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The lag phase of wound repair
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Tim Lag Phase
o1" W o u l l d R e p a i r
A Compnrison of l-eukoeytic anti Corneal Connective Tissue Enzyme Activities * YrIf~C;INIA T,.
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,lol~z E. H"ee[,:s lllemorial, f.aboralor?], Dep(~.rtme.,~l q[ O]du}~(dmo]o{].~l, [.l.nivor.viQ) ~ Orego;~ Jlledie, l. ,Nchool, ['ortla ~(l, Ore.qo,~, U,,S'..4. ( [¢eeeieed ~ /bpr[I .106'U, and ~'~ rccised for*;a 20 .lgebr,uar?/1,76"2 ) ] g n z y m o aetivil, ies d o v e h ) p i n g ill i n v a d i r ~ g i n f h u u n l ~ t t o r y cells ~ n d lit eonneeLive, t,isslm eell.~ (sl, r o i n a l ceils a n d fil)l'obiltsls) dlll'illl~ t h e " l a g p l i t , s o " t)f A%'Ollllt[ l'tqmil" Jl'l'¢(, linen .~budit'.d. .'~-~-N'ti('lt.obTcl*lsc~ {I,11(£ .% I~)rn'ln,|l'n-ri.slst~tiii,, (sy~lltifJe- iliitl 1)h(.>i~.ylh3,~.ll'll~'.ii'le.soil.~,lt,ivo "<~xidasel i k e " l'elt(:[;.iOll we, r e / n v e s b / g a t e d liislfi(:liolni(:~tlly [1l l'gtl, i;.Ol'llell<] WOllil(]8 o|" (i, | 2 , ] , l , 4 8 j 06 i~ll(t ](J8 h r d u r a t i o n . '_l'he cl~t'lie~6 iit;'aeli/i{( ~'hiLo b l o o d cells ( p o | y n ) o r l i h o l l u c : l e l t [ " ]o.ul¢.oeyLcs 0,11d l n o i t o lttl(.:leitr l¢:Uk(ioy~,(s.q) ,,,,'(.,t'O n(,gSO.t,Jvo f o r i.lOI;]l D-lilic:leobi(l,:lst: illtc.[ b|lO fOl'lll&iili-resi~llatlt; '"oxb:hi.se-liRe"' t%~ael,i¢~n. A n / n t e l i s c /'ol'nlalin-resist~m~ "oxidg~.uc-lik(;" z'ci~ct.ioli wets f o l l l l d ill IJ¢)Lill l)oiy#norpiif)llll(sJ,{~.of ,q,;l(t Illonollll(:lt'al" lellkl)(<',yf.l,s ill -°{-ill" wollzi(ls. ".l_']lO liOl'll'llll]]' liogailJve corlie~l) .sl,roll):l] ct.'lls. ;l.s wt.q] n.s bllta t:lJJl,lle]illlll alll] o l l d o L l l o l i l l l l l , ~]so beelllllt' i n t o n s ( d 3, r i c h ill YoJ'mMJl)-rl:sisl.alll, " o x i d m s e - l J k e " ricqivit.y w i t h i n 2-1 h r a f t e r illjtll'y. "J.'hi,~ ,'lc.Livit,x., l)eggl, n t o (]ei'rease in ell coil L.vj)es lit; 9(i h r a, ii([ oli13" ii, fi.lhlt, o r n o g l t t i v o retl, t;b.t~ill w a s l ' o u n d at, 7 di~y.~. El?he o r d e r o f appen, l'anee o f 5-1nlcleot.idi~se. lit b|lt', v&l'iolls cell t,y/ies w&s: c o n n e c t ; l e t ; t;issut: cells ( G - I 2 hi'J; j)o|vn~orpllOl'l.uele~,l.r I o u k o o y t e s (24 ]lr), l l l o n o n u ( - l e a r lmlkoe3-te~; ( 2 4 - 4 8 hi'j, ~tnd epil.helial c(;llu (O(i hr). T i m aet;ivibN in , l l o f t.hose cell l,ylm8 (except; l;he epit, helhiill) f'tl'S~, a p p e , ~ r o d in t h e cells Ioe0,1ed itL o f I l e a r Lh('. W¢)llll(] e d g e allt¢| g r a d u a l l y Sl)l'ead t . h r ( m g h inosl, ¢1t"t h e covne~tl sli'mliit h N 4S hi'. l~R:t.weeli 4 a l l d "7 dri.v.~ t h e O-nll~:h,ol.il'hlso ~tol.ivJty itl all ~:(:11 t y t ) e s })e(..lil/le. r(,sLrit'lt,d t.n I.|l(." w o u l l ( i e d g e . rl'h('. II'lOl'O IILlll!i.'/'OtlS [he illtl~l.llllllll.bor.v cells w e r e . t i m m o r e illiCit,s(; be(sCtlll(J [he-~.(~llZy/ll() rea(;t.ions i n bol,h loui~.ot, y t e s ~l,n d iit ('orllt,.tl s t r o n l ; t l c,~lls. .lb a p p e a i ' e d th,q,t; t;hei-e wits soille ac:t,ivil,y (,i. i.(.,:t.(:{lOll oecill'l'liig ,'it, t h e %'t'
1. Introduction T h e ]),'oeess of w o u n d r e p a i r , when s t u d i e d at the. cellular level, reveals n r e m a r k a b l e ,series o[" c.,}mi)ges in t h e (:nzsqlmlogy, pl]ysiology a n d morpholog3" of c o m m c t i v e l,issue cells in the w o u n d avc-a witl,in a few iJ~"a f ( e r i n j u r y ( W e i m a r , 1957, 1958, 1959, 1960, 1962" W e i m a r a n d ]faraguehi> l.qG5a,b, 1966; W e i n m r , Davis ~md ] l a r a g u c h i , 1965). 1-1; Ires been t h e p u r p o s e of our series of s t u d i e s o n wmlud healing to (let.ermine th,: nn l;m'e of Llm morl>hologJcal, }~ioe}lemieal a n d pllysio]ogical eyelets oeem'ring d u r i n g t.ll(. so-called big or la(ette p/,ase and to r'ehtto. Li,.'m wlJ(.'rdver ]Jossible into cause-aml-effeet re.laLionshi]~s ( W e i m a r , 1060). ])u.nphy a n d Udulm (19.55), on the I):tsis of t h e i r fis)clblg ol7 n rapid 1)reduction of g r o u n d s u b s t a n c e w i t h i n I2-2,1 hr a f t e r i u j u r y , h n v e a p t l y romuned t.he }a.'g l~)ease ns the . ' n b s t r a t e phase of w o u n d repair. * ~i'his i u v e s t i g a g i o n w a s . s . p p o r t e , I b y a P I f 8 r e s e a r c h grant, (.'/788) li'o,n the .N*ati,mal [n.~titute o f .N'euvolrJgi~:~d Disc'~ts~'s ~lcL,'l ]llilxdiiess, U.,% l'ubli,: ll~,.all.h S e r v i c e . A fi,)rl, ion o f t,hi~ p:qn!r was reixd /,efiJro tl,e ~Vo.~tet't! l~Cgi*,Cl~tl (':(,¢ll'ot'('tZc'(" ~t! l)eve|~l)ment.al llicth~gy art, [ : r i d a y ] l ~ t r b . r . a l M'~y I!R;3. 283
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Full t h i c k n e s s c e n t r a l c o r n e a l w o u n d s were m a d e w i t h a S w a n n e e d l e knife in adult, :llbino ( S p r a g u c - l ) : l w l o y ) rals (apl)roximat.ely 2 0 0 - 3 0 0 g ) u n d e r b r i e f ( [ - 2 m i n ) e t h e r ~lnesthesia. T h e anim~lls wore dec:~pit, t,cd a t 0, 6, 1 2, 24, ,18, 91; ~l.ll(l 168 hr. T h e eyes were i m m e . d i a t e l y e n u c : l e a t e d , fixed a n d s t o r e d in liquid n i t r o g e n u n t i l used. A m i n i m u m ot" 8 e y e s were. sLudied for e a c h t;ime p e r i o d . T h e w h i t e blood cells were d i f f e r e n t i a t e d a n d t~he i n t e n s i t y of t h e h i s t o e h e m i c a l r e a c t i o n w a s e v a l u a t e d in e o u n t e r s t a i n e d s e c t i o n s u n d e r oil i m m e r s i o n (>~1000) in an area i m m e d i a t e l y a d j a c e n t to t h e w o u n d ( 0 - 1 5 0 t , ) a n d in a n .nro.a about, :V)0-500/t a w a y f r o m t,he w o u n d . 5 - N u c l e o t i d a s c w a s c a r r i e d ont. a f t e r , t h e m e t . h o d of W a c h s t e i n a n d Meise], (196l), N o v i k o f l ' . . . D r u c k e r : S h i n a m l 0,Jldfischer (1.961), m o d i f i e d by W e i m a r a n d H a r a g u e h i (1965b). T h e in~:ubation m i x t u r e c o n s i s t e d of :30 m | of a 7 8 . 7 . m g / 1 0 0 ml s o l u t i o n of a d e n o s i n e - 5 - p h o s p h a t e ( S i g m a Cllemieal Co., ,¢%. L o u i s , Missouri); 30 ml of 0-2 .~t s t a n d a r d T r i s b u f f e r ( p I [ 7-2); 7-5 ml of 0.1 .~1 MnCI~ (A, h m e d a n d iteis, 1958; B o y e r , l m r d y a n d Myrbiick-, 1961): 3-0 ml of g l a s s - d i s t i l l e d H 2 0 ; a n d ,t-5 ml of 2~/o P b ( N 0 3 ) e. T h e i n c u b a t i o n m i x t u r e was freshly n , a d e for e a c h e x p e r i m e n t a m t filteredit.hrongh ~,Vhatman no. 40 filt,er p a p e r before it was used. T h e c o n t r o l c o n s i s t e d of t h e abo'vc, i i l c u b a t i o n m i x t u r e m i n u s a d e n o s i n e 5- p 1"~o s p h a t e. F r o z e n sect.ions w e r e c u t :~t 30 i~ in "l c r ) ' o s t a ~ ( l [ a r r i s ] t e f r i g e r a t i o n Co., Caml)ri(lg~, Massachusett.~,) a n d . p l a c e d di reetlv in t h e s u b s t r a t e s o l u t i o n . T h e s e c t i o n s w e r e s u n k wit.h a ~la..ss n e e d l e a n d i n e u b a t . e d for 5 hr at. 37-5°0. b~ollowing incubat, ion t h e .~ccLions w e r e p l a c e d in 3 7 % f o r m , l i n for 30 rain, p,sse,! t h r , m g h ' t w o c h a n g e s of glass-all.stilled w a t e r , a n d t h e n i n t o ~}~ (v/v) ~ m l m o n i u m sulfide for 2 rain. T i m react.ion p r o d u c t f o r m e d was b r o w n . A f t e r r i n s i n g in glass-,listille53 w a t e r , t,he st:(:liol,~s w e r e c o u n t e r s t . a i n e d in u n e x t r a c t e d m e t h y l g r e e n (0-2°/0) in 0-2 .~t a c e t a t e bufl'~2r (pl[. -1-0) for 25 rain. ( N o r m a l c o r n e a s t o o k t h e met.hyl g r e e n e o u n t e r s t , a i n m u c h m o r e i n t e n s e l y t h a n t h e w o u n d e d c o r n e a s d i d a n d t h e t i m e for e o u n t e r s t . a i n i n g h a d ~o be s h o r t e n e d to 1 0 - 1 5 rain.) F o r p u r e p r e p a r a t , ions of m e t h y l g r e e n . rel)eale,1 e x t r a c ' t i o n s of t h e d y e ~lre m a d e w i t h c h l o r o f o r m . T h e u n e x t , r a c t e d d y e , h o w e v e r . i'n t,ho preparal, ions d e s c r i b e d in t.his l)al)er p r o v i d e d e x c e l l e n t n u c l e a r d e t a i l . Tim see/.imls were n e x t d e s t . a i n e d in '2 rinses of 10 see ,-ach in a b s o l u t e e t h y l a l c o h o l , c l e a r e d in t o l u e n e fc~r l 0 r a i n a n d in e e d a r w o o , l oil for 10 rain. a n d m o u n t e d in P e r m o u n t . I~or t h e formalin-r~:sistant, oxldase-I~kt re.:~ction t h e e y t o c h r o m e oxid'~se m e t h o d of P;urst.one (1959) a n d \ V e i m a r a n d l l a r a g u c h i (1965a, 1966) w a s u s e d o n f o r m a l i n - f i x e d sect ions (1 hr, 10~/o neui, ral f o r m a l i n ) u s i n g x - p h e n y l - v - p h e n y l e n e d i a m i n e w i t h ] - h y d r o x y 2-napht.hoic. a c i d as c o u p l e r . F r o z e t t s e c t i o n s were cut. a g 20 ~x in t h e c r y o s t a t . T h r e e t y p e s ~f s~,ctions were u s e d : . ( l ) f r e s h - f r o z e n ; (2) f r e s h - f r o z e n , s e c t i o n e d , t h e n fixed 1½ hr a t rnc,m te~nl~er:,t.ure in 10°'; n e u t r a l f o r m a l i n ; a n d (3) c o r n e a s fixed fl,r 2,1 h r in ice-cold P;alc~.r's c:deiu~n-fln'm~,l. T i m s e c t i o n s w,'re i n e u l m t e , l for 1-.1 h r "~t 87"5°0. All s~:el.i,ms,
T H E LAC; P I I A S E Oi" WOUNI) R I,;I'AII¢
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including those which had been fixed in formalin before inculmtion, were routinely t.ransfcrred to 1% cobaltous aeetate-10°/o formalin f,,r I h r after incubation. They were next rinsed t,hrough several changes of glass-distilled water and countcrstaitled in 5layer's e a r m a l u m o,olut.ion (earminie acid in aqueous potassium a l u m i n u m sulfate) (Gurr, 1956) for ] 0 rain. The earmalum solution used was less than 1 week ohl because solut, ions stored for longer periods of time caused considerable fi,ling of the rc(l(lish-brown-formalinresistanC "oxi(lasc-like" reaction product. Following counl,crstaining, the sections were rinsed in glass-distilled water and m o u n t e d in either glychrogel (I,illie, 195,1) or glycerine jelly. The corneas were .~cctioncd coronally with t.he woun,I celttered as much as possible. In such sections, the cells appear flat, spread out, and are considerably easier to identify t-han in sngitally cut sections (Weimar and l laraguchi, 19651)). A m i n i m u m of 8 eyes were studied for each time period evaluated. Frozen se(-tions were cut in a cr)'ostat. Complete details of the m e t h o d s have, been published (Weimar a n d Ha:'-iguchi, 19651), 1966). P h o t o m i c r o g r a p h s were taken with t,he Leit.z O r t h o m a t av.tomatie cameca using K o d a chrome II daylighl, film. 3. R e s u l t s and D i s c u s s i o n
An e x t e n s i v e e v a l u a t i o n of 5-nncleotida,".,-, formalin-resistant, " o x i d a s c - l i k e " a cf,i v i t y a n d succinic d e h y d r o g e n a s c activit.ies in t h e corneal s t r o n m l cells a n d fibroblast,s in h e a l i n g c o r n e a l w o u n d s h as been prese n ted els,-wltere (\Vei~na r a n, 1 l-I arag, tchi, 1965b, 1966). A s u r p r i s i n g o b s e r v a t i o n in connect.ion w i t h t h o s e s t u d i e s was that. llm in~'ading w h i t e blood cells of all t y p e s a p p e a r e d to un(lergo t h e same. v l t e r a t i o n s ira e n z y m e a c t i v i t i e s as did t h e c o r n e a l epit, hclial, s t r o m a l , a n d c,~dot.helial cells followi~lg wounding. To fi2rther e v a h m te this e x t r e m e l y i m p o r t a n t poi,t. 5-nlwlcot i(lase a n d t h e f o r m a l i n r e s i s t a n t " o x i d a s e - l i k e ' reaction were s t u d i e d in detail since b o t h were t o t a l l y negat i v e in l h e e a r l y i n v a d i n g blood cells. Succinic d e h y d r o g e n a s e , on t h e o t h e r h a n d , a.lreadv p o s i t i v e to v a r i o u s d e g r e e s in t h e d i f f e r e n t i n v a d i n g blood cells, was nob c o n s i d e r e d ira t h e s e studies. D i f f e r e n t i a t i o n of t h e i n v a d i n g m o n o n u c l e a r w h i t e blood cells and t h e c o r n e a l fibrocytes from each ot.hcr was possible because of p r o f o u n d tliffercnees in n u c l e a r s t a i n i n g . T h e nuclei of i n v a d i n g blood cells of all ty,q)es s t a i n e d m u c h more i " ensely t h a n did t h e nuclei of t h e s t r o m a l fibrocytes. It) areas a w a y from tim w o u n d t h e p r e s e n c e o f i n t e n s e l y s t a i n i n g , e n z y m c - r i c h m o n o n t m l e a r w h i t e blood cells aJ~¢l n m c r o p h a g e s f r e q u e n t l y t o t a l l y o v e r s h a d o w e d a n d o b s c u r e d t h e s t r o m a l cells w i t h t h e i r often v e r y w e a k e n z y m e a c t i v i t i e s (Plate l). I n this p r e s e n t a t i o n no atXempt will be m a d e to (listingqtish bet.ween m o n o e y t e s and l y m p h o c y t e s since, as t h e y mig-rate into t h e tissues, t h e y b e c o m e g r a d u a l l y a l t e r e d in a p p e a r a n c e , r e n d e r i n g t h e i r d i f f e r e n t i a t i o n difficult, if not i m p o s s i b l e ( G i l m a n a n d Wright,.. 1966).
5-Nucleotidasc Normal. T h e e p i t h e l i a l , e n d o t h e l i a l a n d s t r o m a l cells of t h e n o r m a l rat cornea did n o t s h o w 5 - n u c l e o t i d a s e a c t i v i t y . An occasiori~l l)olynlorl~honuclear leukocyte, was s o n l e t i m c s f o u n d ira t h e n o r m a l connective, tts.~ue s t r o m a , but, no 5 - n u c l e o t i d a s e a c t i v i t y was e v e r d e t e c t e d in such l e u k o c y t e s . 6- and 12-hr wounds. At 6 attd 12 hr a f t e r i n j u r y i n c r e a s i n g m r m b e r s of ],olymorlJhon u c l e a r cells were p r e s e n t t h r o u g h o u t t h e c o n n e c t i v e tissue s t r o m a o f t h e w o u n d e d
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('yes. Tln:se cells were also clliefly nc~al,ivc all.hml~h weakly ]~osit,ivc cells ~mca.sim~nl[y were el)served t~l~Lhe w m t n d ettgc. T}m nmil(muc]enr lellkocytes which began to a l q m a r at~ t, lds Lime were. n e g a t i v e . 24-hr ,wo.undx. W o u n d s o1" 2,l-hr clurnli(m cculld lu,. divi.) cov'lleas in wJtic}, tl~e ilxflafllnlator'y reaction w~ts ~mly lno~ler~t.ely a d v a , m e d . "J.'he exl,e.lll, of ~tcve.lol~lm,~lt of 5-111tcle()t.i(lase, a c t i v i l y in { he i n v a t l i n g whit.e blood cells n nd in 1,he c o n n e c t i v e t ismm cells of t.lm s{.ronm was d i r e c t l y r e l a t e d {o l,he degree of develolmmnL of |.he J n f t a m l n a t o r y react.ion. P o l 3 a n o r p h o n u e l c a r IcukoeyLes rich in 5-1~ucleoLidase ac{,ivil,y were foun(I th ro~lgllcJ~ll, the sLronut in t,hose c o r n e a s in wit|oh ~ltunerotts w h i t e Mood cells were 1,'esent, ( I?la.l e I ). N e g a t i v e pol3:morpl,on~mh-ar lcukocyt.es were. n s u a l l y foltnd i~t l h e strol//~t a w a y fl'om t h e wmtnd edge, bug Lhese cells became, i n c r e a s i n g l y ]~osilive l o w a r d s |.he woun,I area. M o n o n u c l e a r blood cells in mmh c o r n e a s we,'e l~egative to m o d e r a t e l y posit.ire in l,he w o u n d area bul, n e g a t i v e a w a y from t,lm w o u n d . In |.hose few eyes in whiell l.lm i n f l a m m a t o r y rencl,ion was q~J~ly p.~rl.y ~leve.lolmd at, 2,t hr t.he 5 - n u c l e o t i d a s e activiLy was rcst~ricted to tim wom~(l arm~. 'l'}m l m l y m o r p t , o mlcle.ar l e u k o c y t e s presenb a w a y from t, lm v ¢ o t t l l d w e r e . u s u n l l y n e g a t i v e o r o n l y w e a k l y positive. T h e react,ion of t.lle commct.ive Lism/e at,rental cells was v e r y s i l n i l a r 1o t.}1:.1.|;of the w h i t e blood cells. :l'he. n~ore a c u t e t.he inflanmm.tory tea|e/iota, the greaL('r was the developmm~t, of 5 - m m l e o t i d a s e acgivil.y in b o t h the fihrolflasts a t t l~e ~,VOlllld e(lge a n d in the. s t r o m a l cells a w a y from the wm~nd area. On I Ira o t h e r lm.ntl, w h e n t h e inf l a n m m t o r y rea~'tJo,~ was o,~ly m n d e r a | c ' l y dcvelol~etl, tim 5-~mcleol.idase acLivit,y in t h e fibrobl'~sl,s at, t.he wouml mlge was nu~ch we;~ker t.l~an ~ts~al, anti (he reacl;ion in Lhe s t r o m a l cells a~way from the w o u n d edge was m~tel~ fainter. .IS-let web,mix. B y ,18 hr a f t e r i n j u r y t h e i n f t a m n m . l o r y react.ic,n was t'airl 3- ~nif o r m l y developed in all of |.he (:yes st~udie(l. T h e )nosla inl.ensc 5-,mcleot.Masc ;tot |vii T of I.he w h i t e blood cells w a s I'o~lmI n.t. this t,ime period. N m m , r o u s a c t i v e mm~onuclear blood cells were found el. |,he woltlld edge. Ttmse cells hint dew:loped large nm:lcoli a n d long 1)recesses of ey! ~Hfla.sm high in 5-nucleol, idase act.|vii.5.; ( P l a l e 2.). T h e area of rich 5 - m m l e o t i d a s e aet.ivil,y a r o u n d anti a w a y fl'on,, t h e w o u n d was also m o r e x~idespread el; ,t8 hr t h a n a.t, a n y o t h e r l.ime l~eriod. Negat.ive p o l y m o r p h o n u c l e a r l e u k o c y t e s were fm~nd o n l y at; t.he l i m b a l borders of t,he corneal sect,ions. T h e a.ctivity in t he st ronm.l ce.lls t'ollowe~l nnleh tile s a m e i m t.t(/rn as tJ~at, of |.he w h i t e blood cells, excel,t, |.hat it:, now e x t e n d e d f r o m t h e w o u n d to the periphe-ry of |,he sect.ions, i n c r e a s i n g in activiLy near tl~e w o u n d . I n areas in whieh boLh posii,ive sl, ronml cells a n d wtfite blood cells were t%und, t h e w h i t e cells were a l w a y s more act, i r e except, a' t.he ~,~'ound edge where the d e v e l o p i n g fibroMascs were exLremely act.lye. W h i t e Mood cells a n d s t r o m a l celia o c c a s i o n a l l y were f o u n d c l e a r l y in contact.. In such i n s t a n c e s 1)ot]~ cell l.ylms possessed gre'~t.er n(-i,ivit.y t,han isola.t.ed cells in l.he s a m e a r e a (Essner, ] 960; Birns, 1960; Seen,an, 19-51; Novikoff, 1960). Corneal st, rome 1 fibroeyges h a v e been p r e v i o u s l y observed to he p a r t i c u l a r l y act.ire in pha.gocytosing p o l y n m r p h o n u e l e a r h:uko(:ytes imLween 24 m~d ,18 h r at'ter i n j u r y (\Veimar, 1958). -k few dixdding cells were fomul n e a r tim w o u n d edge at.. 48 h r a.l'ter i n j u r y . "l:hese cells :tlso t e n d e d to h a v e a mo,'e i~tense 5-nucleoticlase neLly|by t,h a n ot.her cells i~ t h e same vicinity. 96-hr wou~Ms. I n cent, rest; to t h e w i d e s p r e a d 5-uucteotAdase act,ivit, y found at, 2,l-hr a n d e s p e c i a l l y i n all cell t y p e s ab ,18 hr there wns a s h a r p reducl;ion in the 5-nueleoLidase
'7-
,D
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I>L.~T~: 1. 5-N'u('l('~)t.idasc r(,action. 2.1-1u' wcmnd. I"~il~tly i)().~itlve e¢)l:nc~J til~r,J¢.vt:t, and izstcnnclv po.~itiv(, polynmrphoma.lcar Ic.ukc~;ytcs apl)r~ximatcly .l{)0 IL f'rqm~ the x~',,u,ul edge..K~)tc tllu ditl'c, rellc(, it1 the' iatcnsitic.~ el" st~duillg (,t" the nu~'I(~i ~,l"the lcuko(,yt~.s.and, o1" the c(~ruc~ll fibres.vie, l~r, wr~i.~h ~m,,.~ nol, in fi)cus al'~., clizvmc.ric}~ cells al, ditl'(.l'cni, level.-', of tc~t~(,s in t.hc tissue sccti~m. (('ountcr.~t:~incd witl~ muthyl ~t'ccn. mlcxtna.tc[l; ;.~ ItI6G.7.) I'L.~'L't.: 2. 5-.X'uclcoti(la~c r(~;~'tion. -18-In' w~,uml. 'l'w,~ int~'~(.'ly positive mumom~cl~'av blood ~.clls (ligl~L nm:l(~i) near woun([ u(h[c. Note Lhe extettsive dcvcl,~pmctxt of new cytoplasm wl,ich is 5.mtcleot.id:~::e lmsitive. (C(mnLcrstaincd wil.h methyl gr('(.m tmt:xt~'a(.tcd; ".: 1666.7.) Pr.XT~; 3. 5-Xuelc~ti(iasc rcm.t.i~,n. 9(i.lu' wouad. Two |msitivc monam~cl(~ar k, uk~)cy(cs near w~t, ml edge. N~te the lWCScnce [~l' mmlc~li. Thc,'c arc IIIIIII(:I'()US C'll;:ylllC-riCll cells at. 4itl'(:r('nt. levels in the, sccti(m and seen as br,~wni.~l~ masses ~mt of l'(~uus in tl~is ph~t~grap|~. (C~mnter.~tained witl~ meth.vl green, u ncxtractcd; "< l(ili(i.7.) I)LATI.: .1. 5-XuclcoLi(la~c ~'ca('ti~m. 9(i.hr ~'om~d. 5-.X'ucl¢:,~tidasc a c t i v i t y de~'clr, ping in t.lw b;~.~al (.(:lls of the (.pil.]mliutn adjacent to l,he wemad, cdg('. (Cemmcrn/:~incd with methyl grccl|, m~cxtra(.tcd; x
1060-7.)
])LATE 5. F,)rmalin-r(.si.~tant ~'oxidasc.likc': rvacti~m. 12.hr wound. Posil.ivc i>t~lym~,rph(,nu(.lua~ le~,kocytc. 4-hr incubati~m. ((Jom~(cr.~taincd with M a v c P s (.arm;dum, .'< l ii(i(i.7.) I>LA'rl.: 6. F~rm;~lia-z'csi.sia~t, "oxid;~se.likc" reac(.io~, i.l.lu" wound. [~tc~u~('l.v po.~itiv(, mo~ormclc~r lcukocyLc (top ce~xtcr) and ath('r leukocyte.-.', which cams~,t be i(lcntificd ;~s t,~ Lyl)C bec;utsc I~f intensity [~f rcauti,m. 4-1~r ir~cl~hation. (Co(mt.c,'.~taim-(l with Mayor's c~rmzdum, ... 1066.7.)
I~LA'rI.~ 7. FoL'nmlin-re.sist.antu % x i d a s e - l i k c " reaction. (,IG-hc ~'~un(l. W e a k l y p~,sitivu p~)lymorl~honuclear Icuk~cytes.-l-hr iI~cubatiou. (Count.crstai~cd wi~h .Mayor's carmalum. >1 l tiG6-7.) P[,,'t't~ 8. I:'onnalia-re.gistan}, "'oxidase-like" reaution. 24.hi" ~votmd. Large positive st~',~mal cells and small, roun¢l, i~tc~scly active p()lymorphor~zclcar Icukoc.vtcs ~tt. tl,c w o u n d edge. 4-In' illcLLI)l,l.tion. (Coul~tcrstai~cd with 3layer's carmalum; × 1666-7.)
'l'lllg
LAG
l ? . l f A S l g OJe " ~ V O U N ] )
l,t.i+.~l'AIl+
287
.c~ivit:y i . areas a w a y rrom the w o u n d b y 96 h r a f t e r i n j u r y . O n l y those w h i t e blood cells presm~t, at; the w o u n d edge were still h i g h l y active. T h e a c t i v i t y in the w h i t e blood cells ,~way th'om t h e wouixd was f a i n t to n o n e x i s t e n t . T h e a c t i v i t y in the c o n n e c t i v e tissue s t r o m a l cells a n d fibroblasts was v e r y s i m i l a r t.o t h a t tbund for the w h i t e blood cells: the f i b r o b l a s t s a t t h e w o u n d edge a n d those which were i n v a d i n g t h e e p i t h e l i u m to b r i d g e t h e w o u n d were t h e m o s t a c t i v e cells in t.he sec£ion. S t r o m a l cells a w a y f r o m t h e w o m l d edge h a d o n l y f a i n t to m o d e r a t e actixdty, m u c h like t h a t f o u n d ia t h e g r o u p of 24-hr w o u n d s w i t h t h e least inflamm a t o r y reaction. .HiS-hr wounds. I n t h e 168-hr wom~ds tlfc 5 - n u e l e o t i d a s e a c t i v i t y was d i s t i n c t l y l i m i t e d to t h e w o u n d edge. h c u k o c y t e s of all tyl)es a t t h e w o l m d edge were still v e r y positive. A c t i v i t y was r a r e l y fom~d in w h i t e blood cells a w a y f r o m tim w o u n d edge. \Vhe~: it occurred, i t w a s u s u a l l y in p o l y m o r p h o n u c l e a r l e u k o c y t e s . q?he 5~nuelcot.idase a c t i v i t y ill t h e c o n n e c t i v e tissue cells was r e s t r i c t e d to the fil~robl,sts t)lmt n o w c o m p l e t e l y b r i d g e d t h e w o u n d a n d ' t o those l y i n g i m n l e d i a t e l y adj~cet,t to ~he wot~nd. T h e r e was no a c t i v i t y in ~he c o n n e c t i v e tissue s t r o m a l cells a w a y fl'om the wouud. T h e epitlmlium, n e g a t i v e a t 48 hr a f t e r i n j u r y , b e g a n to develop some 5-nucleotidase a c t i v i t y b y 96 h r a f t e r i n j u r y . A t 96 hr, t h i s a c t i v i t y , was folmd o n l y in t h e epithelial basal ecll l a y e r l y i n g i m m e d i a t e l y a d j a c c n t to t h e w o m l d edge ( P l a t e 4). A t 168 ln: the epithelial a c t i v i t y h a d ga'eaLly increased a n d , irt some instances, e x t e n d e d some d i s t a n c e from t h e w o u n d ( W e i m a r a n d t I a r a g t t c l d , 1965b). 'I'be order of a p p e a r a n c e of 5-nucleotidase a c t i v i t y , in t h e v a r i o u s cell t:Kpcs was as Collows: s t r o m a l cells (6-12 hr); p o l y m o r p h o n u c l e a r letd¢o@tes (24 In'); m o n o n u c l e a r blood cells (24-48 hr); a n d e p i t h e l i a l cells (96 hr). T h e a c t i v i t y in all of these cell t y p e s (with the exception, of com'sc, of t h e e p i t h e l i a l cells) first a p p e a r e d in those cells a t or n e a r ~he w m m d edge a n d g-radually s p r e a d tKrough m o s t of t h e corneal st.roma by 48 hr a f t e r i n j u r y . Ag 96 ha" a definite localization of t h i s enz)nne a c t i v i t y t o t h e v i c i n i t y of the womxd w a s i n d i c a t e d b y t h e s h a r p drop in a c t i v i t y a w a y from the w m m d m)d increased a c t i v i t y a t t h e wom~d edge. T h e a c t i v i t y in t h e s t r o m a l c o n n e c t i v e ~issuc cells a n d w h i t e blood cells was r e s t r i c t e d to t h e w o u n d area a t 7 days after injury. A. g e n e r a l s u m m a r y of t h e p a t t e r n s of a c t i v i t y of 5 - n u c l e o t i d a s e is sho~m in T a b l e I. 'l:he a c t i v i t y of t h e fibroblasts a t the w o u n d edge was so i n t e n s e t h a t p h o t o m i c r o g r a p h s o f t h e w h i t e cells in t h i s regiotl were n o t feasible. I n o r d e r to a v o i d possible en'or in i n t e r p r e t a t i o n d u e to excess r e a c t i o n p r o d u c t f r o m t h e a c t i v e f i b r o b l a s t s a t t h e w o t m d edge, tim e v a l u a t i o n s of t h e w h i t e blood cells a t t h e w o l m d edge were a c t u a l l y m a d e a shor~ d i s t a n c e a w a y f r o m t h e a r e a of t h e m o s t i n t e n s e f i b r o b l a s t a c t i v i t y . I n order t-o e v a l u a t e a cell it w a s r e q u i r e d t h a t t h e a c t i v i t y ~ o r lack of i ~ - - i n t h e cell b e i n g s t u d i e d q t a d to be so d i s t i n c t l y e v i d e n t t h a t no q u e s t i o n of i n t e r l ) r e t a t i o n would arise. I n s o m e i n s t a n c e s , especially in t h e p a r t of t h e microscopic field n e a r e s t t h e w o u n d , t h e color of the cellular c y t o p l a s m b l e n d e d w i t h t h e g e n e r a l color of t h e baekg~'otmd al~d sltch cells were n o t e v a l u a t e d . Tlfis p r e c a u t i o n p r o b a b l y was n o t n e c e s s a r y because a few n e g a t i v e cells were formal in s u c h areas (especially a t 96 hr a n d 7 d a y s a f t e r i n j m ' y ) a n d these h a d a d i s t i n c t l y clear u n c o l o r e d c y t o p l a s m free of t h e b r o w n g r a n u l e s i n d i c a t i v e of 5 - n u c l e o t i d a s e a e t i v i W. I t a p p e a r e d t h a t some a c t i v i t y or reactioll o c c u r r e d - a t t h e w o u n d or in tile vicilfity of t h e w o u n d , w h i c h influenced all t h e ceils w i t h i n i t s r a n g e to d e v e l o p or to a c t i v a t e 5-nucleotidase. T h e effective r a n g e of t h i s wom~d a c t i o n was a p p a r e n t l y w i d e s p r e a d
Stromal eel]s and fibroblasts
Mononuclear blood ceils
nuclear ]eulcooytes
Vo?ymorpho.
4-/-
Wound edge
+/-
--
350500/z away
+
+/--
Wound edge
12 hr
+
--
350500/1 away
350500/z away
4. + ~-+ / + +
+4-4-++/--
4- Positive.
+ + + -I- 1+ -I-
÷4-+++
Wound edge
24 Lc
-- Negative.
+ +-i- + 4-
4-+4--,'--4-
4-++4-÷
Wom)d edge
Wound lmriod 3rd)-500~ away
+ 4-
+++-F-I-/+
.18 hr
5-Nucleotida,~e activity ctt various tin~c periods after woundi~g
T,tm.F, [
++1+
-}-+÷4-+
4-++
+/-++++
+ + 4- 4- 4-
+++
7 days 350Wound 500/z edge away
+
350500/z away
+-,~'4- + +
Wound edge
96 hr
---4
>
7~
'['Hlg LAG
I*I[ASE
0]?
%VOUND
I/,EPAII%
289
during tim fi ,'st a p p r o x i m a t e 48 hr and then g'rad'ually decreased in area until a.t 7 days it was limited to the wound edge where there was a high c o n c e n t r a t i o n of positive cells of all types. The slower d e v e l o p m e n t of 5-nucleotidase a c t i v i t y in the epithelium niight be interpreted as being due to a longer l a t e n t period of activation. F o r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction
The same general p a t t e r n of a c t i v a t i o n or d e v e l o p m e n t of new enzyme a c t i v i t y in the w h i t e blood cells of all types in wom~ded corneas was confirmed with a n o t h e r a p p a r e n t e n z y m e ~ a t b r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction, which we ha~-e n o t y e t fldly identified (Weimar and I-Iara~mhi, ]965a, 1966). I n the case of the formalinvesis~..ant, " o x i d a s e - l i k c " reaction, however, t h e a c t i v i t y in the fibroblasts a t the wouml edge suddenly decreased after 96 hr to become negative or only faintly positive a t 7 days. The same p a t t e r n was followed by the w h i t e blood cells.
F o r m a l i n - r e s i s t a n t " oxid(rse-like" ~'eaction. at v a r i o u s t i m e ~)eriods after w o u . n d i ~ %%routx(lperiod Cell t y p e
6 hr
] 2 hr
24 h r
48 h r
96 h r
7 days
Polymorphonuelear leukocytes
--/4-
+
-b + + q- -t-
-F 4- 4- 4- -I-
4- + +
-F"
_Mouonuelear b l o o d cells
--
q-"
+4-+4--t--
- F + ~ - L,,- ,
4-++
+/-
+
++++
++++
4---I-, +
+[--
l,'ibrobtasts a n d s~,romal cells
+/-'b
-I-- ] ? o s i t i v e .
-
-- ~egative.
The a c t i v i t y of this formalin-resistant " o x i d a s e - l i k e " reaction was more widespread than t h a t or 5-nucleotidase. I t appeared i n t e n s e l y in all cells (leukocytes, s t r o m a l ceils, endo+helimn and epithelium) t h r o u g h o u t the e n t i r e wolmded cornea b y 24 hr after i n j u r y (Plate 8) a n d almost t o t a l l y disappeared bel~weetl 4 and 7 days after injury. A s u m m a r y of the a c t i v i t y p a t t e r n s fom~d a t various time periods after i n j u r y is presented in Table II. As ~\dth 5-nucleotidase, the first white blood cells f o u n d in the w o u n d e d cornea were also negative fpr t h e f o r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction. B y 12 h r after injury, however, t h e polynnorphonuclea~ leukocytes were iaxtensely positive (Table V). At 24 hr after wounding, t h e p o l y m o r p h o n u c l e a r leu_kocytes a n d m o n o n u c l e a r blood cells (Table VI) were so i n t e n s e l y stained t h a t t h e y §imply appeared as " b l a c k masses" and t h e {ncubation period h a d to be s h o r t e n e d to p e r m i t t h e i r differentiation. I~t c o n t r a s t to 5-nucleotidase, the formalhl-resistant " o x i d a s e - l i k e " reaction showed a det~tttite decrease in all cell t y p e s a t 96 h r after i n j u r y (Table VII) and was only slightly positive or negative at 7 days after i n j u r y . It. is n o t possible, of com'se, to determine from these d a t a if the enzyme activities described were newly .formed or if t h e y were a c t i v a t e d . Cohn a n d Hirsch l a v e described 1"
..°90
V.
I,. W:igIMA]~. AN])
K.
]-I. ] I A R A C : U C I I I
the activation or liberation of a m l m b e r of enzymes, similar lo lysosoln;~i cnzynms a n d including an acid mmlcotidase (Cohn and tIirsch, 1960a), by r u p t u r e of the cell granules of p o l ~ n o r p h o m t c l e a r leukocy-tcs either mechanically or by phagocytosis (Cohn and 1-1irsch, 1960a,b). The total enzyme content of the cells remained the santo although the free or active enzyme was increased. The cells were obtained from peritoneal exudatcs ,1 hr a f t e r the i r r i t a n t was introduced. In general the activit.y observed in the pol~norp/~ommlcar Icukoc~.vtes in tim corneal wounds developed between 12 a n d 24 hr after injury, t[owevcr, some ~;~,sitive polymorphonuclear lcukocytes were observed in 2 eyes, which had an unusually well developed i n f l a m m a t o r y reaction a t 6 hr. Several observations point to a possible new formation of the 5-nuclcotidasc t,hat we observed. (1) LPhe mononuclear blood cells a t the wound edge developed a b u n d a n t new cytoplasm, which was v e r y rich in 5-nuclcotidase activity. (2) The fibroblasts t h a t originated a t the wound edge fronl the pre-existing negative stromal cells also developed large quantities of new cytol)lasm, which was il~tensely positive. (3) The epithelial cells developed 5-mtclcotidasc aet;ivity a t first only in chc basal cell layer, which is genetically a p p a r e n t l y the m o s t undiirerentiated (l.i'ell and 3J[cllanby, 1953; Billingham and Silvers, 1963). R e b u c k and coworkers have recorded enhanced peroxidase (l.lebuc]¢ and Mon'~glmn, 1948; Rebuck, Monto, Monaghan and Riddle, 1958; ]~ebuck and Crowlcy, 1955) and c y t o c h r o m e oxidase (Rebuck, 19,62; l~ebuck, h'Ioato, M o n a g h m t . a n d ]~iddle, 1958.; R e b u c k and C~'owlcy, 1955) activities in cxudate white blood cells in " s k i n window" preparations. I n a recent series of articles 5Vulff has recorded in detail, also in intlamr e c t o r y e x u d a t e white blood cells in " s k i n w i n d o w " prcI)arations, enhanced or new d e v e l o p m e n t of transglucosylase (\Vltlff, 1962); sltccinic dehydrogcnase (~V~tlff, 1963a); mitochondrial a-glycerophosphate dehydrogenase (Wulft, 1963a); reduced nicotinandde-adenine dimmleotidc a n d reduced nicotinamide-adenine dimmlcotide phosphate-diaphorascs (\Vulff, 1963a), nicotinamidc-adcnine dinucleotidc-linkcd lactic, malic, and isocitric dehydrogenascs, a.-glyeerophosphate, and ghttamic dchydrogcnases (~Vulff, 1963b); uridine diphosphate glucose-glycogen transglycosylase (\Vulff, 1963c); as well as nonspccific esterases (Wulff, 1963d), and acid and alkaline phosp}mtases (Wulff, I963d). Biochemical studies ~'ill be required to determine w h e t h e r the incrcased enzyme reactions observed in these wounded corneas a c t u a l l y rel)resent newly formed or a c t i v a t e d enzymes. The cellular functions of 5-nucleotidasc and of t,he formalinresistant "oxidase-like" enzyme are unl~town. I-Ieace it is diltleult to evahtate the significance of the a p p e a r a n c e of these systems dm'ing womad healing. I t has been proposed, however, t h a t adenosine-5'-phosphate (the s u b s t r a t e for 5-nucleotidase, n. reversible dega'adative enzyzne) Inay be a " r e s e r v o i r " for " a c t i v e " adenine, a precursor of I~NA synthesis (Brown and Roll, 1955). The point we wish to nmke in tiffs presentation is t h a t there is some characteristic or action of the wound itself t h a t influences all cells ~ithiJ~. its range to increasc Cheb" enzyme actixdties even during the so-called lag or l a t e n t p]msc of wom~d repair. P e r h a p s b y focusing ore" a t t e n t i o n on the n a t u r e a n d m e c h a n i s m of action of the iflfluence t h a t the wound a p p a r e n t l y exerts on the surrounding cell population, we m a y hope to find the means by which the syuxthctic and rel)air activities ot" wound healing are set; in motion.
TILE LAG I.'IlASE 01," WOUND I~,EI'ALII.
291
ACKNOWLEDGMI']NTS The ~uthors wish to thank Myrtle Davis for her tcclmica] assistance. REFERENCES Ahmcd, Z. a n(l lleis, J. 1~. (1958). Ltiochem. J. 69, 386. l~illiagham, R.. E. and Silvers, W. K. (1963). N. E. J. ,Med. 268, 539. Bims, M. (lfl60). Exptl Cell Rc,s..20, 202. Boycr, P. D., L.~rcly, H. aad Myrbiick, K. (1961). ]a The Enzymes, 2mL cd., 5, p. 645. Academic Press, ] Ira., Now York. ISr, wl~, G. B. and Roll, P. M. (1955). llx The Nucleic Acid~, 2, p. 389. Ed. I)y Chargaff, J']. and I);~vi(lson, .}. N. Academic i?rcss, ]nc., New York. Burst.oat, M. S. (1959). J. i]istochem. Cytochem. 7, 112. CoJm, Z. A. ~md Hirsch, J, (:l. (1960~r). J. Exp. 2][exl. 112, 983. Cohu, Z. A. alld ]lirsch, 3'. G. (I960b). J. Exp. )lied. 112, 1015. l)tml)by, J. ]g. and Udupa., K. hr. (1955). N . E . J . Med. 263, 847, .Essncr. E. (1960). J. Biophys, J~ioehem. Cytol. 7, 329. Fcfl, H. B. and 5[el]anby, ]~. (1953). J. Physiol. 119, 470. (:ilman, T. attd Wright, L. J. (1966). Natm'e 209, 1086. ('urr, E. (1956). A Practical ~lhtnual of Medicxtl a~ul ]3iological Staird~tg TcchMCucs, 2:td. ed. lrffcrsciettcc iPublishcrs, fac., New York. Lillic, ]¢. D. (i954). Histopalhologic Yl'echnio and Practical Hidochcmislry, l~lakiston Coml.)atty, New York. Novikof]'. A. B. (1960). I It l)evch)ping.Cell ,Yy.~l,:ms altd Their Control, p. I¢i7. Ed. by Rudnick, D. I~om,ld Press Compa~w, New .York. Novikoff. A. B., [)tucker, J.. ,'Shi~, \V. and Goldtischor, S. (1961). J. llislochcm. Cylochcm. 9, 434. Rcbucl~. 3. \\r. (L952). Fed. l'roc. 11, 42,t. I~cbuck, J. \V. ~ad Crowley, J. 3][. (1955). Ann. N . Y . Acqd. 8ci. 59, 757. l~cbuck. J. W. aad Monaght~t'~, E. A. (1948). lee¢lera~io~ Proo. 7, 277. l~cbuck, J. \V,, Moato, R. \.\[., Monagl~,~n, E. A. ~md Riddle, J. M. (I958). :I~tn. N . Y . Acad. Sci. "73, 8. Scam~n, C. R. (196l). J. Biophys. Biochem. Cylol. 9, 243. Wachstcia. M. and 51eisc}. E. (1957). Am. J. cli n. Pathol. 27, 13. \\'ei:nar, V. (! 957)..d m. J. Ophtl~dmol. 44, 173. Weimar, V, (1958). Arch. Ophthalmol. (Chicago) 60, 93. Weimar, V. (1959)../,.,b:ptl Cell lb/s, 18, 1. Weimar, V. (1960). l a The Tra~parency of The Corsica, p. 111. Ed. by l)uko-Ehlcr, Sir Stewart a.ltd Dorkiz~% E. S. Clmrlcs C. Thomas, Springfield illinois. \Vciru~r, V. (1962). Incesl. Ophlhalmol. 1,226. Weim~u', V., Davis, 51~.and Haro,guchi, K. (L965). Exptl Ceil Res. 39, 10. V. a~ld Haraguchi, K. ]-[. (1965a). J. Hislochem. Cytochem. 13, 239. Weimar, V. and Ha.r~guchi, K. ]-[. (1965b). I.nve.s'l. Ophttgdmol. 4, 853, \Vcimar. V. ~md ffar~guchi, K. H. (1966). Invest. Ophthalmol. 5, 14. Wulff, H. R. (L962). _dcla Haematol. 28, 86. \Vulfl', H. R. (i963a). Acta Haemalol. 29, 208. \Vt|lff, ]-[. 1~,. (1963b). ,,tcht Haen~loI. 30, 16. \'Vulfi', HI. R.. (1.. 3c). Acta ltae~atoL 30, 123. \.Vt,lfl, 1-[. l~. (1963d). Acta Ihzem.atol. 30, 159.
Tim Lag Phase
o1" W o u l l d R e p a i r
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1. Introduction T h e ]),'oeess of w o u n d r e p a i r , when s t u d i e d at the. cellular level, reveals n r e m a r k a b l e ,series o[" c.,}mi)ges in t h e (:nzsqlmlogy, pl]ysiology a n d morpholog3" of c o m m c t i v e l,issue cells in the w o u n d avc-a witl,in a few iJ~"a f ( e r i n j u r y ( W e i m a r , 1957, 1958, 1959, 1960, 1962" W e i m a r a n d ]faraguehi> l.qG5a,b, 1966; W e i n m r , Davis ~md ] l a r a g u c h i , 1965). 1-1; Ires been t h e p u r p o s e of our series of s t u d i e s o n wmlud healing to (let.ermine th,: nn l;m'e of Llm morl>hologJcal, }~ioe}lemieal a n d pllysio]ogical eyelets oeem'ring d u r i n g t.ll(. so-called big or la(ette p/,ase and to r'ehtto. Li,.'m wlJ(.'rdver ]Jossible into cause-aml-effeet re.laLionshi]~s ( W e i m a r , 1060). ])u.nphy a n d Udulm (19.55), on the I):tsis of t h e i r fis)clblg ol7 n rapid 1)reduction of g r o u n d s u b s t a n c e w i t h i n I2-2,1 hr a f t e r i u j u r y , h n v e a p t l y romuned t.he }a.'g l~)ease ns the . ' n b s t r a t e phase of w o u n d repair. * ~i'his i u v e s t i g a g i o n w a s . s . p p o r t e , I b y a P I f 8 r e s e a r c h grant, (.'/788) li'o,n the .N*ati,mal [n.~titute o f .N'euvolrJgi~:~d Disc'~ts~'s ~lcL,'l ]llilxdiiess, U.,% l'ubli,: ll~,.all.h S e r v i c e . A fi,)rl, ion o f t,hi~ p:qn!r was reixd /,efiJro tl,e ~Vo.~tet't! l~Cgi*,Cl~tl (':(,¢ll'ot'('tZc'(" ~t! l)eve|~l)ment.al llicth~gy art, [ : r i d a y ] l ~ t r b . r . a l M'~y I!R;3. 283
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Full t h i c k n e s s c e n t r a l c o r n e a l w o u n d s were m a d e w i t h a S w a n n e e d l e knife in adult, :llbino ( S p r a g u c - l ) : l w l o y ) rals (apl)roximat.ely 2 0 0 - 3 0 0 g ) u n d e r b r i e f ( [ - 2 m i n ) e t h e r ~lnesthesia. T h e anim~lls wore dec:~pit, t,cd a t 0, 6, 1 2, 24, ,18, 91; ~l.ll(l 168 hr. T h e eyes were i m m e . d i a t e l y e n u c : l e a t e d , fixed a n d s t o r e d in liquid n i t r o g e n u n t i l used. A m i n i m u m ot" 8 e y e s were. sLudied for e a c h t;ime p e r i o d . T h e w h i t e blood cells were d i f f e r e n t i a t e d a n d t~he i n t e n s i t y of t h e h i s t o e h e m i c a l r e a c t i o n w a s e v a l u a t e d in e o u n t e r s t a i n e d s e c t i o n s u n d e r oil i m m e r s i o n (>~1000) in an area i m m e d i a t e l y a d j a c e n t to t h e w o u n d ( 0 - 1 5 0 t , ) a n d in a n .nro.a about, :V)0-500/t a w a y f r o m t,he w o u n d . 5 - N u c l e o t i d a s c w a s c a r r i e d ont. a f t e r , t h e m e t . h o d of W a c h s t e i n a n d Meise], (196l), N o v i k o f l ' . . . D r u c k e r : S h i n a m l 0,Jldfischer (1.961), m o d i f i e d by W e i m a r a n d H a r a g u e h i (1965b). T h e in~:ubation m i x t u r e c o n s i s t e d of :30 m | of a 7 8 . 7 . m g / 1 0 0 ml s o l u t i o n of a d e n o s i n e - 5 - p h o s p h a t e ( S i g m a Cllemieal Co., ,¢%. L o u i s , Missouri); 30 ml of 0-2 .~t s t a n d a r d T r i s b u f f e r ( p I [ 7-2); 7-5 ml of 0.1 .~1 MnCI~ (A, h m e d a n d iteis, 1958; B o y e r , l m r d y a n d Myrbiick-, 1961): 3-0 ml of g l a s s - d i s t i l l e d H 2 0 ; a n d ,t-5 ml of 2~/o P b ( N 0 3 ) e. T h e i n c u b a t i o n m i x t u r e was freshly n , a d e for e a c h e x p e r i m e n t a m t filteredit.hrongh ~,Vhatman no. 40 filt,er p a p e r before it was used. T h e c o n t r o l c o n s i s t e d of t h e abo'vc, i i l c u b a t i o n m i x t u r e m i n u s a d e n o s i n e 5- p 1"~o s p h a t e. F r o z e n sect.ions w e r e c u t :~t 30 i~ in "l c r ) ' o s t a ~ ( l [ a r r i s ] t e f r i g e r a t i o n Co., Caml)ri(lg~, Massachusett.~,) a n d . p l a c e d di reetlv in t h e s u b s t r a t e s o l u t i o n . T h e s e c t i o n s w e r e s u n k wit.h a ~la..ss n e e d l e a n d i n e u b a t . e d for 5 hr at. 37-5°0. b~ollowing incubat, ion t h e .~ccLions w e r e p l a c e d in 3 7 % f o r m , l i n for 30 rain, p,sse,! t h r , m g h ' t w o c h a n g e s of glass-all.stilled w a t e r , a n d t h e n i n t o ~}~ (v/v) ~ m l m o n i u m sulfide for 2 rain. T i m react.ion p r o d u c t f o r m e d was b r o w n . A f t e r r i n s i n g in glass-,listille53 w a t e r , t,he st:(:liol,~s w e r e c o u n t e r s t . a i n e d in u n e x t r a c t e d m e t h y l g r e e n (0-2°/0) in 0-2 .~t a c e t a t e bufl'~2r (pl[. -1-0) for 25 rain. ( N o r m a l c o r n e a s t o o k t h e met.hyl g r e e n e o u n t e r s t , a i n m u c h m o r e i n t e n s e l y t h a n t h e w o u n d e d c o r n e a s d i d a n d t h e t i m e for e o u n t e r s t . a i n i n g h a d ~o be s h o r t e n e d to 1 0 - 1 5 rain.) F o r p u r e p r e p a r a t , ions of m e t h y l g r e e n . rel)eale,1 e x t r a c ' t i o n s of t h e d y e ~lre m a d e w i t h c h l o r o f o r m . T h e u n e x t , r a c t e d d y e , h o w e v e r . i'n t,ho preparal, ions d e s c r i b e d in t.his l)al)er p r o v i d e d e x c e l l e n t n u c l e a r d e t a i l . Tim see/.imls were n e x t d e s t . a i n e d in '2 rinses of 10 see ,-ach in a b s o l u t e e t h y l a l c o h o l , c l e a r e d in t o l u e n e fc~r l 0 r a i n a n d in e e d a r w o o , l oil for 10 rain. a n d m o u n t e d in P e r m o u n t . I~or t h e formalin-r~:sistant, oxldase-I~kt re.:~ction t h e e y t o c h r o m e oxid'~se m e t h o d of P;urst.one (1959) a n d \ V e i m a r a n d l l a r a g u c h i (1965a, 1966) w a s u s e d o n f o r m a l i n - f i x e d sect ions (1 hr, 10~/o neui, ral f o r m a l i n ) u s i n g x - p h e n y l - v - p h e n y l e n e d i a m i n e w i t h ] - h y d r o x y 2-napht.hoic. a c i d as c o u p l e r . F r o z e t t s e c t i o n s were cut. a g 20 ~x in t h e c r y o s t a t . T h r e e t y p e s ~f s~,ctions were u s e d : . ( l ) f r e s h - f r o z e n ; (2) f r e s h - f r o z e n , s e c t i o n e d , t h e n fixed 1½ hr a t rnc,m te~nl~er:,t.ure in 10°'; n e u t r a l f o r m a l i n ; a n d (3) c o r n e a s fixed fl,r 2,1 h r in ice-cold P;alc~.r's c:deiu~n-fln'm~,l. T i m s e c t i o n s w,'re i n e u l m t e , l for 1-.1 h r "~t 87"5°0. All s~:el.i,ms,
T H E LAC; P I I A S E Oi" WOUNI) R I,;I'AII¢
21~5
including those which had been fixed in formalin before inculmtion, were routinely t.ransfcrred to 1% cobaltous aeetate-10°/o formalin f,,r I h r after incubation. They were next rinsed t,hrough several changes of glass-distilled water and countcrstaitled in 5layer's e a r m a l u m o,olut.ion (earminie acid in aqueous potassium a l u m i n u m sulfate) (Gurr, 1956) for ] 0 rain. The earmalum solution used was less than 1 week ohl because solut, ions stored for longer periods of time caused considerable fi,ling of the rc(l(lish-brown-formalinresistanC "oxi(lasc-like" reaction product. Following counl,crstaining, the sections were rinsed in glass-distilled water and m o u n t e d in either glychrogel (I,illie, 195,1) or glycerine jelly. The corneas were .~cctioncd coronally with t.he woun,I celttered as much as possible. In such sections, the cells appear flat, spread out, and are considerably easier to identify t-han in sngitally cut sections (Weimar and l laraguchi, 19651)). A m i n i m u m of 8 eyes were studied for each time period evaluated. Frozen se(-tions were cut in a cr)'ostat. Complete details of the m e t h o d s have, been published (Weimar a n d Ha:'-iguchi, 19651), 1966). P h o t o m i c r o g r a p h s were taken with t,he Leit.z O r t h o m a t av.tomatie cameca using K o d a chrome II daylighl, film. 3. R e s u l t s and D i s c u s s i o n
An e x t e n s i v e e v a l u a t i o n of 5-nncleotida,".,-, formalin-resistant, " o x i d a s c - l i k e " a cf,i v i t y a n d succinic d e h y d r o g e n a s c activit.ies in t h e corneal s t r o n m l cells a n d fibroblast,s in h e a l i n g c o r n e a l w o u n d s h as been prese n ted els,-wltere (\Vei~na r a n, 1 l-I arag, tchi, 1965b, 1966). A s u r p r i s i n g o b s e r v a t i o n in connect.ion w i t h t h o s e s t u d i e s was that. llm in~'ading w h i t e blood cells of all t y p e s a p p e a r e d to un(lergo t h e same. v l t e r a t i o n s ira e n z y m e a c t i v i t i e s as did t h e c o r n e a l epit, hclial, s t r o m a l , a n d c,~dot.helial cells followi~lg wounding. To fi2rther e v a h m te this e x t r e m e l y i m p o r t a n t poi,t. 5-nlwlcot i(lase a n d t h e f o r m a l i n r e s i s t a n t " o x i d a s e - l i k e ' reaction were s t u d i e d in detail since b o t h were t o t a l l y negat i v e in l h e e a r l y i n v a d i n g blood cells. Succinic d e h y d r o g e n a s e , on t h e o t h e r h a n d , a.lreadv p o s i t i v e to v a r i o u s d e g r e e s in t h e d i f f e r e n t i n v a d i n g blood cells, was nob c o n s i d e r e d ira t h e s e studies. D i f f e r e n t i a t i o n of t h e i n v a d i n g m o n o n u c l e a r w h i t e blood cells and t h e c o r n e a l fibrocytes from each ot.hcr was possible because of p r o f o u n d tliffercnees in n u c l e a r s t a i n i n g . T h e nuclei of i n v a d i n g blood cells of all ty,q)es s t a i n e d m u c h more i " ensely t h a n did t h e nuclei of t h e s t r o m a l fibrocytes. It) areas a w a y from tim w o u n d t h e p r e s e n c e o f i n t e n s e l y s t a i n i n g , e n z y m c - r i c h m o n o n t m l e a r w h i t e blood cells aJ~¢l n m c r o p h a g e s f r e q u e n t l y t o t a l l y o v e r s h a d o w e d a n d o b s c u r e d t h e s t r o m a l cells w i t h t h e i r often v e r y w e a k e n z y m e a c t i v i t i e s (Plate l). I n this p r e s e n t a t i o n no atXempt will be m a d e to (listingqtish bet.ween m o n o e y t e s and l y m p h o c y t e s since, as t h e y mig-rate into t h e tissues, t h e y b e c o m e g r a d u a l l y a l t e r e d in a p p e a r a n c e , r e n d e r i n g t h e i r d i f f e r e n t i a t i o n difficult, if not i m p o s s i b l e ( G i l m a n a n d Wright,.. 1966).
5-Nucleotidasc Normal. T h e e p i t h e l i a l , e n d o t h e l i a l a n d s t r o m a l cells of t h e n o r m a l rat cornea did n o t s h o w 5 - n u c l e o t i d a s e a c t i v i t y . An occasiori~l l)olynlorl~honuclear leukocyte, was s o n l e t i m c s f o u n d ira t h e n o r m a l connective, tts.~ue s t r o m a , but, no 5 - n u c l e o t i d a s e a c t i v i t y was e v e r d e t e c t e d in such l e u k o c y t e s . 6- and 12-hr wounds. At 6 attd 12 hr a f t e r i n j u r y i n c r e a s i n g m r m b e r s of ],olymorlJhon u c l e a r cells were p r e s e n t t h r o u g h o u t t h e c o n n e c t i v e tissue s t r o m a o f t h e w o u n d e d
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I>L.~T~: 1. 5-N'u('l('~)t.idasc r(,action. 2.1-1u' wcmnd. I"~il~tly i)().~itlve e¢)l:nc~J til~r,J¢.vt:t, and izstcnnclv po.~itiv(, polynmrphoma.lcar Ic.ukc~;ytcs apl)r~ximatcly .l{)0 IL f'rqm~ the x~',,u,ul edge..K~)tc tllu ditl'c, rellc(, it1 the' iatcnsitic.~ el" st~duillg (,t" the nu~'I(~i ~,l"the lcuko(,yt~.s.and, o1" the c(~ruc~ll fibres.vie, l~r, wr~i.~h ~m,,.~ nol, in fi)cus al'~., clizvmc.ric}~ cells al, ditl'(.l'cni, level.-', of tc~t~(,s in t.hc tissue sccti~m. (('ountcr.~t:~incd witl~ muthyl ~t'ccn. mlcxtna.tc[l; ;.~ ItI6G.7.) I'L.~'L't.: 2. 5-.X'uclcoti(la~c r(~;~'tion. -18-In' w~,uml. 'l'w,~ int~'~(.'ly positive mumom~cl~'av blood ~.clls (ligl~L nm:l(~i) near woun([ u(h[c. Note Lhe extettsive dcvcl,~pmctxt of new cytoplasm wl,ich is 5.mtcleot.id:~::e lmsitive. (C(mnLcrstaincd wil.h methyl gr('(.m tmt:xt~'a(.tcd; ".: 1666.7.) Pr.XT~; 3. 5-Xuelc~ti(iasc rcm.t.i~,n. 9(i.lu' wouad. Two |msitivc monam~cl(~ar k, uk~)cy(cs near w~t, ml edge. N~te the lWCScnce [~l' mmlc~li. Thc,'c arc IIIIIII(:I'()US C'll;:ylllC-riCll cells at. 4itl'(:r('nt. levels in the, sccti(m and seen as br,~wni.~l~ masses ~mt of l'(~uus in tl~is ph~t~grap|~. (C~mnter.~tained witl~ meth.vl green, u ncxtractcd; "< l(ili(i.7.) I)LATI.: .1. 5-XuclcoLi(la~c ~'ca('ti~m. 9(i.hr ~'om~d. 5-.X'ucl¢:,~tidasc a c t i v i t y de~'clr, ping in t.lw b;~.~al (.(:lls of the (.pil.]mliutn adjacent to l,he wemad, cdg('. (Cemmcrn/:~incd with methyl grccl|, m~cxtra(.tcd; x
1060-7.)
])LATE 5. F,)rmalin-r(.si.~tant ~'oxidasc.likc': rvacti~m. 12.hr wound. Posil.ivc i>t~lym~,rph(,nu(.lua~ le~,kocytc. 4-hr incubati~m. ((Jom~(cr.~taincd with M a v c P s (.arm;dum, .'< l ii(i(i.7.) I>LA'rl.: 6. F~rm;~lia-z'csi.sia~t, "oxid;~se.likc" reac(.io~, i.l.lu" wound. [~tc~u~('l.v po.~itiv(, mo~ormclc~r lcukocyLc (top ce~xtcr) and ath('r leukocyte.-.', which cams~,t be i(lcntificd ;~s t,~ Lyl)C bec;utsc I~f intensity [~f rcauti,m. 4-1~r ir~cl~hation. (Co(mt.c,'.~taim-(l with Mayor's c~rmzdum, ... 1066.7.)
I~LA'rI.~ 7. FoL'nmlin-re.sist.antu % x i d a s e - l i k c " reaction. (,IG-hc ~'~un(l. W e a k l y p~,sitivu p~)lymorl~honuclear Icuk~cytes.-l-hr iI~cubatiou. (Count.crstai~cd wi~h .Mayor's carmalum. >1 l tiG6-7.) P[,,'t't~ 8. I:'onnalia-re.gistan}, "'oxidase-like" reaution. 24.hi" ~votmd. Large positive st~',~mal cells and small, roun¢l, i~tc~scly active p()lymorphor~zclcar Icukoc.vtcs ~tt. tl,c w o u n d edge. 4-In' illcLLI)l,l.tion. (Coul~tcrstai~cd with 3layer's carmalum; × 1666-7.)
'l'lllg
LAG
l ? . l f A S l g OJe " ~ V O U N ] )
l,t.i+.~l'AIl+
287
.c~ivit:y i . areas a w a y rrom the w o u n d b y 96 h r a f t e r i n j u r y . O n l y those w h i t e blood cells presm~t, at; the w o u n d edge were still h i g h l y active. T h e a c t i v i t y in the w h i t e blood cells ,~way th'om t h e wouixd was f a i n t to n o n e x i s t e n t . T h e a c t i v i t y in the c o n n e c t i v e tissue s t r o m a l cells a n d fibroblasts was v e r y s i m i l a r t.o t h a t tbund for the w h i t e blood cells: the f i b r o b l a s t s a t t h e w o u n d edge a n d those which were i n v a d i n g t h e e p i t h e l i u m to b r i d g e t h e w o u n d were t h e m o s t a c t i v e cells in t.he sec£ion. S t r o m a l cells a w a y f r o m t h e w o m l d edge h a d o n l y f a i n t to m o d e r a t e actixdty, m u c h like t h a t f o u n d ia t h e g r o u p of 24-hr w o u n d s w i t h t h e least inflamm a t o r y reaction. .HiS-hr wounds. I n t h e 168-hr wom~ds tlfc 5 - n u e l e o t i d a s e a c t i v i t y was d i s t i n c t l y l i m i t e d to t h e w o u n d edge. h c u k o c y t e s of all tyl)es a t t h e w o l m d edge were still v e r y positive. A c t i v i t y was r a r e l y fom~d in w h i t e blood cells a w a y f r o m tim w o u n d edge. \Vhe~: it occurred, i t w a s u s u a l l y in p o l y m o r p h o n u c l e a r l e u k o c y t e s . q?he 5~nuelcot.idase a c t i v i t y ill t h e c o n n e c t i v e tissue cells was r e s t r i c t e d to the fil~robl,sts t)lmt n o w c o m p l e t e l y b r i d g e d t h e w o u n d a n d ' t o those l y i n g i m n l e d i a t e l y adj~cet,t to ~he wot~nd. T h e r e was no a c t i v i t y in ~he c o n n e c t i v e tissue s t r o m a l cells a w a y fl'om the wouud. T h e epitlmlium, n e g a t i v e a t 48 hr a f t e r i n j u r y , b e g a n to develop some 5-nucleotidase a c t i v i t y b y 96 h r a f t e r i n j u r y . A t 96 hr, t h i s a c t i v i t y , was folmd o n l y in t h e epithelial basal ecll l a y e r l y i n g i m m e d i a t e l y a d j a c c n t to t h e w o m l d edge ( P l a t e 4). A t 168 ln: the epithelial a c t i v i t y h a d ga'eaLly increased a n d , irt some instances, e x t e n d e d some d i s t a n c e from t h e w o u n d ( W e i m a r a n d t I a r a g t t c l d , 1965b). 'I'be order of a p p e a r a n c e of 5-nucleotidase a c t i v i t y , in t h e v a r i o u s cell t:Kpcs was as Collows: s t r o m a l cells (6-12 hr); p o l y m o r p h o n u c l e a r letd¢o@tes (24 In'); m o n o n u c l e a r blood cells (24-48 hr); a n d e p i t h e l i a l cells (96 hr). T h e a c t i v i t y in all of these cell t y p e s (with the exception, of com'sc, of t h e e p i t h e l i a l cells) first a p p e a r e d in those cells a t or n e a r ~he w m m d edge a n d g-radually s p r e a d tKrough m o s t of t h e corneal st.roma by 48 hr a f t e r i n j u r y . Ag 96 ha" a definite localization of t h i s enz)nne a c t i v i t y t o t h e v i c i n i t y of the womxd w a s i n d i c a t e d b y t h e s h a r p drop in a c t i v i t y a w a y from the w m m d m)d increased a c t i v i t y a t t h e wom~d edge. T h e a c t i v i t y in t h e s t r o m a l c o n n e c t i v e ~issuc cells a n d w h i t e blood cells was r e s t r i c t e d to t h e w o u n d area a t 7 days after injury. A. g e n e r a l s u m m a r y of t h e p a t t e r n s of a c t i v i t y of 5 - n u c l e o t i d a s e is sho~m in T a b l e I. 'l:he a c t i v i t y of t h e fibroblasts a t the w o u n d edge was so i n t e n s e t h a t p h o t o m i c r o g r a p h s o f t h e w h i t e cells in t h i s regiotl were n o t feasible. I n o r d e r to a v o i d possible en'or in i n t e r p r e t a t i o n d u e to excess r e a c t i o n p r o d u c t f r o m t h e a c t i v e f i b r o b l a s t s a t t h e w o t m d edge, tim e v a l u a t i o n s of t h e w h i t e blood cells a t t h e w o l m d edge were a c t u a l l y m a d e a shor~ d i s t a n c e a w a y f r o m t h e a r e a of t h e m o s t i n t e n s e f i b r o b l a s t a c t i v i t y . I n order t-o e v a l u a t e a cell it w a s r e q u i r e d t h a t t h e a c t i v i t y ~ o r lack of i ~ - - i n t h e cell b e i n g s t u d i e d q t a d to be so d i s t i n c t l y e v i d e n t t h a t no q u e s t i o n of i n t e r l ) r e t a t i o n would arise. I n s o m e i n s t a n c e s , especially in t h e p a r t of t h e microscopic field n e a r e s t t h e w o u n d , t h e color of the cellular c y t o p l a s m b l e n d e d w i t h t h e g e n e r a l color of t h e baekg~'otmd al~d sltch cells were n o t e v a l u a t e d . Tlfis p r e c a u t i o n p r o b a b l y was n o t n e c e s s a r y because a few n e g a t i v e cells were formal in s u c h areas (especially a t 96 hr a n d 7 d a y s a f t e r i n j m ' y ) a n d these h a d a d i s t i n c t l y clear u n c o l o r e d c y t o p l a s m free of t h e b r o w n g r a n u l e s i n d i c a t i v e of 5 - n u c l e o t i d a s e a e t i v i W. I t a p p e a r e d t h a t some a c t i v i t y or reactioll o c c u r r e d - a t t h e w o u n d or in tile vicilfity of t h e w o u n d , w h i c h influenced all t h e ceils w i t h i n i t s r a n g e to d e v e l o p or to a c t i v a t e 5-nucleotidase. T h e effective r a n g e of t h i s wom~d a c t i o n was a p p a r e n t l y w i d e s p r e a d
Stromal eel]s and fibroblasts
Mononuclear blood ceils
nuclear ]eulcooytes
Vo?ymorpho.
4-/-
Wound edge
+/-
--
350500/z away
+
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Wound edge
12 hr
+
--
350500/1 away
350500/z away
4. + ~-+ / + +
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+ + + -I- 1+ -I-
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Wound edge
24 Lc
-- Negative.
+ +-i- + 4-
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4-++4-÷
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Wound lmriod 3rd)-500~ away
+ 4-
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.18 hr
5-Nucleotida,~e activity ctt various tin~c periods after woundi~g
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+
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+-,~'4- + +
Wound edge
96 hr
---4
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I/,EPAII%
289
during tim fi ,'st a p p r o x i m a t e 48 hr and then g'rad'ually decreased in area until a.t 7 days it was limited to the wound edge where there was a high c o n c e n t r a t i o n of positive cells of all types. The slower d e v e l o p m e n t of 5-nucleotidase a c t i v i t y in the epithelium niight be interpreted as being due to a longer l a t e n t period of activation. F o r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction
The same general p a t t e r n of a c t i v a t i o n or d e v e l o p m e n t of new enzyme a c t i v i t y in the w h i t e blood cells of all types in wom~ded corneas was confirmed with a n o t h e r a p p a r e n t e n z y m e ~ a t b r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction, which we ha~-e n o t y e t fldly identified (Weimar and I-Iara~mhi, ]965a, 1966). I n the case of the formalinvesis~..ant, " o x i d a s e - l i k c " reaction, however, t h e a c t i v i t y in the fibroblasts a t the wouml edge suddenly decreased after 96 hr to become negative or only faintly positive a t 7 days. The same p a t t e r n was followed by the w h i t e blood cells.
F o r m a l i n - r e s i s t a n t " oxid(rse-like" ~'eaction. at v a r i o u s t i m e ~)eriods after w o u . n d i ~ %%routx(lperiod Cell t y p e
6 hr
] 2 hr
24 h r
48 h r
96 h r
7 days
Polymorphonuelear leukocytes
--/4-
+
-b + + q- -t-
-F 4- 4- 4- -I-
4- + +
-F"
_Mouonuelear b l o o d cells
--
q-"
+4-+4--t--
- F + ~ - L,,- ,
4-++
+/-
+
++++
++++
4---I-, +
+[--
l,'ibrobtasts a n d s~,romal cells
+/-'b
-I-- ] ? o s i t i v e .
-
-- ~egative.
The a c t i v i t y of this formalin-resistant " o x i d a s e - l i k e " reaction was more widespread than t h a t or 5-nucleotidase. I t appeared i n t e n s e l y in all cells (leukocytes, s t r o m a l ceils, endo+helimn and epithelium) t h r o u g h o u t the e n t i r e wolmded cornea b y 24 hr after i n j u r y (Plate 8) a n d almost t o t a l l y disappeared bel~weetl 4 and 7 days after injury. A s u m m a r y of the a c t i v i t y p a t t e r n s fom~d a t various time periods after i n j u r y is presented in Table II. As ~\dth 5-nucleotidase, the first white blood cells f o u n d in the w o u n d e d cornea were also negative fpr t h e f o r m a l i n - r e s i s t a n t " o x i d a s e - l i k e " reaction. B y 12 h r after injury, however, t h e polynnorphonuclea~ leukocytes were iaxtensely positive (Table V). At 24 hr after wounding, t h e p o l y m o r p h o n u c l e a r leu_kocytes a n d m o n o n u c l e a r blood cells (Table VI) were so i n t e n s e l y stained t h a t t h e y §imply appeared as " b l a c k masses" and t h e {ncubation period h a d to be s h o r t e n e d to p e r m i t t h e i r differentiation. I~t c o n t r a s t to 5-nucleotidase, the formalhl-resistant " o x i d a s e - l i k e " reaction showed a det~tttite decrease in all cell t y p e s a t 96 h r after i n j u r y (Table VII) and was only slightly positive or negative at 7 days after i n j u r y . It. is n o t possible, of com'se, to determine from these d a t a if the enzyme activities described were newly .formed or if t h e y were a c t i v a t e d . Cohn a n d Hirsch l a v e described 1"
..°90
V.
I,. W:igIMA]~. AN])
K.
]-I. ] I A R A C : U C I I I
the activation or liberation of a m l m b e r of enzymes, similar lo lysosoln;~i cnzynms a n d including an acid mmlcotidase (Cohn and tIirsch, 1960a), by r u p t u r e of the cell granules of p o l ~ n o r p h o m t c l e a r leukocy-tcs either mechanically or by phagocytosis (Cohn and 1-1irsch, 1960a,b). The total enzyme content of the cells remained the santo although the free or active enzyme was increased. The cells were obtained from peritoneal exudatcs ,1 hr a f t e r the i r r i t a n t was introduced. In general the activit.y observed in the pol~norp/~ommlcar Icukoc~.vtes in tim corneal wounds developed between 12 a n d 24 hr after injury, t[owevcr, some ~;~,sitive polymorphonuclear lcukocytes were observed in 2 eyes, which had an unusually well developed i n f l a m m a t o r y reaction a t 6 hr. Several observations point to a possible new formation of the 5-nuclcotidasc t,hat we observed. (1) LPhe mononuclear blood cells a t the wound edge developed a b u n d a n t new cytoplasm, which was v e r y rich in 5-nuclcotidase activity. (2) The fibroblasts t h a t originated a t the wound edge fronl the pre-existing negative stromal cells also developed large quantities of new cytol)lasm, which was il~tensely positive. (3) The epithelial cells developed 5-mtclcotidasc aet;ivity a t first only in chc basal cell layer, which is genetically a p p a r e n t l y the m o s t undiirerentiated (l.i'ell and 3J[cllanby, 1953; Billingham and Silvers, 1963). R e b u c k and coworkers have recorded enhanced peroxidase (l.lebuc]¢ and Mon'~glmn, 1948; Rebuck, Monto, Monaghan and Riddle, 1958; ]~ebuck and Crowlcy, 1955) and c y t o c h r o m e oxidase (Rebuck, 19,62; l~ebuck, h'Ioato, M o n a g h m t . a n d ]~iddle, 1958.; R e b u c k and C~'owlcy, 1955) activities in cxudate white blood cells in " s k i n window" preparations. I n a recent series of articles 5Vulff has recorded in detail, also in intlamr e c t o r y e x u d a t e white blood cells in " s k i n w i n d o w " prcI)arations, enhanced or new d e v e l o p m e n t of transglucosylase (\Vltlff, 1962); sltccinic dehydrogcnase (~V~tlff, 1963a); mitochondrial a-glycerophosphate dehydrogenase (Wulft, 1963a); reduced nicotinandde-adenine dimmleotidc a n d reduced nicotinamide-adenine dimmlcotide phosphate-diaphorascs (\Vulff, 1963a), nicotinamidc-adcnine dinucleotidc-linkcd lactic, malic, and isocitric dehydrogenascs, a.-glyeerophosphate, and ghttamic dchydrogcnases (~Vulff, 1963b); uridine diphosphate glucose-glycogen transglycosylase (\Vulff, 1963c); as well as nonspccific esterases (Wulff, 1963d), and acid and alkaline phosp}mtases (Wulff, I963d). Biochemical studies ~'ill be required to determine w h e t h e r the incrcased enzyme reactions observed in these wounded corneas a c t u a l l y rel)resent newly formed or a c t i v a t e d enzymes. The cellular functions of 5-nucleotidasc and of t,he formalinresistant "oxidase-like" enzyme are unl~town. I-Ieace it is diltleult to evahtate the significance of the a p p e a r a n c e of these systems dm'ing womad healing. I t has been proposed, however, t h a t adenosine-5'-phosphate (the s u b s t r a t e for 5-nucleotidase, n. reversible dega'adative enzyzne) Inay be a " r e s e r v o i r " for " a c t i v e " adenine, a precursor of I~NA synthesis (Brown and Roll, 1955). The point we wish to nmke in tiffs presentation is t h a t there is some characteristic or action of the wound itself t h a t influences all cells ~ithiJ~. its range to increasc Cheb" enzyme actixdties even during the so-called lag or l a t e n t p]msc of wom~d repair. P e r h a p s b y focusing ore" a t t e n t i o n on the n a t u r e a n d m e c h a n i s m of action of the iflfluence t h a t the wound a p p a r e n t l y exerts on the surrounding cell population, we m a y hope to find the means by which the syuxthctic and rel)air activities ot" wound healing are set; in motion.
TILE LAG I.'IlASE 01," WOUND I~,EI'ALII.
291
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