the basic mechanisnm under- lying the corneal potential .

Donn, Maurice and Mills (1959a,b) s tudied the potent ia l difference and resul t ing short-circuit current in the excised r abb i t cornea. The measured potent ia l was between 15 and 40 inV. Recent ly , th is t issue has been invest igated by a match simpler tech- nique t han t h a t used by D o n n e t al. (1959a), and lower potent ia ls (5-6 mV) were found (Green, 1965, 1966). This quan t i t a t ive difference can be ascribed to differences in the techniques, including the problem of s t i r r ing in the appa ra tu s used b y Don n e t a l . (1959a). I t can also be ascribed to the fac t t h a t the measurements of potent ia l difference were made 30 cm from ei ther surface of the m e m bra ne af te r passage of the bat tdng solution thxough long, nar row polyethylene tubes (Donn et aI., 1959~), whereas in recent exper iments (Green, 1965, 1966), the potent ia l difference was measured with KCl-agar bridges of polyethylene tub ing placed 1 hum from each surface of the membrane . The potent ia l between these bridges was measured through pa i red calomel electrodes and a 10X4-ohm ,_'nput impedance miUivoltmeter. Despi te these differences, all the authors agree, f rom their studies on ion t racer iluxcs, t h a t sodi~m is ac t ive ly t ranspor ted f rom the t ea r fihn to the aqueow.q humor of the cornea, a l though differences do exist in the measurements of quan t i t a t ive amounts of sodiuan passing th rough the membrane .

More recently, i t has been suggested t h a t s tress on the certain par t ic ipates in t h e occurrence of a large t ranscorneal potent ial (K ikkawa , 1966a,b). As this problem o f

* Th i s iJavestigation was s u p p o r t e d b y a P ~ S resesxoh ~ t (N'B 04854) f r o m ~he ~ a t l o r m l ~ t o o f Neurologioa l D i seases a n d BIfiadness, U.S . Publ io H e a l t h S e r v i c ~

93

94 K. GREEN

the possibi l i ty t h a t d is tor t ion of the tissue may lead to a l terat ions in the po ten t ia l has also occurred to the au tho r of this paper, some re levant observat ions are herein presented.

2. M a t e r i a l and M e t h o d s

The corneas oz adult albino rabbits were removed and bathed in a modified Krebs bicarbonate Ringer's solution as described previously (Green, 1965). Some corneas were mounted in modified Ussing chambers (Ussing and Zerahn, 1951). All experiments were performed a t 25°C.

Potentials were measured with a high-impedance millivoltmeter (pH Meter 4c; Radio- meter, Copenhagen, Denma~:k) via large-diameter polyethylene tubes (1.8 ram, inside diameter) filled with an agar-potassium chloridc mixture ( 3 ~ w/v agar in saturated potassium chloride). Corrections were made for junction potentials before and after each measurement. The short-circuit current was determined via agar bri~lges and silver-silver chloride cells through a conventional circuit (Ussing and ZeJrahn, 1951).

All the values quoted here are the mean of at least 8 de'cerminations.

3. Results

The imtent ia ls of corneas mounted in the chamber in such a way t h a t no pressure was exer ted on t h e m and protec ted from short-circuit ing between the ba th ing fluids b y silicone grease were 5-6 mV (250 observations; Green, :~965, 1966). Some corneas were moun ted in the m a n n e r described above and were lei~ for a t least 1 hr to reach equil ibri l ,m. After th ls t ime a sealed polyethylene tube (3 ram, outside diameter) was placext in the chamber in such a way t h a t i t pushed on the endothel ium, causing the cornea to be s t re tched anter ior ly; no change in poten t ia l was observed over 1 hr. Similar ly wi th o ther corneas, the polyethylene tube was placed in such a way t ha t i t pushed on the cornea f rom the epithelial surface so t h a t the radius of curvature was opposi te to t h a t normal ly seen. This d is tor t ion was main ta ined for periods of 1 hr; here again, no effect on the po ten t ia l was observed. Dur ing both membrane distort ions, no change in the short~circuit current was observed. I n addi t ion, some corneas were placed in the chamber bu t were moun ted wi th the epi thel ium under compression and the endothe l ium stretched; t h a t is, the cornea was placed between the chambers in a m a n n e r reverse to t h a t no rmal ly employed. Here again, no changes were observed in e i ther the po ten t ia l difference or the resul t ing short-circuit current . Physica l s t re tching of the cornea, therefore, did not cause a l tera t ions in ei ther ion fluxes (as measured by short~circuit current) or potent ia ls .

I t was found t h a t c lamping the cornea between chambers whose opposite curved surfa~.s were d ry and ungreased caused a va r i e t y of changes in the po ten t ia l and t h a t these changes were r e l a t ed to t he degree of damage ~ f l i c t ed on the cornea. These changes in po ten t ia l were always a decrease in value; for example, when the cornea was c lamped very t igh t ly , the Luci te blocks tended to cut in to the tissue, causing a ma rked loss of potent ia l .

Corneas hav ing radi i of curva ture equal to those of in vivo corneas were also moun ted on the concave surface of one chamber (the concave surface facing upward) so t h a t t he ep!thefial surface faced upward. No ba th ing solutions were employed, and t h e p o t e n t i a l was measured between t h e layers of adsorbed fluid on each surface of thelcornea. I n the in terva ls be tween the measure lnents of potent ia l , which were made every 15m~n , the cornea was placed in a Ringer ' s solution. Once ag,~in, values of 5 - 6 m Y were observed over periods of 1 hr. Once the possibi l i ty of sho~ ing th rough

C O R N E A L P O T E N T I A L 95

the fluids a t the edge of the scleral ring was prevented, s table potent ials were readily measurable. Under these conditions, i t is obvious tha t no stress could be exerted on the cornea; the potent ia l difference of the membrane , however, remained unaltered.

To invest igate fur ther the possible effect of tissue dis tor t ion on the potent ial , some corneas were left in Ringer ' s solution for 1 hr and then removed from the solution; the scleral r ing was l ight ly dried and greased. The cornea was then placed over the end of one of the agar bridges t ha t was held facing upward, and the potent ia l was measured between the two fluid fihns. Tjte measurement of potent ia l was accept~,d as valid i f i t was stable over a period of 1 rain; normally, the potent ia l remahled un- changed af ter the cornea was placed over the electrode. Under these conditions, the cornea hung loosely over the end of the agar bridge. No difference was found hetwcen the potent ia ls measured with the endothe l ium facing u p w a r d - - t h a t is, with the epi- thelial surface under greater s t ress- -or facing dowa t w a r d - - t h a t is, with the endo- thelial surface raider greater stress. These potent ia ls were also between 5 and 6 inV. Even when the cornea was subjected to physical s t re tching by pull ing on the scleral

"ring under ei ther of these conditions, no changes in the potent ia l were observed. No t)otential difference existed across corneas mainta ined at, 4°C, and no potent ials

were measured even under condit ions in which the cornea, was held between opposite surfaces of the Lucite chambers. This finding was true for corneas mounted in the normal manner and also for those mounted in a manner such t h a t the endothel ium was made to be concave and the epi thel ium, convex.

4. Discuss ion

Tile findings reported here suggest t h a t tile effects of stress on the t ranscorneal potent ia l are negligible, since potent ia ls of similar values were measured in both clamped and unclamped corneas. In the work presented here, as well as in prey|otto work (Green, 1965, 1966), tile corneal potent ia l was between 5 and 6 mY, even when the cornea was clamped, at 25, 30 and 35°C. Below 25°C, however, the potent ia l decreased mtti l a tempera ture of about 4°C was reached, a t which point the potent ia l disappeared. These findings, however, s tand in contradis t inct ion to the findings of Kikkawa (1966a,b), which suggested t h a t streas did par t ic ipate in the origin of large t ranscorneal potent ials .

A potent ia l difference exists between two solutions of different ionic concentrat ions when the solutions are separated by a membrane, and this has been shown to be related to ion t ranspor t (Ussh~g, 1949; Ussing and Zerahn, 1951). The large changes in po ten t ia l seen by Kikkawa (1966b) occurred within 30 rain of "c lamping" and thus must indicate t h a t large changes occur in the ion ac t iv i ty coefficients (or concentrat ion) in the ba th ing solution. Since the electrodes were in a solution and not in direct contac t with the corneal tissue, i t is possible to e l iminate any piezoelectric effect, such as is seen in bone tissue when i t is subjected to stress (Basset t and Becker, 1962). In the l a t t e r case, when a bone is ben t in such a way t h a t one side is expanded and the o ther compressed, a small po ten t ia l of 500-1,000/zV (0-5-1.0 mV) occurs, ~ i t h the compressed area being negative. The po la r i ty of thin vol tage is dependent on the direct ion of bending, the compressed area always being negat ive, and is in a s teady s ta te for only 60 sec before dissipat ion. I t is t hough t t h a t t h i . effect is brought abou t by a stress es tabl ished between the collagen and the ground substance.

I f such a stress react ion were exerted in the cornea, i t is l ikely t h a t i t would be. s imilar to t h a t seen in bone, bu t the repor ted corneal effect (K£1t~awa, 1966a.b) is

96 K. GREEN

both a more persistent and a higher voltage than that expected on the basis of the experiments on bone. The corneal effect is most probably, thereibre, related to a change in the ionic concentrations of the bathh~g fluids. It would be of interest to know what changes occur either in the flux rates of the major ions or in the concentrations of these ions in the two'bat.hing solutions lmder the experimental conditions used by Ki]~kawa.

The large transcorneal potential found by Donn et ah (1959a,b) must be ascribed, therefore, to some aspect, of the experimental technique other than the use of a clamped corne~. The presence of long, narrow polyethylene tubes filled with Ringer's solution .introduces a large electrical resistance (about 2 megohms) between the tissue and the recording electrodes. Any variation in the length of such tubes intro- duces large changes in resistance between the bulk solution next to the membrane and the point at which the potenvial is measured. Owing to these factors, the measure- ment of a potential at a distance of 30 cm from either side of the tissue 1ruder these conditions is a measurement of a modified potential. Unless allowance is made for this great electrical resistance between the membrane and the electrodes (whose conductivity is high), the potential measured may not reflect the tam potential of the membrane.

The agreement between the measured potenti:~J and the potential calculated from the sodium fluxes is extremely good (Dony~ et al., 1959b). Agreement also exists between the measured and calculated potential even when *~he potential is calculated using the sodimn and chloride fluxes found throughout the period of change in flux rates during the initial hour folloxring corneal excision (Green, 1965). This agreement is such that, in view of the lack of chsnge in potential seen in the experiments reported here after application of stress to the cornea, all the measured potential from a rabbit cornea car, be explained by the presence of an active traTmport of sodium from the tear film to the aqueous humor. Thus, despite the differences found in the magnitude of the potential (Donnet al., 1959b; Green, 1965), a difference that may be ascribed to differences in techniques, the authors are agreed upon the origin of the potential~ that is, an active transport system.

R E F E R E N C E S

Bassett, C A. L. and Becker, R. O. (1962). 8dence 137, 1063. bonn, A., Maurioe, D. M. and Mills, N. L. (1959a). Arch. OphtMl~l. (Chicago) 62, 741. bonn, A., Maurice, D. M. and MiIls, N. L. (1959b). Arch. Ophthalz~ol. (CMcago) 62, 748. Green, K. (1965). Au~. J. Physiol. 209, 1311. Green, K. (19e6). E ~ a ~ye P~. 5, 106. glklraws, Y. :(1966a). E~1~tl .Eye P~s~ 5, 2L Kikkawa, Y. (1966b). ~ ' ~ Eye lies. 5, 31. u ~ , H. H. 0949). A ~ P ~ , 8 ~ . 17, 1. Ussing, H. H. and Zerahn, K. (1951). Acta Physiol. ,.~cand. 23, 110.