Promotion of Nerve Regeneration in Peripheral Nerve by Short-CourseFK506 after End-to-Side Neurorrhaphy
Bin Chen, M.D., Ph.D.,* Youxin Song, M.D., Ph.D.,† and Zhongjun Liu, M.D.*,1
*Department of Orthopedics, Peking University Third Hospital, Beijing, China; †Department of Trauma and Orthopedics, People’sHospital of Peking University, Beijing, China
Submitted for publication December 5, 2007
doi:10.1016/j.jss.2008.03.032
Background/Aims. To discuss the feasibility of pe-ripheral nerve injury treated by end-to-side neuror-rhaphy in clinic and to evaluate the effect of short-course FK506 on promoting nerve regeneration afterend-to-side neurorrhaphy.
Methods. Thirty adult male Sprague Dawley rats wererandomly divided into 3 groups: Group A, 10 rats receivedend-to-end anastomosis; Group B, 10 rats received end-to-side neurorrhaphy; Group C, 10 rats received thesame operation as Group B. After operation, rats inGroups B and C received muscle injection with salinewater (1 mg/kg · d�1) and FK506 (1 mg/kg · d�1), respec-tively, both for 4 wk. Histological and morphologicalexaminations were performed 12 wk after the operation.In the 2nd, 4th, 6th, 8th, and 12th wk after operation,function recovery analysis was performed.
Results. The results of histological and immuno-chemistry study (the total number of Schwann cellsand the axon numbers at the distal stump of the per-oneal nerve, wet weight of extensor digitorum longusmuscle) suggested that there were significant differ-ences between Group B (saline water group) andGroup C (FK506 group) (P < 0.05), also between GroupA (end-to-end group) and Group C (P < 0.05). Therewere statistically significant differences in functionrecovery (peroneal functional index and sciatic func-tional index) between Groups B and C (P < 0.05) andalso between Groups A and C (P < 0.05).
It is generally acknowledged that end-to-end neuror-rhaphy is the most desirable technique for the repair ofsectioned nerves. But in cases of long segmental nervedefects, where only the distal segment is available forrepair and well-established conventional nerve graft-ing procedures cannot be done or have not workedsatisfactorily, the possibility of end-to-end repair isabsent. Under these conditions, coaptation of the prox-imal end of the distal segment to the lateral face of ahealthy adjacent nerve may provide a viable means ofreinnervation, with the advantage that it does not in-flict any additional damage or sacrifice any donor nerve[1–4]. However, there have been many experimentaland clinical studies with conflicting results. Some au-thors suggested end-to-side coaptation as an alterna-tive method in nerve repair, whereas others reportedlimited motor and sensory recovery [5–8].
FK506, first used in organ transplantation as an im-munosuppressive drug [9], has been characterized bypromoting neurite outgrowth in vitro and enhances neu-roregeneration in peripheral nerve injury. The purpose ofthis study was to investigate end-to-side repair in thelower extremity with special emphasis on functional re-covery, source, type, and extent of regenerating fibersfrom the donor nerve and the effect of FK506 on func-tional recovery after terminolateral neurorrhaphy.
MATERIALS AND METHODS
Surgical Procedure
The use of animals was approved by the Animal Care and UseCommittee of Beijing University.
Thirty male Sprague Dawley rats weighing 200–225 g were ran-domly divided into 3 groups. Shortly before the operation, they wereinstructed to walk on the walking track, and normal preoperative
hind paw prints were then obtained, identified, and stored (walking
304 JOURNAL OF SURGICAL RESEARCH: VOL. 152, NO. 2, APRIL 2009
analysis described below). After that, the rats were initially anes-thetized with an intraperitoneal injection of pentobarbital sodium(30 mg/kg), then shaved and washed with antiseptic solution beforepositioning for surgery. After a longitudinal incision was made on themedial side of the right lower limb extending from midthigh tomidcalf, the sciatic, tibial, and peroneal nerves were exposed andisolated. The peroneal nerve was carefully dissected under magnifi-cation with a surgical microscope (16�), then sharply transectedapproximately 5 mm distal to the bifurcation of the sciatic nerve.
In Group A (10 rats), transected peroneal nerves were reconnectedby conventional end-to-end epineurial technique with 4 stitches of10-0 nylon.
In Groups B and C (10 rats in each), a 1-mm-wide segment of theepineurium was resected at the prepared site of nerve coaptation ofthe tibial nerve. The distal stump of the common peroneal nerve wassutured into the tibial nerve trunk through the window with 3 to 4stitches of 10-0 nylon suture as end-to-side neurorrhaphy. The proximalstump of peroneal nerve was carefully buried into the adjacent muscleand fixed with 8-0 nylon sutures to prevent the occurrence of sponta-neous regeneration from the proximal stump to the distal stump.
Following wound closure with 4-0 silk suture, all of the above ratswere placed in cages and handled in compliance with the Guide forthe Care and Use of Laboratory Animals.
After operation, rats in Group B received muscle injection withsaline water (1 mg/kg · d�1), and Group C received muscle injectionwith FK506 (tacrolimus, 1 mg/kg · d�1; Fujisawa Pharmaceuticals,Osaka, Japan), both for 4 wk. FK506 was dissolved in saline and keptin a refrigerator at 4°C. The general condition of the animals wasobserved daily for 12 wk after the operation.
Footprint Recording and Analysis
At 2, 4, 6, 8, and 12 wk after the operation, nerve function recoverywas assessed in Groups A, B, and C by using the sciatic nerve
FIG. 1. Walking track of footprints (A, Group A; B, Group B; C,Group C). The gaits of the 3 groups showed flexion contracture of thepaws (“drop foot”) and adduction of the toes. The footprints of thosewere narrow compared with normal ones. (Color version of figure isavailable online.)
* In the 12th wk, there was a significant difference between Group
order of A�C�B.
functional index (SFI), tibial functional index (TFI), and peronealfunctional index (PFI) [10–12]. The procedure was repeated when anunsatisfactory result was obtained. Walking tracks for the modelrats were 8.2 � 42 cm cardboard with walls and were darkened at 1end. Paper was put at the bottom of the track to act as film, and themodel rat’s hind limb was painted with black ink and allowed to walkdown the track. On each piece of paper, several prints of the feetcould be seen. For the footprint, we measured its length (PL), thedistance from the 1st to the 5th toes (TS), and the distance from the2nd to the 4th toes (IT). These measurements were made for eachrat’s walking track and recorded as experimental (prefix E) or nor-mal (prefix N). The data (NPL EPL NTS ETS NIT EIT) were fed intothe computer. The following formulas were derived by Bain et al.,who calculated the SFI, PFI, and TFI as follows:
Twelve weeks after operation, animals were perfused through theaorta with phosphate-buffered saline (PBS; pH 7.6) followed by 300–500 mL of fixative composed of 4% paraformaldehyde in PBS (pH 7.4)after an overdose of pentobarbital anesthesia (120 mg/kg). Wetweight of bilateral extensor digitorum longus muscle was recordedfor each group by harvesting the muscle without the tendon, gentlyblotting the surface with absorbent paper to remove any blood orserum, and promptly weighing each muscle. The results were ex-pressed as a percentage of that of the normal contralateral muscle.
Histological and Morphometric Studies
After musle samples were obtained, the sciatic nerves includingtibial and peroneal nerves of all groups were entirely removedthrough a posterolateral approach to the thigh. The peronealnerve was separated from the sciatic trunk. A 10-mm-long seg-ment was obtained from the peroneal nerve at the site 5 mm distalto the distal end-to-end repair site in the specimen of Group A. InGroups B and C, the nerve tissue sample of the 10-mm-longsegment was taken at the site 5 mm distal to the distal end-to-sidecoaptation site.
Nerve tissues harvested were postfixed in 4% glutaraldehyde in0.1 M phosphate buffer at pH 7.4 for 12 h at room temperature. Afterthat, each sample was immersed in graded sucrose solution (from15% to 30% in 0.1 M phosphate buffer) for cryoprotection. A series of7-�m-thick transected serial sections were cut on a cryostat mic-
and C (P � 0.05), also between Groups A and C (P � 0.05), with an
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305CHEN, SONG, AND LIU: FK506 AND E-to-S NEURORRHAPHY
rotome (Frigocut; Leica, Bensheim, Germany) at �20°C. All of thesections were divided into 2 parts for immunochemistry and hema-toxylin and eosin stain at the same time.
Steps of Immunochemistry Stain
Sections took polyclonal anti-s100 (Chemicon, Temecula, CA) andmonoclonal anti-neurofilament 200 (Sigma, St. Louis, MO) as theprimary antibody. Second antibody used in order is 1:200 FITC-conjugated rabbit anti-rat IgG (Sigma) and 1:200 CY3-conjugatedmouse anti-rat IgG (Sigma). The sections were fixed with freezingacetone for 20 min, then washed 3 times for 5 min each with 0.01 MPBS containing 0.3% Triton X-100. After that, they were incubatedin 10% normal goat serum for 1 h at room temperature and incu-bated in primary antibody (1:200) in 0.01 M PBS over 8 h in humiditychamber. After washing with PBS and 0.3% Triton X-100 3 times,the sections were incubated in second antibody in PBS for 1 h atroom temperature. After the same washing procedure, sections werecoverslipped. Then, the density and size of regenerative axons andthe total number of Schwann cells and myelinated axons at the distalstump of the peroneal nerves were qualitatively and quantitativelyevaluated under a light microscope equipped with a video cameralinked to a microcomputer, respectively.
The muscle tissues were fixed in 10% buffered formalin andstained with hematoxylin and eosin for light microscopy.
Statistical Analysis
The rats were randomized. The measurements analyzed the ef-fects of treatment. All parameters were expressed as means � SD.Possible differences between the groups were evaluated using one-way analysis of variance. Statistical significance was set at P � 0.05.
RESULTS
Gait Analysis
No animals in the 3 groups showed any sign of in-fection and foot ulceration at any time throughout theexperiment.
FIG. 2. PFI behavior according to grouping, which was similar tothat of SFI. In the 12th wk, the function recovery of Group C wassignificantly better than Group B (P � 0.05). There was also signif-icant difference between Group A and Group C (P � 0.05). (Colorversion of figure is available online.)
* In the 12th wk, there was a significant difference between Group
order of A�C�B.
Normal gait was recorded as the hind paw toes fullyspread in each group before operation. While morbidone showed flexion contracture of the paw (“drop foot”)and adduction of the toes, after the common peronealnerve was injured (Fig. 1). The end scores of PFI valuesat wk 12 were �40.61 � 7.71 in Group A (end-to-endgroup), �56.65 � 5.10 in Group B (saline water group),and �49.26 � 8.84 in Group C (FK506 group), respec-tively. Statistically, the difference between Groups Band C was significant (P � 0.05), and the differencebetween Group A and Group C was also significant (P� 0.05) (Table 1; Fig. 2). The SFI values were the samecondition within the 3 groups (Table 2; Fig. 3).
The TFI scores determined in footprints at wk 12were �29.94 � 2.30, �29.24 � 2.30, and �30.66 �3.98, respectively, for Groups A, B, and C. There wereno significant differences in all of the groups (P � 0.05)(Table 3; Fig. 4).
Wet Weight of Extensor Digitorum Longus Muscle
In Groups A, B, and C, muscle atrophy was obviouson the experimental side. At wk 12, muscle wet wasabout 64.17 � 7.91% of the normal side in Group A(end-to-end group), 39.33 � 9.67% in Group B (salinewater group), and 53.33 � 5.96% in Group C (FK506group). Group C exhibited significantly greater muscleweight than Group B (P � 0.05). There was also asignificant difference between Group A and Group C(P � 0.05; Table 4; Figs. 5–9).
and C (P � 0.05), also between Groups A and C (P � 0.05), with an
FIG. 3. SFI behavior according to grouping. The SFI progres-sively improved, evolving from a moderate to a slight dysfunction,worst in Group B. In the 12th wk, there was significant differencebetween Groups A and C (P � 0.05). The function recovery of GroupC was significantly better than Group B (P � 0.05). (Color version offigure is available online.)
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Histological and Morphometric Studies
Light microscopy of the distal end of the peronealnerves revealed Wallerian degeneration, axonal at-rophy, and regeneration in Groups A, B, and C in the12th wk. The qualitative evaluation of the lympho-cytic infiltration on hematoxylin and eosin-stainedsections of the peroneal nerves revealed differencesbetween Groups A, B, and C.
From the appearance of the thickness of myelinsheath, Group A was similar to Group C, but superior toGroup B. The quantitative analysis of myelinated axonsrevealed significant difference between Groups B and C(P � 0.05), but not between Groups A and C (P � 0.05;Table 5; Figs. 6 and 8). For the total number of Schwanncells at the distal end of peroneal nerves, there weresignificant differences between Groups A and B (P �0.05), also between Groups A and C (P � 0.05), with anorder of C�A�B (Table 6; Figs. 7 and 8).
In Groups A, B, and C, the appearance of the mic-rotome section of the extensor digitorum longus mus-cles revealed varying degree of muscle degeneration,where polymorphic fibers and connective tissue in-creased, instead of normal muscle fibers. A lesser de-gree of these changes was found in Groups A and C,compared with Group B (Fig. 9).
DISCUSSION
The present study, which attached peroneal nervesto intact tibial nerves, reached three main conclusions.First, end-to-side nerve repair can be used for nerve
* In the 12th wk, there were no significant differences between th
FIG. 4. TFI behavior according to grouping. The TFI values of the3 groups did not change significantly with time. At any time point, therewas no significant difference in Groups A, B, and C (P � 0.05). (Color
version of figure is available online.)
repair in the lower extremity of the rat. Second, whenusing functional recovery to assess regeneration, therewas statistically significant difference between GroupB (saline group) and Group C (FK506 group), whichrevealed that FK506 facilitated the nerve repair afterend-to-side neurorrhaphy. Finally, there was no histo-logical evidence of significant injury to the intact tibialnerve after this kind of neurorrhaphy. Taken together,the results of this study suggest that end-to-side neu-rorrhaphy technique combined with FK506 may offer apotential alternative in the repair of nerve gap defects.
In this study, because all of the proximal stumps ofthe peroneal nerves (end-to-side groups) were well bur-ied in situ into adjacent muscles during operations, therisk that regenerating fibers could bridge the gap andreach the distal stump sutured directly onto the lateralaspect of the tibial nerve was minimized.
Analysis of Functional Recovery
The clinically relevant outcome is end organ func-tional recovery, which is the ultimate test of nerveregeneration [13]. Functional evaluation showed thatthe PFI improved in both Groups B and C, but therewas significant difference in PFI figures between the 2groups at the final evaluation 12 wk after the operation(�56.65 and �49.26, respectively), although there wasalso significant difference between Groups C and A(�49.26 and �40.61, respectively). PFI method is justsuitable for evaluation of a complete lesion of the per-oneal nerve which produces a short and narrow foot-print due to the lack of dorsal flexion of the ankle andextension of the toes [14]. There was no significantdifference in TFI figures among all groups, showingthat the end-to-side repair might not have impairedfunction of the tibial nerve.
* Indicates value significantly different from Group C (P � 0.05).
307CHEN, SONG, AND LIU: FK506 AND E-to-S NEURORRHAPHY
The statistical difference of the extensor digitorumlongus muscle weights between Groups B and C wassignificant, showing consistence with functional recov-ery. The relative loss of digitorum longus muscleweight has been shown to closely reflect the degree ofdenervation [15].
Although the design of the extensor digitorum lon-gus muscle histological qualitative analysis in thisstudy was not sensitive enough to detect a correlationbetween the significant differences in wet muscleweights and the gross appearance of histological sec-tions, it confirmed the functional and morphologicalevidence of regeneration. The muscle sections of allgroups displayed signs of denervation atrophy, show-ing small angulated fibers with increased fibrosis. Theextent of denervation in Group C (FK506 group) wascomparable to that in Group A (end-to-end group), andwas more serious in Group B (saline group).
From the results of this study, the degree of axonalsprouting did not correlate with functional recovery. Apossible explanation is differential sprouting of sen-sory and motor nerves with increased sprouting of sen-sory nerves influencing the absolute axon number. Al-ternatively, motor end plate resorption or anotherdownstream effect could be limiting reinnervation [13].
FIG. 5. In the 12th wk, Group C exhibited significantly greaterwet weight of the extensor digirorum longus muscles than Group B(P � 0.05). There was also a significant difference between Group Aand Group C (P � 0.05). (Color version of figure is available online.)
FIG. 6. In the 12th wk, the total number of myelinated axons ofGroup A was similar to that of Group C (P � 0.05). There was asignificant difference between Group B and Group C (P � 0.05).
(Color version of figure is available online.)
Mechanism of FK506
FK506 (tacrolimus), a neutral macrolide isolatedfrom Streptomyces tsukubaensis [16], is a new FDA-approved immunosuppressant used for prevention ofallograft rejection in, for example, liver and kidneytransplantations. However, at present, scholars havefound that FK506 accelerates nerve regeneration invivo and increases neurite elongation in vitro. FK506 isinactive by itself and requires binding to an FK506binding protein-12 (FKBP-12), or immunophilin, foractivation. In this regard, FK506 is analogous to cyclo-sporin A, which must bind to its immunophilin (cyclo-philin A) to display activity. This FK506–FKBP com-plex inhibits the activity of the serine/threonineprotein phosphatase 2B (calcineurin), the basis for theimmunosuppressant action of FK506. The discoverythat immunophilins are also present in the nervoussystem introduces a new level of complexity in theregulation of neuronal function [17]. Immunophilins are agroup of proteins that serve as receptors for the immuno-suppressant drugs cyclosporin A and FK506. The immu-nophilin designated FK-506 binding protein-12 (FKBP-12) is concentrated more than 10 times higher in thebrain than in immune tissues. Sciatic nerve crushmarkedly augments expression of FKBP-12 mRNA inlumbar motor neurons and dorsal root ganglia neuro-nal cells. Increased FKBP-12 expression appearslinked to regeneration [18].
An experiment by Grand et al. indicated that thecombination of FK506 treatment with cold preserva-tion of nerve allografts resulted in functional and his-tomorphometric recovery superior to that with nerveisograft alone[19]. Daily administration of low-doseFK506 enhances peripheral nerve recovery after tran-section injury [20]. Treatment with FK506 improvedthe rate of functional recovery after nerve resectionand autograft repair [21]. FK506 accelerates recoveryfrom tibial nerve injury [22]. Systemic administrationof low-dose FK506 facilitates peripheral nerve recovery
FIG. 7. The total number of Schwann cells. In the 12th wk, therewas a significant difference between Group A and Group B(P � 0.05), also between Group A and Group C (P � 0.05), with anorder of C�A�B. (Color version of figure is available online.)
and regeneration after nerve grafting [23].
is
308 JOURNAL OF SURGICAL RESEARCH: VOL. 152, NO. 2, APRIL 2009
The effect of FK506 on nerve growth is significantlygreater than that of CsA [24]. Brenner et al. [25] foundthat, when combined with anti-CD40L mAb, low-doseFK506 enhances nerve regeneration without disruptingimmune unresponsiveness. Ellis et al. [26] thought, fallbelow the threshold for immunosuppression, FK506could potentially enhance nerve regeneration while min-imizing toxicity. The striking potency of these agents(nonimmunosuppressive analogues of the immunosup-pressive drugs, FK506), their bioavailability, and the dis-sociation of neurotrophic from immunosuppressant ac-tions argue for their therapeutic relevance in thetreatment of neurodegenerative diseases [27].
The functional results of Group C were significantlybetter than those of Group B (P � 0.05), which wasrelated to the effect of FK506 to potentially limit theextent of motor endplate loss and muscle atrophy. Itacted directly on the neuron (as opposed to the dener-vated distal nerve stump) to accelerate and promoteaxonal regeneration of neurons [28] and significantlyincreased glial cell line-derived neurotrophic factorcontent in the substantia nigra at the same time. Inaddition, FK506 increased striatal brain-derived neu-rotrophic factor content significantly [29]. All of theabove finally limited permanent functional loss.
Compared with Group B (saline group) in this study,the total number of myelinated axons of Group C(FK506 group) was significantly higher (P � 0.05),similar to that of Group A (end-to-end group) (P �0.05). This suggested 2 conclusions: (1) In addition toan effect on rate of axonal elongation, FK506 canimprove functional recovery of denervated targets byincreasing both regenerative and collateral reinner-vation [30]. (2) When 1 neuron regenerates in a regen-
FIG. 8. Light microscopy immunochemistry sections of distal stuatrophy, and regeneration in the 3 groups in the 12th wk. From theto Group C (C), but superior to Group B (B). (Color version of figure
FIG. 9. Histology studies of the extensor digitorum longus mumuscle degenervation, where polymorphic fibers and connective tiss
changes was found in Group A (A) and Group C (C), compared with Gr
erative distal stump, it can maintain more than 1 col-lateral [31]. FK506 can improve the number of thecollaterals.
Despite the results of myelinated axons, the outcomeof the functional recovery in Group C could not com-pare with that in Group A. We hypothesized 3 points:(1) The contacting area of nerve to nerve of end-to-sideneurorrhaphy is less than that of end-to-end repair.For the former, the nerve fibers can be stimulated tosprout only near the site of the end-to-side anastomo-sis. Yan et al. [32] used a new end-to-side repair tech-nique, called helicoids neurorrhaphy, which markedlyincreased the area from which axons can sprout intothe recipient nerve. At 11 mo after surgery, musclevolume, tetanic force, and moist weight of EDL muscleswere significantly higher in the helicoids groups. (2)Despite the effect of “nerve collaterals amplification,”the number is limited so that 1 axon can maintainfinally in a regenerated peripheral nerve [31]. Manysprouts that did not arrive at effectors became invalidfibers. (3) Of all of the collateral sprouts, the proportionof sensory axons, which later would be invalid fibers, ishigher than that of motor ones [33].
End-to-Side Mechanism
At present, the mechanism of nerve regeneration afterend-to-side neurorrhaphy is still controversial. Viterbo etal. [3] concluded that, following nerve damage, Walleriandegeneration occurred distal to the injured site. Thenerve fiber is degenerated. Schwann cells are activatedand the expression of nerve growth factor is increased.The donor nerve sprouts from and grows into the distalstump of recipient nerve under the induction of factors
s of peroneal nerves (400�) revealed Wallerian degeneration, axonalearance of the thickness of myelin sheath, Group A (A) was similaravailable online.)
s (hematoxylin and eosin stain, 200�) revealed varying degree ofincreased, instead of normal muscle fibers. A lesser degree of these
mpapp
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oup B (B). (Color version of figure is available online.)
309CHEN, SONG, AND LIU: FK506 AND E-to-S NEURORRHAPHY
mentioned above, thereby to regain the control of thereceptor and make it neurotized [14].
However, Rovak et al. [34] believe that the normalnerve cannot sprout; the neurotization of the nerve re-ceptor is because the donor nerve is somehow damagedwhen the end-to-side neurorrhaphy is performed; there-fore, the donor nerve generates some axon during its ownrepair process, which grow into the recipient nerve andcause the neurotization of the nerve receptor muscle tosome extent.
Lundborg et al. [35] proved that, after performingthe end-to-side neurorrhaphy of the distal stump ofperoneal nerve to tibial nerve, electronic stimulationon the proximal stump of tibial nerve can cause thetibialis anterior muscle to constrict, which indicates thatthe regenerated nerve comes from tibial nerve. The resultof immunochemistry testing also supports this conclu-sion. They believe that the mechanism of common per-oneal nerve regeneration is not that the tibial nervestem is injured, then generates lateral sprouts whileanastomosing the nerves. If that were the case, thenerve fibers denaturalized could be observed atthe distal stump of tibial nerve. Following an end-to-side neurorrhaphy of the distal stump of peronealnerve to tibial nerve, the muscle strength of the tibialisanterior muscle controlled by the common peronealnerve has recovered to 60% of normal muscle strength.Under this circumstance, the nerve fibers denatural-ized inside the tibial nerve stem should be many.
Our experiment has also proved that the number ofnerve fibers at distal and proximal stump of tibialnerve stem, the area of nerve trunk, and the nerve fiberdensity have no significant difference from the normalnerve stem. Moreover, the TFI score also has no sig-nificant difference within Groups A, B, and C. Weconclude therefore that, although it is impossible to notinjure the normal tibial nerve at the end-to-side nerveneurorrhaphy in that the tibial nerve grows into thecommon peroneal nerve, the lateral sprout coming intobeing should be the main mechanism.
Some other scholars believe that, following an end-to-side nerve neurorrhaphy, morphological nerve re-generation can be proved inside the distal stump ofthe damaged nerve. However, this mechanism is notcaused by the lateral sprouting from the donor nerve.When the recipient nerve is sectioned, there is nerve
TABLE 5
Total Number of Myelinated Axons
Group Values (mean � SD)
A 410.60 � 74.88B 201.00 � 58.75*C 388.80 � 63.48
* Indicates value significantly different from Group C (P � 0.05).
degeneration and retraction at the proximal stump of
its axons. Furthermore, during the process of the re-generation and growth of the proximal stump of axons,some nerve fibers grow into the effectors of recipientnerve through the passage after the end-to-side nerveneurorrhaphy, which makes them reneurotized [36].
In a study by Zhang et al. [37], true blue (acted as acytoplasmic marker) and diamidino yellow (acted as anuclear marker) were injected respectively into tibialisanterior and gastrocnemius muscles of rats which pero-neal nerves were sectioned and sutured to tibial nervesby end-to-side neurorrhaphy. Double-labeling neuronswere observed in L3–L6 serial sections of the lumbarspinal cord and dorsal root ganglia, demonstrating that 1parent nerve fiber can emanate another axon by collat-eral sprouting following end-to-side neurorrhaphy.
We believe that tibial and common peroneal nervefibers spread inside the sciatic nerve and are packed byperineurium, respectively; during the process of the re-traction and regeneration of the proximal stump of thecommon peroneal nerve fibers, they can hardly spreadinto the tibial nerve breaking their own perineurium.Therefore, the 3rd mechanism is impossible to occur andthe other two coexist to make some contribution to thecommon peroneal nerve functioning recovery. What per-centage the lateral sprouting should be in the contribu-tion needs to be investigated by our additional experi-ments, which makes this study less sufficient. Theexperimental results with FK506 application are betterthan the group without application, which indicates thatit has the stimulating effect on the regeneration of theperipheral nerve axon. The results of FK506 applicationsuggest that this method has certain potential applyingvalue in the clinical practice and also proves the validityof end-to-side neurorrhaphy.
CONCLUSION
Although many questions regarding the end-to-sidenerve repair still remain unanswered, we concludethat the technique resulted in a reliable degree of mor-phological regeneration and functional recovery for theperoneal nerve, especially combined with FK506. Thistechnique has a potential for application in humans,with the advantage that it does not require a nervegraft to be performed for most of the situations that we
TABLE 6
The Total Number of Schwann Cells
Group Values (mean � SD)
A 412.60 � 62.28B 314.80 � 51.81*C 477.20 � 56.76*
* Indicates value significantly different from Group C (P � 0.05).
310 JOURNAL OF SURGICAL RESEARCH: VOL. 152, NO. 2, APRIL 2009
can imagine and, as far as we could detect, does notdown-grade the donor nerve function.
ACKNOWLEDGMENTS
We thank Professor Heping Cheng for providing a laser scanningconfocal microscope for use in this study.
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