British JournalofPlastic Surgery (2000), 53, 149 154 �9 2000 The British Association of Plastic Surgeons DOI: 10.1054/bjps. 1999.3252

BRITISH JOURNAL OF ~ PLASTIC SURGERY

Selection of donor nerves - an important factor in end-to-side neurorrhaphy

B. S. Lutz, D. C. C. Chuang, J.-C. Hsu*, S.-F. Ma t and F. C. Wei

Department of Plastic and Reconstructive Surgery, J-Microsurgical Research Laboratory, and *Department of Anaesthesiology, Chang Gung Memorial Hospital, College of Medicine and Chang Gung University, Taipei, Taiwan

SUMMARY. We have examined the effects of end-to-side neurorrhaphy on peripheral nerve regeneration using the median nerve as recipient nerve and either the antagonistic radial nerve or the agonistic ulnar nerve as donor nerves in rat upper limbs. A perineural window was created in all cases. Motor recovery up to 16 weeks post- operation was tested with the grasping test. No recovery of motor function was evident after end-to-side neurorrhaphy of the median nerve to the antagonistic radial nerve, whereas six of eight rats with end-to-side neu- rorrhaphy to the agonistic ulnar nerve achieved 367 g + 47 g grasping power as compared to 526 g + 6 g in end-to- end coapted control animals. No significant difference in flexor digitorum subl imus-motor nerve conduction velocity was found among all three groups. Radial nerve stimulation produced simultaneous contraction of both extensor and flexor muscles of the lower a rm that disabled any coordinated movement of the paw. Histology (toluidine blue, acetylcholinesterase-stain) showed multiple regenerated (motor)-axons distal to the coaptation site in the median nerve. Reinnervation of the median nerve solely by the respective donor nerve was demon- strated by a retrograde double labelling technique. These results show that averaged 70'70 muscle power as com- pared to end-to-end neurorrhaphy with well coordinated muscle function can be achieved by axonal sprouting through end-to-side neurorrhaphy if an agonistic nerve is used as donor nerve. However, satisfying results are unpredictable. Antagonistic nerves show the ability to induce axonal regeneration, but no useful function can be expected. �9 2000 Harcourt Publishers Ltd

Keywords: nerve regeneration, end-to-side neurorrhaphy, antagonist muscle groups, experimental, rat.

Reinnervation of a transected peripheral nerve by coapting it in an end-to-side fashion to a healthy donor nerve was described at the beginning of this century by Ballance ~ and Harris. 2 However, owing to unsatisfactory results this technique was abandoned until 1992 when Viterbo 3 reintroduced this method of nerve repair. Since then, various studies + ~2 have shown histological and/or electrophysiological evidence of both sensory and motor axon regeneration after end-to-side coaptation. This phenomenon may result from collateral sprouting induced by neurotrophic factors. 5 Such factors released from the end-to-side implanted nerve stump cause the uninjured donor-nerve axons to give rise to a sprout which emerges from its side and passes down the attached distal segment while the original homonymous nerve still carries on its original target organ. Collateral sprouting from uninjured axons induced by several neurotrophic or nerve promoting factors such as nerve growth factor, 13,14 brain-derived nerve growth factor] s growth associated protein-43,16 ciliary neurotrophic factor and basic fibroblast growth fac to r , 17 insulin-like growth factor, ~8,~9 antiproteases, 2~ or gangliosides 2~ has been demonstrated. These same factors and substances play an important role during axonal regeneration with most of them released by Schwann cells after nerve injury, especially at the distal nerve stump. ~,22 2s

In these cases in which collateral axonal sprouts from the donor nerve are able to provide sufficient

reinnervation of the end-to-side coapted recipient nerve, a simultaneous movement of muscles inner- vated by the donor nerve and muscles reinnervated by the recipient nerve is to be expected. Simultaneous co- contractions of flexor and extensor muscles, however, impair any useful function of the respective muscles. 26 No functional motor recovery in walking track analy- sis was found after end-to-side neurorrhaphy using the antagonistic peroneal and posterior tibial nerves in the rat. 27 Similar results followed end-to-side coaptation of the rat median nerve to itself, using an ulnar nerve graft. 2s

Our study was conducted using rat median nerve as recipient nerve and either the agonistic ulnar nerve or the antagonistic radial nerve as donor nerves. Our aim was to evaluate recovery of motor function after direct end-to-side neurorrhaphy and to define suitable condi- tions for successful end-to-side neurorrhaphy.

Materials and methods

Animals'

Twenty-one male Sprague-Dawley rats weighing 300 g were used in this experiment. They were anaesthetised with intraperitoneally applied ketamine (40 mg/kg/rat) and atropine (0.04mg/kg/rat). During the whole experiment, the rats had water and standard chow

149

150 British Journal of Plastic Surgery

ad libitum and were treated according to the standards for the use of laboratory animals established by the Institute of Laboratory Animal Resources, US National Academy of Sciences.

examination the respective donor and median nerves were removed for histological evaluation.

Histological studies

Operations

In 16 rats the left median nerve was transected at left upper arm level, 1.5 cm resected, and the distal stump coapted in end-to-side fashion either to the radial nerve (group A = 8) or to the ulnar nerve (group B = 8) through a perineural window. The proximal stump of the median nerve was curved back and implanted into the pectoralis major muscle. In group A, additionally a 2.5 cm segment of the ulnar nerve was resected. Although the ulnar nerve does not show any additional innervation of the muscles that are innervated by the median nerve in the rat, 29 the resec- tion was done as a safety procedure. In five rats, which served as controls (group C), the median nerve was transected and recoapted in end-to-end fashion. 11/0 nylon sutures were used in all cases. On the contralat- eral limb, both median and ulnar nerves were resected at upper arm level for the subsequently planned grasp- ing test.

Gmsping test

A handy pull and push gauge (model AK-1, Algol, Japan) with a grip at its upper end was used. The rats were gently lifted by the tail and allowed to grasp the grip of the gauge as healthy rats naturally do when lifted up in this way. They were lifted with increasing firmness until they loosened their grip. Each rat was tested three times. Only a four digit grasp with the elbows in extension was accepted as a valid test. The value registered by the gauge at the moment of loosen- ing of the grip was documented. All three values were averaged. This test was described by Bertelli 3~ to per- mit observation of the onset of functional recovery and its time related improvement. It was performed after onset of grip function, then again after 12 and 16 weeks in all 21 rats.

Motor nerve conduction velocity ( MNCV)

In six animals of groups A and B, and in all five ani- mals of group C, 16 weeks post-operation motor nerve conduction velocity was measured in the flexor digito- rum sublimus muscle (FDS). In the anaesthetised rats, reincision was made on the left upper limb. The nerves and the FDS were exposed and dissected. Stimulating eIectrodes were placed on the respective donor nerve proximal to the nerve coaptation site. A recording elec- trode was inserted in the FDS. MNCV was determined by the difference in latency when triggering a muscle response from each stimulating electrode. 31 Equipment used was a Caldwell, Quantum 84 instrument. During the whole procedure the animals were kept warm by a heating mat. Nerve and muscle were kept moist using 0.9% saline solution. After having finished the

The excised nerve segments were fixed overnight in a solution of 2.5% glutaraldehyde in PBS at 4~ After postfixation in 1% osmium tetroxide and dehydration in a series of ethanol solutions, the specimens were embedded in Agar 100 resin. One micron transverse sections of the median and donor nerves were cut from the distal and proximal segments, while longitudinal sections were cut from the attachment site. The sections were stained with toluidine blue and examined with a light microscope. Distal nerve segments from the median nerve and muscle branches to the FDS/FDP muscles were prepared for acetylcholinesterase stain. 32

Retrograde double labelling technique

The fluorescent retrograde axonal tracers Fast Blue (FB, Sigma, USA) and Diamidino Yellow (DY, Sigma, USA) were employed to prove that axonal outgrowth after end-to-side neurorrhaphy results from collateral sprouting of the donor nerve. This investigation was performed 16 weeks post-operation in two rats of group A and B respectively. FB was injected into the median nerve using a Hamilton syringe, 1 gl of 3% dye respectively. Similarly, t gl 0.2% DY was injected into the respective donor nerve. Injection was performed in both cases slowly distal to the elbow. To avoid contam- ination of adjacent tissue or leakage of the dye, the surrounding tissue was covered with filter paper insu- lated with Vaseline. After a transit time of 12 days, the animals were deeply anaesthetised and transcardially perfused with 500 ml warm Ringer solution + heparin 1000IE/kg/rat followed by 500ml ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer. The cervical spinal cords, C5 to Thl , were removed and placed in 30% sucrose/4% paraformaldehyde solution for 24 h. Longitudinal serial frozen sections (35 gm) were cut with the cryostat and mounted on slides. Epifluorescence microscopy (Nikon filter, DM 430, V- 2A, BA 450) was used to count the motoneurons: 1. yellow nucleus = single labelled DY, 2. blue cytoplasm = single labelled FB, and 3. yellow/whitish nucleus and blue cytoplasm = double labelled DY/FB. Only clearly visible and complete neurons were counted. Double labelled motoneurons (Fig. 1) indicate reinnervation of the median nerve by the respective donor nerve. DY single labelled motoneurons (Fig. 1) indicate axons innervated by the respective donor nerve only. FB single labelled motoneurons indicate reinnervation of the median nerve by '?' which might be a hint for distal contamination by regenerating nerve fibres from the proximal median nerve stump.

Statistics

Paired and unpaired Student's t-test (Jandel Scientific, Sigma Plot for Windows) was employed. A value of P < 0.05 was considered significant.

End-to-side neurorrhaphy 151

Figure 1--Two FB/DY-double labelled motoneurons with blue cytoplasm and yellow-whitish nucleus (arrow) and one single DY-labelled motoneuron with yellow nucleus (arrowhead) in the ventral horn of a rat from group A. The multiple small bodies are glial cells (x 140).

Table 1 Results of: 1. grasping test (12, 16 weeks) and 2. MNCV (16 weeks) of the flexor digitorum sublimus muscle in groups A, B, C.

Group No. Test Results

1. Grasping test positive Grasping power (g) (12 weeks) (16 weeks)

A 8 - - B 8 6 255 + 68* 367 + 47* C 5 5 437+ 16 5 2 6 + 6

2. M N C V (re~s) (16 weeks) A 6 51.8 _+ 25.8 B 6 32.0 +_ 28.3 C 5 32.5 _+ 13.6

*Statistically significant.

Results

Grasping test

Onset of digit flexion was observed in all animals of group B and C after 4-5 weeks with ability to perform the grasping test after an average of 5 weeks in all rats of group C and 6 weeks in six rats of group B. Two rats of group B were able to grasp, but lacked any grasp power in the grasping test. Animals in group A did not regain a clear grasping movement, thus no grasping test could be performed in these eight ani- mals. Results after 12 and 16 weeks are shown in Table 1 and reveal a significantly stronger grasp power in the control group. However, after 16 weeks end-to-side neuro-rrhaphy in six rats of group B achieved an aver- aged grasp power of 70% as compared to end-to-end neurorrhaphy in group C.

Motor nerve conduction velocity ( MNCV)

Results of M N C V of the FDS muscle in rats of group A, B and C showed no significant difference (Table 1).

Interestingly, a simultaneous contraction of both extensor and flexor muscles occurred after stimulation of the proximal radial nerve that disabled any coordi- nated movement of the wrist or paw. Stimulating the radial nerve proximal to the coaptation site after hav- ing cut the radial nerve distally produced a good and coordinated flexion of the wrist and paw.

Histology

Multiple nerve fibres were evident in the median nerve 2 cm distal to the coaptation site in the experimental groups with no signs of Wallerian degeneration at the coaptation site. Regenerated axons in a motor branch to the flexor muscles are shown in Figure 2. The same branch after acetylcholinesterase stain demonstrates regenerated motor axons (Fig. 3).

Retrograde double labelling

Almost all labelled neurons were located laterally in the grey matter of the ventral horn close to the white

152 British Journal of Plastic Surgery

Discussion

Figure 2--Branch of the median nerve to the flexor muscles (FDS, FDR FCR) showing regenerated axons in group B (toluidine blue, x 650).

Figure 3--Same branch as in Figure 2. Acetylcholinesterase stain demonstrating multiple regenerated motor axons (x 200). AChE stained myelinated axons are indicated by dark spots in their centre (arrowhead)~

matter as well as in the dorsal root ganglia from C6 to ThI with the majority of neurons distributed to C6/7. No contralateral neurons were labelled. In the ventral horn, double labelled neurons were of lesser amount than in the dorsal root ganglia and were found to form clusters (Fig. 4). Ratio of DY/FB-double labelled motoneurons to DY-single labelled neurons was 10:3.3 in the ventral horn and 10:7 in the dorsal root ganglia. No FB-single labelled motoneurons were found in the spinal cord, thus excluding reinnervation of the median nerve by its proximal stump after end-to-side neurorrhaphy.

Several studies investigating end-to-side neuro- rrhaphy have demonstrated axonal outgrowth from the donor nerve into the recipient nerve histologically or immunocytochemically 3-L2 and by electrophysiolog- ical tests. 4-7,9,~~ This phenomenon has given rise to several questions:

1. Does an injury to the donor nerve during end- to-side coaptation induce axonal regeneration from these injured axons into the recipient nerve?

2. Does the demonstrated axonal outgrowth into the recipient nerve result f rom regenerating axons from the proximal stump of the recipient nerve itself?

3. I f the axonal outgrowth indeed results from true collateral sprouting from axons of the donor nerve into the recipient nerve, does this sprout- ing provide sufficient reinnervation of the recipi- ent nerve's targets?

Histological assessment of the coaptation site in our animals excluded injury to the donor nerve, although a perineural window had been created. This was consistent with the observations of others, t~ Possible axonal outgrowth by regenerating axons from the original proximal stump of the median nerve was excluded by a retrograde double labelling technique as described in the results section. Thus, the obsetwed reinnervation of the distal median nerve is regarded as being solely due to collateral sprouting from healthy axons of the respective donor nerves.

In two studies, completed functional tests after end-to-side neurorrhaphy have been published with both of them 27,28 demonstrating poor functional recov- ery. In contrast to these experiments, our results after direct end-to-side coaptation of the median nerve to the agonistic ulnar nerve showed acceptable functional motor recovery in six out of eight rats. Although the grasping power was significantly lower than after end- to-end coaptation and a high standard deviation showed a wide recovery range , averaged 70% grasping power as compared to end-to-end coaptation was achieved by end-to-side neurorrhaphy. Tham j2 found 60% contractile force of the gastrocnemius muscle after having coapted a muscle branch end-to-side to its parent tibial nerve which resembles Lundborg's results. 5 Tarasidis, 27 coapting rat posterior tibial nerve end-to-side to the peroneal nerve found no significant differences in mean conduction velocity between exper- imental groups and control data which is consistent with our results and those of other studies, j~ However, walking track analysis in his experiment indicated poor functional recovery. In 1994, Lundborg 5 observed simultaneous co-contractions of the tibialis anterior and the gastrocnemius muscles during electrostimula- tion of the proximal tibial nerve after end-to-side coap- tation of rat peroneal nerve to the antagonistic tibial nerve. Co-contractions of two antagonistic muscles impair any useful function. 26 Therefore, even when end-to-side neurorrhaphy is able to provide sufficient reinnervation of a recipient nerve, an experimental model employing two antagonistic nerves cannot

End-to-side neurorrhaphy 153

Figure 4~Three clearly FB/DY-double labelled (arrow) and multiple DY-single labelled (arrowhead) motoneurons forming cluster-like formations in the ventral horn of the spinal cord in a rat of group B (x 70).

demons t ra te any funct ional recovery. In our experi- ment , despi te excellent results in m o t o r nerve conduc- t ion velocity, end- to-s ide coap ta t ion o f the med ian nerve to the antagonis t ic radial nerve showed a lack o f any useful function. Bertelli 's mode l 28 employed the med ian nerve coapt ing it to itself, using an u lnar nerve graft. The median nerve was t ransected at the uppe r a rm level, its p roximal s tump implan ted to the rat equivalent o f the h u m a n latissimus dorsi muscle. A n u lna r nerve graft, harvested f rom the same limb, was coap ted end- to-s ide to the ep ineur ium o f the p rox imal med ian nerve and end- to-end to the distal med ian nerve stump. N o funct ional m o t o r or sensory recovery has been demons t ra ted . His to logica l evaluat ions are pending. As a possible reason for these p o o r funct ional results we suggest the following: axonal regenerat ion o f the t ransected d o n o r nerve was directed towards the muscle in which the p r o x i m a l s tump was implanted . Owing to the long dis tance f rom the proximal med ian nerve to the distal med ian nerve stump, fur ther compl i - ca ted by using a nerve graft, neuro t roph ic a t t rac t ion f rom the distal nerve s tump to the p rox imal nerve was p robab ly too weak to induce any axonal sprout ing in the proximal med ian nerve after epineura l end- to-s ide coapta t ion . This is consis tent with the f indings o f A1- Qa t t an 33 who did no t f ind any regenerat ing axons af ter epineura l end- to-s ide neuror rhaphy in nerve grafts.

As demons t r a t ed by G o r d o n , 34 20% m o t o n e u r o n s o f the or iginal m o t o n e u r o n poo l are sufficient for muscle funct ion because m o t o r units can enlarge up to abou t five t imes their or iginal size, This can expla in the p h e n o m e n o n o f 60-70% recovered muscle power af ter end- to-s ide neu ro r rhaphy which was demon- s t ra ted in our and o ther studies, 5,12 a l though the num- ber o f re innervat ing m o t o n e u r o n s in the ventra l ho rn o f the spinal cord was found to be marked ly lower than after end- to -end neurorrhaphy27

Conclusions

1. End- to -s ide neu ro r rhaphy in the rat can achieve an average 70% of muscle power as c o m p a r e d to end- to -end neu ro r rhaphy i f an agonis t ic d o n o r nerve is employed . However, sat isfying results are unpredictable .

2. Antagon i s t i c nerves as d o n o r nerves show the abil i ty o f axona l regenera t ion, but no useful funct ion can be expected because o f resul t ing co-cont rac t ions .

3. A x o n a l regenera t ion in end- to-s ide neu ro r rha - phy results f rom col la tera l sprout ing o f hea l thy d o n o r nerve axons, mos t p robab ly induced by neu ro t roph ic factors released f rom the a t t ached dis ta l nerve stump.

Acknowledgements The authors wish to express their thanks to Ms Shu-Hui Lee for her excellent assistance in carrying out this study. Many thanks also to M. Schr6der MD, Professor and Chairman of the Department of Neuropathology, University Hospital of Aachen, Germany, for his helpful evaluation of the histological specimen.

Barbara S. Lutz was supported by a grant of Deutsche Forschungs-gesellschaft (DFG) 649; the project was supported by a Chang Gung Medical Research Grant.

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T h e A u t h o r s

Barbara S. Lutz M/D, Microsurgical Fellow, David C. C. Chuang MD, Consultant Plastic Surgeon and Professor of Surgery, Department of Plastic and Reconstructive Surgery

Jee-Ching Hsu MD, Consultant Anaesthetist, Department of Anaesthesiology,

Sbwu-Fan Ma PbD, Director of Microvascular/Neural Laboratory

Fu-Cban Wei MD, FACS, Consultant Plastic Surgeon, Professor and Chairman, Vice-Superintendent of CGM-Hospital

Chang Gung Memorial Hospital, College of Medicine & Chang Gung University, Taipei, Taiwan, R.O.C.

Correspondence to Dr Barbara S. Lutz, Senior Consultant, Department of Hand and Plastic Surgery, University Hospital Umea, S-90185 Umea, Sweden.

Paper received 7 December 1998. Accepted 13 September 1999, after revision.