Behavioural Brain Research 235 (2012) 263–272
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Research report
Neuroprotective effects of agmatine in mice infused with a single intranasal
administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
a a,b b c c
Filipe C. Matheus , Aderbal S. Aguiar Jr. , Adalberto A. Castro , Jardel G. Villarinho , Juliano Ferreira ,
d e f b a,
Cláudia P. Figueiredo , Roger Walz , Adair R.S. Santos , Carla I. Tasca , Rui D.S. Prediger ∗
a
Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88049-900 Florianópolis, SC, Brazil
b
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88040-900 Florianópolis, SC, Brazil
c
Departamento de Química, Universidade Federal de Santa Maria, UFSM, Santa Maria, RS 97105-900, Brazil
d
Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brazil
e
Departamento de Clínica Médica, Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazil
f
Laboratório de Neurobiologia da Dor e Inflamac¸ ão, Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC,
88040-900 Florianópolis, SC, Brazil
h i g h l i g h t s
! Agmatine increased the survival rate of aging mice infused intranasally with MPTP.
! Agmatine improved social memory and motor impairments induced by i.n. MPTP administration.
! Agmatine protected against the dopaminergic cell loss induced by i.n. MPTP administration.
! Agmatine prevented MPTP-induced decrease of hippocampal glutamate uptake in aging mice.
! Agmatine may represent a new therapeutic tool for the management of cognitive and motor symptoms of Parkinson’s disease.
a r t i c l e i n f o a b s t r a c t
Article history: We have recently demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-
Received 24 May 2012
tetrahydropyridine (MPTP) suffered impairments in olfactory, cognitive, emotional and motor functions
Received in revised form 9 August 2012
associated with time-dependent disruption of dopaminergic neurotransmission in different brain struc-
Accepted 12 August 2012
tures conceivably analogous to those observed during different stages of Parkinson’s disease (PD).
Available online 17 August 2012
Agmatine, an endogenous arginine metabolite, has been proposed as a novel neuromodulator that plays
protective roles in several models of neuronal cellular damage. In the present study we demonstrated
Keywords:
that repeated treatment with agmatine (30 mg/kg, i.p.) during 5 consecutive days increased the sur-
Agmatine
vival rate (from 40% to 80%) of 15-month-old C57BL/6 female mice infused with a single intranasal (i.n.)
Parkinson’s disease
1-Methyl-4-phenyl-1,2,3,6 administration of MPTP (1 mg/nostril), improving the general neurological status of the surviving animals.
tetrahydropyridine (MPTP) Moreover, pretreatment with agmatine was found to attenuate short-term social memory and locomo-
Intranasal tor activity impairments observed at different periods after i.n. MPTP administration. These behavioral
Aging mice benefits of exogenous agmatine administration were accompanied by a protection against the MPTP-
Non-motor symptoms
induced decrease of hippocampal glutamate uptake and loss of dopaminergic neurons in the substantia
nigra pars compacta of aging mice, without altering brain monoamine oxidase B (MAO-B) activity. These
results provide new insights in experimental models of PD, indicating that agmatine represents a poten-
tial therapeutic tool for the management of cognitive and motor symptoms of PD, together with its
neuroprotective effects. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
Parkinson’s disease (PD) is a debilitating disease primarily
characterized by the progressive loss of neuromelanin-containing
dopaminergic neurons in the substantia nigra pars compacta (SNpc)
with presence of eosinophillic, intracytoplasmic, proteinaceous
Corresponding author at: Departamento de Farmacologia, Universidade Federal
∗ inclusions termed as Lewy bodies and dystrophic Lewy neurites
de Santa Catarina, Campus Trindade, 88049-900 Florianópolis, SC, Brazil.
in surviving neurons [37]. At the time of diagnosis, patients typi-
Tel.: +55 48 3721 9491; fax: +55 48 3337 5479.
E-mail address: [email protected] (R.D.S. Prediger). cally display an array of motor impairments including bradykinesia,
0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.08.017
264 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272
resting tremor, rigidity, and postural instability. Although most of It has been well established that aging is the most prominent risk
the typical motor impairments are due to the loss of nigrostriatal factor for PD. However, preclinical studies addressing the behav-
dopaminergic neurons, PD affects multiple neuronal systems both ioral and neurochemical effects of dopaminergic neurotoxins such
centrally and peripherally, leading to a constellation of non-motor as MPTP have been widely performed in young adult animals which
symptoms including olfactory deficits, affective disorders, memory may represent one possible explanation for the limited success
impairments, as well as autonomic and digestive dysfunction [13]. on translational research in PD. Therefore, the aim of the present
These non-motor features of PD do not meaningfully respond to study was to evaluate the potential of the repeated administration
dopaminergic medication and are a challenge to the clinical man- of exogenous agmatine to prevent behavioral and neurochemical
agement of PD [13]. changes induced by a single i.n. MPTP administration in aging mice.
Numerous epidemiological and experimental studies suggest
that exposure to agricultural chemicals, viruses, metals, and other 2. Materials and methods
toxins contribute to its pathogenesis (for review see [19,56]).
2.1. Animals
In some cases such agents conceivably enter the brain via the
olfactory neuroepithelium, a concept termed the olfactory vector Experiments were conducted using 15-month-old female C57BL/6 mice weigh-
hypothesis [22,56]. In this context, we have recently proposed a ing 25–35 g purchased from the Multidisciplinary Center for Biological Research
(CEMIB, São Paulo, Brazil). The animals were kept in collective cages (5 animals per
new experimental model of PD consisting of a single intranasal
cage) and maintained in a room under controlled temperature (23 1 ◦C) and 12 h
(i.n.) administration of the proneurotoxin 1-methyl-4-phenyl- ±
light cycle (lights on 7:00 AM), with free access to food and water. The animals were
1,2,3,6-tetrahydropyridine (MPTP) in rodents [12,50,57–59]. Young
treated, manipulated and euthanized according to the “Principles of Laboratory Ani-
adult Wistar rats and C57BL/6 mice (3–6-months-old) treated mal Care” (NIH publication no. 80-23, revised 1996) and approved by the Committee
intranasally with MPTP suffer impairments in olfactory, cognitive, on the Ethics of Animal Experiments of the Federal University of Santa Catarina
(CEUA/UFSC; www.ceua.ufsc.br; protocol 23080.019002/2009-71). All efforts were
emotional and motor functions conceivably analogous to those
made to minimize the number of animals used and their suffering.
observed during different stages of PD. Such infusion causes time-
dependent loss of tyrosine hydroxylase (TH) in the olfactory bulb
2.2. Drugs and treatment
and SNpc, resulting in significant dopamine depletion in differ-
ent brain areas [57–59]. We have also identified some pathogenic The animals were allocated to the following groups: (i) control + control (n = 13),
mechanisms possibly involved in the neurodegeneration induced (ii) control + MPTP (n = 20), (iii) agmatine + control (n = 14) or (iv) agmatine + MPTP
(n = 17). The variation in animal’s body weights was considered and counterbal-
by i.n. administration of MPTP including mitochondrial dysfunc-
anced across the four groups. Agmatine sulfate (Sigma Chemicals Co., USA) was
tion, oxidative stress, activation of apoptotic cell death mechanisms
freshly dissolved in 0.9% NaCl (saline) to a final concentration of 3 mg/ml before
and glutamatergic excitotoxicity (for review see [57]). Therefore, each daily treatment. Animals were administered by i.p. route once daily for 5 con-
the i.n. MPTP administration seems to represent a valuable rodent secutive days with control solution (saline) or agmatine (30 mg/kg) in a volume of
0.1 ml/10 g of body weight. The dose of agmatine utilized was chosen based on previ-
model for testing novel drugs for both motor and non-motor symp-
ous studies conducted in our laboratory [28,54,64]. One hour after the last injection
toms relief as well as the discovery of compounds to modify the
of agmatine, the animals were infused intranasally with a single bilateral dose of
course of PD.
MPTP (1 mg/nostril) or control solution (saline) (Fig. 1).
On the other hand, there is increasing evidence that alter- MPTP HCl (Sigma Chemicals Co., USA) was administered by i.n. route according
ations in glutamatergic neurotransmission have a pivotal role in to the procedure previously described [21] and modified in our laboratory [58].
Briefly, mice were lightly anaesthetized with isoflurane 0.96% (0.75 CAM; Abbot
the pathophysiology of PD [10,63]. For instance, dopaminergic
Laboratórios do Brasil Ltda., RJ, Brazil) using a vaporizer system (SurgiVet Inc., WI,
neurons in the SNpc receive moderate excitatory glutamater-
USA) and a 7 mm piece of PE-10 tubing was inserted through the nostrils. The tubing
gic neurons input from the subthalamic nucleus (STN) [33]. It is was connected to a peristaltic pump set at a flow rate of 12.5 l/min. The MPTP HCl
postulated that overstimulation of glutamate receptors on nigral was dissolved in saline at a concentration of 20 mg/ml, after which it was infused for
4 min (1 mg/nostril). The control solution consisted of saline. Animals were given a
dopaminergic neurons may be involved in the neuronal degenera-
1 min interval to regain normal respiratory function and then this procedure was
tion and progression of PD [10,63]. Moreover, it was demonstrated
repeated with infusions administered through the contralateral nostrils.
a pronounced increase in the extracellular levels of glutamate
in the substantia nigra of mice treated chronically with MPTP
2.3. Survival analysis
[48]. Indeed, recent studies have demonstrated that d-cycloserine,
a partial agonist of the glycine binding site of the N-methyl-d- The animals’ survival rate in each group was assessed throughout the experi-
mental protocol period. No death was observed within the 5 days of repeated i.p.
aspartate (NMDA) receptor, improves mnemonic impairments and
treatment with agmatine or control (data not shown). The number of deaths was
anxiety-like behaviors observed in MPTP-lesioned monkeys [65]
monitoring until the 21st day after the i.n. MPTP administration for later assembly
and rats [38,69]. Finally, there are promising findings from clinical
of the survival curve.
[1,23] and preclinical studies of the efficacy of the NMDA recep-
tor antagonist memantine for the treatment of PD [43]. Thus, drugs 2.4. Behavioral tests
modulating the function of glutamate NMDA receptors may have
During a period of 3–19 days after the i.n. administration of MPTP, the animals
beneficial effects in PD therapy.
were submitted to a battery of behavioral paradigms that included the activity cham-
In this context emerges agmatine, a polyamine that is synthe-
ber, social recognition, neurological severity score and open-field tasks (Fig. 1). The
sized after decarboxylation of l-arginine by arginine decarboxylase
time point for the performance of each behavioral task was chosen based on previ-
and it is hydrolyzed to putrescine by enzyme agmatinase and that ous studies using the i.n. MPTP model [12,50,57]. All tests were carried out between
has been recently proposed as neuromodulator [34,61]. In the brain, 9:00 and 14:00 h and they were scored by the same rater in an observation room
where the mice had been habituated for at least 1 h before the beginning of the tests.
agmatine is stored in synaptic vesicles [34] and is released by
2+ Behavior was monitored through a video camera positioned above the apparatuses
Ca -dependent depolarization [61] with multiple molecular tar-
and the images were later analyzed with the ANY Maze video tracking (Stoelting
gets proposed, including the binding and blockage of glutamate Co., Wood Dale, IL, USA) by an experienced experimenter who was unaware of the
NMDA receptors [70]. Age-related changes in agmatine levels in experimental group of the animals tested.
various brain structures have been described indicating potential
involvement of agmatine in aging process [45]. Of high impor- 2.4.1. Activity chambers
In order to assess early effects of MPTP on locomotor activity, the animals were
tance, recent studies have demonstrated the protective effects
tested 3 days after i.n. MPTP administration in activity chambers. The chambers were
of exogenous agmatine administration in animal models of neu-
made of black fiberglass (50 cm 25 cm 15 cm) and the experiments were per-
× ×
rodegenerative disorders such as PD [15,16,30] and Alzheimer’s formed in a sound-attenuated room under low-intensity light (12 lux). Each mouse
disease [7]. was placed in the center of the apparatus and the total distance traveled (m) and
F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272 265
Fig. 1. Time course of behavioral and neurochemical tests following the pretreatment (during 5 consecutive days) with control (saline) or agmatine (30 mg/kg, i.p.) and a
single intranasal (i.n.) administration of control (saline) or MPTP (1 mg/nostril) in 15-month-old female C57BL/6 mice.
the average speed (m/s) were registered during 5 min. Chambers were cleaned with Technology, USA) as described previously [58]. Following quenching of endogenous
10% ethanol between animals. peroxidase with 3% hydrogen peroxide in methanol for 20 min, high tempera-
ture antigen retrieval was performed by immersion of the slides in a water bath
2.4.2. Social recognition at 95–98 ◦C in 10 mM trisodium citrate buffer pH 6.0, for 45 min. After overnight
Short-term social memory was assessed 7 days after i.n. MPTP administration incubation at 4 ◦C with primary antibodies, the slides were washed with PBS and
with the social recognition task previously evaluated in our laboratory [58]. Juve- incubated with the appropriate biotinylated secondary antibody, and then pro-
nile female C57BL/6 mice (25–30 days old) served as social stimuli for the adult cessed using the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA,
mice in the social recognition task. The adult animals were isolated in individual USA) according to the manufacturer’s instructions. The sections were washed in PBS,
cages during seven days before the task. All juveniles were isolated in individual and the visualization was completed by using 3,3-diaminobenzidine (DAB) (Dako
cages for 20 min prior to the beginning of the experiment. The social recognition Cytomation) in chromogen solution and counterstained with Harris’s hematoxylin.
task consisted of two successive presentations (5 min each), separated by an inter- Tissues from the four groups were placed on the same slide and processed under the
trial interval of 30 min, where the juvenile was placed in the home cage of the adult same conditions. We included negative control that consisted in replace the primary
mouse and the time spent by the adult in investigating the juvenile (nosing, sniffing, antibody by nonimmune serum in equivalent concentration.
grooming, or pawing) was recorded in the two presentations. Time spent in social The number of TH-stained positive cells in the SNpc was assessed at three levels
investigation by the adult mouse was measured and then expressed for each ani- between coordinates 2.75 mm and 2.92 mm with respect to the bregma. Three
− −
mal as the ratio of the second exposure to the first exposure (Ratio of Investigation alternate 4 m paraffin sections with an individual distance of 60 m of each sec-
≈
Duration (RID)). A reduction in RID reflects a decrease in investigation behavior dur- tion were obtained, and the number of TH-stained positive cells was determined
ing the second encounter, demonstrating the recognition ability of the adult mouse. upon visual inspection at the SNpc with optical microscope (Eclipse 50I; Nikon,
This transformation was chosen in order to minimize day-to-day variations on the Melville, NY) by using a counting grid at 400 magnification. Results were expressed
× 2
baseline of performance and to equalize variances among different groups [12]. as mean number of TH-stained positive cells per mm from three tissue sections.
3
2.4.3. Neurological severity score 2.6. l-[ H]glutamate uptake
Thirteen days after i.n. administration of MPTP, mice performed the 10-point
neurological severity score (NSS), a composite behavioral scale designed to measure The glutamate uptake assay was evaluated as previously described [55]. The
the general neurological state previously described [25] and recently evaluated in mice were decapitated 21 days after i.n. MPTP administration and the hippocampi
our laboratory [66]. Mice were assessed for the following items: presence of paresis, were quickly removed and stored in KRB (in mM = 122 NaCl, 3 KCl, 1.3 CaCl2, 1.2
inability to walk straight, impairment of seeking behavior, absence of perceptible MgSO4, 0.4 KH2PO4, 25 NaHCO3 and 10 d-glucose) previously aerated with carbogen
startle reflex, inability to exit a 30 cm diameter circle, inability to walk on 3, 2, and (95% O2–5% CO2) to reach pH 7.4. The tissue sections (400 m thick) were obtained
1 cm wide beams, and inability to balance on a 0.7 cm-wide beam and a 0.5 cm- using a tissue slicer (Mcilwain Tissue Chopper, Australia). On average, five slices from
diameter round beam for at least 10 s. If mice showed the impairment described by the middle of the hippocampus were obtained and placed in a 96-well multiwell
an item, a value of 1 was added to total NSS score. Higher scores in the NSS indicate plate containing KRB, pH 7.4. The slices were incubated for 1 h before the gluta-
greater neurological impairment [66]. mate uptake assays were performed. After incubation, the hippocampal slices were
washed for 15 min at 37 ◦C in a Hank’s balanced salt solution (HBSS), composition in
2.4.4. Open field mM: 1.29 CaCl2, 136.9 NaCl, 5.36 KCl, 0.65 MgSO4, 0.27 Na2HPO4, 1.1 KH2PO4, and 5
3
The spontaneous locomotor activity of the animals was evaluated in an open- HEPES. Uptake was assessed using 0.33 Ci/ml l-[ H]glutamate with 100 m unla-
field arena at 19 days after i.n. MPTP administration. The apparatus, made of wood beled glutamate in a final volume of 300 l. Incubation was immediately stopped
covered with impermeable Formica, had a black floor of 50 cm 50 cm (divided after 7 min by discarding the incubation medium and the slices were submitted to
×
by white lines into 9 squares of equal size) and transparent walls, 40 cm high. Each two ice-cold washes with 1 ml of HBSS. The slices were solubilized by adding a solu-
mouse was placed in the center of the open field and the numbers of squares crossed tion with 0.1% NaOH/0.01% SDS and incubated overnight. Aliquots of slice lysates
3
and rearing were registered during 5 min. The apparatus was cleaned with ethanol were taken for determination of the intracellular content of l-[ H]glutamate by scin-
solution (10% v/v) and dried with paper towels after each trial in order to avoid odor tillation counting. Sodium-independent uptake was determined by using choline
impregnation. chloride instead of sodium chloride in the HBSS. Unspecific sodium-independent
uptake was subtracted from total uptake to obtain the specific sodium-dependent
3
glutamate uptake. Results were expressed as nmol of l-[ H]glutamate taken up per
2.5. Immunohistochemistry for tyrosine hydroxylase (TH)
milligram of protein per minute.
For the investigation of possible neuroprotective effects of agmatine against the
loss of dopaminergic neurons induced by i.n. MPTP administration, five animals of 2.7. Monoamine oxidase (MAO) assay
each group were intracardially perfused with 4% paraformaldehyde in physiologi-
cal saline (NaCl 0.9%) at 21 days after MPTP treatment. Brains were collected and To investigate whether the treatment with agmatine may interfere with the gen-
+
fixed in a phosphate buffered saline (PBS) solution containing 4% paraformalde- eration of the toxic metabolite 1-methyl-4-phenylpyridinium (MPP ) from MPTP,
hyde for 24 h at room temperature, dehydrated by graded ethanol, and embedded agmatine was tested for its in vitro inhibitory potential on mouse MAO-A and MAO-
in paraffin. Immunoreactivity of TH-positive neurons in the substantia nigra pars B activities in brain mitochondrial homogenates by a fluorometric method using
compacta (SNpc) was assessed on paraffin tissue sections (4 m), using the anti-TH kynuramine as substrate, as previously described [46]. Brain (all regions without
monoclonal antibody (1:300, catalog MAB318, Millipore/Chemicon International, cerebellum) mitochondria from 15-month-old female C57BL/6 mice were isolated
266 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272
Fig. 2. Effects of the pretreatment (during 5 consecutive days) with control (i.p.)
or agmatine (30 mg/kg, i.p.) on survival rate of 15-month-old female C57BL/6 mice
infused intranasally with control or MPTP (1 mg/nostril). The lines represent the
Fig. 3. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or
percentage survival animals of each group over the course of 21 days after the
agmatine (30 mg/kg, i.p.) on the social recognition memory of 15-month-old female
i.n. MPTP administration. * P < 0.05 compared to the percentage of survival of the
C57BL/6 mice evaluated in the social recognition task 7 days after i.n. infusion of
control/control group. # P < 0.05 compared to the percentage of survival of the
MPTP (1 mg/nostril). Data are expressed as the mean S.E.M. of RIDs (i.e. the ratio
agmatine/MPTP group (Breslow–Gehan–Wilcoxon test). ±
of the second exposure to the first exposure) when the same juvenile was exposed
for 5 min with an interval of 30 min [control/control (n = 10); control/MPTP (n = 9);
according to the method of Naoi et al. [51]. The obtained mitochondrial pellet was agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P 0.05 compared to the
≤
suspended in 10 mM sodium phosphate buffer (pH 7.4) to 100–300 mg/mL and control/control group. #P 0.05 compared to the control/MPTP group (two-way
≤
then was used for assay. Briefly, assays were performed in duplicate in a final ANOVA followed by Newman–Keuls test).
volume of 500 l containing 0.5 mg of protein and incubated at 37 ◦C for 30 min.
Activities of the MAO-A and MAO-B isoforms were isolated pharmacologically by
P 0.001] and their interaction [F1,33 = 8,44, P 0.01] in the ratio of
incorporating 250 nM selegiline (selective MAO-B inhibitor) or 250 nM clorgyline ≤ ≤
investigation duration (RID).
(selective MAO-A inhibitor) into the reaction mixture. The reaction mixture (con-
taining mitochondrial fractions, agmatine and inhibitors) was pre-incubated at 37 ◦C Post hoc comparisons indicated that the i.n. MPTP treatment
for 5 min and the reaction was started by addition of 50 l of kynuramine (90 m promoted a significant increase in the RID when the same juvenile
for MAO-A and 60 m for MAO-B). Agmatine was tested in a concentration range
was re-exposed 30 min after the first encounter, indicating a dis-
of 0.01–1000 m and the results were expressed as percentage of control (tube
ruption in the short-term social recognition ability of aging mice
without agmatine).
caused by i.n. MPTP infusion. Repeated treatment with agmatine
2.8. Statistical analysis prevented the deficit in social recognition ability induced by i.n.
MPTP administration, causing a significant reduction in the RID
Data for Neurological Severity Score are shown as median (interquartile
when the familiar juvenile was re-exposed after 30 min (Fig. 3).
range) and comparisons between groups were performed by Kruskal–Wallis non-
parametric test followed by Dunn’s multiple comparison tests. Statistical analysis
of survival curves were performed with the Cox–Mantel test (log-rank) followed 3.3. Effects of agmatine on the general neurological state of aging
by Gehan–Breslow–Wilcoxon test. The rest of data was checked for normality
mice infused intranasally with MPTP
of frequency distribution with the Kolmogorov–Smirnov test and expressed as
mean standard error of mean (S.E.M.). In this case, Student’s t-test and analysis of
± The general neurological state of the animals was evaluated
variance (ANOVA) were applied when appropriate, as informed in the results section
and figure legends. Following significant ANOVAs, multiple post hoc comparisons by the NSS at 13 days after i.n. MPTP administration. As illus-
were performed using the Newman–Keuls test. The accepted level of significance trated in Fig. 4, Kruskal–Wallis non-parametric test followed by
®
for all tests was P 0.05. All tests were performed using the Statistica software
≤ Dunn’s post hoc tests indicated that mice from the control/MPTP
package (Stat Soft Inc., USA).
group scored significantly higher [P 0.05] than the control/control
≤
group. Of high importance, this increase in NSS score induced by
3. Results
i.n. MPTP administration was prevented by the pretreatment with
3.1. Effects of agmatine on the survival rate of aging mice infused
intranasally with MPTP
As can be seen in Fig. 2, log-rank Mantel–Cox test followed
by Gehan–Breslow–Wilcoxon test indicated a lower percentage of
surviving animals in the control/MPTP group when compared to
the control/MPTP group (treatment factor: P 0.05). The statistical
≤
analysis performed point-by-point indicated an increased mortal-
ity (about 50%) of MPTP-infused mice from the 1st to the 13th day
after treatment. Remarkably, repeated treatment with agmatine
(30 mg/kg, i.p.) during 5 consecutive days increased significantly
the survival rate of MPTP-treated aging mice to about 75% (Fig. 2)
(interaction factor: P 0.05).
≤
3.2. Effects of agmatine on the social recognition memory of
Fig. 4. Effects of the pretreatment (during 5 consecutive days) with control
aging mice infused intranasally with MPTP
(i.p.) or agmatine (30 mg/kg, i.p.) on the general neurological state of 15-month-
old female C57BL/6 mice evaluated in the Neurological Severity Score 13 days
The results for the effects of i.n. administration of MPTP after i.n. infusion of control or MPTP (1 mg/nostril). The results are shown as
median (interquartile ranges) of NSS points [control/control (n = 9); control/MPTP
(1 mg/nostril) on the short-term social recognition memory of
(n = 8); agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P 0.05 compared
aging mice pretreated with control or agmatine (30 mg/kg) are ≤
to the control/control group. #P 0.05 compared to the control/MPTP group
≤
illustrated in Fig. 3. Two-way ANOVA revealed significant effects for
(Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparison
the pretreatment [F = 10.32, P 0.01], treatment [F = 20.72; tests). 1,33 ≤ 1,33
F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272 267
Fig. 5. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or agmatine (30 mg/kg, i.p.) on the spontaneous locomotor activity of 15-month-old female
C57BL/6 mice evaluated during 5 min in the activity chambers and open field at 3 and 19 days, respectively, after i.n. infusion of control or MPTP (1 mg/nostril). Data are
expressed as the mean S.E.M. of the total distance traveled (A) and average speed (B) in the activity chambers [control/control (n = 10); control/MPTP (n = 9); agmatine/control
±
(n = 10); and agmatine/MPTP (n = 9)]; and number of crossings (C) and rearing (D) in the open field [control/control (n = 9); control/MPTP (n = 7); agmatine/control (n = 10);
and agmatine/MPTP (n = 9)]. *P 0.05 compared to the control/control group. #P 0.05 compared to the control/MPTP group (two-way ANOVA followed by Newman–Keuls ≤ ≤
test).
agmatine (30 mg/kg, i.p.), suggesting a protective effect of agmatine rearings induced by i.n. MPTP administration was prevented by the
against the neurological impairments induced by MPTP in aging pretreatment with agmatine (Fig. 5D). Therefore, the locomotor
mice (Fig. 4). activity of aging mice was only disrupted at later periods after i.n.
MPTP administration which was prevented by pretreatment with
3.4. Effects of agmatine on the spontaneous locomotor activity of agmatine (30 mg/kg, i.p.).
aging mice infused intranasally with MPTP
The results of locomotor activity of aging mice evaluated for 3.5. Agmatine prevents the loss of dopaminergic neurons induced
5 min in the activity chambers and open field arena at 3 and 19 days, by i.n. MPTP administration in aging mice
respectively, after i.n. MPTP (1 mg/nostril) administration are sum-
marized in Fig. 5. Two-way ANOVA revealed no significant effects With the purpose of determining the relationship between the
for the main factors and their interaction in the total distance trav- motor impairments observed in the open field at later periods after
eled [pretreatment: F1,33 = 1.21; P = 0.28; treatment: F1,33 = 0.45; i.n. MPTP administration in aging mice and dopaminergic cell death
P = 0.48; interaction: F1,33 = 0.28; P = 0.60] (Fig. 5A) and the average in the nigrostriatal pathway, the evaluation for TH-positive cells in
speed [pretreatment: F1,33 = 1.31; P = 0.29; treatment: F1,33 = 0.28; the substantia nigra was performed 21 days after i.n. administration
P = 0.60; interaction: F1,33 = 0.32; P = 0.60] (Fig. 5B) evaluated in the of MPTP by immunohistochemistry. Fig. 6A–D shows representa-
activity chambers. tive photomicrographs of TH immunohistochemistry in the ventral
On the other hand, two-way ANOVA revealed significant effects mesencephalon containing SNpc.
for the treatment factor [F = 5.86; P 0.05] and the interaction As can be seen in Fig. 6E, two-way ANOVA revealed signif-
1,29 ≤
between pretreatment and treatment [F = 4.26; P 0.05], but not icant effects for the treatment factor [F1,16 = 8.73; P 0.01] and
1,29 ≤ ≤
for the pretreatment factor [F1,29 = 1.26; P = 0.27], in the number the interaction between pretreatment and treatment [F1,16 = 4.52;
P 0.05], but not for the pretreatment factor [F = 0.52; P = 0.48],
of squares crossed in the open field. Post hoc comparisons indi- ≤ 1,16
cated a significant reduction in the number of crossings in the in the number of TH-positive cells in the SNpc. Subsequent
MPTP/control group that was not observed in the agmatine/MPTP Newman–Keuls tests showed that the i.n. administration of MPTP
group (Fig. 5C). induced a pronounced reduction (about 50%) of TH immunostain-
Regarding the number of rearings, two-way ANOVA revealed ing in the SNpc of aging mice when compared to the control/control
F group (P 0.05). Of high importance, the pretreatment with agma-
significant effects for the main factors [pretreatment: 1,29 = 4.23; ≤
P 0.05; treatment: F = 6.70; P 0.05] and their inter- tine (30 mg/kg, i.p.) was able to attenuate the loss of TH-positive
≤ 1,29 ≤
action [F = 4,23; P 0.05] in this parameter. Subsequent neurons in the SNpc of MPTP-treated mice when compared to con-
1,29 ≤
Newman–Keuls test indicated that the reduction in the number of trol/control group (Fig. 6E).
268 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272
Fig. 6. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or agmatine (30 mg/kg, i.p.) on tyrosine hydroxylase (TH)-positive cells in the substantia
nigra pars compacta (SNpc) of 15-month-old female C57BL/6 mice evaluated through immunohistochemistry at 21 days after i.n. infusion of control or MPTP (1 mg/nostril).
(A–D) Representative images of TH immunostaining in the ventral mesencephalon containing SNpc of animals (Scale bar = 200 m). (E) Relative quantification of the number
2
of TH-positive neurons per mm in SNpc of mice. Values represent the mean SEM (n = 5 animals per group). *P 0.05 compared to the control/control group (two-way
± ≤
ANOVA followed by Newman–Keuls test).
3.6. Agmatine prevents decreased hippocampal glutamate uptake uptake was measured in the hippocampus of aging mice 21 days
induced by i.n. MPTP administration in aging mice after i.n. MPTP administration.
As illustrated in Fig. 7, the i.n. MPTP administration sig-
Glutamate clearance from extracellular space is an important nificantly decrease [F = 5.16; P 0.05] the glutamate uptake
1,14 ≤
mechanism related to the reduction of glutamate excitotoxicity. into the hippocampus of aging mice. The pretreatment with
With the purpose of determining possible alterations in gluta- agmatine did not alter per se the basal hippocampal glutamate
matergic neurotransmission following MPTP treatment, glutamate uptake [F1,14 = 2.62; P = 0.70], but it prevented the MPTP-induced
F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272 269
the CNS including the dopaminergic neurodegeneration induced by
PD-mimetic toxins such as MPTP [30] and rotenone [15,16].
The current data corroborates the neuroprotective potential
of agmatine in PD since it attenuated the dopaminergic cell
loss in the SNpc of aging mice infused intranasally with MPTP
(1 mg/nostril). Moreover, the present study provides the first
preclinical data demonstrating that repeated treatment with agma-
tine (30 mg/kg, i.p.) improves short-term memory and motor
impairments displayed by MPTP-treated aging mice. Finally, the
observed behavioral benefits of exogenous agmatine treatment
were accompanied by the prevention of MPTP-induced decrease
of hippocampal glutamate uptake.
Recent studies performed by our group and others with the
administration of exogenous agmatine in laboratory animals have
Fig. 7. Effects of the pretreatment (during 5 consecutive days) with control (i.p.)
3 identified several relevant functions of this substance that are of
or agmatine (30 mg/kg, i.p.) on hippocampal l-[ H]glutamate uptake in 15-month-
old female C57BL/6 mice infused intranasally with control or MPTP (1 mg/nostril). At potential therapeutic importance, including anticonvulsant [17],
the 21th day after i.n. MPTP administration, mice were sacrificed and the hippocam- antinociceptive [28,54,64], anxiolytic [32] and antidepressant-like
pal slices processed for glutamate uptake assay in vitro as described in Section 2.
[73,74] actions. Moreover, age-related changes in agmatine levels
Values are expressed as mean SEM [control/control (n = 4); control/MPTP (n = 3);
± in various brain structures have been demonstrated, thus indicating
agmatine/control (n = 5); and agmatine/MPTP (n = 4)]. P 0.05 compared to the con-
≤ the potential involvement of agmatine in aging process [45].
trol/control group (two-way ANOVA followed by Newman–Keuls test).
In the present study, the repeated treatment with agmatine
(30 mg/kg, i.p.) during 5 consecutive days was able to attenuate
reduction of glutamate uptake [interaction factor: F1,14 = 4.88; significantly the mortality of MPTP-treated aging mice. The acute
P 0.05] (Fig. 7). toxicity effects of MPTP are largely attributed to peripheral mecha-
≤ +
nisms [40]. For instance, MPTP and its toxic metabolite MPP have
been shown to have a variety of peripheral effects including cardiac
3.7. Effects of agmatine on the monoamine oxidase activity
noradrenaline depletion [2], adrenal noradrenaline and dopamine
release [2], hypothermia [27] and neuromuscular blockade via
As can be seen in Fig. 8, Student’s t-tests indicated that the
binding to nicotinic acetylcholine receptors [39]. It must be con-
current tested concentrations of agmatine (0.01–1000 m) had
ceded that, at present stage, it not possible to determine the exact
no significant effect on either MAO-A [t = 1.30, P = 0.25] or MAO-
site of action and molecular mechanisms underlying agmatine
B [t = 0.96, P = 0.38] activities in the mouse brain mitochondrial
homogenates. attenuated the mortality of MPTP-infused aging mice. Neverthe-
less, based on previous literature demonstrating that agmatine
inhibits sympathetically-induced tachycardic responses [14] and
4. Discussion
noradrealine release [60], a speculative hypothesis is that agmatine
may prevent the alterations in the cardiovascular system verified
Therapeutic strategies that slow or stop the neurodegenerative
following MPTP administration.
process of PD are expected to have a major impact on the treatment
Regarding PD symptoms, an increasing number of studies have
of this disease [47]. The current hypothesis about the mechanisms
demonstrated that PD seems to be a multidimensional disease, and
by which neurons come into necrotic or apoptotic process of degen-
besides motor deficits, it is associated with a number of senso-
eration has led to belief that the use of drugs modulating the
rial, cognitive and emotional disturbances that result in a loss in
function of glutamate NMDA receptors may have beneficial effects
quality of life of the individuals [13]. In this context, in a recent
in PD therapy [10,63]. In this context, there is increasing evidence
series of studies we demonstrated that a single i.n. infusion of MPTP
of the neuprotective effects of agmatine, which among other possi-
in rodents produces diverse signs of PD such as impairments in
ble targets blockades NMDA receptors, against different insults of
olfactory, cognitive, emotional and motor functions [12,50,57–59].
Moreover, the i.n. MPTP administration seems to affect the rodents’
brain in a region- and time-dependent manner (for review see
[57]). For instance, we have observed increased susceptibility of
olfactory bulb to i.n. MPTP toxicity, with a marked reduction in
TH-positive neurons and dopamine depletion occurring 24 h after
i.n. MPTP infusion. On the other hand, these alterations were only
observed later (14–21 days after intransal MPTP administration) in
the SNpc and striatum. Therefore, the existence of compensatory
mechanisms, such as the increase in the number of TH-positive
striatal cells and a downregulation of dopamine uptake in surviv-
ing dopaminergic fibers in the striatum [8], may be responsible for
the lack of striatal changes until 14 days post-MPTP. Consistent
with these observations, the current findings indicated no signifi-
cant alterations in the total distance traveled and the average speed
by aging mice in the activity chambers at 3 days after i.n. MPTP
administration.
Since the i.n. administration of MPTP does not cause, at least at
initial periods, gross motor alterations that would preclude assess-
Fig. 8. Effects of agmatine on mouse brain mitochondrial MAO-A and MAO-B activ-
ment of cognitive functions, we investigated the impact of i.n.
ities in vitro. Agmatine was tested in a concentration range of 0.01–1000 m. The
MPTP administration on social recognition memory of aging mice.
values represent the mean S.E.M. of three individuals experiments, performed in
±
duplicate. Aging mice infused with MPTP (1 mg/nostril) spent significantly
270 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272
more time investigating the juvenile during the second presenta- preventing the locomotor impairments in the open field and the
tion than they did in the first encounter, suggesting an impaired decrease of the TH immunoreactivity in the SNpc induced by i.n.
ability to recognize the juvenile mouse after a short time. Previ- infusion of MPTP in aging mice. These results corroborate recent
ous studies from our group have demonstrated that rodents treated findings on agmatine neuroprotection in cellular models of PD-like
intranasally with MPTP performed normally in the long-term reten- neurodegeneration [15,16]. Moreover, Gilad et al. [30] published
tion session (24 h after training) of the inhibitory avoidance task a pioneer study demonstrating that the treatment with agmatine
[12,58] and in the spatial reference memory version of the water (100 mg/kg, i.p.) during 5 days attenuated MPTP-induced reduction
maze [59]. In contrast, MPTP-infused animals displayed a poor per- of synaptosomal dopamine uptake when administered 8 h after
formance in the short-term retention session (1.5 h after training) MPTP injection (40 mg/kg, i.p., for 2 days). Taken together, these
of the inhibitory avoidance task [12,58] as well as in the working results suggest that agmatine may represent a potential disease-
memory version of the water maze [59]. These findings are consis- modifying therapy for PD.
tent with the view of human studies suggesting that PD patients It is well known that MAO-B inhibition reduces the generation
+
present cognitive deficits mainly in working memory and short- of the MPP from MPTP, protecting against the dopaminergic cell
term memory tasks with long-term spatial (declarative) memories death in the SNpc [36]. In accordance with previous literature [30],
mostly spared [11]. we observed that agmatine does not interfere with MAO-B activity
Of high importance, the present findings demonstrate that the on mouse brain mitochondrial homogenates, indicating that MAO-
+
pretreatment with agmatine (30 mg/kg, i.p.) during 5 consecutive B-catalyzed conversion of MPTP to MPP is not affected. However,
+
days was able to prevent the short-term social memory deficits the evaluation of the time-course of MPP kinetic in the mouse
of MPTP-treated aging mice. Therefore, from these limited results brain after i.n. MPTP administration constitutes a very interesting
it appears that agmatine might be particularly useful to restore field that requires additional research.
memory processes in PD. The current results are in accord with The neuroprotective effects of agmatine may result from dif-
previous findings indicating the cognitive-enhancing properties of ferent mechanisms including blocking of NMDA receptors [70],
agmatine in diverse animal models of brain injury. For instance, the inhibition of nitric oxide synthase (NOS) [29], oxygen radical
repeated administration of agmatine (5–40 mg/kg, i.p.) prevented scavenging [6] and protection against mitochondrial membrane
learning and memory impairments in rodents induced by infusion potential collapse [4–6]. However, the sequence of events lead-
of aggregated beta-amyloid(25-35) peptide [7], lipopolysaccha- ing to the protective effects of agmatine against cell damage has
ride [71], streptozotocin [9] and scopolamine [49]. Moreover, not been fully elucidated. Here we observed that agmatine was
recent studies [44,45,67] have demonstrated that spatial learning able to prevent the decrease of hippocampal glutamate uptake in
in rodents induces elevation in agmatine levels at synapses in the aging mice following i.n. MPTP administration. Corroborating our
rat hippocampus, providing further evidence of its participation in findings, previous studies [18,35] have demonstrated that MPTP
learning and memory processes. decreases glutamate uptake by astrocytes in cell culture. Therefore,
Additionally, in the present study we observed a later (19 days one possible mechanism by which agmatine may exert protective
after i.n. MPTP administration) reduction in the locomotor activ- effects against MPTP neurotoxicity may be due to the modulation of
ity of MPTP-treated 15-month-old female C57BL/6 mice evaluated glutamate reuptake into neural cells, the main mechanism respon-
in the open field that was accompanied by a marked reduction sible for decreasing extracellular glutamate levels, thus attenuating
(about 50%) of TH-positive neurons in the SNpc. These findings glutamate neurotoxicity.
contrast with our previous study demonstrating that young adult
(6-months-old) male C57BL/6 mice infused intranasally with the
same dose of MPTP do not present gross motor alterations [58]. 5. Conclusions
Therefore, the current data indicates that the age and gender of
the animals represent important factors that modulate the appear- The present findings reinforce the i.n. MPTP administration as a
ance of motor symptoms in the i.n. MPTP model of PD. Reinforcing valuable rodent model for testing novel palliative and neuroprotec-
the current findings, previous studies have demonstrated that tive compounds for PD and demonstrate that the age of the animals
dopamine-depleting effects and motor impairments induced by represent an important factor that modulate the appearance of
i.p. MPTP administration in mice are age- [41,52] and gender- motor symptoms in this model. More importantly, the present
dependent [3,26,41,53,68]. study provides the first preclinical data indicating that repeated
Epidemiological studies have shown a prevalence of PD in men systemic treatment with agmatine prevents short-term memory
compared to women [20]. In women, the age at onset of PD cor- and motor impairments as well as dopaminergic cell loss in the
relates with the end of the fertile life [62]. However, results from SNpc of aging mice submitted to an experimental model of PD.
previous preclinical studies addressing gender-related differences These results provide new insights in experimental models of PD,
on MPTP toxicity have been inconsistent, with increased suscep- indicating that agmatine may represent a new therapeutic tool for
tibility to MPTP-induced behavioral and neurochemical changes the management of cognitive and motor symptoms of PD, together
been described for both male [3,26] and female [41,53,68] mice. with its neuroprotective potential.
Interestingly, Unzeta et al. [68] observed significant differences in
MAO-A and MAO-B activities during the oestrous cycle as well as
between adult male and female mice. Since the neurotoxic effects Acknowledgements
+
of MPTP depend on its conversion to the MPP by MAO-B, the
observed differences in MAO-B activity may be involved in the This work was supported by grants from Conselho Nacional
gender-related effects of MPTP. Therefore, the use of female mice de Desenvolvimento Científico e Tecnológico (CNPq), Coordenac¸ ão
with 15-months-old in the present study, which at this age show de Aperfeic¸ oamento de Pessoal de Nível Superior (CAPES), Pro-
cessation of estrous cycle with very low levels of estrogen [24], grama de Apoio aos Núcleos de Excelência (PRONEX – Project
attenuates the hormonal variability which could interfere with NENASC), Fundac¸ ão de Apoio à Pesquisa do Estado de Santa Cata-
MPTP toxicity. rina (FAPESC), FINEP (Financiadora de Estudos e Projetos-IBN-Net
Of high importance, the administration of agmatine demon- #01.06.0842-00) and INCT (Instituto Nacional de Ciência e Tec-
strated once again its neuroprotective properties as previously nologia) for Excitotoxicity and Neuroprotection. FCM and AAC
described in several models of neuronal damage [31,42,72], receive scholarships from CNPq. CPF, JF, RW, ARSS, CIT and RDP are
F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263–272 271
supported by research fellowships from CNPq. The authors have no [25] Flierl MA, Stahel PF, Beauchamp KM, Morgan SJ, Smith WR, Shohami E. Mouse
closed head injury model induced by a weight-drop device. Nature Protocols
financial or personal conflicts of interest related to this work.
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