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SCIENTIFIC ARTICLE
Comparison of the Performance of Chronically Versus
Freshly Denervated Autograft in Nerve Repair
Jonathan Isaacs, MD, Scott Adams, MD, Satya Mallu, MD, Kerry Loveland, MD, Zachary Sandbulte, BA
Purpose Graft choice is one of the few variables over which the surgeon has control whenreconstructing nerve gaps. Because repair of chronically denervated nerves generally yieldsinferior recovery, we hypothesized that the use of chronically denervated nerve tissue as agraft source may compromise axonal regeneration and clinical results.
Methods A total of 45 immature female Sprague-Dawley rats underwent transection of oneperoneal nerve before being divided into 3 experimental groups: group A (n � 15) hadacutely denervated nerve graft, group B (n � 15) had 2-month denervated nerve graft, andgroup C (n � 15) had 4-month denervated nerve graft. We included 10 additional rats as asham group. After 2 months, groups A and B underwent removal of 1 cm of the contralateralperoneal nerve. For group A, this section of nerve was immediately sutured back in place toserve as a model for acute denervation. For group B, the defect was repaired with a 1-cmgraft from the distal stump of the previously transected (denervated) peroneal nerve. GroupC underwent the same procedure as group B, but after an additional 2 months. After 8 weeksof regeneration time, the 3 experimental groups and the sham group underwent testing. Weassessed twitch contraction forces of the reinnervated extensor digitorum longus before weharvested the muscle belly for morphologic measurements. Histological nerve tissue eval-uation assessed axonal regeneration.
Results We detected no statistical differences for mean muscle contraction strengths betweenthe experimental groups; nevertheless, the reinnervated extensor digitorum longus musclebellies from the 4-month denervated nerve graft group were statistically smaller than musclesfrom the other 2 experimental groups (p � .05). Axon counts decreased, whereas axondiameters increased in direct correlation with the length of time of graft denervation (p �.05). No difference in axon myelination was found between experimental groups.
Conclusions Prolonged denervation of nerve graft material compromised both axon and reinner-vated muscle recovery in this rodent model. (J Hand Surg 2010;35A:2001–2007. © 2010Published by Elsevier Inc. on behalf of the American Society for Surgery of the Hand.)
Key words Autograft, delayed nerve repair, nerve grafting.
os
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ERVE GRAFTING ACROSS DEFECTS is an acceptedtechnique of nerve repair when primary ten-sionless approximation of nerve ends is im-
ossible. Modern grafting techniques result in improved
From the Division of Hand Surgery, Department of Orthopaedic Surgery, Virginia Commonwealth Uni-versity Health Systems, Richmond, VA.
Received for publication February 7, 2010; accepted in revised form July 29, 2010.
Supported in part by a Basic Science Grant from the American Federation for Surgery of the Hand.
No benefits in any form have been received or will be received related directly or indirectly to the
subject of this article.utcomes compared with primary repairs under ten-ion1–6; yet, consistently good results remain elusive.7
Among the areas of research aimed at improving theeliability of this technique, nerve selection has received
orresponding author: Jonathan Isaacs, MD, Department of Orthopaedic Surgery, Virginia Com-onwealth University, 1200 East Broad Street, PO Box 980153, Richmond, VA 23298; e-mail:
363-5023/10/35A12-0014$36.00/0oi:10.1016/j.jhsa.2010.07.037
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© Published by Elsevier, Inc. on behalf of the ASSH. � 2001
2002 CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT
recent attention. Nichols et al. showed that motor nervesmake better grafts than sensory nerves for motor nerverepairs.8 One question, however, that has been conspicu-ously overlooked involves the length of time the donornerve has been without functioning axons before beingharvested as graft material. Although the sural nerve isarguably the most commonly used graft source, othersources including the superficial radial nerve or even theulnar nerve are recommended in certain situations. Forsevere or global brachial plexus injuries, the ulnar nervemay have suffered serious or irreversible damage, and ifleft in situ, it will have no or minimal chance of sponta-neous axonal regeneration or functional recovery and canbe used as nerve graft material.9,10 For isolated high radialnerve injuries, motor recovery takes precedence; if a largedefect is present, harvesting the functionless superficialradial nerve not only provides needed graft material, it alsoeliminates a less functionally important escape route forvaluable regenerating axons.11 Likewise, when repairing asciatic nerve defect, motor recovery and plantar sensationare clear priorities. The ipsilateral sural nerve provides aminimally morbid and easily accessible graft source.12
All harvested nerve grafts become denervated assoon as they are harvested. However, these strategies alluse nerve grafts that have been typically denervated forsome length of time. This additional denervation periodmay be detrimental to the effectiveness of this tissue asnerve graft material. The purpose of this study was touse an animal model to determine whether chronicallydenervated nerve graft performs notably differentlyfrom freshly denervated autograft.
MATERIALS AND METHODSIn this study, we used 55 immature, 3-month-old femaleSprague-Dawley rats weighing 120 to 150 g. Our insti-tution’s animal utilization committee approved all ex-periments in accordance with national guidelines, andthe animals were housed in a temperature- and humid-ity-controlled room with a 12-hour day–night cyclewith free access to food. The rats were divided into 4groups: freshly denervated nerve graft (group A, n �15), 2-month denervated nerve graft (group B, n � 15),4-month denervated nerve graft (group C, n � 15), andsham (n � 10). We induced anesthesia with 5% isoflu-rane administered via nose cone inhalation and main-tained it with 2% to 3% isoflurane. We maintained andregulated the core body temperature using a heating padfor all procedures.
Using an aseptic technique, we used a standard bi-ceps femoris semitendinosus muscle splitting approachto expose the right sciatic nerve and major branches.
For all experimental groups, the peroneal nerve wasJHS �Vol A, De
transected at the sciatic nerve bifurcation and the cutnerve ends were buried in surrounding muscle to avoidinadvertent spontaneous axonal regeneration. Allwounds were irrigated and closed with 4-0 nylon. Post-operative pain control was maintained using 0.5 mg/kgbuprenorphine subcutaneously and 272 mg/100 mLacetaminophen in water orally.
After 2 months, we performed the second survivalsurgery for groups A (n � 15) and B (n � 15). Usingthe same technique as above, we located the previouslytransected right peroneal nerve and obtained a 10-mmsegment of the distal stump. The left sciatic, peroneal,and tibial nerves were then exposed and 1 cm of the leftperoneal nerve was excised. For group A (freshly de-nervated nerve graft), this segment was immediatelysutured back in place. For group B (2-month denervatednerve graft), the 1-cm segment of the right (previouslytransected) peroneal nerve distal stump was sutured intothe left peroneal nerve defect. All nerve repairs were doneusing surgical microscope visualization and using two orthree 10-0 sutures. We then closed the wounds and animalrecovery and care continued as outlined above.
After another 2 months, group C (4-month dener-vated nerve graft) underwent the identical procedure asgroup B. The sham group underwent exposure of bilat-eral sciatic, tibial, and peroneal nerves only.
All rodents recovered for 8 weeks before undergoingtesting. The left (grafted) side was exposed as before andall branches other than the peroneal nerve were transected.We isolated the extensor digitorum longus muscle andattached its distal tendon to a force transducer. Supramaxi-mal stimulation (5 V, 1 Hz) was applied to the proximalsciatic nerve and the subsequent twitch contraction forcewas converted to digital data (PowerLab data acquisitionsystem; ADInstruments Inc., Colorado Springs, CO). Weperformed this test 3 times with 2-minute recovery inter-vals. The extensor digitorum longus was then harvested,weighed, and measured (cross-sectional area). All datawere stored on an Apple iBook (Cupertino, CA) andanalysis took place using Microsoft Excel (Redmond,WA): one-way analysis of variance for comparison ofmultiple groups and Student’s t-test for head-to-head com-parisons, with significance set at a p value of less than .05.Sections from the middle of the graft (or equivalent in thesham group) were then excised, stored in 4% formalde-hyde, and subsequently stained with toluidine blue forhistological analysis using ImageJ 1.42 software (NationalInstitutes of Health Web site, http://rsb.info.nih.gov/ij/download.html). After testing was completed, all animalswere killed by intraperitoneal injection of 150 mg Euthasol
(Virbac AH, Inc., Fort Worth, TX).cember
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CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT 2003
RESULTSMean muscle contraction forces for the reinnervatedextensor digitorum longus were 58% of normal ratmuscle for the fresh nerve graft group (group A). Thecontraction force was 57% for the 2-month denervatedgroup (group B) and 64% for the 4-month denervatedgroup (group C). There was no statistical difference incontraction force between any of the experimentalgroups (p � .55) but all experimental groups except forthe sham group were statistically significant (Fig. 1).We found no statistically significant difference betweenthe fresh nerve graft, 2-month denervated, and shamgroups in muscle belly diameter, but there was asignificant decrease in muscle diameter for the
FIGURE 1: Muscle contraction strength. We found no statistichad a statistically significant decrease in muscle contraction com
FIGURE 2: Mean muscle belly diameter: group A, 3.09 mm;mm. The p values reflect significant differences between group
4-month denervated group compared with all other
JHS �Vol A, De
groups (Fig. 2). The 4-month denervated group alsoshowed a significant decrease in muscle belly weightcompared with the other experimental groups, andthere was also a significant difference between allexperimental groups versus the sham group (Fig. 3).
Histological analysis of the nerve sections (Fig. 4)revealed a statistical difference in terms of axon count,axon cross-sectional area, and g ratio (axon diameterdivided by total nerve fiber diameter) in favor of thesham group compared with the other 3 groups (exceptfor group C when we examined the axon cross-sectional area). We found a statistically significant dif-ference between groups A, B, and C in terms of adecreasing number of axons for each respective group
significant difference among groups A, B, and C. All groupsd with the sham group.
p B, 3.18 mm; group C, 2.72 mm; and the sham group, 3.11d other groups.
ally
grou
(Fig. 5). However, we noted an increase in axon diam-
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m gr
2004 CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT
eter (cross-sectional area) progressing from group Athrough group C (Fig. 6). There was no statisticallysignificant difference in g ratio among the 3 experimen-
FIGURE 3: Mean muscle weight: group A, 0.078 g; group B,values reflect significant differences between experimental grou
FIGURE 4: Representative histology of nerve sections fro
tal groups (Fig. 7).
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DISCUSSIONThe goal of this study was to evaluate the effect ofprolonged denervation on the quality of potential nerve
8 g; group C, 0.070 g; and the sham group, 0.1024 g. The pd the sham group.
oups A, B, C and D the sham group (magnification �40).
0.07
graft. Despite recent advances with nerve allograft and
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CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT 2005
artificial nerve conduits, autologous nerve grafting re-mains the reference standard reconstructive option forlarge nerve gaps. Many factors, including ease of har-vest, donor morbidity, and quality of graft material,must be considered in choosing the donor nerve. Usingnerve tissue that is already denervated and is of lowfunctional priority seems to meet many of these criteria.Classic examples of this scenario include using thesuperficial radial nerve to graft a proximal radial nerve
FIGURE 5: Axon counts: group A, 2,792; group B, 1,990; gsignificant differences among groups.
FIGURE 6: Average axonal cross-sectional area: group A, 13.group, 42.37 �m2. The p values reflect significant differencebetween group C and the sham group.
injury, the ulnar nerve to reconstruct a brachial plexus
JHS �Vol A, De
injury when the lower trunk is ruptured, or for lowerextremity injuries, the ipsilateral sural nerve to recon-struct a proximal sciatic nerve injury. In all of thesecases, the graft material is in the same surgical field,may be sacrificed, and may even eliminate superfluousaxonal regeneration pathways. However, this chroni-cally denervated nerve tissue is not physiologicallyidentical to uninvolved nerve graft. Loss of axonalcontact and Wallerian degeneration occurs when the
C, 1,313; and the sham group, 3,629. The p values reflect
m2; group B, 21.52 �m2; group C, 34.23 �m2; and the shamong experimental groups. There was no significant difference
roup
70 �
s am
nerve is cut—either at the time of injury or at the time
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2006 CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT
of graft harvest. This can be a difference of manymonths in some situations.
The findings of this study suggest that nerve graftsubjected to prolonged denervation compromises ax-onal regeneration, as demonstrated primarily by signif-icantly decreasing axonal counts. Similar findings havebeen noted in studies focused more specifically ondelayed nerve repair. This effect may be related to theessential role of Schwann cells in maintaining axonalregeneration in the distal stump of an injurednerve.13–15 Prolonged absence of axonal contact withthese cells, as seen in the denervated nerve graft groupsof this study, has been shown to result in eventual lossof neurotrophic potential.16–19 This is considered to beone of the primary causes of impaired recovery seenafter prolonged denervation.20,21
As the axon numbers decreased in our experimentalgroups, the axons present appeared larger and moremature. Sulaiman and Gordon made a similar observa-tion in a delayed rodent nerve repair model.21 Theyconcluded that although decreasing axonal counts wererelated to the “deterioration of the Schwann cell envi-ronment,” the Schwann cells retained the ability toremyelinate the axons that did regenerate.21 Myelina-tion, as indicated by the g ratio in our study, was notaffected by the length of time these Schwann cells wereallowed to deteriorate; this supports the concept that,although they were fewer in number, the axons that didregenerate were functional.22
We also assessed the reinnervated muscles as an
FIGURE 7: The g ratio: group A, 0.76; group B, 0.73; groupdifferences between experimental groups and the sham group.
indirect measurement of functional recovery. Although
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all experimental groups demonstrated weaker contrac-tion forces compared with the sham group, the lack ofstatistical difference between experimental groups wassurprising. Axon counts have been shown to correlatewith muscle contraction forces.23 Compensatory largermotor units as each of the regenerating axons attemptedto reinnervate as many muscle fibers as possible mayexplain this relative preservation of contraction strength,although a more stringent strength testing protocol such astetanic contraction measurements might have revealedmore obvious deficits in muscle function. This ob-servation is also supported by the decreased weightand diameter of reinnervated muscle found for the4-month denervated graft group compared with theother 2 experimental groups.
Animal studies such as this one are inherently flawedby the superior regenerative capacity of rodents com-pared with human beings. A recently described “blow-through” effect in this animal model suggests that re-gardless of experimental variables, if too much time isallowed for nerve regeneration, enough axons willeventually regenerate to skew study outcomes.24 Thesweet spot between functional recovery and this blow-through effect is still being worked out. We chose the8-week recovery time used in this study to fall withinthis time range, although serial testing would be neces-sary to confirm this. Even if our findings were skewedby this effect, they suggest that using nerve graft thathas been subject to prolonged denervation periods maynot be as effective as fresh nerve graft. Whether the
71; and the sham group, 0.56. The p values reflect significant
C, 0.advantages of using fresh nerve graft in favor of less
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CHRONICALLY AND FRESHLY DENERVATED AUTOGRAFT 2007
optimal (although easily available nerve tissue) out-weigh the associated increased donor morbidity is amore difficult question to answer. Likewise, the exactperiod of time that nerve tissue needs to be denervatedbefore becoming an unacceptable graft option is notknown and would require substantially greater andmore exhaustive study. Until then, the surgeon mustcontinue to weigh the pros and cons of all reconstruc-tive options, including the choice of graft material.
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