Novas observações sôbre as fibras nervosas que repopulam o segmento distal de nervos com mais de seis meses de secção completa.

FURTHER DISCUSSION ON THE "NEW-GROWING" NERVE-FIBRES

WHICH REPOPULATE THE DISTAL SEGMENT OF NERVES

COM-PLETELY SEPARATED FROM THE PROXIMAL STUMP FOR MORE

THAN S I X MONTHS

E R O S A B R A N T E S E R H A R T * C E C I L J O S É R E Z Z E **

T h e scope of this paper is t o present and discuss the different

histological findings, recently reported by us before the V I I I International C o n

-gress of A n a t o m i s t s , Wiesbaden, G e r m a n y and the V I n t e r n a t i o n a l Con-gress

of Neuropathology, Zurich, which w e r e identified a m o n g t h e nerve-fibres

that repopulate the distal and intermediate segments of nerves, maintained

undisturbed in their n a t u r a l connective tissue bed, and completely separated

from the proximal s t u m p for more t h a n six months ( E r h a r t and E r h a r t

.

,

E r h a r t

, E r h a r t and R e z z e

.

) .

T h e above referred histological findings resemble some of the

progres-sive stages of the W a l l e r i a n degeneration and, for what we have seen in our

experimental observations, they show u p as a suggestive sequence of stages

w h i c h seem t o indicate a possible g r a d u a l reorganization and further

re-generation of nerve-fibres in isolated stumps.

M A T E R I A L A N D M E T H O D S

T h e serial h i s t o l o g i c a l preparations, l o n g i t u d i n a l l y sectioned 8 u, s i l v e r i m -p r e g n a t e d by D e C a s t r o , considered in t h e -present re-port w e r e selected f r o m the m a t e r i a l w e h a v e recorded and a n a l i s e d d u r i n g these last n i n e y e a r s w h i l e study-ing the n e r v e fibres w h i c h repopulate, u n d e r different e x p e r i m e n t a l conditions, the distal and i n t e r m e d i a t e s e g m e n t s of dog fibular, intercostal, phrenic a n d u l n a r nerves completely separated from the p r o x i m a l s t u m p for more t h a n six m o n t h s . O b v i o u s l y t h e slide selection w a s based on t h e presence of the p a r t i c u l a r h i s t o l o g i c a l pictures in w h i c h w e were interested. T h e y were identified in the i n t e r m e d i a t e a n d or distal nerve segments of e l e v e n different dogs.

R E S U L T S

W h i l e s t u d y i n g t h e n e r v e fibres w h i c h repopulate, u n d e r different e x p e r i m e n t a l conditions, isolated n e r v e segments, we observed now a n d then in the silver pre-parations, six a n d e i g h t m o n t h s post-operatively, a m o n g the n e r v e fibres w i t h

n o r m a l a p p e a r a n c e , different h i s t o l o g i c a l pictures resembling some s t a g e s of the W a l l e r i a n degeneration, but of whose n a t u r e or s i g n i f i c a n c e we had n o idea. A s u g g e s t i v e sequence of these h i s t o l o g i c a l findings showed up clearly, for the first time, in t w o specially w e l l impregnated, completely isolated nerve stumps, t w e l v e m o n t h s post-operatively. W i t h a shedule stablished the revision of o u r e x p e r i m e n t a l m a t e r i a l w a s done, a n d the formerly isolated findings could be put together. T h e y d e m o n s t r a t e d the e x i s t e n c e of s u g g e s t i v e progressive stages of s o m e t h i n g as a develop-ment a n d m a t u r a t i o n w h i c h w o u l d e x p l a i n , perhaps, a possible g r a d u a l r e o r g a n i z a t i o n and r e g e n e r a t i o n of nerve-fibres in isolated nerve segments, m a i n t a i n e d undisturbed on their n a t u r a l e n v i r o n m e n t a n d c o m p l e t e l y separated from the p r o x i m a l s t u m p for m o r e t h a n six m o n t h s .

F i g u r e s 1-5 depict e x a m p l e s of w h a t could be considered as some of the m a i n s t a g e s of the suggested progressive r e o r g a n i z a t i o n referred a b o v e . F i g u r e 1 shows j u s t narrow e l o n g a t e d p a r a l l e l strands w i t h a light c o n c e n t r a t i o n of a silver i m -pregnated " m a t e r i a l " e v e n l y distributed a l o n g them. I n F i g u r e s 2 to 5 the con-c e n t r a t i o n of the silver i m p r e g n a t e d " m a t e r i a l " is progressively higher, first a t i r r e g u l a r t h a n at r e g u l a r i n t e r v a l s a n d there is, furthermore, a tendency for a m a t u r a t i o n of t h e linear strands; the boundaries become g r a d u a l l y more distinct; a x o n s w i t h n o r m a l a p p e a r a n c e a r e seen side by side to these s u g g e s t i v e s t a g e s of nerve-fibres in " r e o r g a n i z a t i o n " . I n F i g u r e s 6 a n d 7 are seen n e w - g r o w i n g a x o n s w i t h their c h a r a c t e r i s t i c beaded a p p e a r a n c e because of the i r r e g u l a r e n l a r g e m e n t s ; in one of them (arrow F i g . 7) a t y p i c a l g r o w i n g bud is evident. F i g u r e 8 shows n o r m a l nerve-fibres w i t h different d i a m e t e r s in t h e d i s t a l segment of a dog u l n a r n e r v e completely separated from the p r o x i m a l s t u m p for fifteen m o n t h s .

D I S C U S S I O N

T h e technical difficulties encountered in demonstrating the origin of

the nerve fibres which repopulate the distal and intermediate segments of

nerves completely separated from the proximal stump for more than six

months, constitute a limiting factor in studying possible patterns of

new-forming isolated nerve-fibres.

It seems based on our former observations t h a t these fibres must have

arisen form a source other than the p r o x i m a l stump, neighbour nerves or

nervi-vasorum ( E r h a r t and R e z z e

) .

Before the end of the fifth m o n t h after nerve transection no remanents

of nerve-fibres could be found in the serial silver preparations of the

inter-mediate or distal segments completely separated from the proximal stump,

because of the W a l l e r i a n degeneration. But, from the sixth month on,

very-few axons with normal appearance were evident. T h e y were progressively

thicker, longer and greater in number in the isolated nerve stumps examined

eight, twelve, fifteen, eighteen, twenty-four and thirty months

postoperatively. T h e y formed inclusively the neuromalike structures ( E r h a r t and R e z

-z e

T h e different histological pictures, w h i c h are now being presented as

suggestive progressive stages of perhaps a certain k i n d of development and

m a t u r a t i o n of nerve-fibres in reorganization, were easier identified in the

elder isolated stumps (twelve or more m o n t h s post-operatively) than in the

more recent ones (six or eight months post-operatively). M a y be, because

in the latter, still poor in nerve-fibres, the "biological recovery" of the

whole system is just beginning, and in the former, usually rich in

nerve-fibres, the "biological recovery" is plain.

These findings and data suggest the existence of something to be

in-vestigated.

I t is accepted t h a t the protein synthesis of neurons seems to occur

ex-clusively in t h e cell bodies, not in the axons ( H y d é n

, Droz and Leblond

)

and there a r e conclusive evidences for the axoplasmic flow theory of W e i s s

(Weiss et a l .

) . A retrograde axoplasmic flow also occurs in accordance

with the observations of L u b i n s k a et a l .

and D a h l s t r o m

, w h o s t a t e s :

— " W h e t h e r or not this centripetally directed transport occurs normally or

is caused by the a x o n a l t r a u m a cannot be decided at present".

I f there is a retrograde axoplasmic flow where should be its protein

synthesis? D e Robertis

suggests that there is some in the nerve terminals

and, after a transection or constriction, even locally in the axons. C o u r t e y

s t a t e d : — " L e bout distal se present c o m m e une portion de neurite isolé

du péricaryon, avec une arborisation termínale à chaqué extrémité, douée

chacune d'une activité propre qui permet l'allongement de la fibre

synchroniquement en directions distale et proximale". Furthermore, S h a n t h a v e e

-rappa and B o u r n e

reported observation based on histochemical results

that the proximal and distal stumps have equivalent metabolism.

Based on such statements and comparing our observation and

histo-logical findings reproduced in the present paper w i t h those of Pinner and

C a m p b e l l

and D a h l s t r ö m

we would suggest for other investigators,

work-ing on the field w i t h specialized histochemical methods, to prove or deny

this proposed eventual progressive reorganization and regeneration of

nerve-fibres in isolated (intermediate and distal) segments completely separated

from the p r o x i m a l s t u m p for more than six months, but maintained in their

natural connective tissues beds.

C a j a l

in 1928 s t a t e d : — " T h e axon is protected and perhaps nourished

by the cells of S c h w a n n and it lives in a milieu of connective tissues rich

in plasma and in capillaries". " I n order that the nutritional process should

be kept up, it is absolutely necessary that some dynamic thing, of whose

n a t u r e we have no idea, should constantly be radiated from the neuronal

soma so as to stimulate the protoplasm and prevent withering".

F r o m C a j a l ' s unknown "dynamic thing", something is known now a

d a y s : the cell protein synthesis, the axoplasmic flow, Levi M o n t a l c i n i

"nerve growth factor". B u t is that a l l ? ! . . .

T h e W a l l e r i a n degeneration is another reality. Nevertheless, if our

supposition is true, it should be more properly nominated Wallerian

des-organization, because what is seen by the usual optical methods, by electron

microscopy and has been demonstrated biochemically, is a desintegration

of the normal nerve structures. B u t what happens w i t h the ultrastructure

constituters once organized and optically identified as axon, myelin, incisures,

nodes, neurilemma and so o n ? . . .

H a s L e v i - M o n t a l c i n i

protein "nerve growth factor" something to do

in such isolated axon regeneration processes?

W h e t h e r or not these questions are just theoretical or will have definite

answers in a near future we cannot decide.

S U M M A R Y

Based on experimental data previously published is presented and

dis-cussed a progressive sequence of histological findings (figs. 1-8) which

sug-gest a possible g r a d u a l reorganization and regeneration of nerve-fibres in

the distal and intermediate nerve segments, completely separated from the

proximal s t u m p for more than six months, but maintained undisturbed in

their natural connective tissues beds.

R E S U M O

Novas observações sôbre as fibras nervosas que repopulam o segmento distal

de nervos com mais de seis meses de secção completa

R E F E R E N C E S

1. C A J A L , S . R A M O N y — D e g e n e r a t i o n a n d regeneration of the n e r v o u s system. T r a n s l a t e d a n d edited by R a o u l M . M a y . O x f o r d U n i v . Press, L o n d o n , 1928. 2. C O U R T E Y , B . — O b s e r v a t i o n s en contraste de phase de l a régéneratio n "per p r i m a m i n t e n t i o n e m " des fibres nerveuses en c u l t u r e chez l'embryon de poulet. C o m p t e s R e n d u s de l ' A s s . A n a t . 117:458-473, 1963. 3. D A H L S T R Ö M , A . — O b s e r v a t i o n s on the a c c u m u l a t i o n of n o r a d r e n a l i n e in the p r o x i m a l a n d distal parts of peripheral adrenergic nerves after compression. J . A n a t . 99:677-689, 1965. 4. D E R O B E R T I S , E . — H i s t o p h y s i o l o g y of Synapses and Neurosecretion. P e r g a m o n Press, O x f o r d -L o n d o n - N e w Y o r k - P a r i s , 1964. 5. D R O Z , B . & -L E B -L O N D , C . P . — A x o n a l m i g r a t i o n of proteins in the c e n t r a l n e r v o u s system and peripheral nerves as shown by a u t o -r a d i o g -r a p h y . J . C o m p . N e u -r o l . 121:325-346, 1963. 6. E R H A R T , M . B . & E R H A R T , E . A . — Sôbre o c o m p o r t a m e n t o das fibras nervosas no côto distal de nervo f i b u l a r de Canis familiaris após t r a n s e c ç ã o t o t a l . F o l i a C l i n i c a et B i o l ó g i c a ( S ã o P a u l o ) 28:98, 1958. 7. E R H A R T , E . A . & E R H A R T , M . B . — N o r m a l a x i s - c y l i n d e r s in t h e distal segment of nerves completely separated from the p r o x i m a l s t u m p for up t o 24 years. A n a t . R e c . 136:189, 1960. 8. E R H A R T , E . A . — N o r m a l nerve-fibres !n t h e distal s e g m e n t of nerves completely separated from the p r o x i m a l s t u m p for more t h a n six m o n t h s . A r q . N e u r o p s i q u i a t . ( S ã o P a u l o ) 20:289306, 1962. 9. E R -H A R T , E . A . & R E Z Z E , C . J . — N e r v e fibres in isolated s e g m e n t s of dog u l n a r nerve a f t e r complete b r a c h i a l p l e x o t o m y a n d p e r i a x i l a r a r t e r y s y m p a t h e c t o m y . A r q . Neuro-siquit. ( S ã o P a u l o ) 23:82-90, 1965. 10. E R H A R T , E . A . & R E Z Z E , C . J . — T h e n e u r o m a - l i k e s t r u c t u r e of the l o n g - t i m e severed a n d isolated n e r v e s t u m p s . A r q . Neuro-psiquiat. ( S ã o P a u l o ) 23:91-94, 1965. 11. E R H A R T , E . A . & R E Z Z E , C . J . — E x p e r i m e n t a l d a t a and p r a c t i c a l results w h i c h modify the present concepts of peripheral nerve fibres degeneration and regeneration. P r o c e e d i n g s of the V I n t e r n a t i o n a l C o n g r e s s of N e u r o p a t h o l o g y : 667-671, 1965. P u b l i s h e d by E x c e r p t a M e d i c a . 12. H Y D É N , H . — T h e N e u r o n . I n T h e Cell, I V , 215323. E d i t e d by B r a chet and M i r s k y . A c a d e m i c Press. N e w Y o r k L o n d o n , 1960. 13. L E V I M O N T A L -C I N I , R . — G r o w t h control of nerve cells by a protein f a c t o r and its a n t i s e r u m . S c i e n c e 143:105-110, 1964. 14. L U B I N S K A , L . et a l . — B i d i r e c t i o n a l m o v e m e n t s of a x o p l a s m in peripheral n e r v e fibres. A c t a B i o l . E x p . V a r . 23:239-247, 1963. ( Q u o t e d from D a h l s t r ö m3

) . 15. P I N N E R , B . & C A M P B E L L , J . B . — A l k a l i n e phosphatase a c t i v i t y of incisures a n d nodes d u r i n g degeneration and r e g e n e r a t i o n of peripheral nerve fibres. E x p . N e u r o l . 12:159-172, 1965. 16. S H A N T H A V E E R A P P A , T . R . & B O U R N E , G . H . — T h e effects of transection of the nerve t r u n k on the p e r i n e u r a l e p i t h e l i u m w i t h special reference to its role in nerve d e g e n e r a t i o n a n d r e g e n e r a t i o n . A n a t . R e c . 150:35-50, 1964. 17. W E I S S , P . ; T A Y L O R , A . C . & P I L L A I , P . A . — T h e nerve fibres as a system in c o n t i n u o u s f l o w : m i c r o c i n e m a t o g r a p h i c a n d e l e c t r o n microscopic demonstrations. S c i e n c e 136:330, 1962.