European Journal of Pain 15 (2011) 286–292

Contents lists available at ScienceDirect

European Journal of Pain

journal homepage: www.EuropeanJournalPain.com

Amelioration of functional, biochemical and molecular deficits by epigallocatechingallate in experimental model of alcoholic neuropathy

Vinod Tiwari, Anurag Kuhad, Kanwaljit Chopra *

Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh 160 014, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 March 2010Received in revised form 1 July 2010Accepted 14 July 2010Available online 10 August 2010

Keywords:Alcoholic neuropathyEpigallocatechin-3-gallateHyperalgesiaTransforming growth factor bTumor necrosis factor a

1090-3801/$36.00 � 2010 European Federation of Intdoi:10.1016/j.ejpain.2010.07.005

* Corresponding author. Tel.: +91 172 2534105; faxE-mail address: dr_chopra_k@yahoo.com (K. Chop

Long term alcohol consumption leads to decreased nociceptive threshold characterized by spontaneousburning pain, hyperalgesia and allodynia. The mechanism involved in this pain includes increased oxida-tive-nitrosative stress, release of pro-inflammatory cytokines and neuronal apoptosis. The present studywas designed to explore the protective effect of epigallocatechin-3-gallate against alcoholic neuropathicpain in rats. Rats fed with alcohol (35%) for 10 weeks showed markedly decreased tail flick latency in tail-immersion test (thermal hyperalgesia), vocalization threshold in Randall–Sellito test (mechanical hyper-algesia) and paw-withdrawal threshold in von-Frey hair test (mechanical allodynia) along with enhancedoxidative-nitrosative stress and inflammatory mediators (TNF-a, IL-1b and TGF-b1 levels). Co-adminis-tration of epigallocatechin-3-gallate (25–100 mg/kg) significantly and dose-dependently prevented func-tional, biochemical and molecular changes associated with alcoholic neuropathy. In conclusion, thecurrent findings suggest the neuroprotective potential of epigallocatechin-3-gallate in attenuating thefunctional, biochemical and molecular alterations associated with alcoholic neuropathy through modu-lation of oxido-inflammatory cascade.

� 2010 European Federation of International Association for the Study of Pain Chapters. Published byElsevier Ltd. All rights reserved.

1. Introduction ropathic pain (Sharma and Sayyed, 2006). Ethanol enhances oxida-

Neuropathic pain refers to the pain caused by a lesion of theperipheral or central nervous system (or both) manifesting as sen-sory signs and symptoms (Backonja, 2003). Abnormal signals arisenot only from injured axons but also from the intact nociceptorsthat share the innervation territory of the injured nerve (Campbelland Meyer, 2006). The peripheral neuropathy is a potentiallyincapacitating complication of chronic consumption of ethanol,characterized by pain and dysesthesias, primarily in the lowerextremities, and is poorly relieved by available therapies (Monforteet al., 1995). Acetaldehyde, one of the most toxic metabolites ofethanol, is reported to have direct neurotoxic effects on neurons(Koike et al., 2001, 2003).

Oxidative stress is known to play a very important role inexperimental animal models of neuropathic pain. Reactive oxygenspecies are involved in the development and maintenance of cap-saicin-induced pain, particularly in the process of central sensitiza-tion in the spinal cord of rats (Lee et al., 2007). A significantdecrease in the activity of anti-oxidant enzymes (superoxide dis-mutase and catalase) and an increase in lipid peroxidation werealso observed in sciatic nerve of diabetic rats with established neu-

ernational Association for the Stud

: +91 172 2541142.ra).

tive stress directly through generation of oxidative free radicalsand lipid peroxidation (Montoliu et al., 1994) and depletion ofendogenous anti-oxidants such as a-tocopherol, glutathione,ascorbate, and vitamin E (McDonough, 2003).

Cytokines are known to play a very important role in the initia-tion, development and maintenance of neuropathic pain (Ignatow-ski et al., 1999; Wolf et al., 2006). TNF-a has been demonstrated asan important mediator of neuropathic pain (Ignatowski et al.,1999). Interleukin-1 has also been implicated in modulation of painperception under various inflammatory conditions (Wolf et al.,2003). In neuronal tissues, TGF-b plays a neurotrophic role in somesituations (Poulsen et al., 1994), while they elicit cell death induc-ing effects in other situations (Krieglstein et al., 2000). TGF-b is alsoknown to play a pivotal role in diabetic neuropathy (Anjaneyuluet al., 2008). However, the exact role of TGF-b and its functionsare not studied and understood in the pathophysiology of alcoholicneuropathy.

Scientific community all around the globe has explored thepotent anti-oxidant and anti-inflammatory properties of epigal-locatechin-3-gallate (EGCG), the main polyphenolic constituentof green tea. EGCG has been shown to inhibit activities of vari-ous pro-inflammatory cytokines (Han, 2003; Li et al., 2004) andactivation of NF-jb (Aktas et al., 2004). Recently, Wang et al.(2009) also reported that pre-treatment of SH-SY5Y cells withEGCG (0.1–10 lM) significantly attenuated the 6-OHDA-induced

y of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292 287

neuronal cell death by inhibiting enhanced oxidative stress.EGCG pre-treatment is also found to ameliorate amyloid beta-induced neurotoxicity via augmentation of anti-oxidant defencecapacity in neuronal cells (Kim et al., 2009).

Thus, the present study was designed with an aim to investigatethe protective potential of EGCG against chronic alcohol-inducedneuropathic pain in rats by assessing various behavioral, biochem-ical and molecular parameters.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (150–200 g) bred in Central AnimalHouse facility of Panjab University were used. The animals werehoused under standard laboratory conditions, maintained on anatural light–dark cycle and had free access to food (AshirwadIndustries, Mohali, India) and water. Adequate measures weretaken to minimise pain or discomfort to animals and experimentswere conducted in accordance with IASP’s guidelines for pain re-search in animals (Committee for Research and Ethical Issues ofthe IASP, 1983; 16: 109–10). Animals were acclimatized to labora-tory conditions before the behavioral tests. All experiments werecarried out between 0900 and 1700 h. The experimental protocolswere approved by the Institutional Animal Ethics Committee ofPanjab University and performed in accordance with the guidelinesfor Control and Supervision of Experimentation on Animals,Government of India.

2.2. Drugs

EGCG was obtained as a gift sample from DSM Nutritional Prod-ucts Ltd, Switzerland. Drugs were prepared freshly before adminis-tration. TNF-a, IL-1b and TGF-b1 ELISA kits were purchased fromR&D Systems, USA. All other chemicals used for biochemical esti-mations were of analytical grade.

2.3. Induction of alcoholic neuropathy

Alcoholic neuropathy was induced by administration of 35% v/vethanol (10 g/kg b.i.d oral gavage) for 10 weeks (Tiwari et al.,2009). The concentration of alcohol was selected on the basis ofstudies performed by other research groups (Kasdallah-Grissaet al., 2006; Cohen et al., 2007).

2.4. Drug treatment schedule

The animals were randomly divided into six experimentalgroups with 5–8 animals in each group. Group I comprised of con-trol animals given double distilled water in place of ethanol by oralgavage; Group II animals were administered ethanol (10 g/kg oralgavage) for 10 weeks; Groups III–V comprised of ethanol and EGCG(25, 50 and 100 mg/kg; oral gavage) treated rats. EGCG was freshlyprepared in double distilled water and administered by oral route1 h before ethanol dosing daily for ten weeks starting from day 1.Group VI (per se group) animals received only EGCG (100 mg/kg;oral gavage). Per se group means the group received only the drugtreatment without ethanol administration. It works as treatmentcontrol. The group is included in order to rule out any effect oftreatment on the control animals. All the behavioral assays weredone by an observer blind to the drug treatment on 6th, 8th and10th week. After 10 weeks, blood was collected and rats were sac-rificed under deep anesthesia and sciatic nerves were isolated. Ser-um and nerve samples were stored at �20 �C until furtherprocessing for the biochemical estimations.

2.5. Behavioral tests

2.5.1. Thermal hyperalgesiaThermal hyperalgesia was assessed by using tail-immersion

test. In this test tail of rat was immersed in a water bath main-tained at 42 �C (Courteix et al., 1993) until tail withdrawal or signsof struggle were observed (cut-off time: 15 s). As this test involveshandling of the animals, one day before the experiment, rats werehandled in the testing environment until they sat quietly in thehand for 15 s (which corresponds to the cut-off time). On the dayof experiment, rats were again handled for 15 s above the waterbath to get the rat used to the condition of the test. No rat shouldshow aversive reaction during handling. After this the tail of the ratwas immersed into the water maintained at 42 �C. The reactiontime (i.e., the time necessary to observe the withdrawal of the tailfrom the bath) was measured 2–3 times in order to obtain two con-secutive values that differed no more than 10%. The tail of the ratwas immediately dried with a soft cellulose paper to avoid tailcooling between two measures. A shortened duration of tail with-drawal indicates thermal hyperalgesia.

2.5.2. Mechanical hyperalgesiaThe nociceptive flexion reflex was quantified using the Randall–

Sellito paw pressure device (IITC, Woodland Hills, USA), which ap-plies a linearly increasing mechanical force (in g) to the dorsum ofthe rat’s hind paw (Taiwo et al., 1989). Nociceptive thresholds, ex-pressed in grams, were applied by increasing pressure to the hindpaw until a squeak (vocalization threshold) was elicited. As thistest involves animal handling, the experimenter gets the rat usedto being handled as following: 3 days before the experiment, ratswere handled without escaping from the hand of the experimenterfor 20 s, two or three times depending on their capacity to be quiet.On the day of the experiment, rats were again handled 2–3 timesfor 20 s. No rats should show aversive reaction during handling.Then, the paw of the rat was placed under the tip and the progres-sive pressure applied until the rat vocalized. The vocalizationthreshold was measured three or four times in order to obtaintwo consecutive values that differed no more than 10%, andrespecting an interval of at least 10 min between two measures.

2.5.3. Mechanical allodyniaRats were placed individually on an elevated mesh (1 cm2 per-

forations) in a clear plastic cage and adapted to the testing environ-ment for at least 15 min. Von-Frey hairs (IITC, Woodland Hills,USA) with calibrated bending forces (in g) of different intensitieswere used to deliver punctuate mechanical stimuli of varyingintensity. Starting with the lowest filament force, von-Frey hairswere applied from below the mesh floor to the plantar surface ofthe hind paw, with sufficient force to cause slight bending againstthe paw, and held for 1 s (Chaplan et al., 1994; Tiwari et al., 2009).Each stimulation was applied five times with an inter-stimulusinterval of 4–5 s. Care was taken to stimulate random locationson the plantar surface. A positive response was noted if the pawwas robustly and immediately withdrawn. Paw-withdrawalthreshold was defined as the minimum pressure required to elicita withdrawal reflex of the paw, at least one time on the five trials.Voluntary movement associated with locomotion was not consid-ered as a withdrawal response. Mechanical allodynia was definedas a significant decrease in withdrawal thresholds to von-Frey hairapplication.

2.6. Biochemical estimations

2.6.1. Sciatic nerve homogenate preparationSciatic nerve samples were rinsed with ice cold saline (0.9% so-

dium chloride) and 10% (w/v) tissue homogenate was prepared in

288 V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292

chilled phosphate buffered saline (pH 7.4) using Potter–Elvehjamtype glass homogenizer. The homogenate was centrifuged at1000g for 10 min at 4 �C to remove nuclei and unbroken cells.The pellet was discarded and a portion of supernatant was againcentrifuged at 12,000g for 20 min to obtain post-mitochondrialsupernatant. The post-mitochondrial supernatant thus obtainedwas used to assay biochemical (lipid perioxidation, reduced gluta-thione, nitrite, and superoxide dismutase activity) and molecular(TNF-a, IL-1b and TGF-b1) parameters.

2.6.2. Estimation of lipid peroxidationThe malondialdehyde content, a measure of lipid peroxidation,

was assayed by the method of Wills (1965). Briefly, 0.5 ml of post-mitochondrial supernatant and 0.5 ml of Tris–HCl were incubatedat 37 �C for 2 h. After incubation, 1 ml of 10% trichloro acetic acidwas added and centrifuged at 1000g for 10 min. To 1 ml of super-natant, 1 ml of 0.67% thiobarbituric acid was added and the tubeswere kept in boiling water for 10 min. After cooling, 1 ml doubledistilled water was added and absorbance was measured at532 nm. Thiobarbituric acid-reactive substances were quantifiedusing an extinction coefficient of 1.56 � 105 M�1 cm�1 and ex-pressed as nmol of malondialdehyde per mg protein. Tissue proteinwas estimated using the Biuret method and the malondialdehydecontent expressed as nmoles/mg protein.

2.6.3. Estimation of reduced glutathioneReduced glutathione was assayed by the method of Jollow et al.

(1974). Briefly, 1.0 ml of post-mitochondrial supernatant (10%)was precipitated with 1.0 ml of sulphosalicylic acid (4%). The sam-ples were kept at 4 �C for at least 1 h and then subjected to centri-fugation at 1200g for 15 min at 4 �C. The assay mixture contained0.1 ml supernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and0.2 ml 5,5,dithiobis (2-nitro benzoic acid) (Ellman’s reagent,0.1 mM, pH 8.0) in a total volume of 3.0 ml. The yellow color devel-oped was read immediately at 412 nm and the reduced GSH levelswere expressed as lmoles/mg protein.

2.6.4. Estimation of superoxide dismutaseSuperoxide dismutase activity was assayed by the method of

Kono (1978). The assay system consisted of 0.1 mM EDTA,50 mM sodium carbonate and 96 mM of nitro blue tetrazolium.In the cuvette, 2 ml of above mixture was taken and to it 0.05 mlof post-mitochondrial supernatant and 0.05 ml of hydroxylaminehydrochloride (adjusted to pH 6.0 with NaOH) were added. Theauto-oxidation of hydroxylamine was observed by measuring thechange in optical density at 560 nm for 2 min at 30/60 s intervals.

2.6.5. Estimation of catalaseCatalase activity was assayed by the method of Claiborne

(1985). Briefly, the assay mixture consisted of 1.95 ml phosphatebuffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and0.05 ml post-mitochondrial supernatant in a final volume of3.0 ml. Changes in absorbance were recorded at 240 nm. Catalaseactivity was calculated in terms of k min�1.

2.6.6. Nitrite estimationNitrite was estimated in the sciatic nerve post-mitochondrial

supernatant using the Greiss reagent and served as an indicatorof nitric oxide production. A measure of 500 ll of Greiss reagent(1:1 solution of 1% sulphanilamide in 5% phosphoric acid and0.1% napthaylamine diamine dihydrochloric acid in water) wasadded to 100 ll of sciatic nerve homogenate and absorbance wasmeasured at 546 nm (Green et al., 1982). Nitrite concentrationwas calculated using a standard curve for sodium nitrite. Nitritelevels were expressed as lg/ml.

2.7. Rat TNF-a, IL-1b and TGF-b1 ELISA

The quantifications of TNF-a, IL-1b and TGF-b1 were done bythe help and instructions provided by R&D Systems QuantikineRat TNF-a, IL-1b and TGF-b1 immunoassay kit. The QuantikineRat TNF-a, IL-1b and TGF-b1 immunoassay is a 4.5 h solid phaseELISA designed to measure rat TNF-a, IL-1b and TGF-b1 levels.The assay employs the sandwich enzyme immunoassay technique.A monoclonal antibody specific for rat TNF-a, IL-1b and TGF-b1 hasbeen pre coated in the microplate. Standards, control and sampleswere pipetted into the wells and any rat TNF-a, IL-1b and TGF-b1present is bound by the immobilized antibody. After washing awayany unbound substance, an enzyme linked polyclonal antibodyspecific for rat TNF-a, IL-1b and TGF-b1 is added to the wells. Fol-lowing a wash to remove any unbound antibody-enzyme reagent,a substrate solution is added to the wells. The enzyme reactionyields a blue product that turns yellow when the stop solution isadded. The intensity of the color measured is in proportion to theamount of rat TNF-a, IL-1b and TGF-b1 bound in the initial steps.The sample values are then read off the standard curve. Valueswere expressed as mean ± S.E.M.

2.8. Statistical analysis

Results were expressed as mean ± S.E.M. The intergroup varia-tion was measured by one way analysis of variance (ANOVA) fol-lowed by Tukey’s test. Statistical significance was considered atP < 0.05. The statistical analysis was done using the SPSS StatisticalSoftware version 14.

3. Results

3.1. Behavioral observations

3.1.1. Modulation of thermal hyperalgesia in tail-immersion testBefore the administration of the ethanol, the mean baseline tail

flick latency (13.40 ± 0.19 s) was not significantly different fromthat in control group (13.20 ± 0.17 s). A significant decrease in tailflick latency (i.e., thermal hyperalgesia) was produced in the etha-nol administered rats after 6 weeks (2.73 ± 0.19, p < 0.05) whichgets further reduced (1.93 ± 0.19, p < 0.05) up to 10th week.Chronic treatment with EGCG (25, 50 and 100 mg/kg) significantlyprevented thermal hyperalgesia in a dose-dependent manner(Fig. 1A). There was no significant change in the mean tail flick la-tency of the control and per se group over the same time period.

3.1.2. Modulation of mechanical hyperalgesia in Randall–Sellito testThe mean baseline paw-withdrawal threshold of ethanol treated

rats (160.33 ± 2.59 gm) was recorded on day 0 before administra-tion of ethanol which was not significantly different from that incontrol group rats (160.35 ± 4.31 gm). A significant decrease inmechanical nociceptive threshold (i.e., mechanical hyperalgesia)was produced in the ethanol treated rats after 6 weeks(80.48 ± 4.85 gm, p < 0.05) and subsequently reduced up to 10thweek (52.98 ± 3.21 gm, p < 0.05). Chronic treatment with EGCG(25, 50 and 100 mg/kg) significantly increased the pain threshold inethanol administered rats in a dose-dependent manner (Fig. 1B).There was no significant change in the mean vocalization thresholdof the control and per se group over the same time period.

3.1.3. Effect on mechanical allodynia in von-Frey hair testThe mean baseline paw-withdrawal threshold (84.60 ± 2.03 gm,

p < 0.05) of ethanol treated rats on day 0 was not significantly dif-ferent from that in control group (84.73 ± 0.95 gm, p < 0.05). After6 weeks of ethanol administration, in response to von-Frey hair

Fig. 1. Effect of chronic treatment with epigallocatechin-3-gallate (EGCG) onreaction time in tail-immersion test (A), mechanical hyperalgesia in Randall–Sellitotest (B) and on mechanical allodynia in von-Frey hair test (C). A significant decreasein tail flick latency in tail-immersion test and paw-withdrawal threshold in RandallSellito and von-Frey hair test was produced in the ethanol administered rats whichwas significantly ameliorated on treatment with EGCG (25, 50 and 100 mg/kg) in adose-dependent manner �different from control group (P < 0.05); #different fromethanol administered group (P < 0.05); $different from one another (P < 0.05) (oneway ANOVA followed by Tukey’s test). CNTL control, E ethanol, EG (25) EGCG(25 mg/kg), EG (50) EGCG (50 mg/kg), EG (100) EGCG (100 mg/kg).

V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292 289

stimulation, a significant decrease (22.99 ± 2.26 gm, p < 0.05) inpaw-withdrawal threshold (i.e., mechanical allodynia) was pro-duced in the ethanol treated rats which gets further reduced(11.34 ± 0.55 gm, p < 0.05) up to 10th week. Treatment with EGCG(25, 50 and 100 mg/kg) significantly and dose-dependently pre-vented the decreased paw-withdrawal threshold. There was nosignificant change in the mean paw-withdrawal threshold of thecontrol and per se group over the same time period.

3.2. Biochemical observations

3.2.1. Effect on ethanol induced changes in lipid peroxidationMalonaldehyde (MDA) levels were increased significantly in the

sciatic nerve of ethanol treated rats (3.58 ± 0.16 nmoles/mg pr,

p < 0.05); as compared to control group (0.70 ± 0.05 nmoles/mg pr). Chronic treatment with EGCG (25, 50 and 100 mg/kg) pro-duced a significant (p < 0.05) and dose-dependent reduction inMDA levels in sciatic nerve of ethanol treated rats (Table 1).

3.2.2. Effect on anti-oxidant profile in the sciatic nerve of ethanoltreated rats

The reduced glutathione levels and enzyme activity of superox-ide dismutase and catalase were significantly decreased in the sci-atic nerve of ethanol treated rats as compared to control group.This reduction in anti-oxidant profile was significantly preventedby EGCG (25, 50 and 100 mg/kg) treatment in sciatic nerve of eth-anol treated rats (Table 1).

3.2.3. Effect on ethanol induced nitrosative stress in the rat sciaticnerve

Nitrite levels were significantly elevated in sciatic nerve of eth-anol treated animals (305.83 ± 4.80 lg/ml, p < 0.05) as comparedto control group (93.50 ± 6.25 lg/ml, p < 0.05). Chronic treatmentwith EGCG (25, 50 and 100 mg/kg) significantly and dose-depen-dently inhibited this increase in nitrite levels in sciatic nerve ofethanol treated rats (Fig. 2).

3.3. Effect on TNF-a, IL-1b and TGF- b1 levels in serum and sciaticnerve of ethanol treated rats

TNF-a, IL-1b and TGF-b1 levels were markedly increased in bothserum (Fig. 3A, C, and E) and sciatic nerve (Fig. 3B, D, and F) of eth-anol treated rats as compared to control and per see group. EGCGtreatment significantly decreased these elevated cytokine levelsboth in serum and sciatic nerve of ethanol treated rats in a dose-dependent manner.

4. Discussion

Various clinical studies suggest the beneficial effects of anti-oxi-dant therapy on signs and symptoms of neuropathic pain condi-tions. Recently, Ranieri et al. (2009) found that 6 week oraltreatment with alpha-lipoic acid and gamma-linolenic acid, the po-tent natural anti-oxidants, in synergy with rehabilitation therapyimproved neuropathic symptoms and deficits in patients withradicular neuropathy. Very recently, Pace et al. (2010) also foundthat treatment with vitamin E (alpha-tocopherol 400 mg/day) for3 months protects against cisplatin-induced peripheral neuropathyin phase III trials. Thus, treatment of neuropathic pain conditionswith natural anti-oxidants seems to hold a promising approach.

In the present study, daily administration of ethanol for10 weeks induced mechanical and thermal hyperalgesia along withtactile allodynia in rats, all of which are symptoms frequentlyoccurring in patients with painful peripheral neuropathy (Scaddinget al., 1982). These results are consistent with previous reports(Dina et al., 2000; Narita et al., 2007) demonstrating neuropathicpain like state in the rats following chronic alcohol consumption.These behavioral deficits induced by chronic ethanol treatmentwere significantly reduced in a dose-dependent manner on dailytreatment with EGCG. Our findings are supported by results fromvarious laboratories. Recently, Yun et al. (2007) found that etha-nol-induced hepatotoxicity and development of a fatty liver wasinhibited on treatment with EGCG. Kim et al. (2009) also reportedthat EGCG treatment inhibited both tetrodotoxin-sensitive andtetrodotoxin-resistant Na(+) currents in rat dorsal root ganglionneurons in a concentration-dependent manner suggesting its po-tential to develop as an analgesic agent. In an another study byHe et al. (2009), four weeks treatment with EGCG (2 mg/kg or6 mg/kg) significantly improved the cognitive deficits induced by

Table 1Effect of epigallocatechin-3-gallate (EGCG) treatment on lipid peroxide, reduced glutathione, superoxide dismutase and catalase levels (mean ± S.E.M.) in sciatic nerve of ethanoladministered rats.

Treatment LPO (nmoles/mg pr) GSH (lmoles/mg pr) SOD (units/mg pr) Catalase (k min�1)

CNTL 0.70 ± 0.05 0.118 ± 0.002 3.13 ± 0.14 4.98 ± 0.30E 3.58 ± 0.16* 0.021 ± 0.002* 0.27 ± 0.01* 0.52 ± 0.05*

E + EG (25) 2.88 ± 0.11#,$ 0.046 ± 0.004#,$ 0.97 ± 0.04# 1.86 ± 0.08#

E + EG (50) 2.36 ± 0.03#,$ 0.063 ± 0.003#,$ 1.22 ± 0.08# 2.16 ± 0.18#

E + EG (100) 1.98 ± 0.06# 0.089 ± 0.004#,$ 1.64 ± 0.06# 3.02 ± 0.28#

EG (100) 0.79 ± 0.06 0.114 ± 0.004 2.90 ± 0.28 4.05 ± 0.41

* Different from control group (P < 0.05).# Different from ethanol administered group (P < 0.05).$ Different from one another (P < 0.05).

Fig. 2. Effect of EGCG treatment on nitrite levels (mean ± S.E.M.) in sciatic nerve ofethanol administered rats. Nitrite levels were significantly increased in ethanoltreated rats which was significantly decreased in dose-dependent manner ontreatment with EGCG (25, 50 and 100 mg/kg). �Different from control group(P < 0.05); #different from ethanol administered group (P < 0.05); $different fromone another (P < 0.05) (one way ANOVA followed by Tukey’s test). CNTL control, Eethanol, EG (25) EGCG (25 mg/kg), EG (50) EGCG (50 mg/kg), EG (100) EGCG(100 mg/kg).

290 V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292

2 week administration of D-galactose in mice as measured in watermaze.

Several studies have suggested that chronic ethanol increasesoxidative damage to proteins, lipids, and DNA (Mansouri et al.,2001; McDonough, 2003). Neural tissues are poor in anti-oxidantdefence enzymes such as catalase and superoxide dismutase andthus are more prone to damage by oxidative stress. Kim et al.(2004) suggested the involvement of endoneural oxidative stressin nerve dysfunction in rats with chronic constriction injury. Reac-tive oxygen species are known to trigger second messengers in-volved in central sensitization of dorsal horn cells (Ali and Salter,2001) or activate spinal glial cells which in turn plays an importantrole in chronic pain (Raghavendra et al., 2003). Reduced glutathi-one is a major low molecular weight scavenger of free radicals incytoplasm and its depletion increases the susceptibility of neuronsto oxidative stress and hyperalgesia (Wullner et al., 1999).

Nitric oxide is also implicated in neuropathic pain (Levy andZochodne, 2004). It sensitizes spinal neurons and contributes tosensitization of central neurons by disinhibition (Lin et al., 1999).Moreover, unfettered production of nitric oxide coupled with defi-cient superoxide dismutase leads to production of notorious perox-ynitrite, which is several times multitude of its parents in terms oftissue toxicity. Rats chronically fed ethanol showed enhanced pro-duction of oxidative markers, such as thiobarbituric acid-reactivesubstances, hydrogen peroxide, and OH� like species (Dicker andCederbaum, 1992). A significant increase in MDA levels, nitrite lev-els and marked decrease in the activity of superoxide dismutase,catalase and reduced glutathione in the sciatic nerve of rats chron-ically fed ethanol suggests the involvement of oxidative and nitro-sative stress in the development of alcoholic neuropathy in rats(Tiwari et al., 2009). In the present study, chronic treatment with

EGCG significantly and dose-dependently attenuated all these bio-chemical alterations. EGCG is known to have neuroprotective ef-fects in wide variety of animal models by inhibition of enhancedoxidative-nitrosative stress and augmentation of anti-oxidant de-fence capacity (Kim et al., 2009; Wang et al., 2009). The EGCGpre-treatment abolished ethanol-induced lipid peroxidation ofthe cell membrane, loss of total superoxide dismutase (SOD) activ-ity and suppressed ethanol-induced gene expressions of Mn– andCu/Zn–SOD (Asaumi et al., 2006). EGCG administration also ledto a dose-dependent decrease in the degree of liver injury andexpression of iNOS and nitrotyrosine in the carbon tetrachloride-treated mice (Chen et al., 2004). EGCG treatment elevated totalsuperoxide dismutase and glutathione peroxidase activities, de-creased malondialdehyde contents and reduced the cell apoptosisindex and expression of cleaved caspase-3 in the hippocampus ofaging mice induced by D-galactose indicating its anti-oxidativeand anti-apoptotic potential (He et al., 2009). Administration ofEGCG also prevented diabetic nephropathy in rats by inhibiting in-creased oxidative stress and protein expressions of COX-2, NF-jbp65 and TGF-b1 in the kidneys of diabetic animals (Yamabeet al., 2006).

TNF-a has been demonstrated as an important mediator of neu-ropathic pain (Ignatowski et al., 1999). Prophylactic treatmentwith TNF-a inhibitors efficiently reduces hyperalgesia suggestingthat TNF-a contribute to the initiation of neuropathic pain (Georgeet al., 1999). Like TNF-a, IL-1b is also known to contribute signifi-cantly in various models of neuropathic pain. Nerve-injury inducedrelease of IL-1b might contribute to the central sensitization asso-ciated with chronic neuropathic pain (Sabrina et al., 2008). Geneticimpairment of interleukin-1 signaling attenuated neuropathicpain, autotomy, and spontaneous ectopic neuronal activity follow-ing nerve injury in mice (Wolf et al., 2006). It has been observed inour study that chronic alcohol administration increased TNF-a andIL-1b both in serum and sciatic nerve and this rise was inhibited byEGCG treatment in a dose-dependent manner.

Transforming growth factors have also been implicated in thegeneration of pathological pain states in both peripheral andcentral nervous system (Lewin and Mendell, 1993). TGF-b is alsoinvolved in the development and progression of diabetic neuropa-thy (Anjaneyulu et al., 2008). But the role of transforming factors inalcoholic neuropathy is still unexplored. In the present study, wefound elevation in TGF-b1 both in serum and sciatic nerve ofchronic alcohol treated rats and this effect was attenuated on treat-ment with EGCG. Ethanol treatment is known to cause an increasein expression of TGF-b1 and CYP2E1 in the centrilobular area ofthe liver and involves increased production of intracellular ROSand depletion of GSH which results in mitochondrial membranedamage and loss of membrane potential followed by apoptosis(Zhuge and Cederbaum, 2006). Acetaldehyde, one of the most toxicmetabolite of ethanol, activated TGF-b signaling by stimulating thesecretion and activation of latent TGF-b1 as well as by inducing the

Fig. 3. Effect of EGCG treatment on serum and nerve TNF-a (A, B), IL-1b (C, D) and TGF-b (E, F) levels in ethanol administered rats. A significant increase in TNF-a, IL-1b andTGF-b1 was found both in serum and sciatic nerve of ethanol treated rats which was significantly decreased in a dose-dependent manner on treatment with EGCG (25, 50 and100 mg/kg). *Different from control group (P < 0.05); #different from ethanol administered group (P < 0.05); $different from one another (P < 0.05) (one way ANOVA followedby Tukey’s test). CNTL control, E ethanol, EG (25) EGCG (25 mg/kg), EG (50) EGCG (50 mg/kg), EG (100) EGCG (100 mg/kg).

V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292 291

expression of the TGF-b-RII gene in cultured hepatic stellate cells(Chen et al., 2002). The same group of workers also reported thatEGCG treatment markedly suppressed the activation of culturedhepatic stellate cell by blocking transforming growth factor-betasignal transduction and by inhibiting the expression of alpha1(I)collagen, fibronectin and alpha-smooth muscle actin genes. EGCGis also found to suppress the ethanol-induced p38 mitogen-acti-vated protein (MAP) kinase phosphorylation, alpha-smooth muscleactin production and activated transforming growth factor-beta1secretion into the medium induced by acetaldehyde (Asaumiet al., 2006).

Thus, it is clear from the above discussion that chronic alcoholadministration for 10 weeks in rats resulted in decreased nocicep-tive threshold by increasing oxido-nitrosative stress, pro-inflam-matory cytokines and TGF-b1 levels both in serum and sciaticnerve of rats and treatment with EGCG prevented all the func-tional, biochemical and molecular deficits in a dose-dependentmanner.

Therefore, it was concluded that anti-oxidant property of EGCGmay be responsible for protecting against the oxidative stress med-iated activation of transforming growth factor-b1 (TGF-b1), animportant mediator of neuropathic pain, possibly by increasing

the endogenous defensive capacity to combat oxidative stress in-duced by chronic alcohol administration. In addition to potentanti-oxidant activity, the suppression of nitrosative stress andcytokine (TNF-a, IL-1b) and TGF-b1 levels both in serum and sciaticnerve by EGCG also contributes significantly in preventing thealcoholic neuropathy in rats.

Disclosure/conflict of interest

The authors declare that there are no personal financial hold-ings that could be perceived as constituting a potential conflict ofinterest.

Acknowledgements

Authors are thankful to DSM Nutritional Products Ltd., Switzer-land for providing gift sample of epigallocatechin-3-gallate. The se-nior research fellowship granted to Mr. Anurag Kuhad and Mr.Vinod Tiwari by Indian Council of Medical Research is also grate-fully acknowledged.

292 V. Tiwari et al. / European Journal of Pain 15 (2011) 286–292

References

Aktas O, Prozorovski T, Smorodchenko A, Savaskan NE, Lauster R, Kloetzel PM, et al.Green tea epigallocatechin-3-gallate mediates T cellular NF-kappa B inhibitionand exerts neuroprotection in autoimmune encephalomyelitis. J Immunol2004;173:5794–6800.

Ali DW, Salter MW. NMDA receptor regulation by Src kinase signalling in excitatorysynaptic transmission and plasticity. Curr Opin Neurobiol 2001;11(3):336–42.

Anjaneyulu M, Berent-Spillson A, Inoue T, Choi J, Cherian K, Russell JW.Transforming growth factor-beta induces cellular injury in experimentaldiabetic neuropathy. Exp Neurol 2008;211(2):469–79.

Asaumi H, Watanabe S, Taguchi M, Tashiro M, Nagashio Y, Nomiyama Y, et al. Greentea polyphenol (�)-epigallocatechin-3-gallate inhibits ethanol-inducedactivation of pancreatic stellate cells. Eur J Clin Invest 2006;36(2):113–22.

Backonja MM. Defining neuropathic pain. Anesth Analg 2003;97(3):785–90.Review. Erratum in: Anesth Analg. 2004;98(1):67.

Campbell JN, Meyer RA. Mechanisms of neuropathic pain. Neuron 2006;52:77–92.Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of

tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55–63.Chen A, Zhang L, Xu J, Tang J. The antioxidant (�)-epigallocatechin-3-gallate inhibits

activated hepatic stellate cell growth and suppresses acetaldehyde-inducedgene expression. Biochem J 2002;368(Pt 3):695–704.

Chen JH, Tipoe GL, Liong EC, So HS, Leung KM, Tom WM, et al. Green tea polyphenolsprevent toxin-induced hepatotoxicity in mice by down-regulating induciblenitric oxide-derived prooxidants. Am J Clin Nutr 2004;80(3):742–51.

Claiborne A. Catalase activity. In: Greenwald RA, editor. Handbook of methods foroxygen radical research. Boca Raton, BR: CRC Press; 1985. p. 283–4.

Cohen AC, Tong M, Wands JR, de la Monte SM. Insulin and insulin-like growth factorresistance with neurodegeneration in an adult chronic ethanol exposure model.Alcohol Clin Exp Res 2007;31(9):1558–73.

Committee for Research and Ethical Issues of the IASP, Ethical standards forinvestigations of experimental pain in animals, Pain 1983;16:109–10.

Courteix C, Eschalier A, Lavarenne J. Streptozocin-induced diabetic rats: behaviouralevidence for a model of chronic pain. Pain 1993;53:81–8.

Dicker E, Cederbaum AI. Increased NADH-dependent production of reactive oxygenintermediates by microsomes after chronic ethanol consumption: comparisonswith NADPH. Arch Biochem Biophys 1992;293:274–80.

Dina OA, Barletta J, Chen X, Mutero A, Martin A, Messing RO, et al. Key role for theepsilon isoform of protein kinase C in painful alcoholic neuropathy in the rat. JNeurosci 2000;20(22):8614–9.

George A, Schmidt C, Weishaupt A, Toyka KV, Sommer C. Serial determination oftumor necrosis factor-alpha content in rat sciatic nerve after chronicconstriction injury. Exp Neurol 1999;160:124–32.

Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem1982;126:131–8.

Han MK. Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic beta-cell damage. Exp Mol Med 2003;35:136–9.

He M, Zhao L, Wei MJ, Yao WF, Zhao HS, Chen FJ. Neuroprotective effects of (�)-epigallocatechin-3-gallate on aging mice induced by D-galactose. Biol PharmBull 2009;32(1):55–60.

Ignatowski TA, Covey WC, Knight PR, Severin CM, Nickola TJ, Spengler RN. Brain-derived TNFa mediates neuropathic pain. Brain Res 1999;841:70–7.

Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene induced livernecrosis: protective role of glutathione and evidence for 3,4-bromobenzenoxideas the hepatotoxic intermediate. Pharmacology 1974;11:151–69.

Kasdallah-Grissa A, Mornagui B, Aouani E, Hammami M, Gharbi N, Kamoun A, et al.Protective effect of resveratrol on ethanol-induced lipid peroxidation in rats.Alcohol Alcohol 2006;41(3):236–9.

Kim CY, Lee C, Park GH, Jang JH. Neuroprotective effect of epigallocatechin-3-gallateagainst beta-amyloid-induced oxidative and nitrosative cell death viaaugmentation of antioxidant defense capacity. Arch Pharm Res 2009;32(6):869–81.

Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, et al. Reactiveoxygen species (ROS) play an important role in a rat model of neuropathic pain.Pain 2004;111(1–2):116–24.

Koike H, Iijima M, Sugiura M, Mori K, Hattori N, Ito H, et al. Alcoholic neuropathy isclinicopathologically distinct from thiamine-deficiency neuropathy. Ann Neurol2003;54(1):19–29.

Koike H, Mori K, Misu K, Hattori N, Ito H, Hirayama M, et al. Painful alcoholicpolyneuropathy with predominant small-fiber loss and normal thiamine status.Neurology 2001;56(12):1727–32.

Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine andan assay for superoxide dismutase. Arch Biochem Biophys 1978;186:189–95.

Krieglstein K, Richter S, Farkas L, Schuster N, Dunker N, Oppenheim RW, et al.Reduction of endogenous transforming growth factors beta preventsontogenetic neuron death. Nat Neurosci 2000;3:1085–90.

Lee I, Kim HK, Kim JH, Chung K, Chung JM. The role of reactive oxygen species incapsaicin-induced mechanical hyperalgesia and in the activities of dorsal hornneurons. Pain 2007;133(1–3):9–17.

Lewin GR, Mendell LM. Nerve growth factor and nociception. Trends Neurosci1993;16:353–9.

Levy D, Zochodne DW. NO pain: potential roles of nitric oxide in neuropathic pain.Pain Practice 2004;4(1):11–8.

Li R, Huang YG, Fang D, Le WD. (�)-Epigallocatechin gallate inhibitslipopolysaccharide-induced microglial activation and protects againstinflammation-mediated dopaminergic neuronal injury. J Neurosci Res2004;78:723–31.

Lin Q, Wu J, Peng YB, Cui M, Willis WD. Nitric oxide-mediated spinal disinhibitioncontributes to the sensitization of primate spinothalamic tract neurons. JNeurophysiol 1999;81(3):1086–94.

Mansouri A, Demeilliers C, Amsellem S, Pessayre D, Fromenty B. Acute ethanoladministration oxidatively damages and depletes mitochondrial DNA in mouseliver, brain, heart, and skeletal muscles: protective effects of antioxidants. JPharmacol Exp Ther 2001;298:737–43.

McDonough KH. Antioxidant nutrients and alcohol. Toxicology 2003;189:89–97.Monforte R, Estruch R, Valls-Solé J, Nicolás J, Villalta J, Urbano-Marquez A.

Autonomic and peripheral neuropathies in patients with chronic alcoholism.A dose-related toxic effect of alcohol. Arch Neurol. 1995;52(1):45–51.

Montoliu C, Valles S, Renau-Piqueras J, Guerri C. Ethanol induced oxygen radicalformation and lipid peroxidation in rat brain: effect of chronic alcoholconsumption. J Neurochem 1994;63:1855–62.

Narita M, Miyoshi K, Narita M, Suzuki T. Involvement of microglia in the ethanol-induced neuropathic pain-like state in the rat. Neurosci Lett 2007;414:21–5.

Pace A, Giannarelli D, Galiè E, Savarese A, Carpano S, Della Giulia M, et al. Vitamin Eneuroprotection for cisplatin neuropathy: a randomized, placebo-controlledtrial. Neurology 2010;74(9):762–6.

Poulsen KT, Armanini MP, Klein RD, Hynes MA, Phillips HS, Rosenthal A. TGF beta 2and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons.Neuron 1994;13:1245–52.

Raghavendra V, Tanga F, Rutkowski MD, DeLeo JA. Anti-hyperalgesic and morphine-sparing actions of propentofylline following peripheral nerve injury in rats:mechanistic implications of spinal glia and proinflammatory cytokines. Pain2003;104(3):655–64.

Ranieri M, Sciuscio M, Cortese AM, Santamato A, Di Teo L, Ianieri G, et al. The use ofalpha-lipoic acid (ALA), gamma linolenic acid (GLA) and rehabilitation in thetreatment of back pain: effect on health-related quality of life. Int JImmunopathol Pharmacol 2009;22(Suppl. 3):45–50.

Sabrina LGV, Van BL, Aaron YL, Kathryn GT, Klaus B, Peter AS. Long-term actions ofinterleukin-1b on delay and tonic firing neurons in rat superficial dorsal hornand their relevance to central sensitization. Mol Pain 2008;4:63.

Scadding JW, Wall PD, Parry CB, Brooks DM. Clinical trial of propranolol in post-traumatic neuralgia. Pain 1982;14(3):283–92.

Sharma SS, Sayyed SG. Effects of trolox on nerve dysfunction, thermal hyperalgesiaand oxidative stress in experimental diabetic neuropathy. Clin Exp PharmacolPhysiol 2006;33(11):1022–8.

Taiwo YO, Coderre TJ, Levine JD. The contribution of training to sensitivity in thenociceptive paw-withdrawal test. Brain Res 1989;487:148–51.

Tiwari V, Kuhad A, Chopra K. Tocotrienol ameliorates behavioral and biochemicalalterations in the rat model of alcoholic neuropathy. Pain 2009;145(1–2):129–35.

Wang L, Xu S, Xu X, Chan P. (�)-Epigallocatechin-3-gallate protects SH-SY5Y cellsagainst 6-OHDA-induced cell death through STAT3 activation. J Alzheimers Dis2009;17(2):295–304.

Wills ED. Mechanism of lipid peroxide formation in animals. Biochem J1965;99:667–76.

Wolf G, Gabay E, Tal M, Yirmiya R, Shavit Y. Genetic impairment of interleukin-1signaling attenuates neuropathic pain, autotomy, and spontaneous ectopicneuronal activity, following nerve injury in mice. Pain 2006;120(3):315–24.

Wolf G, Yirmiya R, Goshen I, Iverfeldt K, Holmlund L, Takeda K, et al. Impairment ofinterleukin-1 (IL-1) signaling reduces basal pain sensitivity in mice: genetic,pharmacological and developmental aspects. Pain 2003;104(3):471–80.

Wullner U, Seyfried J, Groscurth P, Beinroth S, Winter S, Gleichmann M, et al.Glutathione depletion and neuronal cell death: the role of reactive oxygenintermediates and mitochondrial function. Brain Res 1999;826(1):53–62.

Yamabe N, Yokozawa T, Oya T, Kim M. Therapeutic potential of (�)-epigallocatechin3-O-gallate on renal damage in diabetic nephropathy model rats. J PharmacolExp Ther 2006;319(1):228–36.

Yun JW, Kim YK, Lee BS, Kim CW, Hyun JS, Baik JH, et al. Effect of dietaryepigallocatechin-3-gallate on cytochrome P450 2E1-dependent alcoholic liverdamage: enhancement of fatty acid oxidation. Biosci Biotechnol Biochem2007;71(12):2999–3006.

Zhuge J, Cederbaum AI. Increased toxicity by transforming growth factor-beta 1 inliver cells overexpressing CYP2E1. Free Radical Biol Med 2006;41(7):1100–12.