research article amelioration of behavioural, biochemical...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 565813 8 pageshttpdxdoiorg1011552013565813

Research ArticleAmelioration of Behavioural Biochemical andNeurophysiological Deficits by Combination of MonosodiumGlutamate with ResveratrolAlpha-Lipoic AcidCoenzymeQ10 in Rat Model of Cisplatin-Induced Peripheral Neuropathy

Naini Bhadri Tejaswi Sanji Hariprasad Madakasira Guggilla and Rema Razdan

Department of Pharmacology Al-Ameen College of Pharmacy Bangalore Karnataka-560027 India

Correspondence should be addressed to Naini Bhadri nainipharmacologygmailcom

Received 31 August 2013 Accepted 2 October 2013

Academic Editors J-T Cheng and S Wu

Copyright copy 2013 Naini Bhadri 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

Cisplatin or cis-diamminedichloroplatinum (II) (CDDP) is a cytotoxic chemotherapeutic agent with dose-dependent peripheralneuropathy as a foremost side effect characterised by ataxia pain and sensory impairment Cumulative drug therapy of CDDPis known to produce severe oxidative damage It mainly targets and accumulates in dorsal root ganglia that in turn cause damageresulting in secondary nerve fibre axonopathy In the present study we investigated the neuroprotective effect of the combination ofmonosodium glutamate (MSG) with three individual antioxidants that is resveratrol alpha-lipoic acid (ALA) and coenzyme Q10(CoQ10) in cisplatin (2mgkg ip twice weekly) induced peripheral neuropathy in rats After 8 weeks of treatment the degree ofneuroprotection was determined by measuring behavioral and electrophysiological properties and sciatic nerve lipid peroxidationas well as glutathione and catalase levelsThe results suggested that pretreatment with the combination ofMSG (500mgkgday po)with resveratrol (10mgkgday ip) or ALA (20mgkgday ip) or CoQ10 (10mgkg weekly thrice ip) exhibited neuroprotectiveeffect The maximum neuroprotection of MSG was observed in the combination with resveratrol

1 Introduction

Cisplatin is an effective antineoplastic drugwidely used in thetreatment of malignancies including ovarian lung oesopha-gus stomach bladder and testicular cancers However its useis limited by its serious adverse effects including peripheralneuropathy and nephrotoxicity [1]The reported incidence ofneuropathy varies between 24 and 92 [2] Characteristicfeatures of cisplatin neurotoxicity aremainly ataxia pain andsensory impairment Symptoms are prominent in patientsreceiving cumulative doses of cisplatin above 300mgm2 or500mgm2 [3] About 20 of patients have shown poorcompliance for the course of cisplatin therapy attributable tosensory neuropathy [4]Themechanism of cisplatin-inducedneuropathy is not completely understood it is preferentiallytaken up in to dorsal root ganglia and is believed to affectthe stem of the nerves leading to loss of sensory neuronsaffecting large myelinated sensory nerve fibers [5] Howeverdata obtained in human studies indicates that cisplatin

treatment induces a fall in plasma antioxidant level becauseof oxidative stress [6] several mechanism including hypoxiainflammation and free radicals induced apoptosis are thoughtto be involved Excessive production of free radicals such assuperoxide anion hydrogen peroxide and hydroxyl radicalsand the occurrence of lipid per oxidation due to oxidativestress are associated with cisplatin-induced neuropathy

Resveratrol (3541015840-trihydroxystilbene) a naturally occur-ring polyphenol found in grapes and red wine has beenreported to have excellent antioxidant activity It not only pre-vents free radicals formation but also attenuates their toxicityby inhibiting the lipid per oxidation [7] It has shown to exertneuroprotective cancer chemopreventive anti-inflammato-ry and cardioprotective activities [8]

Coenzyme Q10 (CoQ10) or Ubiquinone is an oil-solublevitamin-like substance present in almost all eukaryotic cellsprimarily in the mitochondria It is a component of the elec-tron transport chain and participates in aerobic cellular res-piration generating energy in the form of ATP [9] In recent

2 The Scientific World Journal

years research also focused on its antioxidant properties inseveral cellular compartments [10] CoQ10 has shown protec-tive effect against neuronal loss in several animal models ofneurodegeneration and the ability of CoQ10 to slow diseaseprogression in clinical trials has been considered to exploreneuroprotective activity in many of the neurodegenerativediseases [11]

Alpha-lipoic acid or 12-dithiolane-3-pentanoic acid(ALA) is a naturally available dithiol compound synthesizedenzymatically in the mitochondrion from octanoic acid [12]ALA reacts with reactive oxygen species such as superoxideradicals hydroxyl radicals hypochlorous acid peroxyl rad-icals and singlet oxygen Administration of ALA has beenshown to be beneficial in several oxidative stress models suchas ischemia-reperfusion injury diabetes cataract formationHIV activation neurodegeneration and radiation injury [13]

Monosodium glutamate (MSG) is the sodium salt ofglutamic acid It is widely used as food additive mainly asflavour enhancer and it is believed to be safe without seriousconcerns It contributes an important role in the biosynthesisof several key amino acids [14] Evidence from clinical andpreclinical studies revealed that it is a major oxidative fuel forthe gut and that dietary glutamate extensively undergoes firstpass metabolism by the intestine It is an important precursorfor synthesis of bioactive molecules including glutathionean endogenous antioxidant and also functions as a keyneurotransmitter [15] Partial investigation ofMSG in clinicaltrial as a neuroprotectant in the early 1980s has shown efficacyin preventing neuropathy induced by vincristine [16] A newclass of glutamate receptors named metabotropic glutamatereceptors (mGluRs) that are coupled to effector systemsthrough GTP proteins (G-proteins) became evident by themajority of most recent research activities Activation of(mGluRs) subtypes usually regulates many cellular activitiesin mammalian systems in various experimental modelsActivation of (mGluRs) subtypes protected cells againstvarious types of neuronal insults such as traumatic braininjury stroke and NMDA excitotoxicity [17] Interestinglyin recent years glutamate has been reported to be efficientin preventing neuropathy caused by many cytotoxic drugs inmice rats and humans with no orminimal apparent intrinsictoxicity and its low cost and oral availability make it attractivefor clinical use as a neuroprotectant [18]

In the present study we further explored its potential inrodents for its neuroprotective activity in presence of otherantioxidants namely resveratrol coenzyme Q10 and ALAin rat model of cisplatin-induced peripheral neuropathy byassessing behavioral biochemical and neurophysiologicalparameters

2 Materials and Methods

21 Reagents All reagents and solvents used were of ana-lytical grade Cisplatin (2mgkg ip twice weekly) was usedto induce peripheral neuropathy by intraperitoneal injection[19] Aqueous solution of MSG (Himadia Labs India) wasused at 500mgkgday po dose [18] Resveratrol (Sami LabsIndia) solution was freshly prepared using 2 vv ethanol ata dose of 10mgkgday ip [7] Suspension of ALA (Sami

Labs India) in sterile saline and NaOH was used at a dose25mgkgday ip [20] Solution of coenzyme Q10 (HimediaLabs India) was dissolved in 2 vv ethanol and was usedat a dose of 10mgkg ipday [21] All solutions were freshlyprepared every day before dosing the animals

22 Animals Animal procedures were done in accordancewith the Institutional Animal Ethics Committee of CPCSEAAdult Wistar rats (175ndash225 g) of both sexes were housed inan aseptic animal room at a temperature of 20ndash24∘C and ahumidity of 40ndash70 with a 12 h light cycle and 12 fresh airchanges per hour in clean paddy husk bedding All animalswere allowed free access to rat chow and water They wereacclimatized for a minimum period of 1 week prior to thebeginning of the study

23 Induction of Peripheral Neuropathy Cisplatin (2mgkgip twice weekly) was given for 8 weeks to induce peripheralneuropathy in rats [19] The dose of cisplatin was decided bythe standardization study done previously

24 Experimental Design The animals were divided into fivegroups (119899 = 6 per group) normal control group (Group1) cisplatin-induced peripheral neuropathic rats (Group 2)MSG (500mgkgday po) + cisplatin + resveratrol (10mgkgday ip) (Group 3) MSG (500mgkgday po) + cisplatin+ CoQ10 (10mgkgday ip) (Group 4) and MSG (500mgkgday po) + cisplatin + ALA (25mgkgday ip) (Group 5)daily for 8 weeks All parameters under behavioral studiesbiochemical estimation and electrophysiology were evalu-ated on the completion of the treatment (Figure 1)

25 Assessment of General Toxicity Body weight was mea-sured before each administration of cisplatin or vehicle andafter administration of drug combination All rats wereexamined daily for clinical signs such as piloerection or hindlimb weakness and to assess general health

26 Behavioral Studies

261 Thermal Hyperalgesia Thermal hyperalgesia wasassessed by tail immersion test It was noted with theimmersion of terminal part of the tail (1 cm) in water main-tained at 50∘C The duration of tail withdrawal reflex orsigns of struggle were recorded as response of heat sensationand a cutoff time of 20 s was maintained [22] Rats wereacclimatized to the testing procedure and handled by theinvestigator during the week before the experiment

262 Grip Strength Assays Grip strength meter was used forevaluating grip strength of animals Before commencementof experiment the animals were acclimatized by placing themon the instrument for few minutes Rats were held by the tailabove the grid of grip strength meter to an almost horizontalposition The base of the tail was then pulled following theaxle of the sensor until it released the gridThe force achievedby the animal was then displayed on the screen and wasrecorded as newtons or kg units [23]

The Scientific World Journal 3

Cisplatin

twice weekly

Behavioralelectrophysiologicalandbiochemical studies

0day 4th week 8th week


(2mgkg ip)

MSG (500mgkgday po)+

resveratrol (10mgkgdaypo) dailyALA

(25mgkgday)

ip(10mgkgday)pocoQ10

Figure 1 Schematic representation of experimental design

263 Rota-Rod Performance Assessment The rota-rod test iswidely used in rodents to assess their ldquominimal neurologicaldefectrdquo such as impaired motor function (eg ataxia) andcoordination The rota-rod unit consists of a rotating rod75mm in diameter which was divided into four parts bycompartmentalization to permit the testing of four rats at atime Briefly in a training session the rats were placed onthe rod that was set to 25 rpm and the performance time thateach rat was able to remain on the rota-rod was recordedThe rats were subjected to three training trials from 3 h to 4 hintervals on two separate days for acclimatization purposesIn the test session the rats were placed on the rota-rod andtheir performance times were recorded [1]

27 Electrophysiology

271 Isolation of Sciatic Nerve Rats were sacrificed by over-dose of ketaminexylazine ip Ratrsquos back was shaved andincision was made at L4ndashL6 spinal segments The sciaticnerves were surgically exposed from sciatic notch to thegastrocnemius tendon and removed carefully impregnatedon fine filter paper to remove any accompanying blood andsoaked for 10 minutes in Ringer-Locke buffer to preventspontaneous firing of the nerve [24]

272 Measurement of Nerve Conduction Velocity (NCV) Ratsciatic nerve having a length of 3 cmndash5 cm was prepared andmounted into the nerve chamber consisting of small quantityof phosphate buffer pH 7 to humidify the compartment Firstpair of 5mmspaced electrodes is connected via the stimulatorcable (MLA 270) to output 1 and 2 of the power lab unitThe recording cable (MLA 285) was connected to the input1 of the power lab and microhooks of the same cable to thedesired electrode wires depending upon the length of thesciatic nerve After recording a set of responses latency wascalculated using lab chart 7 software Distance between thestimulating electrode and recording electrode was measuredNerve conduction velocity (msec) was calculated using thefollowing formula

NCV = Distance (mm) latency (1)

28 Biochemical Estimation

281 Sciatic Nerve Homogenate Preparation Sciatic nervesamples were rinsed with ice cold saline homogenized inchilled phosphate buffer (pH 74) and used for the estimationof lipid per oxidation (MDA levels) reduced glutathione andcatalase [25]

282 Estimation of Lipid per Oxidation The extent of lipidper oxidation in terms of thiobarbituric acid reactive sub-stances (TBARS) formation was measured according to themethod of Esterbauer and Cheeseman Tissue extracts weremixed separately with 1mL TCA (20) and 2mL TBA(067) and heated for 1 h at 100∘C After cooling theprecipitate was removed by centrifugationThe absorbance ofeach samplewasmeasured at 535 nmusing a blank containingall the reagents except the sample As 99 TBARS aremalondialdehyde (MDA) therefore TBARS concentrationsof the samples were calculated using the extinction coefficientof MDA that is 156 times 105Mminus1 cmminus1 and were expressed as120583M of malondialdehyde per mg protein [26]

283 Estimation of Reduced Glutathione Reduced glu-tathione was assayed by the method of [27] Briefly 10mLof sciatic nerve homogenate (10 wv) was precipitated with10mL of sulphosalicylic acid (4) The samples were kept at40∘C for at least 1 h and then subjected to centrifugation at1200 g for 15min at 40∘CThe assay mixture contained 01mLsupernatant 27mL phosphate buffer (01M pH 74) and02mL 55dithiobis (2-nitro benzoic acid) (Ellmanrsquos reagent01mM pH80) in a total volume of 30mLTheyellow colourdeveloped was read immediately at 412 nm and the reducedGSH levels were expressed as 120583gmg protein

284 Estimation of Catalase Catalase was estimated by themethod of Aebi [28] which is a quantitative spectroscopicmethod developed for following the breakdown of H

2O2at

240 nm in unit timeThe sample readings were taken by plac-ing 1mL of phosphate buffer and 100 120583L of tissue homogenatein the reference cuvette and 1mL of H

2O2and 100 120583L of

homogenate in the test cuvette in the spectrophotometer For


each measurement the reading was taken at 240 nm 1minafter placing the cuvettes in Shimadzu spectrophotometer

29 Statistical Analysis Results were expressed as mean plusmnSEM The intergroup variation was measured by one-wayanalysis of variance (ANOVA) followed by Tukeyrsquos multiplecomparison test The statistical analysis was done using theGraph pad prism software version 50 Probability values 119875 lt005 were considered to be significant

3 Results

31 Assessment of General Toxicity No deterioration ingeneral status was observed no alterations in the bodytemperature and no abnormal clinical signs were observedNo rats in the cisplatin control group died during the courseof the experiment Cisplatin treated rats (bw 186 plusmn 155 g)showed significant reduction in body weight as comparedwith the control group (bw 216 plusmn 178 g) Treatment with acombination of MSG + resveratrol (bw 202 plusmn 255 g) MSG +coenzyme Q10 (bw 205 plusmn 29 g) and MSG + ALA (bw 204plusmn 332 g) did not produce any major effect on body weight ofthe treated rats as compared to the control group (Figure 2)

32 Behavioral Testing

321 Effect of Drug Treatment on NociceptiveThreshold in TailImmersion (WarmWater) Test At the end of the 4th and 8thweeks cisplatin group exhibited significant decrease in painthreshold fromnoxious stimuli as compared with control rats(119875 lt 001) MSG + resveratrol MSG + Co Q10 and MSG +ALA administration for 8 weeks significantly increased thepain threshold as compared with control neuropathic rats(119875 lt 001 Figure 3)

322 Modulation of Muscle Hyperalgesia Using Grip StrengthAssay Administration of MSG + resveratrol MSG + CoQ10and MSG + ALA for 8 weeks significantly improved thelatency of grip strength when comparedwith cisplatin treatedrats Treatment with the combination of MSG + resveratrolandMSG+ALAwas themost effective (119875 lt 001) (Figure 4)

323 Modulation of Motor Coordination Using Rota-Rod TestCisplatin-induced neuropathy impaired motor coordinationas evaluated by the walking time on a rotating rod and thenumber of falls (rota-rod test) Indeed normal rats wereable to balance on the rotating rod for 1207 plusmn 033 sec ascompared with cisplatin treated group (119875 lt 0001) Motorcoordination was significantly improved after administrationof the combinations of MSG + resveratrol MSG + CoQ10and MSG + ALA when compared with cisplatin treated ratsCombination of MSG + resveratrol treatment was the mosteffective (119875 lt 005) (Figure 5)

33 Effect of Cisplatin on Nerve Conduction Velocity Eightweeks after neuropathy neuropathic rats showed significantdecrease in NCV as compared with the normal groupTreatment with combination of MSG + resveratrol MSG +CoQ10 and MSG + ALA reversed the nerve conduction

0da

y

4th

wee

k

8th

wee

k

50

100

150

200

250

VehicleCisplatinMSG + resveratrol + cisplatin

MSG + ALA + cisplatin

Wei

ght (

g)

lowast

lowastlowastlowast

MSG + CoQ10 + cisplatin

Figure 2 Effect of chronic treatment with combination ofMSG andresveratrolALACo Q10 on body weight lowast(119875 lt 005) lowastlowastlowast(119875 lt0001) versus control group (119875 lt 005) versus cisplatin treatedgroup

0da

y

4th

wee

k

8th

wee

k

0

5

10

15

20

lowastlowastlowast

lowastlowast



Late

ncy

(sec

)


Figure 3 Effect of chronic treatment with combination ofMSG andresveratrolALACoQ10 on thermal hyperalgesia in cisplatin treatedrats lowastlowast(119875 lt 0001) lowastlowastlowast(119875 lt 0001) versus control group (119875 lt005) (119875 lt 001) versus cisplatin treated group

in neuropathic rats and significantly improved nerve con-duction velocity when compared with cisplatin treated ratsMSG + resveratrol andMSG+ALA treatments were themosteffective (Figure 6)


0

2

4

6G

rip fo

rce (

N)


MSG + ALA + cisplatinMSG + CoQ10 + cisplatin

lowastlowastlowast

Figure 4 Effect of chronic treatment with combination ofMSG andresveratrolALACo Q10 on grip strength in cisplatin treated ratslowastlowastlowast(119875 lt 0001) versus control group (119875 lt 005) (119875 lt 001)versus cisplatin treated group

0da

y

4th

wee

k

8th

wee

k

60

80

100

120

lowastlowastlowastlowastlowast

Late

ncy

(sec

)




Figure 5 Effect of chronic treatment with combination ofMSG andresveratrolALACo Q10 on motor coordination in cisplatin treatedrats lowastlowast(119875 lt 001) lowastlowastlowast(119875 lt 0001) versus control group (119875 lt 005)versus cisplatin treated group


341 Effect of Cisplatin-Induced Changes on Lipid per Oxi-dation After administration of cisplatin neuropathic ratsshowed significant increase in MDA levels in sciatic nerve ascompared with the normal group Treatment with the combi-nation ofMSG+ resveratrolMSG+CoQ10 andMSG+ALAat the 4th and 8th weeks showed a significant decrease when

40

45

50

55

60

Ner

ve co

nduc

tion

velo

city

(ms

)



lowastlowastlowast


Figure 6 Effect of chronic treatment with combination of MSGand resveratrolALACoQ10 on nerve conduction velocity in sciaticnerve of cisplatin treated rats lowastlowastlowast(119875 lt 0001) versus control group(119875 lt 005) (119875 lt 001) versus cisplatin treated group

compared with cisplatin treated group MSG + resveratroltreatment was the most effective in reducing the MDA level(Table 1)

342 Effect of Cisplatin onReducedGlutathione Neuropathicrats showed significant decrease in GSH levels in the sciaticnerve as compared with normal group Treatment with thecombination of MSG + resveratrol MSG + CoQ10 andMSG + ALA showed a significance increase in GSH levelCombination of MSG + resveratrol was the most effectiveamong all combinations (Table 1)

343 Effect of Cisplatin-Induced Changes on Catalase ActivitySciatic nerve catalase activity was significantly increased inthe 8th week after administration of the combinations ofMSG + resveratrol MSG + CoQ10 and MSG + ALA ascompared to cisplatin treated rats in which a lower level ofcatalase activity was observed (Table 1)

4 Discussion

Although cisplatin is an effective antineoplastic drug its use islimited by its serious adverse effects including peripheral neu-ropathy and nephrotoxicity [1] It exhibits its toxic effects oncell by inducing oxidative stress and loss of sensory neuronsIn this study the model of cisplatin-induced peripheral neu-ropathy is used to assess the effectiveness of the few putativecombinations of neuroprotective agents namely glutamateresveratrol ALA and CoQ10 Rat model is used as it isqualitatively similar to that of humans It is reported that cis-platin predominantly accumulated in the dorsal root gangliacausing nucleolar damage and also affecting Schwann cellswhich play a vital role in nerve development and regenerationthere by signalling apoptosis [29] Cisplatin has also been


Table 1 Effect of chronic treatmentwith combination ofMSGand resveratrolALACoQ10 on levels ofMDA (amarker of lipid peroxidation)reduced glutathion and catalase in sciatic nerve of cisplatin treated rats

SL NO Group MDA(120583Mmg protein)

GSH(120583gmg protein)

Catalase(Umg protein)

1 Vehicle 152 plusmn 009 7034 plusmn 033 0106 plusmn 0010

2 Cisplatin 276 plusmn 019lowastlowastlowast

5766 plusmn 069lowastlowastlowast


3 Cisplatin + MSG + Res 212 plusmn 008

6172 plusmn 119

0091 plusmn 0006

4 Cisplatin + MSG + Co Q10 222 plusmn 004

6063 plusmn 047

0088 plusmn 0008

5 Cisplatin + MSG + ALA 232 plusmn 002

6076 plusmn 049

0095 plusmn 0010

lowastlowastlowast(119875 lt 0001) versus control group (119875 lt 005) (119875 lt 001) versus cisplatin treated group

reported to cause ROS generation and causes lipid per oxida-tion Recent pieces of evidence suggest that cisplatin reducesperipheral nerve blood supply through potent angiogeniceffect whichmay predispose the nerve damage [3] Significantloss of body weight motor coordination abnormality and adecrease of muscle power could be observed at the 4th weekand kept on progressively increasing till the end of the study(8th week) These parameters appeared to be sensitive andreliable to demonstrate neuropathic conditions Reduction innerve conduction velocity at the end of treatment confirmedabnormal functions of nerve Thus peripheral neuropathywas successfully induced in rats and this model can be usedfor the screening of potential neuroprotective drugs for theprevention of cisplatin-induced peripheral neuropathy

In the present study treatment with a combination ofMSG (500mgkgday po) with resveratrol (10mgkgdayip)ALA (20mgkgday ip)Co Q10 (10mgkgday ip)significantly alleviated cisplatin-induced peripheral neuropa-thy in rats Rats treated with the combination exhibitedless decrease in body weight decreased thermal hyperalge-sia improved motor coordination increased grip strengthimproved nerve conduction velocity and higher levels ofantioxidant enzymes compared with cisplatin treated rats

Resveratrol has been shown to have significant antiox-idant and anti-inflammatory properties and found to bea neuroprotective agent against excitotoxicity [30] Reportssuggest that resveratrol corrects nerve blood flow deficits dueto direct vasodilatation and it has already been shown toproduce vasodilatation by both endothelial dependent andendothelium independent pathways [9]The neuroprotectiverole of resveratrol in our study could probably be due toits antioxidant activity and improvements in nerve bloodflow and restored motor nerve conduction velocity deficitsNerve blood flow needs to be measured to further strengthenthe mechanism of resveratrol in our study ALA is a potentantioxidant and unlike other antioxidants it is both fat- andwater-soluble which works throughout the body ALA playsa key role in most crucial biochemical functions of mito-chondria thus it can help with maintaining the mitochon-drial membrane potential differential by controlling substrateavailability for respiratory chain and regulating the redoxstate Several studies provided evidence thatALA is protectivein diseases characterized by mitochondrial impairment likediabetic neuropathy [31] ALA is capable of scavenging a vari-ety of reactive oxygen species It scavenges hydroxyl radicalsand hypochlorous acid and also terminates singlet oxygen In

our study ALA was able to improve the levels of endogenousantioxidants and decreased the lipid per oxidation Thesefindings could explain the neuroprotective role of ALA

CoQ10 is a crucial component of the oxidative phospho-rylation system in themitochondria as it is involved in oxida-tive phosphorylation of various products of nutrients suchas fatty acids and carbohydrates where energy derived fromreduced equivalents is converted into ATP to drive cellularmachinery and biosynthetic processes [32] Recent studiesexplored the new biochemical functions of CoQ10 includingthe fact that it is considered to be an essential antioxi-dant regenerates other antioxidants stimulates cell growthand inhibits cell death CoQ10 protects phospholipids andmitochondrial membrane proteins from per oxidation andprotects DNA against oxidative damage there by functions asintracellular antioxidant [33] In our study CoQ10 was able toreverse the cisplatin-induced oxidative damage by increasingthe expression of endogenous antioxidants preventing lipidper oxidation and thus exhibiting neuroprotective activity incisplatin treated rats

Glutamate is involved in the modulation of microtubulefunctions which affect intracellular transportation Theseprocesses are important for axons which require all sub-stances from the cell body for biosynthesis [16] Our studyshows that glutamate in combination with ALA resveratroland Co Q10 has significant neuroprotective effects in cisplat-in-induced neuropathy by improving endogenous antioxi-dant levels and probably by exerting an indirect effect onmicrotubules after interacting with a receptor found only onneural cells other possible links in this pathway include localgrowth factor release or alteration of intracellular calmodulinand ATP which have been shown to be regulators of micro-tubule formation and stability [18] However the neuropro-tective effect of glutamate can be further explained andexplored by measuring the calmodulin and ATP

5 Conclusion

In conclusion we have demonstrated that MSG resveratrolALA and CoQ10 combination protected against cisplatin-induced neurotoxicity by increasing endogenous antioxi-dants preventing lipid per oxidation and improving NCVThus these results would encourage future clinical trials ofthese agents in cancer patients to whom coadministration ofneuroprotectants is required to prevent neurotoxicity


Conflict of Interests

The authors declare no competing conflict of interests

Acknowledgments

The authors are thankful to the administration of Al-AmeenCollege of Pharmacy Bangalore India for the financialassistance and facilities provided to the candidates

References

[1] K Hori N Ozaki S Suzuki and Y Sugiura ldquoUpregulations ofP2X3 and ASIC3 involve in hyperalgesia induced by cisplatinadministration in ratsrdquo Pain vol 149 no 2 pp 393ndash405 2010

[2] J E Mollman D J Glover W M Hogan and R E FurmanldquoCisplatin neuropathy risk factors prognosis and protectionby WR-2721rdquo Cancer vol 61 no 11 pp 2192ndash2195 1988

[3] S B Park A V Krishnan C S-Y Lin D Goldstein MFriedlander and M C Kiernan ldquoMechanisms underlyingchemotherapy-induced neurotoxicity and the potential for neu-roprotective strategiesrdquo Current Medicinal Chemistry vol 15no 29 pp 3081ndash3094 2008

[4] E S McDonald K R Randon A Knight and A JWindebankldquoCisplatin preferentially binds to DNA in dorsal root ganglionneurons in vitro and in vivo a potential mechanism forneurotoxicityrdquo Neurobiology of Disease vol 18 no 2 pp 305ndash313 2005

[5] A Alaedini Z Xiang H Kim Y-J Sung and N Latov ldquoUp-regulation of apoptosis and regeneration genes in the dorsalroot ganglia during cisplatin treatmentrdquo Experimental Neurolo-gy vol 210 no 2 pp 368ndash374 2008

[6] A Pace A Savarese M Picardo et al ldquoNeuroprotective effectof vitamin E supplementation in patients treated with cisplatinchemotherapyrdquo Journal of Clinical Oncology vol 21 no 5 pp927ndash931 2003

[7] A Kumar R K Kaundal S Iyer and S S Sharma ldquoEffects ofresveratrol on nerve functions oxidative stress and DNA frag-mentation in experimental diabetic neuropathyrdquo Life Sciencesvol 80 no 13 pp 1236ndash1244 2007

[8] H I Kim T H Kim and J-H Song ldquoResveratrol inhibits Na+currents in rat dorsal root ganglion neuronsrdquo Brain Researchvol 1045 no 1-2 pp 134ndash141 2005

[9] L Ernster andGDallner ldquoBiochemical physiological andmed-ical aspects of ubiquinone functionrdquo Biochimica et BiophysicaActa vol 1271 no 1 pp 195ndash204 1995

[10] G P Littarru and P Langsjoen ldquoCoenzyme Q10 and statinsbiochemical and clinical implicationsrdquo Mitochondrion vol 7pp S168ndashS174 2007

[11] W R Galpern and M E Cudkowicz ldquoCoenzyme Q treatmentof neurodegenerative diseases of agingrdquo Mitochondrion vol 7pp S146ndashS153 2007

[12] K P Shay R F Moreau E J Smith A R Smith and T MHagen ldquoAlpha-lipoic acid as a dietary supplement molecularmechanisms and therapeutic potentialrdquo Biochimica et Biophys-ica Acta vol 1790 no 10 pp 1149ndash1160 2009

[13] L Packer E HWitt and H J Tritschler ldquoAlpha-lipoic acid as abiological antioxidantrdquo Free Radical Biology and Medicine vol19 no 2 pp 227ndash250 1995

[14] B E Cairns X Dong M K Mann et al ldquoSystemic admin-istration of monosodium glutamate elevates intramuscular

glutamate levels and sensitizes rat masseter muscle afferentfibersrdquo Pain vol 132 no 1-2 pp 33ndash41 2007

[15] D G Burin and B Stoll ldquoMetabolic fate and function of dietaryglutamate in the gutrdquoTheAmerican Journal of ClinicalNutritionvol 90 no 3 pp 850Sndash856S 2009

[16] F M Boyle H R Wheeler and G M Shenfield ldquoAmeliorationof experimental cisplatin and paclitaxel neuropathy with glu-tamaterdquo Journal of Neuro-Oncology vol 41 no 2 pp 107ndash1161999

[17] M Anjaneyulu A Berent-Spillson and J W Russell ldquoMetabo-tropic glutamate receptors (mGluRs) and diabetic neuropathyrdquoCurrent Drug Targets vol 9 no 1 pp 85ndash93 2008

[18] T Arkaravichien N Sattayasai S Daduang and J SattayasaildquoDose-dependent effects of glutamate in pyridoxine-inducedneuropathyrdquo Food and Chemical Toxicology vol 41 no 10 pp1375ndash1380 2003

[19] R Bianchi M Brines G Lauria et al ldquoProtective effect oferythropoietin and its carbamylated derivative in experimentalcisplatin peripheral neurotoxicityrdquo Clinical Cancer Researchvol 12 no 8 pp 2607ndash2612 2006

[20] L P Rybak K Husain C Whitworth and S M Somani ldquoDosedependent protection by lipoic acid against cisplatin-inducedototoxicity in rats antioxidant defense systemrdquo ToxicologicalSciences vol 47 no 2 pp 195ndash202 1999

[21] T Ishrat M B Khan M N Hoda et al ldquoCoenzyme Q10 mod-ulates cognitive impairment against intracerebroventricularinjection of streptozotocin in ratsrdquo Behavioural Brain Researchvol 171 no 1 pp 9ndash16 2006

[22] R Necker and R F Hellon ldquoNoxious thermal input from the rattail modulation by descending inhibitory influencesrdquo Pain vol4 pp 231ndash242 1978

[23] M A Ansari S J Ahmad R Khanum and M Akhtar ldquoPhar-macological investigation of protective effects of Nigella sativaoil in experimental diabetic neuropathy in ratsrdquo Indian Journalof Pharmaceutical Education and Research vol 43 no 2 pp166ndash176 2009

[24] S G Liasson ldquoNerve conduction changes in experimentaldiabetesrdquo Journal of Clinical Investigation vol 43 no 12 pp2353ndash2358 1964

[25] V Tiwari A Kuhad and K Chopra ldquoTocotrienol amelioratesbehavioral and biochemical alterations in the rat model ofalcoholic neuropathyrdquo Pain vol 145 no 1-2 pp 129ndash135 2009

[26] Y Jiang C Guo M R Vasko and M R Kelley ldquoImplicationsof apurinicapyrimidinic endonuclease in reactive oxygen sig-naling response after cisplatin treatment of dorsal root ganglionneuronsrdquo Cancer Research vol 68 no 15 pp 6425ndash6434 2008

[27] R Van Doorn C-M Leijdekkers and P T Henderson ldquoSyner-gistic effects of phorone on the hepatotoxicity of bromobenzeneand paracetamol in micerdquo Toxicology vol 11 no 3 pp 225ndash2331978

[28] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[29] J M Garcia J P Cata P M Dougherty and R G SmithldquoGhrelin prevents cisplatin-induced mechanical hyperalgesiaand cachexiardquo Endocrinology vol 149 no 2 pp 455ndash460 2008

[30] AKumar and S S Sharma ldquoNF-120581B inhibitory action of resvera-trol a probable mechanism of neuroprotection in experimentaldiabetic neuropathyrdquo Biochemical and Biophysical ResearchCommunications vol 394 no 2 pp 360ndash365 2010

[31] G Melli M Taiana F Camozzi et al ldquoAlpha-lipoic acid pre-vents mitochondrial damage and neurotoxicity in experimental


chemotherapy neuropathyrdquo Experimental Neurology vol 214no 2 pp 276ndash284 2008

[32] F L Crane Y Hatefi and R L Lester CWidmer ldquoIsolation of aquinone from beef heart mitochondriardquo Biochimica et Biophys-ica Acta vol 25 no 1 pp 220ndash221 1957

[33] F L Crane ldquoBiochemical functions of coenzyme Q10rdquo Journalof the American College of Nutrition vol 20 no 6 pp 591ndash5982001

Submit your manuscripts athttpwwwhindawicom

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ToxinsJournal of

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Autoimmune Diseases


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MEDIATORSINFLAMMATION

of


years research also focused on its antioxidant properties inseveral cellular compartments [10] CoQ10 has shown protec-tive effect against neuronal loss in several animal models ofneurodegeneration and the ability of CoQ10 to slow diseaseprogression in clinical trials has been considered to exploreneuroprotective activity in many of the neurodegenerativediseases [11]

Alpha-lipoic acid or 12-dithiolane-3-pentanoic acid(ALA) is a naturally available dithiol compound synthesizedenzymatically in the mitochondrion from octanoic acid [12]ALA reacts with reactive oxygen species such as superoxideradicals hydroxyl radicals hypochlorous acid peroxyl rad-icals and singlet oxygen Administration of ALA has beenshown to be beneficial in several oxidative stress models suchas ischemia-reperfusion injury diabetes cataract formationHIV activation neurodegeneration and radiation injury [13]

Monosodium glutamate (MSG) is the sodium salt ofglutamic acid It is widely used as food additive mainly asflavour enhancer and it is believed to be safe without seriousconcerns It contributes an important role in the biosynthesisof several key amino acids [14] Evidence from clinical andpreclinical studies revealed that it is a major oxidative fuel forthe gut and that dietary glutamate extensively undergoes firstpass metabolism by the intestine It is an important precursorfor synthesis of bioactive molecules including glutathionean endogenous antioxidant and also functions as a keyneurotransmitter [15] Partial investigation ofMSG in clinicaltrial as a neuroprotectant in the early 1980s has shown efficacyin preventing neuropathy induced by vincristine [16] A newclass of glutamate receptors named metabotropic glutamatereceptors (mGluRs) that are coupled to effector systemsthrough GTP proteins (G-proteins) became evident by themajority of most recent research activities Activation of(mGluRs) subtypes usually regulates many cellular activitiesin mammalian systems in various experimental modelsActivation of (mGluRs) subtypes protected cells againstvarious types of neuronal insults such as traumatic braininjury stroke and NMDA excitotoxicity [17] Interestinglyin recent years glutamate has been reported to be efficientin preventing neuropathy caused by many cytotoxic drugs inmice rats and humans with no orminimal apparent intrinsictoxicity and its low cost and oral availability make it attractivefor clinical use as a neuroprotectant [18]

In the present study we further explored its potential inrodents for its neuroprotective activity in presence of otherantioxidants namely resveratrol coenzyme Q10 and ALAin rat model of cisplatin-induced peripheral neuropathy byassessing behavioral biochemical and neurophysiologicalparameters

2 Materials and Methods

21 Reagents All reagents and solvents used were of ana-lytical grade Cisplatin (2mgkg ip twice weekly) was usedto induce peripheral neuropathy by intraperitoneal injection[19] Aqueous solution of MSG (Himadia Labs India) wasused at 500mgkgday po dose [18] Resveratrol (Sami LabsIndia) solution was freshly prepared using 2 vv ethanol ata dose of 10mgkgday ip [7] Suspension of ALA (Sami

Labs India) in sterile saline and NaOH was used at a dose25mgkgday ip [20] Solution of coenzyme Q10 (HimediaLabs India) was dissolved in 2 vv ethanol and was usedat a dose of 10mgkg ipday [21] All solutions were freshlyprepared every day before dosing the animals

22 Animals Animal procedures were done in accordancewith the Institutional Animal Ethics Committee of CPCSEAAdult Wistar rats (175ndash225 g) of both sexes were housed inan aseptic animal room at a temperature of 20ndash24∘C and ahumidity of 40ndash70 with a 12 h light cycle and 12 fresh airchanges per hour in clean paddy husk bedding All animalswere allowed free access to rat chow and water They wereacclimatized for a minimum period of 1 week prior to thebeginning of the study

23 Induction of Peripheral Neuropathy Cisplatin (2mgkgip twice weekly) was given for 8 weeks to induce peripheralneuropathy in rats [19] The dose of cisplatin was decided bythe standardization study done previously

24 Experimental Design The animals were divided into fivegroups (119899 = 6 per group) normal control group (Group1) cisplatin-induced peripheral neuropathic rats (Group 2)MSG (500mgkgday po) + cisplatin + resveratrol (10mgkgday ip) (Group 3) MSG (500mgkgday po) + cisplatin+ CoQ10 (10mgkgday ip) (Group 4) and MSG (500mgkgday po) + cisplatin + ALA (25mgkgday ip) (Group 5)daily for 8 weeks All parameters under behavioral studiesbiochemical estimation and electrophysiology were evalu-ated on the completion of the treatment (Figure 1)

25 Assessment of General Toxicity Body weight was mea-sured before each administration of cisplatin or vehicle andafter administration of drug combination All rats wereexamined daily for clinical signs such as piloerection or hindlimb weakness and to assess general health

26 Behavioral Studies

261 Thermal Hyperalgesia Thermal hyperalgesia wasassessed by tail immersion test It was noted with theimmersion of terminal part of the tail (1 cm) in water main-tained at 50∘C The duration of tail withdrawal reflex orsigns of struggle were recorded as response of heat sensationand a cutoff time of 20 s was maintained [22] Rats wereacclimatized to the testing procedure and handled by theinvestigator during the week before the experiment

262 Grip Strength Assays Grip strength meter was used forevaluating grip strength of animals Before commencementof experiment the animals were acclimatized by placing themon the instrument for few minutes Rats were held by the tailabove the grid of grip strength meter to an almost horizontalposition The base of the tail was then pulled following theaxle of the sensor until it released the gridThe force achievedby the animal was then displayed on the screen and wasrecorded as newtons or kg units [23]


Cisplatin

twice weekly




(2mgkg ip)



(25mgkgday)













2O2at


2O2and 100 120583L of





3 Results







0da

y

4th

wee

k

8th

wee

k

50

100

150

200

250



Wei

ght (

g)

lowast

lowastlowastlowast



0da

y

4th

wee

k

8th

wee

k

0

5

10

15

20

lowastlowastlowast

lowastlowast



Late

ncy

(sec

)





0

2

4

6G

rip fo

rce (

N)



lowastlowastlowast


0da

y

4th

wee

k

8th

wee

k

60

80

100

120


Late

ncy

(sec

)







40

45

50

55

60

Ner

ve co

nduc

tion

velo

city

(ms

)



lowastlowastlowast






4 Discussion












6172 plusmn 119

0091 plusmn 0006


6063 plusmn 047

0088 plusmn 0008


6076 plusmn 049

0095 plusmn 0010








5 Conclusion





Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Cisplatin

twice weekly




(2mgkg ip)



(25mgkgday)













2O2at


2O2and 100 120583L of





3 Results







0da

y

4th

wee

k

8th

wee

k

50

100

150

200

250



Wei

ght (

g)

lowast

lowastlowastlowast



0da

y

4th

wee

k

8th

wee

k

0

5

10

15

20

lowastlowastlowast

lowastlowast



Late

ncy

(sec

)





0

2

4

6G

rip fo

rce (

N)



lowastlowastlowast


0da

y

4th

wee

k

8th

wee

k

60

80

100

120


Late

ncy

(sec

)







40

45

50

55

60

Ner

ve co

nduc

tion

velo

city

(ms

)



lowastlowastlowast






4 Discussion












6172 plusmn 119

0091 plusmn 0006


6063 plusmn 047

0088 plusmn 0008


6076 plusmn 049

0095 plusmn 0010








5 Conclusion





Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




3 Results







0da

y

4th

wee

k

8th

wee

k

50

100

150

200

250



Wei

ght (

g)

lowast

lowastlowastlowast



0da

y

4th

wee

k

8th

wee

k

0

5

10

15

20

lowastlowastlowast

lowastlowast



Late

ncy

(sec

)





0

2

4

6G

rip fo

rce (

N)



lowastlowastlowast


0da

y

4th

wee

k

8th

wee

k

60

80

100

120


Late

ncy

(sec

)







40

45

50

55

60

Ner

ve co

nduc

tion

velo

city

(ms

)



lowastlowastlowast






4 Discussion












6172 plusmn 119

0091 plusmn 0006


6063 plusmn 047

0088 plusmn 0008


6076 plusmn 049

0095 plusmn 0010








5 Conclusion





Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

2

4

6G

rip fo

rce (

N)



lowastlowastlowast


0da

y

4th

wee

k

8th

wee

k

60

80

100

120


Late

ncy

(sec

)







40

45

50

55

60

Ner

ve co

nduc

tion

velo

city

(ms

)



lowastlowastlowast






4 Discussion












6172 plusmn 119

0091 plusmn 0006


6063 plusmn 047

0088 plusmn 0008


6076 plusmn 049

0095 plusmn 0010








5 Conclusion





Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of











6172 plusmn 119

0091 plusmn 0006


6063 plusmn 047

0088 plusmn 0008


6076 plusmn 049

0095 plusmn 0010








5 Conclusion





Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article amelioration of behavioural, biochemical...

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