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Research Report

The effect of autogenous vein grafts on nerve repair withsize discrepancy in rats: An electrophysiologicaland stereological analysis

Murat Acara, Ahmet Karacalara, Mustafa Ayyildizb, Bunyami Unalc, Sinan Canand,Erdal Agarb, Suleyman Kaplane,⁎aDepartment of Plastic, Reconstructive and Aesthetic Surgery, Ondokuz Mayis University School of Medicine, TR-55139 Samsun, TurkeybDepartment of Physiology, Ondokuz Mayis University School of Medicine, TR-55139 Samsun, TurkeycDepartment of Histology and Embryology, Ataturk University School of Medicine, TR-25100 Erzurum, TurkeydDepartment of Physiology, Başkent University School of Medicine, TR-06530 Ankara, TurkeyeDepartment of Histology and Embryology, Ondokuz Mayıs University School of Medicine, TR-55139 Samsun, Turkey

A R T I C L E I N F O

⁎ Corresponding author. Department of HistTurkey. Fax: +90 362 312 19 19x2265.

E-mail address: skaplan@omu.edu.tr (S. K

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.01.013

A B S T R A C T

Article history:Accepted 3 January 2008Available online 18 January 2008

Aside from anatomical repairs, the reestablishment of sensory and motor innervations forproper functional recovery is one of the fundamental objectives of reconstructive surgery.Theheterotopic transfer of autologous tissues is likely to result in a size discrepancy betweenthe donor and recipient nerves, which will have a negative influence on regeneration.Twenty Wistar albino female rats were used in a study that was divided into two maingroups: tibial–peroneal (TP) and peroneal–tibial repair (PT). Both types of nerves wereexposed on the hind legs with the nerves cut on the right side, while the proximal stump ofthe tibial nerve and distal stump of the peroneal nerve were sutured to each other. Thesegroups are also called end-to-end neurorrhaphy groups (EtoE). On the left side, the tibial andperoneal nerves were cut on the same level as on the right side. After the end-to-endepineural suturing of the nerve, the vein graft was slid over to the repair zone underirrigation. These are called the vein graft group (VG). All processes mentioned above werealso done for the PT group. On the 90th postoperative day, anesthetized animals were fixedprone on a board, with the nerves carefully dissected for electrophysiological recording.Stereological methods for an estimation of the total number of myelinated fiber, a meanaxonal cross-section area and the thickness of the myelin sheet were used. In TP and PTgroups, nerve conduction velocities were found to be higher within the VG group.Nevertheless; the difference was only significant in the PT group. In both TP and PTgroups, the increase in the axon number, axon area and myelin thickness were statisticallydifferent in favor of the vein graft sides. An appearance of vacuoles and degeneratedpertinaciousmaterialwithin themyelin sheath of EtoE sideswas seen. Ahistomorphologicalexamination of the sections proximal to, from, and distal to the repair zone over threemonths revealed less epineural scarring, a thinner epineurium,more regenerated axons and

Keywords:Tibial nervePeroneal nerveVein graftElectrophysiologyStereology

ology and Embryology, Ondokuz Mayıs University School of Medicine, TR-55139 Samsun,

aplan).

er B.V. All rights reserved.

Fig. 1 – A) Amicrograph belongs to a regeneasily dissected from the adjacent tissue,belongs to a regenerated nerve that wassince it has no adherences to the neighbo

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fewer inflammatory cells in groups where vein grafting was used, because the vein graftprovided additional mechanical and chemical support in the size discrepancy of the nerveregeneration.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The peripheral nervous system provides communication withthe central nervous system and the environment (Fawcett andKeynes, 1990; Galtrey et al., 2007). Data acquired from theexternal environment is transmitted to the brain via peripheralnerves, and following evaluation; appropriate responses aretransmitted to the respective muscles via peripheral motornerves. Any interruption to this vital cyclemaynegatively affectthe quality of life. Functional recovery after damage and repairto the peripheral nerve is not only dependant on the vitality andregeneration of the injured nerve, but also on the correct orient-ation of the motor, sensory and autonomous fibers to theirtarget organs (Fawcett andKeynes, 1990; Lundborg, 2003). So far,the optimal functional recovery following nerve damage isobtained by tension-free microsurgical coaptation of the cutends with fascicular alignment (Sunderland et al., 2004). Never-theless, due to the different quantities of fascicles, primaryepineural repairs of nerves with size discrepancy fail to achieveappropriate coaptation, thus precluding the desired outcome(Lundborg, 2003). We used the autologous vein graft (Kelleheret al., 2001; Meek and Coert, 2002) to prevent the sprouting ofexcessive nerve buds from the repair zone in the study. Basedupon recent developments in the field of reconstructive micro-surgery, the reestablishment of innervations of transferredtissues was included in the primary objectives, along with theclosure of the defect (Evans et al., 1994). In primary repair, thecorrect alignment of the Bungner bands cannot be achievedeven when themost meticulous microsurgical technique is putto use (Evans et al., 1991).

The contributionof autogenousveingraftingprovidesmech-anical and chemical support, thus aiding nerve regeneration(Wanget al., 1993;Xu et al., 1998; Zhang et al., 2002).On theotherhand, new biodegradable materials for providing mechanicaland/or chemical support for nerve regeneration has been rapid-ly introduced in recent years (Chen et al., 2000; Ciardelli andChiono, 2006; Lietz et al., 2006a,b; Marchesi et al., 2007). Guidetubes of various types –collagen, silicon or fibronectin– filled

erated nerve that was asince it hasmany of adhapplied to vein graft tecr tissue and neuroma,

with Schwann cells, stem cells, nerve growth factors, laminin, asolution either of acidic or basic fibroblast growth factor areattractive as an alternative therapy to nerve grafts (Chen et al.,2000; Yannas and Hill, 2004; Lietz et al., 2006a,b; Marchesi et al.,2007). To our knowledge, no study was found concerning nerverepair with size discrepancies. We employed this techniqueclinically on size-discrepant digital nerves and observed posi-tive results (Karacalar and Ozcan, 1999; Karacalar et al., 2000).Therefore, it was assumed that this technique would overcomesize discrepancy, aiding axonal regeneration.

Switching the proximal and distal cut ends of tibial and pero-neal nerves on both legs created twomain groups. Nerves on theright leg were repaired with the primary epineural sheet, whileautologous vein grafting was performed for nerves on the otherleg. To test our hypothesis,weused electrophysiological analysison nerve potentials to evaluate physiological recovery as well asstereological methods to estimate themorphometric features ofthemyelinated fiber to see any indicator ofmorphological repair.

2. Results

2.1. Gross and morphological evaluation

In tibial–peroneal end to end group (TP-EtoE) and peroneal–tibial end to end group (PT-EtoE), the repair zone was strictlyadhered to the surrounding tissues, and is also found to beexceedingly difficult to dissect. Another feature of thesegroups was numerous neuromas inhabiting the repair zones(Fig. 1A). At the left side, the dissections were easily made andthe vein graft was readily separable from the surroundingtissues. None of the subjects in these groups displayed neuro-ma formation (Fig. 1B).

2.2. Histopathological findings

The nuclei of muscle cells of the vein graft surrounding thenerve is seen (Fig. 2A). If the vein graft is absent around

pplied to end-to-end neurorrhaphy technique. It could not beerences to the neighbor tissue and neuroma. B) Amicrographhnique. It could be easily dissected from the adjacent tissue,vg, vein graft.

Fig. 2 – A) A vein graft covered the nerve is seen. Arrows show the nuclei of muscle cells of the vein wall. B) Sprout out of nervefibers and neuroma are seen in the control group (end to end neurorrhaphy group). C) A nerve fascicule that is covered byperineurium (arrowheads show this sheet). D) Many nerve fascicules that are covered by perineurium are seen. The flatten cellsof one perineural sheets that contribute to blood–brain barrier was shown (Arrows show the cells of this sheet). epn,epineurium, Cresyl violet staining. (For interpretation of the references to colour in this figure legend, the reader is referred tothe web version of this article.)

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the nerve that is observed in the control group, sprouting outof many nerve fibers and neuroma formation can be seen(Fig. 2B). A successful nerve regeneration following transectionwas observed. The flattened cells of the perineural sheetswhich contribute to theblood–brainbarrier are seen (Fig. 2C, D).In the EtoE groups, significant myelin sheet degenerationwas found (Fig. 3A). After transection, many vacuoles due tomyelin sheet degeneration were visible in the EtoE groups(Fig. 3B).

Fig. 3 – A) Pronounced myelin sheet degeneration is seen in the nin the large nerve fiber in comparison of the small nerve fiber. ArroB) Many of vacuoles in the myelin sheet are seen in the nerve fibmyelin degeneration such vacuolization is observed in the myela, axon, Cresyl violet staining. (For interpretation of the referencweb version of this article.)

2.3. Electrophysiological findings

Although themean nerve conduction velocity of the vein graftside (VG) was better than EtoE side, the difference was notsignificant in TP-VG group. In the peroneal–tibial group (PT),the conduction velocity of the VG side was higher, and thedifference was significant ( p<0.05) Fig. 4.

The amplitudes of the compound action potentials elicitedin EtoE and VG sides of TP groups were depicted in Fig. 5. A

erve belong to the control group. Myelin demolish is obviousws show the damagedmyelin and its debris in the axoplasm.ers belong to the control group. It is suggested that beforein sheet. Arrows show the vacuoles in the myelin sheet.es to colour in this figure legend, the reader is referred to the

Fig. 4 – Mean nerve conduction velocity of TP and PT groups. In the PT group, the conduction velocity of the VG side wassignificantly different from the EtoE. TP-EtoE, tibial–peroneal end to end neurorrhaphy group; TP-VG, tibial–peroneal vein graftgroup; PT-EtoE, peroneal–tibial end to end neurorrhaphy group; peroneal–tibial vein graft group, (Mean±SEM), *p<0.05.

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significant decrease incompound action potential amplitudeson VG side was found ( p<0.05). Amplitude values for EtoE andVG sides for PT groups were not significant.

2.4. Stereological evaluation findings

In TP group, myelinated fiber number of EtoE side in the pro-ximal tibial segment and distal peroneal segment were givenin Fig. 6. The difference was statistically significant in favor ofthe VG side ( p<0.05). In PT group, myelinated fiber number ofEtoE side in the proximal peroneal segment and in the distaltibial segment was given in Fig. 6. The differences were statis-tically significant in favor of the VG side ( p<0.05).

Cross-sectional axon areas (CSAA) of EtoE side in theproximal tibial segment and in the distal peroneal segment for

Fig. 5 – Mean compound action potential amplitude of TP and PTwas significantly lower from the EtoE. There are no significant dibetween the TP groups. TP-EtoE, tibial–peroneal end to end neurPT-EtoE, peroneal–tibial end to end neurorrhaphy group; perone

tibial–peroneal group and also for the VG side were given inFig. 7. Although this assessment shows a decreasing in axonalarea, but this was not raised to a significant level. CSAA of EtoEand VG sides for peroneal–tibial groups were seen Fig. 7. It wasfound that the increase of the axon areas trespassing therepair zone was significantly higher in VG side in comparisonof the EtoE side ( p<0.05).

Cross-sectional areas of myelin sheets (CSAM) for EtoE sidein the proximal tibial and in the distal peroneal segmentswereshown in Fig. 8. There was no significant difference in theCSAM of proximal stumps between EtoE and VG sides (Fig. 8).But a significant difference in CSAM of distal stump of nerveswas only seen in comparison of EtoE and VG sides ( p<0.05).

Myelin thickness (MT) for EtoE side in the proximal tibialand in the distal peroneal segments was shown in Fig. 9. There

groups. In the TP group, the potential amplitude of the VG sidefferences in terms of amplitude of compound action potentialorrhaphy group; TP-VG, tibial–peroneal vein graft group;al–tibial vein graft group, (Mean±SEM), *p<0.05.

Fig. 6 – Mean total myelinated axon number of TP and PT is seen. In distal part of TP and PT groups, the axon number of the VGgroups were significantly different from the EtoE groups. TP-EtoE, tibial–peroneal end to end neurorrhaphy group;TP-VG, tibial–peroneal vein graft group; PT-EtoE, peroneal–tibial end to end neurorrhaphy group; peroneal–tibial vein graftgroup, (Mean±SEM), *p<0.05.

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was no significant difference in the MT of proximal stumpsbetween EtoE and VG sides (Fig. 9). Similarly, no significantdifference was found in comparison of distal stump of EtoEand VG sides.

3. Discussion

Nerve autografts retain their rank as the most appropriateoption for repairing defects in a peripheral nerve (Smahel andJentsch, 1986; Tos et al., 2000; Keskin et al., 2004). Nevertheless,

Fig. 7 – Mean cross-section area of myelinated axon TP and PT imyelinated axon of the VG group was significantly different fromneurorrhaphy group; TP-VG, tibial–peroneal vein graft group; PTperoneal–tibial vein graft group, (Mean±SEM), *p<0.05.

the limited availability of expandable nerve grafts ultimatelylimits their use (Strauch et al., 1996; Lundborg, 2003). Nerveautograft types currently in use are truncal, cable, as well asinterfascicular and free vascularized types. Truncal and cablegrafts are not widely preferred, and indications for vascular-ized nerve grafts are vaguely established. Meanwhile, inter-fascicular nerve autografts are now the most preferred optionamong nerve grafts, and have gained the “Golden Standard”merit, though the functional results are still far from perfect.Researchers are currently focusing on discovering additionalmethods to facilitate axonal regeneration and orientation for

s seen. In distal part of PT groups, the cross-section area ofthe EtoE group. TP-EtoE, tibial–peroneal end to end

-EtoE, peroneal–tibial end to end neurorrhaphy group;

Fig. 8 – Mean cross-section area ofmyelin of TP and PT is seen. In distal part of PT groups, the cross-section area ofmyelin of theVG group was significantly different from the EtoE group. TP-EtoE, tibial–peroneal end to end neurorrhaphy group; TP-VG,tibial–peroneal vein graft group; PT-EtoE, peroneal–tibial end to end neurorrhaphy group; peroneal–tibial vein graft group,(Mean±SEM), *p<0.05.

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primary nerve repairs as well as nerve defect reconstructions(O'Sullivan et al., 1998). In this study, we aimed to minimizescar tissue formation at the nerve end, and functionaldeterioration by establishing an appropriate orientation ofdifferently quantified axons of size-discrepant nerve ends.Nerves repaired with appropriate anatomical orientation andmicrosurgical techniques yielded perfect histological, mor-phological and electrophysiological results.

At present, nerve autografting is considered the most suc-cessful method for nerve repair. On the other hand, differentkinds of biodegradablematerials that support peripheral nerveregeneration have been introduced (Battiston et al., 2005; Chenet al., 2000, 2005; Ciardelli andChiono, 2006; Lietz et al., 2006a,b;

Fig. 9 – Mean myelin thickness of TP and PT is seen. In distal pahigher than EtoE group but differences were not raised to a statineurorrhaphy group; TP-VG, tibial–peroneal vein graft group; PTperoneal–tibial vein graft group, (Mean±SEM).

Marchesi et al., 2007). These bio-engineered grafts, composedof collagenand certain synthetic biodegradable polymers, are apromising alternative to nerve autografting since they canincorporate all the newly developed strategies for nerveregeneration (Yannas and Hill, 2004; Ciardelli and Chiono,2006). In addition, tube fillings with suspensions of Schwanncells, a solution either of acidic or basic fibroblast growth factorshows a high-regenerative activity for the nerve (Yannas andHill, 2004). We know that wrapping collagen around an EtoEsite and tubes improves the orientation of the axons (Dahlinand Lundborg, 2001; Watanabe et al., 2001). It has been testedon the vein graft with silicone; collagen andMillipore wrappedaround the epineural neurorrhaphy zone, and reported that of

rt of TP and PT groups, myelin thickness of the VG group wasstically significant level. TP-EtoE, tibial–peroneal end to end-EtoE, peroneal–tibial end to end neurorrhaphy group;

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those materials, silicone yielded the best results (Chiu et al.,1982; ChiuandStrauch, 1990;Dubuissonet al., 1993;DahlinandLundborg, 2001; Karacaoğlu et al., 2001; Diaz et al., 2004).Looking at it closely, when silicone-unsheathed repairs werecompared with standard repaired controls, better electrophy-siological results were found on the trial side (Strauch et al.,1996; Lundborg, 2003).

Xu et al. examined the histological effects of wrapping avein graft around repaired peripheral nerves (Xu et al., 2000).For this purpose, they used three groups. In the first group, thenerve was isolated, irrigated and closed. In the second, venacava inferior allograft preserved in glutaraldehyde was used,and in the third, the repair site was protected with a contra-lateral femoral vein autograft (Xu et al., 2000). An examinationof sections proximal to, from, and distal to the repair zone overthe following three months revealed less epineural scarring,thinner epineurium, a higher number of regenerated axon andalso fewer inflammatory cells in the vein graft group. At thesame time, while the group inwhich glutaraldehyde preservedthe allograft displayed significant epineural scarring and neu-ral adhesions, no excessive inflammatory reactionwas seen inthe group in which autograft was used. Moreover, neuromaformation was absent in the autograft group.

The venous wall has a very high collagen content thatallows a suitable environment for axon regeneration. In anexperimental study with vein grafts, it was seen that on the30thday, the endothelial cells remained intact, and on the 90thday, while they couldn't define the endothelium, the mediaand smooth muscle cells were still present (Tang et al., 1993).Similarly, on the 90th postoperative day, our specimensrevealed intact media and smooth muscle cells, while theendothelial cells were no longer visible. Vein grafts appeared,directing the regenerating axons to protect the axons frominvading scar tissue and provide metabolic assistance to re-generation via trophic factors. While precluding scar invasion,the vein wall does not prevent blood vessels from reaching therepair zone. Blood vessels pierce through the vein wall forcontribution to nerve regeneration (Lundborg, 1975; Williamset al., 1983; Wang et al., 1993; Hudson et al., 1999). The use ofautogenous vein graft for overcoming size discrepancy pro-vided the basis for this study;while the vein graft also providedadditional mechanical and chemical support. To our knowl-edge, there is no study concerning the use of vein grafting forrepairing nerves with size discrepancies. A significant incre-ment of nerve fibers in the distal part of vein graft groups inboth TP-VG and PT-VG groups compared to their proximalstumps, suggests that covering the neurorrhaphy site with anautogenic sheet like the vein, enhances nerve regeneration.The possible reasons for the this kind of protection of nervefibers from degeneration have been explained in previousstudies (Tang et al., 1993;Wang et al., 1993; Hudson et al., 1999).

In the TP and PT groups, nerve transmission rates werefound to be higher in the VG group. However, the differencewas significant only in the PT group. Nerve transmission rateswere better in both VG groups, suggesting that the axons inthese groups had responded well to the chemotactic signalingof their target organs; thus resulting in an improved quality ofregeneration. The trial sides of TP and PT groups resulted inhigher axon passage ratios through the repair zone. Theseeffects may be attributed to various factors, including an

elaboration of trophic factors from the endothelial andsmooth muscle cells, prevention of leakage of trophic factors,prevention of scar tissue invasion, and prevention of thesprouting of outnumbering axons from the larger nerve stump(Chiu and Strauch, 1990; Geuna et al., 2000; Xu et al., 2000;Geuna et al., 2006). In both groups, the increase in axon areasand myelin thickness has been found statistically different infavor of the trial sides, suggesting an improved quality ofaxons, which trespassed into the repair zone in VG sides,along with better transmission rates of these axons. Theappearance of vacuoles and degenerated pertinacious mate-rial within the myelin sheath of the control side shows theextent of the axonal degeneration.

A decrease in the transmission rates, as seen in the controlsides, were consistent with the histomorphological findings.The nerveswith lower transmission rates also failed to displayproper axonal intensity and homogeneity distal to the repairzone. Moreover, a decrease in CSAA and myelin degenerationwas often encountered. This finding may also be attributed tothe axons' striving for the Schwann-cell conduit to the less-resistant surrounding tissues, and thus getting devoid oftrophic–taxic influence of the target organ. Appropriate signal-ing between the target organ and the regenerating axons isonly possible when appropriate signaling pathways are re-established. It may be concluded that such a pathway could beestablished via the autogenous vein graft, which was sup-ported by our electrophysiological and histomorphologicalfindings. In other words, it may be said that our observation,showing the exchange of proximal and distal nerve stumps(TP vs PT), doesnot lead to a significant variation in thenumberof regenerated nerve fibers and does not deserve particularmention. Previous studies have proposed that the number ofregenerated nerve fibers depend either on the size (i.e. fibernumber) of theproximalnerveor on the size of thedistal nerve.Results of the present study, in agreement with recent results(Lee et al., 2007), suggest that both these factors contribute todetermining the total number of regenerated axons.

4. Conclusions

Histomorphological examination of sections proximal to,from, and distal to the repair zone following three monthsrevealed less epineural scarring, thinner epineurium, moreregenerated axons and fewer inflammatory cells in the groupwhere vein graft was used. The appearance of vacuoles anddegenerated pertinacious material within the myelin sheathof the control group shows the extent of axonal degenerationin the control sides.

5. Experimental procedure

Twenty Wistar adult albino female rats, kept in individualcages under constant laboratory conditions, were used. Sur-gical operations were carried out under general anesthesia, by150 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Istan-bul, Turkey). Surgerical procedures under an operation micro-scope (16×, Zeiss, Oberkochen, Germany) were performed bythe same surgeon.

Fig. 10 – A) End-to-end neurorrhaphy between tibial and peroneal nerves. Some nerve fibers sprout out to peripherally due todifference of nerves diameter (arrows). Tibial nerve is seen on the left side, peroneal seen on the right side of micrograph. B) Avein graft covers the anastomosis line between tibial (left side) and peroneal (right side) nerves. C) End-to-end neurorrhaphybetween peroneal (left side) and tibial (right side) nerves. Some nerve fibers sprout out to peripherally due to difference ofnerves diameter. Arrows shows junction between two nerves. D) A vein graft covers the anastomosis line between peroneal(left side) and (right side) tibial nerves; arrows show the vein graft (vg).

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Each ratwas first fixedonto theoperationboard.Theskinandthe subcutis of neck were incised and then external jugular veinwas exposed. A vein segment was dissected off the surroundingtissue. Afterwards, the vein segment was tied off via two 5/0 silkligatures. The vein graft's relatively large proximal end was cutloose, and a 22G IV cannula was incorporated within under theoperatingmicroscope. The lumen of the graft was irrigated withheparin solution, followed by transection of the distal end of thegraft, leaving it wrapped around the cannula.

The position of the animal was changed to prone in order toexpose thenerve. Following incisionof theskin, the sciaticnervewas exposed via blunt dissection without compromising itsexternal vascular support. The nerve was dissected until thetibial, peroneal and sural branches were exposed. For nerverepairs, 10/0 roundbodied, BV75-3, 3.75mm,3/8 cnonabsorbablemonofilament blue polypropylene was used. After surgicaloperation, the muscle and skin were closed with 4/0 vicryl and4/0 silk, respectively. Each animal was then placed into its cage,with freeaccess to foodandwateruntil 90thdayof their surgery.All work performed in accordance with the terms of the Animal(Scientific Procedures) Act 1986 (project no. 70/4210) and thenumber of animals used was kept to a minimum.

5.1. Groups: Tibial–peroneal (TP) repair group (1)

The sciatic, peroneal and tibial nerves were exposed on eitherhind leg of the rats. These nerves were cut 2 cm distal to thetrifurcation. On the right side, the proximal stump of the tibialnerve and distal stump of the peroneal nerve were sutured toeach other via 4 epineural 10/0 prolene sutures, which were 90°

apart (end-to-end neurorrhaphy, (TP-EtoE)) (Fig. 10A). The distalpartof tibial andproximal sideofperonealnerveswerecauterizedand then sutured by 10/0 prolene. After this, free parts of thenerves were then taken away from the nerve neurorrhaphy site.These procedures prevent both neuroma formation at the freestumpsof nerves and the parasitic neurotisation (Fig. 11).Whenasmaller diameter nerve like peroneal nerve is sutured to a greaterone like tibial nerve, small-sizednervealways centeredaccordingto the larger nerve (Figs. 10A, C).

In the left side (vein graft, (VG)), the tibial and peronealnerves were again cut on the same level. Previously harvestedvein graft was transferred onto the micro forceps by holdingthe tip of the cannula and sliding the graft over the irrigatedcannula and forceps. Afterwards, epineurium of the distalstump of the peroneal nerve was held by the micro forceps,which still held the vein graft. The field was again irrigated,and the vein was slid over the nerve. After end-to-endepineural suturation of the nerve by two 10/0 prolene sutures180° apart, the vein graft was slid over the repair zone. Toprevent the migration of the graft, which can be caused by themovement of the animal, each endwas fixed to the underlyingepineurium by two prolene sutures, (TP-VG) (Fig. 10B).

5.2. Peroneal–tibial (PT) repair group (2)

On the right side, the proximal stump of the peroneal nerveand distal stump of the tibial nerve were sutured to each otheras done in TP (PT-EtoE) (Fig. 10C).

On the left side, the vein graft was similarly slid over thedistal stump of the tibial nerve. After end-to-end epineural

Fig. 11 – Schematic drawing of the surgery applied for these experiments. (A) For TP group, the proximal stump of the tibial nerveanddistal stumpof theperonealnerveweresutured toeachotheron theright leg (TP-EtoE). (B)On the left legTPgroup, the tibial andperoneal nerves were again cut on the same level. The proximal stump of the tibial nerve and distal stump of the peroneal nervewere sutured to eachother and then the veingraftwas slidover thenerves. Topreventunwantedmovements of thegraft, each endwas fixed to the underlying epineurium by two prolene sutures (TP-VG). The distal part of tibial and proximal part of peronealnerves were cauterized and sutured by 10/0 prolene. After this, free parts of the nerves were then taken away from the nerveneurorrhaphy site. (C) For PTgroup, the proximal stumpofperoneal nerve anddistal stumpof the tibial nervewere sutured to eachother on the right leg (PT-EtoE). (D) For the left leg of PT group, the peroneal and tibial nerveswere again cut on the same level. Theproximal stump of the peroneal nerve and distal stump of the tibial nervewere sutured to each other than vein graft was slid overthe nerves (PT-VG). Again, each end was fixed to the underlying epineurium by two prolene sutures to fix the graft in place. Thedistal part of peroneal andproximal part of tibial nerveswere cauterized and then sutured by 10/0 prolene. Free parts of the nerveswere then taken away from the nerve neurorrhaphy site. TP-EtoE, tibial–peroneal end to end group; TP-VG, tibial–peroneal veingraft group; PT-EtoE, peroneal–tibial end to end group; VG-PT, vein graft peroneal–tibial group; T, tibial nerve; P, peroneal nerve.

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suturation of the nerves by two 10/0 prolene sutures 180°apart, the vein graft was slide over the repair zone underirrigation (PT-VG). Each end of the graft was again fixed to theunderlying epineurium by sutures (Fig. 10D).

On the 90th postoperative day, after intraperitoneal keta-mineanesthesia, animalswere fixedproneon theboardand thegluteal muscle was re-exposed through previous incision. Thenervewas carefully dissected off its adhesions and the fieldwasexposed. Thenervewashookedat theproximal anddistal to therepair zone by silver tipped bipolar electrodes and data wasrecorded. The bipolar stimulating electrode was placed underthe proximal peroneal nerve and the recording electrode wasplaced distal to the repair site. Both nerve stimulation andbipolar electrical potentials were recorded with PowerLab/4SP(AD Instruments, Australia). The data acquired via bipolar re-cording electrodes were fed to BioAmp unit (AD Instruments,Australia), which was connected to the PowerLab unit. Thedigitizeddatawere savedvia theScopeV3.6.10 (AD Instruments,Australia) software. The electrical stimulations to the nerveswere again delivered via the stimulator unit on the PowerLab/4SPmodule.Differential square impulses (1–10V),whosevoltage

values were adjusted via the software, were transmitted to theproximal side of the repairs. The mean distance between thestimulating and recording electrodes was 18.99 mm. The ad-justments used throughout the recording process were as fol-lows: Sampling time: 20 ms, sampling resolution: 2560 sample/20 ms, Hi-cut: 1 Hz, Lo-cut: 200 Hz, 50 Hz Notch filter.

6. Stereological analysis

Following electrophysiological recordings, the entire tibial andperoneal nerves, including the repaired segment, were re-moved en bloc from all rats and than fixed with 2.5% gluta-raldehyde in 0.1M phosphate buffer for 4 h. After fixation, theywere rinsed in phosphate buffer and then three tissue blockswere cut approximately 1 mm long one proximal to the graft,one distal to graft and one from the mid-segment of the graft.After this, specimens were post fixed in 1% osmium tetroxideand then processed for electron microscopy. Semi-thin sec-tions were cut by ultramicrotome than stained with toluidineblue (Robinson and Gray, 1996).

Fig. 12 – An unbiased counting frame superimposed on thearea of nerve section. The left and bottom lines of the frame areexclusion lines (in red color); i.e. any myelinated nerve fibershit by those lines were not counted (*). Conversely, if a nervefibre profile is in contact with right and upper lines of thecounting frame (arrowheads) and/or located inside the frame, itis included in the counting. a, axon; Cresyl violet staining. (Forinterpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

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Stereological analyses of the nerveswere done according toprinciples describedpreviously (Gundersen, 1978; Larsen, 1998;Geuna et al., 2001; Unal et al., 2005; Turgut et al., 2005). Astereological workstation that is controlled by software (CAST-GRID®-Computer Assisted Stereological Toolbox-Olympus,Copenhagen, Denmark) was used to control measure andrecord the data (Fig. 12). Mean total myelinated fiber number(FN), cross-section area of axon (CSAA), cross-section area ofmyelin (CSAM) and myelin thickness (MT) were estimated.

All data was first checked with a normal distribution test.The groups displayed a homogenous distribution among them-selves. The mean and standard derivation of each individualgroups were calculated via Paired Samples Test. Due to themultiplicity of data varieties, and the shortness of the datawhich each individual group can provide, statistical analysisbetween the groupswere done viaWilcoxon Signed Ranks Test.A value of p<0.05 indicates a significant difference.

Acknowledgment

We would like to thank Dr. Serdar Colakoglu for his excellenttechnical support.

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