tos_owen et al. 2000 tissue specificity in rat peripheral nerve regeneration through combined...

8/12/2019 Tos_Owen Et Al. 2000 Tissue Specificity in Rat Peripheral Nerve Regeneration Through Combined Skeletal Muscle

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TISSUE SPECIFICITY IN RAT PERIPHERAL NERVE REGENERATIONTHROUGH COMBINED SKELETAL MUSCLE AND VEINCONDUIT GRAFTS

PIERLUIGI TOS, M.D.,

1,2

* BRUNO BATTISTON, M.D.,

1

STEFANO GEUNA, M.D.,3

MARIA G. GIACOBINI-ROBECCHI, M.D.,3 MARK A. HILL, M.D.,4

MARCO LANZETTA, M.D.,2 and EARL R. OWEN, M.D.2

Diffusible factors from the distal stumps of transected periph-eral nerves exert a neurotropic effect on regenerating nervesin vivo (specificity). This morphological study was designedto investigate the existence of tissue specificity in peripheralnerve fiber regeneration through a graft of vein filled withfresh skeletal muscle. This tubulization technique demon-strated experimental and clinical results similar to those ob-tained with traditional autologous nerve grafts. Specifically,we used Y-shaped grafts to assess the orientation pattern ofregenerating axons in the distal stump tissue. Animal modelswere divided into four experimental groups. The proximalpart of the Y-shaped conduit was sutured to a severed tibialnerve in all experiments. The two distal stumps were suturedto different targets: group A to two intact nerves (tibial andperoneal), group B to an intact nerve and an unvascularizedtendon, group C to an intact nerve and a vascularized ten-don, and group D to a nerve graft and an unvascularized

tendon. Morphological evaluation by light and electron mi-croscopy was conducted in the distal forks of the Y-shapedtube. Data showed that almost all regenerating nerve fibersspontaneously oriented towards the nerve tissue (attached ornot to the peripheral innervation field), showing a good mor-phological pattern of regeneration in both the early and latephases of regeneration. When the distal choice was repre-sented by a tendon (vascularized or not), very few nervefibers were detected in the corresponding distal fork of theY-shaped graft. These results show that, using the muscle-vein-combined grafting technique, regenerating axons areable to correctly grow and orientate within the basementmembranes of the graft guided by the neurotropic lure of thedistal nerve stump.

2000 Wiley-Liss, Inc.

MICROSURGERY 20:6571 2000

A peripheral nerve with a long segment defect requires a

grafting conduit for a functional repair.1

A graft should havethe following characteristics: it should supply a metaboli-

cally active environment to support axon regeneration and

progression; it should provide protection against scar inva-

sion; it should guide the regeneration to the distal stump of

the nerve; if a tube is used, it should also allow the free

orientation of growing axons along the tube. Previous stud-

ies on rats demonstrated that vein conduits filled with fresh

skeletal muscle provide histological and functional results

similar to those obtained with traditional nerve grafts in

cases with a substance loss of up to 2 cm.2 This technique

has been used in clinics since 1993 and produces good

functional results for both sensory and mixed nerves.36

Thevein guides the regeneration and the muscle prevents vein

collapse. Moreover, the muscle provides an adequate ad-

hesion for the advancing sprouts by means of neurite-

promoting factors present in its basal laminae (laminin and

fibronectin).7

Classical studies812 have shown that diffusible factors

from the distal stumps of transected peripheral nerves exert

a neurotropic/chemiotactic effect on regenerating nerve fi-

bers in vivo. The specificity of the neurotropic effect on

nerve regeneration indicates the ultimate extent of accuracy

in reinnervation of peripheral targets. Specificity may be

further defined into one of the following classes: tissue

specificity, i.e., the preferential reinnervation of axons to-wards distal nerve tissue rather than other tissues; end-organ

specificity, i.e., the preferential reinnervation of motor and

sensory fibers to their original target organ; and topographic

specificity, i.e., the preferential growth of axons belonging

to certain topographic components of a nerve trunk towards

corresponding distal regions.

Several authors investigated the tissue specificity in

vivo, adopting slightly different models. These have in-

cluded a Y-shaped artery taken from heterogenic13 or syn-

1

Gruppo Interdivisionale di Microchirurgia (G.I.M), Ospedale C.T.O., Turin,Italy

2Microsearch Foundation of Australia, Sydney, Australia

3Dipartimento di Scienze Cliniche e Biologiche, Ospedale San Luigi, Universitadi Torino, Turin, Italy

4Cell Biology Laboratory, School of Anatomy, University of NSW, Sydney,Australia

*Correspondence to: Pierluigi Tos, M.D., First Orthopaedic Department, Uni-versity of Turin, C.T.O. Hospital, Via Zuretti 29, 10126 Turin, Italy.E-mail: [email protected]

Received 9 February 1999; Accepted 25 September 1999

2000 Wiley-Liss, Inc.


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genic animals,14 a Y-shaped silicon chamber,1517 and a

Y-shaped predegenerated muscle.18 Except for the results of

the classical study by Weiss and Taylor,13 these studies

suggest that the growth and orientation of regenerating

axons are influenced by factors present in the distal stump

and that nerves preferentially grow towards nerves rather

than towards other tissues.15,19

Several suggestions have been offered to explain the

variations from the study reported by Weiss and Taylor.13

Lundborg et al.20 and Anderson and Turmaine14 proposed

the existence of antigenic properties of the vascular allo-

graft. Politis et al.15 reported that axonal growth could be

influenced by the endothelium of the vessels and the per-

meability of the artery wall to external factors. In this view,

endothelial cells have been demonstrated to deliver trophic

factors21 or neurite-promoting agents, such as laminin, both

in vitro7 and in vivo.22 Finally, the blood within the artery

(used to prevent it from collapsing) may have influenced the

diffusion of chemical attractants in the system.

Many studies have addressed the issue of specificity in

peripheral nerve regeneration. However, we investigated

tissue specificity within an 8-mm-long Y-shaped graft of

vein filled with fresh muscle, a new grafting technique that

has proved to be a good surgical alternative to the classical

fresh nerve autograft for the repair of peripheral nerve de-

fects.36 In particular, we assessed (1) whether regenerating

axons can specifically orientate and grow along the basal

membranes of the conduit under the influence of the distal

stump, (2) whether factors delivered by the graft material

(e.g., endothelial cells and fresh muscle fibers) and/or vein

permeability influence the growth pattern to a greater de-

gree than the distal neurotropic lure, and (3) whether the

vascularization of the target tissue influences tissue speci-

ficity.

MATERIALS AND METHODS

Animals

Twenty-four male Wistar rats (200250 g) were housed

in individual raised lid cages with fibrecycle pellet bedding.

Food and water were supplied ad libitum. Approval and

consent for our study were obtained from The Microsearch

Foundation of Australia Institutions Research and Animal

Ethical Review Committees.

Surgical Procedures

Anesthesia was induced and maintained with halothane

(4% during induction, 2% thereafter) using a standard an-

esthetic machine. Surgery was performed using an OPMI 7

(Zeiss, Thornwood, NY) operating microscope.

An oblique skin incision was made in the anterior part of

the left thigh, exposing the site of bifurcation of the femoral

vein into the epigastric and femoralis profunda. A 12-mm-

long section was carefully dissected as a Y-shaped tube.

Two thin strips of muscle fiber (8 mm long and 2 mm in

diameter, dissected from the adductor muscle) were then

inserted into the vein. The skin was closed using resorbable

4/0 sutures.

A posterior incision was then carried out on the right

thigh, exposing the trifurcation of the sciatic nerve into the

peroneal, tibial, and sural nerves. The tibial nerve was tran-

sected and 5 mm of nerve resected. The proximal segmentwas introduced 12 mm into a channel of the vein. The

distal targets were introduced 2 mm into the two distal

channels of the vein and then sutured.

Animals were divided into four experimental groups.

All groups had the tibial nerve resected and its proximal

segment sutured 2 mm into one channel of a 12-mm-long

Y-shaped vein filled with fresh skeletal muscle. The distal

target tissues were introduced 2 mm into the remaining

channels so that the distance between the proximal nerve

and the distal tissue was 8 mm.

Target tissue was composed of either two distal intact

nerves (group A), a distal intact nerve and tendon (group B),

a distal intact nerve and vascularized tendon (group C), or anerve graft and tendon (group D) (Fig. 1). Animals in group

A (n 8) had the peroneal nerve transected. After resecting

approximately 2 cm of nerve, the distal portion of the pe-

roneal nerve was inserted (2 mm) into one fork of the vein

and the distal transected portion of the tibial nerve inserted

into the other fork (Fig. 2A). The proximal end of the tran-

sected peroneal nerve was not connected. Animals in group

B (n 8) had the distal stump of the transected tibial nerve

and a 10-mm-long tendon (extensor digitorum, previously

Figure 1. Schematic drawing illustrating the experimental de-

sign of this study. The proximal fork of the Y-shaped graft was

sutured to a severed tibial nerve in all groups. The two distal

forks of the graft were sutured to different distal stumps. Intact

nerve (groups A,B,C) refers to a nerve that maintains its end-

organ connection and vascularization, whereas the nerve graft

(group D) refers to a nerve that is disconnected from its periph-

ery and devascularized.

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harvested from the left foot) inserted inside the two forks of

the distal segment of the muscle-vein-combined graft (Fig.

2B). For group C animals (n 4), the distal targets were

represented by an intact tibial nerve and a vascularized ten-

don (distal part of the adductor brevis muscle). Group D (n

4) animals had the two distal forks of the Y-shaped

conduit sutured to a 1-cm-long nerve graft on one side and

a 10-mm-long tendon (extensor digitorum) on the other

side. The graft was harvested from the distal segment of the

tibial nerve and was totally removed from its blood supply.

All microsurgical procedures used 9-0 monofilament

nylon sutures (three or four stitches for each stump) to at-tach the nerve and/or tendon to the muscle-vein-combined

conduit. Following this microsurgery, the graft was posi-

tioned without kinking in the soft tissue and the distal part

of the avascular tendons or nerve grafts was sutured to a

near muscle. The angle of the two distal stumps was ap-

proximately 3040. The muscle and skin of the right thigh

was closed using 4/0 resorbable sutures.

Four rats from both groups A and B were sacrificed at

week 4 and the other four animals were sacrificed at week

12. Animals in groups C and D were sacrificed at week 12.

Nerves were removed, fixed, and prepared for light and

electron microscopy examination.

Histology: Tissue Preparation

The Y-shaped grafts were fixed in a solution containing

2.5% purified glutaraldehyde and 0,5% sucrose in 0.1 MSorensen phosphate buffer (pH 7.2) for 8 h. Specimens were

then washed in a solution containing 1.5% sucrose in 0.1 M

Sorensen phosphate buffer (pH 7.2) for 612 h. After post-

fixation in 2% osmium tetroxide, grafts were dehydrated

and embedded in Glauerts embedding mixture consisting

of equal parts of Araldite M and the Araldite Harter, HY

964 (Merck, Darmstad, Germany), to which was added 2%

of the accelerator 964, DY 064 (Merck). The plasticizer

dibutyl phthalate was added in a quantity of 0.5%.

Morphological Assessment of Regeneration

Regeneration of the nerve fibers in the two distal forks

of the Y-shaped graft was assessed by means of light and

electron microscopy. A series of 2-m-thick cross-sections

were cut using an Ultratome III ultramicrotome (LKB,

Bromma, Sweden). Sections were taken from various posi-

tions for each of the two distal forks of the Y-shaped cham-

ber. All sections were stained according to the Richardson

method (Nissl toluidine blue) for light microscopy exami-

nation.

Immediately following the series of semithin sections,

ultrathin sections were cut using a diamond knife mounted

on the same LKB ultramicrotome. These sections were

stained with saturated aqueous solution of uranyl acetate

and lead citrate solution and examined using an EM-410electron microscope (Philips, Eindhoven, Holland) for

qualitative ultrastructural analysis.

Quantitative Morphological Analysis

Quantitative morphological assessment of myelinated

nerve fibers was conducted on a series of semithin sections

cut at the midportion of the distal forks of the grafts, which

were prepared and stained as described above. The sections

were analyzed using a Zeiss Videoplan Image Processing

System composed of a camera and a Digicad (GTCO,

Munchen, Germany) graphic tablet connected to a Laborlux

S (Leitz, Wetzlar, Germany) light microscope; the graphic

tablet was connected to an IBM 286 PC computer. Thissystem reproduced microscopic images (obtained through a

100 oil-immersion Leitz objective) on the monitor at a

final magnification of 3,720. This permitted accurate rec-

ognition, counting, and measurement of myelinated nerve

fibers (repeat measurements 4%).

In all Y-shaped grafts, one section of the series cut from

each of its two distal forks was analyzed. In these sections,

10 microscopic fields were selected by a systematic random

sampling technique.23 The number of myelinated nerve fi-

Figure 2. Surgical procedure. The Y-shaped muscle-vein-combined

graft at the end of the operation. The proximal fork has been sutured

in all groups to a tibial nerve. A: Group A: both distal forks were

sutured to intact nerves (peroneal and tibial nerves). B:Group B: one

of the two distal forks was connected to a tendon and the other one

to an intact nerve (tibial nerve).

Tissue Specificity in Nerve Regeneration 67


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bers was then counted for each sampled field (area 1,680m2) and their density calculated. The total number of fi-

bers was estimated by multiplying the fiber density by the

total cross-sectional area of the whole nerve. Finally, the

average diameter of myelinated nerve fibers was obtained

by measuring the widest diameter (inclusive of the myelin

sheath) of at least 100 fibers in each section.

Statistical comparisons of estimates of total number,

density, and mean size of the myelinated nerve fibers were

subjected to analysis of variance (ANOVA) as described by

Glantz24 using the BASIC program run on an IBM 486 PC

computer.P < 0.05 was considered statistically significant.

RESULTS

Group A

The gross appearance of the two forks of the graft con-

nected with two intact nerves (tibial and peroneal) at weeks

4 and 12 after surgery (Fig. 3A). The gross appearance of

both forks was similar to a healthy nerve. Light microscopy

of the Y-shaped graft conduit showed that nerve fibers re-

generated towards both distal stumps. The morphological

pattern of nerve regeneration showed a typical alternation of

myelinated nerve fibers and very few muscle fibers (Fig.

4A). Nerve fibers, both myelinated and unmyelinated, were

organized into fascicles (Fig. 5). Some rare muscle fibers

still persisted among regenerated nerve fibers even at 12

weeks postoperatively (Fig. 5, arrow). Quantitative estima-

tion of the number of myelinated nerve fibers (Table 1)

showed that the total number of fibers regenerated towards

the tibial stump (6,268 607) was significantly (P < 0.05)greater than that of the fibers reaching the peroneal stump

(3,398 1,102). Estimates of mean density and size were

not significantly different between the two forks. The sum

of the total number of myelinated nerve fibers that regen-

erated towards the two different nervous peripheries in

group A animals (Table 1) was 9,916 1,144, a value that

is not significantly different (P> 0.1) compared to the total

number of regenerated fibers counted in groups B, C, and D

(Table 2).

Figure 3. Gross findings. A:Nerve vs. nerve (group A): the appear-

ance of both distal forks of the graft was similar to a nerve. B:Nerve

vs. tendon (groups B,C,D): in all groups, the distal fork of the Y-

shaped graft connected to the tendon was much thinner than the fork

connected to a nerve.Figure 4. Photomicrograph of semithin sections (stained with tolu-

idine blue) from the distal graft fork connected to a nerve (A) and to

a tendon (B). A: A good pattern of regeneration is detectable 12weeks after surgery; some muscle fibers (arrows) are still present.

B:Rare nerve fibers between the vein wall and the muscle fibers,

organized in minifascicles, are detectable (950; bar = 10 m).

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Group B

Macroscopical examination, 4 and 12 weeks after sur-

gery, of the distal portion of the Y-shaped conduit suggested

no regrowth of nerve tissue towards the unvascularized ten-

don when this represented one of the two distal choices (Fig.

3B). At light microscopy examination, occasional myelin-

ated nerve fibers could be identified in the fork connected to

the unvascularized tendon (Fig. 4B). These myelinated fi-

bers were located in the context of the vein wall and/or

spread among the muscle fibers especially in the peripheral

part of the graft. Ultrastructural analysis showed the pres-

ence of a dense extracellular matrix occupied by many dis-

organized collagen fibers and some fibroblasts. In the fork

connected to the intact nerve, we always observed a typical

morphological pattern of nerve regeneration. Table 2 shows

the quantitative estimation of the total number (8,706

2,041), mean density (24,932 2,545), and size (4.30 0.37

m) of myelinated fibers regenerated in the distal forks

connected to the intact nerves of group B, at 12 weeks

postoperatively.

Group C

Macroscopical appearance at week 12 postoperatively

of the distal portion of the Y-shaped conduit when one of

the two distal choices is represented by a vascularized ten-

don suggested no regrowth of nerve tissue towards the ten-

don (Fig. 3B). Table 2 shows the quantitative estimation of

the total number (8,797 2,405), mean density (21,387

1,307), and diameter (4.37 0.60 m) of myelinated fibers

regenerated in the distal forks connected to the intact nerves

of group C, at 12 weeks postoperatively. Statistical com-

parison showed no significant (P> 0.1) difference between

groups B and C (intact tibial nerves) for all the three mor-

phometrical parameters.

Group D

The gross appearance at week 12 of the distal portion ofthe Y-shaped conduit when one of the two distal choices is

represented by an unvascularized tendon and the other one

by a nerve graft showed that the fork sutured to the unvas-

cularized tendon was always much thinner than the fork

sutured to the nerve graft (Fig. 3B). Regeneration towards

the nerve graft appeared to form a neuroma with dense scar

tissue noted at the distal part of the graft. Quantitative es-

timates of myelinated nerve fibers regenerated in the distal

forks connected to the nerve grafts of group D are reported

in Table 2. Statistical evaluation showed that the total num-

ber (9,878 1,461) and mean density (22,032 1,683) of

fibers were not significantly (P> 0.1) different when com-

pared to data from both groups B and C. A trend (P 0.057) towards a smaller size was observed in the regener-

ated nerve fibers of this group (3.55 0.44 m) in com-

parison to the other groups.

DISCUSSION AND CONCLUSIONS

The various levels of specificity of regenerating periph-

eral nerve fibers have been addressed by a number of stud-

ies. In particular, tissue specificity was usually investigated

by the Y-chamber experimental model.1322 Using this ex-

perimental model, our data showed that axons regenerating

within a new biological grafting technique (e.g., the muscle-vein-combined autograft) can orientate and grow, along the

basal membranes of the conduit, towards a nervous periph-

ery when an alternative distal target is given. Besides pro-

viding further evidence of the existence of a neurotropic

effect exerted by the distal stump on the outgrowth of re-

generating axons, our results show that factors delivered by

the graft endothelial cells and muscle fibers, and/or factors

from outside the vein, did not influence the growth pattern

to a greater degree than the distal neurotropic lure. In fact,

fibers only occasionally grow into the distal fork connected

to a tendon (groups B, C, and D), even if this part of the

graft is composed of the same material (muscle and vein) as

the other parts of the graft. This evidence provides a strongsupport to the real effectiveness of the muscle-vein-com-

bined grafting procedure for the repair of peripheral nerve

lesions.

Our demonstration of the neurotropic attraction exerted

by the distal stump of the Y-shaped graft on regenerating

axons is in contrast with the experiments by Weiss and

Taylor,13 and in accordance with the results of more recent

studies conducted on tissue specificity of regenerating pe-

ripheral nerve fibers.1422 Therefore, our data suggest that

Figure 5. Electron micrograph of the distal fork of the Y-shaped graft

connected to a nervous periphery. Fascicles of myelinated and un-

myelinated nerve fibers show the typical features of regeneratedfibers. Note the presence of muscle fibers close to the nerve fibers

(arrow) (3,500; bar = 5 m).



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the concept of contact guidance introduced on the basis of

the study by Weiss and Taylor13 should be reappraised.

Data presented in this study also demonstrate that the

vascularization of the target tissue did not influence the

specificity of axon regeneration. In fact, vascularization of

the tendon (group C) sutured to one of the two forks of the

graft did not determine regeneration of nerve fibers towards

that periphery. On the other hand, when the distal choice

was represented by a nerve graft (group D), i.e., unvascu-

larized, the neurotropic lure on the regenerating fibers is still

detectable. It may be concluded that tropic factors that exert

chemiotactic influence on regenerating axons originate form

the target tissue (the nervous tissue) and are not modified byblood circulation. It is still unclear as to whether vascular-

ization of the distal nervous end-organ and/or its connection

to the periphery (intact nerve vs nerve graft) influences the

maturation of nerve regeneration. In fact, our data showed

only a trend (P< 0.1) towards smaller sizes in nerve fibers

regenerating towards the nerve graft (group D) compared

with those regenerating towards intact nerves (groups A, B,

and C).

This study was designed to gain data on tissue specific-

ity. Therefore, the experiments did not allow investigation

of other levels of specifity, such as end-organ and topo-

graphic.25 Moreover, our experiments were not designed to

gain data on the pruning hypothesis by Brushart and col-leagues2629, which states that regenerated axons reaching

an inappropriate target will be progressively pruned back.

The Y-shaped model allowed us to verify that the muscle-

vein-combined conduit graft permits distal targets to exert a

specific attraction on regenerating axons. Electron micro-

scope observation provided ultrastructural evidence that the

quality of regeneration was similar to that observed in pre-

vious studies using straight biological chambers of the same

type.46 Lundborg et al.30 demonstrated in clinical trials that

the existence of a structured gap, measuring 34 mm,

between the stumps of sectioned median and ulnar nerves

could improve nerve repair by facilitating axonal matching

due to neurotropic distal attraction. We designed this study

to include a gap of 8 mm between the stumps of the severed

nerve. The possibility of the regenerating axons to freely

orientate along basal membranes of the muscle-vein-com-

bined conduit, enabling a correct regeneration pattern, may

explain our good clinical results.46 With traditional nerve

grafting techniques, the surgeon somehow forces the orien-

tation of regenerating axons. In fact, along the muscle-vein-

combined grafts, chemiotrop(h)ic substances originating

from the distal nervous stump can reach the regeneratingaxons which are free to be specifically guided to their cor-

rect target periphery, aiding in correct orientation. Further

experiments are needed to confirm if the same results on

neurotropism occur over longer distances employing this

grafting technique.

ACKNOWLEDGMENTS

The authors are grateful to Prof. David Tracey for his

support and for finding the facilities for processing the

samples and data. They also thank Prof. Renzo Guglielmone

for useful suggestions on manuscript preparation, Dr. Feng-

Chun He (Xian) for valuable suggestions and help, and Dr.

Maurizio Calcagni and Tim Cushway for useful comments

on the preparation of samples, collection of data, and manu-

script preparation.

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Table 1.Comparison of Total Number, Mean Density (no./mm2), and Mean Size of Myelinated

Nerve Fibers From Group A (Distal Forks of the Graft Connected to Two Intact Nerves)

Group A

Tibial Peroneal Total

Total number of myelinated fibers 6,268 607a 3,398 1,102 9,916 1,144

Density of fibers (no./mm2) 21,861 4,490 26,431 5,577 24,171 4,165

Size of fibers (m) 4.17 0.69 4.09 0.41 4.13 0.46

aValues are means S.D.

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Group B

(intact nerve)

Group C

(intact nerve)

Group D

(nerve graft)

Total number of myelinated fibers 8,706 2,041a 8,797 2,405 9,878 1,461

Density of fibers (no./mm2) 24,932 2,545 21,387 1,307 22,032 1,683

Size of fibers (m) 4.30 0.37 4.37 0.60 3.55 0.44

aValues are means S.D.

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tos_owen et al. 2000 tissue specificity in rat peripheral nerve regeneration through combined...

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