Journal of Plastic, Reconstructive & Aesthetic Surgery (2008) 61, 1357e1367

A new model of end-to-side nerve graft for multiplebranch reconstruction: end-to-side cross-face nervegraft in rats*

Ken Matsuda a,*, Masao Kakibuchi b, Tateki Kubo a, Koichi Tomita a,Toshihiro Fujiwara a, Ryo Hattori a, Kenji Yano a, Ko Hosokawa a

a Department of Plastic Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japanb Department of Plastic Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

Received 11 September 2007; accepted 10 April 2008

KEYWORDSEnd-to-sideneurorrhaphy;Nerve graft;Facial nervereconstruction;Cross-face nerve graft;Multiple branchreconstruction;Rat facial nerve

* This work was partly presented at50th council meeting of Japan Societyin the products used in the experimeJapan and the Research Grant from th

* Corresponding author. Address: DepOsaka 565-0871, Japan. Tel.: þ81 6 6

E-mail address: matsuda@psurg.m

1748-6815/$-seefrontmatterª2008Britdoi:10.1016/j.bjps.2008.04.013

Summary Background: The effectiveness of end-to-side nerve graft for multiple branch re-construction was confirmed using a new rat four-branch facial nerve reconstruction model withend-to-side cross-face nerve graft.Methods: Forty Lewis rats were randomly divided into four groups with different reconstruc-tion methods for four branches (bilateral buccal and marginal branches) of the bilateral facialnerves as follows: group I (n Z 12), single sciatic nerve graft with end-to-side neurorrhaphy;group II (n Z 12), four cable grafts using two sciatic and two ulnar nerves with end-to-end neu-rorrhaphy; group III (n Z 8), no repair; and group IV (n Z 8), sham operation. The four groupswere compared with double retrograde tracing of the facial nucleus, electrophysiological andhistomorphometrical assessment of the reconstructed facial nerve.Results: Although there were no significant differences between groups I and II in the electro-physiological tests, group I showed more uniform and better reinnervation in the histomorpho-metrical assessment. Retrograde tracing of facial nucleus revealed significantly higher numberof double-labeled neurons in group I although the total number of labeled neurons was notdifferent between the two groups.Conclusions: End-to-side nerve graft shows a good functional recovery, requires less graft, andis easy to perform. With the availability of the side of the nerve graft itself as a nerve

the 14th research council meeting of Japan Society of Plastic and Reconstructive surgery 2005 andof Plastic and Reconstructive surgery 2007. Disclosure: None of the authors have financial interest

ntal work. This work was supported by a research Grant from the General Insurance Association ofe Japanese Ministry of Education, Science, Sports and Culture (16591793, 16791089, and 18791320).artment of Plastic Surgery, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita-city,

879 6056; fax: þ81 6 6879 6059.ed.osaka-u.ac.jp (K. Matsuda).

ishAssociationofPlastic,ReconstructiveandAestheticSurgeons.PublishedbyElsevierLtd.All rightsreserved.

1358 K. Matsuda et al.

coaptation site, it can be an effective alternative in facial nerve reconstruction and be of greatvalue in various kinds of peripheral nerve surgery.ª 2008 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published byElsevier Ltd. All rights reserved.

End-to-side neurorrhaphy is not a new concept. Initialreports on the clinical application of this concept dateback to the early 1900s.1,2 The idea was not widely ac-cepted at the time and was forgotten for a long time. SinceViterbo et al. reintroduced this concept in an experimentalmodel,3 end-to-side neurorrhaphy models have been adop-ted by many research groups and this technique has beenused widely in clinical practice. Clinically, it is employedto induce collateral sprouting from the healthy donor nerveand minimize the donor nerve morbidity when an appropri-ate proximal end is not available. Unlike this concept, weused the end-to-side nerve graft technique for multiplebranch reconstruction of facial nerve defect.4 There aresome clinical applications5,6 and experimental models7e9

that used multiple recipient nerves attached to one donornerves via end-to-side neurorrhaphies, however our tech-nique has a different concept using the side of the graftednerve itself, not of the intact donor nerve. In our method,the distal end of each branch was coapted to the side of thenerve graft via an epineurial window. The aims of ourmethod in facial nerve reconstruction are to reduce thetechnical difficulty of the proximal end-to-end neurorrha-phy and to increase the regeneration efficiency with lessgraft requirement as compared to the conventional end-to-end cable graft method. The functional recovery of ourclinical case with four branch reconstruction was quite sat-isfactory,4 and its regeneration mechanism and usefulnesswere partly confirmed by using the rat sciatic nerve tran-section models with two branch reconstruction.10 However,in most clinical cases of extensive facial nerve defect, re-construction of three or more branches is required. In orderto investigate the regeneration process and efficiency inmultiple branch reconstruction with our method, we devel-oped a new rat model with end-to-side cross-face nervegraft for four branch reconstruction.

Materials and methods

Operative procedure

Approval for this study was obtained from our institution’sanimal care and ethics committee. Forty adult male Lewisrats with a mean weight of 220 g (range: 210e250 g) wereused in this study. All surgical procedures were carried outby the same surgeon, using microsurgical techniques. Theanimals were prepared for aseptic surgery and anesthetizedwith intraperitoneal sodium pentobarbital (5 mg/100 g BW).The bilateral facial nerves were exposed by cheek incisions,and 15-mm-long segments of the nerve including their trunkand all branches were excised. The left facial nerve trunkwas ligated with 5/0 nylon suture and buried into the sur-rounding soft tissue to prevent auto-reinnervation withthe distal stumps. In group I (n Z 12, end-to-side group),

a 7-cm sciatic nerve graft harvested from syngeneic donorwas sutured to the proximal end of the right facial nervetrunk in an end-to-end fashion. The distal ends of the ipsi-lateral marginal mandibular and buccal branches werecoapted to the side of the sciatic nerve graft via an epineu-rial window. Then, the graft was guided to the contralateralside through a subcutaneous tunnel created at the foreheadregion, and the contralateral buccal branch was coapted tothe side of the nerve graft via an epineurial window, and thedistal end of the graft was sutured to the marginal mandib-ular branch in an end-to-end fashion. In group II (n Z 12,end-to-end group), two sciatic nerve grafts and two ulnarnerve grafts from a syngeneic donor were used as four cablegrafts to reconstruct the four branches; the ipsilateral andcontralateral buccal and marginal mandibular branches.Two sciatic nerve grafts were guided through different sub-cutaneous tunnels to avoid axonal contamination. Along thenerve graft, the distances between right facial nerve trunkand each coaptation site were 1.5 cm, 2.5 cm, 5.5 cm and6.5 cm in Group I, and 1.5 cm, 2 cm, 5 cm and 6 cm in GroupII (ipsilateral marginal mandibular, buccal and contralateralbuccal, marginal mandibular branch respectively). All nervecoaptations were done with four epineurial 10-0 nylonstitches and covered with a surrounding soft tissue to reduceaberrant reinnervation. In group III (n Z 8, no repair group),the proximal stump of the right facial nerve was also ligated.In group IV (n Z 8, sham operation group), the bilateralfacial nerves were only exposed but not excised. The skinwas closed with 5/0 nylon suture in all animals (Figure 1).

Observation of vibrissal whisking

Under normal physiological conditions, the mystacialvibrissae of the rat are erect in anterior orientation. Theirsimultaneous sweepings known as ‘whisking’ or ‘sniffing’are provided by protraction and retraction of the vibrissalhairs by the piloerector muscles, which are innervated bythe buccal branch of facial nerve.11 Following transectionof the facial nerve, the vibrissae drop motionless and ac-quire a caudal orientation. As piloerector muscles are rein-nervated, vibrissae gradually rise and whisking movementsappear again. The ipsilateral and contralateral vibrissalwhisking movements were observed weekly as a rough eval-uation of reinnervation.

Electrophysiological test

Twelve weeks postoperatively, the animals were anesthe-tized with intraperitoneal sodium pentobarbital (5 mg/100 g BW). Via a cheek incision, a bipolar stimulatoryelectrode was placed on the right facial nerve trunk. Thecollector electrodes were situated in whisker pad musclesfor the buccal branch and in the depressor anguli oris

Figure 1 Schematic diagrams demonstrating the procedures in each group. The nerve grafts are indicated in yellow (bu, buccalbranch; mm, marginal mandibular branch; T, trunk of the facial nerve).

End-to-side cross-face nerve graft in rats 1359

muscle for the marginal mandibular branch. Stimulationwas made by PowerLab system (PowerLab 2/20, AD Instru-ments, Castle Hill, New South Wales, Australia) with a con-stant voltage isolated stimulator (DS2A- MK. II, Digitimer,Hertfordshire, England). Electric stimulation was a squarepulse with a duration of 0.1 ms and a frequency of 1 Hz.To set the supramaximum level, stimulation was graduallyintensified from 0 V, resulting about 3 V in most animals.The latency time from stimulation to the first onset ofthe first negative deflection was recorded. The distancesbetween stimulation site and collector electrode were setto 40 mm (for buccal branch) and 30 mm (for marginal man-dibular branch) in the ipsilateral side and 70 mm (for buccalbranch) and 60 mm (for marginal mandibular branch) in thecontralateral side. The conduction velocity in the nervegraft was calculated from these distances and the meanlatency time of 16 stimulations. All recording data werestored in a personal computer and analyzed with Scope(version 3.6.12) software (AD Instruments).

Retrograde double labeling of the facial nucleus

Two fluorescent retrograde axonal tracers; namely, trueblue (TB; Sigma Chemical, St. Louis, MO, USA), anddiamidino yellow (DY; Sigma Chemical), were dissolved in

distilled water to make a 2% suspension and stored at 4 �Cin the dark. After the electrophysiological examination,2 ml of 2% TB and DY solutions were directly injected intoeither the right or left buccal and marginal mandibularbranches, respectively. The injections were given viaa 10-ml Hamilton microsyringe. Following the injection,the site was wiped with a swab, flushed with sterile 0.9%saline, the injection site was closed with 10/0 nylonsutures, and the wound was closed by 5/0 nylon sutures.After allowing 7 days for retrograde transport, the animalswere perfused transcardially with 200 ml of 0.9% saline,followed by 500 ml of ice-cold 4% paraformaldehyde in0.1 M phosphate buffer. After perfusion, the whole brainwas dissected and removed, placed overnight in 4% para-formaldehyde in 0.1 M phosphate buffer for post-fixationand stored overnight in 30% phosphate-buffered sucrosesolution. Then brain stem was cut on a cryostat in40 mm-thick serial coronal sections through the facial nu-cleus. After drying for 30 min, the sections were mountedand examined with an ultraviolet fluorescence microscope(Zeiss Axioplan 2, Carl Zeiss, Goettingen, Germany) usinga combined filter set (487902-0000). Since TB labels thecytoplasm and DY labels the nucleus, only those TB-labeled profiles with nuclei in the plane of section werecounted to avoid double-counting. At first as the pilot

1360 K. Matsuda et al.

study, in group IV (n Z 8), all sections which contain la-beled profiles were numbered from cranial to caudal direc-tion. Then every fifth sections were picked up and fivegroups (starting number at 1,2,3,4 and 5) were made.Summarized numbers of TB or DY-labeled cells in eachgroup were compared to the summarized numbers in theall sections by using Spearman’s correlation coefficienttest. Regarding definitely strong correlation (r Z 0.91 to0.99 for TB count and r Z 0.90 to 0.95 for DY count) be-tween them and the homogenous neuronal density of ratfacial nucleus,12 summarized numbers in every fifthsections were used in all experimental groups for labeledcell counting. The results are presented as summarizednumbers of cells and in percentages of the total numberof labeled cells.

Histomorphometrical examination of thereconstructed branches and nerve grafts

After perfusion, 5-mm-long segments of the reconstructeddistal branches and nerve grafts were harvested. In group I,nerve graft samples were harvested from eight sites asfollows: four from the nerve graft and four from thereconstructed distal branches. In group II, eight sampleswere taken as follows: one from each nerve graft and eachreconstructed distal branch (Figure 2). In groups III and IV,four samples were taken from the right buccal and marginalmandibular branches. After post-fixation in 10% formalin in0.1 M phosphate buffer overnight, the nerve specimenswere fixed with 1% osmium tetroxide for 24 hours, then de-hydrated, and embedded in paraffin. An ultramicrotomewas used to obtain 1-mm cross-sections of the embeddednerve. The sections were stained with 1% toluidine bluefor light microscopy. The microscopic images were digitizedon a gray scale using an automated digital analysis software(Image J 1.33u, developed at the National Institute ofHealth, available at http://rsb.info.nih.gov/ij/). Eachspecimen was evaluated for the total number of fibres,neural tissue area (% nerve) and fibre diameter as describedelsewhere.13,14

Figure 2 The sites where the nerve specimens were harvested. I(red, each number indicates the site in Tables 2 and 3), and anot(blue). In groups III and IV, the ipsilateral buccal and marginal ma

Statistics

The nerve conduction velocity, numbers of labeled cells inthe facial nucleus and myelinated fibre numbers, fibrediameter, and fibre area (% nerve) were recorded as themean� SD. One-factor analysis of variance (ANOVA) wasused to evaluate the difference between the group means.When ANOVA showed statistical significance, specific groupmean comparisons were performed using the Turkey multi-ple comparison test. Statistical computations were per-formed using a computer provided with Microsoft Excel(Microsoft, Seattle, WA, USA) and Statcel 2 (OMS publishing,Saitama, Japan) software. A value of p< 0.05 was consid-ered significant.

Results

Observation of vibrissal whisking

In groups I and II, vibrissal whisking movements appearedon the ipsilateral side at 3 to 4 weeks postoperatively, andon the contralateral side at 5 to 7 weeks postoperatively.These movements were less rhythmical than in group IV,which showed significant whisking movement throughoutthe study. In group III, no significant whisking wasobserved.

Electrophysiological tests

There were no significant differences in the conductionvelocity between group I and group II. They were signifi-cantly lower than in Group IV. Group III showed no responsein all animals (Figure 3).

Retrograde double labeling of the facial nucleus

The results are presented in Table 1. In groups I and II,the percentages of TB- and DY-labeled neurons in eachgroup showed no significant differences, although the

n groups I and II, four samples were taken from the nerve grafther four samples were taken from the reconstructed branchesndibular branch were harvested.

Figure 3 Comparison of the nerve conduction velocity be-tween the trunk and the whisker pad (bu) or angulus oris(mm) in the ipsilateral or contralateral side. There was no sig-nificant difference between groups I and II.

End-to-side cross-face nerve graft in rats 1361

double-labeled neurons in group I were significantlyhigher than those in groups II and IV. The total numberof labeled cells was not significantly different betweengroups I and II. In group IV, the numbers of TB- andDY-labeled cells were significantly higher than in group Ior II, and the TB-labeled cells were localized in thelateral facial subnucleus and the DY-labeled cells werein the intermediate facial subnucleus. In groups I andII, these mytopic organizations in the subnuclei disap-peared, and motor neurons retrogradely labeled by thetwo tracers were scattered throughout the whole facialnucleus (Figure 4), indicating misdirected regenerationin the reinnervation process (i.e. the facial muscleswere partly reinnervated by motor neurons which hadoriginally innervated other facial muscles). There werefew labeled neurons in the contralateral facial nucleus,which showed minimal aberrant reinnervation from the li-gated contralateral facial nerve trunk.

Table 1 Counting of true blue (TB)-, diamidino yellow (DY) -lab

Group Ipsilateral

TB DY

Cell count (% ofthe total numberof labeled cells)

I 118.4� 28.9(63.9� 15.6%)

65.2� 7.2(35.2� 3.9%)

II 103.2� 20.2(58.3� 11.4%)

81.2� 13.0(38.2� 5.1%)

III 2.0� 0.4(100.0� 20.0%)

0 (0%)

IV 333.3� 30.1(53.0� 4.8%)

295.5� 23.3(47.0� 3.7%)

The values are expressed as means� SD.(Ipsilateral, the tracers were injected into the right buccal and marginto the left buccal and marginal mandibular branch).* p< 0.05 as compared with group II.

Histomorphometrical examination of thereconstructed branches and nerve grafts

1) Reconstructed branches

The results are presented in Table 2 and Figure 5. Group Ishowed better reinnervation than group II in the most distalbranch; i.e. the marginal mandibular branch, on the con-tralateral side. In group I, the buccal branch on the contra-lateral side seemed to be less reinnervated than the otherbranches in the group. In group II, the branches on the con-tralateral side were less reinnervated than on the ipsilat-eral side, and there was no significant difference betweenthe buccal branch and the marginal mandibular branch onthe same side.

2) Nerve graft

The results in group I are presented in Table 3 and Figure 5.Fibre width examination revealed no significant differencein four sites, but the fibre number in site 4 was significantlysmaller than in the other sites. As the examined sitebecame more distally, % nerve decreased linearly. Table 4and Figure 5 show the results in group II. There were signif-icant differences in the fibre width and % nerve betweenthe ipsilateral side and the contralateral side, while fibrenumber examination showed no significant differences inall sites. In group II, the short ulnar nerve grafts on theipsilateral side were well-reinnervated than the long sciaticnerve grafts on the contralateral side.

Discussion

In the rodent models of peripheral nerve reconstruction andregeneration, it is important to insulate and reduce theaberrant reinnervation from the proximal stump or sur-rounding regions, since such reinnervation occurs moreeasily than in humans and it prevents accurate evaluationfor the clinical applicability in humans. Regeneration fromthe proximal stump along and outside of the epineurium15

may have additional regeneration effects within a short

eled, and double-labeled (DL) cells in the right facial nucleus

Contralateral

DL TB DY DL

1.6� 1.3(0.9� 0.7%)

103.2� 20.2(61.6� 10.6%)

67.5� 9.0(37.2� 6.0%)

6.2� 2.8*(3.5� 1.6%)*

2.6� 2.2(1.2� 1.0%)

114.4� 15.2(62.8� 8.3%)

67.2� 6.1(36.9� 3.3%)

0.6� 1.3(0.3� 0.7%)

0 (0%) e e e

0.5� 0.6(0.1� 0.1%)

e e e

inal mandibular branch; Contralateral, the tracers were injected

Figure 4 The labeled cells in the right facial nucleus with ultraviolet fluorescence microscopy. In groups I and II, the mytopicorganizations into subnuclei disappeared, but it was manifest in group IV. Scale bar Z 150 mm.

1362 K. Matsuda et al.

distance in the animal model. However, it is not reliable forthe clinical situations with long nerve defects in humans.Regarding the distance of regeneration along the epineu-rium, 1.5 cm15 or 2 cm16 is quite possible although the

Table 2 Histomorphometrical examination of the reconstructe

Group Ipsilateral side

Marginal mandibular

Fibre diameter (m mm) I 3.94� 0.42II 3.70� 0.22III 3.19� 0.45IV 7.47� 0.52

Fibre number I 2670� 970II 3225� 441III 60� 51IV 2050� 157

% Nerve (%) I 48.0� 8.3II 47.2� 7.7III 0.8� 0.6IV 87.6� 3.5

The values are expressed as means� SD.* p< 0.05 as compared with group II.z p< 0.05 as compared with the another side.x p< 0.05 as compared with the buccal branch in the same side.

maximum possible distance is still unclear. To avoid regen-eration along or from outside of the epineurium and toincrease the specificity, some methods e.g., insulating bysilastic tube17,18 or wrapping it with pedicled fat flap19 or

d branches

Contralateral side

Buccal Buccal Marginal mandibular

3.90� 0.29 3.59� 0.22 3.53� 0.22z3.79� 0.42 3.35� 0.26z 3.40� 0.22z3.01� 0.14 e e

7.22� 0.31 e e

2421� 740 1490� 532z 2193� 786*, x2658� 659 1473� 467z 1396� 188z

95� 92 e e

2555� 400 e e

36.5� 10.6 20.2� 8.2z 28.5� 4.0*, z, x44.6� 5.5 17.5� 5.2z 21.0� 3.9z1.8� 1.4 e e

86.5� 2.8 e e

Table 3 Histomorphometrical examination of the nerve grafts in group I

Graft site in group I 1 2 3 4

Fibre diameter (m mm) 4.04� 0.71 3.75� 0.46 3.11� 0.31 3.51� 0.26Fibre number 10779� 3650 9020� 1802 10309� 2478 4093� 1941*% Nerve (%) 44.9� 6.6 40.8� 5.2 28.8� 5.7 22.5� 6.3

All sites are shown in Figure 2.The values are expressed as means� SD.* p< 0.01 as compared with the other sites.

End-to-side cross-face nerve graft in rats 1363

fascia graft,20 have been introduced. However, the mostpreferable and reliable solution for this problem is tomake the nerve gap to be reconstructed as long as possible.In small animal models, it is sometimes difficult, and theproblems are solved by using the contralateral side.17,21

In our model, the contralateral facial nerve brancheswere reconstructed using long sciatic nerve graft. The graftlength was 7 cm, and the reconstructed nerve defectseemed long enough to evaluate the clinical applicabilityof this method in humans. Some myelinated axons were ob-served outside of the graft at sites 1 and 2, but they werefew at sites 3 and 4 in group I (Figure 6). This occurred prob-ably because some aberrant reinnervation or axonalcontamination existed on the ipsilateral side, but this wasextremely less on the contralateral side. A similar phenom-enon was observed in group II; i.e., some myelinated axonswere observed outside the ulnar nerve grafts on the ipsilat-eral side, but they were few around the sciatic nerve graftson the contralateral side. These results indicate that regen-eration along and outside of the epineurium is likely tooccur in a short distance and less in a long one. Regardingthe nerve defects to be reconstructed in humans, our study

Figure 5 Transverse sections of the reconstructed branches andshowed more uniform regeneration in the four branches. In group IIthan on the ipsilateral side. In the assessment of the nerve graft inbecame less reinnervated. In group II, the short ulnar nerve grafts oatic nerve grafts on the contralateral side. The arrows and numberbuccal branch; mm, marginal mandibular branch) Toluidine blue st

showed that more accurate assessment could be done onthe contralateral side of our model; i.e. a more distantregion from the proximal trunk.

In the double retrograde neuronal tracing study, thechoice of retrograde tracers requires a great consideration.Each tracer should have clearly different extraction wave-length, color or different cellular distribution in the labeledcell. Also the stability (progressive fading of fluorescenceand transfer to adjacent non-labeled cells) and toxicity ofthe tracers should be considered especially for long-surviving models.22 In this study, TB and DY were used.The combination of TB or Fast Blue (FB; Sigma Chemical,St. Louis, MO, USA) with DY has been used by manygroups.19,23e27 Since TB and FB labels cytoplasm and DYlabels nucleus, double-labeled cells are easily detected asgreenish blue cytoplasm with bright nucleus under theultraviolet filter set. Although the labeling efficiency ofDY is lower than that of TB25 or FB26 and the ‘blockingeffect’ between these tracers in the subsequent applica-tion was shown,25,26 DY is ‘excited’ by TB once they arein the same neuronal cell body,27 which could help todetect the double-labeled cell.

the nerve grafts in group I (left) and group II (right). Group I, the branches on the contralateral side were less reinnervatedgroup I, as the examined site became more distally, the graftn the ipsilateral side were well-reinnervated than the long sci-s show the site where the nerve samples were harvested. (bu,ain, scale bar Z 10 mm.

Table 4 Histomorphometrical examination of the nerve grafts in group II

Graft site in group II 1 2 3 4

Fibre diameter (m mm) 4.32� 0.39 4.11� 0.23 3.13� 0.25 2.97� 0.27Fibre number 3462� 996 4284� 881 4510� 1062 4012� 1184% Nerve (%) 57.2� 6.3 50.2� 8.3 20.4� 4.2* 21.2� 4.3*

All sites are shown in Figure 2.The values are expressed as means� SD.* p< 0.01 as compared with sites 1 and 2.

1364 K. Matsuda et al.

We injected TB and DY into the buccal branch and themarginal mandibular branch respectively, in either theipsilateral or contralateral side. This is because we alreadyknew the low-rate retrograde double-labeling of motor andsensory neuron with the end-to-side nerve graft,10 whichrepresents the low-rate occurrence of double innervationin this method. Furthermore, few facial neurons may dou-ble-innervate bilateral facial muscles, considering the dif-ference in the distance from the proximal stump. Bothgroup I and group II showed good retrograde labeling, andthere was no significant difference between the two groupsin the total number of labeled neurons. Regarding signifi-cant difference of the number of double-labeled neuronsbetween two groups, it might be due to the axonal branch-ing or collateral sprouting in the end-to-side nerve graft,10

however the percentage of it is still low (i.e. around 3%both in this study and the previous one), indicating it isnot a main regeneration way.

As regards the surgical technique, our end-to-side nervegraft method is clearly different from ‘ordinary’ end-to-side neurorrhaphy method3,9,24,28e35 (recipient nervecoaptation to the side of intact donor nerve), to use theside of the nerve graft itself. In our method, regeneratedaxons come into the nerve graft via proximal end-to-endneurorrhaphy with the donor nerve and then they are dis-tributed to multiple recipient nerves via end-to-side neuro-rrhaphy site(s) and the most distal end-to-end one. In ourprevious study, we concluded that the main regenerationprocess of the recipient nerve which is attached to theside of the nerve graft is not by axonal branching or collat-eral sprouting but by terminal axonal growth from the do-nor nerve.10 Thus the regeneration process of this method

Figure 6 Transverse sections of the end-to-side nerve graft in grsite 1 (left), but they were few in site 4 (right). Toluidine blue sta

seems to be different from that of ‘ordinary’ end-to-sideneurorrhaphy3,9,24,28e34 that is used to induce collateralsprouting or axonal branching from the intact donor nerve.Several authors have confirmed that the efficiency of theend-to-side neurorrhaphy increases with the creation ofepineurial window to the donor nerve at the coaptationsite than the no-window group,24,30,31 and with perineurialwindow28,29,32 or partial neurectomy,28,33,36 it results inbetter regeneration than epineurial window group. Theseresults indicate that the regeneration of the recipientnerve is likely to be affected by the amount of damagedone to the donor nerve. Thus better regeneration seemsto be achieved by terminal axonal growth from the injuredsite of the donor nerve, not by ‘true’ collateral sproutingof the donor axons.37 Even without creating any windowto the donor nerve, regeneration to the recipient nerveis confirmed experimentally38 and the regenerating axonsseems to be able to perforate the epineurium,31 it seemspractical to create the window in the coaptation site forbetter result in clinical applications. Regarding our model,whether the efficiency of the end-to-side neurorrhaphycould be changed by the depth of the window(s) createdat the coaptation site(s) of the nerve graft is still unclear,however it might have some impact to the axonal distribu-tion from the donor nerve to the multiple recipient nerves.For more proper clinical application, further study is neededto elucidate the factors which regulate the axonal distribu-tion to multiple recipient nerves in the end-to-side nervegraft.

Another problem is the misdirected or deviant reinner-vation which should not be ignored considering the func-tional recovery.14,39 As the retrograde tracing study of the

oup I. Many myelinated axons outside of the graft were seen inin, scale bar Z 50 mm.

End-to-side cross-face nerve graft in rats 1365

facial nucleus showed, the misdirected reinnervation wasconfirmed by the lack of the mytopic organizations intothe subnuclei. Once the recipient nerve and its suppliedmuscle is reinnervated by the antagonistic nerve, it resultsin a poor functional recovery.40 A limitation of this end-to-side nerve graft method is the impossibility of eliminat-ing misdirected regeneration. Therefore, this methodseems to be suitable for facial nerve or sensory nervereconstruction rather than motor nerve reconstruction inthe limbs since facial muscles are less antagonistic. Inperipheral nerve reconstruction using the nerve graft(s),the misdirected reinnervation is difficult to regulate consid-ering that even on using multiple cable grafts with refinedmicrosurgical technique, it cannot be avoided com-pletely.41,42 Especially in the facial nerve, it could appearas a synkinesia even after recovery from non-mechanicalinjury to the facial nerve, e.g., Bell’s palsy.43 The patho-genesis of synkinesia is still unknown, but it has been attrib-uted to not only the misdirected regenerating axons butalso to changes in a more proximal region, e.g., hyperexcit-ability of facial nucleus and/or brain-stem reflexes44e47 andinterneuronal signalling.48 As misdirected reinnervationcould not be completely avoided by any method and itcould be treated with surgical measures49 or botulinumtoxin A50; it is practical to increase the number of regener-ating axons from the proximal stump into the nerve graftfor more neural input and better facial reinnervation. Inthis regard, our method has an advantage to improve theefficiency by simple end-to-end neurorrhaphy in the proxi-mal coaptation site. Our histomorphometrical examinationshowed that even in the most distal branch (i.e., the con-tralateral marginal mandibular branch, which is the fourthbranch from the proximal trunk), better reinnervation wasnoticed in group I than in group II. In addition, more uni-form regeneration occurred in the four branches in groupI rather than in group II, and this may be beneficial formultiple branch reconstruction in facial nerve. The resultsof the present study are also consistent with our previousreports,4,10 showing the effectiveness of the end-to-sidenerve graft. Especially, it revealed the availability of theside of the nerve graft itself as a nerve coaptation site in

Figure 7 An intraoperative photograph of the clinical applicationmatic diagram (right). The end-to-side nerve graft was placed on thdible. (t, temporal branch; z, zygomatic branch; bu, buccal branc

the clinical practice, and it is technically easy and less graftis required for multiple branch reconstruction. The fascicu-lar graft delivered from the same nerve graft51 might bevaluable to reduce the graft requirement, however, evenon using a single fascicular graft, two or more recipientnerves could be reconstructed with this method. In ourmodel, equivalent or more favorable reinnervation wasobtained using only about one-third of the nerve graft,and as the number of the branches to be reconstructed in-creases, this merit should be emphasized. Moreover, it willhave a great value for the recent facial nerve reconstruc-tion concept, connecting multiple neural sources withnerve grafts52,53 and inducing the ‘axonal supercharging’effect54 to the facial muscles. An additional merit inclinical practice is the site where the nerve graft is placed.After wide resection of malignant parotid tumor, the mandi-ble is often exposed. By the conventional cable graftmethod, the nerve grafts are often placed along the shortestcourse, partly on the exposed mandible, which is a poorly-vascularized bed for the grafts. With our method, thegraft could be placed on the surrounding well-vascularizedsoft tissue, diverting from the exposed mandible (Figure 7).Placement of the nerve graft on the well-vascularized softtissue could be advantageous for the graft survival55,56

that induces good functional recovery of the facial muscles.In this study, we presented the effectiveness of end-to-

side nerve graft method using the new rat cross-face nervegraft model. Many merits of this end-to-side nerve graftmethod were confirmed in the four branch reconstructionmodel with long sciatic nerve graft. This method hasseveral advantages: (1) the technical ease and certaintyof proximal neurorrhaphy can increase the reinnervationefficiency, (2) the minimum graft requirement reduces thedonor site morbidity, and (3) the nerve graft can be placedon the well-vascularized soft tissue in clinical cases offacial nerve reconstruction. With the availability of the sideof the nerve graft itself, we believe that end-to-side nervegraft method can be an effective alternative in facial nervereconstruction and be of great value in various kinds ofperipheral nerve surgery, especially for multiple branchreconstruction.

in a patient with malignant parotid tumor (left) and its sche-e well-vascularized soft tissue, diverting from the exposed man-h; mm, marginal mandibular branch).

1366 K. Matsuda et al.

Acknowledgment

The authors thank Prof. Masaya Tohyama, Department ofAnatomy and Neuroscience, Graduate School of Medicine,Osaka University, for his support in this study.

This work was supported by a research Grant from theGeneral Insurance Association of Japan and the ResearchGrant from the Japanese Ministry of Education, Science,Sports and Culture (16591793, 16791089, and 18791320).

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