Nerve regeneration over a 25 mm gap in rat sciatic nerves using tubes containing blood vessels: The possibility of clinical application

Abstract

This study was undertaken to investigate the effect of including vessels in a tube used to promote nerve regeneration across a gap. A tube containing sural vessels was designed in a rat model and interposed between the proximal and distal stumps of a divided sciatic nerve, leaving a 25 mm gap. At 12 weeks, a few myelinated axons were seen at the most distal parts of regenerated nerves in 6 out of 10 rats, none of which evoked action potentials in the tibialis anterior muscle, but by 24 weeks all the rats had developed neural tissue in the tubes, which evoked action potentials in the muscle. The vessels within the tube enhanced nerve regeneration and its distance up 25 mm. This type of vessel-containing tube would be useful for the repair of divided human peripheral nerves with long gaps, almost equivalent to or slightly longer than the maximum length over which nerve fibres can regenerate through a unvascularised unmodified tube.

Full text

Nerve regeneration over a 25 mm gap in rat sciatic nerves using
tubes containing blood vessels: the possibility of clinical application
R. Kakinoki, N. Nishijima, Y. Ueba, M. Oka, T. Yamamuro, T. Nakamura
Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan
Accepted: 10 October 1996
Summary. This study was undertaken to in-
vestigate the effect of including vessels in a tube
used to promote nerve regeneration across a gap. A
tube containing sural vessels was designed in a rat
model and interposed between the proximal and
distal stumps of a divided sciatic nerve, leaving a
25 mm gap. At 12 weeks, a few myelinated axons
were seen at the most distal parts of regenerated
nerves in 6 out of 10 rats, none of which evoked
action potentials in the tibialis anterior muscle, but
by 24 weeks all the rats had developed neural tis-
sue in the tubes, which evoked action potentials in
the muscle. The vessels within the tube enhanced
nerve regeneration and its distance up 25 mm. This
type of vessel-containing tube would be useful for
the repair of divided human peripheral nerves with
long gaps, almost equivalent to or slightly longer
than the maximum length over which nerve fibres
can regenerate through a unvascularised un-
modified tube.
ReÂsumeÂ. Plusieurs auteurs ont rapporte que la
repousse maximum des axones du nerf sciatique de
rat, dans un tube, est d'environ 10 mm. La preÂsente
eÂtude a eÂteÂreÂaliseÂe pour confirmer que la mise en
place d'un tube dans un vaisseau permet aux fibres
nerveuses d'atteindre une reÂgeÂneÂration de plus de
10 mm par cette technique de tubulation. Chez un
rat, une veine surale contenant un tube a eÂte uti-
liseÂe et interposeÂe entre les moignons sectionneÂs
d'un nerf sciatique avec'un espace de 25 mm. A la
12
eÁme
semaine, quelques axones myeÂliniseÂs ont eÂteÂ
observeÂs aÁ la partie la plus distale des nerfs reÂgeÂ-
neÂreÂs chez 6 rats sur 10. Aucun de ceux-ci n'a puÃ
donner de potentiels d'action dans les muscles ti-
biaux anteÂrieurs. ApreÁs 24 semaines, tous les rats
ont deÂveloppe une repousse neuronale dans les
tubes, qui ont donne des potentiels d'action dans
les muscles tibiaux anteÂrieurs. Cette chambre in-
travasculaire augmente la reÂgeÂneÂration des nerfs
ainsi que la distance de reÂgeÂneÂration jusqu'aÁ25mm
pour un nerf sciatique de rat. Un vaisseau conte-
nant un tube est consideÂre comme eÂtant clinique-
ment, utile pour la reÂparation de la section de nerf
peÂripheÂrique avec un espace important, qui sont
eÂquivalents ou leÂgeÁrement supeÂrieurs, au maximum
de longueur dont les nerfs peuvent repousser dans
des tubes nonvasculariseÂs classiques.
Introduction
The repair of divided peripheral nerves with gaps
between the stumps presents a surgical problem.
Autogenous nerve grafting is widely accepted, but
there are drawbacks such as the defect created at
the donor site, the limits of length and thickness of
the graft, and the possibility of misdirection of the
axons. Furthermore, the results are not always sa-
tisfactory [22, 23].
Another method has been practised since the
beginning of the 19th century [4, 5, 7, 26]. Axons
can grow across a gap through a tube-like material
(tubulation) which does not produce the defects
associated with nerve grafting. Nerve fibres which
have regenerated through tubulation have been
Reprint request to: R. Kakinoki, Department of Orthopedic
Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan
International Orthopaedics (SICOT) (1997) 21: 332 ± 336
Orthopaedics
International
Ó Springer-Verlag 1997

reported to show several specific qualities for re-
generation, such as tissue [12], topographic [20]
and motor-sensory [1, 2] specificities. Tubulation
may also reduce the possibility of axon misdirec-
tion.
There is a limit to the distance across which
nerves can regenerate in unmodified empty tubes
which is reported to be about 10 mm for rat sciatic
nerves [9, 10, 11]. The axons of these nerves can
extend across a gap of 15 mm to 20 mm using
tubes containing dialysed plasma [13], collagen-
glycosaminoglycan polysaccharide matrix [32],
laminin-containing gel [17, 18] or collagen matrix
[17].
We directed our attention to the value of vessels
in a tube designed to contain sural vessels and
which would encompass a 25 mm gap in sciatic
nerves in the rat, and studied whether axons could
regenerate through the tube for this distance. We
also discuss the possibility of using such a tube in
patients.
Materials and methods
Twenty adult male Sprague-Dawley rats (15± 18 weeks old,
weighing 330 ±365 g) were used. After anaesthesia by an in-
traperitoneal injection of pentobarbital sodium (Nembutal,
40 mg/kg body weight), the left leg was shaved and prepared
for operation. The sural neurovascular bundle was exposed
333R. Kakinoki et al.: Nerve regeneration in rat sciatic nerves
Fig. 1 A ±C. Diagrams showing the operative procedures
involving the preparation of a silicone rubber tube for a
25 mm gap. SUV, sural vessels; SUN, sural nerve; SN, sciatic
nerve; PN, peroneal nerve; TN, tibial nerve, and F, monitor
flap
Fig. 2. The sural vessels elevated at the popliteal fossa. SUV,
sural vessels; PN, peroneal nerve; TN, tibial nerve
Fig. 3. Photograph taken just before wound closure of a vessel-
containing tube with a 25 mm gap. Arrows show the sural
vessels inserted into a 28 mm silicone rubber tube

from the popliteal fossa to the ankle, and the nerve was se-
parated from its accompanying vessels and removed com-
pletely (Fig. 1 a). A 10´5 mm myocutaneous flap vascularised
by the sural vessels was harvested at the posterior surface of
the ankle and elevated from the lower leg (Fig. 2). The left
sciatic nerve was then exposed from piriformis to the popliteal
fossa through a gluteal muscle-splitting approach, and a 20 mm
segment removed (Fig. 2 B). The myocutaneous flap with its
pedicle was mobilised up to the thigh. The sural vessels were
inserted into the silicone tube through a preformed longitudinal
slit. The epineurium of the proximal and distal sciatic stumps
were sutured to the 28 mm silicone rubber tube leaving a gap
of 25 mm between the stumps using a 10 ± 0 monofilament
nylon (Fig. 1 C). The slit was sealed with a small amount of
silicone rubber (Fig. 3). The myocutaneous flap was sutured to
the buttock and the skin closed. The flap was used to monitor
the patency of the sural vessels in the tube, and its colour was
checked every day.
Electrophysiological study
Twelve and 24 weeks after operation, the animals were an-
aesthetised, the left sciatic nerve exposed distal to piriformis
and stimulated with a bipolar electrode. A pair of needle
electrodes was inserted into the tibialis anterior muscle and the
presence of an evoked action potential recorded.
Histological study
The tube and adjavent nerve were removed together and fixed
in 2.5% (v/v) glutaraldehyde, postfixed with 2% (v/v) osmic
acid and embedded in epoxy resin. Transverse sections,
1±2 mm thick, were taken from the most proximal part (Sp),
the middle part (Sm) and the most distal part (Sd) of each
regenerated nerve. Each was stained with 0.5% (w/v) toluidine
blue and examined by light microscopy.
Results
No rat died during operation or in the follow up
period. Two developed necrosis of the monitor
flaps and were excluded. Ten and 8 rats were sa-
crificed at 12 and 24 weeks, respectively.
At 12 weeks, neural tissue had developed in
every silicone rubber tube (Fig. 4) and the vessels
were patent in all 10 rats. Myelinated axons were
present in the proximal and middle portions in
every rat, and in the distal part in 6 out of 10. No
action potentials were evoked in the tibialis ante-
rior muscle of any rat because the numbers of
axons which had regenerated in the 6 rats were too
small.
At 24 weeks, the 8 rats examined had developed
neural tissue in the tubes, and action potentials
were evoked in their tibialis anterior muscles.
Myelinated axons were present in every part of
each regenerated nerve in every rat (Fig. 5). In 6 of
the 8, the vessels had become necrotic, although
the monitor flaps were viable.
334 R. Kakinoki et al.: Nerve regeneration in rat sciatic nerves
Fig. 4. The appearance of a nerve which has regenerated
through a vessel-containing tube with a 25 mm gap at
24 weeks. Sp, section of the proximal part; Sm, the middle
part; Sd, the distal part
Fig. 5 A, B. Light micrographs of transverse sections of the
most distal part of a nerve which has regenerated through a
vessel-containing tube with a 25 mm gap, 24 weeks after the
operation. A An arrow indicates the remnants of the necrotic
vessels, ´400. B ´800, the bar at the bottom right
corner = 10 mm

Discussion
The present study has demonstrated that axons are
able to regenerate across a 25 mm gap and re-
innervate the tibialis anterior muscle 6 months
after operation using a silicone rubber tube con-
taining vessels. It is not possible to exceed this
distance in our model because the maximum
length of the sural vessels is about 30 mm.
Mackinnon and Dellon investigated spontaneous
nerve regeneration in rats, and the mean distance
across which axons regenerated was 23.7+6.4 mm
at 5 months, after removal of a 4.5 cm segment of
sciatic nerve [11]. The nerves had regenerated in a
tube-like space surrounded by well vascularised
muscle fascia. Vascularity enabled axons to extend
up to 45 mm.
Vascularity is one factor which is critical for
determining the distance of axon regeneration in
tubes. Comparing the results of a vessel-containing
tube with a 25 mm gap with a 10 mm gap in a
similar tube [6], the number and mean diameter of
axons in the 25 mm gap were 1500 and 1.9 mmat
24 weeks compared with 8000 and 2.4 mm in the
10 mm gap. This is due to the decrease of neuro-
tropism from the distal nerve stumps which de-
creases as the distance between the stumps in-
creases [10, 19, 21, 24]. Neurotropism is weaker in
tubes with a 25 mm gap than in those with a 10 mm
gap. Vessels within a tube enable axons to re-
generate for a longer distance, but cannot increase
their number or diameter of the axons [6].
Tubes containing vessels are considered to be
useful clinically for the repair of divided nerves.
As peripheral nerves usually accompany blood
vessels forming neurovascular bundles, it is not
difficult to include vessels in the tubulation, even
in the presence of scar tissue [25].
Regeneration is quicker in tubes containing
blood vessels; re-innervation can be achieved
earlier than through unvascularised tubes [6, 8],
and the axons will extend further. The distance will
vary in different species [13], and the length across
which axons can extend is 10 mm in rat sciatic
nerves [9, 10, 11]. Mackinnon et al. reported the
successful repair of 3 ± 5 cm gaps in primates [13,
16]. Other studies have shown that satisfactory
results follow repair of human digital nerve gaps of
less than 3 cm using unvascularised tubes, such as
autogenous veins or biodegradable PGA (poly-
glycolic acid) tubes [3, 15, 23, 14, 28]. A gap of
25 mm in rats corresponds to a longer distance in
man, and so gaps in nerves could be bridged over a
longer distance using tubes containing vessels.
Tang et al. used autogenous veins as nerve
conduits clinically and reported success with gaps
of 0.5 ± 3 cm in digital nerves, and of 2.5 ± 4.5 cm
in ulnar nerves, but failed with a 5 cm gap in a
median nerve [25]. The critical length with this
method was considered to be 3 cm for digital
nerves and 4.5 cm for trunk nerves. A tube con-
taining vessels should allow axons to bridge a gap
of more than 5 cm in human digital nerves, but the
number of axons will decrease as the gap in-
creases. These tubes should therefore be used in
man for repair of peripheral nerves with gaps less
than, or slightly longer, than the critical length for
axon regeneration in unvascularised tubes, the aim
being to accelerate the rate rather than improving
the distance of axon regeneration.
In the present study, the inserted vessels were
patent in every rat at 12 weeks, but they had be-
come necrotic in 75% at 24 weeks, although there
was good nerve regeneration within the tubes. The
blood supply of the neural tissue gradually swit-
ched from the previously inserted vessels to ca-
pillaries which spread from both stumps [6]. The
monitor flap survived with its blood supply from
the inserted vessels for up to 12 weeks, although it
began to be nourished by collateral vessels from
the surrounding tissue by 24 weeks. When this
type of tube is used in man, the blood flow in the
included vessels will not be reduced because per-
ipheral vessels connect with other vessels to form a
vascular network.
We have just begun to repair divided human
digital nerves using vessel-containing tubes. Re-
covery of the repaired nerves has been satisfactory,
although the longest follow up is less than
6 months.
We have used silicone tubes to act as a barrier
for immigrating scar tissue into the lumen of tubes,
for the emigration of neurochemical factors se-
creted from nerve stumps, and as a space for
forming fibrin matrix which would act as a scaf-
fold for nerve growth [29, 30]. Biodegradable or
biological tubes should be used clinically because
silicone rubber tubes have to be removed after
several months, since they might affect joint
movement and induce synovitis.
A longer follow-up and improvement of the
material used for the tubes is needed. Nevertheless,
we conclude that vessel-containing tubes can ac-
celerate nerve regeneration and extend its distance.
Clinically this can be useful for repairing divided
nerves with gaps which are almost the same, or
slightly longer, than the length across which re-
generation can occur in unvascularised unmodified
tubes.
335R. Kakinoki et al.: Nerve regeneration in rat sciatic nerves

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