COMPARISON OF REGENERATION RESULTS OF PREFABRICATED NERVE GRAFT, AUTOGENOUS NERVE GRAFT, AND VEIN GRAFT IN REPAIR OF NERVE DEFECTS HUSEYIN KARAGOZ, M.D.,1* ERSIN ULKUR, M.D.,2 FATIH UYGUR, M.D.,2 MEHMET GUNEY SENOL, M.D.,3 MEHMET YAPAR, M.D.,4 PINAR TURAN, M.D.,5 and BAHATTIN CELIKOZ, M.D.2
The purpose of this study was to evaluate the effectivity of prefabricated nerve grafts in the repairing nerve defect and to compare them with the autogenous nerve graft and vein graft. Four groups were created, each containing 10 rats. First, nerve prefabrication was carried out in groups I and II during 8 weeks. For this purpose, jugular vein graft was sutured to the epineural windows on the peroneal and tibial nerve at the right side in an end-to-side fashion. To create neurotrophic stimulus, partial incision was performed on the nerves in group I, and gene therapy was performed by plasmid injecting to the adjacent muscles in group II. At the end of the eighth week, prefabricated nerve grafts, jugular vein, and the axons passing through it were taken. Then, gap was created on the left peroneal nerve in all groups. Defect on the peroneal nerve was repaired by using the prefabricated nerve grafts in groups I and II, the autogenous nerve graft in group III, and the vein in group IV. Assessment of nerve regeneration was performed by using electromyography. Morphological assessment was performed after follow-up period. According to electrophysiological and morphological results, the results of first three groups were similar. There was no statistically significant difference between three groups. Prefabricated nerve graft is as effective as autogenous nerve graft, C 2008 Wiley-Liss, Inc. Microsurgery and it can be used in the repair of nerve defects as autogenous nerve graft as an alternative. V 29:138–143, 2009.
Proper
end-to-end nerve repair is the treatment of choice in reconstruction of peripheral nerve injuries, because precise microneurosurgical alignment of fascicles is critical to the complete rehabilitation of injured and denervated tissues. However, restoration of nerve function where trauma has caused a defect in the peripheral nerve remains a challenging problem in reconstructive surgery.1 Although a lot of methods have been reported, autogenous nerve grafting has been the standard technique for repairing nerve defect. The results are satisfactory but this technique causes significant donor site morbidity such as scarring, neuroma formation, and loss of sensation. The morbidity of sacrificing normal, functioning nerve as grafts has stimulated marked interest in alternative methods of bridging a nerve gap.2 There are some experimental and clinical studies using conduits for repair of nerve gap.3–5 In our preliminary study, we successfully created a prefabricated nerve by using an autogenous vein bridging between two healthy nerves.6 We carried
1 Department of Plastic and Reconstructive Surgery, Maresal Cakmak Military Hospital, Erzurum, Turkey 2 Department of Plastic and Reconstructive Surgery, Gulhane Military Medical Academy, Haydarpasa Training Hospital, Istanbul, Turkey 3 Department of Neurology, Gulhane Military Medical Academy, Haydarpasa Training Hospital, Istanbul, Turkey 4 Department of Virology, Faculty of Medicine, Gulhane Military Medical Academy, Ankara, Turkey 5 Department of Histology and Embryology, Marmara University, Medical School, Istanbul, Turkey *Correspondence to: Huseyin Karagoz, M.D., Maresal Cakmak Asker Hastanesi, Plastik Cerrahi Servisi, 25070 Erzurum, Turkey. E-mail:
[email protected] Received 11 June 2008; Accepted 8 September 2008 Published online 22 October 2008 in Wiley InterScience (www.interscience.wiley. com). DOI 10.1002/micr.20586
C V
2008 Wiley-Liss, Inc.
out partial nerve cutting in one of the groups, and gene therapy by using VEGF in the other group for the effect of neurotrophic stimulus from the distal stump of the nerve. Thus, we created a new alternative of autogenous nerve graft as effective as autogenous nerve graft but it does not cause donor site morbidity. The purpose of this study was to evaluate the effectivity of prefabricated nerve grafts in the repairing nerve defect and to compare them with the autogenous nerve graft and vein graft. MATERIALS AND METHODS
Forty male Wistar rats weighing 250–300 g were used in this study. Food and water were supplied ad libitum. Approval and consent were obtained from The Gulhane Military Medical Academy Institution’s Research and Animal Ethical Review Committees. Under anesthesia with intraperitoneal 100 mg/kg ketamine and 15 mg/ kg xylasine injection, all surgical procedures were carried out by the same surgeon by using a sterile microsurgical technique. Four groups were created, each containing 10 rats. First, nerve prefabrication was carried out during 8 weeks in groups I and II, and then, repair of nerve defects was performed in both groups. Group I (Partial Incision Group)
The left jugular vein was exposed through median neck incision. A 1-cm segment of vein was resected and rinsed in saline solution. Then at the right side, the three major fascicles of the sciatic nerves were exposed by a gluteal muscle-splitting incision and separated by dissection to a point about 1 cm above the trifurcation. Partial
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Figure 1. Sutured vein graft to the epineural windows between tibial and peroneal nerves (A), sutured prefabricated nerve graft to the peroneal nerve defect (B).
incision ( 25% of the nerve) was performed on the both tibial and peroneal nerves after opening 1-mm diameter epineural window and fascicular injury was carried out. Then, jugular vein was sutured to the epineural windows on the tibial and peroneal nerves in an end-to-side fashion as described in the preliminary study6 (Fig. 1A). After 8 weeks waiting time, rats were anesthetized, and prefabricated grafts including jugular vein graft, which was sutured epineural windows between tibial and peroneal nerves, were taken. At the left side, the three major fascicles of the sciatic nerve were exposed by a gluteal muscle-splitting incision and separated by dissection to a point about 1 cm above the trifurcation. First, electromyography was performed to calculate the latency and the maximal amplitude for healthy peroneal nerve in rats. Then, a 1-cm long nerve gap was created by sectioning from the 1 cm distal to the trifurcation point on the peroneal nerve. Prefabricated nerve graft sectioned from the right side was sutured to the peroneal nerve defect with four 10-0 nylon sutures in an end-to-end fashion (Fig. 1B).
sented by Prof. Patrick Abeischer) in the Switzerland. The transformation to Escherichia coli was carried out. Qiafilter Plasmid Giga Kit (Qiagen, USA) was used for increased of plasmid from transformed E. coli. Amount of plasmids were determined with UV spectrophotometer. The concentration of plasmid was 1.5 mg/ml and it was ready for inoculation to rats. Apart from group I, VEGF gene therapy was performed by 0.3 cc (including 500 lg DNA) plasmid injecting to the adjacent adductor group muscles as described in the preliminary study.6 After 8 weeks waiting time, rats were anesthetized, and prefabricated grafts including jugular vein graft, which was sutured epineural windows between tibial and peroneal nerves, were taken. At the left side, after the electrophysiological assessment, a 1-cm long nerve gap was created on the peroneal nerve as in group I. Prefabricated nerve graft sectioned from the right side was sutured to the peroneal nerve defect with four 10-0 nylon sutures in an end-to-end fashion. Group III (Nerve Group)
Group II (Gene Therapy Group)
In this group, jugular vein was harvested for graft prefabrication as in group I, but fascicular injury was not carried out on neither tibial nor peroneal nerves. A 1-mm diameter epineural windows 1 cm distal to the trifurcation point on the tibial and peroneal nerves were opened. Jugular vein was sutured to the epineural windows on the tibial and peroneal nerves with four 10-0 nylon sutures in an end-to-side fashion. The plasmids included VEGF [pPI-hVEGF (165)NEO-DHFR] were obtained from research center named Ecole Polytecnique Fe´de´rale de Lausanne (EPFL, repre-
In this group, at the left side, after the electrophysiological assessment, a 1-cm long nerve gap was created on the peroneal nerve as in groups I and II. Nerve defect was repaired by using autogenous nerve graft sectioned from same localization on the peroneal nerve. The right sides were left untouched. Group IV (Vein Group)
Jugular vein was harvested as in group I, and then, the electrophysiological assessment was carried out and, a 1-cm long nerve gap was created on the peroneal nerve as in group III at the left side. Nerve defect was repaired Microsurgery DOI 10.1002/micr
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Karagoz et al. Table 1. Mean Latency and Amplitude of Electrophysiological Assessment at Postoperative 8 Week Group I (partial incision)
II (gene therapy)
III (nerve)
IV (vein)
1.51 6 0.03 8.12 6 2.68
1.57 6 0.10 7.98 6 2.68
1.41 6 0.10 8.34 6 0.07
1.89 6 0.10 5.77 6 1.41
Latency (ms) Amplitude (mV)
by using jugular vein graft. The right sides were left untouched.
Kruskal-Wallis test. In all instances, probability values were two-tailed, with P < 0.05 were considered significant.
Nerve Conduction Studies
RESULTS
Nerve conduction studies were performed in left lower extremity using a commercial electromyography device (Synergy, Medelec, UK). Peroneal nerve was used for the motor conduction velocities with the near nerve technique. Platinum electrodes were placed 5 mm proximal from the proximal repair line and 5 mm distal from the distal repair line were stimulated by passing a constant current pulse of 0.1 milliseconds. To place the electrode close to the peroneal nerve, the recording electrode is used to stimulate and the motor response is picked up from the anterior tibial muscle with needle electrodes. The evoked compound muscle action potentials of the anterior tibial muscle were recorded. Calculations were made for nerve conduction velocity, with latency taken as the first inflection from baseline and peak action potential as the maximal amplitude from the baseline. Nerve conduction studies were performed twice in all groups. First, the latency and the maximal amplitude for healthy rats’ peroneal nerves were calculated in all groups. Second, the latency and the maximal amplitude at the end of the postoperative eighth week were calculated. Morphologic Studies
At the end of the 8 weeks, all rats were anesthetized, and their left sides were explored. As soon as the postoperative electromyography values were obtained, specimens including a 1-cm long peroneal nerve from 5 mm distal of the distal suture line were taken for histologic evaluation. After fixation and tissue processing, specimens of all groups were stained with Toluidine blue and observed with an Olympus BH-2 (Tokyo, Japan) photomicroscope to determine the total number of myelinated nerve fibers and mean fiber diameter, using Image Analyzer (Digital Prioris, XL-511:IPS 4.02, Alcatel, TITN, Answer). Statistical Analysis
The package program SPSS (Statistical Package for Social Sciences for Windows 13.0) was used for statistical analysis. Data were assessed for normality, and values were analyzed by using the Mann-Whitney U test and Microsurgery DOI 10.1002/micr
All rats remained healthy throughout the study, without evidence of automutilation or foot ulceration. Electrophysiological Assessment
According to preoperative electromyography results, mean latency was 1.29 6 0.53 milliseconds and mean amplitude was 9.07 6 2.75 mV. The mean values of all groups at postoperative eighth week are shown in Table 1. The latencies of the group I (partial incision group), group II (gene therapy group), and group III (nerve group) were significantly shorter than group IV (vein group). The amplitudes of the group I (partial incision group), group II (gene therapy group), and group III (nerve group) were significantly higher than group IV (vein group). There was no statistical significant difference among the first three groups (P > 0.05). Histologic Results
According to microscopic evaluation, there were so many myelinated fibers had large diameters in group III (nerve group) as predicted (Fig. 2A). The myelinated fibers of group IV (vein group) were less organized and fewer than other groups, while the results of microscopic evaluation in both group I (partial incision group) and group II (gene therapy group) were similar to group III (nerve group) (Figs. 2B–2D). The mean numbers of myelinated fibers of all groups are shown in Table 2. The differences between group I (partial incision group) and group II (gene therapy group), group I (partial incision group) and group III (nerve group), and group II (gene therapy group) and group III (nerve group) were not statistically significant (P > 0.05); however, the differences between group IV (vein group) and other groups (group I, group II, group III) were statistically significant (P < 0.05). The mean fiber diameters of all groups are also shown in Table 2. The differences between group I (partial incision group) and group II (gene therapy group), group I (partial incision group) and group III (nerve group), and group II (gene therapy group) and group III (nerve group) were
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Figure 2. So many myelinated fibers had large diameters in nerve group (A); A lot of myelinated fibers in partial incision group (B), and in gene therapy group (C). The myelinated fibers less organized and fewer than other groups in vein group (D). (Toluidine Blue, original magnification, 320). Table 2. Number of Myelinated Axons and Mean Fiber Diameters Group
Number of axons Fiber diameters (lm)
I (partial incision)
II (gene therapy)
III (nerve)
IV (vein)
1293.7 6 229.1 1.00 6 0.22
1123 6 111.7 0.98 6 0.33
1341.2 6 44.5 1.03 6 0.09
785.6 6 193.7 0.64 6 0.21
not statistically significant (P > 0.05); however, the differences between group IV (vein group) and other groups (group I, group II, group III) were statistically significant (P < 0.05). DISCUSSION
Autogenous nerve grafting has been the standard and more successful technique than others for repairing nerve defect. But this technique causes significant donor site morbidity such as scarring, neuroma formation, and loss of sensation although the results are satisfactory.7–9 Because of this reason, alternative methods like arterial grafts, vein grafts, and artificial tubes filled with growth factors are
searched instead of autogenous sensory nerve for optimum nerve regeneration.10–14 But, there is no technique can used instead of autogenous nerve graft as yet. Although the most of the researchers thought that the requirements for sufficient nerve regeneration were provided theoretically in alternative studies, in fact they were not consider importance of the Schwann cells and own basal laminas from our point of view. Geuna et al. emphasized this subject as the grafted muscle fibers provided a pathway to growing axons as well as to migratory Schwann cells with the basal lamina, which represent the key component for nerve regeneration.15 Schwann cells may play a critical role in creating a permissive climate for regrowth of injured nerves, underMicrosurgery DOI 10.1002/micr
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Karagoz et al.
going mitosis in response to nerve trauma and nerve gaps after axotomy for early phase of nerve regeneration.16 When a nerve is injured, changes occur both proximal and distal to the injury. The axon distal to the damaged nerve becomes degenerated, but the Schwann cell and their basal lamina remain intact.17 In the proximal segment, each nerve fiber will attempt to regenerate by sprouting many new fibers that form a regenerating unit. At the tip of each regenerating fiber is a growth cone with multiple filopodia. These filopodia move distally by sampling the environment and advancing along the basal lamina of the Schwann cells (also called the bands of Bungner). Schwann cells also provide growth factors and the basal lamina, which is the scaffolding for the regenerating units. This process is a critical step in facilitating nerve regeneration and growth. Grafted segments of nerve are similar to distal segments because they undergo Wallerian degeneration.18 We thought that if we want to create a conduit as effective as autogenous nerve graft, we have to create the conduit as similar as the nerve graft. In the preliminary study, it is shown that the prefabricated nerve can be achieved by using autogenous vein as conduit between two healthy nerves, in addition making partial incision on the nerves and performing gene therapy.6 The vein was sutured to the nerve in an end-to-side fashion in this study. The lateroterminal neurorrhaphy has known since 19th century,19 and Viterbo reported that the end-to-side repair could work, allowing the growth of axons laterally into the end of the attached nerve, conducting the electric stimuli, and maintaining adequate trophism of the corresponding muscles.20 Ulkur et al. reported that the use of graft in the end-to-side neuroraphy is possible, and although the classic nerve graft may be used, the vein graft is also a good alternative to the nerve graft.14 Some effects like neurotrophic factors stimulate axonal sprouting in end-to side neuroraphy cases. Partial nerve cutting and VEGF gene therapy were carried out in preliminary study, because their neurotrophic effects on axonal sprouting from epineural window of the nerve. In the group of partial nerve incision, signs of the tibial and peroneal nerve paralysis seemed initially in walking track analysis were improved gradually and completely disappeared at the sixth week.6 As a result, nerve graft prefabrication could be achieved by partial nerve cutting without permanent nerve deformity. Although there was no sequela on the nerves after healing period, making injury on the healthy nerve may be controversial in clinical practice. Therefore, VEGF gene therapy was used as neurotrophic stimulant in the other group. VEGF can compensate increased metabolic necessity by stimulating to production of new capillary vessels.21 Metabolic necessity of the peripheral nerve is increased because of elongation Microsurgery DOI 10.1002/micr
with axonal regeneration and myelinization around the axon. High blood perfusion can compensate to increased metabolic necessity, therefore new capillary vessels important for nerve regeneration.22 There are positive effects of VEGF to axonal growth, and Schwann cell survive and proliferation in addition to angiogenesis promoting effects.21,23 VEGF was studied instead of nerve growth factor (NGF) because NGFs’ negative effects were reported when used pharmacological dose.24 Plasmid DNA must integrate with living recipient cell DNA for protein expression. Gene therapy was applied by direct injection into adjacent muscles because of the striated muscle cells protein expression characteristic after received plasmid DNA. We were not performed administration of exogenous vascular endothelial growth factor, because exogenous growth factor administration has been shown to have difficulty. The limited success of most purified growth factors may be because of properties such as short half-life, low bioavailability, enzymatic inactivation, and the need for carrier molecules.25 For this reason, gene therapy of VEGF was used instead of exogenous administration. There was no systemic and local side effect.6 Because nerve prefabrication could not succeeded in the group without partial incision and VEGF gene therapy in the preliminary study, this group was not studied in this study. Prefabricated nerve graft with axons in an autogenous sheath (vein) was seemed same a true nerve histologically. But, we were not sure whether signs like autogenous nerve graft will be obtained when use for repair of the nerve defect. Because of this, in this study, we intended to comparison of regeneration results of prefabricated nerve grafts, autogenous nerve graft, and vein graft in repair of nerve defects. According to electrophysiological and morphological results, although the best results were obtained in the nerve group (group III), the results of both partial incision group (group I) and gene therapy group (group II) were similar to nerve group (group III). There was no statistically significant difference between three groups. Thus, we can say that prefabricated nerve graft is as effective as autogenous nerve graft, and it can be used in the repair of nerve defect as autogenous nerve graft alternatively. Additionally, there is no donor site morbidity different from autogenous nerve graft, and this is an important advantage. The first question flashed in minds is ‘‘how the prefabricated nerve graft can be used in clinical practice?’’ It is obvious that using in acute cases is not possible because of the process of the prefabrication required time, and the time depends on graft length. However, we know that defect will be occurred when lesion excised in chronic cases like neuroma in-continuity, and we can
Graft Alternatives for Nerve Defects
carry out prefabrication by using two healthy nerve and a vein at the same localization previously (for example, the case of neuroma in-continuity on the median nerve; median nerve, ulnar nerve and whichever superficial vein can used for process of the prefabrication). Therefore, we can repair the defect by prefabricated nerve graft effectively similar to autogenous nerve graft without donor site morbidity. Even if it was shown that tibial and peroneal function indexes were returned completely normal after sixth weeks in partial incision group,6 incision on the healthy nerve does not preferable to repair of nerve defect in clinical practice. Therefore, prefabrication should be carried out by gene therapy without any incision as far as we are concerned. Actually, there were some limitations of the experimental model in this study. We do not know how long prefabricated nerve graft can produce maximally, and what time is needed for longer graft prefabrication. Further experimental studies should be performed with primate model before clinical practice. As a conclusion, the prefabricated nerve graft is effective as well as autogenous nerve graft, but it does not cause donor site morbidity, and it can be used in repair of nerve defect as autogenous nerve alternative.
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Microsurgery DOI 10.1002/micr
