Rutin Protects Dopaminergic Neurons from Oxidative Stressin an Animal Model of Parkinson’s Disease

Mohd. Moshahid Khan • Syed Shadab Raza •

Hayate Javed • Ajmal Ahmad • Andleeb Khan •

Farah Islam • Mohammed M. Safhi • Fakhrul Islam

Received: 14 May 2010 / Revised: 26 November 2011 / Accepted: 29 November 2011 / Published online: 23 December 2011

� Springer Science+Business Media, LLC 2011

Abstract This study was undertaken to investigate the

neuroprotective effects of rutin (vitamin P) on 6-hydroxy-

dopamine (6-OHDA)-induced Parkinson’s disease (PD) in

rats. Oxidative stress and inflammation is an important

event, play a crucial role in neurodegenerative diseases.

Rutin has been shown to have antioxidant and anti-inflam-

matory actions, and thus was tested for its beneficial effects

using 6-OHDA-induced PD rat model. Male Wistar rats

were pre-treated with rutin (25 mg/kg bwt, orally) for

3 weeks and subjected to unilateral intrastriatal injection of

6-OHDA (10 lg in 0.1% ascorbic acid in normal saline).

Three weeks after 6-OHDA infusion, rats were tested for

neurobehavioral activity, and were killed after 4 weeks of

6-OHDA infusion for the estimation of thiobarbituric acid

reactive substances, glutathione, and its dependent enzymes

(glutathione peroxidase and glutathione reductase), dopa-

mine (DA) and its metabolite 3,4-dihydroxyphenyl acetic

acid. The increase in 6-OHDA-induced rotations and defi-

cits in locomotor activity and motor coordination and

decrease in antioxidant level, DA content and its metabolite

and increase in the number of dopaminergic D2 receptors in

striatum were protected significantly with lesioned group

pre-treated with rutin. These findings were further sup-

ported by the histopathological and immunohistochemical

findings in the substantia nigra that showed that rutin pro-

tected neurons from deleterious effects of 6-OHDA. These

results suggest that the consumption of rutin, which is novel

vitamin, may have the possibility of protective effect

against the neurological disorder such as PD.

Keywords Rutin � 6-Hydroxydopamine �Behavioral activity � Oxidative stress � Inflammation �Parkinson’s disease

Introduction

Parkinson’s disease (PD) is a debilitating neurological

disorder which is accompanied by motor deficit caused by

loss of dopaminergic neurons in the striatum, substantia

nigra and nigrostriatal pathway of the brain, considerably

impair the quality of life of PD patients. The cause of

dopaminergic cell death in PD remains unknown, but both

oxidative and nitrasative stress may contribute to neuronal

degeneration and have been intimately linked to other

components of neurodegenerative processes, such as

Neurotoxicology Laboratory—Fund for the Improvement of Science

and Technology Sponsored by DST and Special Assistance

Programme Sponsored by UGC.

Mohd. Moshahid Khan � S. S. Raza � H. Javed �A. Ahmad � A. Khan � Fakhrul Islam

Neurotoxicology Laboratory, Department of Medical

Elementology & Toxicology, Jamia Hamdard (Hamdard

University), Hamdard Nagar, New Delhi 110062, India

Mohd. Moshahid Khan

Department of Neurology, Carver College of Medicine,

University of Iowa, Iowa, IA, USA

A. Ahmad

Department of Neurology, Georgia Health Science University,

Augusta, GA, USA

Farah Islam

Department of Biotechnology, Faculty of Pharmacy,

Jamia Hamdard (Hamdard University), Hamdard Nagar,

New Delhi 110062, India

M. M. Safhi � Fakhrul Islam (&)

Neuroscience and Toxicology Unit, Faculty of Pharmacy,

Jazan University, Gizan, Kingdom of Saudi Arabia

e-mail: fislam2001@yahoo.co.in

123

Neurotox Res (2012) 22:1–15

DOI 10.1007/s12640-011-9295-2

inflammation and cell death (Jenner 2003; Gao et al. 2008;

Hirsch and Hunot 2009).

Brain, besides being rich in lipids and polyunsaturated

fatty acids (PUFA), consumes most of the oxygen, thus it

remains on higher oxidative damage. Oxidative damage to

lipid, fatty acid and protein (protein carbonyl formation)

can lead to structural and functional disruption of the cell

membrane, inactivation of enzymes and finally cell death.

Earlier, our research group investigated and reported

the preventive effect of certain antioxidants on different

experimental models of neurodegeneration (Zafar et al.

2003; Ahmad et al. 2005; Ishrat et al. 2009). Thus, treat-

ment with antioxidant may boost the system to defend

against the oxidative threats.

Inflammation has recently been implicated as a critical

mechanism for the progressive neurodegeneration in PD

(Tansey et al. 2007; Joglar et al. 2009; Hirsch and Hunot

2009). Microglia, the resident innate immune cells, plays

a major role in the inflammatory process in the brain.

Activated microglia releases various pro-inflammatory

cytokines, pro-inflammatory enzymes, cyclooxygenase-2

(COX-2), inducible nitric oxide synthase (iNOS), and the

levels of nitric oxide (NO) and superoxides, which have

deleterious effects on dopaminergic neurons (Hirsch et al.

2003; Xue et al. 2007; Jin et al. 2008; Li et al. 2009).

Moreover, suppressing neuroinflammation with anti-

inflammatory drugs mitigates dopaminergic neurodegen-

eration in various experimental models of PD (Choi et al.

2005; Jin et al. 2008).

The administration of 6-hydroxydopamine (6-OHDA)

into the brain of the rat produces a well established model

of PD (Kirik et al. 1998; Blum et al. 2001; Deumens et al.

2002; Blandini et al. 2008). 6-OHDA selectively destroys

the dopaminergic nigrostriatal pathway by inducing oxi-

dative stress, which can lead to induction of inflammation

and finally cell death. The unilateral, intrastriatal injection

of 6-OHDA induces pronounced behavioural asymmetries,

biochemical and histological deficits similar to PD.

Rutin is a member of bioflavonoids also called vitamin P

with antioxidant, anti-inflammatory, antiallergenic, antivi-

ral and anticarcinogenic properties and has been demon-

strated to scavenge superoxide radicals (La Casa et al.

2006; Kamalakkannan and Prince 2006; Bishnoi et al.

2007). In humans, it attaches to the iron ion (Fe2?), pre-

venting it from binding to hydrogen peroxide, which would

otherwise create a highly reactive free radicals that may

damage cells (Afanas’av et al. 1989). Rutin intake from

natural food sources, such as ‘soba’ noodles or groats,

might be effective in retarding memory dysfunction

resulting from hippocampal pyramidal neuron loss such as

in Alzheimer’s disease (Pu et al. 2004; Koda et al. 2008).

Recently, Khan et al. (2009) has investigated and reported

the neuroprotective effect of rutin on ischaemia/reperfusion

injury. Possibly, this is for the first time, it is evident from

this study, that rutin pre-treatment evokes neuroprotection

to the degenerating dopaminergic neurons and that led us to

study its neuroprotective role in the Parkinsonian rats.

Experimental Procedures

Chemicals

6-OHDA, apomorphine hydrochloride, oxidized glutathi-

one (GSSG), reduced glutathione (GSH), 5,50-dithio-bis-

2-nitrobenzoic acid (DTNB), nicotinamide adenine dinu-

cleotide phosphate reduced form (NADPH), 1-chloro-2,

4-dinitrobenzene (CDNB), dopamine (DA), 3,4-dihydroxy-

phenyl acetic acid (DOPAC), 3,4-dihydroxybenzylamine

(DHBA), rutin, heptane sulfonic acid, bovine serum albumin

(BSA), thiobarbituric acid (TBA), ethylene-diamine tetra-

acetic acid (EDTA), anti-mouse IgG (Jackson Immuno

Research Laboratories Inc, West Groove, PA), antibody of

inducible nitric oxide synthase (iNOS), diaminobenzidine

and haloperidol were purchased from Sigma-Aldrich Co.

Pvt. Ltd. India. H3-Spiperone was procured from New

England Nuclear (NEN) Boston, MA, USA. Other chemicals

were analytical reagent grade.

Animals and Treatments

Male Wistar rats were obtained from Central Animal

House of Jamia Hamdard (Hamdard University), New

Delhi, weighing 250 ± 10 g and aged 80–90 days at the

start of the experiment were used. Rats were housed in

groups of four animals per cage under controlled envi-

ronmental conditions (25 ± 2�C and a 12 h light/dark

cycle) and had free access to food and water ad libitum.

The experiments were in accordance with the guidelines of

the Animal Ethics Committee of Jamia Hamdard (Hamdard

University), New Delhi, India.

Experimental Design

Three sets of experiments were performed to evaluate the

neuroprotective potential of rutin. Experiment 1 was con-

ducted to evaluate the pre-treatment effect of rutin (25 mg/

kg/day orally in saline) for 3 weeks before the 6-OHDA

infusion on the content of thiobarbituric acid reactive

substances (TBARS), H2O2, protein carbonyl (PC), gluta-

thione (GSH) and for the assays of antioxidant enzymes.

The rats were divided into four groups, each having eight

animals. The first group served as sham (S) and vehicle

(saline, orally) was given; group 2 was vehicle-treated

lesioned group (L), received 2 ll of 6-OHDA (5 lg/ll in

0.1% ascorbic acid saline) by stereotaxic injection into the

2 Neurotox Res (2012) 22:1–15

123

striatum; group 3 rats received rutin for 3 weeks before

6-OHDA lesioning (R ? L) and the group 4 received

25 mg/kg rutin orally for 3 weeks, received 2 ll saline by

stereotaxic injection (R ? S).

Experiment 2 was conducted to evaluate the pre-treatment

effect of rutin (25 mg/kg/day orally in saline) for 3 weeks

before 6-OHDA infusion on dopaminergic D2 receptor

binding density and content of DA and its metabolite,

DOPAC, in the striatum. The rats were divided into four

groups, as in experiment 1, each having eight animals.

Experiment 3 was carried out to examine the pre-treat-

ment effect of rutin (25 mg/kg/day orally in saline) for

3 weeks before 6-OHDA infusion on histopathological

changes, iNOS expression, NO and cytokines level. The

rats were divided into four groups, as in experiment 1, each

having eight animals. The behavioral parameters were

performed in all the experiments.

Drug Administration

We examined the effects of different doses of rutin on

6-OHDA induces neurodegeneration in pilot studies to

determine the optimal dose of rutin that provides the most

neuroprotection against degeneration. On the basis of these

findings, rats were pre-treated orally with 25 mg/kg rutin

once daily for 21 days. The same dose of rutin was used in

previous experiment (Khan et al. 2009).

Intrastriatal Administration of 6-OHDA (Lesioning)

Rats were anaesthetized with 400 mg/kg chloral hydrate

intraperitoneally (i.p.) and mounted on a stereotactic stand.

The skin overlying the skull was cut to expose the bregma,

and the coordinates of the striatum (Paxinos and Watson

1982) were measured accurately as antero-posterior 0.5 mm,

lateral 2.5 mm and dorso-ventral 4.5 mm relative to bregma

and ventral from dura with the tooth bar set at 0 mm. Uni-

lateral striatal DA neuronal degeneration was induced in rats

by stereotaxic injections of 10 lg 6-OHDA/2 ll in 0.1% in

ascorbic acid saline using 5 ll Hamilton syringe into the

right striatum. The sham was treated in the same way except

2 ll saline was injected in place of 6-OHDA. The injection

rate was 0.5 ll/min and the needle was kept in place for an

additional 5.0 min before being slowly retracted.

Post-Operative Care

Recovery from anaesthesia took 4–5 h. The rats were kept

in a well-ventilated room at 25 ± 3�C in individual cages

until they gained full consciousness and then were housed

together in a group of four animals per cage. Food and

water was kept inside the cages for the first week so that

animals could easily access it without any physical trauma

due to overhead surgery. Then the animals were treated

normally with food, water and the bedding of the cages

changed daily as usual.

Behavioral Testing

Apomorphine-Induced Rotations

The use of a DA agonist, such as apomorphine is extremely

effective in measuring rotational asymmetry in unilateral

lesioned animals. Rats were subcutaneously injected with

0.5 mg/kg bwt apomorphine hydrochloride to investigate the

rotational asymmetry (Ahmad et al. 2005). The total number

of contralateral turns was counted over a 5-min period.

Rota Rod (Muscular Coordination)

Omni Rotor (Omnitech Electronics, Inc, Columbus, OH,

USA) was used to evaluate the muscular coordination skill on

day 22 of 6-OHDA injection (Rozas et al. 1998). The Rota rod

unit consists of a rotating rod, 75-mm diameter, which was

divided into four parts by compartmentalization to permit the

testing of four rats at a time. After twice daily training for two

successive days (8 rpm on the first day and 10 rpm on second

day) the rotational speed was increased to 15 rpm on the third

day in a test session. The time for each rat to remain on the

rotating bar was recorded for three trials for each rat, at 5 min

intervals with maximum trial length of 180 s per trial.

Narrow Beam Maze (NBM)

The narrow beam is a 105-cm long wooden beam, 4 cm

wide and 3-cm tall. The beam was suspended 80 cm from

the ground by wooden supports at either end. The wooden

supports at the ‘starting’ end of the beam formed a sheer

drop whilst a platform with food pellet located at the other

end. At the start end of the beam, a line was drawn 20 cm

from the end of the beam. During a test, the rat was placed

entirely within this 20 cm starting zone facing its home

cage and a stopwatch started immediately upon release of

the animal. The animals were trained on the NBM for 10

trials per day with 1-min interval. The journey time

between starting end to opposite end of beam was calcu-

lated (Allbutt and Henderson 2007).

Spontaneous Locomotor Activity (SLA)

SLA was monitored in a computerized Video Path Ana-

lyzer (Coulbourn Instruments, Allentown, PA, USA) con-

sists of a chamber (50 9 50 9 35 cm), a video camera

fixed over the chamber by an adjacent rod, an activity

monitor, a programmer/processor and a printer which helps

in quantification of locomotor activity as described

Neurotox Res (2012) 22:1–15 3

123

previously by us (Ahmad et al. 2005). The activity chamber

was furnished with black paper to provide contrast on the

screen. On day 21, after 6-OHDA injection, rats were

individually placed in the chamber, acclimatized for 5 min

and their locomotor activity scores were recorded for

15 min. The data was analysed for the intervals (min), wall

hugging (s), locomotion (s), rest (s), rearing (s), stereo

events (number) and distance travelled (cm). The activity

chamber was swabbed with 10% alcohol before each use to

avoid the interference due to animal odours. Results were

expressed in terms of activity/15 min.

Tissue Preparation

Animals for biochemical assays were killed by cervical

dislocation, and the brains were removed, to dissect stria-

tum and then homogenised in (5% w/v) in 0.01 M phos-

phate buffer (pH 7.0) having 10 ll/ml protease arrests

[5 mM leupeptin, 1.5 mM aprotinin, 2 mM phenyl ethyl-

sulfonylfluoride (PMSF), 3 mM peptastatin A, 10 mM

EDTA, 0.1 mM EGTA, 1 mM benzamidine and 0.04%

butylated hydroxytoluene (BHT)]. Homogenates were

centrifuged at 1,0009g for 5 min at 4�C to remove debris.

This supernatant (S1) was used for the estimation of

TBARS and H2O2 levels. This supernatant was further

centrifuged at 10,5009g for 20 min at 4�C to get post-

mitochondrial supernatant (PMS) (S2) which was used for

the estimation of GSH and antioxidant enzymes.

Biochemical Studies

TBARS Assay

The method of Utely et al. (1967), as modified by Islam et al.

(2002), was used to estimate the rate of lipid peroxidation.

The homogenate 0.25 ml was pipetted out in test tube and

incubated at 37 ± 1�C in a metabolic water bath shaker for

60 min at 120 cycles to and fro; another 0.25 ml was pipetted

in test tube and placed at 0�C incubation. After 1 h of incu-

bation, 0.5 ml of 5% chilled TCA was added to each tube

followed by 0.25 ml of 0.67% TBA and mixed thoroughly

after each addition. The mixture was centrifuged at

4,0009g for 10 min. Thereafter, supernatant was transferred

to another test tube and placed in the boiling water bath for

10 min, after cooling the test tubes the absorbance of the

colour was read at 535 nm. The rate of lipid peroxidation was

expressed as nmol TBARS formed/h/mg protein, using a

molar extinction coefficient of 1.56 9 105 M-1 cm-1.

Assay for Hydrogen Peroxide (H2O2)

The level of H2O2 was measured by the method of Jiang et al.

(1992). In brief, 0.1 ml of tissue homogenate was treated

with 0.9 ml of Fox reagent (88 mg BHT, 7.6 mg xylenol

orange, 9.8 mg ammonium sulphate, 90 ml of methanol and

10 ml 250 mM sulphuric acid) and incubated at 37�C for

30 min. The colour sample was then read at 560 nm in a

spectrophotometer (UV-1601, Shimadzu Japan). Hydro-

peroxide was expressed as nmol H2O2 mg-1 protein, using a

molar extinction coefficient of 1.5 9 104 M-1 cm-1.

Estimation of Protein Carbonyl (PC)

Protein oxidation was estimated by determination of pro-

tein carbonyl content according to the method of Levine

et al. (1990) with slight modification. The tissue homoge-

nate (0.25 ml) was mixed with an equal volume of 20%

TCA. Thereafter, 0.25 ml of 10 mM 2,4-dinitrophenylhy-

drazine (DNPH) in 2.0 M HCl was added and allowed to

stand at room temperature for 1 h, with vortexing every

10–15 min. Then, 0.5 ml of 20% TCA was added and

centrifuged at 11,0009g for 5 min. The supernatant was

discarded and pellet was washed 3 times with 1 ml of

ethanol–ethyl acetate (1:1) to remove free reagent. The

sample was allowed to stand for 10 min before centrifu-

gation and the supernatant was discarded each time. Pre-

cipitated protein was redissolved in 0.6 ml guanidine

hydrochloride solution within 15 min at 37–50�C and then

centrifuged at 11,0009g for 5 min to remove any insoluble

material. The carbonyl content of the supernatant was

measured spectrophotometrically at 370 nm. The results

were expressed as nmol of DNPH incorporated/mg protein

using molar extinction coefficient of 22 9 103 M-1 cm-1.

Reduced Glutathione (GSH)

GSH was determined by the method of Jollow et al. (1974).

In brief, 0.2 ml of PMS was precipitated with 0.2 ml of 4%

sulfosalicylic acid. The sample was kept at 4�C for at least

1 h and then centrifuged at 1,5009g for 10 min at 4�C. The

assay mixture contained 0.1 ml of filtered aliquot, 1.7 ml

phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB. Sam-

ples were read immediately at 412 nm. GSH was calculated

in terms of nmol DTNB conjugate formed/mg protein using a

molar extinction coefficient of 13.6 9 103 M-1 cm-1.

Glutathione Peroxidase (GPx)

GPx activity was estimated according to the procedure

described by Mohandas et al. (1984). The reaction mixture

consisted of phosphate buffer (0.05 M, pH 7.0), EDTA

(1 mM), sodium azide (1 mM), GR (1 EU/ml), glutathione

(1 mM), NADPH (0.2 mM), hydrogen peroxide

(0.25 mM) and 0.1 ml of PMS in the final volume of 2 ml.

The disappearance of NADPH at 340 nm was recorded at

room temperature and the enzyme activity calculated as

4 Neurotox Res (2012) 22:1–15

123

nmol NADPH oxidized/min/mg/protein using molar

extinction coefficient of 6.22 9 103 M-1 cm-1.

Glutathione Reductase

GR activity was assayed by the method of Carlberg and

Mannervik (1975) as modified by Mohandas et al. (1984).

The assay mixture consisted of phosphate buffer (0.1 M,

pH 7.6), NADPH (0.1 mM), EDTA (0.5 mM), GSSG

(1 mM) and 0.05 ml of PMS in total volume of 1 ml. The

enzyme activity was quantitated by measuring the disap-

pearance of NADPH at 340 nm and was calculated as nmol

NADPH oxidized/min/mg protein using molar extinction

coefficient of 6.22 9 103 M-1 cm-1.

Superoxide Dismutase (SOD)

SOD activity was measured spectrophotometrically as

described previously by Stevens et al. (2000) by monitor-

ing the protection of autooxidation of (-)-epinephrine at

pH 10.4 for 5 min at 480 nm. The reaction mixture

contained glycine buffer (50 mM, pH, 10.4), 1 mM

(-)-epinephrine and 0.2 ml of PMS in a total volume

of 1.0 ml. The reaction was initiated by the addition of

(-)-epinephrine. The enzyme activity was calculated in

terms of nmol (-)-epinephrine protected from oxidation/

min/mg protein using molar extinction coefficient of

4.02 9 103 M-1 cm-1.

Catalase Activity (CAT)

CAT was assayed by the method of Claiborne (1985). In

brief, the assay mixture consisted of 0.05 M phosphate

buffer (pH 7.0), 0.019 M H2O2 and 0.05 ml PMS in a total

volume of 3.0 ml. The change in absorbance was recorded

at 240 nm. CAT was calculated in terms of nmol H2O2

consumed/min/mg protein.

Determination of Nitric (NO) Levels

The assay was performed as described by Misko et al.

(1993) with slight modification. To 890 ll of Tris–HCl

buffer (200 mM, pH 7.2), 50 ll PMS and 100 ll freshly

prepared 2,3-diaminonaphthalene (0.05 mg/ml in 6.2 M

HCl) were added and mixed immediately. After 10 min

incubation at 20�C, the reaction was terminated with 10 ll

NaOH (2.8 N). The intensity of the fluorescent signal

produced by the product was maximized by the addition of

base formation of 2,3-diaminonaphthotriazole and mea-

sured after 5 min at excitation 365 nm and emission

450 nm with slit width of 25% against the standard curve.

The standard curve of nitrite was constructed using

different concentration. NO was expressed as pmol of

nitrite/mg protein.

DA Analysis by HPLC

The rats were sacrificed, brains removed and the striatum

dissected out in a cold chamber at 4�C. The striatum were

weighed immediately, frozen and stored at -80�C until use.

The brain tissues were sonicated in ice-cold 0.4 N perchloric

acid containing 100 ng/ml DHBA as internal standard. The

homogenates were centrifuged at 15,0009g for 15 min at

4�C. The supernatant was filtered with 0.2-lm membrane

and an aliquot was injected in the loop of 20 ll for the

measurement of the concentrations of DA and its metabo-

lites, 3,4-dihydrophenyl acetic acid (DOPAC) using high-

performance liquid chromatography (HPLC, Waters) with

an electrochemical detector, as reported previously by us

(Zafar et al. 2003). The mobile phase consisted of 0.1 M

potassium phosphate (pH 4.0), 10% methanol and 1.0 mM

heptane sulfonic acid. The concentrations of DA and its

metabolite DOPAC were calculated using a standard curve

generated by determining ratio between these known

amounts of the amine or its metabolites and a constant

amount of internal standard DHBA and represented as ng/mg

of tissue.

Determination of Dopaminergic D2 Receptor Binding

The binding assay was performed by the method of

Agrawal et al. (1995). After tissue homogenates, the pellet

was resuspended in an equal amount of the buffer and

homogenized by hand and centrifuged at 10,0009g for

15 min. Supernatant was discarded and pellet resuspended

in the same amount of said buffer. In brief, the incubation

mixture of 1.0 ml consisted of synaptic membrane along

with 1.0 nM 1-phenyl-4-[H3] spiperone in 40 mM Tris–

HCl (pH 7.4). A parallel incubation was carried out in the

presence of 1.0 lM haloperidol to ascertain non-specific

binding. The assay was run in triplicate. Reaction mixture

was incubated for 15 min at 37�C, terminated by cooling at

4�C and filtered through glass fibre-filters (GF/C, What-

man) through Millipore Filtration Assembly. The filter

discs were washed rapidly with 2 9 5 ml cold Tris–HCl

buffer (40 mM, pH 7.4), and transferred to scintillation

vials and dried properly. After adding 10.0 ml scintillation

cocktail to vials, the radioactivity was counted in a

b-Scintillation Counter (WALLAC-1410) with an effi-

ciency of 50% for tritium. Specific binding was calculated

by subtracting non-specific binding from total binding

obtained in the absence of haloperidol. Results were

expressed as pmol [H3] spiperone bound/mg protein.

Neurotox Res (2012) 22:1–15 5

123

Histopathological Studies

Brains of rats pre-treated with rutin were removed quickly

and perfused according to the method of Ahmad et al.

(2005). Coronal sections of 3-mm thickness were cut and

embedded in paraffin. Sections of 5-lm thickness were cut

in the coronal plane and stained with haematoxylin and

eosin.

Immunohistochemical Analysis

Immunohistochemistry of substantia nigra pars compacta

(SNpc) was carried out following the method of Ahmad

et al. (2005). The rats from each group were anesthetized

with chloral hydrate (400 mg/kg i.p.) and perfused tran-

scardially with 0.1 M phosphate-buffer saline (PBS, pH

7.2), followed by 4% paraformaldehyde in PBS for fixation

of the tissue. Brains were removed and post-fixed in the

same fixative for 24 h followed by the transfer to 10, 20

and 30% sucrose in PBS. Serial coronal sections of 10 lm

thickness were cut in cryostat (Leica, Germany). Endoge-

nous 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 0.5% BSA and 0.1% Triton X-100. The slides

were washed with PBS and the sections were overlaid with

anti iNOS antibody of dilution 1:100 and incubated for 2 h

in a humid chamber at 4�C. The slides were washed again

to remove the unbound antibodies and incubated with

20.0 ll solution of biotinylated anti-mouse IgG of dilution

1:5,000 for 2 h at 4�C in the humid chamber. The slides

were exposed to streptavidin peroxidase and the labelled

sites were visualized with a solution of diaminobenzidine

and hydrogen peroxide. Finally, the sections were dehy-

drated and cover slipped, viewed under a microscope, and

photomicrographs were taken.

Measurement of Cytokines

Commercially available rat TNF-a and rat IL-1b kits

(eBioscience, BD Bioscience, USA) with high sensitivity

were used to quantify these cytokines according to the

manufacturers’ instructions. Homogenates (n = 6 per

group) were analyzed and each sample was in duplicate.

Cytokine levels were quantified from linear standard

curves, with sensitivity of 5 pg/ml for TNF-a and 8 pg/ml

for IL-1b. Activity was calculated as pg/ml.

Determination of Protein

Protein was determined by the method of Lowry et al.

(1951) using BSA as a standard.

Statistics

Results are expressed as mean ± SEM ANOVA with

Tukey–Kramer post hoc analysis was used to analyze dif-

ferences between the groups. The P \ 0.05 was considered

as significant.

Results

Behavioral Observations

Three weeks after 6-OHDA injection, apomorphine-

induced contra lateral rotations were tested and rutin pre-

treatment affected the number of rotations (Fig. 1a). Rutin

(25 mg/kg/bwt) pre-treated for 21 days significantly

(P \ 0.05) decreased the rotations in the 6-OHDA-lesioned

group (R ? L) as compared to the vehicle-treated 6-OHDA

lesioned (L) animals. No significant change was observed

in the rutin alone pre-treatment sham group (R ? S) as

compared to the sham (S) group.

As measured by rota rod, a significant depletion

(P \ 0.001) in muscle coordination skill in lesioned

(L) group as compared to S group was observed (Fig. 1b).

Rutin (25 mg/kg bwt) was found to be effective in partial

recovery of muscular coordination in R ? L group as

compared to L group. No significant differences were

observed between the R ? S group and the S group.

A significant decrease in balance, motor co-ordination

and working performance in 6-OHDA-lesioned rats

(Fig. 1c) were observed as compared with sham which was

significantly (P \ 0.01) protected in rutin pre-treated

groups (R ? L). No significant difference was observed in

R ? S group as compared to sham. Animals in the lesion

group were taken more time to reach the platform as

compared to sham group. Rutin pre-treatment decreased

the total time required to cross the beam and get to the

platform.

Spontaneous Locomotor Activity

A significant decrease in average speed was observed in the

L group as compared to the sham (S) group, but rutin

supplementation increased the speed significantly (P \0.05) in the R ? L group as compared to the L group.

Distance travelled was decreased significantly in L group

as compared to S group, and it was increased significantly

(P \ 0.05) in rutin pre-treated groups, R ? L, as compared

to L group. A significant (P \ 0.05) recovery in locomo-

tion was observed in the R ? L group as compared to the L

group. However, rutin alone treated group exhibited no

significant change in the motor activity when compared to

S group (Table 1).

6 Neurotox Res (2012) 22:1–15

123

Biochemical Observations

Effect of Rutin on TBARS and H2O2 Content

The effect of rutin on TBARS and H2O2 content was

measured to demonstrate the oxidative damage in lesion

group animals and their protection. There was no signifi-

cant alteration in TBARS and H2O2 content in R ? S

group as compared to S group, whilst it was elevated

significantly (P \ 0.01) in L group, as compared to S group

(Fig. 2a, b). Rutin pre-treatment prevented the apparent

increase in lipid peroxidation and H2O2 content.

Effect of Rutin on the Indices of Protein Oxidation

Protein oxidation was assessed by the determination

of protein carbonyl content in the samples of striatum. In-

trastriatal injection of 6-OHDA induced a significant

120

160

200

#12

14

16 *

#

0

40

80

Mu

scu

lar

coo

rdin

atio

n s

kill

(sec

)

*

0

2

4

6

8

10

Ro

tati

on

s/ 5

min

ute

s

Sham Lesion R+L R+SSham Lesion R+L R+S

25

30*

5

10

15

20

Tim

e (s

ec)

#

0Sham Lesion R+L R+S

ab

c

Fig. 1 Effect of rutin (R) on apomorphine-induced contralateral

rotation (a), muscular coordination skill by rota-rod (b) and motor

coordination by narrow beam test (c). 6-OHDA administration

significantly increased the rotation (*P \ 0.01) and decreased the

muscular coordination skill (*P \ 0.001) and motor coordination

(*P \ 0.001) in lesion (L) group as compared to sham group. Pre-

treatment with rutin significantly decreased rotation (#P \ 0.05) and

increased the muscular coordination skill (#P \ 0.01) and motor

coordination (#P \ 0.01) in R ? L group as compared to lesion

group. Values are expressed as mean ± SEM of eight animals

Table 1 Effect of rutin on locomotor activity

Groups Locomotion time (s) (%) Rest time (s) (%) Distance (cm) (%) Average speed (%)

Sham 234.8 ± 15.4 459.2 ± 44.75 3284.41 ± 91.20 223.76 ± 18.65

Lesion 105.6 ± 8.15*

(-55.02)

733.52 ± 66.82*

(?59.73)

1818.68 ± 67.23*

(-44.62)

117.6 ± 7.37*

(-47.44)

R ? L 168.34 ± 10.74#

(?59.41)

601.46 ± 47.39

(-18)

2685 ± 74.96#

(?47.63)

172.22 ± 14.14#

(?46.44)

R ? S 221.27 ± 12.34

(-5.76)

451.34 ± 46.24

(-1.71)

3189.78 ± 84.72

(?2.88)

227.78 ± 19.41

(?1.79)

Values are expressed as mean ± SEM of eight animals. Significance was determined by ANOVA followed by Dunnett’s test: *P \ 0.05 L

versus S group, #P \ 0.05, R ? L versus L group. Values in parentheses are percentage increase (?) or decrease (-) as compared to S or L

group

Neurotox Res (2012) 22:1–15 7

123

(P \ 0.05) increase in protein carbonyl content. Pre-treat-

ment with rutin, however, reduced the 6-OHDA-induced

increase in protein carbonyl content significantly (P \ 0.01)

in the striatum. There was no statistical significant reduction

in protein carbonyl content in rat pre-treated with rutin alone

in R ? S group (Fig. 2c).

Effect of Rutin on GSH Level

The effect of rutin on GSH content in the striatum is shown

in Fig. 3. GSH content was reduced significantly

(P \ 0.01) in L group, as compared to the S group. The

decrease in GSH content was significantly protected in the

R ? L group as compared to the L group. No significant

change was observed between the drug-treated sham group

(R ? S) and sham group.

Effect of Rutin on Antioxidant Enzymes activity

in Parkinsonian Rats

The activities of antioxidant enzymes (GPx, GR, CAT and

SOD) in R ? S group were not attenuated significantly, as

compared to sham. But the activities of these enzymes

were decreased significantly in L group as compared to S

group (Fig. 4). On the other hand, rutin administration in

R ? L group preserved the activities of these enzymes

significantly as compared to L group.

5

6*

15

18

21*

1

2

3

4 #

3

6

9

12

nm

ol H

2O2/

mg

pro

tein

#

0Sham Lesion R+L R+Sn

mo

l TB

AR

S f

orm

ed/h

r/m

g p

rote

in

0Sham Lesion R+L R+S

70

80

*

20

30

40

50

60

#

0

10

Sham Lesion R+L R+S

nm

ol c

arb

on

yl/m

g p

rote

in

ab

c

Fig. 2 Effect of rutin pre-treatment on the content of TBARS (a),

H2O2 (b) and protein carbonyl (c) in the striatum of 6-OHDA-treated

rats. Intrastriatal 6-OHDA administration significantly increased the

content of TBARS (*P \ 0.01), H2O2 (*P \ 0.01) and protein

carbonyl (*P \ 0.05) in lesion group as compared to sham (S) group

rat. Pre-treatment of rutin significantly reduced TBARS (#P \ 0.05),

H2O2 (#P \ 0.05) and PC (#P \ 0.01) content in R ? L group as

compared to L group. Data are expressed as mean ± SEM (n = 8)

40

50

60

70

*

#

10

20

30

nm

ol D

TN

B c

on

jug

ate

fo

rmed

/mg

pro

tein

0Sham Lesion R+L R+S

Fig. 3 Effect of pre-treatment of rutin on the content of GSH in

the striatum of 6-OHDA-treated rats. Values are expressed as mean ±

SEM (n = 8). GSH level was significantly decreased in lesion group

as compared to sham group. Rutin pre-treatment has significantly

(#P \ 0.05) protected the level of GSH in R ? L group as compared

to lesion group. Unit of GSH is expressed as nmol DTNB conjugate

formed/mg protein. * P \ 0.01, L versus sham; #P \ 0.05; R ? L

versus L

8 Neurotox Res (2012) 22:1–15

123

Effect of Rutin on Brain DA Metabolism

Figure 5a shows the content of DA and its metabolite

DOPAC. Intrastriatal injection of 6-OHDA cause significant

decrease in the level of DA and its metabolite DOPAC. Pre-

treatment with rutin in R ? L group protected the level of

DA and DOPAC significantly (P \ 0.01) as compared to the

L group. No significant change was observed in rutin pre-

treatment sham group (R ? S) as compared to sham group.

Dopaminergic D2 Receptor Binding

6-OHDA lesioning resulted an increased in DA-D2

receptor binding, which was due to the increased density

and maximum number of binding sites. The results

revealed significant increase (P \ 0.01) in DA-D2 receptor

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

group. R ? L group attenuated DA receptor binding when

compared to the lesioned group. No significant change was

observed in the rutin pre-treated sham group (R ? S) as

compared to sham group (Fig. 5b).

Effect of Rutin on NO Level

NO level was significantly elevated (P \ 0.01) in lesion

group when compared with sham group, and it was

significantly (P \ 0.05) decreased in rutin pre-treated

lesioned group (R ? L) when compared with lesion

group (Fig. 6). No significant change was observed in

rutin pre-treated sham group (R ? S) as compared to

sham group.

Effect of Rutin on Histopathological Changes

No perivascular collection of cells was detected in the

vehicle-treated sham group. Such perivascular cells were

widely distributed and formed clusters around dopaminer-

gic neurons after 6-OHDA infusion. In the rutin pre-treated

group, the collection of perivascular cells was decreased

significantly as compared to lesioned group rats (Fig. 7).

Rutin treatment did not show any remarkable effects on

iNOS expression in the R ? S compared with the S group

(data not shown).

200

250

300

350

400

*

#

300

400

500

600

#

*

0

50

100

150

nm

ol N

AD

PH

oxi

dis

ed/m

in/m

g p

rote

in

0

100

200

nm

ol N

AD

PH

oxi

dis

ed/m

in/m

g p

rote

in250300350400

##

4

5

6

7

8

O2c

on

sum

ed#

050

100150200

nm

ol o

f ep

inep

hri

ne

pro

tect

ed f

rom

o

xid

atio

n/m

in/ m

g p

rote

in

*

0

1

2

3

Sham Lesion R+L R+S Sham Lesion R+L R+S

Sham Lesion R+L R+SSham Lesion R+L R+S

nm

ol o

f H

2

/min

/mg

pro

tein

*

a b

cd

Fig. 4 Effect of rutin pre-treatment on the activities of antioxidant

enzymes GPx (a), GR (b), SOD (c) and catalase (d) in the striatum of

6-OHDA-treated rats. The activities of antioxidant enzymes were

decreased significantly in lesion group as compared to S group. The

pre-treatment with rutin has protected their activities significantly in

R ? L group as compared to L group. Values are expressed as

mean ± SEM of eight animals. *P \ 0.05, Lesion vs. Sham group;#P \ 0.05, ##P \ 0.01, Lesion vs. R ? L group

Neurotox Res (2012) 22:1–15 9

123

Effect of Rutin on Inducible Nitric Oxide Synthase

Expression

Increased expression of iNOS immunostaining was observed

in lesion group animals after intrastriatal injection of

6-OHDA. The elevation of iNOS expression was signifi-

cantly decreased following rutin pre-treatment in R ? L

group as compared to L group (Fig. 8). Rutin treatment did

not show any remarkable effects on iNOS expression in the

R ? S compared with the S group (data not shown).

Effect of Rutin on TNF-a and IL-1b

Inflammation in PD is mediated by cytokines including

TNF-a and IL-1b. TNF-a and IL-1b levels were elevated

significantly (P \ 0.001) in 6-OHDA-lesioned group rats.

Rutin protected the R ? L group from the rise in circu-

lating TNF-a and IL-1b production (Table 2).

Discussion

This study demonstrates the potential protective effects of

rutin from toxicity of the nigrostriatal dopaminergic neurons

induced by 6-OHDA injection. Intrastriatal injection of

6-OHDA potentiated both the motor deficit accompanied

with oxidative damage and inflammatory response with loss

of DA. These findings corroborate with earlier reports (Sauer

and Oertel 1994; Ahmad et al. 2005; Jin et al. 2008). Rutin

attenuated behavioral, biochemical and histological param-

eters after 6-OHDA lesioning, confirming the bioactivity of

exogenously applied rutin in an animal PD model. Rutin

neuroprotective effects suggest that it is a powerful antiox-

idant and anti-inflammatory agent, corroborating previous

studies (Gupta et al. 2003; Bishnoi et al. 2007; Pu et al. 2007;

Koda et al. 2009; Khan et al. 2009).

It has been demonstrated that dopaminergic neurons in

PD are especially vulnerable to oxidative stress (Zafar et al.

2003; Jenner 2007; Sanchez-Iglesias et al. 2009). Over-

production of free radicals such as superoxide and perox-

ynitrite cause an imbalance in the redox environment of

cells, and react with proteins and nucleic acids to alter their

functions, or induce lipid peroxidation, leading to eventual

cell death. Therefore, scavenging free radicals and pre-

venting lipid peroxidation, which are the main effects of

rutin, can directly suppress oxidative damage and inflam-

matory response.

250

300

*8

10

DA

DOPAC

50

100

150

200

3 H-s

pip

ero

ne

bo

un

d/m

g p

rote

in

#

2

4

6

*

#

*#

0

pm

ol 0

Sham Lesion R+L R+SSham Lesion R+L R+Sng

DA

an

d D

OP

AC

/mg

tis

sue

ab

Fig. 5 a Effect of pre-treatment of rutin on the level of DA and

DOPAC in the striatum of 6-OHDA-treated rats. The 6-OHDA

infusion led to a significant (*P \ 0.01) decrease in the level of DA

and DOPAC in lesion group as compared with the sham group. Pre-

treatment with rutin significantly (#P \ 0.01) protected the level of

DA and DOPAC in the R ? L group as compared with the L group.

b Effect of rutin on dopaminergic D2 receptor binding. The 6-OHDA

infusion led to a significant (*P \ 0.01) increase in D2 receptor

binding in the lesion (L) group as compared with the sham (S) group.

Pre-treatment with rutin significantly (#P \ 0.05) decreased the

receptor binding in the R ? L group as compared with the L group.

Values are expressed as mean ± SEM (n = 8)

400

450

500*

#

150

200

250

300

350

0

50

100

Sham Lesion R+L R+S

pm

ol o

f n

itri

te /m

g p

rote

in

Fig. 6 Effect of rutin on NO level. The 6-OHDA infusion led to a

significant increase in NO level in the lesion (L) group as compared

with the sham (S) group. Pre-treatment with rutin significantly

decrease the NO level in the R ? L group as compared with the L

group. Values are expressed as mean ± SEM of eight animals.

*P \ 0.01, L versus sham; #P \ 0.05, R ? L versus L

10 Neurotox Res (2012) 22:1–15

123

GSH is the major antioxidant in the brain which buffers

free radicals in brain tissue. It eliminates H2O2 and organic

peroxides by GPxs (Meister 1988). During detoxification,

oxy-radicals are reduced by GPx at the expense of GSH to

form glutathione disulfide (GSSG). GSH is regenerated by

redox recycling, in which GSSG is reduced to GSH by GR

with a consumption of one NADPH. SOD converts

superoxide into H2O2 (Freeman and Crapo 1982). The

catalase, which is found at very low activity in the brain,

detoxifies H2O2 to H2O. It is established that all of these

antioxidant defenses are inter-related (Sun 1990) and a

disturbance in one may disturb the balance in all. A

reduction in the level of GSH may impair H2O2 clearance

and promote formation of •OH, the most toxic molecule to

the brain, leading to more oxidant load and further oxida-

tive damage (Dringen 2000). In this study, 6-OHDA infu-

sion caused an overproduction of free radicals which, in

turn, caused oxidative damages to membrane lipids and

protein levels, and ultimately lead to a decrease in GSH

and the antioxidant enzymes, SOD and catalase. This

Fig. 7 Representative photomicrograph of coronal sections of sub-

stantia nigra stained with haematoxylin and eosin (H&E), showing

effects of 6-OHDA and rutin. Low (9100) and high (9400) power

photomicrograph of SNpc of brain from lesioned group (c, d) animal

showing a large cluster of perivascular cells near a blood vessel (BV),

whilst the lesioned group pre-treated with rutin (e, f) has shown small

clusters of perivascular cells. However, the sham group [a (9100),

b (9400)] has shown uniform distribution of cells

Neurotox Res (2012) 22:1–15 11

123

oxidative neuronal damage in 6-OHDA-treated rats is

consistent with previous reports (Zafar et al. 2003; Ahmad

et al. 2005; Chaturvedi et al. 2006). Moreover, rutin sup-

plementation significantly reduced all the alterations in the

markers of oxidative damage in the striatum of 6-OHDA

infused rats. Recently, khan et al. (2009) and Bishnoi et al.

(2007) have reported that rutin is a potent antioxidant and

may help to enhance the status of endogenous antioxidant

systems and reduce glutathione, and may protect from

oxidative damage.

Fig. 8 The effect of 21 days of pre-treatment of rutin on iNOS

expression in ipsilateral SNpc in rats lesioned by a single injection of

10.0 lg 6-OHDA. The profound expression of iNOS was observed in

lesioned group [c (9100), d (9400)] compared to sham group, whilst

the lesioned group pre-treated with rutin (e, f) has shown a moderate

staining of iNOS. However, the sham group (a, b) has shown almost

negligible staining

Table 2 Effect of rutin on cytokines level

Groups TNF-a IL-1b

Sham 18.09 ± 2.01 83.08 ± 6.16

Lesion 43.66 ± 4.22* 197.74 ± 13.62*

R ? L 30.12 ± 1.78# 154.62 ± 11.93#

Values are expressed as mean ± SEM of eight animals. Significance

was determined by ANOVA followed by Tukey–Kramer test:

*P \ 0.001 L versus S group, #P \ 0.01, R ? L versus L group.

Concentration was calculated as pg/ml

12 Neurotox Res (2012) 22:1–15

123

Depletion in GSH content and enhancement of LPO

leads to the degeneration of nigrostriatal neurons and,

subsequently, leads to a reduction in the content of cate-

cholamine and increase the population of DA receptors

(Schwarting and Hudson 1996; Chaturvedi et al. 2006) and

we are of the opinion that it happens, as a compensatory

mechanism to trap and utilize almost every available

molecule of DA. In this study, the increase in the D2

receptor population in striatum due to 6-OHDA lesioning

was significantly prevented by pre-treatment with rutin.

This finding is in agreement with earlier studies carried out

by us (Ahmad et al. 2005) and others (Agrawal et al. 1995;

Chaturvedi et al. 2006).

The injection of 6-OHDA into the striatum results in

depletion of DA, thereby resulting in a number of behav-

ioral deficits mimicking aspects of PD (Deumens et al.

2002; Cannon et al. 2005; Ogura et al. 2005). The behav-

ioral assessment is a more powerful endpoint in evaluating

neuroprotection. The behavioral testing data in this study

provides a sensitive evaluation of the ability of rutin to

provide protection in this PD model. Apomorphine-

induced contralateral rotation in 6-OHDA-lesioned rats is a

reliable marker for the nigrostriatal DA depletion. We

report here an appreciable decrease in drug-induced rota-

tion with increase in DA and DOPAC level, and significant

increase in locomotor activity in terms of locomotion,

distance travelled, average speed and motor coordination

skill by narrow beam test following pre-treatment with

rutin. Our findings are in harmony with the earlier studies

carried out by us and others, where motor deficits in Par-

kinsonian rats have been attenuated by antioxidant sup-

plementation (Zafar et al. 2003; Ahmad et al. 2005;

Chaturvedi et al. 2006; Jin et al. 2008).

Inflammation has recently emerged as a key player in

the pathogenesis of PD, and the possibility that modulate

the inflammatory response may interfere with the pro-

gression of the disease is attracting increasing interest

(Hirsch et al. 2005; Little et al. 2011). Microglial activation

is considered as a rapid cellular response to inflammation

(Koprich et al. 2008). Activation of microglia induces

cytotoxic mediators such as NO and inflammatory cyto-

kines, which may contribute to the PD progression. NO

production resulting from induced NOS2 gene expression

and subsequent iNOS enzyme activation is a primary

contributor to the inflammatory response. Cytokines are

implicated, with the observation of TNF-a and IL-1b ele-

vation in 6-OHDA-treated rats (Mogi et al. 1999; Koprich

et al. 2008; Jin et al. 2008). These cytokines may have

deleterious effects through several different mechanisms.

Cytokines also directly bind to their receptors on the cell

surfaces on dopaminergic neurons, for example, the TNF-areceptor. Once activated these cytokine receptors could

trigger intracellular death-related signalling pathways. In

turn, this sequence of events results in increased pro-

inflammatory cytokine production, enhancement of leuco-

cyte infiltration into the brain and upregulation of adhesion

molecules that contribute to both necrotic and apoptotic

cell death (del Zoppo et al. 2000). Therefore, cell death in

PD relates directly to a substantial increase in microglia

activation. Thus, neuroprotective effect of rutin is associ-

ated with concomitant reduction of the associated

microglia response, iNOS expression and cytokines level at

the sites of neurodegeneration. The inhibition of microglia

cell activation, iNOS expression and cytokine level show

that rutin has anti-inflammatory activity. It could also be

useful in slowing neuronal death and therefore, halting

progression of the disease.

Our results suggest that rutin has antioxidant and anti-

inflammatory properties which might delay the onset and

slow the progression of PD by protecting 6-OHDA-induced

alterations in behavioral, biochemical and histopatholo-

gical parameters in rats. Further understanding the mech-

anism underlying the neuroprotection of rutin will provide

an avenue to disclose both the pathogenesis and therapeutic

mechanisms underlying PD. Thus, rutin may be considered

a potential candidate in the armamentarium of drugs for

prophylactic treatment in patients who are prone to PD.

Acknowledgments The authors thank the Department of Ayurveda,

Yoga & Naturopathy, Unani, Siddha and Homoeopathy (AYUSH),

Ministry of Health and Family Welfare, Government of India, New

Delhi, for financial assistance. We greatly acknowledge Ms. Lorie

Leo, Department of Internal Medicine, University of Iowa, for

reviewing and editing this manuscript. Technical assistance of

Dharamvir Singh is gratefully appreciated.

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