ROLE OF A MUSCLE TARGET ORGAN ON THE REGENERATION OF MOTOR NERVE FIBRES IN LONG NERVE GRAFTS: A SYNOPSIS OF EXPERIMENTAL AND CLINICAL DATA MANFRED FREY, M.D., RUPERT KOLLER, M.D., CHRISTIAN LIEGL, M.D., WOLFGANG HAPPAK, M.D., and HELMUT GRUBER, M.D.

The importance of the muscle target organ as a promoting factor for nerve fibre regeneration in nerve grafts is still a subject of controversial discussion. In the last few years we published uniform results of experimental series in sheep and rabbits in which we investigated the biology of nerve fibre regeneration in long nerve autografts without connection to a target organ. Surprisingly, we found excellent regeneration of myelinated nerve fibres without the influence of a target or- gan through the whole length of the nerve graft, with an in- crease in the number of nerve fibres up to fourfold at the distal end. In the sheep series the additional contact with a muscle target organ for 6 months had a variable effect on the fibre population in the distal end of the nerve graft. The re-

sults of our planimetric analyses of nerve biopsies in patients with facial paralysis, who were treated by cross-face nerve grafting and free muscle transplantation, are extremely diver- gent from the results of our experimental series. Instead of an increase, we found a decrease in number of regenerating nerve fibres arriving at the distal end of the cross-face nerve graft down to 20%. Species-specific differences in the biol- ogy of nerve regeneration are discussed, together with their implications for the complex clinical situation of nerve grafting with or without the influence of a target organ.

0 1996 Wiley-Liss, Inc. MICROSURGERY 17:80-88 1996

In clinical practice two situations, present the question of whether the assumed positive influence of a muscle target organ on nerve regeneration is more important than the advantages of a two-stage procedure. One is the necessity of bridging a long distance by nerve grafts. Cross-face nerve grafting, for example, is intended to reinnervate the dener- vated facial muscles of the paralysed side through long nerve grafts interposed between the proximal stumps of fa- cial nerve branches on the healthy side and the distal stumps of the peripheral facial nerve branches on the paralysed side. Anderl' proposes principally a two-stage procedure to improve the chances of the regenerating nerve fibres cross- ing the second suture line. In his opinion this factor is more important than a promoting effect on nerve regeneration within the nerve graft by the target organ, which would be activated at the time of nerve grafting in the case of a one-stage procedure. Mackinnon et aL2 showed this posi- tive effect in a cross-face nerve grafting model in primates. Although this model seems to be near to human biology, the reliability of this study is limited by the extremely low

number of observations. For clinical application the proof of the better approach is still missing. Later in this article we will argue why we prefer a one-stage to a two-stage procedure.

The second situation is a functional reconstruction, in which not only the nerve has to be reconstructed, but also the muscle-the target organ itself. It is easily accepted that the risk of progressive and irreversible atrophy in the trans- posed or transplanted muscle is more important than a pos- itive effect on nerve regeneration by connecting the trans- ferred muscle immediately to the nerve graft in the same operation. This opinion is supported by one of our experi- mental studies in sheep.3 The group with nerve grafting and connection to a muscle in the same operation had the worst functional result. For human conditions a comparative study is difficult to perform, and a definitive answer is still ab- sent.

In rare cases we have the chance of comparing data from experimental studies with data from clinical studies. By taking every opportunity to obtain biopsies from the nerves and the muscles involved in neuromuscular reconstructions, we have gathered more and more quantitative insights into clinical regenerative processes ,4 and thus we understand the

From the Division of Plastic and Reconstructive Surgery, Department of Sur- gely (M.F., R.K., W.H.) and the Third Department of Anatomy (C.L., H.G.), Medical School, University of Vienna, Vienna, Austria.

Address reprint requests to Prof. Dr. Manfred Frey at the Klinische Abteilung fur Wiederherstellungs und Plastische Chirurgie, Universitatsklinik fur Chirur- gie, Wahringer Gurtel 18-20, A-1 090 Vienna, Austria.

Received for publication June 27, 1995; accepted September 31, 1995.

0 1996 Wiley-Liss, Inc.

principal difference between experimental and clinical data. Nevertheless one of these multiple factors in complex neu- romuscular reconstructions might clarify whether the re-

the extensive surgical effort is worthwhile. gained function is clinically useful and therefore whether

The purpose of this paper is to review the experimental

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Muscle Target Organ and Nerve Regeneration 81

cia1 nerve branches of the healthy side, original nerve fibre population in the nerve grafts guiding the regenerating fi- bres from the healthy to the paralysed side, regenerated and myelinated nerve fibres in the distal end of the cross-face nerve graft at the time of connection to the free muscle transplant and the pattern of muscle fibres in the gracilis muscle at the time of transplantation and after functional recovery. Seven patients with this kind of complete evalu- ation were included in this study.

and clinical data on regenerative processes in long nerve grafts with or without the influence of the muscular target organ, in an attempt to improve our understanding of the interactions between the nerve graft and the target organ and to facilitate the decision-making process in the clinical prac- tice of these complex reconstruction^.^-^

MATERIALS AND METHODS

Experimental Study in Sheep In 15 adult sheep, the saphenous nerve (28 5 1.8 cm)

was used for ipsilateral or cross-nerve grafting; the nerve was sutured to the proximal stump of the cut motor-nerve branch of the vastus medalis m ~ s c l e . ~ The distal end of the nerve graft was left without a target organ. Semithin cross sections of normal vastus nerves and saphenous grafts and of the distal ends of the grafts were analysed by computer- assisted planimetry 3, 6, 9, 12 and 18 months after the nerve grafting procedure. Changes in the distal end of the nerve graft induced by adding the influence of the rectus femoris muscle as a target organ for a further 6 months were investigated by comparison of the morphometric data.

Histological and Morphometric Assessment Histology and histomorphometry methods were the

same in the different studies and are described in detail in the original paper^.^-^ The nerve biopsies were fixed in 3% glutaraldehyde, post-fixed in 2% buffered osmium tetrox- ide, and embedded in Epon. For quantitative evaluation, 2 pm semithin cross sections were cut on an ultramicrotome and prepared for computer-assisted planimetry . The follow- ing parameters were evaluated for each of the specimens:

1 . Number and area of regular fascicles 2. Total number of myelinated nerve fibres 3. Diameter of myelinated nerve fibres (diameter of a

Experimental Study in Rabbits circle of the same area, as determined for the cross section of the nerve fibre) In 30 rabbits, both saphenous nerves were harvested as

autografts and coapted to the branch of the rectus femoris muscle without connection to any distal target muscle.6 The graft from the right thigh was led to the contralateral ex- tremity (crossover grafting). The graft on the left side re- mained on the same extremity (ipisilateral grafting). The animals were separated into four groups and were sacrificed 3 , 6 , 9 and 12 months after grafting. Specimens of the grafts and the donor motor branches were harvested for histomor- phometric examination. The aim of this study was mainly to support the findings of the experimental series in sheep by using a large number of animals. The performance of an ipsilateral and a crossover nerve graft in the same animal should elucidate possible differences in nerve regeneration between ipsilateral and crossover grafts.

Cross sections of the muscle biopsies were stained for ATPase activity after acid preincubation at pH 4.3 to dif- ferentiate between the different fibre types. The slow type I fibres were dark and intensively stained; and the fast type I1 muscle fibres were light. Besides the number and the di- ameter of the muscle fibres, the distributions between type I and type I1 fibres were evaluated by computer-assisted planimetry.

RESULTS AND DISCUSSION Selected results of our three studies will be presented so

that similarity or divergence becomes evident. On this basis implications for the clinical situations will be developed in the conclusion.

Study in Patients Experimental Study in Sheep Whenever possible, muscle and nerve biopsies were

taken in patients with irreversible unilateral facial palsy treated by cross-face nerve grafting and free gracilis muscle transplantation with microneurovascular ana~tomoses.~ In the first operation nerve biopsies were usually performed at the proximal stump of the healthy facial nerve branches and the sural nerve grafts were used for cross-face grafting. The distal end of the cross-face nerve graft and the gracilis mus- cle was biopsied during the muscle transplantation proce- dure 10-14 months later. During the final corrective pro- cedures, access became possible to the functioning muscle transplant. Planimetric analyses of cross sections provided us with the following information: fibre input from the fa-

The idea of this model was to use a motor nerve branch as a source of reinnervation, a procedure which offers enough fibres to grow into the nerve graft. The vastus nerve was selected not only because of its vicinity to the rectus nerve, but also because, with an average of 1,872 (SEM: 174.3) myelinated nerve fibres, it represents ideal conditions for reinnvervating the rectus femoris muscle, which is supplied by the rectus nerve with comparable fibre counts on the cross section (Fig. 1).3 The guiding structures of the saphenous nerve graft, which have about twice the number of nerve fibres at the time of nerve grafting, are not expected to present limitations. The median diameter of the healthy nerve fibres in the saphenous graft is about one-

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Muscle Target Organ and Nerve Regeneration 83

What are the changes in the population of regenerated fibres at the distal end of the nerve graft caused by the influence of the target muscle organ connected to the graft for 6 months? We would have expected this additional pro- moting factor to cause an increase in number of fibres, but the results are extremely inhomogenous. In the ipsilateral group as well as in the crossover group we found increases or decreases without any correlation to the interval without target muscle (Fig. 3). It is difficult to interpret the tendency toward an increase in median diameter in the ipsilateral group and a decrease in the contralateral group (Fig. 4). The more important question seems to be which of the regener- ated nerve fibres we found in the distal end of the graft innervated muscle fibres and what is the fate of the nerve fibres which had no contact with the muscle. Are they re- sponsible for this decrease in number and diameter?

Other strong factors like the second suture line or the distal muscle nerve segment, in which a promoting influ- ence is concentrated on the muscle target organ alone, make an interpretation difficult.

Experimental Study in Rabbits By using a similar model in a greater number of rabbits

and comparing ipsilateral and crossover nerve grafting within the same animal, we hoped to obtain more homoge- neous results and to revise the divergent results in ipsilateral and crossover nerve grafting.6 If results in sheep and in rabbits are compared, the principle differences in nerve re- generation (like different velocities of fibre growth or the quite different distances for generation) should be consid- ered. In our experiments in rabbits the results were similar to those of the sheep series concerning the relationship of the average number and diameter of nerve fibres along the nerve grafting model. The average number of regenerated nerve fibres was again three- to fourfold the number offered by the supplying rectus nerve. An increase in number of regenerated fibres could be only noted between the 3 month and the 6 month group. After that time the number did not change (Fig. 5) . A secondary decrease after a prolonged period without a target organ was not observed. A steady state between a small amount of microscopically visible degenerating fibres and compensatory regenerating fibres is responsible for this plateau.

The averages of the mean nerve fibre diameters were in a comparable range: original rectus nerve 10.05 pm, 4.43 bm in the proximal part of the original saphenous nerve grafts and 3.64 pm in the distal part; regenerated fibres in the distal part of the ipsilateral graft 3.34 pm and 3.77 bm for the crossover grafts (12 months after nerve grafting).6 The extensive alterations in the proximal supplying motor nerve observed completely changed our understanding of the effect of nerve grafting in the proximal direction. Far beyond the well-known Wallerian degeneration to the next

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Figure 3. Experimental series in sheep. Total number of myelinated nerve fibres in the distal end of the nerve graft after different regen- eration periods (filled bars), compared with the total number 6 months later, after connection to a target muscle for these respective time periods (hatched bars). lpsilateral (A) versus contralateral group (5). (From Frey et al. with permission from Thieme Medical Publishers, Inc.)

few internodes proximal to the transsection site the fibre number amounted to double; at the same time the fibre diameters decreased from about 10 pm to about 6 pm at the site of our biopsy 1 cm proximal to the nerve suture. There- fore the nerve grafting procedure does not only alter the morphology of the grafted nerve but also has a tremendous retrograde effect on the supplying motor branch. Differ- ences between the ipsilateral and the crossover grafts were found only until 3 months after grafting (Fig. 6). In this early state of regeneration, the crossover grafts contained significantly fewer fibres than the ipsilateral grafts. Greater mechanical stress to the crossover grafts seemed to be re- sponsible for this initial difference, which was no longer visible in the 6 month group.

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Figure 4. Experimental series in sheep. Median diameter of myelin- ated nerve fibres in the distal end of the nerve graft after different regeneration periods (filled bars), compared with the median diam- eter 6 months later, after connection to a target muscle for these respective time periods (hatched bars). lpsilateral (A) versus con- tralateral group (B).

The results of the experimental series in rabbits confirm the findings in sheep. The only finding that was not cor- roborated was the different regenerative capacity of ipsilat- era1 and crossover grafts. The greater number of animals and the faster regenerative processes in rabbits clarified that after an initial period of minor regeneration the crossover nerve grafts showed no difference.

Study in Patients Access to biopsies of nerves and muscles in patients is

limited, because they must be taken on occasion of the planned operations and the biopsy is not allowed to reduce the functional result of the reconstruction in any way. Nev- ertheless, the remaining possibilities for analysing human nerve and muscle biopsies are of great interest (realising

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Figure 5. Experimental series in rabbits. Summary of morphometric data concerning the average number of myelinated nerve fibres ob- served after ipsilateral grafting, pooling all animals of one group. A statistically significant difference (Student's t-test, P < 0.05) between the number of fibres seen in the grafts (TPL proximal and TPL distal) in group 1 and those in the other groups was observed. From 6 to 12 months after grafting, there are no significant changes. In addition, there are significant differences in each group between the original motor nerve (N. rectus) and the same nerve harvested at the final experiments (N. rectus Ff).

that results of animal esperiments are species specific and must be applied to human conditions with great care). His- tomorphometric assessment of cross sections of the healthy facial nerve branches shows a fibre input between 1 ,OOO and 2,000 fibres, with an average diameter of around 9 pm; the sural nerve graft contains between 2,000 and 2,500 thinner fibres, with an average diameter of around 5.5 pm, The diameter of the regenerated nerve fibres in the distal end of the cross-face nerve graft at the time of muscle transplan- tation is around 2.5 pm, in a similar relation to the diameter of the fibres of the source of reinnervation as in the exper- imental series (example in Fig. 7). In strong contrast to the results in sheep or rabbits, the number of regenerated nerve fibres in the distal end of the cross-face nerve graft is lim- ited to 100-200 myelinated fibres. This means that only 10-20% of the original fibres were present 10-14 months after nerve grafting. Unfortunately there is no further pos- sibility of insight into the later process of nerve regenera- tion. It is not known whether the number of regenerated nerve fibres will increase after the distal end of the cross- face nerve graft is connected to the gracilis muscle graft, or whether the promoting influence by the muscle target is significantly different from that in the animal models.

Our interest must focus on the final functional result. Documentation of the functional results of dynamic recon-

Muscle Target Organ and Nerve Regeneration 85

regenerated nerve fibres in the muscle nerve behind the second suture line, as we could show only in the sheep experiments. In our patients we gained more information about the reinnervation of the gracilis muscle graft by his- tomorphometric analyses of biopsies of the functioning muscle transplants. Type grouping and changed proportions of slow and fast contracting muscle fibres in the reinner- vated muscle graft showed us the strong influence of the characteristics of the facial nerve branches used for cross- face innervation (Fig. 8). Cadaver studies showed com- pletely different patterns of muscle fibre types in normal facial muscles. Therefore what kind of fibre pattern will be superimposed on the muscle graft depends on the facial nerve branch selected on the healthy side. The transplanted gracilis muscle may thus remain a fast contracting muscle or may be transformed into a slow contracting and more fa- tigue-resistant muscle (Fig. 8).

SYNOPSIS GROUP l ( 3 Months) Average Number of Flbers lpsllateral and Cross-over

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Figure 6. Experimental series in rabbits. Average number of myelin- ated nerve fibres observed in the original motor nerve branch and in other specimens harvested after different regeneration periods. Comparison between the ipsilaterai and the crossover grafting pro- cedure, pooling all animals of one group, in which grafts wuld be harvested biiaterally. A: Three months after nerve grafting, marked differences in the proximal and statistically significant differences in the distal part of the graft (TPL distal). 6: Twelve months after nerve grafting. In the nerve grafts (TPL proximal and TPL distal), no sta- tistically significant differences concerning the morphometric data were observed.

structions in the face is extremely difficult and calls for international ~tandardisation.~,~ Interestingly, the final functional result of the reinnervated muscle target was not parallel to the number of regenerated nerve fibres in the distal end of the nerve graft. This was true not only for the experimental series in sheep but also for our patients. A more precise prognosis is only given by the number of

CONCLUSIONS By carefully combining the data from experimental and

clinical studies, the following conclusions with significant relevance for clinical practice can be drawn:

1. Significant nerve fibre regeneration is possible in nerve grafts even without contact with the muscle target organ. A two-stage procedure is recommended for interpo- sitioning extremely long nerve grafts and for combining nerve grafting with muscle transfers. If interpositioning of a long nerve graft is performed as a one-stage procedure, the overlength has to be calculated in case of secondary resec- tion and renewal of the nerve suture at the distal end of the nerve graft.

2. Only a low number of nerve fibres in the proximal supplying nerve stump have a chance at arriving at the muscle target after they have grown through a long nerve graft in humans. To prevent the necessity of a nerve graft to reinnervate a muscle transplant, alternative techniques might be applied. One possibility is to place the transferred muscle more proximal to obtain direct coaptation between the proximal stump and the distal muscle nerve. The kinetic disadvantages of such a heterotopic muscle transplantation are far outweighed by the better reinnervation and func- tional recovery of the muscle. The other possibility is to use a muscle graft with a very long muscle nerve, which can be dissected far proximally. The gap is not covered by a nerve graft, but by the longer muscle nerve. Preventing a nerve graft must be considered much more important than the longer distance for nerve regeneration through the muscle nerve after muscle transplantation.

3. There are still enough clinical situations in which nerve grafting over long distances cannot be avoided. There is no doubt that very long nerve grafts can also be used successfully. No limits were found for the length of the graft. Even in extremely long nerve grafts the number of

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Figure 7. Semithin cross sections (left side) and corresponding his- tograms (right side) of nerve biopsies in one patient with unilateral facial paralysis. A: Branch of the facial nerve on the healthy side used for cross-face innervation. 8: Sural nerve used as a graff at the

time of the cross-face nerve grafting operation. C: Distal end of the cross-face nerve graft at the time of muscle transplantation 70 months after nerve grafting. (From Frey et with permission from Little, Brown and Company, Boston, MA.)

regenerating fibres does not decrease from proximal to dis- tal. Assuming that regeneration is more limited in human nerve grafts than in several animal models, the fibre input

from the proximal nerve stump becomes of crucial impor- tance. In these clinical situations branches with a high fibre content should be selected for reinnervation.

Muscle Target Organ and Nerve Regeneration 87

Figure 8. Study in patients treated for facial paralysis by free gracilis contracting, light fibres). 0: Normal buccinator muscle. E: Function- muscle transplantation. Muscle cross sections stained for ATPase ing gracilis muscle graft of another patient 10 months after trans- after acid pre-incubation at pH 4.3. A: Normal gracilis muscle at the plantation (see similarity of the fibre type pattern in D with predom- time of transplantation. 6: Normal levator anguli oris muscle. C: inantly slow contracting, dark fibres). (From Frey et with Functioning gracilis muscle graft 10 months after transplantation permissions from Little, Brown and Company, Boston, MA.) (see similarity of the fibre type pattern in B with predominantly fast

4. Crossover nerve grafting seems to be as successful as ipsilateral nerve grafting. Nerve grafting to the contralateral side of the body is a well-accepted method in the face, but

little experience has been reported for the extremities, pos- sibly because of fear of movement coordination problems. Weakening of the healthy side, together with an uncertain

88 Frey et al.

result, is often the main argument against this method, which will gain more and more acceptance in the future.

5 . A positive influence of the muscle target organ on nerve regeneration in a nerve graft is difficult to prove, because regeneration develops very well in the nerve graft without any target organ. An additional promoting factor seems to be delivered more by the distal muscle nerve seg- ment than by the target muscle itself. Comparison of nerve regeneration with and without the influence of the target muscle is extremely difficult, especially in men. We also have no chance to obtain morphologic data on the additional changes in a human nerve graft. Therefore, we always in- volve the muscle target organ in our patients, as an addi- tional promoting factor, when no disadvantage can be shown. For example, within the first year we treat irrevers- ible facial palsy with cross-face nerve grafts with overlength from the buccal and the temporal branches of the healthy side to the corresponding distal branches of the paralyzed side. By that one-stage procedure the long nerve grafts are exposed to the influence of the denervated facial muscles from the beginning. If there is no reinnervation or func- tional recovery of the facial muscles detectable after 1-1 /2 years, the overlength of the cross-face nerve graft is still used to hook up a free muscle graft to the distal end without any disadvantages. These considerations must be part of the discussion with the patient from the beginning. Otherwise the patient will not accept further extensive surgical treat- ment. Replacement of a nonfunctioning muscle target by a free muscle transplant must be included in the plan before any nerve grafting procedure.

6. Immediately after the nerve grafting procedure the nerve fibres grow from the supplying proximal nerve into the nerve graft and will reach the distal end of the graft after several months. No negative effect was observed in grafts left alone for a longer period which was necessary to reach a plateau in the number and average diameter of the regen- erated nerve fibres. In clinical practice this result enables us

to recommend waiting long enough before muscle grafting. Therefore we can expect progressive nerve fibre regenera- tion up to the end of the nerve graft, but we do not have to fear a loss of nerve fibres by waiting too long.

7. The biology of nerve regeneration is highly species specific. Experimental studies help us to understand the interactions of different factors, but they can never replace detailed clinical studies, which include standardised docu- mentation of the lesion, the therapeutic measures and the functional r e ~ o v e r y . ~ ~

REFERENCES 1 . Anderl H: Reconstruction of the face through cross-face nerve trans-

plantation in facial paralysis. Chirurgia Plastica 2: 17-45, 1973. 2. Mackinnon SE, Dellon AL, Hunter DA: Histological assessment of

the effects of the distal nerve in determining regeneration across a nerve graft. Microsurgery 9:46-51, 1988.

3. Frey M, Gruber H, Happak W, et al.: Ipsilateral and cross-over elongation of the motor nerve by nerve grafting: An experimental study in sheep. Plastic and Reconstructive Surgery 85:77-89, 1990.

4. Frey M, Happak W, Girsch W, et al.: Histomorphometric studies in patients with facial palsy treated by functional muscle transplantation: New aspects for the surgical concept. Annals of Plastic Surgery 26: 370-379, 1991.

5. Frey M, Koller R, Gruber I, et al.: Time course of histomorphometric alterations in nerve grafts without connection to a muscle target organ: An experimental study in sheep. Journal of Reconstructive Microsur- gery 8:345-357, 1992.

6. Koller R, Frey M, Meier U, et al.: Fibre regeneration in nerve grafts without connection to a target muscle: An experimental study in rab- bits. Microsurgery 14:s 16-526, 1993.

7. Carpenter MS, Sulin J: Human Neuroanatomy. Baltimore, Williams & Wilkins, 1983, p 124.

8 . Frey M, Sing D, Harii K, et al.: Free muscle transplantation for treatment of facial palsy. First experiences with the International Mus- cle Transplant Registry. European Journal of Plastic Surgery 14:212- 218, 1991.

9. Frey M, Jenny A, Giovanoli P, Stussi E Development of a new documentation system for facial movements as a basis for the Inter- national Registry for Neuromuscular Reconstruction in the Face. Plas- tic and Reconstructive Surgery 93:1334-1349, 1994.

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