Potential neuroprotective effect of ibuprofen, insights...

Potential neuroprotective effect of ibuprofen,

insights from the mice model of Parkinson’s

disease

Maciej Œwi¹tkiewicz1, Ma³gorzata Zaremba1, Ilona Joniec1,Andrzej Cz³onkowski1, Iwona Kurkowska-Jastrzêbska2

1Department of Experimental and Clinical Pharmacology, Medical University of Warsaw,Krakowskie Przedmieœcie 26/28, PL 00-927 Warszawa, Poland

22nd Department of Neurology, Institute of Psychiatry and Neurology, Sobieskiego 9, PL 02-957 Warszawa, Poland

Correspondence: Maciej Œwi¹tkiewicz, e-mail: [email protected]

Abstract:

Background: Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. An inflammatory reaction seems to

be involved in the pathological process in PD. Prospective clinical studies with various nonsteroidal anti-inflammatory drugs

(NSAIDs) have shown that ibuprofen decreases the risk of PD. In the present study we investigated the influence of ibuprofen on do-

paminergic neuron injury in the mice model of PD.

Methods: Twelve-month-old male C57Bl mice were injected with MPTP together with various doses of ibuprofen (10, 30 or

50 mg/kg), administered 1 h before MPTP injection for 7 consecutive days. Evaluation concerned dopamine content in the striatum,

tyrosine hydroxylase (TH) protein and a-synuclein expression measured 7 and 21 days post MPTP administration (dpa).

Results: MPTP caused injury to dopaminergic neuron endings in the striatum: dopamine content decreased by about 90% 7 dpa and

by 85% 21 dpa; TH protein expression diminished by 21% 7 dpa; a-synuclein level decreased by 10 and 26% 7 and 21 dpa, respec-

tively. Ibuprofen administration to mice treated with MPTP significantly increased the level of dopamine in the striatum 7 and

21 dpa. It also prevented TH protein decrease and increased a-synuclein level 21 dpa.

Conclusions: Ibuprofen was shown to protect neurons against MPTP-induced injury in the striatum. The possible mechanism of the

neuroprotective effect of ibuprofen might be associated with decreased dopamine turnover and cyclooxygenases inhibition resulting

in lower reactive oxygen species formation.

Key words:

MPTP, neuroinflammation, neurodegeneration, ibuprofen, a-synuclein, Parkinson’s disease

Introduction

Parkinson’s disease (PD) is one of the most common

neurodegenerative diseases. The detailed mechanisms

underlying the pathological changes in PD are not

clearly understood. An important role in the pathogene-

sis of progressive neurodegeneration is attributed to mi-

croglial and astroglial activation and lymphocytic in-

filtration, observed in the area of impaired neurons.

This local reaction leads to sustained, chronic neuro-

inflammation, which is suggested to drive the pro-

gressive neurodegeneration in PD [2, 26].

The role of inflammation in the pathogenesis of PD

and the use of nonsteroidal anti-inflammatory drugs

Pharmacological Reports, 2013, 65, 1227�1236 1227

Pharmacological Reports2013, 65, 1227�1236ISSN 1734-1140

Copyright © 2013by Institute of PharmacologyPolish Academy of Sciences

(NSAIDs) as neuroprotective agents remain areas of

intense research. A number of experimental and clini-

cal studies have suggested that NSAIDs may have

a therapeutic role in PD [2–4, 15, 16, 18, 22, 23, 25].

The analysis, which combined the results of several

large epidemiologic studies, has suggested that the

use of non-aspirin NSAIDs is associated with a 15%

reduction in the risk of PD. In addition, a greater re-

duction in the risk has been observed in case of regu-

lar (29% reduction) and long-term use of NSAIDs

(21% reduction), consistent with a dose-response rela-

tion. By contrast, no protective effect has been ob-

served for aspirin or acetaminophen [22]. Prospective

clinical studies with NSAIDs have shown that ibupro-

fen in particular decreases the risk of PD [16]. In

a meta-analysis of a large cohort of people in the

USA, users of non-aspirin NSAIDs, and in particular

ibuprofen, have shown a lower risk of PD than non-

users [18]. A meta-analysis of all available prospec-

tive studies has also shown that ibuprofen users have

an approximately 30% lower risk of PD than non-

users [23].

Animal experiments with PD models have shown

that several NSAIDs help to protect against dopamin-

ergic neuron degeneration, dopamine (DA) depletion

and diminished locomotor activity caused by neuro-

toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) [5, 15, 19, 20, 28, 36, 42]. In our previous

studies, also based on the MPTP mice model, ibupro-

fen dose-dependently alleviated the loss of striatal do-

pamine without toxic influence on dopaminergic neu-

rons, while other NSAIDs (e.g., indomethacin) ap-

peared to be toxic in high doses [28, 29]. However,

results vary across parkinsonian models, and some

experiments do not identify specific NSAIDs (e.g., di-

clofenac, ibuprofen, indomethacin and dexametha-

sone) as unequivocal neuroprotectants for PD [5, 36].

In the present study, we investigated the influence

of ibuprofen on the nigrostriatal system, using one of

the best known models of PD – the MPTP mice

model. When administered peripherally, MPTP

crosses the blood-brain barrier and quite selectively

injures dopaminergic neurons of the substantia nigra

(SN). This leads to a decrease in DA content and de-

pletion of the number of neuronal endings in the stria-

tum and causes neuroinflammation in the impaired

structure. We showed that ibuprofen administered in

the phase of acute injury had some protective abilities

against MPTP-induced damage and modified dopa-

mine metabolism in the striatum.

Materials and Methods

Animals

Male C57BL mice (12-month-old) were studied. The

animals were housed in plastic cages with free access

to food and water. They were maintained on a 12-h

light/dark cycle at a temperature of 25 ± 2°C. Experi-

ments were conducted without causing unnecessary

harm to the animals and were approved by the Local

Ethics Committee.

The mice were divided into 8 groups: an MPTP

group (receiving MPTP toxin), a control group

(receiving 0.9% solution of NaCl according to the

pattern of MPTP administration), three ibuprofen

groups – IBF (receiving ibuprofen in a dosage of 10,

30 or 50 mg/kg) and three IBF + MPTP groups

(receiving MPTP toxin and ibuprofen in a dosage of

10, 30 or 50 mg/kg). The animals were sacrificed

7 and 21 days after MPTP intoxication (6 animals per

time point) by spinal cord dislocation. Their brains

were rapidly removed and the striata were dissected

and frozen at –80°C.

MPTP administration

MPTP-hydrochloride (Sigma, USA) was dissolved in

0.9% solution of NaCl (2 mg/ml) and administered

intraperitoneally in four doses of 10 mg/kg each, at

one hour intervals, to a total dose of 40 mg/kg.

Ibuprofen administration

Ibuprofen (Sigma, USA) was dissolved in 0.9% NaCl

solution just before injection. It was administered in

three doses of 10, 30 and 50 mg/kg starting one day

before MPTP treatment and was given every day for 7

consecutive days.

HPLC

The striata were dissected out on an ice-cold plate.

Each tissue sample was rapidly weighed and frozen

(–80°C) for future analysis. Tissues were homoge-

nized in ice-cold 0.1 M HClO4 and centrifuged

(13,000 × g, 15 min) to precipitate proteins. The su-

pernatant was removed, filtered (0.2 µm pore size;

Whatman, USA) and examined for dopamine (DA;

standard supplied by RBI) and its metabolites content:

1228 Pharmacological Reports, 2013, 65, 1227�1236

3,4-dihydroxyphenylacetic acid (DOPAC; RBI, USA)

and homovanillic acid (HVA; Sigma, Germany), using

HPLC with electrochemical detection and glassy car-

bon electrode. The electrochemical potential was set

at 0.8 V with respect to the Ag/AgCl reference elec-

trode. The chromatographic system consisted of an

autosampler automatic injector (Knauer Basic Mara-

thon), a pump (Mini-Star K-500; Knauer, Germany),

and an electrochemical detector (L-3500A; Merck,

Germany). The mobile phase comprised 31 mM so-

dium phosphate (Sigma), 58 mM citric acid (Sigma),

1 mM octane sulfonic acid (Aldrich, Germany),

27 µM ethylenediaminetetraacetic acid (EDTA; Sigma)

in deionized (18.3 mW) purified water containing

12% acetonitrile (Merck) and 1% methanol (Merck).

Monoamines were separated using a C-18 column

(250 × 4 mm reverse phase; Nucleosil; 5 µm particle

size; Macherey-Nagel, Germany) and a mobile phase

flow rate maintained at 0.8 ml/min. The samples were

quantified by comparison with standard solutions of

known concentration using HPLC software, and the

area under the peaks was measured. Data were col-

lected and analyzed using Eurochrom 2000 for Win-

dows (Knauer). Contents of dopamine and its metabo-

lites were expressed as pg/mg fresh tissue.

Evaluation of MPP+ level by HPLC

In order to assess 1-methyl-4-phenyl-tetrahydropy-

ridinium ion (MPP+) level in the striatum, additional

groups of mice (five animals each) received three dif-

ferent doses of ibuprofen before MPTP, which was

administered according to the standard scheme for the

experiment to the total dose of 40 mg/kg. Two hours

after the last MPTP injection the mice were killed by

cervical dislocation and the striata were prepared as

described above. HPLC analysis of MPP+ was per-

formed immediately.

The mobile phase contained 0.14 mol sodium dihy-

drogen phosphate adjusted to pH 2.5 with H3PO4 in 1 :

l of HPLC grade water with 300 ml of acetonitrile,

and was delivered at a rate of 0.5 ml/min onto a re-

versed-phase column (125 × 3 mm with precolumn

5 × 3 mm; Nucleosil 120-3 C-18; Macherey-Nagel,

Germany). The UV detector was set at a wavelength

of 295 nm. Twenty microliter aliquots were injected

into the column. Data were calculated by an external

standard calibration.

Western blot analysis

The striatum samples were homogenized with a mi-

cropestle (Eppendorf) in 1.5 ml of lysis buffer

(150 mM NaCl, 50 mM Tris (pH 8.0), 1% Igepal

CA-630, 0.10% sodium dodecyl sulfate, 0.50% de-

oxycholic acid sodium salt, 0.10 mg/ml phenylmeth-

ylsulfonyl fluoride, 1.0 mM sodium orthovanadate,

10 ng/ml aprotinin and 25 ng/ml pepstatin) at 0–4°C.

They were subsequently incubated for 30 min on ice.

Homogenates were centrifuged at 10,000 × g for

20 min at 4°C. The aliquots of supernatants were

taken for total protein analysis (Bradford Reagent,

Sigma). For electrophoresis, 60 µg/ml of a homoge-

nized sample was taken and added to SDS sample

buffer at a ratio of 1 : 1.

Proteins were electrophoretically transferred to ni-

trocellulose membranes using the iBlot® Dry Blot-

ting System (Invitrogen). The membranes were subse-

quently stained to reconfirm equal loading by

Ponceau-S staining and washed with distilled water.

After blocking for 1.5 h with 5% nonfat dry milk/

0.5% Tween-20 in Tris-buffered saline, the mem-

branes were incubated with polyclonal rabbit anti-

a-synuclein (1 : 500, Millipore) or polyclonal rabbit

anti-tyrosine hydroxylase antibodies diluted in TBST

containing 5% nonfat dry milk (1 : 500, Millipore)

overnight at 4°C. The membranes were subsequently

washed three times (for 10 min. each) in TBST and

incubated with secondary antibodies conjugated to

horseradish peroxidase electrophoresed on 10%

SDS-polyacrylamide gels in Mini Protean II Dual

Slab Cell (Bio-Rad) diluted in TBST containing 5%

nonfat dry milk (1 : 2,000, Amersham) for 2 h. Per-

oxidase activity was visualized using the ECL western

blotting detection system (Amersham) according to

the manufacturer’s instruction. Each gel contained

lanes from each time point and control brains. The

densities of each band were analyzed with a software

program (Syngen) and expressed in terms of their ra-

tio to control lanes.

Results

Dopamine content in the striatum

Dopamine level decreased markedly to 8% of the con-

trol level on the 7th day post MPTP administration


Ibuprofen in the MPTP mice model of Parkinson’s diseaseMaciej �wi¹tkiewicz et al.

(dpa) and rose to 15% on the 21st day, indicating the

process of dopamine content restoration (Fig. 1). Ibu-

profen treatment prevented a decline in the dopamine

content measured on the 7th dpa, in the dose of

30 mg/kg alone, and the dopamine level fell to only

16% of the control level (p < 0.05). Although the ibu-

profen treatment was stopped on the 7th day, the group

of animals receiving ibuprofen showed better dopa-

mine content restoration on the 21st day. The dopa-

mine content represented 15% of the control level in

animals treated with MPTP alone and about 25% in

animals additionally treated with 10, 30 and 50 mg/kg

of ibuprofen (Fig. 1).

Ibuprofen alone increased the dopamine level by

about 20% on the 21st dpa in the doses of 30 mg/kg

and 50 mg/kg (p < 0.01).

Dopamine turnover

The levels of dopamine metabolites DOPAC and HVA

were correlated to the dopamine level in every exam-

ined group on the 7th and 21st dpa. The dopamine turn-

over (DOPAC/DA ratio and HVA/DA ratio) was

markedly changed by MPTP intoxication and in-

creased 2.2 times (DOPAC/DA) and 7.4 times

(HVA/DA) compared to control on the 7th dpa (p <

0.01). The DOPAC/DA ratio returned to the control

level on the 21st dpa, while the HVA/DA ratio re-

mained slightly elevated (p < 0.02) (Fig. 2). Ibupro-

fen alone influenced dopamine turnover, in particular,

it increased the DOPAC/DA ratio in the dose of

10 mg/kg (p < 0.03) and led to an insignificant in-

crease in the dose of 30 mg/kg compared to control. It

increased also HVA/DA ratio in the doses of 10 mg/kg

(insignificantly) and of 30 mg/kg (p < 0.05). Interest-

ingly, on the 21st dpa, we observed diminished

DOPAC/DA ratios in the group of animals receiving

ibuprofen in doses of 10 and 30 mg/kg, when

HVA/DA ratios returned to the control level. When

added to MPTP, ibuprofen markedly decreased both

the DOPAC/DA and HVA/DA ratios in the doses of

30 and 50 mg/kg compared to the MPTP group on the

7th dpa (p < 0.05). There was no difference between

the DOPAC/DA and HVA/DA ratios between the

MPTP and IBF + MPTP groups on the 21st dpa, apart

from a slightly lower HVA/DA ratio in the IBF50 +

MPTP group than in the MPTP group (p < 0.05).

TH protein expression

TH protein expression in the striatum decreased after

MPTP treatment by 21% on the 7th dpa (p < 0.02) and

returned to the control level on the 21st dpa (Fig. 3).

Ibuprofen administration to animals receiving MPTP

completely prevented the decline in TH protein con-

tent on the 7th dpa when given in the doses of 10 and

30 mg/kg (p < 0.05). Ibuprofen in the dose of

50 mg/kg had no such effect. TH protein expression in

the MPTP group was comparable to the control level

on the 21st dpa, and ibuprofen did not influence its


0

2000

4000

6000

8000

10000

12000

14000

16000

18000

cont

rol

IBF10

IBF30

IBF50

MPTP

IBF10

+m

ptp

IBF30

+m

ptp

IBF50

+m

ptp

7 days 21 daysDA pg/ml

*

*

* * * **

**

##

##*

Fig. 1. Dopamine (DA) content in the striatum in mice intoxicated with MPTP and treated with ibuprofen 7 and 21 days after MPTP administra-tion. Bars show the mean value ± SEM for all groups of animals treated with various doses of ibuprofen, MPTP or ibuprofen with MPTP. Asterisk(*) indicates significant differences compared to control; hash (#) indicates significant difference compared to MPTP group on the same day

level on this day. Ibuprofen administered alone did

not change TH expression in the striatum at any time

point (Fig. 3).

a-Synuclein expression

a-Synuclein expression in the striatum decreased

after MPTP treatment by about 10 and 26% compared

to the control level on the 7th and 21st dpa, respec-

tively (p < 0.01) (Fig. 4). The group of animals

treated with ibuprofen and MPTP showed a slight de-

pletion of a-synuclein content on the 7th day, similar

to the MPTP group. However, on the 21st dpa, a-syn-

uclein level was higher in animals receiving 10 and 30

mg/kg of ibuprofen compared to animals treated with

MPTP alone (p < 0.05). Ibuprofen administered alone

decreased a-synuclein expression only in the dose of

50 mg/kg on the 21st dpa (p < 0.01).

MPP+ evaluation

MPTP administration resulted in MPP+ formation at

a level of 4.9 ± 0.54 ng/mg wet tissue weight.



0

0.2

0.4

0.6

0.8

1

1.2

cont

rol

IBF10

IBF30

IBF50

MPTP

IBF10

+m

ptp

IBF30

+m

ptp

IBF50

+m

ptp

7 days 21 daysDOPAC/DA

*

* *

*

*

*

*#

0

0.2

0.4

0.6

0.8

1

1.2

cont

rol

IBF10

IBF30

IBF50

MPTP

IBF10

+m

ptp

IBF30

+m

ptp

IBF50

+m

ptp

7 days 21 days

*

*

*

*

* **

HVA/DA

*

*

*

*

* **

*# #

#

Fig. 2. Dopamine (DA) turnover in the striatum in mice intoxicated with MPTP and treated with ibuprofen 7 and 21 days after MPTP administra-tion. Bars show the mean value ± SEM for all groups of animals treated with various doses of ibuprofen, MPTP or ibuprofen with MPTP. Asterisk(*) indicates significant differences compared to control; hash (#) indicates significant difference compared to MPTP group on the same day

The mice receiving ibuprofen (10, 30 and 50 mg/ kg)

before MPTP injection produced MPP+ levels compara-

ble to those of MPTP alone but with a tendency to in-

crease depending on the ibuprofen dose. The dose of 100

mg/kg increased MPP+ production of 69% (p < 0.05) and

was thus excluded from other experiments (Fig. 5).


0

20

40

60

80

100

120

140

contro

l

IBF1

0

IBF3

0

IBF5

0

MPT

P

IBF1

0+

mptp

IBF3

0+

mptp

IBF5

0+

mptp

7 days 21 days

%o

fc

on

tro

l

Fig. 4. a-Synuclein protein expression in the striatum in mice intoxicated with MPTP and treated with ibuprofen 7 and 21 days after MPTP ad-ministration. Bars show the mean value ± SEM for all groups of animals treated with various doses of ibuprofen, MPTP or ibuprofen with MPTP.Asterisk (*) indicates significant differences compared to control; hash (#) indicates significant difference compared to MPTP group on thesame day

Fig. 3. TH protein expression in the striatum in mice intoxicated with MPTP and treated with ibuprofen 7 and 21 days after MPTP administration.Bars show the mean value ± SEM for all groups of animals treated with various doses of ibuprofen, MPTP or ibuprofen with MPTP. Asterisk (*) in-dicates significant differences compared to control; hash (#) indicates significant difference compared to MPTP group on the same day

Discussion

In the present study, we demonstrated that administra-

tion of ibuprofen to mice treated with MPTP signifi-

cantly accelerated the recovery of DA level in the stria-

tum, prevented TH protein depletion and enhanced

a-synuclein expression. The protective effect of ibu-

profen was not dependent on MPTP conversion to ion

MPP+, which was responsible for MPTP toxicity. The

protection was less evident in the early phase of injury

than in the late phase and was dependent on the dose of

the drug. Ibuprofen probably diminished the injury of

neuron endings in striatum (prevented TH protein de-

pletion), stimulated the phase of recovery and regen-

eration and in this way prevented MPTP toxicity.

Ibuprofen is commonly known as an anti-inflam-

matory agent whose main mechanism of action con-

cerns the inhibition of cyclooxygenase (COX) activity

and prostaglandins production. Ibuprofen inhibits

equally constitutive COX-1 and inducible COX-2 re-

sponsible for inflammation. Both COXs have been

shown to play an important role in neuroinflammation

in the brain. COX-1 is suggested to be an important

player in microglial activation and neurodegenera-

tion; COX-2 may mediate neurotoxic or anti-inflam-

matory effects depending on the stimulus and type of

injured cells [1]. The COX-inhibiting mechanism of

action may be involved in the protective effect of ibu-

profen and other NSAIDs. In some experiments, pre-

treatment with NSAIDs led to partial or total protec-

tion against MPTP toxicity [5, 19, 34, 36, 41, 42]. The

mechanisms of their protective effects have been

linked with inhibition of inflammation (decreased

production of prostaglandins) and with decreased

COX-dependent formation of ROS during the acute

phase of MPTP injury. Ibuprofen has been shown to

protect dopaminergic neurons from 6-OHDA toxicity

in doses that block neuronal COX. The effect has

been correlated with lower prostaglandin production

[11]. On the other hand, MPP+ induced injury of do-

paminergic neurons has also been prevented by ibu-

profen but linked to decreased ROS production [26].

ROS formation is suggested to be involved in pro-

gressive dopaminergic neuron damage in PD [39]. DA

metabolism generates a huge amount of oxygen spe-

cies and is likely responsible for the particular suscep-

tibility of dopaminergic neurons to this kind of injury.

Some hypotheses suggest that primary neurodegen-

eration in the nigrostriatal pathways leads to increased

dopamine turnover and triggers ROS generation,

which in turn contributes to neuronal impairment

[17]. MPTP administration directly increases ROS

production by inhibiting enzymes of the respiratory

chain, and additionally, by increasing dopamine turn-

over – a compensatory mechanism triggered by DA

depletion. Increased ROS formation has been shown

to contribute to dopaminergic neuron injury after

MPTP administration [41, 42]. We showed that ibu-

profen decreased DA turnover in groups of animals

receiving MPTP, which was connected with better re-

covery of DA level in the striatum. It is possible that

lower DA turnover results in lower production of

ROS, contributing to the protective effect of the drug.

Ibuprofen has been shown to decrease ROS produc-



0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

MPTP

40 m

g/kg

IBF

10 m

g/kg

+M

PTP

IBF

30 m

g/kg

+M

PTP

IBF

50 m

g/kg

+M

PTP

IBF

100

mg/

kg +

MPTP

MPP + ng/ml tissue*

Fig. 5. MPP+ content in thestriatum measured 2 h after MPTPadministration in mice treated withvarious doses of ibuprofen beforeMPTP intoxication. Bars show themean value ± SEM. Significantdifference compared to control isindicated by asterisk (*)

tion and damage to dopaminergic neurons injured by

manganese [32] or MPP+ [26].

Ibuprofen is not known to influence the metabo-

lism of catecholamines. We observed increased MPP+

formation when ibuprofen was added to MPTP regi-

men, which may indicate augmented MAO-B activity.

The effect was also visible as an increased DO-

PAC/DA and HVA/DA ratios on the 7th day in animals

receiving ibuprofen alone in the doses of 10 and 30

mg/kg. However, the effect was not so clearly present

in animals receiving higher doses of ibuprofen and

was reverse in animals treated additionally with

MPTP (decreased DOPAC/DA and HVA/DA ratios).

The opposite effects of the various doses of ibuprofen

on DA metabolism are difficult to explain. One of the

possibilities is that ibuprofen may dose-dependently

influence the activation of the enzymes involved in

dopamine breakdown. Ibuprofen administered to ani-

mals with not-injured nigrostriatal system had same

tendency to increase dopamine metabolism. After dis-

continuation of ibuprofen administration, a dopamine

metabolism slightly slowed down (decreased DO-

PAC/DA ratios on the 21st dpa), what may be ex-

plained by the mechanism compensating the previous

acceleration. On the other hand, ibuprofen in higher

doses might inhibit a compensatory enhancement of

dopamine turnover in the injured neurons. It has been

shown that other NSAID, indomethacin, prevent an

increase of dopamine turnover in rat striatum follow-

ing systemic lipopolysaccharide administration [31].

The interesting observation was that ibuprofen al-

most completely prevented TH protein depletion on

the 7th dpa but did not increased dopamine level on

this day. We also noted that the expression of TH pro-

tein returned to the control level after MPTP admini-

stration together with much smaller increase of the

dopamine content. The discrepancy of the restoration

of TH protein and dopamine levels after MPTP-

caused injury has been noted by other authors and has

been explained by the sensitivity of the methods [43].

TH protein restoration, however, does not necessarily

means that the neurotransmitters are already pro-

duced. The fast return of TH protein to the control

level indicated, that some striatal neurons were only

injured and did not died following MPTP treatment.

Apart from the inflammatory reaction, a large part

of the process of neurodegeneration in PD is attrib-

uted to insoluble inclusion bodies of a-synuclein,

which cause dysfunction of DA neurons in the SN and

diminish DA content in the striatum. According to

some hypotheses, a-synuclein oligomers trigger syn-

apse damage and inhibit the release of neurotransmit-

ter before any injury to the cell occurs, which could

explain the early symptoms of PD. However, the

physiological function of a-synuclein has not yet to

be fully characterized. Recent evidence suggests that

a-synuclein is also a constitutive, pre-synaptic protein

that regulates synaptic vesicle formation and neuro-

transmitter release (including DA) [7, 8, 10, 21, 30].

a-Synuclein may also cause the activation of adaptive

compensatory mechanisms that protect neurons [9].

During pathological processes, a-synuclein has the

ability to bind free radicals, such as free iron or iron-

containing radicals and oxidized dopamine, thus pre-

venting their damaging actions [38].

Following MPTP administration in our animal

model, a-synuclein level in the striatum was de-

creased and did not return to the control level as TH

protein did on the 21st dpa. This phenomenon indi-

cated that the level of a-synuclein corresponded to the

injury to dopaminergic neuron terminals in the stria-

tum, which confirmed its function as a synaptic pro-

tein. It also showed that presynaptic endings might

suffer from a long-term or even permanent injury af-

ter MPTP administration. Ibuprofen treatment pre-

vented TH protein depletion, which could suggest that

the majority of neuronal endings in the striatum were

not damaged by MPTP treatment in these groups of

mice. The decrease in a-synuclein level on the 7th dpa

and its subsequent restoration following ibuprofen

treatment on the 21st dpa, might also confirm only

partial injury to nerve endings. In this context, ibupro-

fen attenuated the injury, giving neurons a possibility

to recover.

Other properties of ibuprofen may also be responsi-

ble for its neuroprotective ability. Apart from COX in-

hibition, ibuprofen has been shown to block cytokine

production through direct inhibition of transcription

factors, such as NF-kB [12, 14], and indirectly

through activation of a peroxisome proliferator-

activated receptor-g (PPARg) [24, 27]. Pioglitazone

and rosiglitazone, compounds that stimulate the acti-

vation of PPARg, have been shown to protect against

MPTP-induced neurodegeneration in a model of PD

induced in rhesus monkeys [40] and mice [6, 35]. Ibu-

profen and other NSAIDs decrease the level of in-

ducible nitric oxide synthase (iNOS) mRNA, which

leads to decreased production of NO [37]. Addition-

ally, it has been shown to protect dopaminergic neu-


rons from NO-induced cell death by scavenging NO

radical [4] as well as from glutamate toxicity [13].

In conclusion, ibuprofen showed a dose-dependent

protective effect against MPTP toxicity in mice. The

short-term treatment during the acute phase of injury

prevented TH protein depletion and the neurodegen-

eration of dopaminergic nerve endings in the striatum.

These effects were confirmed in our study by faster

restoration of DA content and an increase in pre-

synaptic protein level (a-synuclein) in the striatum.

The possible mechanism of the neuroprotective effect

of ibuprofen might be associated with decreased DA

turnover and COX inhibition resulting in lower reac-

tive oxygen species formation and less injury. How-

ever, further studies are needed to confirm the possi-

ble role of ibuprofen as a neuroprotective agent in the

model of neurodegenerative disease.

References:

1. Aid S, Bosetti F: Targeting cyclooxygenases -1 and -2 in

neuroinflammation: therapeutic implications. Biochimie,

2011, 93, 46–51.

2. Asanuma M, Miyazaki I: Common anti-inflammatory

drugs are potentially therapeutic for Parkinson’s disease?

Exp Neurol, 2007, 206, 172–178.

3. Asanuma M, Miyazaki I: Nonsteroidal anti-inflammatory

drugs in Parkinson’s disease: possible involvement of

quinone formation. Expert Rev Neurother, 2006, 6,

1313–1325.

4. Asanuma M, Nishibayashi-Asanuma S, Miyazaki I,

Kohno M, Ogawa N: Neuroprotective effects of non-

steroidal anti-inflammatory drugs by direct scavenging of

nitric oxide radicals. J Neurochem, 2001, 76, 1895–1904.

5. Aubin N, Curet O, Deffois A, Carter C: Aspirin and sali-

cylate protect against MPTP-induced dopamine deple-

tion in mice. J Neurochem, 1998, 71, 1635–1642.

6. Barbiero JK, Santiago RM, Lima MM, Ariza D, Morais

LH, Andreatini R, Vital MA: Acute but not chronic ad-

ministration of pioglitazone promoted behavioral and

neurochemical protective effects in the MPTP model of

Parkinson’s disease. Behav Brain Res, 2011, 216,

186–92.

7. Ben Gedalya T, Loeb V, Israeli E, Altschuler Y, Selkoe

DJ, Sharon R: a-Synuclein and polyunsaturated fatty ac-

ids promote clathrin-mediated endocytosis and synaptic

vesicle recycling. Traffic, 2009, 10, 218–234.

8. Bonini NM, Giasson BI: Snaring the function of a-syn-

uclein. Cell, 2005, 123, 359–361.

9. Cabeza-Arvelaiz Y, Fleming SM, Richter F, Masliah E,

Chesselet MF, Schiestl RH: Analysis of striatal transcrip-

tome in mice overexpressing human wild-type alpha-

synuclein supports synaptic dysfunction and suggests

mechanisms of neuroprotection for striatal neurons. Mol

Neurodegener, 2011, 13, 83–99.

10. Cabin DE, Shimazu K, Murphy D, Cole NB, Gottschalk

W, McIlwain KL, Orrison B et al.: Synaptic vesicle de-

pletion correlates with attenuated synaptic responses to

prolonged repetitive stimulation in mice lacking a-syn-

uclein. J Neurosci, 2002, 22, 8797–8807.

11. Carrasco E, Casper D, Werner P: Dopaminergic neuro-

toxicity by 6-OHDA and MPP+: differential requirement

for neuronal cyclooxygenase activity. J Neurosci Res,

2005, 81, 121–131.

12. Carta AR, Pisanu A, Carboni E: Do PPAR-gamma ago-

nists have a future in Parkinson’s disease therapy? Park-

insons Dis, 2011, 2011, 689181.

13. Casper D, Yaparpalvi U, Rempel N, Werner P: Ibuprofen

protects dopaminergic neurons against glutamate toxicity

in vitro. Neurosci Lett, 2000, 289, 201–204.

14. Chávez E, Castro-Sánchez L, Shibayama M, Tsutsumi V,

Pérez Salazar E, Moreno MG, Muriel P: Effects of acetyl

salycilic acid and ibuprofen in chronic liver damage in-

duced by CCl4. J Appl Toxicol, 2012, 32, 51–59.

15. Chen H, Jacobs E, Schwarzschild MA, McCullough ML,

Calle EE, Thun MJ, Ascherio A: Nonsteroidal antiin-

flammatory drug use and the risk for Parkinson’s disease.

Ann Neurol, 2005, 58, 963–967.

16. Chen H, Zhang SM, Hernán MA, Schwarzschild MA,

Willett WC, Colditz GA, Speizer FE, Ascherio A: Non-

steroidal anti-inflammatory drugs and the risk of Parkin-

son disease. Arch Neurol, 2003, 60, 1059–1064.

17. Chu CY, Liu YL, Chiu HC, Jee SH. Dopamine-induced

apoptosis in human melanocytes involves generation of reac-

tive oxygen species. Br J Dermatol, 2006, 154, 1071–1079.

18. Esposito E, Di Matteo V, Benigno A, Pierucci M, Cresci-

manno G, Di Giovanni G: Non-steroidal anti-inflam-

matory drugs in Parkinson’s disease. Exp Neurol, 2007,

205, 295–312.

19. Feng Z-H, Wang T-G, Li D-D, Fung P, Wilson BC, Liu

B, Ali SF et al.: Cyclooxygenase-2-deficient mice are re-

sistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-

induced damage of dopaminergic neurons in the substan-

tia nigra. Neurosci Lett, 2002, 329, 354–358.

20. Ferger B, Teismann P, Earl CD, Kuschinsky K, Oertel

WH: Salicylate protects against MPTP-induced impair-

ments in dopaminergic neurotransmission at the striatal

and nigral level in mice. Naunyn Schmiedebergs Arch

Pharmacol, 1999, 360, 256–261.

21. Fortin DL, Nemani VM, Nakamura K, Edwards RH:

The behavior of a-synuclein in neurons. Mov Disord,

2010, 25, Suppl 1, S21–26.

22. Gange JJ, Power MC: Anti-inflammatory drugs and risk

of Parkinson disease. A meta-analysis. Neurology, 2010

74, 995–1002.

23. Gao X, Chen H, Schwarzschild M, Ascherio A: Use

of ibuprofen and risk of Parkinson disease. Neurology,

2011, 76, 863–869.

24. Heneka MT, Sastre M, Dumitrescu-Ozimek L, Hanke A,

Dewachter I, Kuiperi C, O’Banion K et al.: Acute treat-

ment with the PPARg agonist pioglitazone and ibuprofen

reduces glial inflammation and Ab1-42 levels in

APPV717I transgenic mice. Brain, 2005, 128, 1442–1453.



25. Hirsch EC, Hunot S: Neuroinflammation in Parkinson’s

disease: a target for neuroprotection? Lancet Neurol,

2009, 8, 382–397.

26. Hsieh YC, Mounsey RB, Teismann P: MPP+-induced

toxicity in the presence of dopamine is mediated by

COX-2 through oxidative stress. Naunyn Schmiedebergs

Arch Pharmacol, 2011, 384, 157–167.

27. Jiang C, Ting AT, Seed B: PPAR-g agonists inhibit pro-

duction of monocyte inflamatory cytokines. Nature,

1998, 391, 82–86.

28. Kurkowska-Jastrzêbska I, Babiuch M, Joniec I,

Przyby³kowski A, Cz³onkowski A, Cz³onkowska A:

Indomethacin protects against nerodegeneration caused

by MPTP intoxication in mice. Int Immunopharmacol,

2002, 2, 1213–1218.

29. Kurkowska-Jastrzêbska I, Cz³onkowski A, Cz³onkowska

A: Ibuprofen and the mouse model of Parkinson’s dis-

ease. Ann Neurol, 2006, 59, 988–989.

30. Madine J, Hughes E, Doig AJ, Middleton DA: The ef-

fects of a-synuclein on phospholipid vesicle integrity:

a study using 31P NMR and electron microscopy. Mol

Membr Biol, 2008, 25, 518–527.

31. Masana MI, Heyes MP, Mefford IN: Indomethacin pre-

vents increased catecholamine turnover in rat brain fol-

lowing systemic endotoxin challenge. Prog Neuropsy-

chopharmacol Biol Psychiatry, 1990, 14, 609–621.

32. Milatovic D, Gupta RC, Yu Y, Zaja-Milatovic S, Aschner

M: Protective effects of antioxidants and anti-

inflammatory agents against manganese-induced oxida-

tive damage and neuronal injury. Toxicol Appl Pharma-

col, 2011, 256, 219–226.

33. Miyamoto A, Hashiguchi Y, Obi T, Ishiguro S, Nishio A:

Ibuprofen or ozagrel increases NO release and l-nitro ar-

ginine induces TXA2 release from cultured porcine basi-

lar arterial endothelial cells. Vascul Pharmacol, 2007, 46,

85–90.

34. Mohanakumar KP, Muralikrishnan D, Thomas B: Neuro-

protection sodium salicylate against 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine- induced neurotoxic-

ity. Brain Res, 2000, 864, 281–290.

35. Quinn LP, Crook B, Hows ME, Vidgeon-Hart M,

Chapman H, Upton N, Medhurst AD, Virley DJ:

The PPARg agonist pioglitazone is effective in

the MPTP mouse model of Parkinson’s disease through

inhibition of monoamine oxidase B. Br J Pharmacol,

2008, 154, 226–233.

36. Sairam K, Saravann K, Benerjee R, Mohanakumar KP:

Non-steroidal anti-inflammatory drug sodium salicylate,

but not diclofenac or celecoxib, protects against

1-methyl-4-phenyl-pyridinum-induced dopaminergic

neurotoxicity in rats. Brain Res, 2003, 966, 245–252.

37. Stratman NC, Carter DB, Sethy VH: Ibuprofen: effect

on inducible nitric oxide synthase. Brain Res Mol Brain

Res, 1997, 50, 107–12

38. Surguchov A: Synucleins: Are they two-edged swords?

J Neurosci Res, 2012, 91, 161–166.

39. Surmeier DJ, Guzman JN, Sanchez-Padilla J, Goldberg

JA: The origins of oxidant stress in Parkinson’s disease

and therapeutic strategies. Antioxid Redox Signal, 2011,

14, 1289–1301.

40. Swanson C, Joers V, Bondarenko V, Brunner K, Sim-

mons H, Ziegler T, Kemnitz J et al.: The PPAR-g agonist

pioglitazone modulates inflammation and induces neuro-

protection in parkinsonian monkeys. J Neuroinflamma-

tion, 2011, 8, 91.

41. Teismann P, Ferger B: Inhbition of the cyclooxygenase

isoenzymes COX-1 and COX-2 provide neuroprotection

in the MPTP – mouse model of Parkinson’s disease. Syn-

apse, 2001, 39, 167–174.

42. Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot

S, Vila M et al.: Cyclooxygenase-2 is instrumental in

Parkinson’s disease neurodegeneration. Proc Natl Acad

Sci USA, 2003, 100, 5473–5478.

43. Yang JL, Chen JS, Yang YF, Chen JC, Lin CH, Chang

RS, Tsao PJ et al.: Neuroprotection effects of retained

acupuncture in neurotoxin-induced Parkinson’s disease

mice. Brain Behavior Immun, 2011, 25, 1452–1459.

Received: March 1, 2012; in the revised form: March 21, 2013;accepted: April 10, 2013.


Potential neuroprotective effect of ibuprofen, insights...

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