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Regeneration across ‘stepping-stone’ nerve grafts
196
Brain Research, 618 (1993) 196-202 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19054
Regeneration across 'stepping-stone' nerve grafts Tomoo
Maeda
b,., Susan Daniel
E. Mackinnon A. Hunter
a, T i m o t h y a and
R.T.
J. Best
Rajiv
Midha
b,**, Peter
J. Evans
b
b
'~Department of Surgery, Washington Unicersity, St. Louis, MO (USA) and b Department of Surgery, University of Toronto, Toronto (Canada) (Accepted 23 February 1993)
Key words: Peripheral nerve; Conduit; Neurotropism; Nerve graft; Nerve regeneration
The ability of small nerve segments interposed between synthetic conduits to increase the total nerve gap distance across which successful nerve regeneration would occur was studied. Fifty adult male Lewis rats were randomized into five groups. In Group I a segment of resected sciatic nerve was repaired by a nerve graft. Group II had alternating silicone tubing/nerve graft/silicone tubing replacement of the resected nerve segment (single stepping stone group). Group III had silicone tubing/nerve graft/silicone tubing/nerve graft/silicone tubing repair of the nerve deficit (double stepping stone). Group IV had a single long silicone conduit repair. Group V control underwent a sham operation. Nerve regeneration was evaluated using walking track pattern analysis, electrophysiologic assessment and histomorphological evaluation. 'Stepping stone nerve grafts' enhanced regeneration across nerve gaps in comparison to a single long conduit, but were inferior to a single long nerve graft. In the repair of long nerve gaps, the use of multiple short conduits with interposed short nerve segments could provide a source of trophic factors to enhance regeneration.
INTRODUCTION
tion across a long gap through multiple short conduits a n d n e r v e s e g m e n t s . D i r e c t c o m p a r i s o n was m a d e to a
Peripheral
nerve
grafting
is t h e
current
surgical
single l o n g c o n d u i t
and
to a single n e r v e
g r a f t to
t h e r a p y for r e p a i r o f l a r g e p e r i p h e r a l n e r v e d e f i c i t s 19.
d e t e r m i n e t h e e f f i c a c y o f t h e ' s t e p p i n g s t o n e ' in sup-
N e r v e g r a f t i n g is l i m i t e d by d o n o r site m o r b i d i t y , in-
porting and enhancing peripheral nerve regeneration.
c l u d i n g a n e w s e n s o r y deficit, scar a n d o c c a s i o n a l l y n e u r o m a p a i n at t h e n e r v e g r a f t d o n o r site. A n a l t e r n a -
MATERIALS AND METHODS
tive to t h e n e r v e g r a f t is a s y n t h e t i c c o n d u i t , w h i c h has been
demonstrated
in s e v e r a l l a b o r a t o r y
studies
to
f a c i l i t a t e s u c c e s s f u l n e r v e r e g e n e r a t i o n o v e r s h o r t dist a n c e s 6"14'15'17'27'28. I n a d d i t i o n to s u p p o r t i n g a x o n a l g r o w t h , n e r v e c o n d u i t s m a y a l l o w for t h e c o r r e c t i o n o f f i b e r m i s a l i g n m e n t by p r o m o t i n g s e l e c t i v e r e i n n e r v a t i o n 3"4'~. H o w e v e r , this e f f e c t is l i m i t e d by t h e l e n g t h o f t h e c o n d u i t 14,15. N e u r o n a l g r o w t h a n d m a t u r a t i o n is h y p o t h e s i z e d to be s u p p o r t e d by n e u r o t r o p h i c f a c t o r s e m a n a t i n g f r o m t h e distal n e r v e s t u m p s'26. T h e r e f o r e , t h e i n t e r p o s i t i o n of nerve segments, or 'stepping stones', between short conduits may facilitate neuronal regeneration through artificial t u b e s o v e r g r e a t e r d i s t a n c e s 29. T h e p u r p o s e o f this study w a s to i n v e s t i g a t e p e r i p h e r a l n e r v e r e g e n e r a -
Animal model and study groups Adult male Lewis rats, weighing between 225-250 g (Harlan Sprague-Dawley, Indianapolis, IN) were randomized into five groups of 10 animals). Lewis rats also provided donor syngeneic nerve grafts. The animals were anesthetized with inhalational ether preanesthetic (Diethyl ether, BDH, Toronto, Canada) followed by intramuscular ketamine hydrochloride (1 /zg/100 g Ketalean, M.T.C. Pharmaceuticals, Cambridge, Canada) and acepromazine maleate (0.1 mg/100 g, Atravet, Ayerst, Montreal, Canada). The right sciatic nerve (SN) was exposed through a gluteal muscle splitting incision. The experimental design is summarized in Fig. 1. In Group I, the conventional nerve graft group, a 12 mm segment of SN was resected, beginning 2 mm distal to the origin of the inferior gluteal nerve. The gap was replaced with a 18 mm interpositional syngeneic nerve graft. All neurorrhaphies were performed by conventional microsurgical technique using 10-0 Dermalon (Ethicon, Somerville, NJ) epineurial sutures. In Group II, a 12 mm segment of SN was resected, and the resultant gap was replaced with a sequence of 8 mm silicone conduits
Correspondence: S.E. Mackinnon, One Barnes Hospital, Plaza Suite 17424, St. Louis, Missouri 63110, USA. Fax: (1) (314) 362-4536. * Present address: Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-Cho, Nishinomiya, Hyogo, 633, Japan. ** Present address: University of Alberta, 306 Buchanan Way, Edmonton Alberta, Canada T6R 2B4.
197 Defect/Gap (mm)
Conduit Number/Length (ram)
Nerve Graft Number/Length (ram)
t2/t8
o/o
1/18
12/18
2/8
!/6
18124
3/8
213
t2/m
t/2o
o/o
Nerve
Group I - nerve graft
...:..
::
,~::::::~:::-
Group
.:~:~:::~:,..
[] -
..
"single s t e p p i n g
Group m -
"double
stone"
stepping stone"
Group IV - conduit
Fig. 1. Experimental design.
(Dow Corning, Midland, MI; inner diameter 1.47 mm, outer diameter 1.96 mm)/6 mm nerve graft/8 mm conduit. The conduits were sutured with a 1 mm inset, with a resultant total nerve gap of 18 mm. This model was termed the single 'stepping stone' nerve graft (single
ss). In Group III, an 18 mm segment of SN was resected and replaced by two single SSs in sequence (double SS), This repair consisted of a conduit (8 mm)/nerve segment (3 m m ) / conduit (8 mm)/nerve segment (3 mm)/conduit (8 mm), thereby bridging a 24 mm gap. In Group IV, the long conduit group, a 12 mm segment of SN was resected and replaced by a 20 mm conduit, yielding a gap length of 18 mm. This conduit length was chosen as the maximum length that could be utilized without producing any kinking at the nerveconduit juncture. Thus, Groups I, II and IV are directly comparable in that the nerve defect (12 mm) and the nerve gap (18 mm) were the same in all 3 groups. By contrast in Group III, the 'double stepping stone', both the nerve defect (18 mm) and nerve gap (24 mm) were considerably longer than in the other experimental groups. In Group V, the sham control group, the SN was mobilized, but left intact. Muscle and skin were closed with 4-0 Ethicon (Johnson & Johnson, Peterborough, Ontario) interrupted sutures in all groups. Functional evaluation A previously described walking track analysis technique was used to evaluate rat hind paw prints l'~°. Briefly, the walking track apparatus consisted of an 8.2 × 42 cm track, with an appropriately sized piece of exposed X-ray film at the bottom. The hind feet of the rat were dipped in X-ray film developer, and the animal was allowed to walk down the track. As the rat ambulated, its hind paw prints appeared. Print length, toe spread and intermediary toe spread were determined. The sciatic function index (SFI) was calculated from these indices21. The tracks were analyzed using a computer-linked digital pen and morphometry software (Bioquant, R&M Biometrics, Nashville, Tennessee). Tracks from unmeasurable cases, resulting from fixed joint contractures and self mutilation, were excluded from analysis. All rats underwent walking track assessment preoperatively for baseline and to familiarize the animals with the technique, and then at 2, 4, 8, 12 and 16 weeks. Electrophysiologic assessment Compound nerve action potentials (CNAP) were used to evaluate axonal regeneration across the repair site. At sacrifice (16 weeks),
CNAPs across the graft were measured using computer assisted electromyography (Advantage, Clark-Davis Medical Inc., London, Canada). The SN was exposed proximally, just distal to the sciatic notch, and distally, beyond the repair site. To preclude electromyographic activity, the anesthetized animals were given a neuromuscular blocking agent (Tubocurarine Chloride, 0.3 mg/kg i.p., Burroughs Wellcome, Inc., Kirkland, Quebec) and ventilated via a tracheotomy. Bipolar hooked platinum wire stimulating and recording electrodes were placed under the sciatic nerve proximally and distally, respectively. A ground electrode was placed in muscle midway between the stimulation and recording electrodes. A stimulus duration of 0.05 ms was applied and after eliciting a supramaximal response, a minimum of four and maximum of ten direct CNAPs per nerve were recorded and averaged. Core body temperature was maintained throughout by an overhead heat lamp and a warm water irrigation blanket. Conduction velocity measurements were based on derived latencies and measured distances and were temperature corrected. Histomorphometric assessment Animals were assessed at 16 weeks. Following electrophysiologic recordings the entire sciatic nerve, including the repaired segment,
v
-~
-6°
t.,.
._~
~ ~\
-8o
O .........
li~
<> . . . . . . . . .
•
12
~6
1
...
-lop 2
4
Time
6
of
B
10
~,t
ossesmenf (weeks)
Fig. 2. Functional assessment of walking track analysis. Animals in the sham operated group maintain normal function as assessed by walking track analysis. Animals from all other experimental groups showed little or no improvement in function with no significant differences noted between groups.
198 was removed en bloc. The adherent granulation tissue was dissected, and a small opening was fashioned in the wall of each tube to facilitate fixative penetration. The specimens were immersed in 3% (w/v) phosphate buffered glutaraldehyde, postfixed in osmium tetroxide, and in Araldite 502 (CIBA-Geigy Canada Ltd., Dorval, Quebec). One ~m semithin cross sections were cut on a LKB III ultramicrotome (LKB-Produkter A.B., Bromma, Sweden) and stained with Toluidine blue for light microscopic examination. The proximal, graft, interpositional and distal nerve segments were evaluated qualitatively by an observer blinded to treatment groups for the overall nerve architecture and degree of regeneration. Morphometric evaluations of each distal nerve segment were performed at 1,000 x magnification through a Leitz Dialux 22 light microscope with automated morphometry software (Leco Instruments, Mississauga, Ontario). Mean fiber diameter, total fiber number and percentage of neural tissue were calculated based on a minimum of 500 fibers from representative fields. Data from animals with no regeneration was included in the morphometric evaluation by assigning an arbitrary value of zero.
and Tukey's pairwise comparisons. All statistical tests were two tailed. Statistics were calculated using CRUNCH (v 3.0) program. RESULTS
General observations Most rats stayed healthy and gained weight during t h e 16 w e e k o b s e r v a t i o n p e r i o d . N o c o m p l i c a t i o n s occ u r r e d in G r o u p I. I n G r o u p
II, 2 o f 10 r a t s d e v e l o p e d
a n i n f e c t i o n a t t h e r e p a i r site. D e s p i t e this, w e l l d e v e l oped bridging tissue through the repair site was noted at sacrifice. In Group
III, 3 o u t o f 10 r a t s d e v e l o p e d
local infection, or the nerve
segment
was dislodged
from the conduit. In a further two rats, no regenerating t i s s u e w a s f o u n d in t h e c o n d u i t s . I n t h e r e m a i n i n g five c a s e s , b r i d g i n g t i s s u e in c o n d u i t s w a s o b s e r v e d a t ex-
Statistical analysis Differences between groups were evaluated by one way analysis of variance (ANOVA) for each of the variables. If the ANOVA demonstrated significant (P < 0.05) overall group effects for any of the variables, then specific group mean comparisons were performed for that variable using Student's t-tests, with a significance level set at 0.05. Individual morphometric indices were analyzed by ANOVA
ploration. In Group IV, there was no tissue inside any of the conduits; in one case bridging tissue was observed outside the tube. In no case was kinking of the conduit
noted. Group
V rats remained
healthy, and
sustained no complications.
Fig. 3. Transverse section distal sciatic nerve (Toluidine blue x512). A: a well myelinated population of nerve fibers from Group 1 experimental group is noted. B: regeneration across the 'single stepping stone' group demonstrates a similar histological pattern to that noted in Group I. C: regeneration across the 'double stepping stone' experimental group shows persistent Wallerian degeneration. D: a poorly myelinated fiber population noted in the single animal in which evidence of nerve regeneration was noted across the single long conduit.
199
Fig. 3 (continued).
Functional evaluation The serial changes in SFI are depicted in Fig. 2. Preoperatively, the SFI in all groups was approximately zero, indicating normal function. Following injury, the SFI decreased to approximately - 1 0 0 , indicating no function. In Group I there was a gradual recovery in the SFI by 8 weeks. However, the recovery was minimal and was not significantly different from Groups II, III and IV. The latter three groups demonstrated no further recovery in the SFI even 16 weeks after surgery. Group V rats had normal function as assessed by SFI throughout the course of the study. Histologic results Qualitative histology revealed discernable differences in regeneration between some groups (Fig. 3). In Group I, the conventional nerve graft, a mature population of well myelinated nerve fibers was observed, often grouped within compartments. There was no evidence of Wallerian degeneration. Group II exhibited similar patterns of regeneration as the conventional nerve graft, with a diverse population of mature
nerve fibers and no Wallerian degeneration. Sections from Group III revealed ongoing Wallerian degeneration and decreased numbers of normally myelinated axons. Group IV showed minimal evidence of regeneration in one animal. Group V demonstrated normal histology. Quantitative histomorphometry was performed on distal nerve segment sections (Figs. 4-6). The mean ( + S.E.M.) diameter of myelinated nerve fibers in the distal segment of the sham operated group was significantly greater than in all other experimental groups ( P < 0.05, Scheffe's). The mean fiber diameter in the group I was similar to Group II, and both were significantly greater than the mean fiber diameter of Group III ( P < 0.05). Minimal regeneration into the distal segment was observed in only one animal in Group IV, precluding statistical analysis. The total number of myelinated nerve fibers in the distal segment of all groups was superior to the long conduit group, in which no regeneration into the distal segment was observed except in one animal. The sham group had significantly more nerve fibers than either
200 Group I or III ( P < 0.05, Scheffe's), but was not significantly different from Group II ( P = 0.21). Groups I, II and III were not significantly different from each other.
cvw Ld
2g
a:
Electrophysiologic results
s! i
ka_
! r
I
• ••• •d
i i
ii
II
III
tV
Y
Fig. 4. The mean (_+ S.E.M.) diameter of myelinated nerve fibers in the distal segment of the sham operated group was significantly greater than in all other experimental groups ( P < 0.05 Scheffe's). The m e a n diameter in the graft (1) group was similar to the conduit (II) group. The mean diameter in the graft and single conduit groups was significantly greater than the mean diameter of the double conduit ( l i d group ( P < 0.05).
1,3ooo 9,3oo i 12fi LJ r'n
8oo3 [
Z
600,3
cY
5300
w
a 803
'oo]
J
•
soc,s F 20(;0 ~
I " 0 0 0 ~-
0.0
I
II
III
~V
V
Fig. 5. The total number of myelinated nerve fibers in the distal segment in the sham group was significantly greater than in Groups I or Ill ( P < 0.05 Scheffe's) but was not significantly different from Group H. Groups I, II and III were not significantly different from each other.
35
7~
5o
~.)
25 20
m~
~5
0.0
II
III
IV
V
Fig. 6. The percentage of the nerve which was occupied by neural tissue in the distal segment s h a m operated group was significantly greater than all other experimental groups ( P < 0.05 Scheffe's). The percentage of neural tissue in Groups I and II was not significantly different. The percentage of neural tissue in Group I was significantly greater than that in Group III ( P <0.001). There was no statistical difference between the percentage of neural tissue in Group II versus Group III.
Sham operated animals (Group V) had a mean CV of 59 m / s . The mean CV of Group I was 42.6 m / s , which was significantly greater than that in all groups but Group V (t-test, P < 0.03). Mean CV of Groups II (29.1 m / s ) and III (27.3 m / s ) were similarly and significantly greater than that of the one animal in Group IV (n = 1) (7.0 m / s ; t-test P = 0.001). DISCUSSION
The peripheral nerve will regenerate across short nerve gaps and even make directional changes in order to find distal nerve TM. The importance of the distal stump in enhancing this regeneration has been emphasized ]3'2°'23'27'31. It is hypothesized that Schwann cells in the distal nerve provide a primary source of neurotrophic factors which are important for axonal elongation. A variety of artificial conduits have been used to bridge peripheral nerve defects 22. The use of these conduits provides an alternative to the conventional autograft repair. Potential advantages of conduits would include avoidance of the requirement of a donor nerve, possible avoidance of neuroma formation at the suture line, potentiation of topographic alignment of the regenerating fibers, and improved matching of graft to proximal and distal stump diameters. Several conduits have been recommended for clinical use, with caution that conduits will support neural regeneration only over relatively short distances ~5']6"2~. Numerous exogenous agents and extracellular matrix (ECM) components have been added to these conduits in an attempt to enhance or increase axonal elongation. It appears that the presence of Schwann cells may be an absolute prerequisite for significant nerve regeneration. Schwann cells have been shown to have a unique role in promoting and regulating axonal growth during regeneration. At least three neurotrophic factors are synthesized by Schwann cells - nerve growth factor (NGF)~L12'30, brain-derived neurotrophic factor24 and ciliary neurotrophic factor 9. They are also responsible for the re-expression of the low affinity component of the NGF after axotomy 7. The appearance of NGF receptors on the surface of the Schwann cell would sequester N G F on Schwann cells. This would provide an environment of NGF-laden Schwann cells to facilitate Schwann cell/axonal interaction during regeneration. Schwann cells are also thought to elaborate adhesion molecules and synthesize basement membrane
201 components 2'5. Surface interactions between adhesion molecules in the Schwann cell membrane and ECM proteins secreted by Schwann cells may occur with binding sites and ECM receptors on the growth cone membrane. We hypothesize that the addition of Schwann cells to the conduit would facilitate axonal regeneration. In this study a source of additional Schwann cells was provided in the form of a short segment of nerve graft. Regeneration across an 18 mm gap improved dramatically with the placement of this 'stepping stone' graft. Schwann cells in these nerve grafts could produce trophic factors, and having lost contact with their axons would be expected to up-regulate the low affinity component of the N G F receptor on their cell surface. The interposition of a short segment of nerve graft could provide a source of trophic factors and ECM components obviating potential problems associated with the addition and delivery of these exogenous factors and agents 25. The interposition of the segment of nerve graft between two short conduits or within a long conduit has a beneficial effect on regenerating axons and should increase the length of successful nerve regeneration. As the use of nerve conduits becomes a clinical reality, we suggest consideration be given for interposing a short segment of proximal donor nerve in the mid portion of the nerve conduit, thus accepting a slightly longer nerve gap but insuring a 'stepping stone' of a potential source of Schwann ceils and trophic factors in the mid portion of the conduit. • In conclusion, the observations in this study suggest that the length of successful nerve regeneration through a conduit may be extended or enhanced by the use of a 'stepping stone' nerve graft segment. Acknowledgements. The authors gratefully acknowledge Judy Wicklund for manuscript preparation. This work was funded by the Medical Research Council of Canada. T.M. received fellowship support from the Department of Orthopaedic Surgery, Hyogo College of Medicine, Nishinomiya, Japan. T.J.B. received fellowship support from the Sunnybrook Trust for Medical Research, Toronto, Canada. P.J.E. and R.M. received fellowship support from the Medical Research Council of Canada.
REFERENCES 1 Bain, J.R., Mackinnon, S.E. and Hunter, D.A., Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat, Plast. Reconstr. Surg., 83 (1989) 129-136. 2 Bixby, J.L., Lilien, J. and Reichardt, L.F., Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro, J. Cell Biol., 107 (1988) 353-361. 3 Brushart, T.M.E. and Seiler, W.A., Selective reinnervation of distal motor stumps by peripheral motor axons, Exp. Neurol., 97 (1987) 289-300.
4 Brushart, T.M.E., Preferential reinnervation of motor nerves by regenerating motor axons, J. Neurosci., 8 (1988) 1026-1031. 5 Bunge, R.P. and Bunge, M.B., Interrelationship between Schwann cell function and extracellular matrix production, Trends Neurosci., 6 (1983) 499-505. 6 Dellon, A.L. and Mackinnon, S.E., An alternative to the management of the short nerve gap, Plast. Reconstr. Surg., 82 (1988) 849-856. 7 DiStefano, P.S. and Johnson, E.M., Nerve growth factor receptors on cultured rat Schwann cells, J. Neurosci., 8 (1988) 231-241. 8 Evans, P.J., Bain, J.R., Mackinnon, S.E., Best, T. and Hunter, D.A., Selective reinnervation: a comparison of recovery following microsuture and conduit nerve repair, Brain Res., 599 (1991) 315-321. 9 Friedman, B., Scherer, S.S., Rudge, J.S., et al., Regulation of ciliary neurotrophic expression in myelin related Schwann cells in vivo, Neuron, 9 (1992) 295-305. 10 Hare, G.M.T., Evans, P.J., Mackinnon, S.E., Best, T.J., Bain, J.R., Szalai, J.R. and Hunter, D.A., Walking track analysis: a long-term assessment of peripheral nerve injury, Plast. Reconstr. Surg., 89 (1992) 251-258. 11 Heumann, R., Korsching, S., Bandtlow, C., Thoenen, H., Changes of NGF synthesis in non-neuronal cells in response to sciatic nerve transection, J. Cell BioL, 104 (1987) 1623-1631. 12 Ikegami, R., Changes of nerve growth factor (NGF) content in injured peripheral nerve during regeneration: local synthesis of NGF by Schwann cells, J. Jpn. Orthop. Assoc., 64 (1990) 612-622. 13 Jenq, C.B. and Coggeshall, R.E., Regeneration of transected rat sciatic nerves after using isolated nerve fragments as distal inserts in silicone tubes, Exp. Neurol., 91 (1986) 154-162. 14 Lundborg, G., Dahlin, L.B., Danielsen, N., Hansson, H.A., Johannesson, A., Longo, F.M., Varon, S. and Engin, D., Nerve regeneration across an extended gap: a neurobiological view of nerve repair and the possible involvement of neuronotrophic factors, J. Hand Surg., 7 (1982) 580-587. 15 Lundborg, G., Longo, F.M. and Varon S., Nerve regeneration model in trophic factors in vivo, Brain Res., 232 (1982) 157-162. 16 Lundborg, G., Nerve Injury and Repair, Churchill Livingstone, 1988, pp. 164-178. 17 Mackinnon, S.E., Dellon, A.L., Hudson, A.R. and Hunter, R.T, Nerve regeneration through a pseudosynovial sheath in a primate model, Plast. Reconstr. Surg., 75 (1985) 833-839. 18 Mackinnon, S.E., Dellon, A.L., Lundborg, G., Hudson, A.R. and Hunter, D.A., A study of neurotropism in a primate model, J. Hand Surg., 11 (1986) 888-894. 19 Mackinnon, S.E. and Dellon, A.L., Surgery of the Peripheral Nerve, Thieme Medical Publishers, New York, 1988, pp. 552-571. 20 Mackinnon, S.E., Dellon, A.L. and Hunter, D.A., A histologic assessment of the effects of the distal nerve in determining nerve regeneration across a nerve graft, Microsurgery, 9 (1988) 46-51. 21 Mackinnon, S.E. and Dellon, A.L., A study of nerve regeneration across synthetic (Maxon T.M.) and biological (collagen) nerve conduits for nerve gaps up to 5 cm in the primate, J. Reconstr. Microsurg., 6 (1990) 117-122. 22 Mackinnon, S.E. and Dellon, A.L., Clinical nerve reconstruction with a bioabsorbable polyglycolic acid tube, Plast. Reconstr. Surg., 85 (1990) 419-424. 23 Mackinnon, S.E. and Dellon A.L., Reinnervation of distal sensory nerve environments by regenerating sensory axons, Neurosci., 46 (1992) 595-603. 24 Meyer, M., Matsuoka, I., Wetmore, C., Olson, L. and Thoenen, H., Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and MGF mRNA, J. Cell Biol. 119 (1992) 45-54. 25 Muller, H.W., Williams, L.R. and Varon, S., Nerve regeneration chamber: evaluation of exogenous agents applied by multiple injections, Brain Res. 413 (1987) 320-326. 26 Ochii, M., Experimental study of orientation of regenerating fibers in the severed peripheral nerve, Hiroshima J. Med. Sci., 31 (1983) 389-406. 27 Politis, M.J., Ederle, K. and Spencer, P.S., Tropism in nerve
202 regeneration: in vivo attraction of regenerating axons by diffusible factors derived from cells in distal nerve stumps of transected peripheral nerve, Brain Res., 253 (1982) 1-12. 28 Seckel, B.R., Ryan, S.E., Gagne, R.G., Chiu, T.H. and Watkins, E., Target specific nerve regeneration through a nerve guide in the rat, Plast. Reconst. Surg., 78 (1986) 793-798. 29 Smahel, J., Stimulative wirkung von isolierten nervensegmenten auf die regeneration peripherer nerven, Handchirurgie, 20 (1988) 3-6.
30 Taniuchi, M, Clark, H.B., Schweitzer, J.B., Johnson, E.M., Expression of nerve growth factor receptor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 31 Williams, L.R,, Powell, H.C., Lundborg, G. and Varon, S,, Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber, Brain Res., 23 (1984) 201-211.
Brain Research, 618 (1993) 196-202 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19054
Regeneration across 'stepping-stone' nerve grafts Tomoo
Maeda
b,., Susan Daniel
E. Mackinnon A. Hunter
a, T i m o t h y a and
R.T.
J. Best
Rajiv
Midha
b,**, Peter
J. Evans
b
b
'~Department of Surgery, Washington Unicersity, St. Louis, MO (USA) and b Department of Surgery, University of Toronto, Toronto (Canada) (Accepted 23 February 1993)
Key words: Peripheral nerve; Conduit; Neurotropism; Nerve graft; Nerve regeneration
The ability of small nerve segments interposed between synthetic conduits to increase the total nerve gap distance across which successful nerve regeneration would occur was studied. Fifty adult male Lewis rats were randomized into five groups. In Group I a segment of resected sciatic nerve was repaired by a nerve graft. Group II had alternating silicone tubing/nerve graft/silicone tubing replacement of the resected nerve segment (single stepping stone group). Group III had silicone tubing/nerve graft/silicone tubing/nerve graft/silicone tubing repair of the nerve deficit (double stepping stone). Group IV had a single long silicone conduit repair. Group V control underwent a sham operation. Nerve regeneration was evaluated using walking track pattern analysis, electrophysiologic assessment and histomorphological evaluation. 'Stepping stone nerve grafts' enhanced regeneration across nerve gaps in comparison to a single long conduit, but were inferior to a single long nerve graft. In the repair of long nerve gaps, the use of multiple short conduits with interposed short nerve segments could provide a source of trophic factors to enhance regeneration.
INTRODUCTION
tion across a long gap through multiple short conduits a n d n e r v e s e g m e n t s . D i r e c t c o m p a r i s o n was m a d e to a
Peripheral
nerve
grafting
is t h e
current
surgical
single l o n g c o n d u i t
and
to a single n e r v e
g r a f t to
t h e r a p y for r e p a i r o f l a r g e p e r i p h e r a l n e r v e d e f i c i t s 19.
d e t e r m i n e t h e e f f i c a c y o f t h e ' s t e p p i n g s t o n e ' in sup-
N e r v e g r a f t i n g is l i m i t e d by d o n o r site m o r b i d i t y , in-
porting and enhancing peripheral nerve regeneration.
c l u d i n g a n e w s e n s o r y deficit, scar a n d o c c a s i o n a l l y n e u r o m a p a i n at t h e n e r v e g r a f t d o n o r site. A n a l t e r n a -
MATERIALS AND METHODS
tive to t h e n e r v e g r a f t is a s y n t h e t i c c o n d u i t , w h i c h has been
demonstrated
in s e v e r a l l a b o r a t o r y
studies
to
f a c i l i t a t e s u c c e s s f u l n e r v e r e g e n e r a t i o n o v e r s h o r t dist a n c e s 6"14'15'17'27'28. I n a d d i t i o n to s u p p o r t i n g a x o n a l g r o w t h , n e r v e c o n d u i t s m a y a l l o w for t h e c o r r e c t i o n o f f i b e r m i s a l i g n m e n t by p r o m o t i n g s e l e c t i v e r e i n n e r v a t i o n 3"4'~. H o w e v e r , this e f f e c t is l i m i t e d by t h e l e n g t h o f t h e c o n d u i t 14,15. N e u r o n a l g r o w t h a n d m a t u r a t i o n is h y p o t h e s i z e d to be s u p p o r t e d by n e u r o t r o p h i c f a c t o r s e m a n a t i n g f r o m t h e distal n e r v e s t u m p s'26. T h e r e f o r e , t h e i n t e r p o s i t i o n of nerve segments, or 'stepping stones', between short conduits may facilitate neuronal regeneration through artificial t u b e s o v e r g r e a t e r d i s t a n c e s 29. T h e p u r p o s e o f this study w a s to i n v e s t i g a t e p e r i p h e r a l n e r v e r e g e n e r a -
Animal model and study groups Adult male Lewis rats, weighing between 225-250 g (Harlan Sprague-Dawley, Indianapolis, IN) were randomized into five groups of 10 animals). Lewis rats also provided donor syngeneic nerve grafts. The animals were anesthetized with inhalational ether preanesthetic (Diethyl ether, BDH, Toronto, Canada) followed by intramuscular ketamine hydrochloride (1 /zg/100 g Ketalean, M.T.C. Pharmaceuticals, Cambridge, Canada) and acepromazine maleate (0.1 mg/100 g, Atravet, Ayerst, Montreal, Canada). The right sciatic nerve (SN) was exposed through a gluteal muscle splitting incision. The experimental design is summarized in Fig. 1. In Group I, the conventional nerve graft group, a 12 mm segment of SN was resected, beginning 2 mm distal to the origin of the inferior gluteal nerve. The gap was replaced with a 18 mm interpositional syngeneic nerve graft. All neurorrhaphies were performed by conventional microsurgical technique using 10-0 Dermalon (Ethicon, Somerville, NJ) epineurial sutures. In Group II, a 12 mm segment of SN was resected, and the resultant gap was replaced with a sequence of 8 mm silicone conduits
Correspondence: S.E. Mackinnon, One Barnes Hospital, Plaza Suite 17424, St. Louis, Missouri 63110, USA. Fax: (1) (314) 362-4536. * Present address: Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-Cho, Nishinomiya, Hyogo, 633, Japan. ** Present address: University of Alberta, 306 Buchanan Way, Edmonton Alberta, Canada T6R 2B4.
197 Defect/Gap (mm)
Conduit Number/Length (ram)
Nerve Graft Number/Length (ram)
t2/t8
o/o
1/18
12/18
2/8
!/6
18124
3/8
213
t2/m
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o/o
Nerve
Group I - nerve graft
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::
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Group
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..
"single s t e p p i n g
Group m -
"double
stone"
stepping stone"
Group IV - conduit
Fig. 1. Experimental design.
(Dow Corning, Midland, MI; inner diameter 1.47 mm, outer diameter 1.96 mm)/6 mm nerve graft/8 mm conduit. The conduits were sutured with a 1 mm inset, with a resultant total nerve gap of 18 mm. This model was termed the single 'stepping stone' nerve graft (single
ss). In Group III, an 18 mm segment of SN was resected and replaced by two single SSs in sequence (double SS), This repair consisted of a conduit (8 mm)/nerve segment (3 m m ) / conduit (8 mm)/nerve segment (3 mm)/conduit (8 mm), thereby bridging a 24 mm gap. In Group IV, the long conduit group, a 12 mm segment of SN was resected and replaced by a 20 mm conduit, yielding a gap length of 18 mm. This conduit length was chosen as the maximum length that could be utilized without producing any kinking at the nerveconduit juncture. Thus, Groups I, II and IV are directly comparable in that the nerve defect (12 mm) and the nerve gap (18 mm) were the same in all 3 groups. By contrast in Group III, the 'double stepping stone', both the nerve defect (18 mm) and nerve gap (24 mm) were considerably longer than in the other experimental groups. In Group V, the sham control group, the SN was mobilized, but left intact. Muscle and skin were closed with 4-0 Ethicon (Johnson & Johnson, Peterborough, Ontario) interrupted sutures in all groups. Functional evaluation A previously described walking track analysis technique was used to evaluate rat hind paw prints l'~°. Briefly, the walking track apparatus consisted of an 8.2 × 42 cm track, with an appropriately sized piece of exposed X-ray film at the bottom. The hind feet of the rat were dipped in X-ray film developer, and the animal was allowed to walk down the track. As the rat ambulated, its hind paw prints appeared. Print length, toe spread and intermediary toe spread were determined. The sciatic function index (SFI) was calculated from these indices21. The tracks were analyzed using a computer-linked digital pen and morphometry software (Bioquant, R&M Biometrics, Nashville, Tennessee). Tracks from unmeasurable cases, resulting from fixed joint contractures and self mutilation, were excluded from analysis. All rats underwent walking track assessment preoperatively for baseline and to familiarize the animals with the technique, and then at 2, 4, 8, 12 and 16 weeks. Electrophysiologic assessment Compound nerve action potentials (CNAP) were used to evaluate axonal regeneration across the repair site. At sacrifice (16 weeks),
CNAPs across the graft were measured using computer assisted electromyography (Advantage, Clark-Davis Medical Inc., London, Canada). The SN was exposed proximally, just distal to the sciatic notch, and distally, beyond the repair site. To preclude electromyographic activity, the anesthetized animals were given a neuromuscular blocking agent (Tubocurarine Chloride, 0.3 mg/kg i.p., Burroughs Wellcome, Inc., Kirkland, Quebec) and ventilated via a tracheotomy. Bipolar hooked platinum wire stimulating and recording electrodes were placed under the sciatic nerve proximally and distally, respectively. A ground electrode was placed in muscle midway between the stimulation and recording electrodes. A stimulus duration of 0.05 ms was applied and after eliciting a supramaximal response, a minimum of four and maximum of ten direct CNAPs per nerve were recorded and averaged. Core body temperature was maintained throughout by an overhead heat lamp and a warm water irrigation blanket. Conduction velocity measurements were based on derived latencies and measured distances and were temperature corrected. Histomorphometric assessment Animals were assessed at 16 weeks. Following electrophysiologic recordings the entire sciatic nerve, including the repaired segment,
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Fig. 2. Functional assessment of walking track analysis. Animals in the sham operated group maintain normal function as assessed by walking track analysis. Animals from all other experimental groups showed little or no improvement in function with no significant differences noted between groups.
198 was removed en bloc. The adherent granulation tissue was dissected, and a small opening was fashioned in the wall of each tube to facilitate fixative penetration. The specimens were immersed in 3% (w/v) phosphate buffered glutaraldehyde, postfixed in osmium tetroxide, and in Araldite 502 (CIBA-Geigy Canada Ltd., Dorval, Quebec). One ~m semithin cross sections were cut on a LKB III ultramicrotome (LKB-Produkter A.B., Bromma, Sweden) and stained with Toluidine blue for light microscopic examination. The proximal, graft, interpositional and distal nerve segments were evaluated qualitatively by an observer blinded to treatment groups for the overall nerve architecture and degree of regeneration. Morphometric evaluations of each distal nerve segment were performed at 1,000 x magnification through a Leitz Dialux 22 light microscope with automated morphometry software (Leco Instruments, Mississauga, Ontario). Mean fiber diameter, total fiber number and percentage of neural tissue were calculated based on a minimum of 500 fibers from representative fields. Data from animals with no regeneration was included in the morphometric evaluation by assigning an arbitrary value of zero.
and Tukey's pairwise comparisons. All statistical tests were two tailed. Statistics were calculated using CRUNCH (v 3.0) program. RESULTS
General observations Most rats stayed healthy and gained weight during t h e 16 w e e k o b s e r v a t i o n p e r i o d . N o c o m p l i c a t i o n s occ u r r e d in G r o u p I. I n G r o u p
II, 2 o f 10 r a t s d e v e l o p e d
a n i n f e c t i o n a t t h e r e p a i r site. D e s p i t e this, w e l l d e v e l oped bridging tissue through the repair site was noted at sacrifice. In Group
III, 3 o u t o f 10 r a t s d e v e l o p e d
local infection, or the nerve
segment
was dislodged
from the conduit. In a further two rats, no regenerating t i s s u e w a s f o u n d in t h e c o n d u i t s . I n t h e r e m a i n i n g five c a s e s , b r i d g i n g t i s s u e in c o n d u i t s w a s o b s e r v e d a t ex-
Statistical analysis Differences between groups were evaluated by one way analysis of variance (ANOVA) for each of the variables. If the ANOVA demonstrated significant (P < 0.05) overall group effects for any of the variables, then specific group mean comparisons were performed for that variable using Student's t-tests, with a significance level set at 0.05. Individual morphometric indices were analyzed by ANOVA
ploration. In Group IV, there was no tissue inside any of the conduits; in one case bridging tissue was observed outside the tube. In no case was kinking of the conduit
noted. Group
V rats remained
healthy, and
sustained no complications.
Fig. 3. Transverse section distal sciatic nerve (Toluidine blue x512). A: a well myelinated population of nerve fibers from Group 1 experimental group is noted. B: regeneration across the 'single stepping stone' group demonstrates a similar histological pattern to that noted in Group I. C: regeneration across the 'double stepping stone' experimental group shows persistent Wallerian degeneration. D: a poorly myelinated fiber population noted in the single animal in which evidence of nerve regeneration was noted across the single long conduit.
199
Fig. 3 (continued).
Functional evaluation The serial changes in SFI are depicted in Fig. 2. Preoperatively, the SFI in all groups was approximately zero, indicating normal function. Following injury, the SFI decreased to approximately - 1 0 0 , indicating no function. In Group I there was a gradual recovery in the SFI by 8 weeks. However, the recovery was minimal and was not significantly different from Groups II, III and IV. The latter three groups demonstrated no further recovery in the SFI even 16 weeks after surgery. Group V rats had normal function as assessed by SFI throughout the course of the study. Histologic results Qualitative histology revealed discernable differences in regeneration between some groups (Fig. 3). In Group I, the conventional nerve graft, a mature population of well myelinated nerve fibers was observed, often grouped within compartments. There was no evidence of Wallerian degeneration. Group II exhibited similar patterns of regeneration as the conventional nerve graft, with a diverse population of mature
nerve fibers and no Wallerian degeneration. Sections from Group III revealed ongoing Wallerian degeneration and decreased numbers of normally myelinated axons. Group IV showed minimal evidence of regeneration in one animal. Group V demonstrated normal histology. Quantitative histomorphometry was performed on distal nerve segment sections (Figs. 4-6). The mean ( + S.E.M.) diameter of myelinated nerve fibers in the distal segment of the sham operated group was significantly greater than in all other experimental groups ( P < 0.05, Scheffe's). The mean fiber diameter in the group I was similar to Group II, and both were significantly greater than the mean fiber diameter of Group III ( P < 0.05). Minimal regeneration into the distal segment was observed in only one animal in Group IV, precluding statistical analysis. The total number of myelinated nerve fibers in the distal segment of all groups was superior to the long conduit group, in which no regeneration into the distal segment was observed except in one animal. The sham group had significantly more nerve fibers than either
200 Group I or III ( P < 0.05, Scheffe's), but was not significantly different from Group II ( P = 0.21). Groups I, II and III were not significantly different from each other.
cvw Ld
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Electrophysiologic results
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I
• ••• •d
i i
ii
II
III
tV
Y
Fig. 4. The mean (_+ S.E.M.) diameter of myelinated nerve fibers in the distal segment of the sham operated group was significantly greater than in all other experimental groups ( P < 0.05 Scheffe's). The m e a n diameter in the graft (1) group was similar to the conduit (II) group. The mean diameter in the graft and single conduit groups was significantly greater than the mean diameter of the double conduit ( l i d group ( P < 0.05).
1,3ooo 9,3oo i 12fi LJ r'n
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Z
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I " 0 0 0 ~-
0.0
I
II
III
~V
V
Fig. 5. The total number of myelinated nerve fibers in the distal segment in the sham group was significantly greater than in Groups I or Ill ( P < 0.05 Scheffe's) but was not significantly different from Group H. Groups I, II and III were not significantly different from each other.
35
7~
5o
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25 20
m~
~5
0.0
II
III
IV
V
Fig. 6. The percentage of the nerve which was occupied by neural tissue in the distal segment s h a m operated group was significantly greater than all other experimental groups ( P < 0.05 Scheffe's). The percentage of neural tissue in Groups I and II was not significantly different. The percentage of neural tissue in Group I was significantly greater than that in Group III ( P <0.001). There was no statistical difference between the percentage of neural tissue in Group II versus Group III.
Sham operated animals (Group V) had a mean CV of 59 m / s . The mean CV of Group I was 42.6 m / s , which was significantly greater than that in all groups but Group V (t-test, P < 0.03). Mean CV of Groups II (29.1 m / s ) and III (27.3 m / s ) were similarly and significantly greater than that of the one animal in Group IV (n = 1) (7.0 m / s ; t-test P = 0.001). DISCUSSION
The peripheral nerve will regenerate across short nerve gaps and even make directional changes in order to find distal nerve TM. The importance of the distal stump in enhancing this regeneration has been emphasized ]3'2°'23'27'31. It is hypothesized that Schwann cells in the distal nerve provide a primary source of neurotrophic factors which are important for axonal elongation. A variety of artificial conduits have been used to bridge peripheral nerve defects 22. The use of these conduits provides an alternative to the conventional autograft repair. Potential advantages of conduits would include avoidance of the requirement of a donor nerve, possible avoidance of neuroma formation at the suture line, potentiation of topographic alignment of the regenerating fibers, and improved matching of graft to proximal and distal stump diameters. Several conduits have been recommended for clinical use, with caution that conduits will support neural regeneration only over relatively short distances ~5']6"2~. Numerous exogenous agents and extracellular matrix (ECM) components have been added to these conduits in an attempt to enhance or increase axonal elongation. It appears that the presence of Schwann cells may be an absolute prerequisite for significant nerve regeneration. Schwann cells have been shown to have a unique role in promoting and regulating axonal growth during regeneration. At least three neurotrophic factors are synthesized by Schwann cells - nerve growth factor (NGF)~L12'30, brain-derived neurotrophic factor24 and ciliary neurotrophic factor 9. They are also responsible for the re-expression of the low affinity component of the NGF after axotomy 7. The appearance of NGF receptors on the surface of the Schwann cell would sequester N G F on Schwann cells. This would provide an environment of NGF-laden Schwann cells to facilitate Schwann cell/axonal interaction during regeneration. Schwann cells are also thought to elaborate adhesion molecules and synthesize basement membrane
201 components 2'5. Surface interactions between adhesion molecules in the Schwann cell membrane and ECM proteins secreted by Schwann cells may occur with binding sites and ECM receptors on the growth cone membrane. We hypothesize that the addition of Schwann cells to the conduit would facilitate axonal regeneration. In this study a source of additional Schwann cells was provided in the form of a short segment of nerve graft. Regeneration across an 18 mm gap improved dramatically with the placement of this 'stepping stone' graft. Schwann cells in these nerve grafts could produce trophic factors, and having lost contact with their axons would be expected to up-regulate the low affinity component of the N G F receptor on their cell surface. The interposition of a short segment of nerve graft could provide a source of trophic factors and ECM components obviating potential problems associated with the addition and delivery of these exogenous factors and agents 25. The interposition of the segment of nerve graft between two short conduits or within a long conduit has a beneficial effect on regenerating axons and should increase the length of successful nerve regeneration. As the use of nerve conduits becomes a clinical reality, we suggest consideration be given for interposing a short segment of proximal donor nerve in the mid portion of the nerve conduit, thus accepting a slightly longer nerve gap but insuring a 'stepping stone' of a potential source of Schwann ceils and trophic factors in the mid portion of the conduit. • In conclusion, the observations in this study suggest that the length of successful nerve regeneration through a conduit may be extended or enhanced by the use of a 'stepping stone' nerve graft segment. Acknowledgements. The authors gratefully acknowledge Judy Wicklund for manuscript preparation. This work was funded by the Medical Research Council of Canada. T.M. received fellowship support from the Department of Orthopaedic Surgery, Hyogo College of Medicine, Nishinomiya, Japan. T.J.B. received fellowship support from the Sunnybrook Trust for Medical Research, Toronto, Canada. P.J.E. and R.M. received fellowship support from the Medical Research Council of Canada.
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30 Taniuchi, M, Clark, H.B., Schweitzer, J.B., Johnson, E.M., Expression of nerve growth factor receptor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties, J. Neurosci., 8 (1988) 664-681. 31 Williams, L.R,, Powell, H.C., Lundborg, G. and Varon, S,, Competence of nerve tissue as distal insert promoting nerve regeneration in a silicone chamber, Brain Res., 23 (1984) 201-211.