NERVE FIBRES IN ISOLATED SEGMENTS OF DOG ULNAR NERVES
AFTER COMPLETE BRACHIAL PLEXOTOMY AND PERIAXILAR
ARTERY SYMPATHECTOMY
EROS ABRANTES ERHART * CECIL J. REZZE * *
W e reported observation of nerve fibres of normal appearance in the distal
segments of human nerves which had been completely transected for years
( E r h a r t
5) . Experimentally we reproduced the phenomenon in dogs (Erhart
and Erhart
6, 7) . Wide gaps between proximal and distal stumps were present
in all instances. N o nerve fibres could be found in the tissues between the
stumps, nor were collateral sprouts observed to come off the proximal intact
part of the nerve. It seemed, therefore, that those fibres of normal appearance
must have arisen from a source other than the proximal stump of the severed
nerve.
M A T E R I A L A N D M E T H O D S
E x p e r i m e n t s w e r e p e r f o r m e d u n d e r g e n e r a l a n e s t h e s i a in ten a d u l t m o n g r e l d o g s . T h e u l n a r n e r v e w a s chosen b e c a u s e i t runs a l o n g c o u r s e in t h e f o r e l i m b w i t h o u t b r a n c h i n g o r e n g a g i n g in i n t e r c o m m u n i c a t i o n s . T h e p r o c e d u r e s w e r e t o be c a r r i e d o u t in f o u r s t a g e s u s i n g the r i g h t side. T h e f o l l o w i n g d e s c r i p t i o n c o r r e s p o n d s t o t h e s t a n d a r d s c h e m e ( f i g . 1 ) :
a ) I n t h e f i r s t s t a g e , the n e r v e w a s d i v i d e d in t w o p l a c e s , j u s t c a u d a l t o t h e b r a n c h i n g of t h e c a u d a l c u t a n e o u s a n t e b r a c h i a l n e r v e , a n d j u s t c a u d a l t o t h e e l b o w , and s e g m e n t s r e m o v e d b e t w e e n d o u b l e b l a c k c o t t o n l i g a t u r e s , as s h o w n in f i g . 1 A . T h e i n t e r m e d i a t e and distal s e g m e n t s w e r e l e f t in p l a c e u n d i s t u r b e d by d i s s e c t i o n . T h e e x c i s e d s e g m e n t s w e r e p r e p a r e d h i s t o l o g i c a l l y f o r c o n t r o l s .
b ) M o r e t h a n a y e a r l a t e r , t h e o p e r a t i v e f i e l d w a s o p e n e d u n d e r a n e s t h e s i a , a n d the l i g a t u r e s i d e n t i f i e d . T h e e x t r e m i t i e s o f t h e p r o x i m a l , i n t e r m e d i a t e and d i s t a l s e g m e n t s w e r e f r e e d g e n t l y a n d e x c i s e d w i t h t h e tissues b r i d g i n g t h e s e g m e n t s . A l l t h i s m a t e r i a l w a s p r e p a r e d h i s t o l o g i c a l l y b y D e C a s t r o s i l v e r i m p r e g n a t i o n , M a s s o n , M a l l o r y c o n n e c t i v e tissue, P a l - W e i g e r t m o d i f i e d by E r h a r t f o r m y e l i n sheaths, a n d H & E g e n e r a l m e t h o d s . N e w l i g a t u r e s w e r e a p p l i e d an t h e w o u n d c l o s e d .
c ) O n e m o n t h l a t e r , a r i g h t 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 w e r e p e r f o r m e d . W e r e e x c i s e d -t h e v e n -t r a l r a m i f r o m C4 t o T j , t r u n k s
and c o r d s of t h e p l e x u s t i l l t h e l e v e l w h e r e a r i s e the m a i n n e r v e s o f t h e l i m b , t h e s t u m p s o f a l l c o m p o n e n t s b e i n g l i g a t e d w i t h w h i t e c o t t o n t h r e a d s . T h e p e r i a r t e r i a l s y m p a t h e c t o m y s e v e r e d 4 t o 5 c m o f t h e a x i l a r a r t e r y . T h e w o u n d w a s c l o s e d .
d ) O n e m o n t h l a t e r , t h e o p e r a t i v e f i e l d w a s a g a i n e x p o s e d u n d e r a n e s t h e s i a and t h e w i d e g a p s b e t w e e n t h e t h r e e s e g m e n t s o f t h e u l n a r n e r v e i d e n t i f i e d . T h e p r o -x i m a l , t h e i n t e r m e d i a t e and t h e d i s t a l s e g m e n t s w e r e e -x c i s e d f o r h i s t o l o g i c a l s t u d y ( f i g . I D ) , as w e l l as s e g m e n t s o f o t h e r t r a n s e c t e d n e r v e s t o be used f o r c o n t r o l s . F r o m t h e ten d o g s s u b j e c t e d t o t h e s t a n d a r d u l n a r n e r v e o p e r a t i o n , f o u r w e r e s u c c e s s f u l l y s u b m i t t e d t o t h e p r o p o s e d s c h e m e , w i t h s o m e p e c u l i a r i t y w h i c h w i l l be c o n -sidered l a t e r o n . O n e d i e d d u r i n g t h e t h i r d o p e r a t i o n . T h r e e died f r o m i n j u r i e s i n c u r r e d as t h e r e s u l t o f f i g h t i n g in t h e k e n n e l . T h e r e m a i n i n g t w o a r e still l i v i n g in o r d e r t o be s u b m i t t e d t o t h e p r o p o s e d s c h e m e , b e i n g e l a p s e d t w o o r m o r e y e a r s a f t e r t h e s t a n d a r d o p e r a t i o n .
R E S U L T S
A f t e r t h e f i r s t o p e r a t i o n a l l t e n d o g s s h o w e d c o m p l e t e m o t o r a n d s e n s o r y loss in t h e t e r r i t o r y s u p p l i e d b y t h e r i g h t u l n a r n e r v e . I t w a s e v i d e n t t h e v i c i o u s p o s t u r e d u e t o t h e d e n e r v a t i o n o f t h e m . f l e x o r c a r p i u l n a r i s .
W h i l e p e r f o r m i n g t h e second o p e r a t i o n a n d t h e n e c r o s c o p y o f t h e t h r e e r e f e r r e d d o g s w h i c h h a v e died p r e c o c i o u s l y , in a l l i n s t a n c e s w i d e g a p s s e p a r a t e d c o m p l e t e l y t h e p r o x i m a l , i n t e r m e d i a t e a n d d i s t a l s e g m e n t s of t h e u l n a r n e r v e . N o g r o s s i n t e r -c o m m u n i -c a t i o n s w i t h o t h e r n e r v e s w e r e e v i d e n t in t h e s u r g i -c a l f i e l d .
W h e n e x c i s i n g d u r i n g this o p e r a t i o n t h e p r o x i m a l e x t r e m i t y o f t h e d i s t a l s t u m p , t h e r e w a s in s o m e cases a d i s c r e e t c o n t r a c t i o n o f t h e m . f l e x o r c a r p i u l n a r i s .
n e r v e fibres c o u l d be found p e n e t r a t i n g t h e e x t r e m i t i e s o f t h e i n t e r m e d i a t e a n d d i s t a l s e g m e n t s , n o r in t h e tissues b r i d g i n g t h e s t u m p s ; d ) N e u r o m a - l i k e s t r u c t u r e s in t h e e x t r e m i t i e s o f t h e i n t e r m e d i a t e a n d distal s t u m p s c l o s e t o t h e s i t e o f s e c t i o n .
T h e h i s t o l o g i c a l p r e p a r a t i o n o f t h e i n t e r m e d i a t e and d i s t a l s e g m e n t s o f t h e d o g s w h i c h h a v e died r e s p e c t i v e l y t h i r t y - f i v e , e i g h t y a n d o n e h u n d r e d a n d s i x t y d a y s a f t e r t h e first o p e r a t i o n , s h o w e d a l l k n o w n c h a r a c t e r i s t i c s o f t h e W a l l e r i a n d e -g e n e r a t i o n .
A s c o n s e q u e n c e o f t h e t h i r d o p e r a t i o n — p l e x o t o m y a n d p e r i a r t e r i a l s y m p a -t h e c -t o m y ( f i g . 1 C ) — -t h e d o g s s h o w e d c o m p l e -t e m o -t o r a n d s e n s o r y loss o f -t h e w h o l e r i g h t f o r e l i m b w i t h a c h a r a c t e r i s t i c v i c i o u s p o s t u r e , plus c l i n i c a l signs o f a n e f f e c t i v e p e r i a r t e r i a l s y m p a t h e c t o m y .
S y m p a t h e c t o m y w a s n o t d o n e in o n e o f t h e d o g s and, as r e f e r r e d b e f o r e , o n e died d u r i n g this o p e r a t i o n . F r o m this l a t t e r a t h o r o u g h e x a m i n a t i o n o f t h e o p e r a t i v e f i e l d w a s c o n d u c t e d . W i d e g a p s w e r e s e p a r a t i n g t h e s t u m p s a n d t h e h i s t o l o g i c a l s e r i a l p r e p a r a t i o n o f t h e i n t e r m e d i a t e and d i s t a l u l n a r n e r v e s e g m e n t s , w h i c h w e r e l e f t in p l a c e u n d i s t u r b e d b y d i s s e c t i o n , s h o w e d n e r v e f i b r e s a n d n e r v e b u n d l e s .
T h e u l n a r n e r v e m a t e r i a l e x c i s e d d u r i n g t h e f o u r t h o p e r a t i o n ( f i g . I D ) r e -v e a l e d t h e f o l l o w i n g : a ) P r o x i m a l s t u m p c o m p l e t e l y d e g e n e r a t e d b e c a u s e o f t h e p l e x o t o m y ; b ) N e r v e f i b r e s a n d b u n d l e s i n t h e i n t e r m e d i a t e a n d d i s t a l u l n a r n e r v e s t u m p s ( f i g . 2C and D ) . S o m e f i b r e s s h o w e d v a r i c o s i t i e s o r f u s i f o r m s w e l l i n g s in o n e o f t h e e x t r e m i t i e s o r disposed a l o n g t h e a x i s - c y l i n d e r ( f i g . 2 E ) , g i v i n g t h e m a b e a d e d a p p e a r a n c e ; c ) N o n e r v e f i b r e s c o u l d be f o u n d p e n e t r a t i n g t h e e x t r e m i -t i e s o f -t h e i n -t e r m e d i a -t e a n d d i s -t a l u l n a r n e r v e s e g m e n -t s .
M e d i a n , r a d i a l and m u s c u l o - c u t a n e o u s n e r v e s e g m e n t s e x c s i s e d f o r c o n t r o l s h o w e d c o m p l e t e W a l l e r i a n d e g e n e r a t i o n .
D I S C U S S I O N
Our experimental findings of nerve fibres with normal appearance in the
distal and intermediate segments of nerves completely separeted from the
proximal stumps for more than six months, discussed by our former papers,
were again confirmed, leading us definetely to a different conclusion of what
is generally accepted by most of the modern authors.
J.B. Campell from the Neurosurgical Laboratory, N e w York University
School of Medicine confirming our findings wrote the following in two letters
addressed to us: April 1962 "In the distal stump, capped with Millipore in
order to preclude access of axons, there were plenty axons". April 1963
"Mi-croscopic study with silver impregnation and myelin stains tend to confirm
all the findings in your paper". Discussing his own findings he suggested:
" I provisionally conclude that these axons must have their origin in the
inner-vation of the blood vessels of the distal stump. Bearing this in mind, I deduced
that they have spread into the Schwann tubules left vacant by the preceding
transection. I do not know whether my conclusions agrees with yours, but
the observation definitely substantiates what you have seen".
Fig. 2 — Photomicrographs of 8 μ sections of dog ulnar nerves submitted to the four stages operation. De Castro silver simpregnation (X700). A and B . Nerve fibres in the intermediate nerve segment excised by the second operation (dog number XII). C and D . Nerve fibres in the intermediate and distal nerve segments excised by the fourth operation (dog number X). E . Nerve fibres with turgescences (neurogenic buddings) in the intermediate nerve segment excised by the fourth
This hypothesis was also previously considered by us in another paper
5,
but these new results seem to exclude the possibility of reinnervation of the
intermediate and distal segments being done by ingrowing sprouts comming
from other nerves or from "nervi-vasorum".
Lee Ching-Yuen
1 1studying the Wallerian degeneration on the electron
microscope states: "From the seventh day onward after resectioning sciatic
nerves (one to three centimeters) of rats and rabbits, all the axoplasmic
organells gradually disappeared from the axon of myelinated fibres. On the
twenty-first day all the organelles were still seen in the axons of unmyelinated
fibres but on the twenty-eigth day only dense particles and the endoplasmatic
reticulum were found. On the thirty-fifth day no axonal remants were
present".
How can one explain than the presence of nerve fibres with normal
appearance in the intermediate and distal stumps of long-time transected and
reoperated ulnar nerve, thirty days after a complete plexo-and sympathectomy
and sixty days after the excision of the stumps extremities (stage B operation) ?
The somatic and sympathetic fibres of the main nerve trunks were
transected and excised by the plexotomy, an the sympathetic fibres which
run toghether with the arterial branches of the axilar artery were mostly
excised by the periarterial sympathectomy. W e know that other nerve
path-ways through arterial anastomoses nets and muscles could be considered, but
why do we find nerve fibres with normal appearance only in the intermediate
and distal ulnar nerve previously operated segments? If those repopulating
nerve fibres came together with vascular branches, or through some other
way, they should be seen also in other nerves. Or the long-time severed
nerve segments present different living conditions! W e know there are the
neurilemma bands waiting for the ingrowing of new nerve-fibres. But why
those nerve fibres begin to appear, and can be found as stated in previous
paper, only six months after the nerve transection, if the distal segment is
kept in its surrounding connective tissue bed! Nerve fibre growing is not
so slow to take six months to reach the upper third of the forearm. Before
the sixth month the nerve stumps were always completely degenerated.
If these axis-cylinders found in the intermediate and distal long-time
severed ulnar nerve segments were ingrowed nerve-fibres, why they did not
degenerate when the stumps were cut, as we did in our four stages ulnar nerve
operation.
the nerve, as a whole, is the less affected part of the structure while
manipu-lating the nerve segments during the operations.
In serial longitudinal and cross-sections w e have followed these
axis-cylinders but, from where they came from, or their origin, we were not able
to find. W e could never, as said before, find nerve-cells along such nerve
segments.
Gamble and Eames
9describing electron microscopy pictures of degenerated
human deep ulnar nerve avulsed from the cord some fifteen months previously,
wrote the following: "Most of these processes are believed to belong to
Schwann cells organized as bands of Büngner but a few have the appearance
of and may be surviving or regenerating axons".
W e hope that these authors interpretation is correct because such electron
microscopy pictures would confirm once more our findings.
With respect to the fusiform swellings found in the extremities or along
the axis-cylinders of the intermediate and distal long-time severed ulnar nerve
segments, they seem to be the same structure described many years ago by
Cajal
3and studied under the electron microscope by Estable and his group
8.
These latter authors state: "It is an experienced fact that besides the
mention-ed terminal growing bud, formative turgescences along the nerve fibre exist,
which can be, in turn, the place of neurogenic budding. This fact would
lead to the inference that the nerve fibre has all along its segments the
potencial property of regeneration or, in a wider sense, of creating all the
structures that normally constitute them". "From a general point of view
regeneration means: the morphological building of new nerve fibres
(proli-feration) and restitution of nerve functions".
In recent papers Blümcke
1, Blümcke and Niedorf
2, Knoche and Blümcke
1 0performing careful histochemical and electron microscopy examinations of
normal and regenerating peripheral nerve fibres, consider also these fusiform
swellings (Wachstumsendkolben — growing end bulbs) as characteristic of
regenerating nerve fibres.
It must be emphasized that w e found axis-cylinders with these "neurogenic
buddings" in the isolated stumps of the ulnar nerve of dogs which were
submitted after the second nerve transections (stage B operation), to a
plexo-and sympathectomy.
How can one explain the presence of axis-cylinders with growing buds
without the trophic action of a cell-body?
Recently Courtey
4studying in tissue culture the regeneration of isolated
and transected nerve fibres of chick embryos under the phase microscope
states: "Le bout distal se présente comme une portion de neurite isolé du
pé-ricaryon, avec une arborisation termínale à chaque extrémité, douée chacune
d'une activité propre qui permet l'allongement de la fibre synchroniquement
en directions distale et proximale".
Could we consider homologous in behaviour, Courtey's distal segment
and our intermediate and distal ulnar nerve segments kept practically
undis-turbed on their surroundings connective tissue bed, in the "millieu" of Cajal?
Courtey described in each extremity of the distal segment "une
arbori-sation terminale"; this structure would be the primordia of the growing bud?
More recently, Levi-Montalcini
1 2suggests that the protein "nerve growth
factor" ( N G F ) which was isolated from some mouse sarcomas, snake venom
and mouse salivary glands, which is a normal constituent of the sympathetic
cells, and is also present in the blood and body fluids of a variety of vertebrates,
man included, may provide new leads to understanding of some neurogenetic
processes. Moreover, she states that granuloma tissue, experimentally
pro-duced in a variety of mammals, releases N G F and suggests, furthermore, that
there is reason to believe that each nerve cell type might be receptive to
only one specific factor.
W e know that after nerve severance there is an intense proliferation of
Schwann cells and epi- peri- and endoneural reactions. Would these repair
processes release some "nerve growth factor" which would be perhaps one
of the keys to lead us to some explanation of our findings?
Another still open question is the explanation of the neuroma-like structures
generally found in the extremities of long-time severed intermediate and distal
nerve segments. This problem will be considered in another paper.
More concrete data about our findings are naturally necessary and the
experiments are going on. Even so, as stated in previous paper, the practical
results obtained up to now in more than one hundred and fifty patients allows
us to go on recommending strongly, based on our observations, that proper
nerve suture or neurolisys of long-time severed nerves by accident are worth
while even after delays of many years.
S U M M A R Y
Experiments were performed in adult dogs in order to study our reported
observation of normal nerve fibres in the intermediate and distal segments
of nerves completely separated from the proximal stump for more than six
months.
The results obtained confirmed once more our previous observation: there
are normal nerve fibres and nerve bundles repopulating the pattern fascicles
of the completely separated intermediate and distal segments of the ulnar
nerve, although all other nerves of the operated limb, including the proximal
stump of the ulnar, show a typical Wallerian degeneration. I t seems,
there-fore, that these fibres of normal appearance must have arisen from a source
other than the proximal ulnar nerve stump, neighbour nerves or
"Nervi-vasorum".
go on recommending strongly, based on our observations, that proper nerve
suture or neurolisys of long-time severed nerves by accident are worth while
even after delays of many years.
R E S U M O
Prosseguindo estudos anteriores que demonstraram a existência de fibras
íntegras no segmento distal de nervos com mais de seis meses de secção
completa, novas experiências foram efetuadas em cães, desta feita sendo
uti-lizado o nervo ulnar, por causa de suas características morfo-funcionais e
topo-gráficas que se enquadravam perfeitamente aos nossos propósitos.
A experimentação foi desenvolvida obedecendo a esquematização
repre-sentada na fig. 1. Os resultados obtidos confirmaram nossos achados
ante-riores, visto que, mesmo após plexotomia braquial total e simpatectomia
pe-riarterial da artéria axilar, continuaram evidentes numerosas fibras nervosas
de aspecto normal nos segmentos intermédios e distal do nervo ulnar mantidos
em seus leitos conetivos naturais, mas completamente separados entre si e do
côto proximal. Essas fibras nervosas, em face da experimentação efetuada,
pa-recem não ser provenientes do côto proximal do nervo ulnar, nem de
interco-municações com nervos vizinhos, nem tãopouco de "nervi-vasorum". Há indícios,
no entanto, que possivelmente tenham se originado à custa de elementos
cons-tituintes de fibras "degeneradas", sob regência conjugada das células de
Schwann e do meio ambiente natural. Os trabalhos prosseguem nesse sentido
em busca de dados mais concretos. Todavia, desejamos ressaltar que os
re-sultados práticos obtidos até o momento em mais de cento e cinqüenta
pa-cientes, segundo informações de vários colegas que têm dado crédito às nossas
pesquisas, vêm demonstrar que muito se poderá fazer em prol da reabilitação
de inválidos, através de reconstruções e recuperação funcional de nervos com
lesões antigas que infelizmente vinham sendo considerados como irreversíveis e
irreparáveis.
R E F E R E N C E S
-ção t o t a l . P r e s e n t e d b e f o r e t h e I I I M e e t i n g o f t h e S o c . B r a s , de A n a t o m i a , P ô r t o A l e g r e , O c t . 1958. 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 ) 28:98-100, 1958/59. 8. E S T A B L E , C ; A C O S T A - F E R R E I R A , W . & S O T E L L O , J. R . — A n e l e c t r o n m i c r o s c o p e s t u d y o f t h e r e g e n e r a t i n g n e r v e f i b r e s . Z e i t . f. Z e l l f o r s c h u n g 46:387-399, 1957. 9. G A M B L E , N . J. & E A M E S , R . A . — E l e c t r o n m i c r o s c o p y o f t h e c o n n e c t i v e tissues o f n o r m a l a n d d e g e n e r a t e d h u m a n p e r i p h e r a l n e r v e s . J. A n a t . , 98:175, 1964. 10. K N O C H E , H . & B L Ü M C K E , S. — L i c h t m i k r o s k o p i s c h e B e o b a c h t u n g e n ü b e r frühe R e g e -n e r a t i o -n s s t a d i e -n p e r i p h e r e r N e r v e -n . Z e i t . f. M i k r o s . A -n a t . F o r s c h u -n g 69:248-278, 1962. 11. L E E C H I N G - Y U E N , J. — E l e c t r o n m i c r o s c o p y o f W a l l e r i a n d e g e n e r a t i o n . J. C o m p . N e u r o l . , 120:65-80, 1963. 12. L E V I - M O N T A L C I N I , R . — G r o w t h c o n t r o l o f n e r v e c e l l s b y a p r o t e i n f a c t o r a n d i t s a n t i s e r u m . S c i e n c e 143:105-110, 1964.
