Parietal cell regeneration in rat gastric mucosal wounds: a quantitative light and electron microscopical study

RAT GASTRIC MUCOSAL WOUNDS

A quantitative light and electron microscopical study

AKADEMISK AVHANDLING

SOM MED VEDERBÖRLIGT TILLSTÅND AV REKTORSÄMBETET VID UMEÅ UNIVERSITET FÖR AVLÄGGANDE AV MEDICINE DOKTORSEXAMEN

KOMMER ATT OFFENTLIGEN FÖRSVARAS I ANATOMISKA INSTITUTIONENS STORA FÖRELÄSNINGSSAL

I UMEÅ, DEN 28 APRIL 1982, KL 0 9 .0 0

av

HÅKAN BLOM

EX . LÄ K A R E

UMEÅ 1982

New S e r i e s No 81 - ISSN 0346-6612

ABSTRACT

PARIETAL CELL REGENERATION IN RAT GASTRIC MUCOSAL WOUNDS A q u a n t i t a t i v e l i g h t and e l e c t r o n m i c r o s c o p i c a l s tu dy

Håkan Blom, M.D. Department of Anatomy, U n i v e r s i t y of Umeå, S-901 87 Umeå, Sweden

The aims o f t h e s t u dy were t o o b t a i n a method wi th which i t would be p o s s i b l e t o produce s t a n d a r d i z e d wounds in t h e g a s t r i c mucosa, and t o f o l l o w t h e r e g e n e r a t i o n of t h e a c i d produci ng p a r i e t a l c e l l s i n t h o s e l e s i o n s d u r i n g d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . Q u a n t i t a t i v e methods a p p l i e d t o l i g h t and e l e c t r o n microscopy were used.

Wounds were c a u t e r i z e d in t h e corpus mucosa in Sprague-Dawley r a t s and i n a d d i t i o n , p y l o r o p l a s t y , t r u n c a l vagotomy with p y l o r o p l a s t y or a n t - rectoiriy were p erf or med. Ot he r groups of r a t s wi th wounds were given l o n g - t e r m t r e a t m e n t wi th p e n t a g a s t r i n or c i m e t i d i n e . S t i m u l a t i o n t e s t s were c a r r i e d out in two groups of wound o p e r a t e d r a t s .

A f t e r d i f f e r e n t p e r i o d s of t i m e t h e ani ma ls were p e r f u s i o n f i x e d and specimens from t h e wounds and normal mucosa b e s i d e t h e wounds were p r e ­ pa re d f o r l i g h t and e l e c t r o n mi cr osc opy. By means of s t e r e o l o g i c a l t e c h n i q u e s , d i f f e r e n t mucosal and c e l l u l a r s t r u c t u r e s were t h en measur­

ed.

P a r i e t a l c e l l s were found in 90 days ol d wounds. At t h i s s t a g e t h e y were immature with l a r g e n u c l e i and few s p e c i a l i z e d c e l l o r g a n e l l e s . In s p i t e o f t h i s a pp e ar a nc e t h e y were a b l e t o respond m o r p h o l o g i c a l l y t o s t i m u l a t i o n and t o s e c r e t e a c i d . With f u r t h e r h e a l i n g t h e morphology of t h e p a r i e t a l c e l l s became normal, but t h e i r volume f r a c t i o n in t h e mucosa remained subnormal. The f r a c t i o n of mucosa oc cupied by e p i t h e l ­

i a l c e l l s a l s o s t a y e d lower t h an normal.

P y l o r o p l a s t y r e s u l t e d in d e c r e a s e d c e l l and n u c l e a r s i z e of both normal and r e g e n e r a t i n g p a r i e t a l c e l l s . In t h e l a t t e r , t h e r e was a l s o a de­

c r e a s e in t h e m i t o c h o n d r i a l volume d e n s i t y . I f a t r u n c a l vagotomy was added t o t h e p y l o r o p l a s t y t h e s e changes d i s a p p e a r e d and, in a d d i t i o n , an i n c r e a s e in p a r i e t a l c e l l volume d e n s i t y was n o t i c e d in t h e normal mucosa.

Antrectorny produced s m a l l e r p a r i e t a l c e l l s , and t h e i r m a t u r a t i o n was d e l a y e d . F u r t h e r m o r e , mucosal t h i c k n e s s d e c r e a s e d . I f p e n t a g a s t r i n was gi ven t o r a t s wi th wounds an i n c r e a s e i n t h e number of p a r i e t a l c e l l s was n o t e d , but m a t u r a t i o n and morphology remained u n a f f e c t e d .

C i m e t i d i n e t r e a t m e n t di d not a f f e c t t h e p a r i e t a l c e l l volume d e n s i t y in wounds o r normal mucosa. However, a l a r g e i n c r e a s e i n t h e s e c r e t o r y s u r f a c e d e n s i t y was n o t i c e d when t h e e f f e c t of t h e l a s t dose had c e a s ­ ed.

Key words: R a t , g a s t r i c mucosa, g a s t r i c wounds, p a r i e t a l c e l l s , r e ­ g e n e r a t i o n , p y l o r o p l a s t y , t r u n c a l vagotomy, a n tr e c t o m y , pen­

t a g a s t r i n , c i m e t i d i n e , c a r b a c h o l i n e , s t e r e o l o g y , l i g h t

mi cr osc op y, e l e c t r o n micr oscopy, p H - s t u d i e s .

New Series No 81

From the Department o f Anatomy, Umeå University, Umeå, Sweden.

PARIETAL CELL REGENERATION IN RAT GASTRIC MUCOSAL WOUNDS

A quantitative light and electron microscopical study

by

Håkan Blom

Umeå 1982

The aims o f t h e s t u d y were t o o b t a i n a method wi t h which i t would be p o s s i b l e t o produce s t a n d a r d i z e d wounds in t h e g a s t r i c mucosa, and t o f o l l o w t h e r e g e n e r a t i o n o f t h e a c i d p r oduci ng p a r i e t a l c e l l s in t h o s e l e s i o n s d u r i n g d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . Q u a n t i t a t i v e methods a p p l i e d t o l i g h t and e l e c t r o n microscopy were used.

Wounds were c a u t e r i z e d in t h e corpus mucosa in Sprague-Dawley r a t s and i n a d d i t i o n , p y l o r o p l a s t y , t r u n c a l vagotomy wi th p y l o r o p l a s t y or a n t ­ rectomy were pe rf or me d. Ot he r groups o f r a t s with wounds were gi ven l o n g - t e r m t r e a t m e n t wi th p e n t a g a s t r i n or c i m e t i d i n e . S t i m u l a t i o n t e s t s were c a r r i e d out in two groups of wound o p e r a t e d r a t s .

A f t e r d i f f e r e n t p e r i o d s of t im e t h e a ni ma ls were p e r f u s i o n f i x e d and speci mens from t h e wounds and normal mucosa b e s i d e t h e wounds were p r e ­ p a r e d f o r l i g h t and e l e c t r o n m ic rosc opy. By means of s t e r e o l o g i c a l t e c h n i q u e s , d i f f e r e n t mucosal and c e l l u l a r s t r u c t u r e s were t h en measur­

ed.

P a r i e t a l c e l l s were found in 90 days ol d wounds. At t h i s s t a g e t h e y were immature wi t h l a r g e n u c l e i and few s p e c i a l i z e d c e l l o r g a n e l l e s . In s p i t e o f t h i s a p p e ar a nc e t h e y were a b l e t o respond m o r p h o l o g i c a l l y t o s t i m u l a t i o n and t o s e c r e t e a c i d . With f u r t h e r h e a l i n g t h e morphology of t h e p a r i e t a l c e l l s became normal, but t h e i r volume f r a c t i o n in t h e mucosa remained subnormal. The f r a c t i o n of mucosa oc cu pi ed by e p i t h e l ­ i a l c e l l s a l s o s t a y e d lower t h a n normal.

P y l o r o p l a s t y r e s u l t e d in d e c r e a s e d c e l l and n u c l e a r s i z e of both normal and r e g e n e r a t i n g p a r i e t a l c e l l s . In t h e l a t t e r , t h e r e was a l s o a d e­

c r e a s e in t h e m i t o c h o n d r i a l volume d e n s i t y . I f a t r u n c a l vagotomy was added t o t h e p y l o r o p l a s t y t h e s e changes d i s a p p e a r e d and, in a d d i t i o n , an i n c r e a s e in p a r i e t a l c e l l voi urne d e n s i t y was n o t i c e d in t h e normal mucosa.

Antrectomy produced s m a l l e r p a r i e t a l c e l l s , and t h e i r m a t u r a t i o n was d e l a y e d . F u r t h e r m o r e , mucosal t h i c k n e s s d e c r e a s e d . I f p e n t a g a s t r i n was g i ve n t o r a t s wi t h wounds an i n c r e a s e i n t h e number of p a r i e t a l c e l l s was n o t e d , but m a t u r a t i o n and morphology remained u n a f f e c t e d .

C i m e t i d i n e t r e a t m e n t di d not a f f e c t t h e p a r i e t a l c e l l volume d e n s i t y in wounds o r normal mucosa. However, a l a r g e i n c r e a s e in t h e s e c r e t o r y s u r f a c e d e n s i t y was n o t i c e d when t h e e f f e c t of t h e l a s t dose had c e a s ­ e d .

Key words: R a t , g a s t r i c mucosa, g a s t r i c wounds, p a r i e t a l c e l l s , r e ­ g e n e r a t i o n , p y l o r o p l a s t y , t r u n c a l vagotomy, a n t r e c t o m y , pen­

t a g a s t r i n , c i m e t i d i n e , c a r b a c h o l i n e , s t e r e o l o g y , l i g h t

m ic r o sc o p y , e l e c t r o n mic rosc opy, p H - s t u d i e s .

Why i s my wound i n c u r a b l e which r e f u s e t h t o be h e a l e d . . .

J e r . 15:18

CONTENTS

REPORTS CONSTITUTING THE THESIS ... 7

INTRODUCTION ... 9

Exp er iment al u l c e r s t u d i e s ... 9

The p a r i e t a l c e l l s ... 11

AIMS OF THE STUDY ... 14

MATERIALS AND METHODS ... 15

Animals ... 15

O p e r a t i o n s ... 15

P o s t o p e r a t i v e c a r e ... 17

Drug a d m i n i s t r a t i o n ... 17

S t i m u l a t i o n t e s t s ... 18

T i s s u e p r e p a r a t i o n ... 19

S t e r e o l o g i c a l a n a l y s e s ... 20

C o r r e c t i o n o f s y s t e m a t i c e r r o r s c o nne ct ed wi th t h e s t e r e o l o g i c a l methods ... 23

S t a t i s t i c a l a n a l y s i s ... 25

RESULTS ... 26

Animal r e c o v e r y a f t e r t h e d i f f e r e n t s u r g i c a l p r o c e d u r e s ... 26

Normal r e g e n e r a t i o n ... 26

The r e s p o n s e t o s t i m u l a t i o n in nor ma lly r e g e n e r a t i ng p a r i e t a l c e l l s ... 28

E f f e c t s o f p y l o r o p l a s t y , p y l o r o p l a s t y in c o mbi na ti on wi t h t r u n c a l vagotomy, and a nt re ct o my ... 28

E f f e c t s of l o n g - t e r m a d m i n i s t r a t i o n of p e n t a g a s t r i n ... 29

E f f e c t s of l o n g - t e r m t r e a t m e n t wi t h C i m et i d i n e ... 30

DISCUSSION ... 31

The u l c e r model ... 31

T i s s u e p r e p a r a t i o n ... 32

The s t e r e o l o g i c a l t e c h n i q u e s ... 33

P a r i e t a l c e l l r e g e n e r a t i o n ... 33

E f f e c t s o f p h y s i o l o g i c a l and ph a rm ac ol o gi ca l s t i m u l a t i o n on t h e p a r i e t a l c e l l r e g e n e r a t i o n ... 35

H2 - r e c e p t o r bl ocka de and p a r i e t a l c e l l r e g e n e r a t i o n and

morphology ... 37

SUMMARY AND CONCLUSIONS ... 38 ACKNOWLEDGEMENTS ... 39 REFERENCES ... 40 PAPER I

Q u a n t i t a t i v e u l t r a s t r u c t u r a l s t u d i e s on p a r i e t a l c e l l r e ­

g e n e r a t i o n i n e x p e r i m e n t a l u l c e r s in r a t g a s t r i c mucosa ... 47 PAPER II

Immature p a r i e t a l c e l l s in h e a l i n g g a s t r i c wounds r espond

t o s t i m u l a t i o n . An e x p e r i m e n t a l s tu d y in r a t s ... 57 PAPER I I I

E f f e c t s o f t r u n c a l vagotomy, p y l o r o p l a s t y , and a n tr e ct o my on p a r i e t a l c e l l r e g e n e r a t i o n in e x p e r i m e n t a l g a s t r i c wounds

in t h e r a t . A l i g h t and e l e c t r o n m i c r o s c o p i c a l s t u d y ... 71 PAPER IV

I s t h e r e a t r o p h i c e f f e c t of p e n t a g a s t r i n on r e g e n e r a t i n g

p a r i e t a l c e l l s in r a t g a s t r i c mucosal wounds? ... 91 PAPER V

C i m e t i d i n e and p a r i e t a l c e l l r e g e n e r a t i o n in e x p er i m en t al wounds i n r a t g a s t r i c mucosa. A l i g h t and e l e c t r o n m i c r o ­

s c o p i c a l s t u dy ... 105

REPORTS CONSTITUTING THE THESIS

T hi s t h e s i s i s based on t h e f o l l o w i n g p u b l i c a t i o n s and m a n u s c r i p t s , which w i l l be r e f e r r e d t o by t h e i r Roman numerals:

I . Blom H. and H e l an de r H . F . : Q u a n t i t a t i v e U l t r a s t r u c t u r a l s t u d i e s on p a r i e t a l c e l l r e g e n e r a t i o n in e x pe r i me n t a l u l c e r s in r a t g a s t r i c mucosa. G a s t r o e n t e r o l o g y 1931: 80: 334 - 343.

I I . B l o m H . : Immature p a r i e t a l c e l l s in h e a l i n g g a s t r i c wounds r espond t o s t i m u l a t i o n . An e x p e r i m e n t a l s t u dy in t h e r a t . D i g e s t i o n , in p r e s s .

I I I . Blom H. , El s t i g H. and H e l an de r H .F .: E f f e c t s of t r u n c a l vagotomy, p y l o r o p l a s t y , and a n t re ct o my on p a r i e t a l c e l l r e g e n e r a t i o n in ex­

p e r i m e n t a l g a s t r i c wounds in t h e r a t . A l i g h t and e l e c t r o n mi cr o­

s c o p i c a l s t u d y . Accepted in Scand J G a s t r o e n t .

IV. Bl omH. and E r i k o i n e n T . : Is t h e r e a t r o p h i c e f f e c t of p e n t a g a s t - r i n on r e g e n e r a t i n g p a r i e t a l c e l l s in r a t g a s t r i c mucosal wounds?

( Su bmi t te d f o r p u b l i c a t i o n ) .

V. B l o m H . : C i m e t i d i n e and p a r i e t a l c e l l r e g e n e r a t i o n in e x p er i m en t al wounds i n r a t g a s t r i c mucosa. A l i g h t and e l e c t r o n m i c r o s c o p i c a l

s t u d y . Accepted in Scand J G a s t r o e n t .

INTRODUCTION

Exp er imen ta l u l c e r s t u d i e s

In 1586 Donati p u b l i s h e d t h e f i r s t c l e a r r e p o r t s on t h e g a s t r i c u l c e r d i s e a s e ( 1 ) . Two c e n t u r i e s l a t e r Abercrombie (2) and C r u v e i l h i e r (3) d e s c r i b e d t h e g a s t r i c l e s i o n s , and t h e y a l s o s u g g e s t e d c a u s a t i v e f a c t o r s . At t h i s t i m e , d i f f e r e n t models f o r s t u d y i n g u l c e r e t i o l o g y and h e a l i n g began t o a p p ea r ( 4 ) , and in 1875 Quincke (5) d e s c r i b e d t h r e e p o s s i b l e ways t o pr oduce g a s t r i c wounds in dogs , v i z . by p r e s s u r e , by l i g a t i o n of v e s s e l s , and by h e at a p p l i e d by hot i r o n s .

Exp er iment al u l c e r s * have s i n c e been produced in many d i f f e r e n t ways ( 6 ) , p a r t l y depending on t h e aims of t h e s t u d y . Thus, i f t h e i n t e r e s t has been f o cu s ed on dr ugs a n d / o r s u r g i c a l p r o c e d u r e s which can p r e v e n t t h e a p p e ar a nc e of mucosal l e s i o n s , methods have been chosen which r e ­ s u l t in a c o n s i s t e n t l y high i n c i d e n c e o f r e a d i l y d i s c e r n i b l e u l c e r s . Such methods i n c l u d e l i g a t i o n of t h e p y l o r u s in r a t s ( t h e so c a l l e d S h a y - r a t , 7 ) , d i f f e r e n t ki nds of s t r e s s (8 - 12) , and a d m i n i s t r a t i o n of v a r i o u s u l c e r o g e n i c d r u g s , such as h i s t a m i n e (13 - 1 4 ) , r e s e r p i n e (15 - 1 7 ) , a d r e n a l i n e (18 - 1 9 ) , c o r t i s o n e (20 - 2 3 ) , and o t h e r a n t i i n f l a m ­ mat ory drugs (16, 24 - 2 8 ) .

All t h e s e methods have in common t h a t t h ey produce s u p e r f i c i a l e r o s i o n s ( 2 9 ) , seldom r e a c h i n g below t h e m u s c u l a r i s mucosæ, r a t h e r t h a n deep p e n e t r a t i n g " c h r o n i c " l e s i o n s r es e m b l i n g human g a s t r i c u l c e r s (30 - 3 2 ) . The number of t h e s e s u p e r f i c i a l l e s i o n s and t h e i r e x a c t l o c a t i o n c a n no t be p r e d i c t e d .

* The t er m u l c e r r e f e r s t o t i s s u e l e s i o n s which a r i s e from t h e a c t i o n

o f c o r r o s i v e a g e n t s , whereas wounds r e s u l t from mechanical t ra u ma . In

e x p e r i m e n t a l r e s e a r c h on g a s t r i c l e s i o n s , mechanical methods of v a r i o u s

k i n d s ( v i d e i n f r a ) a r e o f t e n employed t o produce t h e p ri ma ry wound in

t h e mucosa. However, t h e s u b s eq ue n t development and u l t i m a t e h e a l i n g of

such wounds a r e o b v i o u s l y i n f l u e n c e d by t h e g a s t r i c j u i c e , i n c l u d i n g

i t s c o r r o s i v e a c i d . Ag a i n s t t h i s background i t i s not e n t i r e l y obvious

w h et h e r t h e t er m g a s t r i c wound or u l c e r should be p r e f e r r e d . Thus, in

t h i s p u b l i c a t i o n t h e t er ms have been used in p a r a l l e l .

S i m i l a r e r o s i o n s can a l s o be produced by s t i m u l a t i o n a n d / o r i n j u r i n g of t h e b r a i n , p r e d o m i n a n t l y t h e hypothalamus ( f o r r evi ew see 3 3 ) .

Thus , i f t h e s e p r o c e d u r e s a r e a p p r o p r i a t e when one i s i n t e r e s t e d in s t u d y i n g how t o p r e v e n t t h e o c c u r r e n c e of mucosal damage, t h ey may be l e s s s u i t a b l e i f u l c e r h e a l i n g t e s t s a r e t o be pe rf or me d, mainly be­

c a u s e t h e y c annot be s t a n d a r d i z e d wi t h r e s p e c t t o l o c a l i z a t i o n and d e p t h .

S i n c e most t h e r a p y c on c er n s h e a l i n g of an a l r e a d y e x i s t i n g l e s i o n , a t t e m p t s have been made t o produce s t a n d a r d i z e d wounds which morpholog­

i c a l l y r es embl e human c h r o n i c u l c e r s . Such wounds can be o bt a i n e d in many d i f f e r e n t ways, but t h e most common p r oc e d u r e i s pr ob ab ly s u r g i c a l e x c i s i o n o f p a r t s of t h e t h e mucosa, u s u a l l y down t o , or i n c l u d i n g , t he m u s c u l a r i s mucosæ (34 - 4 0 ) . C a u t e r i z a t i o n by hot i r o n s (41 - 46) and l i g a t i o n o f blood v e s s e l s (47) have a l s o been employed. In 1969 Takagi e t a l . (48) d e s c r i b e d a method " f o r p r o d u c t i o n of c h r o n i c g a s t r i c u l ­ c e r s " by i n j e c t i o n s of a c e t i c a c i d i n t o t h e s u b s e r o s a l l a y e r s of t he g l a n d u l a r mucosa, and t h i s p r oc e d u r e has l a t e r been modif ied and used a l s o by o t h e r s (49 - 5 1 ) . Recent models i n c l u d e p h o t o c o a g u l a t i o n by l a s e r (52 - 53) and f r e e z i n g o f t h e mucosa by means of a cryoprobe

( 5 4 ) .

In most u l c e r h e a l i n g s t u d i e s t h e p r i n c i p a l i n t e r e s t has been t o d e t e r ­ mine how l ong t i m e i t t a k e s u n t i l t h e wound i s c ove re d by e p i t h e l i u m and how t h i s t im e can be a f f e c t e d . Less i n t e r e s t has been devoted t o t h e e v e n t s o c c u r r i n g on t h e c e l l u l a r , or s u b c e l l u l a r , l e v e l du ri ng r e ­

g e n e r a t i o n . The m a j o r i t y o f s t u d i e s p u b l i s h e d on t h i s s u b j e c t a re based on o b s e r v a t i o n s made by l i g h t microscopy (34 - 38, 40 - 47, 49, 53 -

54) and onl y a few i n v e s t i g a t o r s have u t i l i z e d t h e e l e c t r o n microscope (39, 55 - 5 6 ) .

At t h e l i g h t m i c r o s c o p i c a l l e v e l i t i s d i f f i c u l t t o d i f f e r e n t i a t e t h e

e p i t h e l i a l c e l l t y p e s in t h e g a s t r i c mucosa wi th a c c u r a c y , and s i n c e

r e g e n e r a t i n g c e l l s in a h e a l i n g wound d i f f e r m o r p h o l o g i c a l l y from t h e i r

normal c o u n t e r p a r t s , l i g h t m i c r o s c o p i c a l i d e n t i f i c a t i o n becomes uncer-

t a i n . Thi s f a c t p r o b a b l y c o n t r i b u t e s to t h e d i v e r g e n t o p i n i o n s e x p r e s s ­ ed by d i f f e r e n t a u t h o r s , e . g . wi t h r e g a r d t o t h e p a r i e t a l c e l l s . Thus, some i n v e s t i g a t o r s b e l i e v e t h a t p a r i e t a l c e l l s a r e p r e s e n t in h e a le d u l c e r s (34, 36 - 39, 42, 44, 47, 5 6 ) , wh il e o t h e r s c l a i m t h a t t h e s e c e l l s never r e a p p e a r (35, 41, 46, 5 4 ) .

In p r e v i o u s u l c e r h e a l i n g s t u d i e s no a t t e m p t s have been made t o q u a n t i ­ t a t e t h e r e g e n e r a i o n of t h e d i f f e r e n t e p i t h e l i a l c e l l t y p e s , and c o n s e ­ q u e n t l y i t has not been p o s s i b l e t o d e c i d e when a l e s i o n i s " c o m p l e t e l y h e a l e d " . F u r t h e r m o r e , up t o d a t e no s y s t e m a t i c i n f o r m a t i o n has been a v a i l a b l e on t h e morphol og ic al e v e n t s o c c u r r i n g w i t h i n t h e c e l l s in h e a l i n g g a s t r i c wounds.

The p a r i e t a l c e l l s

The p a r i e t a l c e l l s - sometimes r e f e r r e d t o as t h e o x y n t i c (from t h e Greek o x y s - a c i d ) c e l l s - a r e found mainly in t h e c o r p u s , or f u n d i c , mucosa in t h e mammalian stomach ( 5 7 ) . The c e l l s a r e r e l a t i v e l y l a r g e ,

i n t h e r a t t h e voi urne has been c a l c u l a t e d t o about 1100 pm^ (58) and i n man t o 5500 pnP ( 5 9 ) , and t h e y a r e e l l i p s o i d a l o r c o n i c a l in s h a p e . They a r e s i t u a t e d p e r i p h e r a l l y along t h e gland t u b u l e s ( p a r i e t a l p o s i t i o n ) , r e s u l t i n g in k n o b - l i k e p r o t r u s i o n s ( 6 0 ) . They a r e c h a r a c t e r ­ i z e d by a system o f s e c r e t o r y c a n a l i c u l i ( F i g . 1 ) , which were f i r s t d e s c r i b e d by Golgi in 1893 ( 6 1 ) . The i n t r a c e l l u l a r l o c a t i o n of t h e s e c a n a l i c u l i was e s t a b l i s h e d by Zimmermann in 1898 ( 62) .

A r ema rk ab ly high amount of m i t o c h o n d r i a , occupying between 22 t o 47%

o f t h e c y t o p l a s m i c volume (58, 63 - 6 6 ) , wi t h d e n s e l y packed c r i s t a e s u g g e s t s a hi gh o x i d a t i v e met abo li sm in t h e p a r i e t a l c e l l s (57) and t h i s f e a t u r e has been u t i l i z e d f o r h i s t o c h e m i c a l i d e n t i f i c a t i o n ( 4 6 ).

Th us , under normal c o n d i t i o n s t h e s u c c i n i c de hydroge na se (SO) a c t i v i t y

i s much h i g h e r in t h e p a r i e t a l c e l l s t han in any o t h e r e p i t h e l i a l c e l l

t y p e in t h e g a s t r i c mucosa ( 6 7 ).

Fi g . 1. Mature p a r i e t a l c e l l from a s t a r v e d r a t . The c e l l i s c o n i c a l in

shape wi th t h e n uc le us (N) s i t u a t e d p e r i p h e r a l l y . There i s an

abundance of m i t o c h o n d r i a (M) and in between t h e s e o r g a n e l l e s

many t u b u l o - v e s i c l e s ( arrow) a r e s een. The c e l l i s not s t i m u l ­

a t e d , h enc e, t h e i n t r a c e l 1u l a r c a n a l i c u l a r system (C) i s p o or l y

d e v e l o p e d .

Anot her c h a r a c t e r ! S t i c f e a t u r e i s t h e p r e s e n c e of a v a r y i n g amount of c y t o p l a s m i c t u b u l e s a n d / o r v e s i c l e s , o f t e n r e f e r r e d t o as t u b u l o - v e s i - c l e s ( 5 7 ) . The t u b u l o - v e s i c l e s and t h e i n t r a c e l l u l a r c a n a l i c u l a r system a r e i n t i m a t e l y i n vo l v e d in t h e s e c r e t o r y p r o c e s s of t h e p a r i e t a l c el I s .

S i n c e t h e s e c r e t o r y p r o d u c t i s h y d r o c h l o r i c a c i d (6 8 ), t h e p a r i e t a l c e l l s a r e d i r e c t l y i n vo l v e d in t h e e t i o l o g y o f t he p e p t i c u l c e r d i s ­ e a s e , and many s t u d i e s have been c a r r i e d out t o c l a r i f y t h e s e c r e t o r y p r o c e s s and how i t can be m o d i f i e d . Morphological i n v e s t i g a t i o n s r eve al t h a t s t i m u l a t i o n r e s u l t s in an i n c r e a s e in t h e a r e a of t h e membrane l i m i t i n g t h e i n t r a c e l 1u l a r c a n a l i c u l i - t h e s e c r e t o r y s u r f a c e - a t which a c i d p r o b ab l y i s produced (6 8 ). This i n c r e a s e i s p a r a l l e l e d by a d e c r e a s e in t h e a r e a of t h e t u b u l o - v e s i c l e s (69 - 7 1) . When s t i m u l a t i o n c e a s e s t h e c e l l r e t u r n s to i t s r e s t i n g s t a t e with many t u b u l o - v e s i c l e s and a r e l a t i v e l y small s e c r e t o r y s u r f a c e a r e a (70 - 7 1) . Based on q u a n t i t a t i v e d a t a i t has been p o s t u l a t e d t h a t t h e t u b u l o - v e s i c u l a r membranes a r e r e v e r s i b l y i n c o r p o r a t e d i n t o t h e c a n a l i c u l a r membrane d u r i n g s t i m u l a t i o n ( 7 2 ) .

V ar i o u s d r u g s , f o r example t h e i ^ - r e c e p t o r a n t a g o n i s t c i r n e t i d i n e , as well as c e r t a i n s u r g i c a l p r o c e d u r e s , v i z . vagotomy and a n t r e c t o m y , can d i m i n i s h a c i d s e c r e t i o n . However, s u r p r i s i n g l y few s t u d i e s have been de v ot ed t o t h e m o rp ho lo gi ca l c ons eq ue n se s of t h e s e t r e a t m e n t s and a v a i l a b l e d a t a a r e c o n t r a d i c t o r y .

Thus , some a u t h o r s have f a i l e d t o d e m o n s t r a t e any s i g n i f i c a n t e f f e c t s o f vagotomy on t h e p a r i e t a l c e l l mass ( 7 3 ) , w hi l e o t h e r s have seen t r a n s i e n t (74 - 75) or c h r o n i c (76) c ha nges .

On t h e o t h e r hand, most i n v e s t i g a t o r s a g r ee t h a t a n t re ct o my r e s u l t s in h y p o p l a s i a of t h e p a r i e t a l c e l l mass. This i s pr o ba bl y an e f f e c t of d e­

c r e a s e d serum g a s t r i n l e v e l s which i s a consequence of an a n tr e ct o my

(77 - 78) . S i n c e g a s t r i n i s known t o e x e r t a t r o p h i c e f f e c t on t h e

g a s t r i c mucosa (78) d e c r e a s e d l e v e l s t hus r e s u l t s in h y p o p l a s i a of t h e

g a s t r o i n t e s t i n a l e p i t h e l i a ( 7 9 ).

I n t r a c e l l u l a r changes a r e minimal in normal p a r i e t a l c e l l s a f t e r vagotomy o r a n t re ct o my ( 5 8 ) , and very l i t t l e i s known about p o s s i b l e e f f e c t s on r e g e n e r a t i n g c e l l s .

In t h e p r e s e n t s e r i e s of s t u d i e s , a s t a n d a r d i z e d method f o r t h e p r odu c­

t i o n o f c h r o n i c g a s t r i c wounds has been d e v e l o p e d , and t h e r e g e n e r a t i o n o f t h e p a r i e t a l c e l l s has been f o l l o we d by means of q u a n t i t a t i v e meth­

ods a p p l i e d t o l i g h t and e l e c t r o n mi cr osc opy. The e f f e c t s of s u r g i c a l and p ha rm ac ol o gi ca l i n t e r f e r e n c e have been i n v e s t i g a t e d .

AIMS OF THE STUDY

A g a i n s t t h e background o u t l i n e d above t h e s p e c i f i c aims of t h e p r e s e n t s t u d y can be summarized as:

- t o d ev el op an u l c e r model wi t h which i t should be p o s s i b l e t o f o l l o w t h e r e g e n e r a t i o n o f e p i t h e l i a l c e l l s in t h e g a s t r i c mucosa under con­

t r o l l e d c o n d i t i o n s by means of q u a n t i t a t i v e t e c h n i q u e s a p p l i e d t o l i g h t and e l e c t r o n microscopy

- t o f o l l o w t h e r e g e n e r a t i o n of t h e a c i d p r oduci ng p a r i e t a l c e l l s under normal c o n d i t i o n s

- t o i n v e s t i g a t e wh et he r r e g e n e r a t i n g p a r i e t a l c e l l s a r e c a p a b l e t o r e ­ spond t o s t i m u l a t i o n

- t o i n v e s t i g a t e i f t r u n c a l vagotomy wi th p y l o r o p l a s t y o r a n tr e ct o my a f f e c t p a r i e t a l c e l l r e g e n e r a t i o n a n d / o r morphology

- t o i n v e s t i g a t e i f p e n t a g a s t r i n or c i m e t i d i n e a d m i n i s t r a t i o n a f f e c t

t h e p a r i e t a l c e l l r e g e n e r a t i o n a n d / o r morphology.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley r a t s ( An ti ci me x, Sweden) were used f o r t h e i n v e s t ­ i g a t i o n s . At t h e b e g i n n i n g of t h e e x p e r i m e n t s t h e y were two t o t h r e e months o l d and wei ghing about 300 g. During t h e e x p e r i m e n t a l p e r i o d t h e y were k ep t s e p a r a t e in normal c a g e s , with f r e e a c c e s s t o w a t e r and a s t a n d a r d l a b o r a t o r y r a t d i e t (ASTRA-EWOS, Sweden).

O p e r a t i o n s

B e f o r e s u r g e r y t h e a n im a ls were f a s t e d f o r 24 hour s in wire-mesh bottom c a g es with f r e e a c c e s s t o w a t e r . A f t e r a n e s t h e s i a wi th sodium p e n t o b a r ­ b i t a l (40 mg/kg) t h e y were s u b j e c t e d t o one or more o f t he f o l l o w i n g o p e r a t i o n s :

C a u t e r i z a t i o n of wounds (I - V). Using a modif ied hemost at ( I ) a wound, 1 .6 cm^ i n s i z e , was c a u t e r i z e d in t h e a n t e r i o r g a s t r i c wall about 3 mm d i s t a l t o t h e t r a n s v e r s e r i d g e . One of t h e shanks of t h e i n s t r u ­ ment was h e a t e d in b o i l i n g w a t e r f o r two m i n u t e s . During t h i s t ime t h e stomach in t h e l a p a r o t o m i z e d animal was t u r n e d i n s i d e out t h ro ug h an i n c i s i o n in t h e rumen. A f t e r c o o l i n g in room t e m p e r a t u r e f o r 20 s econds t h e h emos tat was lo ck ed over t h e g a s t r i c wall wi th t h e hot shank in c o n t a c t wi th t h e mucosa. The i n t i t a l t e m p e r a t u r e d u r i n g c a u t e r i z a t i o n was 70°C and t h e p r e s s u r e a p p l i e d over t h e g a s t r i c wall was c o n s t a n t . A f t e r one minute t h e i n s t r u m e n t was removed, and t o f a c i l i t a t e r e c o g n i ­ t i o n two s i l k s u t u r e s (6 - 0) were put in t h e wound e dg es . The ruminai i n c i s i o n was t h e n c l o s e d and e v e n t u a l f u r t h e r s u r g e r y was c a r r i e d o u t .

P y l o r o p l a s t y ( I I I ) . P y l o r o p l a s t y (58, 80) was performed by making a

l o n g i t u d i n a l i n c i s i o n , about 7 mm l o n g , in t h e p y l o r o- d u od e n al j u n c t i o n

and c l o s i n g t h e opening t r a n s v e r s a l l y wi th s i l k s u t u r e s ( F i g . 2A). This

o p e r a t i o n p r e v e n t s t h e g a s t r i c s t a s i s which f o l l o w s t r u n c a l vagotomy

( 3 1 ) .

F i g . 2. Schemat ic drawings p r e s e n t i n g t h e d i f f e r e n t o p e r a t i o n s pe rf or m­

ed in a d d i t i o n t o c a u t e r i z a t i o n . A: p y l o r o p l a s t y with t r u n c a l

vagotomy. B: a n tr e c t o m y with gastr oduodenost omy (ad modum B i l l ­

r o t h I ) .

Tr uncal vagotomy ( I I I ) . Both vagal t r u n c s were c ut and about 1 cm of them was removed ( 8 2 ) . To e l i m i n a t e any r ema ini ng nerve f i b e r s , t h e muscl e l a y e r s o f t h e esophagus were a l s o c ut j u s t above t h e c a r d i a (58)

( F i g . 2A). The c omp l et en e ss of t h e vagotomy was checked l i g h t m ic r o­

s c o p i c a l l y in p a r a f f i n embedded t i s s u e t ak e n a t t h e t im e of s a c r i f i c e . P y l o r o p l a s t y was always performed t o g e t h e r with t h e vagotomy.

Antrectomy ( I I I ) . The d i s t a l p a r t o f t he stomach and t h e f i r s t 5 mm of t h e duodenum were removed. The r e s e c t e d p o r t i o n of t h e stomach was l i m i t e d by a l i n e from t h e c a r d i a on t h e l e s s e r c u r v a t u r e t o halfway between t h e p y l o r u s and t h e rumen on t h e g r e a t e r c u r v a t u r e . G a s t r o i n ­ t e s t i n a l c o n t i n u i t y was r e e s t a b l i s h e d by an end t o end g a s t r o d u o d e n o s - torrçy (83) ( F i g . 2B). Care was t a k en not t o d i s t u r b t h e a r t e r i a l o r n e r v e su pp ly t o t h e g a s t r i c remnant.

Sham o p e r a t i o n s ( I ) . One group of a ni ma ls was sham o p e r a t e d . The ab­

domen was i n c i s e d and t h e stomach opened t h rough t h e rumen. The mucosa was t hen exposed by t u r n i n g t h e stomach i n s i d e o u t . A f t e r one minute t h e wounds were s u t u r e d .

P o s t o p e r a t i v e c a r e

The a n t r e c t o m i z e d a n im a ls were f a s t e d a l s o a f t e r o p e r a t i o n , and d u r i n g t h i s t ime t h e y r e c e i v e d d a i l y s u bc ut ane ous i n j e c t i o n s wi th 10 ml of Tyrode s o l u t i o n . A f t e r t h r e e days t h e s e r a t s were put on normal d i e t . All t h e o t h e r o p e r a t e d a ni ma ls were r e t u r n e d t o c a g e s , and allowed food and w a t e r i mmed ia te ly a f t e r o p e r a t i o n .

Duri ng t h e e n t i r e e x p e r i m e n t a l p e r i o d a l l r a t s were weighed weekl y, and i f t h e r e were any s i g n s of abnormal weight i n c r e a s e t h e y were d i s c a r d ­ ed.

Drug a d m i n i s t r a t i o n

C i m e t i d i n e (V). The C i m e t i d i n e (Smith, Kl i ne & Fr e nc h, no. 92334) was

d i s s o l v e d in 1 M HC1. Water was t h e n added t o make t h e drug c o n c e n t r a ­

t i o n 25 mg/ml and t h e pH was a d j u s t e d t o around 6 w i t h 0 . 1 H NaOH. The

s o l u t i o n was a d m i n i s t e r e d o r a l l y t w i c e d a i l y ( a t 8 am and 5 pm) t h ro u g h a stomach t u b e , and t h e dose was 75 mg/kg of c i m e t i d i n e each t ime (84 - 8 6 ) . The t r e a t m e n t c o n t i n u e d f o r 130 d a y s . As c o n t r o l s , one group of r a t s was given w a t e r .

P e n t a g a s t r i n ( IV). P e n t a g a s t r i n ( P ep t a v l o n ^ I C I ) was a d m i n i s t e r e d as s u b c u t a n e o u s i n j e c t i o n s t w i c e d a i l y . The hormone, 250 pg/kg ( 8 7 ) , was mixed wi th 16% h y dr o l y z e d g e l a t i n ( C a l c i t o n i n D i l u e n t B, p r e p a r e d by Armour P h a r m a c e u t i c a l Co. ) i mmedi at el y b e f o r e i n j e c t i o n . A c o n t r o l

group r e c e i v e d t h e g e l a t i n d i l u t e d wi th p h y s i o l o g i c a l s a l i n e .

G a s t r i n a n a l y s e s were performed on blood samples t a k e n from t h e a ni ma ls i mm ed i at el y b e f o r e t i s s u e f i x a t i o n . The blood was c e n t r i f u g e d and t h e plasma f r e e z e - d r i e d . The g a s t r i n a s s a y s were t h en performed by Dr. J . F . R e h f e l d , Âr hu s, Denmark.

S t i m u l a t i o n t e s t s

Morphol ogi cal s t u d i e s ( I I ) . Rat s with 90 days old wounds, f a s t e d f o r 24 h o u r s , were us ed. The a ni ma ls were put i n t o Bollman cages and two c a t h ­ e t e r s were p l ac e d s u b c u t a n e o u s l y . P e n t a g a s t r i n , 20 nmol/ kg, h, and c a r - b a c h o l i n e , 110 n mo l/ kg ,h ( He la nde r & S u n d e l l , t o be p u b l i s h e d ) or R i n g e r ' s s o l u t i o n was i n f u s e d c o n t i n u o u s l y d u r i n g one h our . A f t e r t h i s t i m e t h e a ni ma ls were r a p i d l y a n e s t h e t i z e d wi th e t h e r , and t h e stomachs f i x e d as t o be d e s c r i b e d .

pH mea s ur em en ts . ( I I ) . Animals with 90 days old wounds were s t i m u l a t e d as above and were t h e n a n e s t h e t i z e d wi th e t h e r . The stomach was opened and t h e mucosa exposed t h ro ug h a ruminai i n c i s i o n . The wound was l o c a l ­ i z e d and wiped once wi th a dry c o t t o n s t i c k . Small c h i p s of pH-paper

(Pehanon, Fluka AG) were t h e n put on t h e mucosal s u r f a c e in t h e wound,

wound edge and d o r s a l g a s t r i c w a l l . The pH was r e g i s t e r e d when t h e

pH- p a p e r , which had a s p e c i f i c c o l o u r f o r each p H - i n t e r v a l , was m o i s t ­

ene d.

T i s s u e p r e p a r a t i o n

F i x a t i o n p r o c e d u r e s (I - V). All a nima ls were f a s t e d in wire mesh b o t ­ tom c ages f o r 24 hour s b e f o r e s a c r i f i c e . Under a n e s t h e s i a t h e abdomen was c u t open and t h e a o r t a expos ed. A metal c a n n u l a , c on ne ct e d t o a p e r i s t a l t i c pump, was i n t r o d u c e d i n t o t h e v e s s e l and a r i n s i n g s o l u ­ t i o n , a m o d if ie d Tyrode b u f f e r (8 8 ) , was pumped i n t o t h e v a s c u l a r s y s ­ tem. At t h e same t i m e t h e p o r t a l vein was opened, t h e i n t e s t i n e s t i e d o f f , and t h e a o r t a above t h e diaphragm clamped. When t h e p e r f u s e d t i s s u e a p p e ar e d empty of b l o o d , p e r f u s i o n was c o n t i n u e d with f i x a t i v e . At t h e same t im e 10 ml of t h e same s o l u t i o n was i n j e c t e d d i r e c t l y i n t o t h e stomach.

The f i x a t i v e c o n s i s t e d of a m i x t u r e of 4% g l u t a r a l d e h y d e , 3% f o r m a l d e ­ hyde, and 0.05% t r i n i t r o p h e n o l in a 0 . 0 5 M N a - c a c o d y l a t e b u f f e r ( 8 9 ).

A f t e r p e r f u s i o n f o r 10 m in ut es t he stomach was c ut open and, under a d i s s e c t i o n m i c r o s c o p e , specimens were t a k en from t h e c e n t e r o f t h e wound and from a c o r r e s p o n d i n g s i t e in t h e d or s a l g a s t r i c w a l l . F i x a ­ t i o n by immersion was t h en c o n t i n u e d f o r two hours in t h e same f i x a ­ t i v e . A f t e r r i n s i n g in 0 . 1 M N a - c a c o d y l a t e b u f f e r , p o s t f i x a t i o n was c a r r i e d out in 1% OSO4 in 0. 1 M N a - c a c o d y l a t e f o r two h o u r s . All s o l u t i o n s were kept a t room t e m p e r a t u r e .

D e h y d r a t i o n was performed in r i s i n g c o n c e n t r a t i o n s of e th a no l and em­

be ddi ng in Fpon 812.

Specimens f o r l i g h t m i c r o s c o p y . Survey s e c t i o n s of whole wounds were made from p a r a f f i n embedded t i s s u e s ( I , I I I ) , f i x e d in 10% n e u t r a l f o r m a l i n . Such s e c t i o n s were s t a i n e d with h e m a t o x y l i n - e o s i n or PAS. For s t e r e o l o g i c a l i n v e s t i g a t i o n s (IV, V), 1 pm t h i c k epon s e c t i o n s con­

t r a s t e d wi th t o i u i d i n e b l u e were used.

Specimens f o r e l e c t r o n m i c r o s c o p y . S e c t i o n s , 60 - 80 nm t h i c k , were c ut

i n a P o r t e r Blum MT2-B microtome and put on formvar c o a t e d one hol e

c o p p e r g r i d s . S e c t i o n t h i c k n e s s was e s t i m a t e d by t h e method d e s c r i b e d

by Small ( 9 0 ) . A f t e r c o n t r a s t i n g with uranyl a c e t a t e and l e ad hydr o xi de t h e s e c t i o n s were s t u d i e d in a P h i l i p s EM 300 o r a JEOL JEM-100CX e l e c t r o n mi cr os cope .

S t e r e o l o g i c a l a n a l y s e s

L i g h t m i c r o s c o p i c a l a n a l y s e s (IV, V). S t e r e o l o g i c a l a n a l y s e s were c a r ­ r i e d out u s i n g o i l immersion o p t i c s a t 1,000 t i m es m a g n i f i c a t i o n . The t e c h n i c a l l y b e s t s e c t i o n from each t i s s u e block was used and in each such s e c t i o n two randomly chosen a r e a s were a n a l y z e d . The a n a l y s e s were p erf or me d by means of an o c c u l a r s q u a r e g r i d wi th about 9, 000 t e s t p o i n t s p e r mm^ o f t i s s u e s e c t i o n . The p o i n t s f a l l i n g ove r p a r i e t a l c e l l s , e p i t h e l i a l c e l l s and whole mucosa ( e x c l u d i n g t h e lamina p r o ­ p r i a ) , r e s p e c t i v e l y , were c o u n t e d . The p a r i e t a l c e l l volume d e n s i t y in t h e mucosa (VVpC)> i * e * t h e f r a c t i o n of mucosa t a k e n up by p a r i e ­ t a l c e l l s , was c a l c u l a t e d a c c o r d i n g t o methods a dopt ed from Weibel

( 9 1 ) . The volume d e n s i t y o f e p i t h e l i a l c e l l s in t h e mucosa (Vye c ) was a l s o a s s e s s e d .

Using an e y e - p i e c e mi cr ome te r t h e mucosal h e i g h t was measured in s e c t ­ i o n s which were c u t p a r a l l e l t o t h e g a s t r i c g l a n d s .

E l e c t r o n m i c r o s c o p i c a l a n a l y s e s (I - V). The t e c h n i c a l l y b e s t s e c t i o n on t h e g r i d was chosen and phot ographed a t a m a g n i f i c a t i o n of about 600 t i m e s . On each s u r v e y , an a r e a l i m i t e d by two p a r a l l e l l i n e s about 50 - 250 pm a p a r t , t h e mucosal s u r f a c e , and t h e bottom of t h e g l an d s was randomly chosen ( F i g . 3 ) . Within such an a r e a a l l p a r i e t a l c e l l s show­

i ng t h e i r n u c l e u s were phot og ra ph ed a t a p rima ry m a g n i f i c a t i o n of about

5 , 0 0 0 . Pa pe r p r i n t s of t h e s e c e l l s ( f i n a l m a g n i f i c a t i o n around

X

11, 000 - 15,000) were coded a t random and s u b j e c t e d t o s t e r e o l o g i c a l a n a l y s e s .

A m u l t i p u r p o s e t e s t g r i d (91) ( F i g . 4) was superi mposed over t h e p a r i e ­

t a l c e l l s and t h e number of p o i n t s f a l l i n g over t h e c e l l , n u c l e u s , and

m i t o c h o n d r i a , r e s p e c t i v e l y , were c o u n t e d . The number of i n t e r s e c t i o n

p o i n t s between t h e t e s t l i n e and t h e s e c r e t o r y s u r f a c e (I - V) and t h e

t u b u l o - v e s i c u l a r s u r f a c e membranes ( I ) were a l s o c o un t e d .

tween t h e g a s t r i c g l an ds (GL) and p i t s (GP) a r e l a t i v e l y

t e n s i v e lamina p r o p r i a (LP) i s s een.

F i g . 4 . The m u l t i p u r p o s e t e s t g r i d which was used f o r t h e s t e r e o l o g i c a l measurements in t h e p a r i e t a l c e l l s , "d" i s t h e l e n g t h o f t h e s h o r t l i n e segment ( se e t h e t e x t ) .

Using t h e s e f i g u r e s t h e m i t o c h o n d r i a l volume d e n s i t y (Vym) was c a l c u l a t e d in r e l a t i o n t o c y t o p l a s m i c volume ( e x c l u d i n g t h e n u c l e u s ) , and t h e n u c l e a r volume d e n s i t y (VVn) i n r e l a t i o n t o c e l l volume.

The p r o f i l e a r e a s of t h e n u c l e i and t h e c e l l s were a l s o c a l c u l a t e d a c c o r d i n g t o t h e f or mu la :

A=<H>2 *ï r (i-i)

where A i s t h e p r o f i l e a r e a (pm^), d t h e l e n g t h of t h e s h o r t l i n e

segment on t h e t e s t g r i d ( F i g . 4 ) , and M t h e m a g n i f i c a t i o n .

I I , IV and V) and t u b u l o - v e s i c u l a r ( I ) membranes were c a l c u l a t e d as f o l 1ows:

P

d

2 X M ^{( 1 . 2 )}

L i s t h e 1 enght of t h e t e s t l i n e and P t h e niimber of t e s t p o i n t s f a l l i n g o ve r t h e c e l l p r o f i l e . The s u r f a c e d e n s i t y , Sy, can t hen be c a l c u l a t e d a c c o r d i n g t o :

where I i s t h e number of i n t e r s e c t i o n p o i n t s between t h e t e s t l i n e and t h e s u r f a c e measured.

Thus, ( 1 . 2 ) and ( 1 . 3 ) g i v e s :

C o r r e c t i o n o f s y s t e m a t i c e r r o r s c on ne ct e d wi th t h e s t e r e o l o g i c a l methods______________________________________________________________________

"Holmes e f f e c t 11. S t e r e o l o g i c a l f or mu la s hold w i t h o u t r e s t r i c t i o n s onl y when t h e s e c t i o n s s t u d i e d a r e i n f i n i t e l y t h i n . What i s r e a l l y i n v e s t ­ i g a t e d in t i s s u e s e c t i o n s i s t h e p r o j e c t i o n i n t o an o b s e r v a t i o n pl ane o f t h e e n t i r e c o n t e n t o f a r e l a t i v e l y t h i c k s l i c e ( i n t h e p r e s e n t s t u d i e s between 50 t o 80 nm).Thi s was r e c o g n i z e d by Holmes in 1927 (92) ( F i g . 5 ) , and he s u g g e s t e d f or mul as t o compensate f o r t h e o v e r e s t i m ­ a t i o n of t h e volume d e n s i t y of t h e p r o f i l e s measured.

I f c o r r e c t i o n s f o r t h e "Holmes e f f e c t " (91, 93) a r e a p p l i e d on t h e d a t a i n Pa pe r I one f i n d s t h a t t h e m i t o c h o n d r i a l volume d e n s i t y might have been o v e r e s t i m a t e d by a f a c t o r of between 13 t o 24% (dependi ng on s e c t i o n t h i c k n e s s ) , and t h e s e c r e t o r y s u r f a c e d e n s i t y by some 25 t o

( 1 . 3 )

I t sho ul d be p o i n t e d out t h a t t h e s e c o r r e c t i o n s f o r t h e "Holmes e f f e c t "

a r e v a l i d onl y when t h e measured s t r u c t u r e s have a h i g h e r d e n s i t y t han t h e s u r r o u n d i n g s t r u c t u r e s . Thi s i s t h e c a se f o r m i t o c h o n d r i a and s u r ­ f a c e membranes.

A A i

^

j

mm PS

!■ ^up ''iÊÈÊÊÈÈ!^

F i g . 5. I f a r e l a t i v e t h i c k s e c t i o n (C) i s c u t from t h e specimen (B) t h e opaque s t r u c t u r e s w i l l be o v e r e s t i m a t e d due t o p r o j e c t i o n o f s l i c e c o n t e n t (Holmes e f f e c t ) . This o v e r e s t i m a t i o n d e c r e a s e s when t h e s e c t i o n s becomes t h i n n e r (A). ( A f t e r Weibel and E l i a s ) .

N u c l e a r volume d e n s i t y o v e r e s t i m a t i o n . To e l i m i n a t e d i f f i c u l t i e s in

i d e n t i f i c a t i o n , onl y such c e l l s which exposed t h e n uc le u s were a n a l y s ­

e d. Thi s p r o c e d u r e r e s u l t s in o v e r e s t i m a t i o n of t h e n u c l e a r volume

d e n s i t y . The e r r o r due t o t h i s b i a s e d s ampl ing p r o c e d u r e has been

a n a l y s e d f o r s p h e r i c a l c e l l s by Konvinski & Kozlowski ( 9 4) , and u s in g

t h e i r methods i t can be c a l c u l a t e d t h a t an e x p e r i m e n t a l l y found n u c l e a r

volume d e n s i t y o f e . g . 14.5% ( I ) shoul d be c o r r e c t e d t o 9.2%. However,

s i n c e t h e p a r i e t a l c e l l s a r e not s p h e r i c a l in s h a pe , but r a t h e r c o n i ­

c a l , i t i s d o u b t f u l whe the r t h i s c o r r e c t i o n i s a p p r o p r i a t e .

For more d e t a i l e d i n f o r m a t i o n on t h e s t e r e o l o g i c a l p r i n c i p l e s , t e c h ­ n i q u e s , and t h e o r i e s s ee r e f e r e n c e s 91 and 95.

E r r o r s due t o t i s s u e p r e p a r a t i o n . F i x a t i o n , d e h y d r a t i o n , embedding and s e c t i o n i n g change t h e morphology of a l l c e l l t y p e s ( 9 6) . No a t t e m p t s have been made t o c o r r e c t f o r t h e s e c ha nges .

The s t e r e o l o g i c a l methods a r e t h u s hampered by s e v e r a l s y s t e m a t i c e r r o r s . None of t h e s e have been c o r r e c t e d f o r i n t h e p r e s e n t s t u d i e s and t h e r e f o r e t h e r e a d e r should pay more a t t e n t i o n t o c ompar is ons be ­ tween t h e d i f f e r e n t groups o f d a t a t h an t o t h e a b s o l u t e f i g u r e s . The v a l u e s a r e comparable s i n c e a l l t i s s u e have been p r o c e s s e d in e x a c t l y t h e same way.

P e r s o n a l e r r o r s . To e s t i m a t e t h e p e r so na l e r r o r of t h e method, d u p l i c ­ a t e measurements were made w i t h i n a s ix- month i n t e r v a l . This was done on a l l measured p a r a m e t e r s in one r a t in P ap e r s I and I I I . The e r r o r in t h e r e l a t i o n t o t h e mean v a l u e s amounted t o between 1 - 3 % f o r t h e c e l l p r o f i l e a r e a , 2 - 2.5% f o r t h e n u c l e a r p r o f i l e a r e a , 2 - 2.5% f o r t h e n u c l e a r volume d e n s i t y , 3 - 9 % f o r t h e m i t o c h o n d r i a l volume d e n s i t y , and 7 and 9% f o r t h e s e c r e t o r y and t u b u l o - v e s i c u l a r s u r f a c e d e n s i t i e s , r e s p e c t i v e l y .

S t a t i s t i c a l a n a l y s i s

S i g n i f i c a n c e of d i f f e r e n c e s between mean v a l ue s was c a l c u l a t e d u s i n g

t h e t w o - t a i l e d S t u d e n t s t - t e s t .

RESULTS

Animal r e c o v e r y a f t e r t h e d i f f e r e n t s u r g i c a l p r o c e d u r e s

The a n im a ls g e n e r a l l y w i t h s t o o d t h e s u r g i c a l i n t e r v e n t i o n s w e l l . When an u l c e r o p e r a t i o n , wi th or w i t h o u t p y l o r o p l a s t y (I - V), or a sham o p e r a t i o n ( I , I I I ) was performed t h e r e was no m o r t a l i t y a t a l l . How­

e v e r , in t h e vagotomy group ( I I I ) t h e m o r t a l i t y was r a t h e r h i g h , about 35%, w i t h most d e a t h s o c c u r i n g l a t e , u s u a l l y more t h an 90 days p o s t - o p e r a t i v e l y . The m o r t a l i t y among t h e a n t r e c t o m i z e d r a t s ( I I I ) was a l s o c o n s i d e r a b l e , but in t h i s group most d e a t h s o c c u r r e d on t h e f i r s t p o s t ­ o p e r a t i v e days .

All ani ma ls were weighed weakly but no s i g n i f i c a n t d i f f e r e n c e was noted i n t h e i n i t i a l and f i n a l w e i g h t s between t h e d i f f e r e n t groups wi th one e x c e p t i o n , t h e c i m e t i d i n e t r e a t e d r a t s (V), which i n c r e a s e d more t h an t h e c o r r e s p o n d i n g c o n t r o l a n i m a l s .

The r a t s used in t h e c i m e t i d i n e s t u dy (V) were o r a l l y i n c u b a t e d t wi ce d a i l y . As a r u l e t h i s t r e a t m e n t was well t o l e r a t e d .

In t h e p e n t a g a s t r i n s tu d y (IV) t h e ani ma ls r e c e i v e d s u bc ut ane ous i n j e c ­ t i o n s t w i c e d a i l y f o r 90 d a y s . No c ut ane ous i n f e c t i o n s or o t h e r a d v e r s e

r e a c t i o n s were seen due t o t h i s p r o c e d u r e .

Normal r e g e n e r a t i o n (I )

The mucosa. Examinati on of t h e wounds 24 hours p o s t o p e r a t i v e l y , r e v e a l ­ ed a l e s i o n which c o r r e s p o n d e d in s i z e t o t h e c a u t e r i z e d a r e a , and t h e e n t i r e mucosa, i n c l u d i n g t h e m u s c u l a r i s mucosae was c o a g u l a t e d . A f t e r one week t h e r e was s t i l l a l a r g e mucosal d e f e c t in t he c e n t e r of t h e wound, bu t in t h e p e r i p h e r y a l a y e r of cuboi da l t o low columnar c e l l s c o n t a i n i n g a v a r y i n g amount of mucous, P A S - p o s i t i v e g r a n u l e s were s e en .

A f t e r 30 days o f h e a l i n g t h e e n t i r e d e f e c t was c ove re d by a mucosa

f or mi ng p y l o r u s - l i k e g l a n d s , and a t t h i s s t a g e t h e most common c e l l

t y p e was a p o o r l y d i f f e r e n t i a t e d " s u r f a c e mucous c e l l " . With f u r t h e r h e a l i n g t h e mucosa became more s i m i l a r t o t h a t found in t h e normal c o r ­ pus r e g i o n . However, t h e p e r c e n t a g e of mucosa t a k e n up by e p i t h e l i a l c e l l s n ev er r e ac h ed normal l e v e l s ( F i g . 6 ) .

% 100

30 60 90 130 260

F i g . 6. Gr aphi c i l l u s t r a t i o n of t h e e p i t h e l i a l and p a r i e t a l c e l l r e ­ g e n e r a t i o n in t h e g a s t r i c wounds (mean +_ S . D . ) .

Open c i r c l e s : e p i t h e l i a l c e l l volume d e n s i t y (% of mucosal volume, not i n c l u d i n g t h e m u s c u l a r i s mucosae). F i l l e d c i r c l e s : p a r i e t a l c e l l volume d e n s i t y (% o f e p i t h e l i a l c e l l volume). The h o r i z o n t a l l i n e s r e p r e s e n t normal mean v a l u e s + S.D.

P a r i e t a l c e l l s were found a f t e r 90 days of h e a l i n g , but t h e t o t a l

volume o f t h e s e c e l l s remained subnormal ( Fi g. 6 ) .

The p a r i e t a l c e l l . The f i r s t p a r i e t a l c e l l s , p o s i t i v e l y i d e n t i f i e d by t h e p r e s e n c e of i n t r a c e l l u l a r c a n a l i c u l i ,. were immature in a pp e a r a n ce w i t h l a r g e n u c l e i , low m i t o c h o n d r i a l volume d e n s i t y and a small t o t a l amount of i n t r a c e l l u l a r membranes. The l a t t e r i s t h e sum of t h e s e c r e ­ t o r y s u r f a c e and t u b u l o - v e s i c u l a r s u r f a c e membrane d e n s i t i e s , and t h e low v a l u e i n t h e s e p a r i e t a l c e l l s was mainly due t o a d e c r e a s e in t h e t u b u l o - v e s i c u l a r s u r f a c e d e n s i t y .

A f t e r 260 d a y s , a l l u l t r a s t r u c t u r a l p e c u l i a r i t i e s seen in t h e e a r l y

"wound p a r i e t a l c e l l s " had d i s a p p e a r e d .

The r e s p o n s e t o s t i m u l a t i o n in n or mal ly r e g e n e r a t i n g p a r i e t a l c e l l s ( I I ) __________________________________________________________________________

The p ur po se with t h i s s t u dy was t o i n v e s t i g a t e , whe the r r e g e n e r a t i n g p a r i e t a l c e l l s in t h e wounds r esponded t o s t i m u l a t i o n . One group of un­

s t i m u l a t e d r a t s was compared wi th a group which had been s t i m u l a t e d .

A f t e r s t i m u l a t i o n wi th p e n t a g a s t r i n and c a r b a c h o l i ne, a t doses which n o r ma l ly s t i m u l a t e t h e p a r i e t a l c e l l s t o near maximal a c i d s e c r e t i o n , t h e r e was a l a r g e i n c r e a s e in t h e s e c r e t o r y s u r f a c e a r e a in both normal and r e g e n e r a t i n g p a r i e t a l c e l l s , t h e i n c r e a s e be e in g 90% and 76% r e ­ s p e c t i v e l y .

Moreover, s t i m u l a t i o n r e s u l t e d in a d e c r e a s e in pH on t h e mucosal s u r ­ f a c e of t h e wounds and t h e normal mucosa b e s i d e t h e wounds. On t h e e l e v a t e d margin s u r r o u n d i n g t h e wounds t h e pH was h i g h , u s u a l l y between 4 and 9, and d i d not change wi th s t i m u l a t i o n .

E f f e c t s o f p y l o r o p l a s t y , p y l o r o p l a s t y in c ombi nati on with t r u n c a l vago- tomy, and a n tr e c t o m y ( I I I ) _________________________________________________

P y l o r o p l a s t y r e s u l t e d in changes in both h e a l i n g and normal mucosa. In

t h e wounds, s m a l l e r p a r i e t a l c e l l s wi th s m a l l e r n u c l e i and lower m i t o ­

c h o n d r i a l volume d e n s i t y were s een. In normal mucosa p y l o r o p l a s t y t e n d ­

ed t o d e c r e a s e t h e s i z e of t h e p a r i e t a l c e l l s . A d e c r e a s e in n u c l e a r

p r o f i l e a r e a was a l s o n ot e d.

In r a t s s u b j e c t e d t o t r u n c a l vagotomy in a d d i t i o n t o p y l o r o p l a s t y , t h e p a r i e t a l c e l l s from normal mucosa were s i g n i f i c a n t l y l a r g e r t h an c e l l s from t h e c o r r e s p o n d i n g p y l o r o p l a s t y group. When compared with p a r i e t a l c e l l s from sham o p e r a t e d a ni ma ls no u l t r a s t r u c t u r a l changes were seen a f t e r 130 days r e g e n e r a t i o n .

However, t r u n c a l vagotomy pl us p y l o r o p l a s t y r e s u l t e d in marked i n c r e a s e i n t h e p a r i e t a l c e l l volume d e n s i t y in normal inucosa, both when compar­

ed t o sham o p e r a t e d and p y l o r o p l a s t y o p e r a t e d a n i m a l s .

In t h e wounds, t r u n c a l vagotomy p l us p y l o r o p l a s t y d i d not c a u se any a l t e r a t i o n s in t h e morphology of t h e p a r i e t a l c e l l s in a d d i t i o n t o t h o s e found a f t e r p y l o r o p l a s t y a l o n e .

The a n t r e c t o m y , by which t h e e n t i r e antrum (and t h e r e b y most of t he g a s t r i n p r o d u ci n g c e l l s ) was removed produced g e n e r a l i z e d changes in bot h r e g e n e r a t i n g and normal mucosa.

In t h e wounds t h e r e was a d e c r e a s e in mucosal h e i g h t and in p a r i e t a l c e l l s i z e . F u r t h e r m o r e , t h e r e was a marked d e c r e a s e in n u c l e a r s i z e and m i t o c h o n d r i a l volume d e n s i t y .

In normal mucosa, a n t re ct o my r e s u l t e d in s i m i l a r changes as t h o s e seen i n t h e wounds. Thus, a d e c r e a s e in mucosal h e i g h t was noted in a d d i t i o n t o a d e c r e a s e d c e l l and n u c l e a r s i z e . F u r t h e r m o r e , a d e c r e a s e in e p i ­ t h e l i a l c e l l volume d e n s i t y was n ot e d.

E f f e c t s o f l o n g - t e r m a d m i n i s t r a t i o n of p e n t a g a s t r i n (IV)

I n j e c t i o n s wi th p e n t a g a s t r i n , t w i ce d a i l y f o r 90 d a y s , a t a dose of 250

pg/ kg r e s u l t e d in marked changes in t h e p a r i e t a l c e l l mass both in

wounds and in normal g a s t r i c mucosa. At both s i t e s , h y p e r p l a s i a was

s e e n , and in t h e wounds t h e r e was a 150% i n c r e a s e in t h e p a r i e t a l c e l l

volume d e n s i t y . In normal inucosa t he i n c r e a s e was 30%.

No changes were o bs er ve d in mucosal h e i g h t or in p a r i e t a l c e l l s i z e and t h e r e f o r e t h e i n c r e a s e in p a r i e t a l c e l l volume d e n s i t y must be t h e r e ­ s u l t of an i n c r e a s e d number of c e l l s .

P e n t a g a s t r i n di d not a f f e c t p a r i e t a l c e l l m a t u r a t i o n or u l t r a s t r u c t u r e d u r i n g t h e e x p e r i m e n t a l p e r i o d .

E f f e c t s o f l o n g - t e r m t r e a t m e n t w i t h C i m e t i d i n e (V)

The dose o f c i m e t i d i n e used in t h i s s t u dy r ed u ce s basal a c i d s e c r e t i o n t o n e a r l y n i l ( 9 7 ) . The l a s t dose was given 15 t o 17 h ou rs p r i o r t o f i x a t i o n o f t h e t i s s u e , and a t t h i s t ime t h e i n h i b i t i n g e f f e c t of t h i s dose would have c ea se d (84, 98 - 9 9 ) . In t h e l i g h t mic rosc ope no a l t e r ­ a t i o n s were obs erve d in t h e p a r i e t a l c e l l mass in t h e c i m e t i d i n e t r e a t ­ ed group compared t o t h e group of r a t s which had r e c e i v e d w a t e r on l y.

However, t h e r e g e n e r a t i n g mucosa in t h e c i m e t i d i n e t r e a t e d r a t s was s i g n i f i c a n t l y t h i c k e r t han in t h e c o n t r o l group.

The u l t r a s t r u c t u r e o f t h e p a r i e t a l c e l l s d i f f e r e d between t h e two groups in one i m p o r t a n t a s p e c t , v i z . t h e s e c r e t o r y s u r f a c e d e n s i t y . The i n t r a c e l 1u l a r c a n a l i c u l a r system in p a r i e t a l c e l l s from a ni ma ls which had been given c i m e t i d i n e was more e x t e n s i v e t h an in c e l l s from c o n t r o l r a t s . Thus, t h e r e was an i n c r e a s e by 76% i n r e g e n e r a t i n g p a r i e t a l c e l l s and by 49% in p a r i e t a l c e l l s from t h e normal ( d o r s a l g a s t r i c w a l l ) mucosa.

The plasma g a s t r i n l e v e l s a t t h e t ime of t i s s u e f i x a t i o n was not s i g n i ­

f i c a n t l y d i f f e r e n t between t h e two gr ou ps .

DISCUSSION

The u l c e r model

Depending on t h e aim, d i f f e r e n t p r o c e d u r e s a r e used t o i nduce wounds in t h e g a s t r i c mucosa (7 - 54 ). In t h e p r e s e n t i n v e s t i g a t i o n s t h e aim was t o d ev el op a model by which t h e r e g e n e r a t i o n of p a r i e t a l c e l l s could be f o l l o w e d under c o n t r o l l e d c o n d i t i o n s . F u r t h e r m o r e , t h e morphology of t h e wounds s ho u ld as c l o s e as p o s s i b l e resemble t he c h r o n i c g a s t r i c u l c e r in man. Such a wound i s c h a r a c t e r i z e d by deep p e n e t r a t i o n , below t h e m u s c u l a r i s mucosæ, and a r e l a t i v e l y slow h e a l i n g r a t e (30, 4 3 ) .

Many methods can be used t o o b t a i n such wounds (40 - 45, 47 - 48, 54) b u t , t h e s t a n d a r d i z a t i o n has o f t e n proved t o be a major o b s t a c l e .

With t h e c a u t e r i z a t i o n method used h e re t h e l e s i o n s were of t h e same s i z e and d epth every t i m e . This could be a ch ie v ed s i n c e t h e two f a c t o r s t h a t c o n t r i b u t e d t o t h e g e n e s i s of t he l e s i o n s , t h e h e a t and t h e p r e s ­ s u r e a p p l i e d by t h e h e m o s t a t , were kept c o n s t a n t .

However, i t must be emphasized t h a t t h i s i s an a r t i f i c i a l l e s i o n , and t h e e t i o l o g i c a l f a c t o r s c o n t r i b u t i n g t o a c l i n i c a l u l c e r a r e t h u s mis­

s i n g . On t h e o t h e r hand, i t can be assumed t h a t in t h e abs enc e of such f a c t o r s , t h e c o n d i t i o n s f o r r e g e n e r a t i o n w i l l p r o b ab l y be o p t i m a l , and t h e wounds sh oul d t h u s p e r mi t d e t a i l e d o b s e r v a t i o n s , under c o n t r o l l e d c o n d i t i o n s , on t h e h e a l i n g p r o c e s s , a l s o a t a s u b c e l l u l a r l e v e l .

A n o t he r p o i n t o f d i s p u t e might be t h e l o c a l i z a t i o n of t h e mucosal l e s i o n . In man, a benign g a s t r i c u l c e r i s p r e f e r e n t i a l l y l o c a t e d t o t h e a n t r u m - c o r p u s b o r d e r on t h e l e s s e r c u r v a t u r e (31) and not in t h e corpus r e g i o n as in t h i s s t u d y . However, t h e main purpose of t h e p r e s e n t i n ­ v e s t i g a t i o n was t o f o l l o w t h e p a r i e t a l c e l l r e g e n e r a t i o n and t h e r e f o r e an a r e a was chosen where t h e p a r i e t a l c e l l d e n s i t y i s maximal ( 100).

In t h e p r e s e n t i n v e s t i g a t i o n r a t s were used as e x p e r i m e n t a l a n i m a l s .

T he re a r e many d i f f e r e n c e s , both a n a t o m i c a l l y and f u n c t i o n a l l y , between