Research ArticleEffect of Frankincense Extract on Nerve Recovery inthe Rat Sciatic Nerve Damage Model
Xiaowen Jiang Jun Ma Qingwei Wei Xinxin Feng Lu Qiao Lin LiuBinqing Zhang and Wenhui Yu
Department of Veterinary Medicine Northeast Agricultural University 59 Mucai Street Xiangfang District Harbin 150030 China
Correspondence should be addressed to Wenhui Yu yuwenhuineaueducn
Received 2 December 2015 Revised 31 January 2016 Accepted 17 March 2016
Academic Editor Kamal D Moudgil
Copyright copy 2016 Xiaowen Jiang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
This study investigated the effect of frankincense extract on peripheral nerve regeneration in a crush injury rat model Forty-eightSprague-Dawley rats were randomly divided into four groups control and frankincense extract low- medium- and high-dosegroups At days 7 14 21 and 28 following the surgery nerve regeneration and functional recovery were evaluated using the sciaticfunctional index (SFI) expression of GAP-43 and the proliferation of Schwann cells (SCs) in vivo and in vitro At day 7 the SFIin the frankincense extract high-dose group was significantly improved compared with the control group After day 14 SFI wassignificantly improved in the medium- and high-dose groups There was no significant difference in GAP-43 expression amongthe groups at day 7 However after day 14 expression of GAP-43 in the high-dose group was higher than that in the control groupHistological evaluation showed that the injured nerve of frankincense extract high-dose group recovered better than the othergroups 28 days after surgery Further S100 immunohistochemical stainingMTT colorimetry and flow cytometry assays all showedthat frankincense extract could promote the proliferation of SCs In conclusion frankincense extract is able to promote sciatic nerveregeneration and improve the function of a crushed sciatic nerve This study provides a new direction for the repair of peripheralnerve injury
1 Introduction
Posttraumatic peripheral nerve repair is a major challengein restorative medicine Peripheral nerve injuries may resultin temporary or life-long neuronal dysfunction that canlead to economic or social disability [1 2] In recent yearspharmacologic agents and immune system modulators havebeen investigated to enhance nerve regeneration [3ndash9] Mosttreatments for peripheral nerve injury achieve recovery to agreat extent in animal models [10] but there are few effectiveclinical drug treatments availableThemost common types ofperipheral axon injuries are lacerations as well as stretch andcompression injuries [2] Following peripheral nerve injuryWallerian degeneration occurs a process of acute myelin andaxonal degeneration in the distal area of the damaged nerveThis process is in connection with macrophage infiltrationSchwan cell proliferation and axonal regrowth [11]
Traditional Chinese medicine has a long history andrich experience in the treatment of peripheral nerve injury
Fewer side effects and its effectiveness for multiple targetshave led to the increasing importance of traditional Chinesemedicine for promoting peripheral nerve regeneration [12ndash14] Frankincense is a fragrant gum from trees of the genusBoswellia (family Burseraceae) and is produced primarilyfrom several varieties found in Somalia Yemen and OmanIt is commonly used to reduce swelling and alleviate the paincaused by inflammatory diseases or tumors and to invigorateblood circulation [15 16] Moreover the boswellic acids iso-lated from frankincense have potential immunomodulatoryeffects [17 18]
In the present study we evaluated the effects of frankin-cense on peripheral nerve regeneration using an establishedrat sciatic nerve injurymodelWe investigated functional andhistologic changes following a sciatic nerve crush injury Therelative expression of GAP-43 was investigated as well as thelevel of SCs The present study provides a theoretical basisfor the development of natural drugs for the treatment ofperipheral nerve injury
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 3617216 8 pageshttpdxdoiorg10115520163617216
2 Evidence-Based Complementary and Alternative Medicine
2 Materials and Methods
21 Animals and Grouping A total of 48 healthy adultmale Sprague-Dawley rats with body weights of 200ndash250 g(purchased from Harbin Medical University) were used inthis studyThe rats weremaintained under specific pathogen-free laboratory conditions on a 12 h lightdark cycle with freeaccess to food andwaterThe rats were randomly divided intofour groups control (119899 = 12) frankincense extract high-dose(119899 = 12) frankincense extract medium-dose (119899 = 12) andfrankincense extract low-dose (119899 = 12)
22 Animal Models The rats were anesthetized with anintraperitoneal injection of 10 chloral hydrate (3mLkg)and then shaved and washed with antiseptic solution beforepositioning for surgery Surgery was performed by clampingthe left sciatic nerve 3 times for 10 s each using 2mm widepincers The distal injury was marked with a 10-0 nylonmicroscopic suture in the epineurium Complete crush wasconfirmed by the presence of a translucent band across thenerve The incision was then closed in layers (muscle andskin) with absorbable sutures All operations were performedon the left hindlimb and the right limb served as a nonop-erated control All surgeries were completed by one personunder aseptic conditions
23 Drug-Delivery Method and Dose The rats were givenfrankincense extract (purchased from Xirsquoan Xu-Huang Bio-Tech Co Ltd) via intragastric administration on the firstday after surgery high-dose group 300mgkgmedium-dosegroup 150mgkg low-dose group 75mgkg The controlgroup was given an equivalent volume of distilled water
24 Observed Index and Methods
241 Sciatic Functional Index Evaluation of the sciatic func-tional index (SFI) was performed on days 7 14 21 and 28following surgery The rats were put on a confined walkingtrack (10 cm times 50 cm) with a dark shelter at one end Whiteoffice paper was placed on the bottom of the track The ratrsquoshindlimbs were dipped in water-soluble blue ink and therat was allowed to walk down the track leaving its hindfootprints on the paper The following measurements weretaken from the footprints (1) the print length (2) the toespread and (3) the intermediary toe spread Using these datathe SFI was calculated using the following formula derived byBain et al [19] (E sciatic nerve crush side N normal side)functional recovery was assessed by calculating the SFI ratiowith the SFI value of 0 set as normal and the SFI value of minus100set as complete injury
SFI = 1095 (ETS minus NTS)NTS
minus
383 (EPL minusNPL)NPL
+
133 (EIT minusNIT)NIT
minus 88
(1)
242 The Expression of GAP-43 in the Injured Nerve Asample of crushed nerve (about 1 cm) was excised on days7 14 21 and 28 following surgery Samples containing
30 120583g of total protein from the injured nerve were subjectedto 12 SDS-PAGE electroblotted onto PVDF membranes(Biosharp) and then blocked with 5 nonfat milk for2 h at 20∘C The membranes with the transferred pro-teins were incubated with the rabbit anti-GAP-43 primaryantibody (1 400 Beijing Biosynthesis Biotechnology CoLtd) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1 1000) asthe secondary antibody Chemiluminescence reaction wascarried out with an ECL kit (Biosharp) for 1min followedby exposure to a Kodak X-Omat radiographic film Similarprocedures were carried out with an anti-120573-actin antibody(1 1000 Beijing ZSGB-Biotechnology Co Ltd) Relativeexpression was calculated as the ratio of the gray value of thetarget protein band to an internal reference band
243 Immunohistochemical Analysis Immunohistochemi-cal analysis of the sciatic nerves was performed on day 28The distal segment of sciatic nerve was removed and fixedin a 4 paraformaldehyde solution for 48 h conventionallydehydrated cleared and embedded in paraffin 4 120583m thickThen dewaxed paraffin sections were treated with 3 hydro-gen peroxide for 20min to block endogenous peroxidasesAfter threewashes the sliceswere repaired for 8min in 001Mcitrate buffer (pH 60) in a microwave The sections wereblocked with bovine serum albumin (BSA) for 20min atroom temperature and incubated at 4∘C overnight with anti-S100B (1 200 Beijing Biosynthesis Biotechnology Co Ltd)The following day the slices were washed with phosphate-buffered saline and then a secondary antibody labeled withbiotin (Beijing ZSGB-Biotechnology Co Ltd) was addedStreptavidin labeled with horseradish peroxidase was addedand slices were incubated at room temperature for 20minThe antigens were visualized with 331015840-diaminobenzidineThe sections were then redyed with hematoxylin for 20 sand fixed with neutral balsam Sections were processed withImage-Pro Plus 60 software
244 Hematoxylin-Eosin Staining At 28 days after surgerythe sciatic nerve at the distal anastomotic site was removedand fixed in 10 neutral formalin for 24 hours dehydratedthrough a graded alcohol series embedded in paraffinand cut into 4 120583m transverse sections The sections weredewaxed with xylene hydrated through a graded alcoholseries stained with hematoxylin treated with acidic alcoholimmersed in running water stained with eosin dehydratedpermeabilized and mounted The tissue was viewed undera light microscope to examine myelin degeneration andregeneration
245 Effect of Frankincense Extract on SCs Proliferation SCsat the 5th passage (purchased from Chinese Academy ofSciences Shanghai China) were used for this experimentCells were randomly divided into experimental and controlgroups Concentrations of frankincense extract in the culturemedium of the experimental groups were 625 125 250500 1000 and 2000mgL Medium of the control groupcontained no frankincense extract SCs (180120583L of 1times 106mL)of every group were placed in a 96-well culture plate with
Evidence-Based Complementary and Alternative Medicine 3
Table 1 Concentration and time effect of frankincense extract on SCs (119899 = 6 119909 plusmn 119904)
Group Concentration The proliferation of SCs (119863490 nm)mgL 12 h 24 h 48 h 72 h 96 h
lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
DMEMcontaining 10 fetal bovine serumThen 20 120583LMTT(5mgmL) was added to each well for 4 h The liquid wasremoved and 200120583L of dimethyl sulfoxide was added onan oscillator for 10min The absorbance of each pore wasdetected at the wavelength of 490 nm in an enzyme-linkedimmunosorbent assay meter
246 Effect of Frankincense Extract on the Cell Cycle ofSCs The same experimental groups as above were usedMedium containing frankincense extract was added to theexperimental group and normal DMEM to the control groupAfter culturing for 48 h SCs of every group were harvestedwashed with phosphate-buffered saline and fixed in 70ethanol overnight Cells were stained with propidium iodide(1mL106 cells) and incubated for 30min at 37∘C The cellcycle of SCs was detected by flow cytometry and analyzedusing Modfit LT software
3 Results
31 SFI The effect of frankincense extract on the SFI wascalculated on days 7 14 21 and 28 after sciatic nerve crushinjury All the groups showed a gradual recovery of sciaticfunction but the sciatic function was worse in the controlgroup (Figure 1) At days 14 and 21 comparing to the controlgroup the neurological function in the frankincense extracthigh-dose group was significantly improved (119901 lt 005) Atday 28 the neurological function was significantly improvedin the medium-dose group and high-dose group comparedwith the control group (119901 lt 005) But there was no obviousdifference in the low-dose group at any time point
32 The Expression of GAP-43 As shown in Figure 2 therewas no obvious difference in the low-dose group comparedwith the control group But at days 14 21 and 28 the expres-sion of GAP-43 in frankincense extract high-dose group wassignificantly higher than control group (119901 lt 005) At day28 comparing to the control group GAP-43 expression wasmuch higher in frankincense extract medium-dose group(119901 lt 001)
33 Immunohistochemical Analysis Expression of S100 infrankincense extract medium- and high-dose groups wassignificantly higher than that in the control group at all timepoints but there was no obvious difference in the low-dosegroup (Figure 3)
SFI
minus100
minus80
minus60
minus40
minus20
0
Days after surgery7d 14d 21d 28d
lowast
lowast
lowastlowastlowastlowast
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
Figure 1 Effects of frankincense extract on the sciatic functionindex 7 14 21 and 28 days after surgery lowastSignificant differencecompared with the control 119901 lt 005 lowastlowastSignificant differencecompared with the control 119901 lt 001
34 Histological Changes in Injured Sciatic Nerve In thecontrol group 28 days after surgery the nerve fiber atinjury site was reconnecting There was distinct Walleriandegeneration and myelin sheath disintegration Vacuoleswere present in nerve fibers and spindle cells proliferated Infrankincense extract low-dose group the nerve fiber at injurysite was reconnecting the adjacent axon degenerated myelinsheath collapsed vacuoles were seen in nerve fibers and SCsproliferated No significant differences were observed in thehistology of the injured sciatic nerve between the low-dosegroup and medium-dose group But in high-dose group 28days after surgery the injured nerve recovered better than theother groups and SCs proliferated obviously (Figure 4)
35 Effect of Frankincense Extract on SCs Less than 48 h afterfrankincense extract treatment frankincense extract couldpromote the proliferation of SCs in a concentration range of625ndash2000mgL (Table 1) Compared with the control groupthere was a significant difference in concentrations rangingfrom 125 to 2000mgL Seventy-two hours after treatment
4 Evidence-Based Complementary and Alternative Medicine
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
lowast
lowastlowast
lowastlowast
GA
P-43
relat
ive e
xpre
ssio
n le
vel
10
08
06
04
02
00
GAP-43
a b c d a b c d a b c d a b c d7d 14d 21d 28d
120573-actin 43kDa
46kDa
Figure 2 Effects of frankincense extract on GAP-43 relative expression level 7 14 21 and 28 days after surgery (a) Frankincense extractcontrol group (b) Frankincense extract low-dose group (c) Frankincense extract medium-dose group (d) Frankincense extract high-dosegroup lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
Table 2 Effect of frankincense extract on the cell cycle of SCs (119899 = 6119909 plusmn 119904 )
500 4867 plusmn 279 2366 plusmn 138lowastlowast 2767 plusmn 353lowastlowastlowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificantdifference compared with the control 119901 lt 001
frankincense extract could promote the proliferation of SCsin concentrations of 125 and 250mgL The result indicatesthat it could promote the proliferation of SCs less than 48 hafter frankincense extract treatment
36 Effect of Frankincense Extract on the Cell Cycle of SCsThe percentage of SCs treated by frankincense extract in theS phase and G
2M phase was significantly higher than in the
control group (Figure 5 Table 2)
4 Discussion
The sciatic nerve crush injury model is a relatively mildnerve injury that is constantly used in studies of nerveregeneration [20ndash22] The SFI is one standard for evaluatingmotor function [23] and it can reflect nerve function after
peripheral nerve injury In our study all groups showeda gradual recovery of sciatic function At day 7 SFI inthe frankincense extract high-dose group was significantlyimproved compared with the control group At days 14 21and 28 the neurological function was significantly improvedin the medium- and high-dose groups compared with thecontrol group However there was no significant differencebetween the low-dose group and the control group at alltime points These results suggest that frankincense extractcan accelerate functional recovery following injury in a dose-dependent manner What is more histological evaluationshowed 28 days after surgery that the injured nerve offrankincense extract high-dose group recovered better thanthe other groups obviously
GAP-43 was originally found in neurons and its expres-sion is particularly high during axonal growth and dur-ing development and regeneration in both the central andperipheral nervous systems [24] During the course of neu-ronal development or regeneration the expression of GAP-43 varies over a 100-fold range from very low levels inresting neurons to high levels in cells undergoing axogenesisor synaptic remodeling [24 25] In the present study wefound that high-dose frankincense extract treatment effec-tively elevated the expression of GAP-43 in the crushednerve on days 14 21 and 28 after surgery compared withdistilled water treatment Thus we postulate that frank-incense extract promotes the regeneration and functionalrecovery of the injured sciatic nerves by upregulatingGAP-43expression
Evidence-Based Complementary and Alternative Medicine 5
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
(m) (n) (o) (p)
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
AOD
150
100
50
0
lowastlowast
lowastlowastlowastlowast
lowastlowast
(q)
Figure 3 S100 expression in rat sciatic nerve (magnification 400x) (a) Control group 7 d (bndashd) Frankincense extract low- medium- andhigh-dose groups 7 d (e) Control group 14 d (fndashh) Frankincense extract low- medium- and high-dose groups 14 d (i) Control group 21 d(jndashl) Frankincense extract low- medium- and high-dose groups 21 d (m) Control group 28 d (nndashp) Frankincense extract low- medium-and high-dose groups 28 d (q) S100-positive cells in normal and crushed nerves lowastlowastSignificant difference comparedwith the control119901 lt 001
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
2 Evidence-Based Complementary and Alternative Medicine
2 Materials and Methods
21 Animals and Grouping A total of 48 healthy adultmale Sprague-Dawley rats with body weights of 200ndash250 g(purchased from Harbin Medical University) were used inthis studyThe rats weremaintained under specific pathogen-free laboratory conditions on a 12 h lightdark cycle with freeaccess to food andwaterThe rats were randomly divided intofour groups control (119899 = 12) frankincense extract high-dose(119899 = 12) frankincense extract medium-dose (119899 = 12) andfrankincense extract low-dose (119899 = 12)
22 Animal Models The rats were anesthetized with anintraperitoneal injection of 10 chloral hydrate (3mLkg)and then shaved and washed with antiseptic solution beforepositioning for surgery Surgery was performed by clampingthe left sciatic nerve 3 times for 10 s each using 2mm widepincers The distal injury was marked with a 10-0 nylonmicroscopic suture in the epineurium Complete crush wasconfirmed by the presence of a translucent band across thenerve The incision was then closed in layers (muscle andskin) with absorbable sutures All operations were performedon the left hindlimb and the right limb served as a nonop-erated control All surgeries were completed by one personunder aseptic conditions
23 Drug-Delivery Method and Dose The rats were givenfrankincense extract (purchased from Xirsquoan Xu-Huang Bio-Tech Co Ltd) via intragastric administration on the firstday after surgery high-dose group 300mgkgmedium-dosegroup 150mgkg low-dose group 75mgkg The controlgroup was given an equivalent volume of distilled water
24 Observed Index and Methods
241 Sciatic Functional Index Evaluation of the sciatic func-tional index (SFI) was performed on days 7 14 21 and 28following surgery The rats were put on a confined walkingtrack (10 cm times 50 cm) with a dark shelter at one end Whiteoffice paper was placed on the bottom of the track The ratrsquoshindlimbs were dipped in water-soluble blue ink and therat was allowed to walk down the track leaving its hindfootprints on the paper The following measurements weretaken from the footprints (1) the print length (2) the toespread and (3) the intermediary toe spread Using these datathe SFI was calculated using the following formula derived byBain et al [19] (E sciatic nerve crush side N normal side)functional recovery was assessed by calculating the SFI ratiowith the SFI value of 0 set as normal and the SFI value of minus100set as complete injury
SFI = 1095 (ETS minus NTS)NTS
minus
383 (EPL minusNPL)NPL
+
133 (EIT minusNIT)NIT
minus 88
(1)
242 The Expression of GAP-43 in the Injured Nerve Asample of crushed nerve (about 1 cm) was excised on days7 14 21 and 28 following surgery Samples containing
30 120583g of total protein from the injured nerve were subjectedto 12 SDS-PAGE electroblotted onto PVDF membranes(Biosharp) and then blocked with 5 nonfat milk for2 h at 20∘C The membranes with the transferred pro-teins were incubated with the rabbit anti-GAP-43 primaryantibody (1 400 Beijing Biosynthesis Biotechnology CoLtd) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1 1000) asthe secondary antibody Chemiluminescence reaction wascarried out with an ECL kit (Biosharp) for 1min followedby exposure to a Kodak X-Omat radiographic film Similarprocedures were carried out with an anti-120573-actin antibody(1 1000 Beijing ZSGB-Biotechnology Co Ltd) Relativeexpression was calculated as the ratio of the gray value of thetarget protein band to an internal reference band
243 Immunohistochemical Analysis Immunohistochemi-cal analysis of the sciatic nerves was performed on day 28The distal segment of sciatic nerve was removed and fixedin a 4 paraformaldehyde solution for 48 h conventionallydehydrated cleared and embedded in paraffin 4 120583m thickThen dewaxed paraffin sections were treated with 3 hydro-gen peroxide for 20min to block endogenous peroxidasesAfter threewashes the sliceswere repaired for 8min in 001Mcitrate buffer (pH 60) in a microwave The sections wereblocked with bovine serum albumin (BSA) for 20min atroom temperature and incubated at 4∘C overnight with anti-S100B (1 200 Beijing Biosynthesis Biotechnology Co Ltd)The following day the slices were washed with phosphate-buffered saline and then a secondary antibody labeled withbiotin (Beijing ZSGB-Biotechnology Co Ltd) was addedStreptavidin labeled with horseradish peroxidase was addedand slices were incubated at room temperature for 20minThe antigens were visualized with 331015840-diaminobenzidineThe sections were then redyed with hematoxylin for 20 sand fixed with neutral balsam Sections were processed withImage-Pro Plus 60 software
244 Hematoxylin-Eosin Staining At 28 days after surgerythe sciatic nerve at the distal anastomotic site was removedand fixed in 10 neutral formalin for 24 hours dehydratedthrough a graded alcohol series embedded in paraffinand cut into 4 120583m transverse sections The sections weredewaxed with xylene hydrated through a graded alcoholseries stained with hematoxylin treated with acidic alcoholimmersed in running water stained with eosin dehydratedpermeabilized and mounted The tissue was viewed undera light microscope to examine myelin degeneration andregeneration
245 Effect of Frankincense Extract on SCs Proliferation SCsat the 5th passage (purchased from Chinese Academy ofSciences Shanghai China) were used for this experimentCells were randomly divided into experimental and controlgroups Concentrations of frankincense extract in the culturemedium of the experimental groups were 625 125 250500 1000 and 2000mgL Medium of the control groupcontained no frankincense extract SCs (180120583L of 1times 106mL)of every group were placed in a 96-well culture plate with
Evidence-Based Complementary and Alternative Medicine 3
Table 1 Concentration and time effect of frankincense extract on SCs (119899 = 6 119909 plusmn 119904)
Group Concentration The proliferation of SCs (119863490 nm)mgL 12 h 24 h 48 h 72 h 96 h
lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
DMEMcontaining 10 fetal bovine serumThen 20 120583LMTT(5mgmL) was added to each well for 4 h The liquid wasremoved and 200120583L of dimethyl sulfoxide was added onan oscillator for 10min The absorbance of each pore wasdetected at the wavelength of 490 nm in an enzyme-linkedimmunosorbent assay meter
246 Effect of Frankincense Extract on the Cell Cycle ofSCs The same experimental groups as above were usedMedium containing frankincense extract was added to theexperimental group and normal DMEM to the control groupAfter culturing for 48 h SCs of every group were harvestedwashed with phosphate-buffered saline and fixed in 70ethanol overnight Cells were stained with propidium iodide(1mL106 cells) and incubated for 30min at 37∘C The cellcycle of SCs was detected by flow cytometry and analyzedusing Modfit LT software
3 Results
31 SFI The effect of frankincense extract on the SFI wascalculated on days 7 14 21 and 28 after sciatic nerve crushinjury All the groups showed a gradual recovery of sciaticfunction but the sciatic function was worse in the controlgroup (Figure 1) At days 14 and 21 comparing to the controlgroup the neurological function in the frankincense extracthigh-dose group was significantly improved (119901 lt 005) Atday 28 the neurological function was significantly improvedin the medium-dose group and high-dose group comparedwith the control group (119901 lt 005) But there was no obviousdifference in the low-dose group at any time point
32 The Expression of GAP-43 As shown in Figure 2 therewas no obvious difference in the low-dose group comparedwith the control group But at days 14 21 and 28 the expres-sion of GAP-43 in frankincense extract high-dose group wassignificantly higher than control group (119901 lt 005) At day28 comparing to the control group GAP-43 expression wasmuch higher in frankincense extract medium-dose group(119901 lt 001)
33 Immunohistochemical Analysis Expression of S100 infrankincense extract medium- and high-dose groups wassignificantly higher than that in the control group at all timepoints but there was no obvious difference in the low-dosegroup (Figure 3)
SFI
minus100
minus80
minus60
minus40
minus20
0
Days after surgery7d 14d 21d 28d
lowast
lowast
lowastlowastlowastlowast
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
Figure 1 Effects of frankincense extract on the sciatic functionindex 7 14 21 and 28 days after surgery lowastSignificant differencecompared with the control 119901 lt 005 lowastlowastSignificant differencecompared with the control 119901 lt 001
34 Histological Changes in Injured Sciatic Nerve In thecontrol group 28 days after surgery the nerve fiber atinjury site was reconnecting There was distinct Walleriandegeneration and myelin sheath disintegration Vacuoleswere present in nerve fibers and spindle cells proliferated Infrankincense extract low-dose group the nerve fiber at injurysite was reconnecting the adjacent axon degenerated myelinsheath collapsed vacuoles were seen in nerve fibers and SCsproliferated No significant differences were observed in thehistology of the injured sciatic nerve between the low-dosegroup and medium-dose group But in high-dose group 28days after surgery the injured nerve recovered better than theother groups and SCs proliferated obviously (Figure 4)
35 Effect of Frankincense Extract on SCs Less than 48 h afterfrankincense extract treatment frankincense extract couldpromote the proliferation of SCs in a concentration range of625ndash2000mgL (Table 1) Compared with the control groupthere was a significant difference in concentrations rangingfrom 125 to 2000mgL Seventy-two hours after treatment
4 Evidence-Based Complementary and Alternative Medicine
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
lowast
lowastlowast
lowastlowast
GA
P-43
relat
ive e
xpre
ssio
n le
vel
10
08
06
04
02
00
GAP-43
a b c d a b c d a b c d a b c d7d 14d 21d 28d
120573-actin 43kDa
46kDa
Figure 2 Effects of frankincense extract on GAP-43 relative expression level 7 14 21 and 28 days after surgery (a) Frankincense extractcontrol group (b) Frankincense extract low-dose group (c) Frankincense extract medium-dose group (d) Frankincense extract high-dosegroup lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
Table 2 Effect of frankincense extract on the cell cycle of SCs (119899 = 6119909 plusmn 119904 )
500 4867 plusmn 279 2366 plusmn 138lowastlowast 2767 plusmn 353lowastlowastlowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificantdifference compared with the control 119901 lt 001
frankincense extract could promote the proliferation of SCsin concentrations of 125 and 250mgL The result indicatesthat it could promote the proliferation of SCs less than 48 hafter frankincense extract treatment
36 Effect of Frankincense Extract on the Cell Cycle of SCsThe percentage of SCs treated by frankincense extract in theS phase and G
2M phase was significantly higher than in the
control group (Figure 5 Table 2)
4 Discussion
The sciatic nerve crush injury model is a relatively mildnerve injury that is constantly used in studies of nerveregeneration [20ndash22] The SFI is one standard for evaluatingmotor function [23] and it can reflect nerve function after
peripheral nerve injury In our study all groups showeda gradual recovery of sciatic function At day 7 SFI inthe frankincense extract high-dose group was significantlyimproved compared with the control group At days 14 21and 28 the neurological function was significantly improvedin the medium- and high-dose groups compared with thecontrol group However there was no significant differencebetween the low-dose group and the control group at alltime points These results suggest that frankincense extractcan accelerate functional recovery following injury in a dose-dependent manner What is more histological evaluationshowed 28 days after surgery that the injured nerve offrankincense extract high-dose group recovered better thanthe other groups obviously
GAP-43 was originally found in neurons and its expres-sion is particularly high during axonal growth and dur-ing development and regeneration in both the central andperipheral nervous systems [24] During the course of neu-ronal development or regeneration the expression of GAP-43 varies over a 100-fold range from very low levels inresting neurons to high levels in cells undergoing axogenesisor synaptic remodeling [24 25] In the present study wefound that high-dose frankincense extract treatment effec-tively elevated the expression of GAP-43 in the crushednerve on days 14 21 and 28 after surgery compared withdistilled water treatment Thus we postulate that frank-incense extract promotes the regeneration and functionalrecovery of the injured sciatic nerves by upregulatingGAP-43expression
Evidence-Based Complementary and Alternative Medicine 5
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
(m) (n) (o) (p)
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
AOD
150
100
50
0
lowastlowast
lowastlowastlowastlowast
lowastlowast
(q)
Figure 3 S100 expression in rat sciatic nerve (magnification 400x) (a) Control group 7 d (bndashd) Frankincense extract low- medium- andhigh-dose groups 7 d (e) Control group 14 d (fndashh) Frankincense extract low- medium- and high-dose groups 14 d (i) Control group 21 d(jndashl) Frankincense extract low- medium- and high-dose groups 21 d (m) Control group 28 d (nndashp) Frankincense extract low- medium-and high-dose groups 28 d (q) S100-positive cells in normal and crushed nerves lowastlowastSignificant difference comparedwith the control119901 lt 001
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
DMEMcontaining 10 fetal bovine serumThen 20 120583LMTT(5mgmL) was added to each well for 4 h The liquid wasremoved and 200120583L of dimethyl sulfoxide was added onan oscillator for 10min The absorbance of each pore wasdetected at the wavelength of 490 nm in an enzyme-linkedimmunosorbent assay meter
246 Effect of Frankincense Extract on the Cell Cycle ofSCs The same experimental groups as above were usedMedium containing frankincense extract was added to theexperimental group and normal DMEM to the control groupAfter culturing for 48 h SCs of every group were harvestedwashed with phosphate-buffered saline and fixed in 70ethanol overnight Cells were stained with propidium iodide(1mL106 cells) and incubated for 30min at 37∘C The cellcycle of SCs was detected by flow cytometry and analyzedusing Modfit LT software
3 Results
31 SFI The effect of frankincense extract on the SFI wascalculated on days 7 14 21 and 28 after sciatic nerve crushinjury All the groups showed a gradual recovery of sciaticfunction but the sciatic function was worse in the controlgroup (Figure 1) At days 14 and 21 comparing to the controlgroup the neurological function in the frankincense extracthigh-dose group was significantly improved (119901 lt 005) Atday 28 the neurological function was significantly improvedin the medium-dose group and high-dose group comparedwith the control group (119901 lt 005) But there was no obviousdifference in the low-dose group at any time point
32 The Expression of GAP-43 As shown in Figure 2 therewas no obvious difference in the low-dose group comparedwith the control group But at days 14 21 and 28 the expres-sion of GAP-43 in frankincense extract high-dose group wassignificantly higher than control group (119901 lt 005) At day28 comparing to the control group GAP-43 expression wasmuch higher in frankincense extract medium-dose group(119901 lt 001)
33 Immunohistochemical Analysis Expression of S100 infrankincense extract medium- and high-dose groups wassignificantly higher than that in the control group at all timepoints but there was no obvious difference in the low-dosegroup (Figure 3)
SFI
minus100
minus80
minus60
minus40
minus20
0
Days after surgery7d 14d 21d 28d
lowast
lowast
lowastlowastlowastlowast
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
Figure 1 Effects of frankincense extract on the sciatic functionindex 7 14 21 and 28 days after surgery lowastSignificant differencecompared with the control 119901 lt 005 lowastlowastSignificant differencecompared with the control 119901 lt 001
34 Histological Changes in Injured Sciatic Nerve In thecontrol group 28 days after surgery the nerve fiber atinjury site was reconnecting There was distinct Walleriandegeneration and myelin sheath disintegration Vacuoleswere present in nerve fibers and spindle cells proliferated Infrankincense extract low-dose group the nerve fiber at injurysite was reconnecting the adjacent axon degenerated myelinsheath collapsed vacuoles were seen in nerve fibers and SCsproliferated No significant differences were observed in thehistology of the injured sciatic nerve between the low-dosegroup and medium-dose group But in high-dose group 28days after surgery the injured nerve recovered better than theother groups and SCs proliferated obviously (Figure 4)
35 Effect of Frankincense Extract on SCs Less than 48 h afterfrankincense extract treatment frankincense extract couldpromote the proliferation of SCs in a concentration range of625ndash2000mgL (Table 1) Compared with the control groupthere was a significant difference in concentrations rangingfrom 125 to 2000mgL Seventy-two hours after treatment
4 Evidence-Based Complementary and Alternative Medicine
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
lowast
lowastlowast
lowastlowast
GA
P-43
relat
ive e
xpre
ssio
n le
vel
10
08
06
04
02
00
GAP-43
a b c d a b c d a b c d a b c d7d 14d 21d 28d
120573-actin 43kDa
46kDa
Figure 2 Effects of frankincense extract on GAP-43 relative expression level 7 14 21 and 28 days after surgery (a) Frankincense extractcontrol group (b) Frankincense extract low-dose group (c) Frankincense extract medium-dose group (d) Frankincense extract high-dosegroup lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
Table 2 Effect of frankincense extract on the cell cycle of SCs (119899 = 6119909 plusmn 119904 )
500 4867 plusmn 279 2366 plusmn 138lowastlowast 2767 plusmn 353lowastlowastlowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificantdifference compared with the control 119901 lt 001
frankincense extract could promote the proliferation of SCsin concentrations of 125 and 250mgL The result indicatesthat it could promote the proliferation of SCs less than 48 hafter frankincense extract treatment
36 Effect of Frankincense Extract on the Cell Cycle of SCsThe percentage of SCs treated by frankincense extract in theS phase and G
2M phase was significantly higher than in the
control group (Figure 5 Table 2)
4 Discussion
The sciatic nerve crush injury model is a relatively mildnerve injury that is constantly used in studies of nerveregeneration [20ndash22] The SFI is one standard for evaluatingmotor function [23] and it can reflect nerve function after
peripheral nerve injury In our study all groups showeda gradual recovery of sciatic function At day 7 SFI inthe frankincense extract high-dose group was significantlyimproved compared with the control group At days 14 21and 28 the neurological function was significantly improvedin the medium- and high-dose groups compared with thecontrol group However there was no significant differencebetween the low-dose group and the control group at alltime points These results suggest that frankincense extractcan accelerate functional recovery following injury in a dose-dependent manner What is more histological evaluationshowed 28 days after surgery that the injured nerve offrankincense extract high-dose group recovered better thanthe other groups obviously
GAP-43 was originally found in neurons and its expres-sion is particularly high during axonal growth and dur-ing development and regeneration in both the central andperipheral nervous systems [24] During the course of neu-ronal development or regeneration the expression of GAP-43 varies over a 100-fold range from very low levels inresting neurons to high levels in cells undergoing axogenesisor synaptic remodeling [24 25] In the present study wefound that high-dose frankincense extract treatment effec-tively elevated the expression of GAP-43 in the crushednerve on days 14 21 and 28 after surgery compared withdistilled water treatment Thus we postulate that frank-incense extract promotes the regeneration and functionalrecovery of the injured sciatic nerves by upregulatingGAP-43expression
Evidence-Based Complementary and Alternative Medicine 5
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
(m) (n) (o) (p)
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
AOD
150
100
50
0
lowastlowast
lowastlowastlowastlowast
lowastlowast
(q)
Figure 3 S100 expression in rat sciatic nerve (magnification 400x) (a) Control group 7 d (bndashd) Frankincense extract low- medium- andhigh-dose groups 7 d (e) Control group 14 d (fndashh) Frankincense extract low- medium- and high-dose groups 14 d (i) Control group 21 d(jndashl) Frankincense extract low- medium- and high-dose groups 21 d (m) Control group 28 d (nndashp) Frankincense extract low- medium-and high-dose groups 28 d (q) S100-positive cells in normal and crushed nerves lowastlowastSignificant difference comparedwith the control119901 lt 001
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
4 Evidence-Based Complementary and Alternative Medicine
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
lowast
lowastlowast
lowastlowast
GA
P-43
relat
ive e
xpre
ssio
n le
vel
10
08
06
04
02
00
GAP-43
a b c d a b c d a b c d a b c d7d 14d 21d 28d
120573-actin 43kDa
46kDa
Figure 2 Effects of frankincense extract on GAP-43 relative expression level 7 14 21 and 28 days after surgery (a) Frankincense extractcontrol group (b) Frankincense extract low-dose group (c) Frankincense extract medium-dose group (d) Frankincense extract high-dosegroup lowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificant difference compared with the control 119901 lt 001
Table 2 Effect of frankincense extract on the cell cycle of SCs (119899 = 6119909 plusmn 119904 )
500 4867 plusmn 279 2366 plusmn 138lowastlowast 2767 plusmn 353lowastlowastlowastSignificant difference compared with the control 119901 lt 005 lowastlowastSignificantdifference compared with the control 119901 lt 001
frankincense extract could promote the proliferation of SCsin concentrations of 125 and 250mgL The result indicatesthat it could promote the proliferation of SCs less than 48 hafter frankincense extract treatment
36 Effect of Frankincense Extract on the Cell Cycle of SCsThe percentage of SCs treated by frankincense extract in theS phase and G
2M phase was significantly higher than in the
control group (Figure 5 Table 2)
4 Discussion
The sciatic nerve crush injury model is a relatively mildnerve injury that is constantly used in studies of nerveregeneration [20ndash22] The SFI is one standard for evaluatingmotor function [23] and it can reflect nerve function after
peripheral nerve injury In our study all groups showeda gradual recovery of sciatic function At day 7 SFI inthe frankincense extract high-dose group was significantlyimproved compared with the control group At days 14 21and 28 the neurological function was significantly improvedin the medium- and high-dose groups compared with thecontrol group However there was no significant differencebetween the low-dose group and the control group at alltime points These results suggest that frankincense extractcan accelerate functional recovery following injury in a dose-dependent manner What is more histological evaluationshowed 28 days after surgery that the injured nerve offrankincense extract high-dose group recovered better thanthe other groups obviously
GAP-43 was originally found in neurons and its expres-sion is particularly high during axonal growth and dur-ing development and regeneration in both the central andperipheral nervous systems [24] During the course of neu-ronal development or regeneration the expression of GAP-43 varies over a 100-fold range from very low levels inresting neurons to high levels in cells undergoing axogenesisor synaptic remodeling [24 25] In the present study wefound that high-dose frankincense extract treatment effec-tively elevated the expression of GAP-43 in the crushednerve on days 14 21 and 28 after surgery compared withdistilled water treatment Thus we postulate that frank-incense extract promotes the regeneration and functionalrecovery of the injured sciatic nerves by upregulatingGAP-43expression
Evidence-Based Complementary and Alternative Medicine 5
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
(m) (n) (o) (p)
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
AOD
150
100
50
0
lowastlowast
lowastlowastlowastlowast
lowastlowast
(q)
Figure 3 S100 expression in rat sciatic nerve (magnification 400x) (a) Control group 7 d (bndashd) Frankincense extract low- medium- andhigh-dose groups 7 d (e) Control group 14 d (fndashh) Frankincense extract low- medium- and high-dose groups 14 d (i) Control group 21 d(jndashl) Frankincense extract low- medium- and high-dose groups 21 d (m) Control group 28 d (nndashp) Frankincense extract low- medium-and high-dose groups 28 d (q) S100-positive cells in normal and crushed nerves lowastlowastSignificant difference comparedwith the control119901 lt 001
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
Evidence-Based Complementary and Alternative Medicine 5
(a) (b) (c) (d)
(e) (f) (g) (h)
(i) (j) (k) (l)
(m) (n) (o) (p)
Days after surgery7d 14d 21d 28d
Frankincense extract control groupFrankincense extract medium-dose groupFrankincense extract low-dose groupFrankincense extract high-dose group
AOD
150
100
50
0
lowastlowast
lowastlowastlowastlowast
lowastlowast
(q)
Figure 3 S100 expression in rat sciatic nerve (magnification 400x) (a) Control group 7 d (bndashd) Frankincense extract low- medium- andhigh-dose groups 7 d (e) Control group 14 d (fndashh) Frankincense extract low- medium- and high-dose groups 14 d (i) Control group 21 d(jndashl) Frankincense extract low- medium- and high-dose groups 21 d (m) Control group 28 d (nndashp) Frankincense extract low- medium-and high-dose groups 28 d (q) S100-positive cells in normal and crushed nerves lowastlowastSignificant difference comparedwith the control119901 lt 001
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
6 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
Figure 4 Histological changes in injured sciatic nerve (magnification 40x) (a) Control group (b) Frankincense extract low-dose group (c)Frankincense extract medium-dose group (d) Frankincense extract high-dose group
SCs are glial cells in the peripheral nervous system Theyprovide a permissive environment for nerve regenerationand express multiple types of neurotrophic factors such asnerve growth factor and brain-derived neurotrophic factorto provide trophic support for axon regeneration [26 27]S100 exists only in glial cells of the central nervous systemand SCs of the peripheral nervous system We observedthe expression of S100 by immunocytochemical staining ofthe injured nerve which reflected the proliferation of SCsAs a result the expression of S100 in all the groups washigher than the control group inferring that frankincenseextract promotes the proliferation of SCs To further studythe effect of frankincense extract on SCs we treated SCswith frankincense extract of different concentrations in vitroFrankincense extract of 125 250 and 500mgL promoted theproliferation of SCs significantly and elevated the percentageof cells in S phase and G
2M phase In SCs G
0G1phase
cells were decreased and S phase and G2M phase cells
increased comparedwith control group Frankincense extractpromotes the growth and the transition from G
0G1phase
to S phase in SCs It can be inferred that the mechanismof how did frankincense extract promote proliferation ofSCs was that frankincense extract promotes the growth andthe transition from G
0G1phase to S phase in SCs It can
shorten the growth retardation stimulate the cell to enter the
active S phase from the stationary G0 phase and increasethe number in G
2M cells leading to the proliferation of
cells
5 Conclusions
Taken together these findings suggest that frankincenseextract may promote the regeneration of crushed sciaticnerve by improving the sciatic nerve function and increasingthe expression of GAP-43 as well as promoting the pro-liferation of SCs However the exact mechanism has notbeen clearly defined Further studies are needed to evaluatethe significance of frankincense extract in peripheral nerveregeneration
Competing Interests
The authors declare that they have no competing interests
Authorsrsquo Contributions
Xiaowen Jiang and Jun Ma contributed equally to this work
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
Evidence-Based Complementary and Alternative Medicine 7
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(a) Control group
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(b) 500mgL
Num
ber
600
400
200
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(c) 250mgL
Num
ber
600
400
200
500
300
100
0
Channels (PI-A)0 50 100 150 200 250
S phaseG0G1G2M
(d) 125mgL
Figure 5 Effect of frankincense extract on the cell cycle of SCs 48 h after frankincense extract treatment Schwann cells were stained with PIfor DNA content analysis by FACS Details of the experiments are given in Section 2
Acknowledgments
This study was supported by Doctoral Research Fund ofNortheast Agricultural University and Postdoctoral Fund ofHeilongjiang Province no LBH-Z11234
References
[1] E O Johnson A B Zoubos and P N Soucacos ldquoRegenerationand repair of peripheral nervesrdquo Injury vol 36 supplement 4pp S24ndashS29 2005
[2] M G Burnett and E L Zager ldquoPathophysiology of peripheralnerve injury a brief reviewrdquo Neurosurgical Focus vol 16 no 5article E1 2004
[3] A M Xavier K G G Serafim D T Higashi et al ldquoSimvastatinimproves morphological and functional recovery of sciaticnerve injury in Wistar ratsrdquo Injury vol 43 no 3 pp 284ndash2892012
[4] J Javanbakht R Hobbenaghi E Hosseini et al ldquoHistopatho-logical investigation of neuroprotective effects of Nigella sativaon motor neurons anterior horn spinal cord after sciatic nervecrush in ratsrdquo Pathologie Biologie vol 61 no 6 pp 250ndash2532013
[5] R Hobbenaghi J Javanbakht S Sadeghzadeh et al ldquoNeu-roprotective effects of Nigella sativa extract on cell death inhippocampal neurons following experimental global cerebralischemia-reperfusion injury in ratsrdquo Journal of the NeurologicalSciences vol 337 no 1-2 pp 74ndash79 2014
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
8 Evidence-Based Complementary and Alternative Medicine
[6] R Hobbenaghi J Javanbakht E Hosseini et al ldquoNeuropatho-logical andneuroprotective features of vitaminB12 on the dorsalspinal ganglion of rats after the experimental crush of sciaticnerve an experimental studyrdquo Diagnostic Pathology vol 8article 123 2013
[7] J Gao S Ma Y Ji J E Wang and J Li ldquoSciatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor an experimental studyrdquo Injury vol 39 no 12 pp1414ndash1420 2008
[8] R Mohammadi Z Esmaeil-Sani and K Amini ldquoEffect of localadministration of insulin-like growth factor I combined withinside-out artery graft onperipheral nerve regenerationrdquo Injuryvol 44 no 10 pp 1295ndash1301 2013
[9] E O Johnson A Charchanti and PN Soucacos ldquoNerve repairexperimental and clinical evaluation of neurotrophic factors inperipheral nerve regenerationrdquo Injury vol 39 supplement 3 ppS37ndashS42 2008
[10] R Deumens A Bozkurt M F Meek et al ldquoRepairing injuredperipheral nerves bridging the gaprdquo Progress in Neurobiologyvol 92 no 3 pp 245ndash276 2010
[11] S Rotshenker ldquoWallerian degeneration the innate-immuneresponse to traumatic nerve injuryrdquo Journal of Neuroinflamma-tion vol 8 article 109 2011
[12] Y Kou Z Wang Z Wu et al ldquoEpimedium extract promotesperipheral nerve regeneration in ratsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 9547986 pages 2013
[13] S Wei X Yin Y Kou and B Jiang ldquoLumbricus extractpromotes the regeneration of injured peripheral nerve in ratsrdquoJournal of Ethnopharmacology vol 123 no 1 pp 51ndash54 2009
[14] E Tamaddonfard A A Farshid E Ahmadian and A Hamid-hoseyni ldquoCrocin enhanced functional recovery after sciaticnerve crush injury in ratsrdquo Iranian Journal of Basic MedicalSciences vol 16 no 1 pp 83ndash90 2013
[15] H Safayhi T Mack J Sabieraj M I Anazodo L R Subra-manian and H P T Ammon ldquoBoswellic acids novel specificnonredox inhibitors of 5-lipoxygenaserdquo Journal of Pharmacol-ogy and ExperimentalTherapeutics vol 261 no 3 pp 1143ndash11461992
[16] A Y Fan L Lao R X Zhang et al ldquoEffects of an acetoneextract of Boswellia carterii Birdw (Burseraceae) gum resin onadjuvant-induced arthritis in lewis ratsrdquo Journal of Ethnophar-macology vol 101 no 1ndash3 pp 104ndash109 2005
[17] M L Sharma A Kaul A Khajuria S Singh and G B SinghldquoImmunomodulatory activity of boswellic acids (pentacyclictriterpene acids) fromBoswellia serratardquoPhytotherapy Researchvol 10 no 2 pp 107ndash112 1996
[18] H P T Ammon ldquoModulation of the immune system byBoswellia serrata extracts and boswellic acidsrdquo Phytomedicinevol 17 no 11 pp 862ndash867 2010
[19] J R Bain S E Mackinnon D A Hunter and S E MackinnonldquoFunctional evaluation of complete sciatic peroneal and pos-terior tibial nerve lesions in the ratrdquo Plastic and ReconstructiveSurgery vol 83 no 1 pp 129ndash136 1989
[20] M Jiang X Zhuge Y Yang X Gu and F Ding ldquoThe promotionof peripheral nerve regeneration by chitooligosaccharides in therat nerve crush injurymodelrdquoNeuroscience Letters vol 454 no3 pp 239ndash243 2009
[21] H Wang J Fang F Hu G Li and H Hong ldquoSeawaterimmersion aggravates sciatic nerve injury in ratsrdquo Experimentaland Therapeutic Medicine vol 9 no 4 pp 1153ndash1160 2015
[22] Y Gong L Gong X Gu and F Ding ldquoChitooligosaccharidespromote peripheral nerve regeneration in a rabbit commonperoneal nerve crush injury modelrdquo Microsurgery vol 29 no8 pp 650ndash656 2009
[23] A Schiaveto De Souza C A da Silva and E A Del BelldquoMethodological evaluation to analyze functional recovery aftersciatic nerve injuryrdquo Journal of Neurotrauma vol 21 no 5 pp627ndash635 2004
[24] J H P Skene ldquoAxonal growth-associated proteinsrdquo AnnualReview of Neuroscience vol 12 pp 127ndash156 1989
[25] L I Benowitz and A Routtenberg ldquoGAP-43 an intrinsicdeterminant of neuronal development and plasticityrdquo Trends inNeurosciences vol 20 no 2 pp 84ndash91 1997
[26] S P Frostick Q Yin and G J Kemp ldquoSchwann cells neu-rotrophic factors and peripheral nerve regenerationrdquo Micro-surgery vol 18 no 7 pp 397ndash405 1998
[27] D D Pearse F C Pereira A E Marcillo et al ldquocAMP andSchwann cells promote axonal growth and functional recoveryafter spinal cord injuryrdquoNature Medicine vol 10 no 6 pp 610ndash616 2004
