Exptl Eye Res (1970) 10, 302-312

E f f e c t o f T o p i c a l A p p l i c a t i o n o f H y d r o c o r t i s o n e o n t h e C o r n e a l T h i c k n e s s

T. IIAIIA*

Departmc~~t of Physiology, In,st itute of Ophthalmoloffy, University of LoTuton, Judxl Street, London, W.C.1, England

(Received 16 31arch 1970, Boston.)

A rabbit cornea treated inten~.i~qy with topical hydrocortisone for 4 daya becomes thizx in comparison with that. df control eye. This appe.ar~ to Ire caum-d by a tx,duetion in tho imhibi- (.ion prczasure of the cornc-'~ rather than a c h a n g e in properties of tho limiting lnyer~.

1. Introduction

I n t h e course o f expe r imen t . s i n t e n d e d to e l u c i d a t e t i le m e c h a n i s m c o n t r o l l i n g t h e n o r m a l h y d r a t i o n of t h e c o r n e a i t w a s no: .ed t.hat., in t.ne r a b b i t , co rnea l thicknr~.,..s c h a n g e d s p o n t a n e o u s l y t h r o u g h o u t t h e d a y . I t was a t first con.~idered t l m t th is might . be a r e s p o n s e to s t ress , a n d in p r e l i m i n a r y t r i a l s , a s t e ro id w ~ apI , l i cd t(,picudty" to ore, eye . As a r e s u l t a t h i n n i n g o f t he c o r n e ~ of t h e eye r e l a t i ve to th(: con t ro l w~.~ not~,d.

T h e i n t r o d u c t o r y gToup of exper iment .~ r e p o r t e d in thi ~ p:~I)er (u)nf i rmed a n d dc t inc , t t.his c h a n g e in c o r n e a l thickne.ss . T h e m a i n e x p e r i m e n t a l ser ies was l~lamwM to i (h ,n t i fv t h e caUSE of t h e t h i n n i n g . It, w a s t h o u g h t that. if t he corne~t I.a~comc.~ t l f inner i t is :t r e su l t e i t h e r of:

(1) r e d u c t i o n o f t h e pass ive i m b i b i t i o n r a t e r e s u l t i n g f r o m : (a) d e c r e a s e in t h e I ) e r m e a b i l i t y o f t h e e p i t h e l i u m or e .ndo the l iu tn : (b) r e d u c t i o n in t h e i m b i b i t i o n p re s su re o f the co rnea l s t r o m a " (c) c h a n g e in t he s t r u c t u r a l force.~ w i t h i n the cornea l s t r o m a r e su l t i ng f rom :~

r ise in i n t r a o c u l a r p ressure as s u g g e s t e d by F, lde r s a m l I l i is~: (1967); or (2) m o r e r a p i d loss of fluid a.~ a cot~ 'e .quence of e i ther"

(a) i n c r e a s e in e v a p o r a t i o n : or (b) i n c r e a s e in t h e a c t i v i t y of t h e e m t o t h e l i a l p m n p .

T h e s e poss ib i l i t i e s w e r e i n v e s t i g a t e d in t u r n by cotnt) 'Lring t he s t e r o M - t r ( ~ t e d eye "Mth i ts c o n t r o l .

: P e r m e a b i l i t y c h a n g e s in t he ce l l u l a r l aye r s were inves t . iga ted w i t h fluore~scein ia t he l i v i n g a n i m a l . I n r i v e m e a s u r e m e n t s a lso w e r e ca r r i ed ou' , to see if t h e s teroi t t ~,ffeeted i n t r a o c u l a r p r e s s u r e , a n d b y c los ing t h e eycli&% to as .~ss t h e in f luence of evapora tAon on t h e d i f f e r ence in t h i c k n e s s .

T h e c o r n e a l t i s s u e w a s r e m o v e d to m e a s u r e t he swe l l i ng p r e s s u r e a n d to e x a m i n e t h e a c t i v i t y o f t h e e n d o t h e l i a l p u m p b y m e a n s of t he t e m p e r a t u r e r eve r sa l effect . S o m e p r e l i m i n a r y c h e m i c a l d e t e r m i n a t i o n s of t h e effect of s t e r o i d on t h e p r o t e i n a n d p o l y s a c c h a r i d e c o n s t i t u e n t s of t h e s t r o m a a lso we re c a r r i e d o u t .

2. Mater ia l s and Methods

Albino or p i g m e n t e d rabb i t s of bo th sexes weigh ing 2.0-2-5 kg were used.

St~r o ~ t r e a ~ The s te ro id d rops used were 0 .5% h y d r o c o r t i s o n e free-alcohol suspens ion in 0-9°/0 NaCI

solut ion. The cont ro l so lu t ion was 0 . 9 3 % NaC1 solut ion. E a c h of these solut ions was d r o p p e d

* Present address: Hara Eye Hospital, ~Nishi 1-1-11, Utgunomiya, Japan 320. 302

I [YDI{OCOfLTISONE AND C O R N E A L THIGI<:NESS 303

on one of ~he eortmas of a r a b b i t eve ry hour f rom 9 a .m. to 6 p .m. , a t o t a l of 10 t imes a. d a y for 4-5 days. I f one co rnea in i t i a l ly a p p e a r e d to be ~hicker i t was chosen to rece ive t he st~:roid drops. Be tween e~mh t rc~t tment the rabb i t s were k e p t in the i r cage a n d fed nor- real ly.

7'hic]¢nes's ~n,.'asurcment ~ f e a s u r e m e n t s were t a k e n with a p a e h o m e t e r (Maurice a n d Giardini , 1951.) a t 10 po in t s

a r o u n d the hor izonta l mer id i an of the cornea . I f s ignif icant d iscrepancies a p p e a r e d in th is g r o u p o f values furt,her r ead ings were taker~ and inc luded in the average . Analys is of several group~ of 10 read ings o b t a i n e d in the course of these e x p e r i m e n t s suggests the s t a n d a r d er ror of the m e a n va lue wa~ on tim order of 0.003 ram, a va lue c o m p a r a b l e to t h a t obt : t ined by Mishimt~ and Maur ice (1961).

I,, mos t cases the an ima l s were s eda t ed with at{ i n t r a m u s c u l a r in jec t ion of 0-5 ml p r o m a z i n c hy, t rochloride (0"05 g,/ml) before m e a s u r e m e n t s were t aken . Local anes thes i a wa.s e:nployc,! o~dy for t o n o m e t r y , for which the h a n d - h e l d u p p l a n a t i o n t o n o m e t e r (Perkir~s, 19a. 0 was u~ed.

Fl,~or,',,.:,:in. Me;tmlre.tuent.~ of l luoreseein c o n c e n t r a t i o n were m a d e wi th a f iuorophoto- me ie r {M;turi,-e. 1963~. The smal les t sensi~.ive window, set wi th in the corneal sect ion, was used f.,r d e t e r m i t , i n g t.be :~tr~:~mal concet,Aration.

E,~ttl,cJrrrt,.'o~+. l~,oth ewes were closed wi th adhes ive t ape to d e t e r m i n e if the c h a n g e in thicknc-~.~ :w~s ~l.ue to cv , .worat ion at.ross the ep i the l ium. Af t e r v a r y i n g per iods t he lids w,:-re o.rwned so t h a t the t.hickne:ss couhl be m e a s u r e d ; this m e a s u r e m e n t took lee.~ t h a n a Itzilltli(.'. t.ttl e~c l t oc~-~tsioll.

Epith,'!ini. !mrmeabilit!l. The seda ted an ima l was wrapped in a c lo th a n d t u r n e d on its side. The upper .~:ld lower lids were l if ted with the fingers to fo rm a con junc t iva l reservoir , t ak ing care trot to ha rm t he epi thel ia l surface of e i t lmr eye. This reservoi r wins filled wi th a solut ion ¢,f t2,1, fluorescein sod ium in a.qo/ sal ine, buffered s~qt.h Tris ( h y d r o x y m e t h y l ) v , . / O

• ,minom,:than,. '. (Tris) to p l [ 7-i , so t t , a t the su r face of the ep i the l ium was c o m p l e t e l y co~'ered. Aft~-r 5 rain the i luorescein solut ion was washed a w a y wi th saline, a n d 20 rain le.t,:r the t tuorescein concen t r i , t ion of the corneal s t r o m a was m e a s u r e d v, fith the fluoro- p h o t o m e t e r . Since t?atche_~ of heav i ly st.ained cornea f r e q u e n t l y occur red in spi te of the prec .u t t ioas t.aket~, read ings were r e s t r i c t ed to a reas where a u n i f o r m l y low va lue was found .

End~hcl ia l permeability. An in jec t ion of 1 ml of 10°/~, f luorescein sod ium was m a d e into the m a r g i n a l ear vein, a n d the cozment.rations of fluorescein in the cornea l s t r o m a a n d aqueous h u m o r of both eyes s u b s e q u e n t l y were m e a s u r e d a t in tervals .

I n vitro experimvnts Imbibi t ion "pressure. A r a b b i t was anes the t i z ed wi th i n t r a v e n o u s 20~/o u r e t h a n e . The

e p i t h e l i u m of one eye was s c r aped off wi th a razor blade, a n d a disk of t issue was p u n c h e d ou t f r o m the cen te r of the co rnea us ing a 6-1 m m t r e p h i n e ; t h e e n d o t h e l i n m was t h e n r u b b e d a w a y wi th sof t a b s o r b e n t paper .

Fo l lowing a r ap id d e t e r m J n a t i o n of i ts wet w e i g h t the s t r o m a l disk was p l aced in t h e i n s t r u m e n t descr ibed b y It :ara a n d Maur ice (unpub l i shed d a t a ) and t h e imb ib i t i on pressure m e a s u r e d . The de l ay b e t w e e n p u n c h i n g ou t t h e cornea a n d the s t a r t of the imbib i t ion p ressure d e t e r t n i n a t i o n was less t h a n a m inu t e . The r e a d i n g was c o m p l e t e d in 30-60 rain; t h e o t h e r co rnea t h e n was m e a s u r e d in t h e s a m e way . I n some cases t h e steroid-trea+.ed co rnea r e a d i n g was t a k e n first, in o thers t he con t ro l cornea .

A f t e r t he imbib i t ion pressl~re m e a s u r e m e n ~ the co rnea was d r i ed a t 105°C for 15 h r a n d i ts dry, w e i g h t measu red .

304 T . t [ A l~ A

Temperature reversal. A rabbi t was killed with in t ravenous urebhane and both eyes were immedia te ly enueleated and stored side by sid(; in a moi.~t, chamber at 4°C for 20--30 hr. ~kfter this, botlx eyes were incubated undor aerobic condit ions ia a moist chamber a t 37°C for 8 hr. The thickness of the corneas was me[~sured at- i n t e r v a l s ~ g e n e r a l l y of 1 hr. In some cases t h e eFithelia of both corneas were scraped off with a razor blade after storage and were incuba ted in the same manner .

Eh~ion of tissue com pone.raz. A disk (6"I mm in diameter) of the bare s t r , t , ,a was i)rc- pared f rom both the control and the s teroid- t reated corneas, as described above, and each was placed in 2 ml of 0-9% NaCl. After an immersion period of ei ther 30 rain or 48 ltr a t 4°C the protein con ten t in 0.5 m! of incubat ion fluid was mea-sured spect rophotometr ica l ly by the me thod of Lowry, Rosebrough, Far r and Randal l (1951), modified to detect 1 iLg of protein. :In abou t 0-2 ml of incubat ion medium, obtained as described above, gluc.osamine was measured speet rophotometr ica l ly by the Elson and Morgan (1933) method, modilled to de tec t 2/zg of material .

f ° g 0

0 . 4 o ~ o o

"'E 0"30 f

E m t I I I 2 3

p-

o o . oi . 8 8 0

! . I | , ! i ~ J .... t ! t _l 4 5 6 7 8 g io ~t t ~ ~3 14

Time (doFs) (a)

O" S O

0.40

I 1 I I I _ 1~,oo 13,oo ,5.oo 17,o0 19,oo

Time (hr) (b)

:Fig. 1. Spontaneotm thickness ehangeus in the u n t r e a t ~ l cornea of a rabbit . (a) Long- te rm change.q ia measuroments ab 9 a;m. da i ly ; (b) diurnal change. O , l~ight oorne~; Q , left oornea.

3 . R e s u l t s

N o r m a l thickness

A l t h o u g h severa l workers lmve c l a i m e d t h a t t h e t h i cknes s of the r a b b i t co rnea r e m a i n s c o n s t a n t over severa l hou r s (Mishima and Maur ice , 1961; Y t t e b o r g a n d :Dohlman, 1965) th is was f o u n d to be t he e x c e p t i o n i n t h e p re sen t e .xperiments. T h e m e a s u r e m e n t s gene ra l l y c h a n g e d bo th w i t h i n a per iod of hou r s a n d f r o m d a y to d a y (Fig. i ) . These m o v e m e n t s were s ign i f ican t ly g r ea t e r t h a n t h e s tandarcl e r ror in t h e m e a n v a l u e of one d e t e r m i n a t i o n : The cause of th is v a r i a t i o n cou ld n o t be a sce r t a ined , s ince i t d id n o t a p p e a r to fo l low a n y zegular p a t t e r n , or to be r e l a t e d to t h e t i m e of day ,

I - [ Y D I t O C O R ' I Y I S O N i , ] A N D C O R N E A L T H I C K N E S S 305

the removal of the rabbi t £rom the cage, or any others factors. I t was considered best always to c.omparc the t rea ted with the control eye, when the effects of topical steroid were studied.

Ej~cct of st,troid on tltick~w.ss The experimental cornea decreased in thickness below the control by an average of

0.019 mm after 4 days ' t r ea tment . The rat io of s teroid- treated to control cornea was 0"95 (Table I). These differences are significant a t tlm P.m0.05 level. This effect was shown more clearly after switching the side of drop appl icat ion (Fig. 2).

T,tia LE I

Effect of steroid on thickne~ss of cornea

Before, t r e a t m e n t . A f t e r 4 d a y s " t r e a t m e n t

C o n t r o l S t e r o h I C o n t r o l S t e r o i d C o n t r o l - s t e r o i d S t e r o i d ( r am) ( m ~ ) ( r am) ( r a m ) ( ram) C o n t r o l × 100

0-362 0-376 0.387 0 ,365 0 .022 94-3 0 .395 0-393 0.381 0-383 0 .002 100.5 0 .400 0 .407 0 .412 0 .399 0 ,013 96-8 0-416 0-419 0,412 0-399 0.013 96-8 0-415 0-,t32 0-392 0-365 0-027 93.1 0 .460 0-,165 0 .439 0-427 0 .012 97-3 0-443 0-457 0 .418 0-397 0.021 95-0 0-429 0-438 0-425 0-398 0 .027 93.6 0-423 0 -4°3 0-400 0 .380 0 .020 95.0 0.393 0-401. 0 .390 0 .380 0 ,010 97-4 0-370 0-3-tO 0.373 0-336 0-037 90-1

0-410 0-414 0-403 0"384 0 .019 95"4 - - 0 - 0 0 9 -F, 0-011 _-}=_0"006 - t -0-007 .-i- 0"003 ::h0"8

V a l u e s f r o m c o n t r o l a n d t r e a t e d eyca o f 11 s e p a r a t e r a b b i t s M e a n ~ S.E.,~L

Et~poraHon In i t ia l a t t empts to introduce the steroid by subconjunct iva l inject ion lead to a large

decrease in thickness. T h a t this was due to a disruption of the oily layer of tear film, as found by Mishima and Maurice (1961), was shown by closing the eyelid, whereupon the thickness retuxned to normal .

When the t r e a t m e n t was given by drops, t ap ing the eyelids closed caused a pro- port ional r i se in the thickness of bo th corneas, the difference between the t rea ted and control eyes being main ta ined (Table I I ; :Fig. 3).

Epithelial yermeability The permeabi l i ty of the epi thel ium to fluorescein was compared ir~ the steroid-

t rea ted and control eyes in 5 animals. No difference between the pene t ra t ion of fluorescein into the cornea on the two sides was noted (Table I I I ) .

306 T , I I A I t A

0 . 4 3

0 . 4 1

- o o

~'~ 0 . 5 9 E

,2 (1}

-5 ! . . . . . . ~_~ 0 . 4 1

0 " 3 9

0 - 3 5

. . . . |

I 2:

I •

o o

I . |

(o} !

0

0

0 0

i._ ~. I 5 4 5

Time (hr) i t )

]£xo. 2. The effect of steroid on the corne~ll thickness. (a) Four th (lay on the initial tr~ttment~ T.ho s tero id- t rea ted cornea became signif icantly th inner 3 days af ter the bovinning of the drops.

O , S tero id- t rea ted eorne~ (right eye); O , control cornea (left eye}. Af te r the fom' th d a y the t r e a t m e n t was dis~.ontinutw3. W h e n the thickness of bot.h corneas became approx ima te ly the..~ame, Om ~ido of the drops was re-

versed. (b) F o u r t h d a y after switching drops. 0 , Control cornea (right eyo); C), s terold- t reated emrnca (left eye). The s teroid- t reated cornea again became significantly thinner.

T A B L F . 1 I

Cha~ges in corneo, l thickness fol lowi~g 1-hr lid closure in co~2rol and treated eyes of 8 rabbits

Control Steroid- t reated

~e~ore Af ter A_ft~r- ~ f t e r Before Af ter A f t e r - Af te r closure clost~re before before closure closure before before

(ram) (mm) (ram) (ram) (ram) (ram)

0-397 0-414 0-017 1-04 0-382 0-393 0-011 1-03 0-,':~57 0-368 0-011 1-03 0"348 0"354 0"006 1-01 0-370 0-390 0.020 1-05 0-355 0.370 0"015 1.(~:t 0-400 0-400 0-000 1 -(~ 0-384 0-396 0-012 1.03 0-411 0-432 0'021 1-05 0"391 0.402 0-011 1.03 0-400 0-408 0"008 1-02 0.393 0-402 0-009 1-02 0-382 0.395 0-013 1-03 0-344 0.359 0"015 1-04 0-403 0-425 0-022 1-05 0"390 0"398 0-008 1-02

0-390 0-404 0-014: 1"03 0-373 0-384 0-0I 1 1-03 =1-0-007 -i-0.007 -~0-003 -4-0-01 + 0 . 0 0 7 ~ 0 . 0 0 7 2:0-001 ::i:O-O0

Mean q- s .~.~.

J t Y D ] I O C O R T I S O N E A N D C O I ~ N E A L T H I C K N E S S 307

Endotheli, d permeability The t ime course of the fluoreseein concentra t ions in the aqueous humor and the

cor~ma of the experimental and control eye was plot ted for a typical exper iment (Fig. 4). No significant difference could be detected on the two sides ei ther dttring the rising or fall ing phase of the corneal concentrat ion.

I t is diflicult to assess exact ly what difference in endothel ial permeabi l i ty could be

0 - 4 0 | e ~ • : ~ e !

0.~9 [-

A H

"<" 0-4; t----

0-39 -

0-~7

0 .35 -

0 ~ O 0 0 Q

@

I I . I

{c)

I

O

O

Q

0

Time (hr) (b)

FI~O. 3. Change~ i~ co rnea l t [f ickneas fo l lowing sfleroid d rops .

Bo~h eye~ closed

1 O

O •

• •

I I

5 6

• , S t e r o i d - t r e a t e d .~.orrma ; 0 , con t ro l co rnea . Ver t i ca l a r r o w ind ioa te~ d rop . (a) F i r a t d a y of t r e ~ t ~ n c n t ; (b ) I i f th d a y o f t r e a t m e n t (no drops) . ~VheL~ n o t e d b o t h eyes were c losed.

" ~ . t B L E I_l_]:

2'luorescvin eo~meneration in vorneal stroma after 5 rnin contact of epithelial surface with 1% solution*

S t e r o i d

Con t ro l S t e r o i d Confirol

4 .30 4~.40 1-02 0-75 0"66 0.88 0-97 1.00 1-03 2-03 1-94 0.96 2-32 2.12 0.91

Mean 0-96

I k T ~ m u m v~lues in ~/m.lo

3 0 8 "J'. .1-I A R A

de t ec t ed by tlris me thod . I t WOlfld appea r from the 4 e x p e r i m e n t s tJmC were carr ied o u t succcssf~Llly, that , a n y difference between the two sides is ~mlil:ely Ix) an~ount- to more t h a n 10%.

7 ° . g -~ o . 8 8

~. 0 . 4 ¢)

F!uoTesce~n ~nJ.

o f 0

o

tP

A,

z~ ill

Ix

~ l _ - , _~;. , __:~_

T,me (he)

~' Io . 4. C h a n g e s in f l uo re sce in c o l ~ c e n t r a t i o n in t h e eyc~ o f a r a b b i t i~f~e~r in t ravc i~ou~ i n j e c t i o n o f 1 ml 1 0 % s o l u t i o n .

A , C o r n e a l ' i 3 t r o m a o f t h e s t e r o i d - t r e a t e d e y e ; L ' , ex)rneal s t x o m a o f t h e eoa~tf<;l e y e ; O , aqtJt, ou~ h u m o r o f t h e s t e r o i d - t x e a t e d e y e ; O , aqucoum h u m o r o f t h e c o n t r o l eye .

E

Q

0 . , 5 - • o

8

0 . 4 - -

0 . 5 - -

0

o

0 - 5 -

0 - 4 - -

! 0 ~

0 0

0 0 • 0

0

I , I . _I .....

( a ) . . . . . . . . .

0

• O O

/ I . . 1 ! J 4 6 IS

T i m e (h r ) (b)

:FIG. 5. T e m p e r a t u r e revers*rl o f t h e c o l d - s w o l l e n c o r n e a s f r o m t h e 2 e y e s o f a rabbi~, a t 37°(3. (a) W i t h e p i t h e l i u m ; (b) w i t h o u $ e p i t h e l i u m , O , S t e r o i d - t r e a t e d c o r n e a ; O , c o n t r o l c o r n e a .

] tY I ) I¢OCOI~ : I ' ISONE A N D C O R N E A L T H I C K N E S S 309

Acti~fity of the e~dothelial pump In 3 successful exper iments the amoun t of swelling after refi ' igeration was ident ical

in the s teroid- t reated and control eye, and bo th eycs re turned to near ly the original thickness value dur ing the subsequent incubat ion a t 37°C.

TABr.E I V

Intraocular pressures of control and steroid-treated eyes of 4 rabbits

C o n t r o l Stera id- i . rea tex] ( m m H g ) ( m m H g )

13"3 12"3 15.3 15-8 / 7 - 7 17 '0 12-0 12.0

M e a n ] 4.6 14"3

.,-. -I00

E -80

~" - 6 0 .o .s2 E

- 4 0

- - 2 0

0

o

0 0

0

o a o o Q o

o

~ I I . I_ I _I I I ! I I

~-0 3-5

Hydration (g H20/g dry wt} :~'3o. 6. R e l a t i o n o f i m b i b i t i o n p r e s s u r e a n d h y d r a t i o n o f t-h¢~ c o r n e a l s t r o m a . E a c h p o i n t f r o m a n

i n d i v i d u a l s amp]e . O , S t e x o i d - t r e a t e d c o r n e a ; O , c o n t r o l comaea.

This was true -~'hether or no t tire epi thel ium was allowed to remain in place (Fig. 5). I t appears ~,hat tshe steroid had. no considerable effect on ei ther t he pe rmeab i l i t y of the endothel ia l layer t o the passive movemen t of aqueous humor or the ac t iv i ty of the endothel ia l pump mechanism. Again, i t is difficult to assess the quan t i t a t ive signi- ficance of these findings.

]ntra~culax pressure There was no significant difference be tween the 2 eyes in 4 an imals (Table IV).

310 T . I I A R A

lmbibit ion pressure The resul ts of the pressure m e a s u r e m e n t s were plot;ted aga ins t the h y d r a t i o n values

(Fig. 6). I t is c lear t h a t the s teroid po in t s t e n d to lie below those of the cont ro l cyt,,.

'1:~, n~,F. V

EJ~ect of steroid on th.ic]c~gs, hydration aT~t imbibition prcssv.re of cornva

S t e r o i d C o n t r o l S t c r o M . t.reat.,.~t "C-~at ~'~)i × 100

D a y s H y d r a t i o n l m b i b i - l I y d r a t i o n [ m b i b i - o f ( m g I L . O / t i o a ( r ag [ i a O / tiot~ I m b i b t -

t r e a t - T h i c k n e s s m g dr ) - pre~,.quro T h i e k n c m s m g d r y pret~aurc H y d r a t i o n tio:~ m e n t ( r a m ) c o r n e a ) ( m m l t g ) ( turn) c o r n e a ) ( m m | t g } p r e ~ n r o

4 0 . 3 6 2 3-1 - - 7 2 0 . 3 5 2 3 .0 - - 61 96-~ 81 .7 4 0 . 3 9 0 3-4 - - 56 0 -380 3.1 - - 5 2 91 .2 92 .9 4 0 . 3 9 0 3-2 ~ 67 0.:172 3-1 - - 62 96 .9 92 .5 4 0 - 3 7 0 3-2 - - 9.1 0 . 3 5 0 3 .0 ..- 77 9 3 . 8 81 ..q .5 0 -400 3 .4 ~ 60 0 . 3 8 0 .1.2 - - 62 .(14. I l ( |3-3 5 0 . 3 7 3 3- l ~ 77 0.3"~6 3 .0 - - 61 90. ~ 79 .2

0-381 3 .2 - - 7 1 0 , : 1 6 2 3 . ! - - 6 3 :., t ".~ 89- l + 0 . 0 0 5 ! 0 - I 4 - 6 : ~ 0 - 0 0 7 _~:0.0 _.~:3 -.~ 0-9 ¢ : 3 . t ;

5 l e a n - - S.F..-~t.

TAm.~ : V I

Protein clotted f rom cx~rnval stroma in salin, al 4°C

C o n t r o l S t e r o i d - Ste-,roid

trcatc~.t C o n t r o l

(a) 3 0 - r a i n i m m e r a i o n

14.7 27-7 1-88 18-4 10"0 0-54 11-9 11-7 0"98 19"5 16-2 0 -83

M e a n 16" 1 16-4 1-06

(b) 4 8 - h r i m m e r s i o n

77"8 60 -0 1 ' 0 2 63"4 61 -6 0 -97 76-9 63-7 0 ' 8 3 59"6 6 4 . 6 1 .08

M e a n 69-4 6 2 . 5 0"98

V a l u e s a r e / z g p r o b e i n / m g d r y c o r n e a f o r s a m p l e o f t i s s u e 6.1 m m in d i a m e t e r .

HYDI¢OCOI~TISONE AND CORNEAL THICKNESS 311

When the ratios of the steroid to control eye for individual animals were considered (Table V) it was found thab both the hydra t ion ~md the imbibi t ion pressuxe of the t reated eyes are significantly lower at the 5% level. The lower hydra t ion was to be ant ic ipated from the lower value of the corneal thickness, assuming no change in dry weight after (;reatment had occurred. '/'he fact t h a t the imbibi t ion pressure is lower, in spite of th.e lesser hydra t ion in the t reated eyes, emphasizes the significance of the result.~.

Eb.m'd material

After a 30-rain immersion there was no appreciable difference in the quan t i ty of protein washed out from steroid and control corneal stroma. When the period was prolonged t(, ,t8 hr nearly ,l t imes more protein was detected, but the rat io of steroid t o (;ontrol remained insignificantly d i fferent from un i ty (Table VI).

Under the conditions of the extract ion there was no detectable amoun t (<2 / zg in 0-2 m l of t.he i l)cu bat ion reed i u m) of glucosam i ne discharged from the s troma into the salin:; solu', ion.

4. D i scus s i on

Tlmre have been several reports concerning the eflhct of topical steroid on the corneM t,hickness. Sunga and I~oneg>ger (1963) and t tone~ger (1965) have reported that. following endothelial damage, a. steroid-treated cornea swelled significantly less t hal~ an untreated cornea. Miller, Peezon and Whi twor th (1965) and J3aum and Levene (19(;8) have reported an increase in thickness accompanied by an. increased in t ra- oc~tlar pressure following 4--8 weeks' t rea tment . An increase in corneal thickness folh)wing intraocular h3~pertension has been reported by Ytteborg and Dohlman (1965). ' the experiment.s described here show t h a t a s l ight but significant fail in the thickness of the cornea occurred after short- term t r ea tmen t with hydrocort isone. The effect was delayed for several days after the s ta r t of the t r ea tment and was not; re lated to a ri~c in ocular tension. The results suggest t h a t a lo~('er imbibi t iou pressure is responsible for the decrease in corneal thickness, and uLtelnpts to demonst~rate either a change in the permeabil i ty of cell layers or in the ac t iv i ty of the pump mechanism were, not successful. The imbiMtion pressure of the stroma, closely related to i ts swelling pressure, is considered to be developed by the mucopolysaccharide present in the grmmd slabstance between the collagen fibrils (Maurice, 1969). I t is generally accepted, furthermore, t ha t steroids exert an influence on mueopolysaccharide as well as protein metabolism (Schwartz, 1966). Al though no serious a t t emp t was made to establish a direct re lat ionship between steroid t r ea tmen t and the properties of the corneal polysaceharide it is evident t ha t a ra t ional basis for the thi tming tha t is found m a y be postulated.

A C K N O W L E D G ~ I E N T S

I wish to thank Dr D. M. MauTice for supervising this study and :Dr D. F. Cole for his useful suggestions.

REFERENCES

~Baum, J. L. and Leveno, Y¢. Z. (1968). Arch. O2~htlutlmol. (Chicago) 79, 366. Ehlers, N. and l~iisse, D. (1967). Acta Ophthal~tol. 45, 809. Elson. L. A. and Morgan, W. T. J'. (1933)..Biochem. J. 27, 18o_,4.

312 T. HARA

Honegger, H. (1965). Arch. OplJthaln~ol. 168, 594. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem. 193, 265. Mauriee, D. ~[. (1963). Exptl Eye Res. 2, 33. Maurice, D. l~I. (1969). In T}~e Eye, vol. 1, 2nd ed. (Ed. by Davsoa, H.). Academic Press, London. Maurice, D. l~I. and Giardini, A. A. (1951). Brit. J. Ophthalmol. 3S, 169. BIiller, D., Peczon, J. D. and ~rhitworth, C. G. (1965). Am. J. Ophthalmol. $9, 31. Mishima, S. and Maurice, D. 5~. (1961). Exptl Eye Ires. 19 6. Perkins, E. S. (1965). Brit. J. OphthalmoL 9~" 591. Schwartz, B. (1966). Interuatio~al Ophthalmology Cliuicz, Uorlicosteroid and the Eye. Little Brown

& Co., Boston. Sunga, H. R. aI1d Honcgger, If. (1963). Bet. Deut. Ophthal~vl. Ges. Heidelberg 56, 444. Ytteborg, J. and Dohlman, C. (1965~, Arch. OTI~halinol. (Chicago) 74, 375.