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Cell migration in the adult rat and rabbit lens
Exptl Eye Res. ( 1 9 6 8 ) 7, 2 4 4 - 2 4 6
Cell Migration
in t h e A d u l t R a t a n d R a b b i t L e n s *
CALVIN HANNA ,~N]) I-IENRY C. ~EATTS
Departme.nt of Pharmaco~.vgy, U niversit?] of ,4r]ean~.~as Medical Center, Little Rock, Arkansas, U.S.A. (Received 28 August 1967, Bosto~O The loctttion of dividing epit.hcli~l cells and the s u b s e q u e n t migratiot~ of these cells int~ the lens subst, a.nee of tim a d u l t r a t trod rabbit¢ were d e t e r m i n e d using ingrac~meral [a.I4g]lAxymidine and autoradiographic techniques. The germ inat.ive zone of the Ions, located just ant,crier to the lens equator, wins an average of five cx~lls wide. One of the.so cells migrated posterioriy each d a y a n d became a new lens cortex fiber in an ~verage of 6 and 8 weeks fi)r the rat and rabbit, respectively. Each new cortex fiber migrated inw~,rd forming a lens fiber w i t h o u t a nucleus in an average of 6 and I0 weeks for the r a t ~nd r~bbit, respectively.
1. Introduction T h e g e r m i t l a t i v e z o n e o f t h e r a t , r a b b i t , a n d h m n a n l e n s is loc,~ted in ~ r a t h e r n a r r o w b a n d o f cells jtLst a n t e r i o r t o t h e l e ~ s e q u a t o r . I n t h i s z o n e o r p r o g e l l i t o r c o m p a r t m e n t are fomld cells undergoing DNA synthesisand m i t o s i s ( i ~ a . m m a n d 0 : B r m n , 1961). U s i n g [ a ] ~ [ ] O l y m i d i n e , H a n n a a n d O'Bric~t ( 1 9 6 0 a , b , 1')61, 1.963) d e t e r m i n c ( t t h a t t h e r a t e o f m i g r a t i o n o f cells f r o m t h e p r o g e n i t o r c o m p a r t m c : n t a n d i n t o t h e l e n s s u b s t a . n c e w a s r e l a t e d t o t h e a g e o f t h e r~tt. A s t h e s e cells r a p i d l y l e f t t h e p r o g e n i t o r c o m 1)artment of the young rat, they were replaced by new cells indicating ~ stead), state renewal system. L i t t l e is k n o w n a b o u t t h e r a t e o f m i g T a t i o n a n d s u b s e q u e n t d i f l ' c r e n t i a t i o n o f et)it h e l i a l c e l l s o f t h e l e n s o f t h e a d u l t a~fimal, i n c l u d i n g m a n ( H a r e m , 1965). T h i s r c l ) o r c det-fils tli~ m i g r a t i o n o f l e n s e p i t h e l i a l ceils a n d t h e i r d i f f c r e x l t i a t i o n i n t o n o n - n u c l e a t e d l e n s f i b e r s i n t h e a d u l t r a t a-nd r a b b i t .
2. Materials and Methods One-year-old albino rats were briefly anesthetized with ether, and 1-year-old albino r a b b i t ~ w e r e b r i e f l y a n e s t h e t i z e d ~ d t h i n t r a v e n o u s 1-5°/o t h i o p e n t a l s o 4 i u m . D u r i n g a n e s t h e s i a a n i n t r a e a m e r a l i n j e c t i o n of 5 t~c of [aHJthymiclil~e in 0.005 m l of w a t e r (10 c / m m o l e ; S c h w a r z . B i o R e s e a r c h , i n c . , Ora n g e b m ' g , N.Y., U.S.A.) w a s m a d e i n t o e a c h e y e . :Rats a n d r a b b i t s w e r e k i l l e d in ])airs, 2 lind 2,1- hr; 1, 2 al~d 3 w e e k s a n d 1, 1½, 2, 2½, 3 a n d 3½ m o n t h s l a t e r . G r o u p s of 4 r a b b i t s a n d r a t s were kille'.t 4, 5, 6 , 6½ a n d 7 m o n t h s l a t e r . T h e e y e s Were r e m o v e d , fixed in f o r m a l i n , e m b e d d e d in p a r a f f i n , a n d s e c t i o n s 5/z in t h i c k n e s s were cut. t h r o u g h t h e s a g i t t a l p l a n e of t h e eye. T h e s e c t i o n s w e r e p a i n t e d ~ d t h K o d a k N T B 3 e m u l s i o n a n d d e v e l o p e d i n K o d a k D-19 1 m o n t h l a t e r (I-Ianna a n d O ' B r i e n , 1960a,b, 1963). T h e s e c t i o n s w e r e s t a i n e d w i t h 1 ~ ) t o l u i d i n e b l u e in 2 % a q u e o u s p h e n o l a n d w i t h 1 % a q u e o u s eosin.
3. Results T h e ~ n t e r i o r t o p o s t e r i o r s p r e a d b e t w e e n t h e a I { - l a b e l e d e p i t h e l i a l cells o f t h e r,~t l e n s ~ v e r a g c d 4 cell w i d t h s . T h i s w a s d e t e r m i n e d f r o m s e c t i o n s 5 tL i n t h i c k n e s s c u t * Tiffs investigation was supported by :PHS research grants (NJ~,-402~I and ~]$-5076) from the National Institute of ~eurological Diseases and :BliD.c~,ess, U.S. Public ~ c a l t h Service.
244
LENS
CELL
5IIGRATION
245
from the sagitta.l plane of the lens, and there was a n average of 1½ aI=l:-labeled cells in each h a l f of the lens scctio,t 2 h r after the injection of [aH]thymidine. T h a t is, in the rat the progenitor compartnml~t is a b o u t 4- cells deep and contahm a b o u t 1½ cells m~dergoi:.,g DNA synthesis. I n lens sections t~om rats ldlled 1-10 weeks after the injectiou of [al:I]thymidinc the ,um~ber of all-labeled cells was n e a r l y the same. An occasional labeled cell was found in the ])roge,litor c o m p a r t m e n t for t h e first 3 weeks. I n time the "~H-labelcd cells were retold fm'ther a11(l f u r t h e r anterior from the progcnit.or c o m p a r t m e n t until the labeled cells were /bund entering the leas cortex a t an average of 6 weeks (Table I). O n the a~,erage, the aII-labeled ceils m i g r a t e d from ~he progenitor c o m l ) a r t m e a t for a distance of a.bout 148 cell widths m 4½ months, or a cell migrated out of the l)rogenitor coml)artment, each day. '.['A ~BLI'-" I
Position of all-labeled cells in the rat a ~ l rabbit lens with time
Animal no.
Time (m(,nths)
A v e r a g e cell w i d t h s f r o m a l { - l a b e l c d cell t o the: start. ¢)f c e n t e r of s t a r t of lens eort.cx lens c o r t e x lens n u c l e u s
J, '2 13, 14 15, 16 19, 20 2I ,~o 23, 24 25-28 29-32
(2 h,') 1 l~ 2-.~ 3 3} 4 5
42 9 0
90 63 4S S 0
14S 112 97 59 50 36 7
~abbits 1, 2 15, IG 17, IS 19, 20 23, 24 25-28 29-32 33-36 37 --40 41-44
(2 Kr) 1~ 2 2½ 3.~. 4 5 6 6½ 7
6,q 15 4 0
132 103 97 5"2 3g 16
2°8 172 161 133 109 78 -53 2l 3
The results using t h e a d u l t r a b b i t were similar to those obtained from the r a t with the excel)tions t h a t : (a) tile progeJtitor c o m p a r t m e n t averaged 5 cells wide and; (b) t h e distances traveled i~l ~he r a b b i t lenses were greater (Table I). 4. Discussion
The rates of migTatioll of lens cells in t h e r a t and r a b b i t were quite similar, i.e. a b o u t a cell width per day. Possibly this is related to the f a c t t h a t b o t h attimals are rodmlts. :Results in m a n - - c o t f l d t h e y be d e t e r m i r m d - - w o u l d be similar because a b o u t l½ ceils in a section 5/z hi tlfickness cut on t h e sagittal plane itmorporate's [aIt]th)nxaidiae ix1 2 hr, a n d t h e progenitor cort~partmellt is not a p p r e c i a b l y h~rger (I-Ia~ma, Bick_nell a n d O'Brien, 1961). This rate of mig,ratiott of epithelial cells does n o t a p p e a r to hold for
246
CALVIN If[ANNA AN]) I[ENRY C. K]",ATTS
the chic'lcen a.ml ]~ossibly rt.:i)tilcs ( H a m ~ ~md ()'Brie~l, 1961; Hnmia, 1965; and t Tanna mtd Keatts, ] 966). The c])it, helia of the adult, rat, all(1 rabbit lens are charact:eriz(,.d by the addition of tlew ccll:~ h'om a sm:dl Zolle of ceils (germimllive zone or progctfitor compal'tment) t h a t r(;l)la(:e those lost I)y continuous diffcrenlial;io~ into new lens libels. '.['h(, ratt, of m.w cell 1)r,)duct,i(m o~: i,urnover in t.he l~rogenit9~' ('Oml);U'tmc~t, c;m he est, imatcd from the rate ot' cell difl'crentiat, iolt into lc~s fibers ir~ the absence of cell loss (Bert:alanff)', 1964). 'L'h(, munber of :~l-I-I;~l)eled cells ilt t,he Lel~s of the ral~ and rabbit, rcm~inc(l ne~lrly coltstal~ between 1 and 10 weclcs after the inj¢~ct,io~ of [al []th)'midirte i~dicating n,) loss of all-labeled cells. Siuce the difl'crent.i:~,t.ing cells migrated one c(:l[ ~vidt,h l)(;r day al~d sin('c the ln'ogenitor (:Oml~artme~tt. is ,'¢-5 cells ~'ide the ttm~o~;t,r t,i~tc is 3-5 days. This turnover tim,, is in good agrce~tc~t, with t.ha~ reported by 5]iIculicict~ and Yottng (1963) for thc l 0-d~y-old rat, a~t(l rel~ )rtcd l:y ][amm (1955) for th~ yotmg mouse of 54 hr and 56 hr, resl)ectivcly, where rh(, turnover time was calculated from the per ccitt of aH-l~fl)elcd celts i~ the le~s 1 hr following ~he injc(:.tiot~ of [a] []thymidi~e. REJ?EI-'iENCES Bertalanfl)'. F. 1). (1964), Lab. lnwst. 13, 871. .Hanna, C.. (1965). Invest Ophthal~ntal. 4, 480. Ha m~a, C., Bicknell, D. S. and O'Bricn, J. 15. (1961). Arch. Ophthalmot. (Chicago) 65, (;96. Harma, C. and ].
Cell Migration
in t h e A d u l t R a t a n d R a b b i t L e n s *
CALVIN HANNA ,~N]) I-IENRY C. ~EATTS
Departme.nt of Pharmaco~.vgy, U niversit?] of ,4r]ean~.~as Medical Center, Little Rock, Arkansas, U.S.A. (Received 28 August 1967, Bosto~O The loctttion of dividing epit.hcli~l cells and the s u b s e q u e n t migratiot~ of these cells int~ the lens subst, a.nee of tim a d u l t r a t trod rabbit¢ were d e t e r m i n e d using ingrac~meral [a.I4g]lAxymidine and autoradiographic techniques. The germ inat.ive zone of the Ions, located just ant,crier to the lens equator, wins an average of five cx~lls wide. One of the.so cells migrated posterioriy each d a y a n d became a new lens cortex fiber in an ~verage of 6 and 8 weeks fi)r the rat and rabbit, respectively. Each new cortex fiber migrated inw~,rd forming a lens fiber w i t h o u t a nucleus in an average of 6 and I0 weeks for the r a t ~nd r~bbit, respectively.
1. Introduction T h e g e r m i t l a t i v e z o n e o f t h e r a t , r a b b i t , a n d h m n a n l e n s is loc,~ted in ~ r a t h e r n a r r o w b a n d o f cells jtLst a n t e r i o r t o t h e l e ~ s e q u a t o r . I n t h i s z o n e o r p r o g e l l i t o r c o m p a r t m e n t are fomld cells undergoing DNA synthesisand m i t o s i s ( i ~ a . m m a n d 0 : B r m n , 1961). U s i n g [ a ] ~ [ ] O l y m i d i n e , H a n n a a n d O'Bric~t ( 1 9 6 0 a , b , 1')61, 1.963) d e t e r m i n c ( t t h a t t h e r a t e o f m i g r a t i o n o f cells f r o m t h e p r o g e n i t o r c o m p a r t m c : n t a n d i n t o t h e l e n s s u b s t a . n c e w a s r e l a t e d t o t h e a g e o f t h e r~tt. A s t h e s e cells r a p i d l y l e f t t h e p r o g e n i t o r c o m 1)artment of the young rat, they were replaced by new cells indicating ~ stead), state renewal system. L i t t l e is k n o w n a b o u t t h e r a t e o f m i g T a t i o n a n d s u b s e q u e n t d i f l ' c r e n t i a t i o n o f et)it h e l i a l c e l l s o f t h e l e n s o f t h e a d u l t a~fimal, i n c l u d i n g m a n ( H a r e m , 1965). T h i s r c l ) o r c det-fils tli~ m i g r a t i o n o f l e n s e p i t h e l i a l ceils a n d t h e i r d i f f c r e x l t i a t i o n i n t o n o n - n u c l e a t e d l e n s f i b e r s i n t h e a d u l t r a t a-nd r a b b i t .
2. Materials and Methods One-year-old albino rats were briefly anesthetized with ether, and 1-year-old albino r a b b i t ~ w e r e b r i e f l y a n e s t h e t i z e d ~ d t h i n t r a v e n o u s 1-5°/o t h i o p e n t a l s o 4 i u m . D u r i n g a n e s t h e s i a a n i n t r a e a m e r a l i n j e c t i o n of 5 t~c of [aHJthymiclil~e in 0.005 m l of w a t e r (10 c / m m o l e ; S c h w a r z . B i o R e s e a r c h , i n c . , Ora n g e b m ' g , N.Y., U.S.A.) w a s m a d e i n t o e a c h e y e . :Rats a n d r a b b i t s w e r e k i l l e d in ])airs, 2 lind 2,1- hr; 1, 2 al~d 3 w e e k s a n d 1, 1½, 2, 2½, 3 a n d 3½ m o n t h s l a t e r . G r o u p s of 4 r a b b i t s a n d r a t s were kille'.t 4, 5, 6 , 6½ a n d 7 m o n t h s l a t e r . T h e e y e s Were r e m o v e d , fixed in f o r m a l i n , e m b e d d e d in p a r a f f i n , a n d s e c t i o n s 5/z in t h i c k n e s s were cut. t h r o u g h t h e s a g i t t a l p l a n e of t h e eye. T h e s e c t i o n s w e r e p a i n t e d ~ d t h K o d a k N T B 3 e m u l s i o n a n d d e v e l o p e d i n K o d a k D-19 1 m o n t h l a t e r (I-Ianna a n d O ' B r i e n , 1960a,b, 1963). T h e s e c t i o n s w e r e s t a i n e d w i t h 1 ~ ) t o l u i d i n e b l u e in 2 % a q u e o u s p h e n o l a n d w i t h 1 % a q u e o u s eosin.
3. Results T h e ~ n t e r i o r t o p o s t e r i o r s p r e a d b e t w e e n t h e a I { - l a b e l e d e p i t h e l i a l cells o f t h e r,~t l e n s ~ v e r a g c d 4 cell w i d t h s . T h i s w a s d e t e r m i n e d f r o m s e c t i o n s 5 tL i n t h i c k n e s s c u t * Tiffs investigation was supported by :PHS research grants (NJ~,-402~I and ~]$-5076) from the National Institute of ~eurological Diseases and :BliD.c~,ess, U.S. Public ~ c a l t h Service.
244
LENS
CELL
5IIGRATION
245
from the sagitta.l plane of the lens, and there was a n average of 1½ aI=l:-labeled cells in each h a l f of the lens scctio,t 2 h r after the injection of [aH]thymidine. T h a t is, in the rat the progenitor compartnml~t is a b o u t 4- cells deep and contahm a b o u t 1½ cells m~dergoi:.,g DNA synthesis. I n lens sections t~om rats ldlled 1-10 weeks after the injectiou of [al:I]thymidinc the ,um~ber of all-labeled cells was n e a r l y the same. An occasional labeled cell was found in the ])roge,litor c o m p a r t m e n t for t h e first 3 weeks. I n time the "~H-labelcd cells were retold fm'ther a11(l f u r t h e r anterior from the progcnit.or c o m p a r t m e n t until the labeled cells were /bund entering the leas cortex a t an average of 6 weeks (Table I). O n the a~,erage, the aII-labeled ceils m i g r a t e d from ~he progenitor c o m l ) a r t m e a t for a distance of a.bout 148 cell widths m 4½ months, or a cell migrated out of the l)rogenitor coml)artment, each day. '.['A ~BLI'-" I
Position of all-labeled cells in the rat a ~ l rabbit lens with time
Animal no.
Time (m(,nths)
A v e r a g e cell w i d t h s f r o m a l { - l a b e l c d cell t o the: start. ¢)f c e n t e r of s t a r t of lens eort.cx lens c o r t e x lens n u c l e u s
J, '2 13, 14 15, 16 19, 20 2I ,~o 23, 24 25-28 29-32
(2 h,') 1 l~ 2-.~ 3 3} 4 5
42 9 0
90 63 4S S 0
14S 112 97 59 50 36 7
~abbits 1, 2 15, IG 17, IS 19, 20 23, 24 25-28 29-32 33-36 37 --40 41-44
(2 Kr) 1~ 2 2½ 3.~. 4 5 6 6½ 7
6,q 15 4 0
132 103 97 5"2 3g 16
2°8 172 161 133 109 78 -53 2l 3
The results using t h e a d u l t r a b b i t were similar to those obtained from the r a t with the excel)tions t h a t : (a) tile progeJtitor c o m p a r t m e n t averaged 5 cells wide and; (b) t h e distances traveled i~l ~he r a b b i t lenses were greater (Table I). 4. Discussion
The rates of migTatioll of lens cells in t h e r a t and r a b b i t were quite similar, i.e. a b o u t a cell width per day. Possibly this is related to the f a c t t h a t b o t h attimals are rodmlts. :Results in m a n - - c o t f l d t h e y be d e t e r m i r m d - - w o u l d be similar because a b o u t l½ ceils in a section 5/z hi tlfickness cut on t h e sagittal plane itmorporate's [aIt]th)nxaidiae ix1 2 hr, a n d t h e progenitor cort~partmellt is not a p p r e c i a b l y h~rger (I-Ia~ma, Bick_nell a n d O'Brien, 1961). This rate of mig,ratiott of epithelial cells does n o t a p p e a r to hold for
246
CALVIN If[ANNA AN]) I[ENRY C. K]",ATTS
the chic'lcen a.ml ]~ossibly rt.:i)tilcs ( H a m ~ ~md ()'Brie~l, 1961; Hnmia, 1965; and t Tanna mtd Keatts, ] 966). The c])it, helia of the adult, rat, all(1 rabbit lens are charact:eriz(,.d by the addition of tlew ccll:~ h'om a sm:dl Zolle of ceils (germimllive zone or progctfitor compal'tment) t h a t r(;l)la(:e those lost I)y continuous diffcrenlial;io~ into new lens libels. '.['h(, ratt, of m.w cell 1)r,)duct,i(m o~: i,urnover in t.he l~rogenit9~' ('Oml);U'tmc~t, c;m he est, imatcd from the rate ot' cell difl'crentiat, iolt into lc~s fibers ir~ the absence of cell loss (Bert:alanff)', 1964). 'L'h(, munber of :~l-I-I;~l)eled cells ilt t,he Lel~s of the ral~ and rabbit, rcm~inc(l ne~lrly coltstal~ between 1 and 10 weclcs after the inj¢~ct,io~ of [al []th)'midirte i~dicating n,) loss of all-labeled cells. Siuce the difl'crent.i:~,t.ing cells migrated one c(:l[ ~vidt,h l)(;r day al~d sin('c the ln'ogenitor (:Oml~artme~tt. is ,'¢-5 cells ~'ide the ttm~o~;t,r t,i~tc is 3-5 days. This turnover tim,, is in good agrce~tc~t, with t.ha~ reported by 5]iIculicict~ and Yottng (1963) for thc l 0-d~y-old rat, a~t(l rel~ )rtcd l:y ][amm (1955) for th~ yotmg mouse of 54 hr and 56 hr, resl)ectivcly, where rh(, turnover time was calculated from the per ccitt of aH-l~fl)elcd celts i~ the le~s 1 hr following ~he injc(:.tiot~ of [a] []thymidi~e. REJ?EI-'iENCES Bertalanfl)'. F. 1). (1964), Lab. lnwst. 13, 871. .Hanna, C.. (1965). Invest Ophthal~ntal. 4, 480. Ha m~a, C., Bicknell, D. S. and O'Bricn, J. 15. (1961). Arch. Ophthalmot. (Chicago) 65, (;96. Harma, C. and ].