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Nerve-muscle sandwich grafts: The importance of schwann cells in peripheral nerve regeneration through muscle basal lamina conduits
N E R V E - M U S C L E S A N D W I C H G R A F T S : T H E I M P O R T A N C E OF SCHWANN CELLS IN PERIPHERAL NERVE REGENERATION THROUGH MUSCLE BASAL LAMINA CONDUITS J. S. CALDER and C. J. GREEN
From the Blond-Mclndoe Medical Research Centre, Queen Victoria Hospital, East Grinstead, Sussex, and the Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK )
An interposed segment of nerve was used to enhance the distimce over which freeze-thawed muscle autografts will support effective peripheral nerve regeneration. Gaps were created in the sciatic nerves of adult Lewis rats. Regeneration through 1 and 1.5 cm freeze-thawed muscle grafts was compared to regeneration through nerve-muscle sandwich grafts in which muscle grafts of equivalent length were divided and a 2 mm segment of the distal nerve sutured between the two halves of the muscle, providing an intermediate depot of Schwann cells. Electrophysiological and morphological evaluation was carried out 40 weeks after operation. Despite lengthening the graft, and having four anastomoses instead of two, this manoeuvre enhanced nerve regeneration over each gap studied and for the 1.5 cm gaps compared favourably with perfect match nerve autografts. In addition, a number of grafts were examined at 7 and 14 days by S l 0 0 immunohistochemistry. Schwann cell migration was seen to proceed both proximally and distally from the intermediate segment at a rate similar to that from the distal stump. It is concluded that sandwich grafts may prove to be effective alternatives to cutaneous nerve grafts for peripheral nerve reconstruction. Journal of Hand Surgery (British and European Volume, 1995) 20B: 4." 423-428
Primary direct suture offers the best hope for useful return of function following peripheral nerve division, the acutely denervated distal stump providing the most favourable terrain for regenerating axons. However, delay to surgical repair or actual loss of nerve tissue will result in a gap between the nerve ends. Seddon (1963) established autologous nerve grafting as the optimal treatment for this type of injury. By using cutaneous nerves from the leg or arm, a bridging graft was provided containing acutely denervated Schwann cells and their associated basal laminae. Extensive mobilization of the nerve ends and excessive tension at the repair site were avoided. There are several drawbacks with this technique, including the sacrifice of a functioning nerve, with loss of sensation, scarring and possible neuroma formation. There may be insufficient graft material to reconstruct more extensive defects. Endoneurial tube size may be small and unable to support regeneration of larger sensory or motor fibres. Several pieces of cutaneous nerve are required to reconstruct mixed nerves of much larger diameter and these m a y suffer from ischaemia and connective tissue formation between the cables. Although .good functional results can be obtained, especially in younger patients, failure is frequent. Attempts to find alternative, non-neural conduits have been numerous (Fields et al, 1989). Recently, denatured muscle autografts have attracted attention as possible alternatives to cutaneous nerve grafts (Fawcett and Keynes, 1986; Glasby et al, 1986), although evidence now exists to suggest muscle graft failure with increasing gap length (Hems and Glasby, 1992; Calder and Norris, 1993). The purpose of this study was to enhance the length over which a freeze-thawed muscle graft will support effective peripheral nerve regeneration by the use of an interposed nerve segment. These "sandwich
grafts" were compared to ordinary freeze-thawed muscle grafts and to nerve autografts, using electrophysiology and morphometric analysis. MATERIALS A N D M E T H O D S Surgery
34 adult Lewis rats of both sexes and of 340 g average weight were used for this study. Anaesthesia was carried out using intramuscular injection of Hypnorm (0.3 ml/Kg), fentanyl citrate (0.315 mg/ml) and fluanisone (10mg/ml; Janssen Pharmaceuticals Ltd) and intraperitoneal injection of diazepam (2.5mg/Kg; Phoenix Pharmaceuticals Ltd). The sciatic nerve was exposed and divided in midlthigh. Sufficient nerve was resected to create gaps of 1 or 1.5 cm. Graft material was obtained from the biceps femoris muscle. The muscle was frozen with a hand-held freeze spray (Lipfreeze, L I P Ltd) then thawed in sterile distilled water at room temperature. This method of denaturing was chosen as it mirrors that employed in clinical practice (Calder and Norris, 1993). Grafts of 1 and 1.5 cm length were fashioned and sutured between the nerve ends (Fig la; n = 6 per gap length). The contralateral leg was then operated on, and a graft of equivalent length inserted. This was then divided at its mid-point and 2 mm beyond the distal anastamoses. This segment was reversed and sutured into place, so creating a sandwich graft--a muscle graft with an interposed nerve segment (Fig lb). In a further group of 6 animals, nerve autografts of 1 or 1.5 cm were inserted. This was done by dividing and suturing the sciatic nerve at 2 points and thus represented a perfect match nerve graft, rarely encountered in clinical practice. 423
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995
trode was connected to one of the retractors in the wound bed. A supramaximal stimulus was provided by an external pulse generator capable of delivering square wave constant current pulses of variable duration and amplitude. The recorded signal was taken to an isolated high input impedance (1015 f~) preamplifier using AD210 isolation and AD549 operational amplifiers (Analog Devices Inc), which produced an amplification of 100 x . The amplified signal was filtered and displayed on a Medelec MS92 oscilloscope (Medelec Ltd, U K ) . Permanent records of wave-forms were produced using heat sensitive graduated roll paper. F r o m the waveforms, apical conduction velocities (ACVs) were calculated for each nerve. On completion of recording, the surface temperature of the nerve near to the stimulating electrodes was measured using a thermocouple thermometer.
Morphometry
Fig 1
(a) Appear/mce of a 1.5 cm freeze-thawed muscle graft at 14 days. Short segments of the proximal and distal sciatic nerve have been resected with the graft ( p = p r o x i m a l , d = d i s t a l ) . (b) Nerve-muscle sandwich graft at 14 days. The intermediate nerve segment is shown by the arrow.
Eight non-operated rats were used to provide control data for normal common peroneal nerves.
Electrophysiology At 40 weeks the animals were anaesthetized again and the sciatic and common peroneal nerves exposed from the sciatic notch to the knee. All other branches of the sciatic nerve were divided. An insulating film of latex rubber was placed beneath the nerve. Compound nerve action potentials were recorded using 2 pairs of bipolar electrodes, of 0.25 m m diameter silver plated copper wire (30 a.w.g., R.S. Components Ltd). The stimulating electrodes were placed proximally on the sciatic nerve with the cathode distal to the anode. The common peroneal nerve was divided at the distal limit of its exposure. The free end was placed on the recording electrodes, which were separated by 5 ram. A portion of the nerve between the recording electrodes was crushed by pressure from a pair of microsurgical forceps in order to obtain a unipolar tracing. A ground reference elec-
After electrophysiological evaluation, the nerves were immersion fixed in 3% (w/v) phosphate buffered glutaraldehyde, then embedded in Spurr's resin. Transverse 0.75 ~tm sections were cut from the common peroneal nerve at a point 5 mm from the trifurcation of the sciatic nerve. An acridine orange stain was used to visualize the myelin sheaths. A manual count was made of the total number of myelinated axons in each common peroneal nerve, using an immediate morphometry system viewed under a light microscope set at x 40 objective magnification (AxioHOME, Carl Zeiss Ltd.). Automated fibre diameter measurements were made using a Kontron 386 computer with purpose designed software. A minimum of 400 fibres were counted per nerve.
Immunohistochemistry In order to study Schwann cell migration from the intermediate segment 1.5 cm grafts were inserted in two further groups of four animals each. At 7 and 14 days, the grafts, along with a segment of the proximal and distal nerve ends, were excised en bloc and fixed immediately in Zamboni's fluid for 6 hours at room temperature. This was followed by several washes in 0.01 M phosphate-buffered 0.15M saline (PBS, pHT.4) containing 15% (w/v) sucrose. After embedding in OCT ( B D H Lab supplies) 15 gm longitudinal sections were cut and collected onto Vectabond coated slides. S100 immunohistochemistry was carried out according to the indirect immunofluorescence method. Sections were immersed in PBS containing 0.2% triton-X for 1 hour, then rinsed in PBS for 3 minutes. To reduce background autofluorescence the slides were then placed in pontamine sky blue for 30 minutes. After further washes in PBS (3 x 5 minutes) the S100 first layer antibody was applied, ( D A K O A/S, Glostrup, Denmark; diluted 1/1200), and
NERVE-MUSCLE
SANDWICH
425
GRAFTS
the slides incubated overnight at 4°C in a humid chamber. The next day, with washes in PBS (3 x 5 minutes) before and after, the second layer antibody was applied ( F I T C conjugated goat anti-rabbit IgG, diluted 1/100 with PBS), and incubated for 1 hour at room temperature in a moist chamber. The sections were mounted in PBS/glycerine and viewed under an epifluorescence microscope.
Statistical analysis Differences between groups for each variable were compared by one way analysis of variance (ANOVA) with a significance level of P < 0.05. Individual group means for apical conduction velocity, myelinated fibre number and diameter were compared using Student's t-tests.
significant (P=0.0164) with functional recovery across a sandwich graft approaching that of a nerve graft (/}=0.078).
Morphometry Morphometric results are summarized in Figures 3 and 4. The mean number of myelinated fibres for normal common / peroneal nerves was 1970±18. More myelinated fibres were observed in the 1 cm nerve graft group but these were of reduced diameter. The differences in fibre numbers between the muscle grafts (711 +270) and sandwich grafts (1335_+224) was significant for the 1.5 cm gaps (P=0.001). There was no significant difference in mean fibre diameters between the muscle grafts and sandwich grafts. 3000
RESULTS
Electrophysiology The range of nerve temperatures was 31.3°C to 33.4°C (mean= 32.11°C), thus there was no requirement to adjust conduction velocities. Mean ACVs for each group are depicted in Figure 2. For the 8 non-operated, normal nerves the mean ACV was 37.7 ms -1. For 1 cm nerve grafts this velocity was significantly slower at 31.3 ms -1 (P=0.007). For the 1 cm gaps, nerve grafts performed significantly better than the muscle grafts (P=0.0007) or the sandwich grafts (P=0.01). There was no significant difference between the muscle and sandwich grafts ( A C V = 25.2 m s - 1 and 27.8 m s - 1 respectively, P = 0.08). However, with the gap length increasing to 1.5 cm, the difference between muscle and sandwich grafts became
I11
2000
t'N [ + e"
1000-
t
0. N
IemNG
IcmMG
IcmSG
1.5emNG
1,5craMG
l.ScmSG
Number of myelinated fibres
Fig 3 50-
40-
~ -
t
Mean number of myelinated fibres in the common peroneal nerves distal to the grafts. For the 1.5 cm gap lengths, significantly more fibres had regenerated through the sandwich grafts than through muscle grafts (P=0.001).
10
I11 308+ 720-
6-
10
7
1
54-
0J
~-
m
1.5cmMG
1.5crnSG
3N
IcmNG
lcmMG
lcmSG
1.5emNG
1.5emMG
1.5cmSG
2
Apical conduction velocities in ms -1
Fig 2
Mean apical conduction velocity for normal nerves (N) was 37.7 ms_ 1. For the reconstructed nerves, perfect match nerve grafts (NG) gave the best functional results, but over the 1.5 cm gaps the difference between nerve grafts and sandwich grafts (SG) was not statistically significant (P = 0.078). Muscle grafts (MG) proved least effective.
0_ N
lcmNG
lcmMG
lemSG
1,5emNG
Diameter of myelinated fibres in pm
Fig 4
Mean diameters of myelinated nerve fibres in #m showed no significant difference between each type of graft.
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995
Immunohistochemistry At 7 days in the muscle grafts, Schwann cells had started to migrate from the proximal and distal stumps. Schwann cells from the proximal stump had migrated about twice as far as those from the distal stump. In the sandwich grafts, Schwann cells were seen also to have migrated in both directions fi'om the intermediate segment at a rate equal to that from the distal stump (Figs 5a and b). At 14 days in the muscle grafts, Schwann cells migrating from the proximal and distal stumps had not met (Fig 6a). In contrast, ,significant numbers of Schwann cells were seen throughout the entire length of each sandwich graft at this time course and they had aligned into bands of Bt~ngner (Fig 6b).
a
DISCUSSION The morphological results reported here show that Schwann cells will migrate in both directions from the interposed segment, at equal rates. In providing a solid
Fig 6
Fig 5
SI00 immunostaining of a sandwich graft at 7 days showing Schwann cells migrating from the intermediate segment into the freeze-thawed muscle graft (a) towards the proximal stump and (b) towards the distal stump.
(a) S100 immunostaining of part of a freeze-thawed muscle graft at 14 days showing a complete absence of Schwann cells. (b) In contrast, by 14 days, immunoreactive Schwann cells had migrated throughout the entire length of the sandwich grafts and had become aligned.
matrix between the nerve ends for cellular outgrowth and organisation, denatured muscle is theoretically more attractive as a graft material than vein or synthetic tubes. By using an interposed nerve segment, the length over which a muscle graft will support effective regeneration was enhanced. Successful peripheral nerve regeneration requires the existence of a growth permitting terrain and factors offering directional guidance for growth cones. The optimal terrain for axonal regeneration is the Schwann cell surface and the synergism between Schwann cells and axons during regeneration is well recognized (Calder et al, 1994). The guiding influence of the distal stump has been demonstrated by numerous studies (Lundborg et al, 1982; Politis et al, 1982; Politis and Spencer, 1983; Scaravilli, 1984; Williams et al, 1984; Kuffler, 1986; Glasby et al, 1988; Weis and Schr6der, 1989; Abernethy et al, 1992). Peripheral nerve regeneration readily occurs across short gaps. As the distance between the nerve ends increases so the neurotropic effect exerted by the distal stump diminishes and it becomes more difficult
NERVE-MUSCLESANDWICHGRAFTS for S c h w a n n cells to m i g r a t e across the g a p f r o m each nerve end. Thus, in the clinical m a n a g e m e n t o f l o n g nerve gaps, no acellular c o n d u i t has p r o v e d effective and the c u t a n e o u s nerve a u t o g r a f t still remains, after several decades, the t r e a t m e n t o f choice. Recently freeze-thawed muscle a u t o g r a f t s have a t t r a c t e d a t t e n t i o n as effective substrates for p e r i p h e r a l nerve r e g e n e r a t i o n following e n c o u r a g i n g e x p e r i m e n t a l and early clinical results ( G l a s b y et al, 1986; G l a s b y , 1990; N o r r i s et al, 1988). H o w e v e r , in a recent r e p o r t of the clinical results o f m i x e d p e r i p h e r a l nerve r e p a i r by this technique, no p a t i e n t achieved an $4 sensory recovery ( M R C g r a d i n g ) a n d m o t o r r e c o v e r y was p o o r (Calder a n d N o r r i s , 1993). These results were d i s a p pointing a n d t e n d e d to suggest graft failure with increasing g a p length, consistent with the w o r k o f H e m s a n d G l a s b y (1992) w h o d e m o n s t r a t e d the failure o f m u s c l e grafts to b r i d g e 5 c m gaps in the p e r i p h e r a l nerves o f rabbits, w h e n c o m p a r e d to nerve grafts. It has b e e n p r o p o s e d t h a t freeze-thawed muscle grafts w o r k b y a b a s a l l a m i n a " n e u r i t e - p r o m o t i n g " effect ( B r y a n et al, 1993). H o w e v e r , studies o f regeneration t h r o u g h nerve grafts suggest t h a t b a s a l l a m i n a alone is n o t a p a r t i c u l a r l y effective s u b s t r a t e c o m p a r e d to the S c h w a n n cell surface ( H a l l , 1986; Sj6berg et al, 1988). M a r t i n i et al (1988) studied cell a d h e s i o n m o l ecule expression during p e r i p h e r a l nerve r e g e n e r a t i o n . A x o n s p r e f e r r e d m c o n t a c t the S c h w a n n cell surface at the S c h w a n n cell/basal l a m i n a interface. L1 a n d N - C A M .~ere localized at these sites. A x o n s were always in contact with living S c h w a n n cells o r o t h e r axons, never exclusively with b a s a l lamina, a n d were u n a b l e to grow long distances on b a s a l l a m i n a a l o n e where the expression o f L1 and N - C A M was p a t c h y . A recent study b y E n v e r a n d H a l l ( t 9 9 4 ) has s h o w n t h a t a x o n s will n o t regenerate into muscle grafts w i t h o u t a c c o m p a n y i n g S c h w a n n cells f r o m the p r o x i m a l stump. They argue t h a t the s a r c o l e m m a l b a s a l l a m i n a a l o n e will n o t s u p p o r t effective p e r i p h e r a l nerve r e g e n e r a t i o n unless the g a p is short e n o u g h to p e r m i t the m i g r a t i o n of S c h w a n n cells across the graft f r o m the nerve ends. A x o n a l o u t g r o w t h a p p e a r e d to be d e p e n d e n t u p o n a large e n o u g h local p o p u l a t i o n o f S c h w a n n cells w h o s e m i g r a t i o n into the graft is critical. M u s c l e grafts m a y therefore be i m p o r t a n t in t h a t they p r o v i d e a solid m a t r i x for S c h w a n n cells to migrate, align a n d p r o d u c e new b a s a l l a m i n a . Feneley et al (1991) also suggest that muscle grafts w o r k b y s u p p o r t i n g S c h w a n n cell m i g r a t i o n a n d t h a t measures to " s p e e d the p o p u l a t i o n o f the graft with S c h w a n n cells, will increase the r a t e o f axon r e g e n e r a t i o n t h r o u g h the grafts a n d p r o b a b l y also increase their m a x i m u m useful length". T h e results o f this s t u d y clearly d e m o n s t r a t e h o w this m a y be achieved. By using a n i n t e r p o s e d segment o f a u t o l o g o u s nerve, r e g e n e r a t i o n was significantly enhanced. This is in agreem e n t with similar studies using i n t e r p o s e d segments in vein grafts for nerve r e c o n s t r u c t i o n ( S m a h e l a n d Jentsch, 1986; M a e d a et al, 1993; T a n g et al, 1993).
427 T h e nerve within a s a n d w i c h graft has the a d v a n t a g e o f m a t c h i n g the d i a m e t e r o f the recipient nerve, in c o n t r a s t to a c u t a n e o u s nerve graft where m u l t i p l e small d i a m e t e r " c a b l e s " have to be inserted. D u r i n g the course o f a s e c o n d a r y p e r i p h e r a l nerve r e c o n s t r u c t i o n the scarred nerve ends are resected a n d the further r e m o v a l o f a s h o r t segment o f h e a l t h y nerve for use as an i n t e r p o s e d segment o u g h t n o t to c o m p r o m i s e f u n c t i o n a l outcome. The p o s s i b l e d i s a d v a n t a g e s o f h a v i n g f o u r a n a s t o m o s e s i n s t e a d o f two, a n d o f increasing the gap length, a p p e a r to have been o u t w e i g h e d b y the beneficial effect o f d e p o t S c h w a n n cells a l o n g the graft, which m i g r a t e in b o t h directions a n d align into b a n d s o f Bt~ngner. Thus, g r o w t h cones o f regenerating axons will e n c o u n t e r a graft o f similar c o m p o s i t i o n to a n acutely d e n e r v a t e d distal nerve segment. T h e use o f an i n t e r p o s e d nerve segment can significantly e n h a n c e the effectiveness o f freeze-thawed muscle grafts, p a r t i c u l a r l y over longer gaps. This e x p e r i m e n t emphasises the f u n d a m e n t a l i m p o r t a n c e o f S c h w a n n cells in p e r i p h e r a l nerve r e g e n e r a t i o n a n d claims o f o p t i m a l r e g e n e r a t i o n t h r o u g h n o n - n e u r a l acellular conduits s h o u l d be viewed with caution. The finding that r e g e n e r a t i o n t h r o u g h s a n d w i c h grafts a p p r o a c h e s t h a t t h r o u g h perfect m a t c h nerve grafts as g a p length increases is significant. This t e c h n i q u e deserves further investigation as a n a l t e r n a t i v e to c u t a n e o u s nerve grafting in the difficult clinical p r o b l e m o f the long nerve gap.
Acknowledgements The authors thank Mr R. Birch of the Peripheral Nerve Injury Unit, Royal National OrthopaedicHospital, Stanmore, Dr S. M. Hall of the Divisionof Anatomyand CellBiology,Guy's Hospital,London and Dr G. Terenghiof the Blond-McIndoeMedicalRese~trchCentre, East Grinsteadfor helpfulcomments during the preparation of this paper. We also thank Mr G. T. Mott of the Departmentof Bioengineering,Mount VernonHospital,Northwood,for technical assistancewith the electrophysiologyequipment and Dr C. Sowterof the Department of Pathology, St Bartholomew'sHospital, London, for help and adviceregardingcomputerizedmorphometry. This workwas fundedby the Ernest KleinwortCharitableTrust and the Rank Foundation.
References ABERNETHY, D. A., RUD, A. and THOMAS, P. K. (1992). Neurotropic influenceof the distalstump of transectedperipheralnerveon axonairegeneration: Absence of topographic specificityin adult nerve. Journal of Anatomy, 180:395-400. BRYAN,D. J., MILLER,R. A., COSTAS,P. D., WANG,K.-K. and SECKEL, B. R. (1993). Immunocytochemistryof skeletalmusclebasal lamina grafts in nerve regeneration.Plasticand ReconstructiveSurgery,92: 927-940. CALDER, J. S. and NORRIS, R. W. (1993). Repair of mixedperipheralnerves using muscleautografts:A preliminarycommunication.BritishJournal of Plastic Surgery,46:557 564. CALDER, J. S., GREEN, C. J. and TERENGHI, G. (1994). Do Schwanncells lead or follow axons? A study of peripheral nerve regeneration through denatured muscleautografts. CellVision, 1: 4:313-318. ENVER, M. K. and HALL,S. M. (1994). Are Schwanncellsessentialfor axonal regeneration into muscle autografts? Neuropathology and Applied Neurobiology,20: 587-598. FAWCETT, J. W. and KEYNES, R. J. (1986). Muscle basal lamina: A new graft material for peripheral nerve repair. Journal of Neurosurgery, 65: 354-363. FENELEY, M. R., FAWCETT,J. W. and KEYNES, R. J. (1991). The role of Schwann cellsin the regenerationof peripheralnerveaxons through muscle basal lamina grafts. ExperimentalNeurology, 114:275 285. FIELDS, R. D., LE BEAU, J. M., LONGO, F. M. and ELLISMAN,M. H. (1989). Nerve regenerationthrough artificialtubular implants.Progressin Neurobiology,33:87 134.
428 GLASBY, M. A., GSCHMEISSNER, S. G., HITCHCOCK, R. J. and HUANG, C. L. (1986). The dependence of nerve regeneration through muscle grafts on the availability and orientation of basement membrane. Journal of Neurocytology, 15: 497-510, GLASBY, M. A., DAVIES, A. H., GATTUSO, J. M., HUANG, C. L-H. and WYATT, J. P. (1988). The effect of distal influences on rat peripheral nerve regeneration through muscle grafts. Neuro-Orthopedics, 6: 61-66. GLASBY, M. A. (1990). Nerve growth in matrices of orientated muscle basement membrane: Developing a new method of nerve repair. Clinical Anatomy, 3: 161-182. HALL, S. M. (1986). The effect of inhibiting Schwann cell mitosis on the re-innervation of acellular autografts in the peripheral nervous system of the mouse. Neuropathology and Applied Neurobiology, 12: 401-414. HEMS, T. E. J. and GLASBY, M. A. (1992). Comparison of different methods of repair of long peripheral nerve defects: An experimental study. British Journal of Plastic Surgery, 45:497 502. KUFFLER, D. P. (1986). Isolated satellite cells of a peripheral nerve direct the growth of regenerating frog axons. Journal of Comparative Neurology, 249: 57-64, LUNDBORG, G., DAHLIN, L. B., DANIELSEN, N. et al (1982). Nerve regeneration in silicone chambers: Influence of gap length and of distal stump components. Experimental Neurology, 76:361 375. MAEDA, T., MACKINNON, S. E., BEST, T. J., EVANS, P. J., HUNTER, D. A. and MIDHA, R. T. R. (1993). Regeneration across "stepping-stone" nerve grafts. Brain Research, 618: 196-202. MARTINI, R. and SCHACHNER, M. (1988). Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelinassociated glycoprotein) in regenerating adult mouse sciatic nerve. Journal of Cell Biology, 106: 1735-1746. NORRIS, R. W., GLASBY, M. A., GATTUSO, J. M. and BOWDEN, R. E. M. (1988). Peripheral nerve repair in humans using muscle autografts: A new technique. Journal of Bone and Joint Surgery, 70B: 530 533.
THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995 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 peripheral 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. SCARAVILLI, F. (1984). The influence of distal environment on peripheral nerve regeneration across a gap. Journal of Neurocytology, 13: 1027-1041. SEDDON, H. J. (1963). Nerve grafting. Journal of Bone and Joint Surgery, 45B: 3: 447-461. SJOBERG, J., KANJE, M. and EDSTROM, A. (1988). Influence of nonneuronal cells on regeneration of the rat sciatic nerve. Brain Research, 453:221 226. SMAHEL, J. and JENTSCH, B. (1986 ). Stimulation of peripheral nerve regeneration by an isolated nerve segment. Annals of Plastic Surgery, 16: 494-501. TANG, J.-B., SONG, Y.-S., ZHU, R.-R. et al. (1993). Reconstruction of long defects in peripheral nerves by vein conduit interposed with normal nerve segments: Report of t0 cases. Journal of Reconstructive Microsurgery, 9: 6: 468. WEIS, J. and SCHR~DER, J. M. (1989). Differential effects of nerve, muscle, and fat tissue on regenerating nerve fibres in vivo. Muscle and Nerve, 12: 723-734. WILLIAMS, L. R., POWELL, H. C., LUNDBORG, G. and VARON, S. (1984). Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber. Brain Research, 293:201 211.
Accepted: 6 March 1995 Mr J. S. Calder, FRCS, Department of Plastic and Reconstructive Surgery, Queen Victoria Hospital, Holtye Road, East Grinstead, Sussex, RH19 3DZ, UK. © 1995 The British Society for Surgery of the Hand
From the Blond-Mclndoe Medical Research Centre, Queen Victoria Hospital, East Grinstead, Sussex, and the Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK )
An interposed segment of nerve was used to enhance the distimce over which freeze-thawed muscle autografts will support effective peripheral nerve regeneration. Gaps were created in the sciatic nerves of adult Lewis rats. Regeneration through 1 and 1.5 cm freeze-thawed muscle grafts was compared to regeneration through nerve-muscle sandwich grafts in which muscle grafts of equivalent length were divided and a 2 mm segment of the distal nerve sutured between the two halves of the muscle, providing an intermediate depot of Schwann cells. Electrophysiological and morphological evaluation was carried out 40 weeks after operation. Despite lengthening the graft, and having four anastomoses instead of two, this manoeuvre enhanced nerve regeneration over each gap studied and for the 1.5 cm gaps compared favourably with perfect match nerve autografts. In addition, a number of grafts were examined at 7 and 14 days by S l 0 0 immunohistochemistry. Schwann cell migration was seen to proceed both proximally and distally from the intermediate segment at a rate similar to that from the distal stump. It is concluded that sandwich grafts may prove to be effective alternatives to cutaneous nerve grafts for peripheral nerve reconstruction. Journal of Hand Surgery (British and European Volume, 1995) 20B: 4." 423-428
Primary direct suture offers the best hope for useful return of function following peripheral nerve division, the acutely denervated distal stump providing the most favourable terrain for regenerating axons. However, delay to surgical repair or actual loss of nerve tissue will result in a gap between the nerve ends. Seddon (1963) established autologous nerve grafting as the optimal treatment for this type of injury. By using cutaneous nerves from the leg or arm, a bridging graft was provided containing acutely denervated Schwann cells and their associated basal laminae. Extensive mobilization of the nerve ends and excessive tension at the repair site were avoided. There are several drawbacks with this technique, including the sacrifice of a functioning nerve, with loss of sensation, scarring and possible neuroma formation. There may be insufficient graft material to reconstruct more extensive defects. Endoneurial tube size may be small and unable to support regeneration of larger sensory or motor fibres. Several pieces of cutaneous nerve are required to reconstruct mixed nerves of much larger diameter and these m a y suffer from ischaemia and connective tissue formation between the cables. Although .good functional results can be obtained, especially in younger patients, failure is frequent. Attempts to find alternative, non-neural conduits have been numerous (Fields et al, 1989). Recently, denatured muscle autografts have attracted attention as possible alternatives to cutaneous nerve grafts (Fawcett and Keynes, 1986; Glasby et al, 1986), although evidence now exists to suggest muscle graft failure with increasing gap length (Hems and Glasby, 1992; Calder and Norris, 1993). The purpose of this study was to enhance the length over which a freeze-thawed muscle graft will support effective peripheral nerve regeneration by the use of an interposed nerve segment. These "sandwich
grafts" were compared to ordinary freeze-thawed muscle grafts and to nerve autografts, using electrophysiology and morphometric analysis. MATERIALS A N D M E T H O D S Surgery
34 adult Lewis rats of both sexes and of 340 g average weight were used for this study. Anaesthesia was carried out using intramuscular injection of Hypnorm (0.3 ml/Kg), fentanyl citrate (0.315 mg/ml) and fluanisone (10mg/ml; Janssen Pharmaceuticals Ltd) and intraperitoneal injection of diazepam (2.5mg/Kg; Phoenix Pharmaceuticals Ltd). The sciatic nerve was exposed and divided in midlthigh. Sufficient nerve was resected to create gaps of 1 or 1.5 cm. Graft material was obtained from the biceps femoris muscle. The muscle was frozen with a hand-held freeze spray (Lipfreeze, L I P Ltd) then thawed in sterile distilled water at room temperature. This method of denaturing was chosen as it mirrors that employed in clinical practice (Calder and Norris, 1993). Grafts of 1 and 1.5 cm length were fashioned and sutured between the nerve ends (Fig la; n = 6 per gap length). The contralateral leg was then operated on, and a graft of equivalent length inserted. This was then divided at its mid-point and 2 mm beyond the distal anastamoses. This segment was reversed and sutured into place, so creating a sandwich graft--a muscle graft with an interposed nerve segment (Fig lb). In a further group of 6 animals, nerve autografts of 1 or 1.5 cm were inserted. This was done by dividing and suturing the sciatic nerve at 2 points and thus represented a perfect match nerve graft, rarely encountered in clinical practice. 423
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995
trode was connected to one of the retractors in the wound bed. A supramaximal stimulus was provided by an external pulse generator capable of delivering square wave constant current pulses of variable duration and amplitude. The recorded signal was taken to an isolated high input impedance (1015 f~) preamplifier using AD210 isolation and AD549 operational amplifiers (Analog Devices Inc), which produced an amplification of 100 x . The amplified signal was filtered and displayed on a Medelec MS92 oscilloscope (Medelec Ltd, U K ) . Permanent records of wave-forms were produced using heat sensitive graduated roll paper. F r o m the waveforms, apical conduction velocities (ACVs) were calculated for each nerve. On completion of recording, the surface temperature of the nerve near to the stimulating electrodes was measured using a thermocouple thermometer.
Morphometry
Fig 1
(a) Appear/mce of a 1.5 cm freeze-thawed muscle graft at 14 days. Short segments of the proximal and distal sciatic nerve have been resected with the graft ( p = p r o x i m a l , d = d i s t a l ) . (b) Nerve-muscle sandwich graft at 14 days. The intermediate nerve segment is shown by the arrow.
Eight non-operated rats were used to provide control data for normal common peroneal nerves.
Electrophysiology At 40 weeks the animals were anaesthetized again and the sciatic and common peroneal nerves exposed from the sciatic notch to the knee. All other branches of the sciatic nerve were divided. An insulating film of latex rubber was placed beneath the nerve. Compound nerve action potentials were recorded using 2 pairs of bipolar electrodes, of 0.25 m m diameter silver plated copper wire (30 a.w.g., R.S. Components Ltd). The stimulating electrodes were placed proximally on the sciatic nerve with the cathode distal to the anode. The common peroneal nerve was divided at the distal limit of its exposure. The free end was placed on the recording electrodes, which were separated by 5 ram. A portion of the nerve between the recording electrodes was crushed by pressure from a pair of microsurgical forceps in order to obtain a unipolar tracing. A ground reference elec-
After electrophysiological evaluation, the nerves were immersion fixed in 3% (w/v) phosphate buffered glutaraldehyde, then embedded in Spurr's resin. Transverse 0.75 ~tm sections were cut from the common peroneal nerve at a point 5 mm from the trifurcation of the sciatic nerve. An acridine orange stain was used to visualize the myelin sheaths. A manual count was made of the total number of myelinated axons in each common peroneal nerve, using an immediate morphometry system viewed under a light microscope set at x 40 objective magnification (AxioHOME, Carl Zeiss Ltd.). Automated fibre diameter measurements were made using a Kontron 386 computer with purpose designed software. A minimum of 400 fibres were counted per nerve.
Immunohistochemistry In order to study Schwann cell migration from the intermediate segment 1.5 cm grafts were inserted in two further groups of four animals each. At 7 and 14 days, the grafts, along with a segment of the proximal and distal nerve ends, were excised en bloc and fixed immediately in Zamboni's fluid for 6 hours at room temperature. This was followed by several washes in 0.01 M phosphate-buffered 0.15M saline (PBS, pHT.4) containing 15% (w/v) sucrose. After embedding in OCT ( B D H Lab supplies) 15 gm longitudinal sections were cut and collected onto Vectabond coated slides. S100 immunohistochemistry was carried out according to the indirect immunofluorescence method. Sections were immersed in PBS containing 0.2% triton-X for 1 hour, then rinsed in PBS for 3 minutes. To reduce background autofluorescence the slides were then placed in pontamine sky blue for 30 minutes. After further washes in PBS (3 x 5 minutes) the S100 first layer antibody was applied, ( D A K O A/S, Glostrup, Denmark; diluted 1/1200), and
NERVE-MUSCLE
SANDWICH
425
GRAFTS
the slides incubated overnight at 4°C in a humid chamber. The next day, with washes in PBS (3 x 5 minutes) before and after, the second layer antibody was applied ( F I T C conjugated goat anti-rabbit IgG, diluted 1/100 with PBS), and incubated for 1 hour at room temperature in a moist chamber. The sections were mounted in PBS/glycerine and viewed under an epifluorescence microscope.
Statistical analysis Differences between groups for each variable were compared by one way analysis of variance (ANOVA) with a significance level of P < 0.05. Individual group means for apical conduction velocity, myelinated fibre number and diameter were compared using Student's t-tests.
significant (P=0.0164) with functional recovery across a sandwich graft approaching that of a nerve graft (/}=0.078).
Morphometry Morphometric results are summarized in Figures 3 and 4. The mean number of myelinated fibres for normal common / peroneal nerves was 1970±18. More myelinated fibres were observed in the 1 cm nerve graft group but these were of reduced diameter. The differences in fibre numbers between the muscle grafts (711 +270) and sandwich grafts (1335_+224) was significant for the 1.5 cm gaps (P=0.001). There was no significant difference in mean fibre diameters between the muscle grafts and sandwich grafts. 3000
RESULTS
Electrophysiology The range of nerve temperatures was 31.3°C to 33.4°C (mean= 32.11°C), thus there was no requirement to adjust conduction velocities. Mean ACVs for each group are depicted in Figure 2. For the 8 non-operated, normal nerves the mean ACV was 37.7 ms -1. For 1 cm nerve grafts this velocity was significantly slower at 31.3 ms -1 (P=0.007). For the 1 cm gaps, nerve grafts performed significantly better than the muscle grafts (P=0.0007) or the sandwich grafts (P=0.01). There was no significant difference between the muscle and sandwich grafts ( A C V = 25.2 m s - 1 and 27.8 m s - 1 respectively, P = 0.08). However, with the gap length increasing to 1.5 cm, the difference between muscle and sandwich grafts became
I11
2000
t'N [ + e"
1000-
t
0. N
IemNG
IcmMG
IcmSG
1.5emNG
1,5craMG
l.ScmSG
Number of myelinated fibres
Fig 3 50-
40-
~ -
t
Mean number of myelinated fibres in the common peroneal nerves distal to the grafts. For the 1.5 cm gap lengths, significantly more fibres had regenerated through the sandwich grafts than through muscle grafts (P=0.001).
10
I11 308+ 720-
6-
10
7
1
54-
0J
~-
m
1.5cmMG
1.5crnSG
3N
IcmNG
lcmMG
lcmSG
1.5emNG
1.5emMG
1.5cmSG
2
Apical conduction velocities in ms -1
Fig 2
Mean apical conduction velocity for normal nerves (N) was 37.7 ms_ 1. For the reconstructed nerves, perfect match nerve grafts (NG) gave the best functional results, but over the 1.5 cm gaps the difference between nerve grafts and sandwich grafts (SG) was not statistically significant (P = 0.078). Muscle grafts (MG) proved least effective.
0_ N
lcmNG
lcmMG
lemSG
1,5emNG
Diameter of myelinated fibres in pm
Fig 4
Mean diameters of myelinated nerve fibres in #m showed no significant difference between each type of graft.
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995
Immunohistochemistry At 7 days in the muscle grafts, Schwann cells had started to migrate from the proximal and distal stumps. Schwann cells from the proximal stump had migrated about twice as far as those from the distal stump. In the sandwich grafts, Schwann cells were seen also to have migrated in both directions fi'om the intermediate segment at a rate equal to that from the distal stump (Figs 5a and b). At 14 days in the muscle grafts, Schwann cells migrating from the proximal and distal stumps had not met (Fig 6a). In contrast, ,significant numbers of Schwann cells were seen throughout the entire length of each sandwich graft at this time course and they had aligned into bands of Bt~ngner (Fig 6b).
a
DISCUSSION The morphological results reported here show that Schwann cells will migrate in both directions from the interposed segment, at equal rates. In providing a solid
Fig 6
Fig 5
SI00 immunostaining of a sandwich graft at 7 days showing Schwann cells migrating from the intermediate segment into the freeze-thawed muscle graft (a) towards the proximal stump and (b) towards the distal stump.
(a) S100 immunostaining of part of a freeze-thawed muscle graft at 14 days showing a complete absence of Schwann cells. (b) In contrast, by 14 days, immunoreactive Schwann cells had migrated throughout the entire length of the sandwich grafts and had become aligned.
matrix between the nerve ends for cellular outgrowth and organisation, denatured muscle is theoretically more attractive as a graft material than vein or synthetic tubes. By using an interposed nerve segment, the length over which a muscle graft will support effective regeneration was enhanced. Successful peripheral nerve regeneration requires the existence of a growth permitting terrain and factors offering directional guidance for growth cones. The optimal terrain for axonal regeneration is the Schwann cell surface and the synergism between Schwann cells and axons during regeneration is well recognized (Calder et al, 1994). The guiding influence of the distal stump has been demonstrated by numerous studies (Lundborg et al, 1982; Politis et al, 1982; Politis and Spencer, 1983; Scaravilli, 1984; Williams et al, 1984; Kuffler, 1986; Glasby et al, 1988; Weis and Schr6der, 1989; Abernethy et al, 1992). Peripheral nerve regeneration readily occurs across short gaps. As the distance between the nerve ends increases so the neurotropic effect exerted by the distal stump diminishes and it becomes more difficult
NERVE-MUSCLESANDWICHGRAFTS for S c h w a n n cells to m i g r a t e across the g a p f r o m each nerve end. Thus, in the clinical m a n a g e m e n t o f l o n g nerve gaps, no acellular c o n d u i t has p r o v e d effective and the c u t a n e o u s nerve a u t o g r a f t still remains, after several decades, the t r e a t m e n t o f choice. Recently freeze-thawed muscle a u t o g r a f t s have a t t r a c t e d a t t e n t i o n as effective substrates for p e r i p h e r a l nerve r e g e n e r a t i o n following e n c o u r a g i n g e x p e r i m e n t a l and early clinical results ( G l a s b y et al, 1986; G l a s b y , 1990; N o r r i s et al, 1988). H o w e v e r , in a recent r e p o r t of the clinical results o f m i x e d p e r i p h e r a l nerve r e p a i r by this technique, no p a t i e n t achieved an $4 sensory recovery ( M R C g r a d i n g ) a n d m o t o r r e c o v e r y was p o o r (Calder a n d N o r r i s , 1993). These results were d i s a p pointing a n d t e n d e d to suggest graft failure with increasing g a p length, consistent with the w o r k o f H e m s a n d G l a s b y (1992) w h o d e m o n s t r a t e d the failure o f m u s c l e grafts to b r i d g e 5 c m gaps in the p e r i p h e r a l nerves o f rabbits, w h e n c o m p a r e d to nerve grafts. It has b e e n p r o p o s e d t h a t freeze-thawed muscle grafts w o r k b y a b a s a l l a m i n a " n e u r i t e - p r o m o t i n g " effect ( B r y a n et al, 1993). H o w e v e r , studies o f regeneration t h r o u g h nerve grafts suggest t h a t b a s a l l a m i n a alone is n o t a p a r t i c u l a r l y effective s u b s t r a t e c o m p a r e d to the S c h w a n n cell surface ( H a l l , 1986; Sj6berg et al, 1988). M a r t i n i et al (1988) studied cell a d h e s i o n m o l ecule expression during p e r i p h e r a l nerve r e g e n e r a t i o n . A x o n s p r e f e r r e d m c o n t a c t the S c h w a n n cell surface at the S c h w a n n cell/basal l a m i n a interface. L1 a n d N - C A M .~ere localized at these sites. A x o n s were always in contact with living S c h w a n n cells o r o t h e r axons, never exclusively with b a s a l lamina, a n d were u n a b l e to grow long distances on b a s a l l a m i n a a l o n e where the expression o f L1 and N - C A M was p a t c h y . A recent study b y E n v e r a n d H a l l ( t 9 9 4 ) has s h o w n t h a t a x o n s will n o t regenerate into muscle grafts w i t h o u t a c c o m p a n y i n g S c h w a n n cells f r o m the p r o x i m a l stump. They argue t h a t the s a r c o l e m m a l b a s a l l a m i n a a l o n e will n o t s u p p o r t effective p e r i p h e r a l nerve r e g e n e r a t i o n unless the g a p is short e n o u g h to p e r m i t the m i g r a t i o n of S c h w a n n cells across the graft f r o m the nerve ends. A x o n a l o u t g r o w t h a p p e a r e d to be d e p e n d e n t u p o n a large e n o u g h local p o p u l a t i o n o f S c h w a n n cells w h o s e m i g r a t i o n into the graft is critical. M u s c l e grafts m a y therefore be i m p o r t a n t in t h a t they p r o v i d e a solid m a t r i x for S c h w a n n cells to migrate, align a n d p r o d u c e new b a s a l l a m i n a . Feneley et al (1991) also suggest that muscle grafts w o r k b y s u p p o r t i n g S c h w a n n cell m i g r a t i o n a n d t h a t measures to " s p e e d the p o p u l a t i o n o f the graft with S c h w a n n cells, will increase the r a t e o f axon r e g e n e r a t i o n t h r o u g h the grafts a n d p r o b a b l y also increase their m a x i m u m useful length". T h e results o f this s t u d y clearly d e m o n s t r a t e h o w this m a y be achieved. By using a n i n t e r p o s e d segment o f a u t o l o g o u s nerve, r e g e n e r a t i o n was significantly enhanced. This is in agreem e n t with similar studies using i n t e r p o s e d segments in vein grafts for nerve r e c o n s t r u c t i o n ( S m a h e l a n d Jentsch, 1986; M a e d a et al, 1993; T a n g et al, 1993).
427 T h e nerve within a s a n d w i c h graft has the a d v a n t a g e o f m a t c h i n g the d i a m e t e r o f the recipient nerve, in c o n t r a s t to a c u t a n e o u s nerve graft where m u l t i p l e small d i a m e t e r " c a b l e s " have to be inserted. D u r i n g the course o f a s e c o n d a r y p e r i p h e r a l nerve r e c o n s t r u c t i o n the scarred nerve ends are resected a n d the further r e m o v a l o f a s h o r t segment o f h e a l t h y nerve for use as an i n t e r p o s e d segment o u g h t n o t to c o m p r o m i s e f u n c t i o n a l outcome. The p o s s i b l e d i s a d v a n t a g e s o f h a v i n g f o u r a n a s t o m o s e s i n s t e a d o f two, a n d o f increasing the gap length, a p p e a r to have been o u t w e i g h e d b y the beneficial effect o f d e p o t S c h w a n n cells a l o n g the graft, which m i g r a t e in b o t h directions a n d align into b a n d s o f Bt~ngner. Thus, g r o w t h cones o f regenerating axons will e n c o u n t e r a graft o f similar c o m p o s i t i o n to a n acutely d e n e r v a t e d distal nerve segment. T h e use o f an i n t e r p o s e d nerve segment can significantly e n h a n c e the effectiveness o f freeze-thawed muscle grafts, p a r t i c u l a r l y over longer gaps. This e x p e r i m e n t emphasises the f u n d a m e n t a l i m p o r t a n c e o f S c h w a n n cells in p e r i p h e r a l nerve r e g e n e r a t i o n a n d claims o f o p t i m a l r e g e n e r a t i o n t h r o u g h n o n - n e u r a l acellular conduits s h o u l d be viewed with caution. The finding that r e g e n e r a t i o n t h r o u g h s a n d w i c h grafts a p p r o a c h e s t h a t t h r o u g h perfect m a t c h nerve grafts as g a p length increases is significant. This t e c h n i q u e deserves further investigation as a n a l t e r n a t i v e to c u t a n e o u s nerve grafting in the difficult clinical p r o b l e m o f the long nerve gap.
Acknowledgements The authors thank Mr R. Birch of the Peripheral Nerve Injury Unit, Royal National OrthopaedicHospital, Stanmore, Dr S. M. Hall of the Divisionof Anatomyand CellBiology,Guy's Hospital,London and Dr G. Terenghiof the Blond-McIndoeMedicalRese~trchCentre, East Grinsteadfor helpfulcomments during the preparation of this paper. We also thank Mr G. T. Mott of the Departmentof Bioengineering,Mount VernonHospital,Northwood,for technical assistancewith the electrophysiologyequipment and Dr C. Sowterof the Department of Pathology, St Bartholomew'sHospital, London, for help and adviceregardingcomputerizedmorphometry. This workwas fundedby the Ernest KleinwortCharitableTrust and the Rank Foundation.
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 4 AUGUST 1995 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 peripheral 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. SCARAVILLI, F. (1984). The influence of distal environment on peripheral nerve regeneration across a gap. Journal of Neurocytology, 13: 1027-1041. SEDDON, H. J. (1963). Nerve grafting. Journal of Bone and Joint Surgery, 45B: 3: 447-461. SJOBERG, J., KANJE, M. and EDSTROM, A. (1988). Influence of nonneuronal cells on regeneration of the rat sciatic nerve. Brain Research, 453:221 226. SMAHEL, J. and JENTSCH, B. (1986 ). Stimulation of peripheral nerve regeneration by an isolated nerve segment. Annals of Plastic Surgery, 16: 494-501. TANG, J.-B., SONG, Y.-S., ZHU, R.-R. et al. (1993). Reconstruction of long defects in peripheral nerves by vein conduit interposed with normal nerve segments: Report of t0 cases. Journal of Reconstructive Microsurgery, 9: 6: 468. WEIS, J. and SCHR~DER, J. M. (1989). Differential effects of nerve, muscle, and fat tissue on regenerating nerve fibres in vivo. Muscle and Nerve, 12: 723-734. WILLIAMS, L. R., POWELL, H. C., LUNDBORG, G. and VARON, S. (1984). Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber. Brain Research, 293:201 211.
Accepted: 6 March 1995 Mr J. S. Calder, FRCS, Department of Plastic and Reconstructive Surgery, Queen Victoria Hospital, Holtye Road, East Grinstead, Sussex, RH19 3DZ, UK. © 1995 The British Society for Surgery of the Hand