ORIGINAL RESEARCH

Effect of Local and Systemic Dimethylsulfoxide onPeripheral Nerve Repair: A Controlled Randomized

Experimental StudyElif Sanli1, Gungor Cagdas Dincel2, Ebru Umay3

1Department of Plastic, Reconstructive and Aesthetic Surgery, Kirikkale University Faculty of Medicine, Kirikkale,Turkey; 2Eskil Vocational High School, Laboratory and Veterinary Science, Aksaray University, Aksaray, Turkey;3Diskapi Yildirim Beyazit Training and Research Hospital, Department of Physical Medicine and Rehabilitation,

University of Health Sciences, Ankara, Turkey

ABSTRACT

Introduction: We investigated the possible beneficial effect of dimethylsulfoxide (DMSO) on peripheral nerverepair in rats. Methods: Seventy rats were divided into four groups: control, sham, DMSO-L, and DMSO-IP.Except in the control group, nerve repair was done at the right sciatic nerve. DMSO was administeredlocally and intraperitoneally for 12weeks to the DMSO-L and DMSO-IP groups, respectively. No therapeuticagent was administered to the other groups. Nerve regeneration was assessed by behavioral, electrophysio-logical, histopathological, and immunohistochemical tests. Results: With the exception of S-100 proteinexpression, all results indicate that DMSO has a beneficial effect on peripheral nerve regeneration.Functional nerve recovery was notably more evident in the DMSO-L than in the DMSO-IP group. Undermacroscopic examination, nerve scores of the regeneration area in the DMSO-L group was also better thanin the others. Discussion: We believe that DMSO can improve peripheral nerve regeneration in rats.

Keywords: dimethylsulfoxide; DMSO; peripheral nerve injury; neurotmesis; nerve; muscle

INTRODUCTION

Peripheral nerve injury causes loss of sensation andfunction [1], and after complete nerve transection,surgical repair is mandatory. However, surgicalrepair is often insufficient for sufficient functionaloutcome, and it causes sequelae in the patient.Dimethylsulfoxide (DMSO) is an antioxidant andanti-inflammatory agent that has been shown toprotect brain tissue against the oxidative injuryproduced by ischemia [2]. Moreover, DMSOdecreases neuronal damage due to chronic ethanolexposure [3]. This agent has also been shown toreduce oxidative damage to the brain after ische-mia/reperfusion and middle-cerebral artery occlu-sion [4, 5]. However, the effect of DMSO onperipheral nerve injury has not been well evaluatedin the literature, therefore, the present study was

designed to evaluate the effectiveness of local andsystemic administration of DMSO on peripheralnerve repair after neurotmesis.

MATERIALS AND METHODS

Ethics

This experimental study was approved by the LocalEthical Committee (Number: 05.03.2015/0021-350).

Experimental Drugs

In this study, 10% DMSO (Micro Therapeutics, Inc.,Irvine, CA) diluted with a saline solution was usedlocally around to the nerve injury site at a dose of

Received 10 May 2019; accepted 12 July 2019.Address correspondence to Elif Sanli, Department of Plastic, Reconstructive and Aesthetic Surgery, Kirikkale University Faculty ofMedicine, Ankara-Kirikkale Road 7th km Yahsihan, 71450 Kirikkale, Turkey. E-mail: drelifsanli@hotmail.com

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/iivs.

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Journal of Investigative Surgery, 0: 1–12, 2019Copyright # 2019 Taylor & Francis Group, LLCISSN: 0894-1939 print / 1521-0553 onlineDOI: 10.1080/08941939.2019.1644403

0.1ml/day. The density of anhydrous liquid DMSOis approximately 1.1 g/ml. The systemic Ld 50 for arat is 13 g/kg [6]. Anesthesia was performed byintraperitoneal administration of 40mg/kg of keta-mine hydrochloride (Ketalar; Pfizer Inc., USA) and5mg/kg xylazine hydrochloride (Rompun 2%; BayerHealthCare AG, Germany).

Animals and Groups

A total of 70 male Wistar albino rats (270–300 g)were randomly divided into four groups as follows:

Control group (neither surgical procedure norexperimental agent was administered to the animals;n: 10)

Sham group (peripheral nerve repairs weredone, but no experimental agent was administeredto the animals; n:20)

DMSO-IP group (peripheral nerve repairs weredone, and 0.1ml anhydrous DMSO diluted in 0.9mlsaline was injected intraperitonealy daily for12weeks; n: 20)

DMSO-L group (peripheral nerve repairs weredone, and 0.1ml anhydrous DMSO diluted in 0.9mlsaline was injected into the injury site daily for12weeks; n: 20)

Surgical Technique

The surgical procedure performed on the animals inthe present study was previously described by Fesliet al. and has been widely used in the literature [7].Sedation anesthesia was performed on all animalsexcept those in the control group. The right sciaticnerve was exposed through a dorsal gluteal splittingincision, transected at 1 cm above the trifurcationpoint, and reconnected with four epineural 9/0sutures (Daylon, Do!gsan, Istanbul, Turkey), exceptin the control group (Figure 1(a)). Nerve repair was

performed under an operating microscope usingaseptic techniques and microsurgical rules. Half ofeach experimental group was euthanized at thefourth week and the other half of each group waseuthanized at the 12th week. The control group waseuthanized at the fourth week. The intact un-repaired nerve tissues in the control group andthe repaired nerve tissues in the other groupswere collected.

Macroscopic Evaluation

At the end of the fourth and 12th weeks, the ratswere euthanized, and the operated nerve tissueswere explored and scored according to the study ofSiemionow et al. [7, 8]. The weight ratios of thegastrocnemius muscles were also obtained. Theweights of the gastrocnemius muscles from theexperimental sides (right leg) were divided by thoseof the normal sides (left leg), and statistical analysiswas performed.

Immunohistochemical Analysis

Immunohistochemistry was performed to investigatenerve growth factor (NGF sc- 365944 Santa CruzBiotechnology, USA), S-100 protein (sc- 53438 SantaCruz Biotechnology, USA) and myelin basic protein(MBP sc- 13914 Santa Cruz Biotechnology, USA)expression. Commercial antibodies were visualizedon 4- to 5-lm-thick paraffin sections using an indir-ect streptavidin/biotin immunoperoxidase kit (HRP;Thermo Scientific, Waltham, MA, USA). All stepswere carried out following the procedure describedby Dincel and Kul [9]. Accordingly, tissue sectionswere placed on adhesive slides, deparaffinized for5min in each of three xylene series, and rehydratedin a graded alcohol series and distilled water.Antigen retrieval was accomplished by boiling

FIGURE 1. (a) The right sciatic nerve was exposed through a dorsal gluteal splitting incision, transected at 1 cm above thetrifurcation point and reconnected with four epineural 9/0 sutures, except in the control group. (b) Macroscopic view of therepaired area of the sham group. There was a difficult sharp dissection performed in the presence of severe adhesion (12th weekof the study). (c) Macroscopic view of the repaired area of the DMSO-IP group. There was mild adhesions around the repair site.Dissection was easily done (12th week of the study). (d) Macroscopic view of the repaired area of the DMSO-L group. There wasnot any adhesions around the repair site and the dissection was done easily (12th week of the study).

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sections on glass slides in citrate buffer (pH 6.0;Thermo Scientific) for 20min. Endogenous peroxidaseactivity was quenched in 3% hydrogen peroxide inabsolute methanol for 7min at room temperature.Sections were rinsed three times with phosphate-buf-fered saline (pH 7.4) for 5min between each step ofthe test. Sections were incubated in blocking serumfor 5min to prevent nonspecific binding. Thereafter,sections were incubated with the 1:100 diluted pri-mary antibody (NGF, S-100 protein and MBP) for60min in a humidified chamber at room temperature.Sections were treated with a biotin-labeled secondaryantibody for 15min and streptavidin-peroxidaseenzyme for 15min at room temperature. Finally, sec-tions were incubated in aminoethyl carbazolechromogen (Thermo Scientific) for 5–10min to inducethe color reaction. Mayer’s hematoxylin was appliedas a counterstain for 1–2min. Thereafter, sectionswere mounted with water-based mounting medium(Thermo Scientific). As a control for nonspecificendogenous peroxidase and biotin activities in eachtest, the primary antibody step was omitted. Sectionswere immediately analyzed. Immunostaining wasevaluated using a binocular microscope (OlympusBX51 microscope equipped with a DP25 camera,Japan) and photographed under a 20! objective.

Histomorphometric Analysis

The density of positive staining was measured usinga computerized image system composed of a LeicaCCD camera DFC420 (Leica Microsystems ImagingSolutions, Ltd., Cambridge, UK), connected to aLecia DM4000 B microscope (Leica MicrosystemsImaging Solutions, Ltd.) and was used according tothe procedure described by Dincel and Kul [9].Accordingly, five representative fields were selectedunder high-power view and consecutive pictureswere captured by the Leica QWin Plus v3 softwareby a 20! objective (Leica Microsystems ImagingSolutions) at a setting identical to the image systemfor analyzing. For examining the staining of eachantibody, we used the same setting for all slides.Integrated optical density of all positive stainings ofNGF, S-100 protein and MBP in each photographwere measured. For the quantification of the meanNGF-, S-100 protein-, and MBP-positive area/totalarea were measured and calculated by Leica QwinPlus on the pictures. All images were collectedunder the same lighting conditions. To avoid obser-ver bias, a blinded investigator quantified all of thesections. Data were statistically described in termsof mean and standard deviation (mean±SD) forarea %. After calculating the proportion (% pixels)of stained area to the whole field, the mean (in %pixels) staining area for each slide was deter-mined [9].

Ultramicrotome Sections Evaluation

Sciatic nerve tissues were embedded in epoxy resin,and 70-nm sections were obtained by ultramicrot-omy. The tissue sections were stained with toluidineblue and evaluated according to the histomorpho-metric analysis method [9].

Electrophysiological Test

Electrophysiological studies were performed tomeasure the amplitude of the compound motoraction potential (CMAP) and conduction velocity(CV). Measurements were taken using a Neuro-MEP-Micro two-channel EMG device (Neurosoft,Russia), and recordings were collected at the fourthand 12th weeks after operation.

Behavioral Evaluation

Functional healing was evaluated by a pinprick test(PPT) and toe-spread test (TST) at the fourth and12th weeks. Scoring was performed from 0 to 3 asdescribed in previous literature [10].

Statistics

The Kolmogorov-Smirnov test was applied to con-firm normal distribution of the sample population inall study parameters (p <.05). For determination ofthe statistical differences (post hoc evaluation)between the groups, the Mann–Whitney U test andthe Bonferroni correction test were performed onall results.

The fourth-week value results of the S-100 pro-tein, MBP area fill, myelinated axon count, clinicaltest results, and nerve scales were not normally dis-tributed and were analyzed using theKruskal–Wallis multiple variant analysis test (p <.05). The value results of the fourth-week NGF, MBParea percentage, and gastrocnemius weight ratioswere normally distributed. Therefore, the one-wayanalysis of variance (ANOVA) test was performedfor all those values (p < .05).

The 12th-week value results of the clinical testsand nerve scales were not normally distributed.Therefore, those results were statistically analyzedusing the Kruskal–Wallis multiple variant analysistest (p < .05). The other results from the 12th weekwere normally distributed, so the ANOVA test wasperformed for all those values (p < .05).

Statistically significant differences in repeatedmeasurements within the group were evaluatedwith the Wilcoxon signed rank test, and the

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Bonferroni correction was used to control possibleType-I errors in intra-group comparisons.Statistically significant differences among the groupswere analyzed with the ANOVA test and post-hocanalysis. Values of p < .05 were considered statistic-ally significant.

RESULTS

Macroscopic Findings

The right gastrocnemius muscle weight ratios of thecontrol group were significantly higher than thoseof the other groups at the fourth week of the study(p¼ .000). Moreover, the muscle ratios in the DMSO-IP and DMSO-L groups were significantly higherthan those in the sham group (Table 1; p¼. p¼ .013,p¼ .003). The weight ratios of the control groupwere higher than those of the sham (p¼ .000) andDMSO-IP groups (p¼ .037), and the DMSO-Lgroup’s weight ratios were higher than the shamgroup’s weight ratios at the 12th week of the study(Table 2; p¼ .04).

The nerve scales were also analyzed at thefourth and 12th weeks of the study. There was nostatistically significant difference between the con-trol and DMSO-L group at the fourth week, and thenerve-scale values were higher in the DMSO-IP(p¼ .000) and sham (p¼ .000) groups than in thecontrol group. Moreover, the values of the shamgroup were higher than those of the DMSO-IP(p¼ .000) and DMSO-L (p¼ .000) groups, while thenerve-scale values of the DMSO-IP group were alsohigher than those of the DMSO-L group (p¼ .000;Table 1). The nerve-scale values of the sham groupwere higher than those of the other groups at the12th week of the study (Table 2, Figure 1(b,c)).

Immunohistochemical Findings

NGF Expression Analysis. There was no statisticallysignificant difference between the DMSO-IP groupand the other groups in terms of area percentagevalues. The area percentage values of NGF expres-sion in the DMSO-L group were higher than thoseof the sham (p¼ .015) and control groups (p¼ .001)

TABLE 1. Descriptive statistical results of the 4th week of the study

Findings Parameters Control Sham DMSO-IP DMSO-L

Macroscopic findings Gastrocnemius muscle weight ratio 1.02 ± 0.14 0.54 ± 0.13 0.59 ± 0.21 0.63 ± 0.18Nerve scale 0.0(0.0–0.0) 3.0(3.0–3.0) 1.0(1.0–2.0) 0.0(0.0–0.0)

Immunohistochemical findings NGF 7.00 ± 2.73 9.58 ± 4.31 15.06 ± 3.63 19.38 ± 10.03S-100 48.28 ± 8.29 18(11–21) 15(11–25) 13(11–15)MBP 36.18 ± 18.89 12.66 ± 9.24 13.74 ± 6.29 11.74 ± 5.48

Ultramicrotome section findings Myelin sheet thickness 4.41 ± 0.34 1.69 ± 0.42 3.65 ± 0.21 3.43 ± 0.07Axon diameter 3.73 ± 0.52 15.62 ± 0.52 7.07 ± 0.52 3.36 ± 0.52Regenerated myelinated axon count 25.80 ± 3.49 8.60 ± 0.55 37.20 ± 4.97 49.80 ± 3.03Myelinated axon count 196.60 ± 5.77 59(54–62) 49(46–54) 82(79–85)

Electrophysiological findings CMAP (mV) 4.86 ± 1.82 0.17 ± 0.55 2.32 ± 0.61 2.51 ± 1.16CV (m/sec) 48.07 ± 4.70 4.15 ± 13.12 42.71 ± 1.79 41.15 ± 1.76

Clinical findings PPT results 3.0(3.0–3.0) 0.0(0.0–0.0) 1.0(1.0–3.0) 1.0(1.0–2.25)TST results 3.0(3.0–3.0) 2.0(1.8–2.0) 2.5(2.0–3.0) 3.0(3.0–3.0)

TABLE 2. Descriptive statistical results of the 12th week of the study

Findings Parameters Sham DMSO-IP DMSO-L

Macroscopic findings Gastrocnemius muscle weight ratio 0.54 ± 0.13 0.74 ± 0.32 0.81 ± 0.21Nerve scale 3.0(3.0–3.0) 0.0(0.0–0.0) 0.0(0.0–0.0)

Immunohistochemical findings NGF 6.93 ± 2.82 20.39 ± 5.42 22.69 ± 6.58S-100 27.02 ± 7.72 16.03 ± 4.21 18.45 ± 5.70MBP 9.78 ± 3.95 26.45 ± 7.88 25.91 ± 8.26

Ultramicrotome section findings Myelin sheet thickness 2.55 ± 0.33 4.71 ± 0.52 5.38 ± 0.90Axon diameter 7.61 ± 0.37 4.23 ± 0.25 5.16 ± 4.62Regenerated myelinated axon count 26.40 ± 3.58 41.60 ± 1.14 35.00 ± 5.38Myelinated axon count 80.80 ± 2.05 66.80 ± 7.82 101.60 ± 5.32

Electrophysiological findings CMAP (mV) 0.46 ± 0.95 3.83 ± 1.84 4.76 ± 1.20CV (m/sec) 12.19 ± 19.66 43.37 ± 6.53 49.12 ± 2.35

Clinical findings PPT results 0.0(0.0–0.0) 2.0(1.0–3.0) 3.0(2.0–3.0)TST results 2.0(0.0–3.0) 3.0(3.0–3.0) 3.0(2.75–3.0)

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at the fourth week of the study (Tables 1 and 3,Figure 2(a–d)), and the area percentage values ofNGF expression in the DMSO-L and DMSO-IPgroups were higher than those of the sham group atthe 12th week of the study (p¼ .000). There was nostatistically significant difference between theDMSO-L and DMSO-IP group (Tables 2 and 4,Figure 2(e,d)).

S-100 Protein Expression Analysis. There was nostatistically significant difference between the shamand the DMSO-L, the sham and the DMSO-IP, orthe DMSO-L and the DMSO-IP groups at the fourthweek of the study. The S-100 protein expressions of

the control group were higher than those of the othergroups (p¼ .000; Tables 1 and 3, Figure 3(a–d)).There was no statistically significant differencebetween the DMSO-L and the DMSO-IP group at the12th week of the study. The S-100 protein expressionvalues of the sham group were higher than those ofthe other groups (p¼ .006 and p¼ .021; Tables 2 and4, Figure 3(e–g)).

MBP Expression Analysis. There was no statisticallysignificant difference between the sham and theDMSO-L, the sham and the DMSO-IP, or theDMSO-L and the DMSO-IP group at the fourthweek. The expression values of MBP were higher in

FIGURE 2. (a) NGF immunoreactivity in the healthy control group. (b) Fairly weak immunoreactivity for NGF in sham group at4th week of the study. (c) Weak immunoreactivity for NGF in DMSO-L group at 4th week of the study. (d) Mildimmunoreactivity for NGF in DMSO-IP group at 4th week of the study. (e) Fairly weak/weak immunoreactivity for NGF in shamgroup at 12th week of the study. (f) Mild immunoreactivity for NGF in DMSO-L group at 12th week of the study. (g) Strongexpression of NGF in DMSO-IP group at 12th week of the study.

FIGURE 3. (a) S-100 protein immunoreactivity in the healthy control group. (b) Strong expression of S-100 protein in shamgroup at 4th week of the study. (c) Strong expression of S-100 protein in DMSO-L group at 4th week of the study. (d) Weak/mildexpression of S-100 protein in DMSO-IP at 4th week of the study. (e) Strong expression of S-100 protein in sham group at 12thweek of the study. (f) Strong expression of S-100 protein in DMSO-L at 12th week of the study. (g) Weak/mild expression of S-100 protein in DMSO-IP at 12th week of the study.

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the control group than in the other groups (p¼ .001;Tables 1 and 3, Figure 4(a–d)). There was no statis-tically significant difference between the DMSO-Land the DMSO-IP group at the 12th week of thestudy. However, the expression values of MBP werehigher in the DMSO-L (p¼ .001) and DMSO-IPgroups (p¼ .000) than in the sham group (Tables 2and 4, Figure 4(e–g)).

Ultramicrotome Sections Findings

Myelin Sheath Thickness. The control group’s val-ues were higher than those of the other groups atthe fourth week of the study (p¼ .000, p¼ .005,p¼ .000) and the DMSO-L and DMSO-IP groups’values were also higher than those of the sham

group (p¼ .000; Tables 1 and 3). There was no statis-tical difference between the control and the DMSO-L, the control and the DMSO-IP, or the DMSO-Land the DMSO-IP group at the 12th week of thestudy, and the control group’s values were higherthan those of the sham group (p¼ .001). The DMSO-L and DMSO-IP groups’ values were also higherthan those of the sham group (p¼ .000; Tables 2 and4; Figure 5).

Myelinated Axon Diameter. The sham group’s val-ues were higher than those of the other groups(p¼ .000). Moreover, the values of the DMSO-L andDMSO-IP groups were higher than those of the con-trol group at the fourth week of the study (p¼ .000;Tables 1 and 3). There was no statistical differencebetween the control and the DMSO-IP group at the

FIGURE 4. (a) MBP immunoreactivity in the healthy control group. (b) Fairly weak immunoreactivity for MBP in sham group at4th week of the study. (c) Weak immunoreactivity for MBP in DMSO-L group at 4th week of the study. (d) Mildimmunoreactivity for MBP in DMSO-IP at 4th week of the study. (e) Fairly weak/weak immunoreactivity for MBP in sham groupat 12th week of the study. (f) Mild immunoreactivity for MBP in DMSO-L at 12th week of the study. (g) Strong expression ofMBP in DMSO-IP at 12th week of the study.

FIGURE 5. (a) Light photomicrograph of the sciatic nerve obtained from a sham-group rat (12th week). Significant damage tomyelinated nerve fibers, myelin sheaths, and axons, which are seen to be moderate compared to the fourth week in the shamgroup. TB bar, 10 lm. (b) Light photomicrograph of the sciatic nerve obtained from a DMSO-treated rat (IP, 12th week). Slightaxonal damage, myelin sheath degeneration, and partially diffuse regenerated axons were seen compared to the DMSO-IP fourth-week group. TB bar, 10 lm. (c) Light photomicrograph of the sciatic nerve obtained from a DMSO-treated rat (L, 12th week).Slight axonal damage, myelin sheath degeneration, and partially diffuse regenerated axons were seen compared to the DMSO-Lfourth-week group. TB bar, 10 lm.

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TABLE 3. Statistical results of 4th week of the study [Bonferroni test and correction were performed after one way ANOVA(p< .05) and Kruskal–Wallis test (p< .012)]

Groups (comparison) Parameters Mean difference p Value

Control/sham Gastrocnemius muscle weight ratio 0.7 .000Nerve scale –2.9 .000NGF –2.5 1S-100 protein 31 .000MBP 23.5 .001Myelin sheet thickness 2.7 .000Axon diameter –11.89 .000Regenerated myelinated axon count 17.2 .000Myelinated axon count 138.4 .009PPT test results 3 .000TST test results 1.2 .000

Control/DMSO-IP Gastrocnemius muscle weight ratio 0.4 .000Nerve scale –1.3 .000NGF –8.1 .079S-100 protein 31 .000MBP 22.5 .001Myelin sheet thickness 0.76 .005Axon diameter –3.34 .000Regenerated myelinated axon count –24 .000Myelinated axon count 146.8 .009PPT test results 1.2 .004TST test results 0.5 .012

Control/DMSO-L Gastrocnemius muscle weight ratio 0.4 .000Nerve scale –1 1NGF –12.4 .001S-100 protein 35 .000MBP 24.5 .001Myelin sheet thickness 0.98 .000Axon diameter –2.63 .000Regenerated myelinated axon count –11.4 .000Myelinated axon count 114.8 .009PPT test results 1.5 .000TST test results 0.2 0.317

Sham/DMSO-IP Gastrocnemius muscle weight ratio –2.3 .013Nerve scale 1.6 .000NGF –5.5 0.496S-100 protein 0.25 1MBP –1.1 1Myelin sheet thickness –1.96 .000Axon diameter 8.54 .000Regenerated myelinated axon count –28.6 .000Myelinated axon count 8.4 .028PPT test results –1.8 .000TST test results –0.7 .007

Sham/DMSO-L Gastrocnemius muscle weight ratio 2.8 .003Nerve scale 2.8 .000NGF –9.8 .015S-100 protein –4.2 1MBP 0.92 1Myelin sheet thickness –1.74 .000Axon diameter 9.26 .000Regenerated myelinated axon count –41.2 .000Myelinated axon count –23.6 .009PPT test results –1.5 .000TST test results –1 .001

DMSO-IP/DMSO-L Gastrocnemius muscle weight ratio 1.2 1Nerve scale 1.2 .000NGF –4.3 0.933S-100 protein 4.2 1MBP 2 1Myelin sheet thickness 0.21 1Axon diameter 0.71 0.271Regenerated myelinated axon count –12.6 .000Myelinated axon count –32 .009PPT test results 0.3 1TST test results –0.3 0.933

Significant results are written in bold.

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TABLE 4. Statistical results of 12th week of the study [Bonferroni test and correction were performed after one way ANOVA(p< .05) and Kruskal–Wallis test (p< .012)]

Groups (Comparison) Parameters Mean difference p Value

Control/sham Gastrocnemius muscle weight ratio 0.5 .000Nerve scale –3 .000NGF 0.007 1S-100 protein 21.2 .000MBP 26.4 .001Myelin sheet thickness 1.86 .001Axon diameter –3.88 .000Regenerated myelinated axon count –0.6 1Myelinated axon count 115.8 .000PPT test results 3 .000TST test results –1.4 .002

Control/DMSO-IP Gastrocnemius muscle weight ratio 0.3 .037Nerve scale –1.1 0.292NGF –13.3 .000S-100 protein 32.2 .000MBP 9.7 0.669Myelin sheet thickness –0.29 1Axon diameter –0.5 0.434Regenerated myelinated axon count –15.8 .000Myelinated axon count 129.8 .000PPT test results 1 .008TST test results 0 1

Control/DMSO-L Gastrocnemius muscle weight ratio 0.2 0.193Nerve scale –0.2 0.317NGF –15.6 .000S-100 protein 29.8 .000MBP 10.2 0.562Myelin sheet thickness –0.96 0.1Axon diameter –1.43 .000Regenerated myelinated axon count –9.2 .007Myelinated axon count 95 .000PPT test results 0.4 1TST test results 0.2 1

Sham/DMSO-IP Gastrocnemius muscle weight ratio –2 0.278Nerve scale 2.9 .000NGF –13.4 .000S-100 protein 11 .006MBP –16.6 .000Myelin sheet thickness –2.15 .000Axon diameter 3.37 .000Regenerated myelinated axon count –15.2 .000Myelinated axon count 14 .007PPT test results –2 .000TST test results –1.4 .003

Sham/DMSO-L Gastrocnemius muscle weight ratio –3 .04Nerve scale 2.8 .000NGF –15.7 .000S-100 protein 8.58 .021MBP –16.6 .001Myelin sheet thickness –0.28 .000Axon diameter 2.45 .000Regenerated myelinated axon count –8.6 .013Myelinated axon count –20.8 .000PPT test results –2.6 .000TST test results –1.2 .015

DMSO-IP/DMSO-L Gastrocnemius muscle weight ratio –0.7 1Nerve scale –0.1 1NGF –2.3 1S-100 protein –2.41 1MBP 0.54 1Myelin sheet thickness –0.67 0.483Axon diameter –0.92 .017Regenerated myelinated axon count 6.6 .076Myelinated axon count –34.8 .000PPT test results –0.6 0.296TST test results 0.2 1

Significant results are written in bold.

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12th week of the study. The sham group’s valueswere higher than those of the control, DMSO-L, andDMSO-IP groups (p¼ .000). Moreover, the values ofthe DMSO-L group were higher than those of thecontrol and DMSO-IP groups (p¼ .000; Tables 2 and4; Figure 5).

Regenerated Myelinated Axon Count. The valueswere higher in the control group than in the shamgroup and higher in both DMSO groups than in thecontrol and sham groups (p¼ .000). Moreover, thevalues of the DMSO-L group were higher than thoseof the DMSO-IP group at the fourth week (p¼ .000;Tables 1 and 3). There was no statistically significantdifference between the control and sham groupsand the DMSO groups. The regenerated axon countswere higher in both DMSO groups than in the con-trol and sham groups at the 12th week of the study(p¼ .000, p¼ .007, p¼ .013; Tables 2 and 4; Figure 5).

Myelinated Axon Count. The myelinated axoncount was higher in the control group than in theother groups (p¼ .009). Also, the DMSO-L grouphad a higher myelinated axon count than the shamand DMSO-IP groups at the fourth week (p¼ .009;Tables 1 and 3). The control group had higher val-ues than the other groups at the 12th week of thestudy (p¼ .000). Also, the DMSO-L group had ahigher myelinated axon count than the DMSO-IPand sham groups (p¼ .000). The values of the shamgroup were higher than those of the DMSO-IPgroup (p¼ .007; Tables 2 and 4; Figure 5).

Electrophysiological Findings

The mean values of the CMAP and CV measure-ments of the sciatic nerves of the rats as recorded atthe end of the fourth and 12th weeks are given inTable 1 and 2. The mean CMAP and CV values inthe sham group were lower than those of the othergroups at the fourth and 12th weeks. The mean val-ues of CMAP in the DMSO-L group were lowerthan in the control group. Conversely, the mean val-ues of CV in the DMSO-L group were not statistic-ally different from those of the control group at the12th week of the study. There was no statisticallysignificant difference in the mean values of CMAPand CV in the DMSO groups at the fourth week ofthe study. However, the mean values of CV werehigher in the DMSO-L group than in the DMSO-IPgroup at the end of the study.

The variation of CMAP and CV values duringthe study was not statistically different in the shamgroup. Conversely, the variation was statistically dif-ferent in the DMSO-L and DMSO-IP groups. Thevariation of CMAP values during the study was

statistically different between the sham and theDMSO-L and DMSO-IP groups, and the sham grouphad lower variation than the DMSO groups.However, the variation of CV values was not statis-tically different between the groups, and there wasno statistically significant difference between theDMSO groups in terms of mean CMAP and CV val-ues variations.

Behavioral Findings

PPT Results. There was no statistical differencebetween the DMSO groups at the fourth week. ThePPT results of the control group were higher thanthose of the other groups (p¼ .000, p¼ .004), andboth DMSO groups had higher PPT results than thesham group (p¼ .000; Tables 1 and 3). There was nostatistical difference between the DMSO groups orbetween the control and DMSO-L group at the 12thweek. The PPT results of the control group werehigher than those of the sham and DMSO-IP groups(p¼ .000, p¼ .008), and both DMSO groups hadhigher PPT results than the sham group (p¼ .000;Tables 2 and 4).

TST Results. There was no statistical differencebetween the DMSO groups or between the controland DMSO-L groups at the fourth week. The TSTresults of the control group were higher than thoseof the sham and DMSO-IP groups (p¼ .000,p¼ .012), and both DMSO groups had higher TSTresults than the sham group (p¼ .007, p¼ .001;Tables 1 and 3). There was no statistical differencebetween the DMSO groups or between the controland either DMSO group at the 12th week. The TSTresults of the control group were higher than thoseof the sham group (p¼ .002), and both DMSOgroups had higher TST results than the sham group(p¼ .003, p¼ .015; Tables 2 and 4).

DISCUSSION

The cell body and target organ are affected by injuryand exhibit molecular and cellular changes afterneurotmesis, and the withdrawal of target-derivedneurotrophic support has been shown to be the big-gest determinant of neuronal survival [11, 12]. Atthe site of injury, the distal stump undergoesWallerian degeneration [13], and in the first 24 h,Schwann cells (SCs) proliferate and switch from amyelinating to a regenerative phenotype. They con-trol the up-regulation of various molecules, anddenervated SCs down-regulate several proteins [14,15]. When macrophages remove the debris at theinjury site, SCs align and form B€ungner bands.

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However, lack of neuronal contact in the distalstump leads to a chronic degeneration of SCs, whichcauses down-regulation of growth factors [16].

An interruption of microcirculation occurs inperipheral nerve injuries [17]. Reperfusion thenbegins and causes the formation of free radicals thatattract lipids and cause lipid peroxidation [17].Moreover, an excessive inflammatory response cancause scar tissue to develop that can mechanicallyrestrict axonal sprouting [18]. A high expression oftransforming growth factor beta (TGF-b) activatesfibroblasts and causes fibrosis [19], and the com-bined effect of ischemia and the mechanical barrierincreases the disruption of nerve regeneration.

Many studies have reported the effect of antioxi-dant and anti-inflammatory agents on peripheralnerve regeneration [17]. However, to the best of ourknowledge the use of DMSO has not been wellinvestigated previously in the literature.

DMSO is analgesic, anti-inflammatory [20–22],antifibrotic [23], a free-radical scavenger [24, 25],and an antioxidant agent. It has been shown to pro-tect the ischemic brain [3], heart [26], and kidney[27] against oxidative injury. Furthermore, it reducesoxidative damage to the brain after ischemia/reper-fusion and middle-cerebral artery occlusion in rats[4, 5]. Therefore, we decided to determine the effect-iveness of DMSO on peripheral nerve repair afterneurotmesis in rats.

In the present study, the muscle weight ratioswere higher in the DMSO groups than in the shamgroup at the fourth week, and the muscle weightratios of the DMSO-L group were higher than thoseof the sham group at the 12th week. Moreover, themuscle weight ratios of the DMSO-L group werenot different from those of the control group at the12th week of the study. These results indicate thatDMSO could enhance structural muscle recovery bypreventing muscle atrophy. Local administration ofDMSO seems to be more effective than systemicadministration for muscle protection.

At the end of the fourth and 12th weeks, everysciatic nerve was re-explored and photographed.Nerve regeneration in the DMSO groups was similarto the control group in the present study, indicatingthat DMSO could be effective in healing and avoid-ing perineural fibrosis by decreasing inflammationaround the surgical area. Smoother nerve regener-ation in the DMSO-L group was also more remark-able than in the other experimental groupsaccording to the nerve scale results.

NGF is a necessary molecule for growth andhealing, and it affects both motor and sensory neu-rons [28–30]. In the present study, DMSO was foundto be beneficial to peripheral nerve regeneration byincreasing NGF around the injured tissues. Localadministration of the drug seems to be especially

effective on NGF expression. It was also found thatDMSO did not affect S-100 protein expressionaround the area of the nerve injury, and we believethat DMSO may not be involved in myelin synthe-sis. At the early stage of nerve healing, DMSO didnot affect MBP expression. However, the expressionof MBP was increased at the 12th week of the study.MBP constitutes the majority of the protein found inthe myelin sheath and helps to stabilize the myelinstructure by serving as a scaffold for the lipid com-ponent [31]. Therefore, DMSO may assist the myeli-nization of the axon segment at the injured area.

The myelin sheath thicknesses were greater inthe DMSO groups than in the sham group at boththe early stages and late period of peripheral nerveregeneration in the present study. We believe thatDMSO could have a positive effect on the myelinsheath by increasing MBP expression in the injuredaxon. Both local and systemic administrations ofDMSO were also found to increase axon diametersof myelinated neurons. However, the axon diametervalues were significantly higher in the DMSO-Lgroup than in the control and other groups in thelate period of regeneration.

Regenerated myelinated axon counts werehigher in the DMSO groups in our study, whichcould represent further evidence of acceleratednerve regeneration, as perfect nerve recoverydepends upon the balance between Walleriandegeneration and nerve regeneration. Myelinatedaxon counts were found to be higher in the DMSO-L group than in the other groups. This result sup-ports the previous findings of the study that localadministration of the drug is more useful for fineperipheral nerve regeneration in rats.

The electrophysiological findings are anotherimportant indicator of the functional recovery ofperipheral nerve injury in the present study. Thevariations of CMAP and CV levels between andwithin the groups during nerve regeneration showthat local administration of DMSO may morestrongly contribute to axonal transmission. It couldbe due to large regenerated myelinated axon countsin both DMSO groups.

Both DMSO groups showed higher PPT andTST results than the sham group at the end of thestudy. Functional recovery after an axonotmesisinvolves not only the number of regenerated axonsbut also the correct guidance of the axons towardtheir appropriate targets [32]. The functional testsalso demonstrated that local or systemic administra-tion of DMSO increases the rate of functional axonalregeneration. However, DMSO is more effectivewhen applied locally. Clinical studies have demon-strated that high-dose systemic DMSO producesvarious adverse effects, such as nausea, vomiting,flushing, fever, chills, dyspnea, cardiac symptoms,

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Journal of Investigative Surgery

transient hypertension, hypotension, anaphylaxis,encephalopathy, amnesia, and seizures [33].Therefore, local administration of the drug may bebetter than systemic use.

Rats could not followed up for long term (e.g., 1year) in the present study because of some technical,financial and ethical problems. Therefore, furtherdetailed long term experimental and clinical studiescould be done in the future.

CONCLUSION

Our study shows that, although DMSO did noteffect S-100 protein expression in the myelin sheath,it did accelerate functional peripheral nerve healingin a rat model by increasing MBP and NGF expres-sion. Moreover, systemic and local DMSO mayimprove nerve healing by reducing inflammationand oxidative stress. However, local application ofDMSO seems to be much more effective than intra-peritoneal administration and offers a safe, simple,and quick method of use.

DECLARATION OF INTEREST

None for Elif Sanli, Gungor Cagdas Dincel and EbruUmay. The authors have read the Journal's positionon issues involved in ethical publication.

FUNDING

The study was funded by the Scientific ResearchProject Fund of our university.

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