Neuroscience 300 (2015) 246–253

THE NONSTEROIDAL ANTIINFLAMMATORY DRUG PIROXICAM REVERSES THE ONSET OF DEPRESSIVE-LIKE BEHAVIOR IN 6-OHDA ANIMAL MODEL OF PARKINSON’S DISEASE R. M. SANTIAGO, a* F. S. TONIN, a J. BARBIERO, a T. ZAMINELLI, a S. L. BOSCHEN, a R. ANDREATINI, a C. DA CUNHA, a M. M. S. LIMA b AND M. A. B. F. VITAL a a

Pharmacology Department, Federal University of Parana´, Brazil

b

Physiology Department, Federal University of Parana´, Brazil

Key words: Parkinson’s disease, depression, 6-OHDA, piroxicam.

INTRODUCTION Depression is one of the most common psychiatric symptoms in patients with Parkinson’s disease (PD; Mayeux, 1990; Cimino et al., 2011), occurring in approximately half of PD patients (Fernandez, 2012) and has also been observed in animal models of PD. In the 1-m ethyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP), rotenone, and 6-hydroxydopamine (6-OHDA) models of PD, animals exhibit depressive-like behavior, such as an increase in immobility time in the modified forced swim test (FST) and anhedonia-like behavior in the sucrose preference test (Tadaiesky et al., 2008; Vuckovic et al., 2008; Santiago et al., 2010). Some authors reported that depression is characterized by activation of the inflammatory response. Several lines of evidence suggest that neuroinflammatory processes could be involved in the development of PD (Hirsch et al., 2012). This hypothesis is supported by an increase in the presence of microglia, proinflammatory cytokines, and cyclooxygenase (COX)-2 in the brains of PD patients (Knott et al., 2000; Nagatsu et al., 2000). Inflammatory processes that are associated with an increase in the expression of the COX-2 enzyme have been implicated in the deleterious cascade of events that lead to neurodegeneration (Knott et al., 2000). Lima et al. (2006) showed that distinct models of PD (e.g., 6-OHDA, MPTP, and lipopolysaccharide) increased the expression of COX in a time-dependent manner, which itself is significant when considering the importance of the neuroinflammatory process in PD. Inflammation plays a major role in degenerative processes in the brain, and antiinflammatory medications have been shown to be potentially effective in reducing such processes in the central nervous system (Teismann and Ferger, 2001). Aarsland et al. (2012) reported that PD-associated depression appears to present with an increase in the activity of inflammation pathways, in which several inflammatory mediators appear to be upregulated. In one study, patients with PD and major depression had lower levels of both cortisol and interleukin-6 (IL-6) in cerebrospinal fluid (CSF) than patients who had only depression (Palhagen et al., 2010). One hypothesis is that treatment with COX-2 inhibitors may be effective in depression in an animal model of PD. Piroxicam is a nonsteroidal antiinflammatory drug

Abstract—Depression is one of the most common psychiatric symptoms in patients with Parkinson’s disease (PD). Some authors have reported that depression is characterized by activation of the inflammatory response. Animal models of PD also present with depressive-like behavior, such as increased immobility time in the modified forced swim test and anhedonia-like behavior in the sucrose preference test. Considering the potential neuroprotective effect of nonsteroidal antiinflammatory drugs in neurodegenerative diseases, the objective of the present study was to investigate the effects of piroxicam on depressivelike behavior in male Wistar rats lesioned with 6-hydroxydopamine (6-OHDA) in the substantia nigra (SN). Antidepressant-like effects were observed after prolonged administration of piroxicam for 21 days. In the forced swim test, the 6-OHDA + saline group exhibited significant reductions in swimming time and increased immobility time compared with the sham + saline. In the sucrose preference test, the 6-OHDA + piroxicam group exhibited no reduction of sucrose preference compared with the sham + saline, with significant effects of treatment and time and a significant treatment  time interaction. 5-Hydroxytryptamine (5-HT) levels significantly decreased in the hippocampus in the 6-OHDA + saline group and not changed in the 6-OHDA + piroxicam group when compared with the sham + saline on day 21. In conclusion, 21-day treatment with piroxicam reversed the onset of depressive-like behavior and prevented the reduction of hippocampal 5-HT levels. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

*Corresponding author. Address: Departamento de Farmacologia, Universidade Federal do Parana´, Brazil. Tel: +55-41-3361-1717; fax: +55-41-3266-2042. E-mail address: [email protected] (R. M. Santiago). Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; 6-OHDA, 6hydroxydopamine; ANOVA, analysis of variance; COX, cyclooxygenase; CSF, cerebrospinal fluid; EDTA, ethylenediaminetetraacetic acid; FST, forced swim test; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; IDO, indoleamine 2,3-dioxygenase; IL-6, interleukin-6; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine; NSAID, nonsteroidal antiinflammatory drug; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PGE2, prostaglandin E2; SN, substantia nigra; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; TNF-a, tumor necrosis factor a. http://dx.doi.org/10.1016/j.neuroscience.2015.05.030 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 246

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253

(NSAID) with analgesic and antipyretic effects. It is widely used to treat various diseases and inflammatory arthropathologies, such as rheumatoid arthritis and osteoarthritis (Dadashzadeh et al., 2002). The main pharmacological effects of NSAIDs are provided by inhibition of COX activity, which is involved in arachidonic acid metabolism and suppresses COX-2 gene expression (Shishodia et al., 2004). Considering the potential neuroprotective effect of NSAIDs in neurodegenerative diseases that directly or indirectly involve inflammation, the overall objective of the present study was to investigate the effects of piroxicam on depressive-like behavior in rats lesioned with 6-OHDA in the substantia nigra (SN).

EXPERIMENTAL PROCEDURES Animals Male Wistar rats (n = 92), weighing 280–320 g, were used. The rats were allowed 1 week to acclimate to the environment before beginning any experimentation. The animals were randomly housed in groups of five in polypropylene cages (41  32  16.5 cm) with wood shavings as bedding. They were maintained in a temperature-controlled room (22 ± 2 °C) on a 12-h/12-h light/dark cycle (lights on at 7:00 AM). The animals had free access to water and food throughout the experiment. The studies were performed in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals, United States National Institutes of Health. The protocol complied with the recommendations of Universidade Federal do Parana´ and was approved by the University Ethics Committee (protocol no. 470). Drugs Piroxicam (10 mg/kg; EMS, Sa˜o Paulo, Brazil) was dissolved in 0.9% saline (vehicle). The vehicle of each drug was administered in respective control rats. The drug was administered orally (p.o.) by gavage in a constant volume of 1.0 ml/kg. The dose of piroxicam was based on previous studies that described its antiinflammatory effects (Murase et al., 2008; Fujita et al., 2012). The neurotoxin 6-OHDA (6 lg/ll; Sigma, St. Louis, MO, USA) was dissolved in artificial CSF supplemented with 0.2% ascorbic acid. The neurotoxin was infused bilaterally in a single dose directly into the SN through stereotaxic surgery. Prolonged treatment The rats were randomly distributed into four groups: sham + saline (n = 7/8), sham + 10 mg/kg piroxicam (n = 7/9), 6-OHDA + saline (n = 7/8), and 6-OHDA + 10 mg/kg piroxicam (n = 6/9). On day 0, all of the animals underwent stereotaxic surgery, in which the experimental groups received bilateral infusions of 6-OHDA in the SN and the sham groups received bilateral infusions of CSF. One hour after surgery, the animals were treated with 10 mg/kg piroxicam or saline (p.o.) once daily for 21 days (from day 0 to 22). The open-field test was conducted on days 1 and 21. The

247

same animals were also tested in the FST on day 22. Another set of animals underwent analogous randomization and underwent the sucrose preference test on day 22. Stereotaxic surgery The neurotoxin 6-OHDA was prepared according to established doses that promote significant dopaminergic neuron loss (Ferro et al., 2005; Lima et al., 2006). The animals were anesthetized with 0.3 ml/kg equitesin, i.p. Bilateral infusions in total volume of 1 ll of 6-OHDA (6 lg/ll) or CSF were performed in the SN using a 27-gauge stainless-steel needle according to the following coordinates: anteroposterior (AP), 5.0 mm from bregma; mediolateral (ML), ±2.1 mm from midline; dorsoventral (DV), 8.0 mm from skull (Paxinos and Watson, 2005). The flow of the injections was controlled by an electronic pump (Harvard Apparatus, Holliston, MA, USA) at a rate of 0.33 ll/min for 3 min. The injection needle was left in the injection side for an additional 2 min to avoid reflux. Sham operations followed the same procedure, but CSF was injected instead of the neurotoxin. Open-field test The apparatus consisted of a round arena (100-cm diameter, 45-cm height), with the floor divided into 19 units. The animals were gently placed on the right side of the open field and allowed to freely explore the area for 5 min. Three locomotor parameters were recorded in this test: locomotion frequency (number of crossings from one unit to another), rearing frequency (number of times the animals stood on their hindpaws), and immobility time (number of seconds of lack of movement during testing). The open field was washed with a 5% water–ethanol solution before behavioral testing to eliminate possible bias caused by odors left by previous rats. Modified FST The procedure was a modification of the method proposed by Porsolt et al. (1978) and Reneric et al. (2002). The test was conducted in two sessions. In the training session, the rats were placed in a tank (25-cm diameter, 65-cm height) that contained water at a temperature of 24 ± 1 °C and depth of 30 cm for 15 min. Twenty-four hours after the training session, the rats were subjected to the FST for 5 min, which was videotaped for subsequent quantification of the following parameters: immobility (i.e., the absence of motion of the entire body and only small movements necessary to keep the animal’s head above the water), climbing (i.e., vigorous movements of the forepaws in and out of the water, usually directed against the wall of the tank), and swimming (i.e., large forepaw movements that displaced water and moved the body around the cylinder and were more than necessary to keep the head above the water). The water was changed after each animal to avoid the influence of urinary or fecal material and temperature (Santiago et al., 2010).

248

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253

Sucrose preference test Sucrose preference is frequently used as a measure of anhedonia in rodents (Papp et al., 1991; Casarotto and Andreatini, 2007; Santiago et al., 2010). Initially, each rat was provided two bottles of water on the extreme sides of the cage during the 24-h training phase to allow the rats to drink from two bottles. After training, the contents of one bottle were randomly switched to contain a 0.5% sucrose solution. The bottles were weighed before being presented to the rats and after 24 h. The sum of water consumption and sucrose consumption was defined as total intake. The percentage of sucrose intake was calculated using the following equation: % sucrose preference = sucrose intake  100/total intake. All of the tests were conducted weekly (i.e., every Tuesday). Determination of dopamine, serotonin, and metabolite concentrations The striatum and hippocampus were rapidly dissected after the FST and stored at 80 °C until neurochemical quantification. The endogenous concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-hydroxytryptamine [5-HT]), and 5-hydroxyindoleacetic acid (5-HIAA) were assayed by reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection. The system consisted of a Synergi Fusion-RP C-18 reverse-phase column (150  4.6-mm inner diameter, 4-lm particle size) fitted with a 4  3.0-mm pre-column (Security Guard Cartridges Fusion-RP), an electrochemical detector (ESA Coulochem III Electrochemical Detector) equipped with a guard cell (ESA 5020) with the electrode set at 350 mV and a dualelectrode analytical cell (ESA 5011A), and a LC-20AT pump (Shimadzu, Kyoto, Japan) equipped with a manual Rheodyne 7725 injector with a 20-ll loop. The column was maintained inside a temperature-controlled oven (Shimadzu, Kyoto, Japan) at 25 °C. The cell contained two chambers in series. Each chamber included a porous graphite coulometric electrode, double counter electrode, and double reference electrode. The oxidizing potentials were set at 100 mV for the first electrode and 450 mV for the second electrode. The tissue samples were homogenized with an ultrasonic cell disrupter (Sonics, Newtown, USA) in 0.1 M perchloric acid that contained 0.02% sodium metabisulfite and an internal standard. After centrifugation at 10,000g for 20 min at 4 °C, 20 ll of the supernatant was injected into the chromatograph. The mobile phase, used at a flow rate of 1 ml/min, had the following composition: 20-g citric acid monohydrate (Merck, Darmstadt, Germany), 200-mg octane-1-sulfonic acid sodium salt (Merck, Darmstadt, Germany), 40-mg ethylenediaminetetraacetic acid (EDTA; Sigma), and 900-ml HPLC-grade water. The pH of the buffer running solution was adjusted to 4.0, which was then filtered through a 0.45-lm filter. Methanol (Merck) was added to give a final composition of 10% methanol (v/v). The neurotransmitter and metabolite concentrations were calculated using standard curves that were generated by determining, in triplicate, the ratios between three

different known amounts of the internal standard. The units are expressed as ng/g of wet tissue (Santiago et al., 2010; Bortolanza et al., 2010). Immunohistochemical and neuronal quantification The animals were intracardially perfused after the sucrose preference test, first with saline and then with 4% formaldehyde as the fixative solution in 0.1 M phosphate buffer (pH 7.4). After tissue extraction for neurochemical purposes, the midbrains were collected and quickly frozen in dry ice. For each rat, 12 sections (each 40-lm thick) were cut on a cryostat in the coronal plane, covering approximately 360 lm ( 4.92 to 5.28 from bregma) of the midbrain (Paxinos and Watson, 2005). These coordinates correspond to the maximal extent of dopaminergic neurons within the SN pars compacta (SNpc). Immunohistochemistry was performed using mouse anti-tyrosine hydroxylase (TH) monoclonal antibody (1:200; catalog no. MAB318, Millipore, Chemicon International, Technology, Billerica, MA, USA). Hightemperature antigen retrieval was performed by immersing the slides in a water bath at 95–98 °C in 10 mM trisodium citrate buffer (pH 6.0) for 45 min. Nonspecific binding was blocked by incubating the sections for 1 h with normal goat serum diluted in phosphate-buffered saline (PBS). After overnight incubation at 4 °C with primary antibodies, the slides were washed with PBS and incubated with the ready-to-use secondary antibody Envision plus (Dako Cytomation, Carpinteria, CA, USA) for 1 h at room temperature. The sections were washed in PBS, and visualization was performed using 3,3-diaminobenzidine (DAB; Dako Cytomation) in chromogen solution, with light counterstaining with Harris’ hematoxylin. Cell counts were performed using ImagePro Express 6 software (Media Cybernetics, Rockville, MD, USA). Each slice was digitized with a digital camera connected to an IX71 microscope (Olympus Optical, Tokyo, Japan). A digital area was created to delimitate the boundaries of the SNpc. For each analysis, the same area was adopted. A ‘‘manual tag’’ tool was used to count the neurons inside the area. All of the counts were performed in images obtained at 40 magnification. A mean number of neurons in the SNpc was obtained for each group, and the results are expressed as a percentage of the sham + saline group (Dombrowski et al., 2010). Statistical analysis The open field was analyzed using a one-way analysis of variance (ANOVA) followed by the Newman–Keuls post hoc test. The FST, sucrose preference test and neurochemical data were analyzed using a two-way ANOVA for independent measures followed by the Bonferroni post hoc test. The level of significance was set at p < 0.05.

RESULTS Behavioral effects of prolonged treatment with piroxicam The 6-OHDA + saline group exhibited a decrease in locomotion frequency (F3,33 = 5.790, p = 0.0030) and

249

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253

rearing frequency (F3,33 = 5.042, p = 0.0060) compared with the sham + saline group 1 day after 6-OHDA infusion (Table 1). The 6-OHDA + saline group exhibited a significant increase in immobility time compared with the sham + saline group 1 day after neurotoxin exposure (F3,33 = 4.977, p = 0.0064). No difference in both parameters (locomotion and rearing frequency) was found between groups 21 days after neurotoxin infusion. In the FST, the 6-OHDA + saline group exhibited an increase in immobility time compared with the sham + saline treatment (F1,23 = 27.85, p < 0.0001) or lesion (F1,23 = 12.67, p = 0.0017) and treatment  lesion interaction (F1,23 = 15.47, p = 0.0007) Fig. 1A and a significant decrease in swimming time compared with the sham + saline group 22 days after 6-OHDA infusion treatment (F1,23 = 5.243, p = 0.0315) or lesion (F1,23 = 2.997, p = 0.0968) and interaction (F1,23 = 6.109, p = 0.0213) Fig. 1B. The neurotoxin did not exert significant effects on climbing behavior treatment (F1,23 = 1.428, p = 0.2442) or lesion (F1,23 = 0.3532, p = 0.5581) and interaction (F1,23 = 0.0005770, p = 0.9814) Fig. 1C. The 6-OHDA + piroxicam was not significantly different from the sham + saline group in the immobility, swimming and climbing time. In the sucrose preference test (Fig. 2), only the 6-OHDA + saline group exhibited a significant reduction of sucrose preference compared with baseline. The 6-OHDA + piroxicam group exhibited no reduction of sucrose preference compared with the sham + saline group 22 days after neurotoxin exposure treatment (F1,29 = 3.970, p = 0.0558) or lesion (F1,29 = 10.07, p = 0.0036) and interaction (F1,29 = 0.9089, p = 0.3483). Determination of monoamine levels after prolonged piroxicam treatment 22 days after 6-OHDA infusion Striatal dopamine levels were significantly reduced in the 6-OHDA + saline and 6-OHDA + piroxicam groups compared with the sham + saline group on day 22 after neurotoxin infusion treatment (F1,28 = 2.402, p = 0.1324) or lesion (F1,28 = 92.25, p < 0.0001) and interaction (F1,28 = 3.035, p = 0.0925) Fig. 3A. The 6-OHDA + saline and 6-OHDA + piroxicam groups exhibited a significant reduction of DOPAC concentrations compared with the sham + saline group

250

A

sham 6-OHDA

swimming (s)

200

*

150 100 50 0 Saline 150

Piroxicam

B

sham

immobility (s)

***

6-OHDA

100

50

0 Saline 100

Piroxicam

C

sham 6-OHDA

climbing (s)

80 60 40 20 0 Saline

Piroxicam

Fig. 1. Effect of prolonged administration of piroxicam (10 mg/kg/day, p.o.) for 22 days after 6-OHDA infusion in the substantia nigra pars compacta. (A) Immobility time. (B) Swimming time. (C) Climbing time. The data are expressed as mean ± SEM (n = 6–7/group). *p < 0.05, *** p < 0.001, compared with sham + saline group (one-way ANOVA followed by Newman–Keuls post hoc test).

on day 22 treatment (F1,28 = 0.002529, p = 0.09602) or lesion (F1,28 = 59.41, p < 0.0001) and interaction (F1,28 = 0.06892, p = 0.7948) Fig. 3B. The HVA concentration significantly decreased in the 6-OHDA + saline and 6-OHDA + piroxicam groups compared with the sham + saline group on day 22

Table 1. Effect of piroxicam on locomotor behavior induced by 6-OHDA Group

Time-point (day)

Locomotion frequency

Rearing frequency

Immobility time (s)

Sham + saline

1 21 1 21 1 21 1 21

57.00 ± 6.63 59.00 ± 2.99 54.00 ± 5.82 50.14 ± 3.59 27.44 ± 4.45⁄⁄ 58.42 ± 4.46 51.9 ± 5.78 64.42 ± 6.15

15.37 ± 2.35 14.14 ± 1.69 15.14 ± 0.76 17.28 ± 3.73 7.22 ± 1.10⁄⁄ 14.28 ± 4.17 11.00 ± 1.89 18.57 ± 2.93

0.12 ± 0.12 0.14 ± 0.14 0.28 ± 0.28 0.28 ± 0.18 2.00 ± 0.62⁄ 0.14 ± 0.14 0.50 ± 0.26 0.33 ± 0.33

Sham + piroxicam 6-OHDA + saline 6-OHDA + piroxicam

The data are expressed as mean ± SEM (n = 7–10/group). *p < 0.05, **p < 0.01, different from sham + saline group (ANOVA followed by Newman–Keuls post hoc test).

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253 5000

100 sham 6-OHDA

80 60

*

40

3000

***

2000

Piroxicam

Saline

Fig. 2. Percentage of sucrose preference 22 days after 6-OHDA infusion in the substantial nigra in animals treated with piroxicam (10 mg/kg, p.o.). The data are expressed as mean ± SEM (n = 8–9/group). *p < 0.05, compared with sham + saline group (two-way ANOVA followed by Bonferroni post hoc test).

dopamine (ng/g)

6-OHDA

0

0 Saline

sham 6-OHDA

1000

Piroxicam

B

sham 6-OHDA

800

5-HIIA (ng/g)

A

600

*** 400 200

4000 0 Saline

***

2000

***

Saline

3000

Piroxicam

B

sham 6-OHDA

2000

***

***

1000

0 Saline 1500

Piroxicam

C

Piroxicam

Fig. 4. Hippocampal concentrations of (A) 5-HT and (B) 5-HIAA 22 days after 6-OHDA infusion in the substantial nigra in animals treated with piroxicam (10 mg/kg, p.o.). The data are expressed as mean ± SEM (n = 7–8/group). ***p < 0.001, compared with sham + saline group (two-way ANOVA followed by Bonferroni post hoc test).

0

DOPAC (ng/g)

sham

1000

20

6000

A

4000

5-HT (ng/g)

sucrose consumption (%)

250

sham

Hippocampal 5-HT levels significantly decreased in the 6-OHDA + saline group compared with the sham + saline group on day 22 treatment (F1,27 = 11.03, p = 0.0026) or lesion (F1,27 = 21.18, p < 0.0001) and interaction (F1,28 = 5.955, p = 0.0215) Fig. 4A. Only the 6-OHDA + saline group exhibited a significant reduction of 5-HIAA levels compared with the sham + saline group on day 22 treatment (F1,27 = 2.109, p = 0.1579) or lesion (F1,27 = 5.183, p = 0.0310) and interaction (F1,28 = 24.36, p < 0.0001) Fig. 4B. Importantly, the 6-OHDA + piroxicam group exhibited no reduction of the levels of serotonin and its metabolite in the hippocampus (Fig. 4A, B).

HVA (ng/g)

6-OHDA 1000

Neuronal quantification in the SNpc

***

***

500

0 Saline

Piroxicam

Fig. 3. Striatal concentrations of (A) DA, (B) DOPAC, and (C) HVA 22 days after 6-OHDA infusion in the substantial nigra in animals treated with piroxicam (10 mg/kg, p.o.). The data are expressed as mean ± SEM (n = 8/group). ***p < 0.001, compared with sham + saline group (two-way ANOVA followed by Bonferroni post hoc test).

treatment (F1,28 = 0.3975, p = 0.5335) (F1,28 = 92.48, p < 0.0001) and (F1,28 = 9.974, p = 0.0038) Fig. 3C.

or lesion interaction

The analysis of the neuronal population revealed that all of the 6-OHDA-treated groups exhibited neuronal loss in the SNpc (Fig. 5A). The quantification of THimmunoreactive neurons indicated that 6-OHDA in the 6-OHDA + saline and 6-OHDA + piroxicam groups reduced the neuronal population in the SNpc by 32.6% compared with the sham + saline group treatment (F1,32 = 0.8242, p = 0.03708) or lesion (F1,32 = 469.6, p < 0.0001) and interaction (F1,28 = 2.336, p = 0.1363; Fig. 5B).

DISCUSSION In the present study, piroxicam reversed depressive-like behavior in rats that received 6-OHDA lesions in the

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253

TH-ir neuronios (% sham+saline)

150

sham 6-OHDA

100

***

***

50

0 Saline

Piroxicam

Fig. 5. Histological analysis of the SNpc at the end of the experiment (22 days after neurotoxin exposure and treatment with 10 mg/kg piroxicam). (A) Representative photomicrographs of TH-immunoreactive neurons in the ventral midbrain in all of the groups. (B) Stereological quantification of the total number of neurons in the SNpc in each group. The data are expressed as a percentage of the control group (n = 9/group). ***p < 0.01, compared with sham + saline group (two-way ANOVA followed by Bonferroni post hoc test).

SNpc. Prolonged treatment with piroxicam for 22 days caused antidepressant-like effects in the FST and returned sucrose preference to baseline levels. The 6-OHDA + piroxicam group also exhibited normal 5-HT levels in the hippocampus, in contrast to a reduction of 5-HT levels in the 6-OHDA + saline group. The hypothesis that depression is related to neuroinflammation is supported by the antiinflammatory effects of many antidepressants that are used in clinical practice. Tricyclic antidepressants and selective serotonin reuptake inhibitors act through different mechanisms and have different neurotransmitter actions, but all of them have antiinflammatory properties (Lu et al., 2010; Maes et al., 2011). Evidence indicates that the neurodegenerative process in clinical depression occurs through inflammatory pathways (CatenaDell’Osso et al., 2011; Kubera et al., 2011; Maes et al., 2011; Leonard and Maes, 2012). The FST was developed by Porsolt et al. (1978) and has been widely used to study antidepressant-like effects. In the present study, we used a modified FST that divided

251

behavior into climbing, swimming, and immobility and found that 6-OHDA-lesioned animals that were treated for 22 days with piroxicam exhibited a decrease in immobility time and increase in swimming time compared with 6-OHDA-lesioned animals that were treated with saline for the same period of time. However, climbing behavior was not different between groups. The increase in swimming time but no changes in climbing time indicate that the observed changes are related to the serotonergic system (Detke et al., 1995), which is consistent with the hypothesis that neurotransmitter deficiency is a cause of depression in PD patients. Malkesman and Weller (2009) reported that mice usually have a preference for sucrose solution when it is supplied with water, but this preference decreases when the animals are exposed to chronic mild stress. Willner (1997) proposed that the reduction of the consumption of a sweet solution (e.g., sucrose, saccharin) in rats is a measure of anhedonia. In the sucrose preference test, the 6-OHDA + saline group exhibited a reduction of sucrose preference on day 22, indicating a state of anhedonia, but the 6-OHDA + piroxicam group presented no reduction of sucrose consumption, indicating that this treatment reversed the anhedonia-like effect of 6-OHDA. Evidence suggests that dysfunctional 5-HT neurotransmission is an important risk factor for depression (Tan et al., 2011). A post-mortem study reported that patients who had PD and depression exhibited the death of serotonergic neurons in the dorsal raphe nucleus compared with nondepressed patients with PD (Paulus and Ellinger, 1991). Studies also suggest that depression in PD is related to a reduction of brain serotonin activity, reflected by a reduction of 5-HIAA levels compared with PD patients without depression (Kostic et al., 1987; Mayeux et al., 1988). Mayeux et al. (1986) reported larger decreases in CSF 5-HIAA levels in PD patients with major depression compared with PD patients without depression. In fact, we observed normal levels of hippocampal 5-HT levels in lesioned animals treated with piroxicam compared with the 6-OHDA + saline group, despite the lack of an effect of this NSAID treatment on striatal dopamine levels. Our results are consistent with Frisina et al. (2009), who described that the etiology of depression in PD may result from alterations in the serotonergic system, which, in turn, is separate from the central dopaminergic system deficiency associated with motor symptoms in PD. Additionally, Schrag (2004) described evidence that correlates changes in physiological levels of 5-HT in depressed patients with PD. Our data corroborate these studies. Prolonged treatment with piroxicam reversed depressive-like behavior and reversed the reduction of hippocampal 5-HT levels. However, piroxicam did not reverse the reduction of striatal dopamine levels or loss of dopaminergic neurons in the SN. The neurochemical findings in the 6-OHDA + saline group are consistent with Santiago et al. (2010). One hypothesis that may explain these results is the relationship between neuroinflammation and the reduction of the neurotransmitter 5-HT. Several

252

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253

mechanisms have been proposed to account for the depressogenic action of cytokines. Some authors have reported that the levels of proinflammatory cytokines are increased in the SN, striatum and CFS patients with PD (Nagatsu et al., 2000; Hirsch et al., 2013). Thus, increases in the concentrations of such proinflammatory cytokines (e.g., IL-1, IL-6, and tumor necrosis factor a [TNF-a]) and prostaglandin E2 (PGE2) have been observed in the blood and CSF of depressed patients (Calabrese et al., 1986; Thomas et al., 2005; Kahl et al., 2006; Dowlati et al., 2010). Another study investigated the levels of IL-1b, IL-6, IL-10, TNF-a, and cortisol in patients with PD and depression, and high levels of TNF-a were associated with depression (Menza et al., 2010). Animal and human studies indicate that cytokines interact with many pathophysiological domains that characterize depression, including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity, and behavior (Miller et al., 2009; Loftis et al., 2010; Song and Wang, 2011; Kuan-Pin, 2012). Some of these cytokines are able to alter 5-HT turnover in multiple brain regions, including the downregulation of 5-HT1A receptors and reduction of 5-HT availability through the activation of the enzyme indoleamine 2,3-dioxygenase (IDO). PGE2 and TNF-a are able to induce IDO activity (Byrne et al., 1986; Braun et al., 2005; Dantzer et al., 2008; O’Connor et al., 2009). This enzyme breaks down the 5-HT precursor tryptophan into kynurenine (Li et al., 2011). Cytokines induce the activation of the tryptophan-degrading enzyme IDO and increase the expression of the serotonin transporter (Miller et al., 2009; Dantzer et al., 2011). The mechanism of action of the antiinflammatory effects of piroxicam involves the inhibition of COX, the ratelimiting enzyme for PG synthesis (Sostres et al., 2010).

CONCLUSION Although the evidence is still limited, the present results suggest that neuroinflammation is involved in depression in PD patients through a reduction of hippocampal 5-HT levels, and treatment with the NSAID piroxicam prevented this reduction. Our data suggest that prolonged treatment with piroxicam prevents the occurrence of depressive-like behavior and reduction of hippocampal 5-HT levels in animals lesioned with 6-OHDA.

CONFLICT OF INTEREST We declare that we have no conflicts of interest.

Acknowledgments—This work was supported by grants from CNPq and CAPES. The funding agencies had no further role in the study design, collection, analysis, and interpretation of the data, writing the report, and decision to submit the paper for publication. RA, CC, MABFV, and MMSL are recipients of CNPq fellowships.

REFERENCES Aarsland D, Pahlhagen S, Ballard CG, Ehrt U, Svenningsson P (2012) Depression in Parkinson disease: epidemiology, mechanisms and management. Nat Rev Neurol 8:35–47. Bortolanza M, Wietzikoski EC, Boschen SL, Dombrowski PA, Latimer M, Maclaren DA, Winn P, Da Cunha C (2010) Functional disconnection of the substantia nigra pars compacta from the pedunculopontine nucleus impairs learning of a conditioned avoidance task. Neurobiol Learn Mem 94(2):229–239. Braun D, Longman RS, Albert ML (2005) A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation. Blood 106(7):2375–2381. Byrne GI, Lehmann LK, Kirschbaum JG, Borden EC, Lee CM, Brown RR (1986) Induction of tryptophan degradation in vitro and in vivo: a c-interferon-stimulated activity. J Interferon Res 6(4):389–396. Calabrese JR, Skwerer RG, Barna B, Gulledge AD, Valenzuela R, Butkus A, Subichin S, Krupp NE (1986) Depression, immunocompetence, and prostaglandins of the E series. Psychiatry Res 17:41–47. Casarotto PC, Andreatini R (2007) Repeated paroxetine treatment reverses anhedonia induced in rats by chronic mild stress or dexamethasone. Eur Neuropsychopharmacol 17(11):735–742. Catena-Dell’Osso M, Bellantuono C, Consoli G, Baroni S, Rotella F, Marazziti D (2011) Inflammatory and neurodegenerative pathways in depression: a new avenue for antidepressant development? Curr Med Chem 18(2):245–255. Cimino CR, Siders CA, Zesiewicz TA (2011) Depressive symptoms in Parkinson disease: degree of association and rate of agreement of clinician-based and self-report measures. J Geriatr Psychiatry Neurol 24(4):199–205. Dadashzadeh S, Vali AM, Rezagholi N (2002) LC determination of piroxicam in human plasma. J Pharm Biomed Anal 28:1201–1204. Dantzer R, O’Connor JC, Freund GG, Johnson RW, Kelley KW (2008) From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9:46–56. Dantzer R, O’Connor JC, Lawson MA, Kelley KW (2011) Inflammation-associated depression: from serotonin to kynurenine. Psychoneuroendocrinology 36(3):426–436. Detke MJ, Rickels M, Lucki I (1995) Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology (Berl) 121:66–72. Dombrowski PA, Carvalho MC, Miyoshi E, Correia D, Bortolanza M, Dos Santos LM, Wietzikoski EC, Eckart MT, Schwarting RK, Branda˜o ML, Da Cunha C (2010) Microdialysis study of striatal dopamine in MPTP-hemilesioned rats challenged with apomorphine and amphetamine. Behav Brain Res 215(1):63–70. Dowlati Y, Herrmann N, Swardfager W, Liu H, Sham L, Reim EK, Lanctoˆt KL (2010) A meta-analysis of cytokines in major depression. Biol Psychiatry 67(5):446–457. Fernandez HH (2012) Nonmotor complications of Parkinson disease. Cleve Clin J Med 79(2):14–18. Ferro MM, Bellissimo MI, Anselmo-Franci JA, Angellucci ME, Canteras NS, Da Cunha C (2005) Comparison of bilaterally 6OHDA- and MPTP-lesioned rats as models of the early phase of Parkinson’s disease: histological, neurochemical, motor and memory alterations. J Neurosci Methods 148:78–87. Frisina PG, Haroutunian V, Libow LS (2009) The neuropathological basis for depression in Parkinson’s disease. Parkinsonism Relat Disord 15:144–148. Fujita I, Okumura T, Sakakibara A, Kita Y (2012) Involvement of inflammation in severe post-operative pain demonstrated by presurgical and post-surgical treatment with piroxicam and ketorolac. J Pharm Pharmacol 64:747–755. Hirsch EC, Vyas S, Hunot S (2012) Neuroinflammation in Parkinson’s disease. Parkinsonism Relat Disord 18(Suppl 1):S210–S212. Hirsch EC, Jenner P, Przedborski S (2013) Pathogenesis of Parkinson’s disease. Mov Disord 28(1):24–30.

R. M. Santiago et al. / Neuroscience 300 (2015) 246–253 Kahl KG, Bens S, Ziegler K, Rudolf S, Dibbelt L, Kordon A, Schweiger U (2006) Cortisol, the cortisol-dehydroepiandrosterone ratio, and pro-inflammatory cytokines in patients with current major depressive disorder comorbid with borderline personality disorder. Biol Psychiatry 59(7):667–671. Knott C, Stern G, Wilkin GP (2000) Inflammatory regulators in Parkinson’s disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2. Mol Cell Neurosci 16:724–739. Kostic VS, Djuricic BM, Covickovic-Sternic N, Bumbasirevic L, Nikolic M, Mrsulja BB (1987) Depression and Parkinson’s disease: possible role of serotonergic mechanisms. J Neurol 234:94–96. Kuan-Pin S (2012) Inflammation in psychopathology of depression: clinical, biological, and therapeutic implications. BioMedicine 2(2):68–74. Kubera M, Obuchowicz E, Goehler L, Brzeszcz J, Maes M (2011) In animal models, psychosocial stress-induced (neuro)inflammation, apoptosis and reduced neurogenesis are associated to the onset of depression. Prog Neuropsychopharmacol Biol Psychiatry 35(3):744–759. Leonard B, Maes M (2012) Mechanistic explanations how cellmediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression. Neurosci Biobehav Rev 36(2):764–785. Li M, Soczynsk JK, Kennedy SH (2011) Inflammatory biomarkers in depression: an opportunity for novel therapeutic interventions. Curr Psychiatry Rep 13:316–320. Lima MMS, Reksidler AB, Zanata SM, Machado HB, Tufik S, Vital MA (2006) Different Parkinsonism models produce a time-dependent induction of COX-2 in the substantia nigra of rats. Brain Res 1101:117–125. Loftis JM, Huckans M, Morasco BJ (2010) Neuroimmune mechanisms of cytokine-induced depression: current theories and novel treatment strategies. Neurobiol Dis 37:519–533. Lu X, Ma L, Ruan L, Kong Y, Mou H, Zhang Z, Wang Z, Wang JM, Le Y (2010) Resveratrol differentially modulates inflammatory responses of microglia and astrocytes. J Neuroinflamm 7:46. Maes M, Leonard B, Fernandez A, Kubera M, Nowak G, Veerhuis R, Gardner A, Ruckoanich P, Geffard M, Altamura C, Galecki P, Berk M (2011) (Neuro)inflammation and neuroprogression as new pathways and drug targets in depression: from antioxidants to kinase inhibitors. Prog Neuropsychopharmacol Biol Psychiatry 35:659–663. Malkesman O, Weller A (2009) Two different putative genetic animal models of childhood depression: a review. Prog Neurobiol 88(3):153–169. Mayeux R, Stern Y, Williams JB, Cote L, Frantz A, Dyrenfurth I (1986) Clinical and biochemical features of depression in Parkinson’s disease. Am J Psychiatry 143:756–759. Mayeux R, Stern Y, Sano M, Williams JB, Cote LJ (1988) The relationship of serotonin to depression in Parkinson’s disease. Mov Disord 3:237–244. Mayeux R (1990) Depression in the patient with Parkinson’s disease. J Clin Psychiatry 51(Suppl):20–23 [discussion 24–25]. Menza M, Dobkin RD, Marin H, Mark MH, Gara M, Bienfait K, Dicke A, Kusnekov A (2010) The role of inflammatory cytokines in cognition and other non-motor symptoms of Parkinson’s disease. Psychosomatics 51:474–479. Miller AH, Maletic V, Raison CL (2009) Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry 65:732–741. Murase A, Okumura T, Sakakibara A, Tonai-Kachi H, Nakao K, Takada J (2008) Effect of prostanoid EP4 receptor antagonist, CJ042,794, in rat models of pain and inflammation. Eur J Pharmacol 580:116–121. Nagatsu T, Mogi M, Ichinose H, Togari A (2000) Changes in cytokines and neurotrophins in Parkinson’s disease. J Neural Transm Suppl 60:277–290.

253

O’Connor JC, Andre C, Wang Y, Lawson MA, Szegedi SS, Lestage J, Castanon N, Kelley KW, Dantzer R (2009) Interferon- and tumor necrosis factor- mediate the upregulation of indoleamine 2,3dioxygenase and the induction of depressive-like behavior in mice in response to bacillus Calmette-Guerin. J Neurosci 29(13):4200–4209. Palhagen S, Qi H, Ma˚rtensson B, Wa˚linder J, Grane´rus AK, Svenningsson P (2010) Monoamines, BDNF, IL-6 and corticosterone in CSF in patients with Parkinson’s disease and major depression. J Neurol 257:524–532. Papp M, Willner P, Muscat R (1991) An animal model of anhedonia: attenuation of sucrose consumption and place preference conditioning by chronic unpredictable mild stress. Psychopharmacology (Berl) 104(2):255–259. Paulus WJ, Ellinger K (1991) The neuropathologic basis of different clinical subgroups of Parkinson’s disease. J Neuropathol Exp Neurol 50:743–755. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates: the new coronal set. 5th ed. San Diego: Academic Press. Porsolt RD, Anton G, Blavet N, Jalfre M (1978) Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol 47(4):379–391. Reneric JP, Bouvard M, Stinus L (2002) In the rat forced swimming test, chronic but not subacute administration of dual 5-HT/NA antidepressant treatments may produce greater effects than selective drugs. Behav Brain Res 136(2):521–532. Santiago RM, Barbieiro J, Lima MMS, Dombrowski PA, Andreatini R, Vital MABF (2010) Depressive-like behaviors alterations induced by intranigral MPTP, 6-OHDA, LPS and rotenone models of Parkinson’s disease are predominantly associated with serotonin and dopamine. Prog Neuropsychopharmacol Biol Psychiatry 34:1104–1114. Schrag A (2004) Psychiatric aspects of Parkinson’s disease: an update. J Neurol 251:795–804. Shishodia S, Koul D, Aggarwal BB (2004) Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-b activation through inhibition of activation of Ib kinase and Akt in human nonsmall cell lung carcinoma: correlation with suppression of COX-2 synthesis. J Immunol 173:2011–2022. Song C, Wang H (2011) Cytokines mediated inflammation and decreased neurogenesis in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry 35:760–768. Sostres C, Gargallo CJ, Arroyo MT, Lanas A (2010) Adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs, aspirin and coxibs) on upper gastrointestinal tract. Best Pract Res Clin Gastroenterol 24(2):121–132. Tadaiesky MT, Dombrowski PA, Figueiredo CP, Cargnin-Ferreira E, Da Cunha C, Takahashi RN (2008) Emotional, cognitive and neurochemical alterations in a premotor stage model of Parkinson’s disease. Neuroscience 156:830–840. Tan SKH, Hartung H, Sharp T, Temel Y (2011) Serotonin-dependent depression in Parkinson’s disease: a role for the subthalamic nucleus? Neuropharmacology 61:387–399. Teismann P, Ferger B (2001) Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2 provide neuroprotection in the MPTP-mouse model of Parkinson’s disease. Synapse 39(2):167–174. Thomas AJ, Davis S, Morris C, Jackson E, Harrison R, O’Brien JT (2005) Increase in interleukin-1 in late-life depression. Am J Psychiatry 162:175–177. Vuckovic MG, Wood RI, Holschneider DP, Abernathy A, Togasaki DM, Smith A, Petzinger GM, Jakowec MW (2008) Memory, mood, dopamine, and serotonin in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-lesioned mouse model of basal ganglia injury. Neurobiol Dis 32:319–327. Willner P (1997) Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134:319–329.

(Accepted 12 May 2015) (Available online 18 May 2015)