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Neurobiology of Disease 22 (2006) 421 – 434

Neuroprotective and neurorescue effect of black tea extract in

6-hydroxydopamine-lesioned rat model of Parkinson’s disease

R.K. Chaturvedi,a S. Shukla,a K. Seth,a S. Chauhan,c C. Sinha,a

Y. Shukla,b and A.K. Agrawala,*

aDevelopmental Toxicology Division, Industrial Toxicology Research Centre, Post Box-80, M.G. Marg Lucknow-226001, IndiabEnvironmental Carcinogenesis Division, Industrial Toxicology Research Centre, Post Box-80, M.G. Marg Lucknow-226001, IndiacSchool of Studies in Botany, Jiwaji University, Gwalior-474001, India

Received 7 July 2005; revised 9 November 2005; accepted 9 December 2005

Available online 9 February 2006

In the present study, an attempt has been made to explore the

neuroprotective and neuroreparative (neurorescue) effect of black tea

extract (BTE) in 6-hydroxydopamine (6-OHDA)-lesioned rat model of

Parkinson’s disease (PD). In the neuroprotective (BTE + 6-OHDA) and

neurorescue (6-OHDA + BTE) experiments, the rats were given 1.5%

BTE orally prior to and after intrastriatal 6-OHDA lesion respectively.

A significant recovery in dddd-amphetamine induced circling behavior

(stereotypy), spontaneous locomotor activity, dopamine (DA)-D2

receptor binding, striatal DA and 3–4 dihydroxy phenyl acetic acid

(DOPAC) level, nigral glutathione level, lipid peroxidation, striatal

superoxide dismutase and catalase activity, antiapoptotic and pro-

apoptotic protein level was evident in BTE + 6-OHDA and 6-OHDA +

BTE groups, as compared to lesioned animals. BTE treatment, either

before or after 6-OHDA administration protected the dopaminergic

neurons, as evident by significantly higher number of surviving

tyrosine hydroxylase immunoreactive (TH-ir) neurons, increased TH

protein level and TH mRNA expression in substantia nigra. However,

the degree of improvement in motor and neurochemical deficits was

more prominent in rats receiving BTE before 6-OHDA. Results suggest

that BTE exerts both neuroprotective and neurorescue effects against

6-OHDA-induced degeneration of the nigrostriatal dopaminergic

system, suggesting that possibly daily intake of BTE may slow down

the PD progression as well as delay the onset of neurodegenerative

processes in PD.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease; Black tea; Polyphenols; Neuroprotection;

Neurorescue; Neurodegeneration

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative

disorder with unknown etiology. Its neuropathology includes

degeneration of dopaminergic nigrostriatal pathway, which is a

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.12.008

* Corresponding author. Fax: +91 522 2628227.

E-mail address: aka33@rediffmail.com (A.K. Agrawal).

Available online on ScienceDirect (www.sciencedirect.com).

cumulative effect of glutathione depletion, iron deposition,

increased lipid peroxidation, oxidative DNA damage, mitochon-

drial dysfunction, excitotoxicity and alterations in antioxidant

enzymes activities (Jenner and Olanow, 1996; Gerlach et al.,

2003). Various aspects of PD, whether being genetic predisposi-

tion, altered neurotransmitter synthesis or alteration in detoxifica-

tion system, ultimately all have been shown to involve a common

cascade of events, which are mediated by oxidative stress (OS)

(Fahn and Sulzer, 2004). Recent reports revealed that, neuro-

degeneration in PD has also been linked to dietary habits, where

deficiency of antioxidant components such as folic acid (Miller,

2002; Zhu, 2004), vitamins (A, C, E and niacin) and selenium in

body have been shown to increase the risk of PD (Hellenbrand et

al., 1996; Paraskevas et al., 2003). Deficiency of these compounds

leads to increase in the level of reactive oxygen species (ROS) and

it has been speculated that OS possibly plays a role not only in

onset of the PD but also in progression of the disease (Zhu, 2004).

In addition to this, OS has also been shown to pose a major

limitation in l-DOPA therapy, as l-DOPA and endogenous

dopamine (DA) gets auto-oxidized forming quinone and ROS

and these may further accelerate the progression of disease (Zhu,

2004; Fahn and Sulzer, 2004; Foster and Hoffer, 2004). To

overcome free radical mediated consequences of disease process

and drug therapies, various antioxidant supplements have been

proposed and have been shown to play an important role in

neuroprotection (Mattson et al., 2002). Reports indicate that

administration of antioxidants with l-DOPA therapy protect

against ROS-induced damage both in vivo and in vitro (Chalimo-

niuk et al., 2004). Similarly, the success of the other therapeutic

approaches including cell replacement therapies, up to a major

extent also depends on conditions of OS prevailing at the time of

transplantation. In this regard, our study as well as others has

shown that the cell survival following transplantation can be

enhanced by administration of antioxidants (Dugan et al., 2001;

Agrawal et al., 2004a).

The progressive neurodegeneration in PD is not halt/slow down

by the currently used drug therapies. Hence, current researches are

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434422

focusing on finding therapies, preferentially natural products

including food supplements which could help in preventing/

delaying the ongoing neurodegeneration in PD (Dawson and

Dawson, 2002). Recently, a novel dietary strategy is proposed that

is specifically tailored toward lowering the risk of human PD,

which includes eating a nutritionally balanced diet that contains

adequate amounts of fruits and vegetables, along with adequate

dietary supplementation of vitamins C, B6, B12 and folate (Zhu,

2004).

Further, supplementation of dietary antioxidants such as

adenosine, selenium, a-tocopherol, h-carotene, ascorbic acid, N-

acetyl cysteine, tea polyphenols and flavonoids have been

shown to protect against OS and exert neuroprotective action

(Riederer et al., 1989; Soto-Otero et al., 2000; Roghani and

Behzadi, 2001; Sweeney et al., 2002; Zafar et al., 2003a,b).

Among these, tea and tea polyphenols have attracted increasing

interest because besides their radical scavenging property (Frei

and Higdon, 2003; Mercer et al., 2005), they have also been

shown to have iron chelating (Rah et al., 2005), anti

inflammatory (Pan et al., 2000), antiapoptotic (Mercer et al.,

2005), antineoplastic (Steele et al., 2000), anticarcinogenic

(Lambert et al., 2005), antimutagenic (Taneja et al., 2003;

Halder et al., 2005) and antiangiogenic properties (Oak et al.,

2005) under in vivo and in vitro condition as well as the ability

to modulate cell signaling (Levites et al., 2002). Moreover, tea

has been shown to possess stronger antioxidant property than

typical antioxidants such as glutathione, ascorbic acid, tocoph-

erol and than that of most reported dietary agents on a daily

basis (Yoshino et al., 1994; Rechner et al., 2002).

Besides this tea (Camellia sinensis) has an added advantage

of being one of the most commonly worldwide consumed

beverages. Green tea (GT), black tea (BT) and their constituents

(catechins, theaflavins, thearubigins and flavonoids) have the

ability to penetrate the blood brain barrier (BBB) and fulfill the

requirement of a potential neuroprotective agent (Nakagawa and

Miyazawa, 1997; Rah et al., 2005; Luczaj and Skrzydlewska,

2004). Further, tea extract and polyphenols have been shown to

attenuate MPTP and 6-OHDA-induced cell death in in vivo and

in vitro studies (Levites et al., 2001, 2002; Nie et al., 2002).

Research, to date, has been mainly focused on GT, whereas

there is not much information available exploring the neuro-

protective potential of BT, the consumption of which amounts to

be 80% of total tea consumption worldwide (Ames et al., 1993).

In addition, it has been reported that in comparison to GT, BT

has an advantage of being highly rich in multimeric polyphenol

(theaflavins), generated during tea leaves fermentation (absent in

GT), which is reported to be strongest antioxidant among

catechins (Dufresne and Farnworth, 2001; Leung et al., 2001).

BT has been shown to be more effective iron chelator than GT,

thus preventing metal catalyzed free radical formation (Hurrell et

al., 1999; Dufresne and Farnworth, 2001). BT has also been

shown to contain about one third the caffeine content of coffee,

which has shown protective effects in 6-OHDA rat model of PD

(Joghataie et al., 2004). In the present investigation, an attempt

has been made to study the neuroprotective and neurorescuing

effect of BT extract (BTE) in 6-OHDA-induced rat model of

PD. Possibly, this is for the first time, it has been evident from

this study, that consumption of BT is capable of imparting

neuroprotection to the degenerating DA neurons on one hand

while on the other hand rescues the degeneration of DA neurons

in rat model of PD.

Materials and methods

Chemicals

6-hydroxydopamine-HBr, d-amphetamine, dopamine (DA),

3,4-dihydroxyphenyl acetic acid (DOPAC), reduced glutathione

(GSH), thiobarbituric acid (TBA), trichloroacetic acid (TCA), 5–5Vdithio-bis-2-nitro benzoic acid (DTNB), phenazine methosulphate

(PMS), nitrobluetetrazolium (NBT), octane sulphonic acid, halo-

peridol, normal goat serum (NGS), 3–3V diaminobenzidine (DAB)

with metal enhancer, primary monoclonal anti-tyrosine hydroxy-

lase (TH), anti h-actin, anti Bcl-2 and anti Bax antibodies, alkaline

phosphatase and peroxidase-linked secondary antibodies, nitrocel-

lulose membrane (0.45 Am) and BCIP/NBT solution (5-bromo-4-

chloro-3-indolyl phosphate/nitrobluetetrazolium) were purchased

from Sigma Chemical Co. (USA). Trizol reagent and RT-PCR kit

were obtained from Gibco BRL (USA). TH primer was procured

from IDT, Coralville (USA). Sodium pentobarbital was procured

from MERCK (Germany). Radio ligand [3H]-spiperone (specific

activity 15.7 Ci/mmol) was obtained from Amersham, (UK) and

GF/C glass microfiber filters were obtained from Whatman (USA).

Dry BT leaves were purchased from Assam (India). All the other

chemicals used in the study were of AR grade, which were

available locally.

Animals and treatment

Female albino rats of Wistar strain (200–250 g, body weight)

were obtained from the Industrial Toxicology Research Centre

animal breeding colony. The animals were housed in plastic

polypropylene cages under standard animal house conditions with

a 12 h light/dark cycle and a temperature of 25 T 2-C. The animals

had free access to drinking water and pellet diet (Hindustan Lever

Laboratory Animal Feed, Kolkata, India). The institutional animal

care and ethical committee approved all procedures of animal

experimentation.

Aqueous black tea extract (BTE) was freshly prepared every

day. For preparing 1.5% BTE, 1.5 g of BT leaves were suspended

in 100 ml of hot water (85-C), brewed for 5 min, cooled to room

temperature and filtered. Rats were given 1.5% BTE ad libitum to

drink instead of water, following the experimental design, as

discussed below.

Group I (Sham)—received 3 Al of 0.2% l-ascorbate saline by

stereotaxic injection into the striatum and drinking water orally for

42 days.

Group II (Lesioned)—received 3 Al of 6-OHDA (4 Ag/Al in0.2% l-ascorbate saline) by stereotaxic injection into the striatum

and drinking water orally for 42 days.

Group III (BTE)—received 1.5% BTE orally for a total 42 days

as a sole source of drinking water.

Group IV (BTE + 6-OHDA)—received BTE treatment for

42 days in total, where 1.5% BTE was given for 21 days prior

to 6-OHDA lesioning (performed on 22nd day) and BTE

treatment continued till the 42nd day thereafter (Neuroprotective

experiment).

Group V (6-OHDA + BTE)—received 1.5% BTE from day 1

after 6-OHDA lesioning (day 0) and continued till the 42nd day

(Neurorescue experiment).

After 42 days, assessment of neuroprotective and neurorescue

potential of BTE was done using neurobehavioral, neurochemical

and immunohistochemical parameters.

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434 423

Intrastriatal 6-OHDA administration (6-OHDA lesion)

Rats were anesthetized by sodium pentobarbital (40 mg /kg

b.w. i.p.) and then mounted in a stereotaxic apparatus (Stoelting

Co. USA). The skull was exposed by making a small incision

in the skull skin. Unilateral striatal DA neuronal degeneration

was induced in rats by stereotaxic injections of 3 Al 6-OHDA

(4 Ag/Al dissolved in 0.2% l-ascorbate saline) using 10

Al Hamilton syringe into the right striatum at following

coordinates (in mm with respect to bregma) AP �0.5, L 2.5,

V 4.5 (Paxinos and Watson, 1998). The injection rate was

maintained at 1 Al/min using an auto injector device. After each

injection, needle was left in place for an additional 5 min for

diffusion before withdrawal. Another group of rats, which were

injected with a 6-OHDA-free solution (0.2% l-ascorbate saline)

in a similar manner, served as sham.

Postoperative care

Recovery from anesthesia took approximately 5 h. The rats

were kept in a well-ventilated room at 25 T 2-C in individual cages

till they gained full consciousness and were then housed together in

a group of four animals per cage. Food was kept inside the cages

during the first week so that animals could easily access it without

any physical trauma due to surgical intervention.

Neurobehavioral studies

d-amphetamine-induced circling behavior

Six rats from each group were assessed for circling behavior

after injecting 5 mg/kg d-amphetamine i.p. following the method

of Shukla et al. (2004). Rotational behavior was recorded after 30

min of injection, assessed for a period of 30 min and is expressed

as number of ipsilateral rotations/30 min.

Spontaneous locomotor activity (SLA)

SLA was monitored in a computerized Optovarimax system

(Columbus Instruments Ohio USA), which helps in eliminating

observer’s bias in quantification of motility following previously

described method (Agrawal et al., 2004a). The Optovarimax is a

horizontal 2 D-activity meter, consisting of two arrays of 15

infrared beams, which are placed perpendicular to each other. The

beams are spaced about 1 in. (25.4 mm) and each beam is very

narrow (3 mm in diameter). It also consists of an activity monitor

and a programmer/processor.

Rats were individually placed in the chamber, acclimatized for

5 min and their locomotor activity scores were recorded for 10 min.

Interruption in the photo beams positioned in parallel inside the

chamber resulted in an activity count, which is processed by the

microcomputer and recorded for data analysis. The activity

chamber was swabbed with 10% ethanol every time to avoid the

interference due to animal odors. Results are expressed in terms of

distance travelled in cm/10 min.

Neurochemical studies

Studies related to oxidative stress

In order to assess free radical mediated effects following 6-

OHDA lesioning and to see free radical scavenging potential of

BTE, estimation of lipid peroxidation (LPO) and reduced

glutathione (GSH) was carried out in ipsilateral substantia nigra

(SN), while enzymatic antioxidants (total superoxide dismutase

and catalase) were estimated in ipsilateral striatum. Rats were

sacrificed by cervical dislocation followed by decapitation and

their brains were dissected quickly on ice pack. Both regions were

dissected, weighed and processed fresh on same day for the

estimation of LPO, GSH, total SOD and catalase as follows.

Lipid peroxidation potential (LPO)

LPO in ipsilateral SN was measured by estimating malonal-

dialdehyde (MDA) levels following the method of Bohme et al.

(1977). In brief, brain homogenate was prepared in 0.15 M KCl

(5%w/v homogenate) and aliquots of 0.6 ml was incubated for 0

and 1 h at 37-C. The reaction was stopped by adding 1.2 ml of

28% w/v TCA and the volume was made up to 3 ml by adding 1.2

ml of water. Following centrifugation at 3000 � g for 10 min,

supernatant was removed and colour was developed by addition of

0.5 ml of 1% w/v TBA dissolved in 0.05 N NaOH to the

supernatant which was further kept in a boiling water bath for 15

min till the appearance of pink colour. The absorbance was read at

532 nm in a spectrophotometer. Results are expressed as nmoles

MDA formed/minute/mg protein.

Reduced glutathione (GSH)

GSH was measured in ipsilateral SN following the method of

Sedlak and Linsay (1968). Briefly, SN tissue was deproteinized

with an equal volume of 10% TCA and was allowed to stand at

4-C for 2 h. The contents were centrifuged at 2000 g for 15 min.

The supernatant was added to 2 ml of 0.4 M Tris buffer (pH 8.9)

containing 0.02 M EDTA (pH 8.9) followed by the addition of 0.01

M DTNB. Finally, the mixture was diluted with 0.5 ml of distilled

water, to make the total mixture to 3 ml and absorbance was read in

a spectrophotometer at 412 nm and results are expressed as AgGSH/gm tissue.

Total superoxide dismutase (SOD) activity

Total SOD was measured in ipsilateral striatal region following

the method of Kakkar et al. (1984). In brief, 3.0 ml of assay

mixture consisted of sodium pyrophosphate buffer, 1.2 ml (0.082

M, pH 8.3), PMS, 0.3 ml (186 AM), NBT, 0.3 ml (300 AM),

NADH, 0.2 ml (780 AM), and 1 ml of 10% striatal tissue

homogenate (prepared in 0.1 M phosphate buffer). The reaction

was initiated by addition of NADH, followed by incubation at

37-C for 90 s. Adding 1.0 ml glacial acetic acid stopped the

reaction and the reaction mixture was vigorously shaken with 4.0

ml of n-butanol. The mixture was allowed to stand for 10 min,

centrifuged for 10 min at 3000 rpm and butanol layer was

separated. The colour intensity of the formazan in butanol layer

was measured at 560 nm against butanol using a spectrophotom-

eter. A mixture without enzyme preparations was run in parallel,

which served as blank. The SOD activity is expressed in nmol

formazan formed/minute/mg protein.

Catalase activity

Catalase activity in striatal region was assayed following the

method of Sinha (1972) using H2O2 as substrate. The reaction

mixture of 1.5 ml consisted of 1.0 ml phosphate buffer (0.01 M, pH

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434424

7.0), 0.4 ml distilled water and 0.1 ml of 10% homogenate

(prepared in 0.1 M phosphate buffer). Reaction was started by

adding 0.5 ml H2O2, incubated at 37-C for 1 min and reaction was

stopped by addition of 2.0 ml of dichromate: acetic acid reagent.

The tubes were immediately kept in a boiling water bath for 15 min

and centrifuged for 10 min (3000 rpm). The green colour

developed during the reaction was read at 570 nm on a

spectrophotometer. Control tubes, devoid of enzyme, were also

processed in parallel. The enzyme activity is expressed as nmol

H2O2 consumed/min/mg protein.

DA–D2 receptor binding

Assay of DA–D2 receptor binding was carried out in

ipsilateral striatum of all experimental groups following the

method of Agrawal et al. (1981). Crude synaptic membrane

fraction was prepared by homogenizing the striatal tissue in 19

volume of pre-chilled 0.32 M sucrose followed by centrifuga-

tion at 50,000 � g for 10 min. The pellet was rehomogenized

in 5 mM Tris–HCl buffer (pH 7.4), in same volume and

centrifuged at a similar speed for 10 min at 4-C. The pellet was

finally suspended in 40 mM Tris–HCl buffer (pH 7.4) and

stored at �20-C till assay. The binding incubation was carried

out in triplicate at 37-C for 15 min using synaptic membrane

fraction (100 Al equivalent to 250–300 Ag protein) with 1 nM

[3H]-spiperone as specific ligand for DA–D2 receptor. Parallel

assay in triplicate using 1 AM unlabelled haloperidol (DA

receptor antagonist) was carried out to determine non-specific

binding. After 15 min incubation at 37-C, the reaction was

terminated by cooling in ice, the contents were filtered through

glass fiber filters (Whatman GF/C, 25 Am pore size) under

vacuum and washed twice with 5 ml cold Tris–HCI buffer. The

filters were dried and radioactivity was counted in 5 ml of

scintillation mixture (0.065 g POPOP, 3.25 g PPO, 26 g

naphthalene, 250 ml toluene, 150 ml methanol and 250 ml 1,4-

dioxane) in LKB Rack h liquid scintillation counter (Packard

Instrument, Germany) having an efficiency of 50% for tritium.

Specific binding was calculated by subtracting non-specific

binding from total binding obtained in absence of haloperidol.

The results are expressed in terms of pmole of ligand bound/gm

protein. Protein was estimated by the method of Lowry et al.

(1951). To evaluate the kinetics of DA–D2 receptors, Scatchard

analysis was performed using varying concentrations (0.1–10

nM) of [3H]-Spiperone. Affinity (Kd) and the maximum number

of binding sites (Bmax) were calculated using linear regression

analysis (Scatchard, 1949).

DA and DOPAC levels

Five rats from each group were sacrificed by decapitation 24

h after the last dose of BT. The brains were removed quickly

and ipsilateral striatum was dissected in cold condition on ice

pack. The striatal tissue levels of DA and its metabolite

DOPAC were measured by high-performance liquid chromatog-

raphy (HPLC, Merck, Germany), using electrochemical detector

(Merck, L-3500 A) following the method of Chaturvedi et al.

(2003). The separation of DA and DOPAC was done using

mobile phase, containing 10% methanol, 32 mM citric acid,

12.5 mM Na2HPO4, 0.5 mM octyl sodium sulphate and 0.05

mM EDTA. Electrochemical detection was done at +0.8 V

using Ag/AgCl reference electrode and keeping the flow rate at

1 ml/min. DA and DOPAC levels were quantified by peak

height comparisons with standards, run on the day of analysis.

The results are expressed in terms of pg DA and DOPAC/gm

tissue.

TH immunohistochemical study

TH immunoreactivity was carried out in substantia nigra pars

compacta (SNpc) following the method of Agrawal et al. (2004b).

The rats from each group were deeply anesthetized with sodium

pentobarbital (40 mg/kg, i.p.) and perfused transcardially with 0.1

M phosphate-buffered saline (PBS, pH 7.2), followed by 4%

paraformaldehyde in PBS for fixation of tissue. Brains were

removed and post-fixed in the same fixative for 24 h followed by

transfer to 10%, 20%, and 30% sucrose (W/V) in PBS. Serial

coronal sections of 20 Am thickness were cut in freezing

microtome (Slee Mainz Co., Germany). Endogenous peroxidase

activity was inhibited by incubating the sections in 0.5% H2O2 in

methanol. Non-specific binding sites were blocked by incubating

the sections in PBS containing 1.5% NGS, 0.5% BSA and 0.1%

Triton X-100. These sections were then incubated for 48 h in

primary antibody (anti-TH antibody, 1:500). After removing the

primary antibody, sections were washed three times with PBS and

incubated in peroxidase linked secondary antibody (1: 200) for 2

h at room temperature followed by three washes with PBS. Colour

for peroxidase linked antibody was developed with DAB as

chromogen. Sections were transferred on to gelatinized glass

slides, dehydrated, cleared, mounted in DPX, cover slipped and

then visualized under microscope.

Image analysis

The total number of TH-ir neurons in the ipsilateral SNpc was

counted in rats of all groups, using the optical dissector (3-

dimensional cell counting) method (West, 1999). The unbiased

stereological cell-counting procedure was applied, where a person

unknown to the experimental design carried out the stereology,

using computerized Leica Qwin image analysis system. The total

ten sections used for counting (fraction of sections being sampled

1/5) covered the entire SN. Every fifth coronal tissue section

through SNpc at three levels: �5.2 mm, �5.5 mm and �5.7 mm

with respect to the bregma (Paxinos and Watson, 1998) was

sampled for TH-ir neurons counting. A contour was drawn around

the SNpc in each of the sections that contained the TH-ir cells.

The criterion for delineating the SNpc from the ventral tegmental

area was localization of the oculomotor nerve root. The ventral

tegmental area was considered to be within and medial to the

rootlets, whereas the SN was considered to be located laterally.

On each section, a 150 � 150 Am grid was randomly placed on

the image. The counting frame area was 45 � 45 Am = 2025 Am2.

Therefore, the area sampling fraction was 2025 / (150 � 150) =

0.09. Counting frame density was 10 counting frames/section.

Mounted section thickness averaged 18 Am, an optical dissector

height was determined at 12 Am following 2 Am top guard zone

and 2 Am bottom guard zone. The TH-ir cells were only counted

if the first recognizable labeled profiles of the cell came into focus

within counting frame. Particles less than 5 Am were excluded

from counting. For cells that intersected the counting frame, those

that intersected the green lines were counted, whereas those that

intersected the red lines and present in guard zones were

excluded. To avoid measuring the same neuron twice, the sections

Table 1

Average black tea extract (BTE) or drinking water intake

Treatment groups Average intake in ml/day

Sham 25 T 4.3

6-OHDA 22 T 2.6

BTE 26 T 3.2

6-OHDA + BTE 24 T 2.9

BTE + 6-OHDA 28 T 3.6

Average intake of 1.5% BTE in BTE, 6-OHDA + BTE and BTE + 6-

OHDA groups and drinking water intake in sham and 6-OHDA groups.

BTE was prepared in drinking water, no significant difference was observed

in the average intake of 1.5% BTE and intake was consistent between

animals of different groups and intragroup. Further, there was no significant

difference in consumption of normal drinking water in sham and 6-OHDA

group as compared to rats receiving 1.5% BTE. Values represent mean T SE

of 30 rats/group.

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434 425

were separated by approximately 100 Am. The total number of

neurons in the SNpc was estimated by multiplying the number of

neurons counted within the sampled regions with the reciprocals

of the fraction of the sectional area sampled and the fraction of

the section thickness sampled (West, 1999). The data represents

mean T SE (n = 5 rats/group).

Level of TH protein, antiapoptotic protein (Bcl-2) and proapoptotic

protein (Bax) by immunoblotting

The level of TH protein, antiapoptotic protein (Bcl-2) and

proapoptotic protein (Bax) were detected by Western blot

analysis in striatum and SN from control and treated rats. In

brief, brain regions were dissected, weighed and homogenized

in 1% sodium dodecyl sulphate (SDS) solution at 4-C using

Teflon/glass homogenizer. The homogenized samples were

boiled at 100-C for 5 min in Laemmli sample buffer and

centrifuged at 14,000 rpm for 10 min at 4-C. Electrophoresis

and Western blotting was carried out according to the method of

Laemmli (1970). Briefly, sample containing 20 Ag protein, per

well were loaded in sodium dodecyl sulphate-polyacrylamide

gel (SDS-PAGE; 5% stacking gel and 10% separating gel) and

were then electroblotted on nitrocellulose membrane (0.45 Am).

The membranes were incubated with the primary anti-TH

antibody (1:1000), anti Bcl-2 antibody (1:2000) and anti Bax

antibody (1:2000 in 10 ml of PBS containing 0.05% Tween-20,

0.5% BSA PBST) for 4 h at room temperature, followed by 3

washes with PBST. Further, the membranes were incubated in

alkaline phosphatase linked secondary antibody (1:30,000) for 1

h. The membranes were washed 3 times with PBST and then

processed for colour development with BCIP/NBT solution. The

densitometry (quantification) of bands was carried out by

measuring the pixel density using computerized gel imaging

system (Hero lab, Germany). The values were normalized to h-actin intensity levels.

TH-mRNA expression by reverse transcription polymerase chain

reaction (RT-PCR)

TH-mRNA level was detected by RT-PCR analysis using

protocol of Chen et al. (2003). RNA was isolated from striatum

and SNpc using trizol reagent and TH and h-actin cDNA was

synthesized by reverse transcription with oligo (dT) primer. The

cDNA samples were subjected to PCR amplification with

specific primers complementary to the coding sequence of rat

TH cDNA 5V-ATG CCC ACC CCC AGC GCC CC-3V and 5V-GAC ACT TTT CTT GGG AAC CA-3V and h-actin 5V-ATTTGG CAC CAC ACT TTC TAC A-3V and 5V-TCA CGC ACG

ATT TCC CTC TCA G-3V. The cycling parameters were

common for both TH and h-actin: denaturation at 94-C for 1

min, annealing at 55-C for 1 min and elongation at 72-C for 2

min (30 cycles). The amplified product (TH-513 bp and h-actin380 bp) was run on 1.5% agarose gel and the ladder marker of

100 bp (100–1000 bp) was run as the standard. To analyze the

relative expression of TH-mRNA, the amount of cDNA was

normalized with respect to signals from ubiquitously expressed

h-actin as an internal reference. The densitometry (quantifica-

tion) of TH bands was carried out by measuring the optical

density using computerized gel imaging system (Hero lab,

Germany). The values were normalized to h-actin intensity

levels.

Statistical analysis

Mean significant difference between treatment groups was

determined using one-way analysis of variance (ANOVA). Prior to

this, homogeneity of variance, between various groups was

ascertained. Further, the effect of individual treatment between

the two groups was assessed by comparison of least significant

differences, taking t values for error DF at 5% level of

significance. Values of P < 0.05 were considered to be statistically

significant. For TH-ir cell counts, inter-group comparisons were

performed with ANOVA.

Results

General observation

No significant change in the body weight was observed

between animals of lesioned and treated group when compared

to sham. As BTE was prepared in drinking water, no significant

difference was observed in the average intake of 1.5% BTE, and

intake was consistent between animals of different groups and

intra-group. Further, there was no significant difference in

consumption of normal drinking water in sham and 6-OHDA

group as compared to rats those received 1.5% BTE (Table 1).

Neurobehavioral studies

In order to understand the extent of neurodegeneration caused

by 6-OHDA and to see the efficacy of BTE in ameliorating

behavioral deficits, we have studied neurobehavioral changes.

Agonist-induced stereotypy was monitored by measuring unilateral

circling behavior, whereas spontaneous locomotor activity (SLA)

was quantified as distance travelled.

d-amphetamine-induced circling behavior

The results of d-amphetamine-induced circling behavior (ste-

reotypy) are summarized in Fig. 1. Amphetamine, a DA receptor

agonist, causes ipsilateral rotations towards the lesioned side in 6-

OHDA-lesioned rats, exhibiting a significant increase (P < 0.001) in

circling behavior when compared to sham-operated rats. Rats

receiving BTE treatment prior to 6-OHDA lesioning (BTE + 6-

OHDA) exhibited significant attenuation in circling behavior by

Fig. 1. d-amphetamine-induced circling behavior in sham, 6-OHDA

lesioned, BTE, 6-OHDA + BTE and BTE + 6-OHDA-treated rats. For

circling behavior rats were challenged with 5 mg/kg b.w. i.p. d-

amphetamine and ipsilateral rotations were counted for 30 min. Significant

increase of d-amphetamine-induced rotations in 6-OHDA-lesioned rats is

evident as compared to sham. A significant decrease (recovery) in rotations

was observed in rats receiving BTE either before (BTE + 6-OHDA) or after

6-OHDA administration (6-OHDA + BTE) as compared to lesioned rats.

Values represent mean T SE of 6 rats. One-way ANOVA ***P < 0.001,

*P < 0.05. a = vs. sham, b = vs. lesion DF (4, 29); F value, 46.59.

Table 3

DA–D2 receptor binding

Treatment

groups

DA–D2 receptor

binding (pmol bound/

g protein)

Scatchard analysis

Kd (nM) Bmax pmol

bound/g protein

Sham 466 T 35.15 0.79 812 T 42.10

6-OHDA 807 T 23.76a,*** 0.39 1269 T 36.15a,***

BTE 432 T 27.80 0.81 1232 T 45.19

6-OHDA + BTE 713 T 27.79b,* 0.60 1102 T 35.21b,*

BTE + 6-OHDA 648 T 35.91b,** 0.70 1027 T 35.12b,**

DA–D2 receptor binding in ipsilateral striatal synaptic membranes of

sham, 6-OHDA lesioned, BTE, 6-OHDA + BTE and BTE + 6-OHDA

treated rats. Significant increase in DA–D2 receptor binding in 6-OHDA-

lesioned rats is evident as compared to sham. A significant decrease

(recovery) of DA–D2 receptor binding was observed in rats receiving BTE

either before (BTE + 6-OHDA) or after 6-OHDA administration (6-OHDA+

BTE) as compared to lesioned rats. Values represent mean T SE of 5 rats.a vs. sham, DF (4, 24); F value, 27.86.b vs. lesion, DF (4, 24); F value, 27.86.

* One-way ANOVA, P < 0.05.

** One-way ANOVA, P < 0.01.

*** One-way ANOVA, P < 0.001.

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434426

51% (P < 0.001) while 6-OHDA + BTE group showed attenuation

by 28% (P < 0.05) in comparison to lesioned rats. No significant

change was observed in the BTE alone treatment group as

compared to sham group. These results suggest the protective role

of BTE against 6-OHDA-induced stereotypy (DF = 4,29 and F

value = 46.59).

Spontaneous locomotor activity

A significant decrease (P < 0.001) in SLA was observed in 6-

OHDA-lesioned group when compared with sham, which was

restored by 45% (P < 0.01) and 31% (P < 0.05) in BTE + 6-OHDA

and 6-OHDA + BTE groups, respectively, as compared to lesioned

rats. However, BTE alone treated group exhibited no significant

Table 2

Spontaneous locomotor activity

Treatment groups Spontaneous locomotor activity

distance travelled in cm/10 min

Sham 1489 T 135

6-OHDA 555 T 61a,***

BTE 1540 T 70

6-OHDA + BTE 841 T 48b,*

BTE + 6-OHDA 977 T 92b,**

Spontaneous locomotor activity in sham, 6-OHDA lesioned, BTE, 6-

OHDA + BTE and BTE + 6-OHDA treated rats. In SLA, locomotor activity

was observed for 10 min. Significant decrease in SLA in 6-OHDA-lesioned

rats is evident as compared to sham. A significant increase (recovery) in

SLAwas observed in rats receiving BTE either before (BTE + 6-OHDA) or

after 6-OHDA administration (6-OHDA + BTE) as compared to lesioned

rats. Values represent mean T SE of 6 rats.a vs. sham, DF (4, 29); F value, 23.62.b vs. lesion, DF (4, 29); F value, 23.62.

* P < 0.05, one-way ANOVA.

** P < 0.01, one-way ANOVA.

*** P < 0.001, one-way ANOVA.

change in the motor activity when compared to sham group (DF =

4, 29 and F value = 23.62) (Table 2).

Neurochemical studies

Degeneration of dopaminergic neurons following 6-OHDA

lesioning results in significant neurochemical alterations such as a

decrease in DA content and reduced tyrosine hydroxylase (TH)

immunoreactivity. Further, to observe the recovery in functional

viability of DA neurons and in order to correlate neurobehavioral

Fig. 2. Dopamine (DA) and 3,4 dihydroxy phenyl acetic acid (DOPAC)

level in ipsilateral striatum of sham, 6-OHDA lesioned, BTE, 6-OHDA +

BTE and BTE + 6-OHDA treated rats. Significant decrease in striatal DA

and DOPAC level in 6-OHDA-lesioned rats is evident as compared to

sham. A significant increase (recovery) in DA and DOPAC level was

observed in rats receiving BTE either before (BTE + 6-OHDA) or after 6-

OHDA administration (6-OHDA + BTE) as compared to lesioned rats.

Values represent mean T SE of 5 rats. One-way ANOVA ***P < 0.001,

**P < 0.01, *P < 0.05. a = vs. sham, b = vs. lesion. DF (4, 24); F value,

18.48 for DA and 12.87 for DOPAC.

Table 4

Total SOD and catalase activity

Treatment

groups

Total SOD activity

(nmol formazan

formed/min/mg protein)

Catalase activity nmol H2O2

consumed/min/mg protein

Sham 7.58 T 0.46 7.79 + 0.53

6-OHDA 2.10 T 0.25a,*** 2.73 + 0.47a,***

BTE 7.66 T 0.41 8.06 + 0.42

6-OHDA + BTE 3.56 T 0.22b,* 4.14 + 0.44b,*

BTE + 6-OHDA 5.18 T 0.41b,*** 5.23 + 0.52b,**

Total superoxide dismutase (SOD) and catalase activity in ipsilateral striatal

region of sham, 6-OHDA lesioned, BTE, 6-OHDA + BTE and BTE + 6-

OHDA treated rats. Significant decrease of SOD and catalase activity in 6-

OHDA lesioned rats is evident as compared to sham. A significant increase

(recovery) in SOD and catalase activity was observed in rats receiving BTE

either before (BTE+6-OHDA)or after 6-OHDAadministrations (6-OHDA+

BTE) as compared to lesioned rats. Values represent mean T SE of 5 rats.a vs. sham, DF (4, 24); F value, 44.66 for SOD and 23.10 for

catalase.b vs. lesioned, DF (4, 24); F value, 44.66 for SOD and 23.10 for

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434 427

changes with neurochemical alterations, [3H]-spiperone binding

reflecting specifically the functional viability of DA neurons/effect

on DA–D2 receptors and estimation of level of DA and its

metabolite DOPAC was done in the striatal region.

DA–D2 receptor binding

Results of DA–D2 receptor binding are summarized in Table 3.

The results revealed significant increase (P < 0.001) in DA–D2

receptor binding in 6-OHDA-lesioned rats as compared to sham

group. 6-OHDA + BTE group was found to exhibit attenuated DA

receptor binding to an extent of 28% (P < 0.05) when compared to

the lesioned group. However, attenuation was more pronounced in

rats of BTE + 6-OHDA group (47%, P < 0.01). No significant

change was observed in the BTE alone treatment group as

compared to the sham group (DF = 4, 24 and F value = 27.86).

The results of Scatchard analysis are given in Table 3. As evident

from the results, 6-OHDA lesioning resulted in an increased DA–

D2 receptor binding, which was due to the increased affinity (1/Kd)

Fig. 3. Lipid peroxidation (LPO) (a) and reduced glutathione (GSH) level

(b) in ipsilateral substantia nigra of sham, 6-OHDA lesioned, BTE, 6-

OHDA + BTE and BTE + 6-OHDA treated rats. Significant increase of

LPO and decrease in GSH level in 6-OHDA-lesioned rats is evident as

compared to sham. A significant decrease in LPO level and increase in

GSH levels were observed in rats receiving BTE either before (BTE + 6-

OHDA) or after 6-OHDA administration (6-OHDA + BTE) as compared to

lesioned rats. Values represent mean T SE of 5 rats. One-way ANOVA

***P < 0.001, **P < 0.01, *P < 0.05. a = vs. sham, b = vs. lesion. DF (4,

24), F value, 49.88 for LPO and 11.72 for GSH.

catalase.

* One-way ANOVA, P < 0.05.

** One-way ANOVA, P < 0.01.

*** One-way ANOVA, P < 0.001.

and maximum number of binding sites (Bmax). Bmax and 1/Kd were

significantly decreased in 6-OHDA + BTE and BTE + 6-OHDA

group.

DA and DOPAC levels

The results are summarized in Fig. 2. A significant decrease in

DA and DOPAC levels was observed in striatal region of 6-OHDA-

lesioned rats (P < 0.001) as compared to sham, indicating significant

loss of DA neurons in lesioned animals. DA and DOPAC level in 6-

OHDA + BTE group was restored significantly by 38% and 36%

respectively (P < 0.05) when compared to lesioned group. However,

BTE + 6-OHDA group exhibited more pronounced and significant

increase in the DA and DOPAC levels (53% and 58% respectively,

P < 0.01) in comparison to lesioned rats indicating functional

viability of DA neurons. No significant change was observed in the

BTE alone treatment group as compared to sham group (DF = 4,24,

F value = 18.48 for DA level and 12.87 for DOPAC level).

Studies related to oxidative stress

LPO and GSH level

A significant increase (P < 0.001) in lipid peroxidation was

observed in 6-OHDA-lesioned group when compared to sham-

operated animals. Rats of BTE + 6-OHDA group exhibited

attenuation in lipid peroxidation by 59% (P < 0.001) while 6-

OHDA + BT group showed attenuation by 30% (P < 0.01) in

comparison to lesioned rats. BTE alone treated group exhibited no

significant change in the LPO level when compared to sham group

(DF = 4, 24 and F value = 49.88) (Fig. 3a).

A significant decrease (P < 0.001) in GSH level was observed

in 6-OHDA-lesioned group when compared to sham group, which

was restored by 42% (P < 0.05) and 70% (P < 0.01) in BTE + 6-

OHDA and 6-OHDA + BTE groups respectively as compared to

lesioned rats. However, BTE alone treated group exhibited no

significant change in the GSH level when compared to sham group

(DF = 4, 24 and F value = 11.72) (Fig. 3b).

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434428

SOD and catalase activity

A significant decrease (P < 0.001) in SOD and catalase

activity was observed in striatum of 6-OHDA-lesioned group

when compared to sham, which was restored significantly by

27% (P < 0.01) and 28% (P < 0.05) respectively in 6-OHDA +

BTE group as compared to lesioned group. BTE + 6-OHDA

group exhibited a significant increase in striatal SOD and

catalase activity by 56% (P < 0.001) and 49% (P < 0.01)

respectively as compared to lesioned rats. BTE alone treated group

exhibited no significant change in these antioxidant enzyme

activities as compared to sham group (DF (4, 24), F value, 44.66

for SOD and 23.10 for catalase) (Table 4).

TH immunohistochemistry

The neuroprotective/neurorescue action of BTE and functional

viability of dopaminergic neurons in the SNpc were further

assessed by mapping the rate-limiting enzyme, TH, for DA-

biosynthesis using monoclonal antibody against TH. In 6-OHDA-

lesioned rats, number of surviving TH-ir neurons was significantly

less (Fig. 4b) as compared to those in the sham group (Fig. 4a). 6-

OHDA + BTE and BTE + 6-OHDA groups exhibited a significant

increase in TH-ir neurons, when compared to lesioned rats (Figs.

Fig. 4. TH-immunoreactive (TH-ir) neurons in substantia nigra (SN) of sham (a–c

OHDA (m–o) treated rats. The TH-ir neurons were significantly degenerated in S

unlesioned, contralateral hemisphere contained numerous TH-ir cells and process

processes. No significant difference in the number of TH-ir neurons was observ

increase in TH-ir neurons number (TH-expression) was observed in 6-OHDA + B

treatment either before or after 6-OHDA lesioning protected the ipsilateral nigral do

TH-ir cells in SNpc. Arrows (b, e, g, k and n) indicate the TH-ir cell body in SNpc

SNpc of different groups. Magnification 1, 10 and 40�. SNpc: substantia nigra par

4d and e). The higher number of TH-ir neurons in SNpc of BTE +

6-OHDA and 6-OHDA + BTE group further suggest the neuro-

protective/neurorescue action of BT on dopaminergic neurons.

However, no significant difference in the number of TH-ir neurons

was observed in BTE alone treated group (Fig. 4c) as compared to

sham group.

In order to quantify total TH-ir neurons count in ipsilateral

SNpc, image analysis (stereology) was performed in TH positive

sections. 6-OHDA lesioning caused a significant decrease (67%,

P < 0.001) in the number of TH-ir neurons (255 T 37) as compared

to sham (776 T 59). It is evident from the results that number of

TH-ir neurons is significantly high in 6-OHDA + BTE (411 T 44,

38%, P < 0.05) and BTE + 6-OHDA (521 T 56, 51%, P < 0.01)

groups as compared to lesioned rats. No significant difference was

observed in BTE alone treated group (795 T 54) as compared to

sham group (Fig. 5).

TH-protein level

6-OHDA administration significantly decreased (P < 0.001)

the striatal TH-protein level (56 kDa) as compared to the sham

group. The level was significantly restored in BTE + 6-OHDA

(P < 0.01) and 6-OHDA + BTE (P < 0.05) groups as compared

to lesioned group. There was no significant difference observed

), 6-OHDA lesioned (d– f), BTE (g– i), 6-OHDA + BTE (j– l) and BTE + 6-

N of 6-OHDA-lesioned rats (d) as compared to sham (a). The SNpc in the

es but the SNpc in the lesioned hemisphere contained few TH-ir cells and

ed in BTE alone treated group (c) as compared to sham (a). A significant

TE (d) and BTE + 6-OHDA group (e) as compared to lesioned group. BTE

paminergic neurons from 6-OHDA. Arrows (d, j and m) indicate the loss of

. Arrows (c, f, i, l and o) indicate the TH-ir cell body in and TH-ir process in

s compacta; SNl: substantia nigra pars lateralis; VTA: ventral tegmental area.

Fig. 5. TH-immunoreactive (TH-ir) neurons count in ipsilateral SNpc in

sham, 6-OHDA lesioned, BTE, 6-OHDA + BTE and BTE + 6-OHDA-

treated rats. Significant decrease of TH-ir neurons in 6-OHDA-lesioned rats

was evident as compared to sham. A significant increase in TH-ir neurons

was observed in rats receiving BTE either before (BTE + 6-OHDA) or after

6-OHDA administration (6-OHDA F BTE) as compared to lesioned rats.

TH-ir neurons in ten sections of ipsilateral SNpc (fraction of sections being

sampled 1/5) from each rat were counted using optical dissector method.

The data represent mean + SE (n = 5 rats/group). One-way ANOVA ***P <

0.001, **P < 0.01, *P < 0.05. a = vs. sham, b = vs. lesioned.

Fig. 6. The expression of TH protein, antiapoptotic protein (Bcl-2) and proapopto

OHDA + BTE and BTE + 6-OHDA treated rats. Equal amount (10 Ag) of protein w

was carried out using anti-TH (56 kDa), anti Bcl-2 (26 kDa) and anti Bax antibo

observed in 6-OHDA-lesioned rats. A significant increase in TH and Bcl-2 protein

after 6-OHDA administration (6-OHDA + BTE) as compared to lesioned rats. The

A significant decrease in Bax protein level was observed in 6-OHDA + BTE an

normalized to internal reference h-actin (43 kDa) and represented as the mean T S

BTE; and lane 5, BTE + 6-OHDA. One-way ANOVA ***P < 0.001, **P < 0.0

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434 429

in TH-protein level in BTE alone treated group as compared to

sham group (Fig. 6).

Antiapoptotic (Bcl-2) and proapoptotic protein (Bax) level

6-OHDA administration significantly (P < 0.001) decreased the

Bcl-2 protein level (26 kDa), while significantly increased (P <

0.001) the Bax protein level (21 kDa) as compared to sham group.

The levels were significantly restored in BTE + 6-OHDA (P <

0.01) and 6-OHDA + BTE (P < 0.05) groups as compared to

lesioned group. There was no significant difference observed in

these protein levels in BTE alone treated group as compared to

sham group (Fig. 6).

TH-mRNA expression

A 513 bp fragment of TH amplified PCR product was observed

in all groups. TH mRNA expression was significantly decreased

(P < 0.001) in 6-OHDA-lesioned group as compared to sham

group. The mRNA expression was significantly restored in BTE +

6-OHDA (P < 0.001) and 6-OHDA + BTE (P < 0.01) groups as

compared to lesioned group. There was no significant difference

observed in TH mRNA expression in BTE alone treated group as

compared to sham group (Fig. 7).

Discussion

Oxidative stress to dopaminergic neurons of SNpc is believed

to be one of the leading causes of neurodegeneration in PD. Thus

ROS scavenging antioxidants may play an important role in the

prevention of PD and combat against OS-induced progressive

tic protein (Bax) by Western blotting in sham, 6-OHDA lesioned, BTE, 6-

ere subjected to 10% SDS-PAGE electrophoresis and Western blot analysis

dy (21 kDa). A significant decrease in TH and Bcl-2 protein content was

level was observed in rats receiving BTE either before (BTE + 6-OHDA) or

level of Bax protein was significantly upregulated in 6-OHDA-lesioned rat.

d BTE + 6-OHDA group as compared to lesioned rats. The values were

E of three experiments. Lane 1, sham; 2, lesioned; 3, BTE; 4, 6-OHDA +

1, *P < 0.05. a = vs. sham, b = vs. lesioned.

Fig. 7. TH-mRNA expression by RT-PCR in sham, 6-OHDA-lesioned,

BTE, 6-OHDA + BTE and BTE + 6-OHDA treated rats. An equal aliquot

of amplified PCR TH (513 bp) and h-actin (380 bp) DNA product was

separated on 1.5% agarose gel and stained with ethidium bromide for

qualitative comparison. 100 bp DNA ladder was run as standard. A

significant decrease in TH-mRNA expression was observed in 6-OHDA-

lesioned rats. A significant increase in TH-mRNA level observed in rats

receiving BTE either before (BTE + 6-OHDA) or after 6-OHDA

administration (6-OHDA + BTE) as compared to lesioned rats. h-actintranscripts are shown as an internal reference for amplification of cDNA

and h-actin specific bands were detected in all groups. The values were

normalized to h-actin and represented as the mean T SE of three

experiment. Lane 1, marker; 2, sham; 3, lesioned; 4, BTE; 5, 6-OHDA +

BTE; and lane 6, BTE + 6-OHDA. One-way ANOVA ***P < 0.001, **P <

0.01. a = vs. sham, b = vs. lesioned.

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434430

neurodegeneration. However, many of these antioxidants are

unable to penetrate the blood brain barrier (BBB), leading to lack

of effectiveness of these antioxidants in PD. Recently GT, BT and

its polyphenols (catechins, theaflavins and thearubigins) have been

shown to penetrate the BBB and possess strong antioxidant (Luczaj

and Skrzydlewska, 2004; Frei and Higdon, 2003) and iron

chelating (Rah et al., 2005) properties and their consumption has

been shown to reduce the risk of many diseases including cancer

(Lambert et al., 2005), stroke (Lee et al., 2003a), coronary heart

disease (Vita, 2005) and neurological disorders (Mandel and

Youdim, 2004). Similarly, an epidemiological study has shown

reduced risk of PD associated with consumption of two cups of tea

per day (Checkoway et al., 2002). Several studies demonstrated the

neuroprotective role of GT and its polyphenols, but there is a

lacuna of knowledge of neuroprotective and neurorescue effect of

BT.

To the best of our updated knowledge, this study is the first to

show neuroprotective and neuroreparative (neurorescue) effect of

BT in 6-OHDA-lesioned rat model of PD. In the present study, we

prepared hemiparkinsonian rat model with 6-OHDA by unilateral

lesioning of the striatum. This progressive lesion yields an animal

model, which resembles the pattern of neurodegeneration and

neuropathology at early stages of PD, in which a portion of the

nigrostriatal projections remain intact, and is ideal to see the

suitability of neuroprotective agents (Carman et al., 1991). In

unilateral striatum 6-OHDA-lesioned model, loss of 60–70% of

TH-ir dopaminergic neuron fibers in striatum and 50–60%

reduction of TH-ir neurons have been shown earlier (Kirik et al.,

1998).

Rats were administered 1.5% BTE orally before and after 6-

OHDA lesion. The BTE dose is based on earlier studies, where

0.5%–3% BTE has been shown to protect against alcohol- and

cigarette smoke-induced oxidative stress and preventing carcino-

genesis and neurodegeneration (Luczaj and Skrzydlewska, 2004;

Misra et al., 2003; Shukla and Taneja, 2002). Active compo-

nents(s), responsible for neuroprotective and neurorescue action of

BTE, are not clearly defined yet. However, earlier identified BTE

components (catechins, theaflavins, thearubigins, flavonols) have

been shown to possess strong antioxidant (Luczaj and Skrzydlew-

ska, 2004; Frei and Higdon, 2003) and iron chelating (Rah et al.,

2005) properties and these constituents and possibly other

unidentified polyphenols may also be involved in neuroprotec-

tive/neurorescue action in this study. Further, 1.5% BTE contains

10–12% dry weight catechins, 3–6% teaflavin, 12–18% thear-

ubigins, 6–8% flavonols, 10–12% phenolics acids and 8–11%

methylxanthines (Dufresne and Farnworth, 2001; Luczaj and

Skrzydlewska, 2004). Moreover, rats receiving 1.5% BTE had no

significant difference in daily BTE intake in different groups in

comparison to the rats receiving only drinking water in sham and

lesioned group (Table 1). Therefore, it can be considered that the

active component(s) of BTE may be reaching to animals of all

experimental groups in identical manner.

In the present study, an increase in amphetamine-induced

rotations and a decrease in locomotor activity in 6-OHDA-lesioned

animals has been observed, which could be closely linked to the

degree of dopaminergic dysfunctioning and deterioration of motor

performance (Kondo et al., 2004). Amphetamine administration

releases endogenous DA and lesioned animals exhibited ipsilateral

rotations to the lesioned side, due to an imbalance in striatal DA

levels of lesioned and unlesioned side (Ungerstedt, 1971). These

rotations are reported to be a reliable indicator of nigrostriatal

dopaminergic depletion (Kondo et al., 2004). Oral BTE adminis-

tration before or after 6-OHDA lesioning, significantly reduces the

amphetamine-induced rotation and increases SLA, showing neuro-

protective/neurorescue effect of BTE on dopaminergic neurons

against 6-OHDA toxicity. The possible mechanism involved in

neuroprotective action of BTE and its polyphenols against 6-

OHDA, is its catechol like structure, since it is known that catechol

containing compounds are potent radical scavengers and chelators

of ferric ion (Mandel and Youdim, 2004). Our findings correlate

well with the earlier studies carried out by others and us, where

motor deficits in parkinsonian rat have been attenuated by

adenosine and selenium (dietary antioxidant) and ginko biloba

(Zafar et al., 2003a,b; Ahmad et al., 2005).

Our results revealed that 6-OHDA increased the lipid perox-

idation and reduced the GSH level in SN, along with reduced

activities of antioxidant enzyme (total SOD and catalase) in

striatum. Administration of BTE alone does not significantly alter

the basal (normal) level of antioxidant enzymes and oxidative

stress markers. However, following the oxidative stress, induced by

6-OHDA, BTE partially restored the level of nigral oxidative

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434 431

markers (LPO and GSH level) and partially restored the impair-

ments in two major ROS metabolizing enzymes (SOD and

catalase) in striatum. Such regulation of oxidative stress markers

and antioxidant enzymes by BTE in the present study may be well

correlated with the previous reports, where black tea has been

shown to restore the level of brain oxidative stress markers and

antioxidative enzymes up to basal level against alcohol intoxication

(Luczaj and Skrzydlewska, 2004). Further, in this study, BTE alone

has not been shown to significantly modulate the basal level of

these markers and antioxidant enzymes (Luczaj and Skrzydlewska,

2004). The reduction of oxidative products in the present study

may be due to the BTE’s fundamental action, as it has been

reported that BTE possess strong radical scavenging (Mercer et al.,

2005), iron chelating (Rah et al., 2005), anti-inflammatory (Pan et

al., 2000) and antiapoptotic properties (Mercer et al., 2005) under

in vivo and in vitro condition, as well as the ability to modulate cell

signaling (Levites et al., 2002). Further, it has also been reported

that BTE act as an antioxidant as well as it increases the level of

antioxidant (Luczaj and Skrzydlewska, 2004; Frei and Higdon,

2003). The neuroprotective and neurorescuing effects of BTE may

involve the regulation of antioxidant enzymes and transcription

factors which may be regulated by epigallocatechin (EGCG), a

polyphenol of BT (Higdon and Frei, 2003). Further, this effect of

BT may correlate well with the earlier studies where tea and tea

polyphenols have been shown to regulate and increase the SOD,

catalase and GSH activity and decrease the lipid peroxidation in in

vivo and in vitro, under conditions of oxidative stress (Komatsu

and Hiramatsu, 2000; Levites et al., 2001; Skrzydlewska et al.,

2002; Higdon and Frei, 2003). Further, EGCG has been shown to

induce the expression of a reporter gene containing the antioxidant

regulatory element present in the promoter of phase II antioxidant

enzymes (Chen et al., 2000). It has also been reported that BT

polyphenol theaflavins possess the ability to prevent formation of

oxygen radicals by inhibiting activity of xanthine oxidase-enzyme

participating in superoxide anion generation (Chen et al., 2000).

Similarly, oral administration of GT or BT inhibited lipid

peroxidation in vitro as well as in vivo studies (Sano et al.,

1995; Lee et al., 2003b). Neuroprotection provided by BT against

oxidative stress is further supported by an earlier study where BT

and its polyphenols were able to prevent changes of natural

microviscocity of cell membrane, which was remarkably increased

under oxidative stress (Halder and Bhaduri, 1998).

Significant increase in DA–D2 receptor binding in 6-

OHDA-lesioned rats as compared to sham, has been reported

to be a consequence of denervated supersensitivity, where loss

of striatal DA terminals have been shown (Agrawal et al.,

1995). Such increase was considered to be a response offered

by residual striatal DA neurons/post synaptic cells towards

mitigating the initial disturbances in nigrostriatal DA loops

(Agrawal et al., 1995). BTE administration before and after 6-

OHDA lesioning significantly decreased the DA–D2 receptor

supersensitivity (binding) in striatum. This decrease in binding

in BTE exposed rats suggests protection to DA neurons against

6-OHDA, and is related to normalization of denervation-related

super sensitivity of DA–D2 receptor in the striatum and could

be correlated with enhanced DA levels in striatum. These

effects are not just a functional benefit, rather an outcome of

the increasing number of functional viable dopaminergic

neurons as evident by the presence of significantly increased

TH-ir neurons, TH protein level and TH-mRNA expression in

SNpc. It can be suggested that BTE preserved the TH-ir

nigrostriatal dopaminergic neurons against neurodegeneration

induced by 6-OHDA. The increase in striatal DA content

following BTE treatment can also be associated with the ability

of BTE to prevent DA degradation or possibly decreased DA

reuptake. The enhanced level of DA in present study, in BTE-

treated rats, could be due to the protective effect of BTE on

dopaminergic neurons and maintenance of DA level. Further,

this could also be due to the reduction in autoxidation of DA

by enhancement of antioxidant enzymes activity in striatum and

SN following BTE treatment, imparting substantial protection to

neurons as shown in this study (Higdon and Frei, 2003).

Similarly tea polyphenols have also been shown to inhibit

catechol-O-methyltransferase (COMT) activity (Lu et al., 2003),

resulting in higher availability of DA as observed in the present

study. These findings are consistent with earlier studies, where

dietary antioxidant, ginko biloba, caffeine and tea polyphenols

have been shown to prevent striatal DA depletion and

dopaminergic neuron loss in neurotoxin-induced animal model

of PD (Levites et al., 2001; Zafar et al., 2003a,b; Ahmad et al.,

2005).

There is also evidence for increased expression of apoptotic

proteins and impairment of Ca2+ homeostasis, which enhances the

neurodegeneration in PD (Blum et al., 2001). In the present study

6-OHDA administration has been shown to reduce the level of

antiapoptotic protein (Bcl-2) and increase the level of proapoptotic

protein (Bax) in the lesion group as compared to sham.

Administration of BTE alone does not significantly alter the basal

(normal) level of antiapoptotic or proapoptotic proteins markers.

However, following the 6-OHDA-induced oxidative stress, BTE

partially restored the level of these markers in BTE + 6-OHDA and

6-OHDA + BTE groups, suggesting an antioxidative/antiapoptotic

role of BTE. Bcl-2 is known to regulate levels of reactive oxygen

species or their intermediates in cells, which is one possible

mechanism of anti-apoptosis (Tyurina et al., 1997). Overexpression

of Bcl-2 can exert an antioxidative effect by raising SOD activity,

GSH levels and ONOO� formation (Lee et al., 2002). These

findings are consistent with the earlier study, where tea poly-

phenols have been shown to induce and suppress the expression of

Bcl-2 and Bax protein respectively in the SN, against MPTP-

induced neurodegeneration (Mandel and Youdim, 2004). Further,

neuroprotection and neurorescue effects offered by BTE, against 6-

OHDA is supported by previous studies, where tea polyphenols

have been shown not only to be neuroprotective against the

induction of apoptosis but also to rescue damaged neurons through

MAP kinases (Chung et al., 2003), PKC, JNK, cell survival genes

activation (Levites et al., 2002; Mandel et al., 2003). Tea

polyphenols also helps in calcium homeostasis (Ishige et al.,

2001) and suppression of pro-apoptotic genes expression (mdm 2,

caspase 1, 3 and Bax) towards providing neuroprotection (Levites

et al., 2002). Further, results of present study are substantiated by

earlier studies where tea polyphenols have been shown to exert

neuroprotective and neurorescue action on dopaminergic and

cholinergic neurons against the h amyloid protein-induced toxicity

and transient forebrain ischemia (Levites et al., 2003; Lee et al.,

2003a). Similarly, the GT polyphenol EGCG has been shown not

only to neurorescue the long-term serum deprived PC-12 cells but

also to promote neurite outgrowth (Reznichenko et al., 2005).

The results presented in this study, if extrapolated to humans,

indicate that regular intake of BT may be helpful in preventing

neurodegeneration as well as slowdown the disease progression.

Further studies to understand the basic mechanism would be worth

R.K. Chaturvedi et al. / Neurobiology of Disease 22 (2006) 421–434432

investigating. Similarly, the active component(s) of BTE respon-

sible for neuroprotective and neurorescue effects need to be

identified in further studies.

Acknowledgments

We are grateful to Prof. Y. K. Gupta, Director, ITRC for his

continuous support during this study. We are also thankful to Dr.

M. M. Ali and Mr. N. Mathur for their guidance in neurobehavioral

studies and statistical analysis, respectively. R.K. Chaturvedi, S.

Shukla and C. Sinha are recipients of Senior Research Fellowship

from CSIR, New Delhi. K. Seth is a recipient of WOS (Women

Scientist Award) from Department of Science and Technology

(DST), New Delhi. Technical assistance of Mr. S. K. Shukla and

Mr. Kailash Chandra is gratefully acknowledged (ITRC Manu-

script communication No-2357).

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