S E L E C T I V E R E I N N E R V A T I O N OF R E G E N E R A T I N G M I X E D NERVE FIBRES ACROSS A SILICONE TUBE GAP Further experimental evidence of neurotropism Z - J . L U O a n d S-B. L U
From the Departments of Orthopaedic Surgery, Air Force GeneralHospital and GeneralHospital of PLA, Beijing, People's Republic of China This study investigated specific regeneration of a mixed motor and sensory nerve by the method of spinal dorsal root ganglions resection. A I0 mm segment of tibial nerve was resected and the nerve ends inserted in a silicone tube. Fourteen weeks later, dorsal root ganglia from L6 to S1 were resected on the experiment side. Twenty weeks later, the regenerating motor nerve fibres of mixed nerves selectively grew into motor branches. The rate of misdirected growth in mixed nerves was less than 6%. These results suggest that regenerating motor and sensory axons of mixed nerves are able to select their distal target organs accurately. Better results may be obtained using the entubulation repair method.
Journal of Hand Surgery (British and European Volume, 1996) 2lB." 5." 660-663 How regenerating axons direct themselves towards their end targets correctly after nerve transection is a complicated problem. Increasing knowledge of neurotropism has made the study of tubulation of interest. Many experiments have demonstrated that regenerating nerves can selectively reinnervate their targets. Non-neural nerve conduits to bridge nerve gaps have been successful in many animal experiments. These conduits provide a closed space for regenerating axons. It seems that regenerating nerves can cross a 10 mm conduit which contains the distal nerve end. Nerve regeneration will not occur in an open-ended conduit or in a conduit where the distal end is blocked by non-neural material (Danielsen et al, 1983; Lundborg and Hansson, 1979; Lundborg et al, 1982; Nachemson et al, 1988; Seckel et al, 1984; Williams et al, 1984). Preferential growth of regenerating nerve fibres towards and into the distal nerve tissue regardless of the position in the chamber have been shown using a preformed rectangular mesothelial chamber to study possible neurotrophic and chemotactic effects on outgrowing axons (Nachemson, 1988). In a study of a Y-shaped nerve guide, the proximal peroneal fascicle of a rat sciatic nerve was inserted into the proximal limb of a Y-shaped chamber, and the distal peroneal and tibial fascicles were placed within the two distal limbs of the same Y. The proximal peroneal nerve grew preferentially to the peroneal fascicle (Seckel et al, 1986). Similar experiments demonstrated that a motor nerve could also grow preferentially to the distal motor stump (Brushart, 1988; Brushart and Seiler, 1987). However, as there has been no precise method of distinguishing motor and sensory axons in mixed peripheral nerves, most of the studies could only observe neurotropism in motor or sensory nerves. There is little information about neurotropism in mixed nerves and no reports about the rate of misdirected growth up to now. We designed an experimental model in which we investigated the regenerating motor and sensory fibres of a mixed nerve across a 10 mm nerve gap bridged by a silicone tube.
MATERIALS AND M E T H O D S Twelve adult dogs weighing 15 to 20 kg were used. (Animal care complied with the guidelines of the National Institutes of Health and national law on the care and use of laboratory animals.) Animals were anaesthetized by intravenous injections of sodium pentobarbital. In the six animals in the experimental group, the tibial nerve of the right lower extremity was exposed by a longitudinal popliteal incision. The tibial nerve was mobilized proximal to its division into motor and sensory branches. The branch to the soleus muscle was cut and ligated. A 10 mm tibial nerve segment, which contained two motor branches (gastrocnemius branches) and one sensory branch (sural nerve), was resected 3 to 4 cm above the entry into gastrocnemius and the nerve ends placed in a 10 mm silicone tube (Fig 1). Fourteen weeks later, the animals were reanaesthetized. The lamina of L6 to S1 were resected through a posterior lumbar approach. The spinal dorsal ganglions (DRGs) of L6 to S1 were exposed and resected. The animals
Fig 1
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Entubulation of tibia1 nerve. The proximal end is at left. The gap is 10 ram.
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were observed for 6 more weeks and then the tibial nerve and its branches were removed. Another six normal animals comprised the control group. Their D R G s from L6 to S1 were resected on the right side. The peripheral nerves of the extremities were not treated. Six weeks later, the right tibial nerve and its branches were removed as in the experimental group. The specimens were placed in 2.5% glutaraldehyde fixative overnight. Then the nerves were post-fixed in 1% osmium tetroxide, and embedded in Epon 812. Onemicron transverse sections were cut and stained with methyl blue and suchsin. The sections were taken 2 mm proximal to the site of entubulation, the middle of the tube, 2 mm distal to the site of entubulation and the tributary of tibial nerve respectively (Fig 2). The total number of nerve fibres in each part of the sections was counted in a computer (IBAS-2000). The number of axons/mm 2 were analysed with the aid of a computer. The motor nerve fibre count in the endmotor and sensory nerve branches were statistically analyzed by using non-parametric two-tailed tests. RESULTS Light microscopic evaluation demonstrated that all myelinated nerve fibres in the sensory branches of the tibial nerve were totally degenerated in the control group. There were no motor nerve fibres in the sural nerve (Fig3a) compared to the motor branches (Fig 3b). Some of the nerve fibres in the gastrocnemius branches had degenerated, but most myelinated fibres (nearly two-thirds of the nerve fibres) were seen clearly. In the experimental group, the axons regenerating through the silicone tube reached its distal end. The growth density of nerve fibres proximal to the site of entubulation was 1627/mm 2, the growth density of nerve fibres distal to the entubulation was 2067/mm 2, the ratio of distal to proximal being 1.5 : 1. The number of motor axons which regenerated into the distal motor branches was significantly greater than the number which regenerated into the distal sensory branch (P<0.01) (Table 1, Proximal Stump
Comparison of motor branches and sensory branches of tibial nerve. (a) Transverse section of the sural nerve shows no myelinated axons 6 weeks after D R G resection. (b) In contrast, the gastrocnemus branch of the tibial nerve contains mostly myelinated axons and partially degenerated axons. (Original magnification x 400.)
Table 1 - - M y e l i n a t e d motor axon counts in the distal branches of the
initial nerve
Control group Experimental group
Regenerated Distal Nerve Stump
Motor nerve branches Mean (SEM)
Sensot'y nerve branch Mean (SEM)
1225 (120)** 1199 (102)**
0 21 (25)
**P<0.01.
m
i I t
Fig 3
I i I
Figs 4a and 4b). The rate of misdirected growth of motor fibres was 0 to 6%. DISCUSSION ,~NMG Silicone Tube
Fig 2
Diagramto show the sites of nerve section.
This study confirms that the regenerating axons of a mixed nerve can cross a 10 mm gap to reach their distal motor and sensory branches. The silicone tube provides the regenerating axons with closed space in which they can choose their direction freely. After resection of DRGs, sensory nerve fibres of peripheral nerves begin
THE JOURNALOF HANDSURGERYVOL.21B No. 5 OCTOBER1996
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Fig 4
(a) Transverse sections of distal sensory nerve branch of tibial nerve after regeneration. (b) Transverse section of distal motor nerve branch of tibial nerve after regeneration. (Original magnification x 400.)
Wallerian degeneration. Six weeks after D R G resection, all nerve fibres in the peripheral nerve were m o t o r axons. All nerve fibres degenerated completely in the sural nerve in the c o n t r o l group, indicating that the sensory branch o f the tibial nerve in the dog is a pure sensory nerve a n d does n o t c o n t a i n a n y m o t o r nerve fibres. This provided a m o d e l for investigation o f selective regeneration of mixed nerve t o w a r d s its distal m o t o r a n d sensory divisions. The n u m b e r of m o t o r axons entering motor branches was m u c h m o r e t h a n the n u m b e r o f motor axons entering sensory b r a n c h e s in the experimental group. This suggests that nerve regeneration has target-specificity due to n e u r o t r o p i s m . It is t h o u g h t that the distal m o t o r nerve or m o t o r end organs attract a n d influence regenerating m o t o r axons t h r o u g h diffusible neurotropic factors ( L u n d b o r g et al, 1982; Politis et al, 1982). It has been d e m o n s t r a t e d that the tropic factors are protein a n d / o r p r o t e i n d e p e n d e n t a n d are in degenerated distal peripheral nerves. N o r m a l nerves do n o t contain these factors or c o n t a i n substances which inactivate them (Politis a n d Spencer, 1983). Some n e u r o t r o p i c factors ( N T F s ) a n d neurite p r o m o t i n g factors ( N P F s )
have been f o u n d in nerve regeneration c h a m b e r s ( L o n g o et al, 1983a; 1983b; 1984). These factors have been p r o v e d to e n h a n c e nerve r e g e n e r a t i o n ( D a v i e s et al, 1985). The m e c h a n i s m of n e u r o t r o p i s m r e m a i n s u n k n o w n . I n o u r experiment, the distance between nerve stumps in the tube was 10 m m a n d the results strongly s u p p o r t tropic regeneration. The m o t o r nerve fibres in a mixed nerve could distinguish their distal m o t o r a n d sensory c o m p o n e n t s a n d reach their e n d targets d u r i n g the regeneration. We call this n e u r o t r o p i c p h e n o m e n o n " F u n c t i o n a l F i b r e Specificity". A t present, most m e t h o d s o f peripheral nerve repair fail to restore n o r m a l m o t o r a n d sensory f u n c t i o n ( M a c k i n n o n a n d Dellon, 1990). The m a i n r e a s o n for this is that the regenerating axons grow into i n a p p r o priate e n d o n e u r i a l tubes. After e x p e r i m e n t a l work, L u n d b o r g a n d his colleagues (1994) have succeeded in clinical repair o f a nerve defect by using a silicone tube. E n t u b u l a t i o n in nerve repair provides nerve fibres with a m i c r o e n v i r o n m e n t in which the regenerating axons are p r o v i d e d with directional guidance. O u r present study confirmed that a short nerve gap bridged b y a silicone tube i m p r o v e d the results of nerve repair b y increasing the p r o p o r t i o n o f regenerating axons which entered the a p p r o p r i a t e e n d o n e u r i a l tube a n d decreased the rate of misdirected growth. W i t h further refinement of nerve guide material, a n d b y a d d i n g effective n e u r o t r o p h i c factors to the inside of the tube, better clinical results s h o u l d be obtained. O u r experimental m e t h o d of D R G resection can distinguish between m o t o r a n d sensory axons in peripheral mixed nerves a n d can be used to calculate the rate o f misdirected growth. It should prove useful for q u a n t i t a t i v e study of nerve r e g e n e r a t i o n a n d neurotropism. References BRUSHARTT M (1988). Preferentialreinnervationof motor nerveby regenerating motor axons. Journalof Neuroseienee,8: 1026-103l. BRUSHARTT M and SEILERW A (1987). Selectivereinnervationof distal motor stumps by peripheral motor axons. ExperimentalNeurology, 97: 289-300. DANIELSENN, DAHLINL B, LEEY F and LUNDBORGG (1983). Axonal growth in mesothelialchambers: the role of the distal nerve segment. ScandinavianJournalof Plasticand ReconstructiveSurgery, 17:119 125. DAVIS G E, VARON S, ENGVALL E and MANTHORPE M (1985). Substratum-bindingneurite-promotingfactors: relationships to laminin. Trends in Neurosciences,8:528 532. LONGO F M, HAYMANE G, DAVISG E et al (1984). Neurite-promoting factors and extracellularmatrix componentsaccumulatingin vivo within nerve regenerationchambers.BrainResearch, 309:105-117. LONGO F M, MANTHORPE M, SKAPER S D, LUNDBORG G and VARON S (1983a). Neuronotrophic activitiesaccumulatein vivo within siliconenerveregenerationchambers.BrainResearch, 261:109-117. LONGO F M, SKAPER S D, MANTHORPE M, WILLIAMS L R, LUNDBORG G and VARON S (1983b). Temporalchangesof neuronotrophic activitiesaccumulatingin vivowithinnerveregenerationchambers. ExperimentalNeurology,81: 756-769. LUNDBORGG and HANSSONH A (1979). Regenerationof peripheralnerve through a preformedtissuespace. BrainResearch, 178:573-576. LUNDBORG G, LONGO F M and VARON S (1982). Nerve regeneration model and trophic factors in vivo. BrainResearch, 232: 157-161. LUNDBORGG, DAHLINL B, DANIELSONNet al (1982). Nerveregeneration in siliconechambers: influenceof gap length and of distal stump components.ExperimentalNeurology,76:361 375. LUNDBORG G, ROSEN B, ABRAHAMSON S O, DAHLIN L and
REINNERVATION ACROSS A SILICONE TUBE G A P D A N I E L S E N N (1994). Tubular repair of the median nerve in the human forearm. Preliminary findings. Journal of H a n d Surgery, 19B: 273 276. M A C K I N N O N S E and D E L L O N A L (1990). Clinical nerve reconstruction with a bioabsorbable polyglycolic acid tube. Plastic and Reconstructive Surgery, 85: 419-424. N A C H E M S O N A K (1988). Axonal regeneration and growth direction in square-shaped mesothelial chambers. Scandinavian Journal of Plastic and Reconstructive Surgery, 22: 199-206. N A C H E M S O N A K, HANSSON H A and L U N D B O R G G (1988). Neurotropism in nerve regeneration: an immunohistochemical study. Acta Physiotogica Scandinavia, 137:139 148. POLITIS M J, EDERLE K and SPENCER P S (1982). Tropism in nerve regeneration in vivo. Attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected nerves. Brain Research, 253:1 12. POLITIS M J and SPENCER P S (1983). An in vivo assay of neurotropic activity. Brain Research, 278: 229-231.
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Accepted after revision: 18 March 1996 Dr Z-J. Luo, Department of Orthopaedic Surgery, Air Force General Hospital, 30, Fu Chen Road, Hai Dian District, Beijing, 100036,People's Republic of China. © 1996 The British Society for Surgery of the Hand