Cannabidiol increases the nociceptive threshold in a preclinical model of Parkinson's disease

Journal Pre-proof Cannabidiol increases the nociceptive threshold in a preclinical model of Parkinson's disease Glauce Crivelaro do Nascimento, Daniele Pereira Ferrari, Francisco Silveira Guimaraes, Elaine Aparecida Del Bel, Mariza Bortolanza, Nilson Carlos FerreiraJunior PII:

S0028-3908(19)30370-3

DOI:

https://doi.org/10.1016/j.neuropharm.2019.107808

Reference:

NP 107808

To appear in:

Neuropharmacology

Received Date: 22 May 2019 Revised Date:

11 September 2019

Accepted Date: 2 October 2019

Please cite this article as: Crivelaro do Nascimento, G., Ferrari, D.P., Guimaraes, F.S., Del Bel, E.A., Bortolanza, M., Ferreira- Junior, N.C., Cannabidiol increases the nociceptive threshold in a preclinical model of Parkinson's disease, Neuropharmacology (2019), doi: https://doi.org/10.1016/ j.neuropharm.2019.107808. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Cannabidiol increases the nociceptive threshold in a preclinical model of Parkinson’s disease

*Glauce Crivelaro do Nascimento1,2; *Daniele Pereira Ferrari3; Francisco Silveira Guimaraes4; Elaine Aparecida Del Bel1,2,3; Mariza Bortolanza1a; Nilson Carlos FerreiraJunior1a

* GCN and DPF are equally responsible for this paper. 1 Department of Basic and Oral Biology, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, 14040-904, Brazil. 2 Department of Physiology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, 14049-900, Brazil. 3 Department of Neuroscience, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, 14049-900, Brazil. 4 Department of Pharmacology, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, 14049-900, Brazil.

a Corresponding authors: MB and NCFJ Department of Oral and Basic Biology School of Dentistry of Ribeirao Preto, University of Sao Paulo Cafe Avenue - FORP - USP – Ribeirao Preto - SP - Brazil - 14049-900 Phone.: + 55-16-33154050 E-mail address: [email protected]; [email protected].

2

1

Abstract

2

Medications that improve pain threshold can be useful in the pharmacotherapy of Parkinson’s

3

disease (PD). Pain is a prevalent PD´s non-motor symptom with a higher prevalence of

4

analgesic drugs prescription for patients. However, specific therapy for PD-related pain are

5

not available. Since the endocannabinoid system is expressed extensively in different levels of

6

pain pathway, drugs designed to target this system have promising therapeutic potential in the

7

modulation of pain. Thus, we examined the effects of the 6-hydroxydopamine- induced PD on

8

nociceptive responses of mice and the influence of cannabidiol (CBD) on 6-

9

hydroxydopamine-induced nociception. Further, we investigated the pathway involved in the

10

analgesic effect of the CBD through the co-administration with a fatty acid amide hydrolase

11

(FAAH) inhibitor, increasing the endogenous anandamide levels, and possible targets from

12

anandamide, i.e., the cannabinoid receptors subtype 1 and 2 (CB1 and CB2) and the transient

13

receptor potential vanilloid type 1 (TRPV1). We report that 6-hydroxydopamine- induced

14

parkinsonism decreases the thermal and mechanical nociceptive threshold, whereas CBD

15

(acute and chronic treatment) reduces this hyperalgesia and allodynia evoked by 6-

16

hydroxydopamine. Moreover, ineffective doses of either FAAH inhibitor or TRPV1 receptor

17

antagonist potentialized the CBD- evoked antinociception while an inverse agonist of the CB1

18

and CB2 receptor prevented the antinociceptive effect of the CBD. Altogether, these results

19

indicate that CBD can be a useful drug to prevent the parkinsonism-induced nociceptive

20

threshold reduction. They also suggest that CB1 and TRPV1 receptors are important for

21

CBD-induced analgesia and that CBD could produce these analgesic effects increasing

22

endogenous anandamide levels.

23 24 25 26 27 28

Keywords:

29

Parkinson’s disease; Pain; Transient receptor potential vanilloid type 1.

30

6-hydroxydopamine;

anandamide;

cannabidiol;

cannabinoid

receptors;

3 11. Introduction 2

Besides originally described as a motor disease, Parkinson´s disease (PD) patients

3

suffer from a variety of non-motor symptoms such as sleep disorders, olfactory dysfunctions,

4

anxiety, depression, and pain (Dauer and Przedborski, 2003; Faivre et al., 2019). All these

5

symptoms have a significant impact on their quality of life (Aarsland and Kramberger, 2015;

6

Calabresi et al., 2006; Nègre-Pagès et al., 2008), and usually appear a long time before the

7

first motor signals (Bezard and Fernagut, 2014; Blanchet and Brefel-Courbon, 2018).

8

Around 60% of PD patients are affected by pain (Barone et al., 2009; Faivre et al.,

9

2019; Politis et al., 2010), which can be directly or partly related to the disease. It can appear

10

as nociceptive, neuropathic, or miscellaneous pain (Wasner and Deuschl, 2012). Also,

11

compared to healthy controls, these patients have lower pain threshold and tolerance

12

(Blanchet and Brefel-Courbon, 2018), developing allodynia (Djaldetti et al., 2004;

13

Schestatsky et al., 2007). However, the mechanisms of PD-associated pain are not completely

14

understood, and this problem has not received much attention from preclinical researchers.

15

Decreased nociceptive threshold has been reported in rodent models of PD after 1-

16

methyl-4-phenyl-1,2,4,5-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA)-

17

induced dopaminergic degeneration (Gómez-Paz et al., 2018; Nascimento et al., 2018;

18

Zengin-Toktas et al., 2013). These findings suggest that the loss of dopamine (DA) in the

19

basal ganglia may be involved in the reduced pain threshold (Wasner and Deuschl, 2012).

20

The degeneration of the nigrostriatal pathway impairs the dopamine-mediated

21

descending pathways, resulting in hyperalgesia (Fil et al., 2013). In this way, Magnusson and

22

Fisher (2000) showed that systemic administration of DA agonists induced hypoalgesia by

23

modulating D2 receptors in the dorsolateral striatum (Magnusson and Fisher, 2000).

24

Furthermore, some studies have pointed out to an increase in the pain threshold after

25

administration of the dopaminergic medication such as levodopa (Brefel-Courbon et al., 2005;

26

Gerdelat-Mas et al., 2007). However, the absence of a total recuperation of these symptoms

27

with dopaminergic therapy suggests that non-dopaminergic mechanisms could also be

28

involved in the appearance or maintenance of pain symptoms (Brefel-Courbon et al., 2005).

29

Despite the higher prevalence of analgesic drugs prescription for PD patients (33%)

30

than for general population (20%), pain is often neglected and insufficiently treated in these

31

patients (Broetz et al., 2007; Blanchet and Brefel-Courbon, 2018). In addition, analgesic drugs

32

frequently cause side effects such as opiate-induced constipation that in turn may exacerbate

33

constipation developed by parkinsonian patients in early stages of the disease (Stocchi and

4 1

Torti, 2017). Therefore, even though the well-established correlation between pain and PD,

2

still there is no specific therapy for the Parkinson's disease-related pain, thus requiring further

3

efforts in investigating effective drugs, with fewer side effects, for pain in this

4

neurodegenerative condition (Seppi et al., 2019).

5

The endocannabinoid system could be a promising target for the treatment of PD-

6

associated pain. This system has now been recognized as presenting great therapeutic

7

potential in the modulation of pain (Guindon and Hohmann, 2009), besides having a

8

neuroprotective effect on neurodegenerative diseases (Chagas et al., 2014). Cannabidiol

9

(CBD) is the principal non-intoxicating phytocannabinoid constituent of the Cannabis sativa

10

plant (Shohami et al., 2011; Russo, 2017). CBD has low affinity for both cannabinoid subtype

11

1 (CB1) and 2 (CB2) receptors (Ligresti et al., 2016), but it can upregulate the levels of the

12

endocannabinoid anandamide (AEA) by inactivation of the Fatty Acid Amide Hydrolase

13

(FAAH) (Campos et al., 2010), the enzyme responsible for anandamide degradation.

14

Besides AEA effects in cannabinoid receptors, AEA is also an agonist of the transient

15

receptor potential vanilloid type 1 (TRPV1) (Campos et al., 2010; dos-Santos- Pereira et al.,

16

2016; Zygmunt et al., 2000). TRPV1 is a channel activated by multiple painful stimuli

17

including noxious heat, pungent chemicals (capsaicin and jellyfish venom), and protons

18

(Kaneko and Szallasi, 2014; Szallasi et al., 2007). Accumulated compelling data have

19

demonstrated the role of TRPV1 in inflammatory and neuropathic pain states (Moran and

20

Szallasi, 2018; Stocchi and Torti, 2017). For instance, TRPV1 activation in nociceptive

21

neurons triggers the release of neuropeptides and transmitters, resulting in perceived pain

22

(Jara-Oseguera et al., 2010).

23

Therefore, CBD may represent a useful pharmacological alternative in the treatment of

24

PD-related pain. Here, we tested the hypothesis that CBD has an antinociceptive effect in a

25

preclinical model of 6-OHDA-induced parkinsonism in mice via FAAH inhibition and

26

indirect activation of the CB1 receptor.

27 282. Materials and Methods

Ethical statement: Male adult C57⁄BL6 mice (FMRP-USP, Ribeirão Preto, Brazil;

29

2.1.

30

20– 25g body weight) were used in this study. All animal experimental procedures were

31

approved by the local Animal Care and Use Committee of the University of São Paulo/Brazil

32

at the Ribeirao Preto campus (Protocol number # 2017.1.369.58.4) and are in accordance with

33

the Guide for the Care and Use of Laboratory Animals of the National Council for the Control

5 1

of Animal Experimentation (CONCEA).

2

2.2.

3

groups of three animals per cage in a controlled environment under a light-dark cycle of

4

12 hours (lights on at 06:00 and off at 18:00h), in a quiet room with controlled temperature

5

(23 ºC ± 1 ºC). All animals had free access to food and water. Animal care, including the

6

manual recording of body weight, observation of food and water intake, and cage changes that

7

occurred daily during the middle of the light cycle. Mice were left undisturbed during their

8

dark cycle.

Experimental animals - Housing and husbandry: C57⁄BL6 mice were housed in

9 10

2.3.

Pharmacological treatments: The following drugs, at doses based on the literature,

11

were used: cannabidiol (CBD, 10, 30 and 100 mg/kg (McPartland et al., 2015)), morphine

12

(MOR, 10 mg/kg (Aksu et al., 2018)), celecoxib (CXB, non-steroidal anti-inflammatory, 20

13

mg/kg, (Zhao et al., 2017)), URB597 (URB, FAAH inhibitor, 0.5 mg/kg, (Campos et al.,

14

2013)), AM251 (AM, CB1 antagonist, 1 mg/kg, (Campos et al., 2013)), Capsazepine (CPZ,

15

TRPV1 antagonist, 5 mg/kg, (dos-Santos-Pereira et al., 2016)) and SCH 336 (SCH, CB2

16

inverse agonist, 2mg/kg, (Lunn et al, 2006). All drugs were administered intraperitoneally.

17 18

2.4.

Sample size: The total number of mice used in this work was 49. All mice were tested

19

in the nociception tests before and after 6-OHDA lesion for determination of hyperalgesia and

20

allodynia time-course (first experimental protocol). Then, they were randomly distributed to

21

vehicle (VEH, n=6), CXB (n=6), MOR (n=6), CBD (n=7 per each dose) groups for the

22

second experimental protocol. After a 3-day drug washout, these mice were randomly

23

redistributed to the following groups: VEH (n=9), URB (n=10), AM (n=10), CPZ (n=10) and

24

SCH (n=10) treatments. Finally, one-week drug washout was performed, and 32 mice were

25

randomly distributed to VEH (n=7), CBD (n=8), CBD+URB (n=8), CBD+AM (n=8),

26

CBD+CPZ (n=8) or CBD+SCH (n=8) groups. In all cases, the animals were re-tested in the

27

nociception tests to ensure drug washout.

28 29

2.5.

30

experimental protocols:

31 32

Drug treatment and experimental design: This work was compounded by four

(1)

First, nociceptive tests were performed before the 6-OHDA lesion (-1 day) and

after 7, 14, and 21 days of the surgery.

6

(2)

1

After 21 days of the surgery, these mice were distributed into VEH, CXB,

2

MOR and CBD10, CBD30 and CBD100 mg/kg for the second experimental protocol. This

3

protocol consisted in evaluating CBD effects on hyperalgesia (by Hot Plate and Tail Flick

4

tests) and allodynia (Acetone Drop and Von Frey tests) responses compared to effects of

5

celecoxib, morphine and saline administration in mice with established motor impairment

6

(Data not shown).

7

(3)

After 3-day of drug washout, we tested the antinociceptive effects of chronic

8

treatment of CBD. Mice received CBD (10 mg/kg) for 14 days, once a day, and then were

9

tested for nociceptive behaviors 60 minutes after the last administration. (4)

10

After 3-day of drug washout, we tested different doses of URB, AM, CPZ and

11

SCH to find ineffective doses of these drugs on hyperalgesia and allodynia responses in

12

parkinsonian mice. These ineffective doses were used in the fourth protocol. (5)

13

Finally, after one week of drug washout, we test the involvement of FAAH,

14

CB1, CB2 and TRPV1 in the antinociceptive effect of the CBD in parkinsonian mice. A pre-

15

treatment with URB, AM, CPZ or SCH was made 30 min before CBD administration (30

16

mg/kg – the least effective dose according to our results). Hyperalgesia and allodynia tests

17

were performed 30 min after CBD administration.

18

After each protocol and drug washout, animals were re-subjected to nociception tests

19

to ensure drug washout (data not shown) and afterward regrouped randomly for performing

20

the following protocol.

21 22

2.6.

Experimental Procedures: All tests were performed at the light phase, in the

23

morning period (8:00-12:00) and at the same place (in the same laboratory). The same trained

24

person performed all the nociception tests. The information about the treatments was withheld

25

from experimenters that performed the hyperalgesia and allodynia tests to reduce bias, thus

26

characterizing a blinded-experiment.

27

2.6.1.

28

on a previous study (dos-Santos-Pereira et al., 2016). 6-Hydroxydopamine (6- OHDA; 2,4,5-

29

Trihydroxyphenethylamine hydrochloride; 2.5 ug/ul diluted in saline 0.9% containing 0.02%

30

ascorbic acid) obtained from Sigma-Aldrich, USA, was used. Third-two animals were

31

anesthetized with 2,2,2- tribromoethanol (250 mg/kg i.p.) and fixed in the stereotaxic

32

apparatus for performing the surgery (David Kopf, model USA, 9:57). Stereotaxic

33

coordinates of the dorsolateral region of striatum were (in mm from Bregma): anteroposterior

6-Hydroxydopamine-lesioned mice model: The 6-OHDA-induced lesion was based

7 1

(AP) = +0.5; lateral-lateral (LL) = -2.3 and dorsoventral (DV) = −3.9 and −3.0 (Paxinos and

2

Franklin, 2001). 6-OHDA-HCl was microinjected in a total volume of 2 µl/injection (2

3

injection places; 1 µL/min). After the microinjection, the cannula was left in place for five

4

additional minutes to prevent reflux of the injected solution. At the end of the surgical

5

procedure, the animals were kept warm by a 60W light bulb until full recovery from

6

anesthesia and then led to the vivarium, where they received amoxicillin (5 mg/ml orally) for

7

three days.

8

2.6.2.

9

inches tall) for 2 min, and the number of times they touch the cylinder wall with the right, left,

10

or both forepaws following rearing was recorded (Schallert et al., 2000). The asymmetry

11

score was calculated as the sum of left (unaffected) forepaw touches + 1/2 bilateral

12

touches/total number of forepaw touches (right + left + bilateral). For the mice that use both

13

forepaws equally, the score would be 0.5. For mice with the complete asymmetry of forepaw

14

use due to severe dopamine depletion affecting the right forepaw, the asymmetry score would

15

be 1 (Data not shown).

16

2.6.3.

17

following sequence: Von Frey, Acetone Drop, Tail Flick, and Hot Plate. This sequence was

18

based on previous studies from our group (do Nascimento and Leite-Panissi, 2014;

19

Nascimento et al., 2018). Specifically, the hot plate test was designed to evaluate the

20

supraspinally-integrated responses (Caggiula et al., 1995; Rubinstein et al., 2002), and it

21

determines changes in latency as an indicator of modifications of the supraspinal pain process.

22

In turn, the tail-flick latency is mediated by a spino-bulbo-spinal circuit (Le Bars et al., 2001),

23

as well as acetone – cold test, which is compared with the tail-flick test, but it uses the paw

24

withdrawal as parameter, avoiding the use of the tail that is an important organ related to

25

thermoregulation. In addition, different effects could be found with mechanical von Frey

26

filament, an important instrument to access the mechanisms of cutaneous stimulation-induced

27

sensory input (Pitcher et al., 1999).

28

2.6.3.1. Hot Plate: Thermal hyperalgesia was assessed with the hot plate test as previously

29

described (Fujii et al., 2019). Mice were placed in a 10-cm wide glass cylinder in a hot plate

30

apparatus (Insight, Wembley, UK) at 55±0.3 °C. The end-point (latency) was characterized

31

by the removal of the paw, followed by clear paw flinching or licking movements. A cut-off

32

of 30 s was established to prevent tissue damage (Kinsey et al., 2011). The latency of the flick

Cylinder test: Animals were placed in a clear plastic cylinder (3.8 inches wide, 6

Nociceptive Behaviors: The nociceptive tests were performed according to the

8 1

response was measured in seconds, and each animal was tested three times with 60 seconds

2

between each trial. The scores were averaged over the 3 trials for the final score of each

3

animal.

4

2.6.3.2.

5

flick test as described previously (Nascimento et al., 2018). Mice were placed at the apparatus

6

where a thermal stimulus was applied to the tail. We measured the latency for tail withdrawal

7

as an indication of the pain threshold. A cutoff time of 9-s was used to prevent tissue damage.

8

The latency of flick response was measured in seconds, and each animal was tested three

9

times, with 60 seconds between each trial. These scores were averaged over the 3 trials for the

10

final animal score. To avoid novelty stress- induced analgesia, animals were acclimated to the

11

test situation two days before the experiment by restraining the mouse in the same apparatus,

12

but without the thermal stimulus.

13

2.6.3.3.

14

as described previously (Brenner et al., 2014). Mice were acclimated for 5 min on a wire

15

mesh. Then, we sprayed acetone (100 µL) onto the plantar surface of the hind paw, without

16

touching the skin. The first 10 seconds of activity were disregarded as a response to the direct

17

application of the droplet. We measured the flinches in response to skin cooling induced by

18

acetone using the following scores: 0- none; 1- rapid withdrawal or abrupt movement of the

19

paw; 2- prolonged withdrawal or repeated movements of the paw; 3- repeated movements

20

followed by licking of the paw.

21

2.6.3.4.

22

force required to elicit a behavioral response, such as the withdrawal of a paw from the

23

applied stimulus (Chaplan et al., 1994; Nascimento et al., 2018, 2013). Employing von Frey

24

filaments (Nylon monofilaments - 0.35 mm diameter, Scientific Anglers™ Full Sinking Fly

25

Fishing Line) from 0.01 to 15.1 g, we first characterized the percent response at each stimulus

26

intensity (calibration). In the experimental day, the mice were placed individually in a cage

27

(10 X 20 X 15 cm) with a meshed metal wire floor (squares of 5 X 5 mm). Mice were

28

successively stimulated starting with the weakest to the strongest filaments (0.05 to 4 g). The

29

sequence of stimuli stopped when the animal reacted with immediate flinching or licking of

30

the hind paw. The force of the last used filament was considered the pain threshold.

31

Tail Flick: The spinally mediated thermal hyperalgesia was assessed by the tail-

Acetone Drop Test: Cold allodynia was assessed by the modified acetone drop test

Von Frey test: Mechanosensitivity can be determined as the threshold amount of

9 1

2.7.

Euthanasia and Tissue Processing: At the end of the behavioral analysis, the animals

2

were deeply anesthetized with urethane (1.5 g/kg, Sigma-Aldrich, St. Louis, MO, USA) and

3

rapidly perfused transcardially with cold 0.9% saline solution containing heparin (200 µl of

4

heparin 25,000 UI/l of solution) and sodium nitrite (1 g/l solution). Animals were then

5

immediately perfused with 4% paraformaldehyde (PFA; pH 7.4; Sigma-Aldrich, St. Louis,

6

MO, USA). Brains were immediately removed, post- fixed in 4% paraformaldehyde for 2 h,

7

and cryoprotected in 30% sucrose solution. Brains were quickly frozen in isopentane (−40 °C,

8

Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C until histological processing.

9

Coronal sections (25 µm) were processed in a freezing microtome (Leica, model CM1850)

10

throughout the rostrocaudal extent of the striatum and substantia nigra compacta. Striatal

11

sections were obtained from rostral (Bregma: +0.86 mm), medial (Bregma +0.02 mm) and

12

caudal areas (Bregma −0.58 mm). Substantia nigra sections were obtained from rostral

13

(Bregma −2.54 mm), medial (Bregma −3.16 mm) and caudal areas (Bregma −3.80 mm).

14

2.8.

Immunohistochemistry: To verify the extension of 6-OHDA lesion, immunostaining

15

was

then

16

immunohistochemical protocols (Gomes et al., 2008) with specific tyrosine hydroxylase (TH,

17

1:2000; Pel Freez, Arkansas, USA) antibody diluted in 0.1 M phosphate-buffered saline (pH

18

7.4), containing 0.15% Triton X-100. After incubation with the primary (48 h) and secondary

19

(1.5 h) antisera, peroxidase reactions were developed with diaminobenzidine. The slices were

20

mounted on slides and coverslipped for microscope observations. Samples from each

21

experimental group were processed at the same time. Quantitative analyses were then

22

performed in striatum and substantia nigra.

carried

out

in

free-floating

sections

with

standard

avidin–

biotin

23 24

2.9.

25

was evaluated by repeated measures analysis of variance (r-ANOVA), with drug and time as

26

factors. The results are expressed as mean ± S.E.M, and post-hoc analysis was performed with

27

the help of the Bonferroni's test. For the analysis of the nociception tests and

28

immunohistochemistry, comparison between different groups was performed using two-way

29

analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test. Values are

30

presented as mean ± S.E.M. Statistical significance was set at p<0.05. All statistics in this

31

study were performed using Prism 6, version 6.0d (GraphPad Software, Inc.).

32

Statistical Analysis: The effect on the time course of 6-OHDA-induced nociception

10 1

3.

Results

2

3.1.

Time-course of 6-OHDA-induced hyperalgesia and allodynia responses

3

In the present study, we first confirmed by immunohistochemistry that 6-OHDA

4

induced PD-like lesions (Figure 1A). We used TH-immunoreactivity (TH-ir) to assess the

5

percentage of the striatal area devoid of TH-ir for all mice used in the experiments. On

6

average, 65% of the striatum was denervated. There was a lesion side effect for all striatal

7

analyzed areas (rostral: F2,42 = 3.3; p<0.05; medial: F2,38 = 12.7; p<0.05; caudal: F2,48 = 6.4;

8

p<0.05, data not shown).

9

To rule out that different lesion intensity would interfere with CBD analgesic effects,

10

the cylinder test was performed. The ratio of forelimb placement [(left- right)/(right + left +

11

both)] of the non-treated group did not differ from drug treatment group (F4,42 = 4.2; p>0.05).

12

As expected, there was no interaction “lesion side × drug treatment” (F4,48 = 13.1; p>0.05,

13

Supplementary Data).

14

We used four nociceptive behavioral tests to measure the time-course of thermal and

15

mechanical pain threshold in mice after 6-OHDA-injection. The hot plate test showed a

16

significantly decrease in thermal latency in mice at 14 and 21 days from surgery (F4,48 = 3. 1;

17

p<0.01; Figure 1C) when compared to basal measures. On the other hand, tail flick latency

18

diminished since the first analyzed period (7 – 21 days) after 6- OHDA lesion (F4,48 = 6.4;

19

p<0.05; Figure 1D). Regarding allodynia responses, there was significantly increase in

20

nociception behavior at 14 and 21 days from surgery when compared to basal measures in

21

both tests (acetone drop, F4,48 = 13.3; p<0.05; Figure E; and von Frey, F4,48 = 4.3; p<0.05;

22

Figure F).

23

These results suggest that the mice model of PD induced by unilateral lesion of the

24

striatum displays thermal and mechanical pain hypersensitivity that start at different time

25

points after the 6-OHDA lesion.

26 27

3.2.

Antinociceptive effects of acute CBD on experimental parkinsonism

28

Hyperalgesia response – Twenty-one days after 6-OHDA injection, we tested the

29

antinociceptive effects of CBD. Three different doses (10, 30, 100 mg/kg i.p.) of CBD were

30

administered 60 min previously to the nociceptive tests. CXB, MOR, and CBD (10, 30 and

31

100 mg/kg) increased hot plate response latency (p<0.05; Figure 2A) when compared to VEH

32

group. Morphine (MOR) also significantly increased this latency when compared to CXB and

33

the three doses of CBD (F3,48 = 5.31 p<0.01; Figure 2A). In addition, the highest dose of CBD

11 1

(100 mg/kg) induced antinociceptive effects in the hot plate test comparing to the other two

2

doses (F3,32 = 6.8; p<0.05; Figure 2A). The tail flick latency was measured to evaluate a

3

hyperalgesia response under spinal control. The results indicated that celecoxib (CXB),

4

morphine (MOR) and the three doses of CBD significantly reduced this thermal hyperalgesia

5

when compared to VEH group (F3,48 = 17.3; p<0.05; Figure 2B). The highest dose of CBD

6

(100 mg/kg) was the most effective in increasing tail flick latency.

7

Allodynia response – There was no change in the withdrawal latency to cold stimulus

8

in the VEH, CXB, and CBD (30 mg/kg) mice groups (p>0.05; Figure 2C). MOR and the

9

doses of 10 and 100 mg/kg of CBD significantly increased this latency (F4,48 = 26.1 p<0.01;

10

Figure 2C). CBD and MOR presented similar antinociceptive effects in this test (p>0.05;

11

Figure 2C). Antinociceptive effects in mechanical allodynia measured by Von Frey method

12

were observed after CXB, MOR and CBD (10 and 100 mg/kg) administration when compared

13

to CBD at 30 mg/kg (F3,32 = 26.2; p<0.05; Figure 2D).

14 15

3.3.

We also tested the antinociceptive effects of chronic treatment with CBD. Mice

16 17

Antinociceptive effects of chronic CBD on experimental parkinsonism

received CBD (10 mg/kg i.p.) either in one single dose or daily dose for 14 days.

18

Hyperalgesia response – Both acute and chronic CBD increased hot plate response

19

latency (p<0.01) when compared to VEH group. The chronic CBD had significantly higher

20

antinociceptive effect compared to acute dose of CBD (F 2,19 = 96.22; p<0.01; Figure 3A). In

21

tail flick, both acute and chronic CBD treatments significantly reduced this thermal

22

hyperalgesia when compared to VEH group (F

23

differences were found between acute and chronic CBD (p>0.05).

2,19

= 14.26; p<0.01; Figure 3B). No

24

Allodynia response – The withdrawal latency for cold stimulus were increased in mice

25

that receive CBD, compared to VEH group (F 2,19 = 8.881; p<0.01; Figure 3C). Similar effects

26

were found between acute and chronic treatment of CBD (p>0.05). The evaluation of

27

mechanical allodynia showed similar antinociceptive effects of both acute and chronic CBD

28

when compared to VEH (F 2,19 =14.06; p<0.05; Figure 3D).

29 30

3.4.

Antinociceptive effects of URB, AM251, Capsazepine and SCH 336 on

31

experimental parkinsonism

32

The progression in understanding the pain pathways have helped in discovering

33

several new pharmacological targets to treat pain: TRPV1 and the cannabinoid receptors

12 1

along with their endogenous agonists (endocannabinoids) are some of them.

2

Hyperalgesia response – Following three days of CBD washout, we tested the effect

3

of URB, AM251, CPZ and SCH in lesioned-mice using the hot plate and tail flick tests. The

4

drugs were administered 60 min previously to reach the plasmatic peak during the nociceptive

5

tests (Deiana et al., 2012). There were no changes in the hot plate and tail flick responses

6

latency in doses tested of VEH, URB, AM, CPZ and SCH in any of the groups (p>0.05;

7

Figure 4A-B).

8

Allodynia response – URB, AM, CPZ and SCH effects were also tested in allodynia

9

responses. There was no change in the withdrawal latency to a cold stimulus (Figure 4C) nor

10

intensity of mechanical allodynia (Figure 4D) measured by Von Frey method in doses tested

11

of VEH, URB, AM, CPZ and SCH (p>0.05).

12 13

3.5.

Involvement of CB1, CB2 FAAH, and TRPV1 in the antinociceptive effects

14

induced by CBD on experimental parkinsonism

15

To test the hypothesis that CBD could act on 6-OHDA-induced nociception via FAAH

16

inhibition, and modulation of CB1, CB2 and TRPV1 receptors, we administrated CBD

17

(30mg/kg) plus each drug related to the cited targets (URB, AM, CPZ and SCH). The

18

hyperalgesia and allodynia responses were evaluated in mice with experimental parkinsonism.

19

For this, the animals were randomly distributed into the groups after a new 3 days period of

20

drug washout. The nociception tests were repeated before the experiment to ensure drug

21

washout.

22

Hyperalgesia response – Pre-treatment with the FAAH inhibitor (URB) and TRPV1

23

antagonist (CPZ) potentiated the effect induced by CBD (interaction: F2,42= 12.6; p<0.05,

24

two-way ANOVA; p<0.05 vs. VEH and CBD; Figure 5A). On the other hand, pre-treatment

25

with the CB1 receptor antagonist AM251 blocked the effects induced by CBD on hot plate

26

response latency (interaction: F2,48= 21.5; p<0.05, two- way ANOVA; p<0.05 vs. VEH and

27

CBD). The CB2 inverse agonist SCH 336 had no influence on antinociceptive effects of CBD

28

(p>0.05; Figure 5A). A similar finding was observed for tail flick latency. Pre-treatment with

29

URB and CPZ potentiated the effect induced by CBD (interaction: F2,42= 20.6; p<0.05, two-

30

way ANOVA; p<0.05 vs. VEH and CBD; Figure 5B), while pre-treatment with AM and SCH

31

blocked the effects induced by CBD on tail flick response latency (interaction: F2,48= 21.5;

32

p<0.05, two-way ANOVA; p<0.05 vs. VEH and CBD; Figure 5B).

33

Allodynia response – Administration of URB or CPZ potentiated the antinociceptive

13 1

effect of CBD (30 mg/kg) on withdrawal latency to a cold stimulus (interaction: F2,50= 6.2;

2

p<0.05, two-way ANOVA; p<0.05 vs. VEH and CBD; Figure 5C). Distinctly, pre-treatment

3

with both AM and SCH blocked the effects induced by CBD in this test (interaction: F2,48=

4

8.7; p<0.05, two-way ANOVA; p<0.05 vs. VEH and CBD; Figure 5C). Ineffective doses on

5

allodynia of the URB or CPZ, when administered with an inefficacious dose of 30 mg/kg of

6

CBD, evoked a synergistic increased in the withdrawal latency to a mechanical stimulus,

7

suggesting similar mechanisms of action of the CBD, URB, and CPZ (interaction: F2,48= 18.1;

8

p<0.05, two-way ANOVA; p<0.05 vs. VEH and CBD; Figure 5D). On the other hand, AM

9

and SCH did not change the lack of effect of the CBD in mechanical allodynia response

10

(p>0.05; Figure 5D).

11 12

4.

Discussion

13

The present results showed that: (i) experimental parkinsonism decreases the

14

nociceptive threshold to thermal and mechanical stimulations, (ii) acute or chronic treatment

15

with CBD decreases the hyperalgesia and allodynia in this condition, (iii) the highest CBD

16

dose (100 mg/kg) induces a similar effect to morphine in parkinsonian mice, (iv) the inverse

17

agonist of the CB1 receptor prevents the antinociceptive effect of CBD while ineffective

18

doses of either a FAAH inhibitor or a TRPV1 antagonist increase the CBD antinociceptive

19

effect. Altogether, these results indicate that acute or chronic treatment with CBD can be

20

useful to reduce parkinsonism-increased nociception. They also suggest that CBD induces

21

antinociception increasing endogenous anandamide levels and acting via CB1 and TRPV1

22

receptors, but not via CB2 receptors.

23

Our data showed an antinociceptive effect of the three analyzed doses of CBD in

24

hyperalgesia responses (hot plate and tail flick tests). For allodynia responses, the lower and

25

higher doses of CBD were significantly effective, whereas an intermediate dose did not work.

26

Moreover, in three of the four performed tests (Figure 2B-D), a similar antinociceptive effect

27

between CBD (100mg/kg) and morphine (MOR) was found. CBD was also compared to

28

celecoxib (CXB), a nonsteroidal anti‐inflammatory drug, which was minimally effective

29

against both thermal and mechanical endpoints, which corroborates previous results

30

(Hosseinzadeh et al., 2017). In our animal model, the highest dose of CBD promoted a better

31

antinociceptive effect than CXB.

32

Hypernociception found in 6-OHDA lesioned animals are in line with several other

33

studies in the field (Charles et al., 2018; Gee et al., 2015; Kaszuba et al., 2017; Zengin-Toktas

34

et al., 2013). Hyperalgesia and allodynia can be mediated by central sensitization at distinct

14 1

central nervous system levels. At note, Charles and colleagues described a hyperexcitability

2

of neurons from lamina V of the spinal cord in 6-OHDA-induced parkinsonism (Charles et

3

al., 2018). The dorsal horn of the spinal cord is the first relay for nociceptive inputs from the

4

periphery. Our data evidence decrease in nociceptive threshold in the first days after the 6-

5

OHDA lesion when tail flick test was performed. Importantly, the tail flick use a noxious heat

6

stimulation of the tail that produces a spinally mediated flexion reflex (Bannon and

7

Malmberg, 2007). In contrast, in the hot-plate test, the final response involves not only spinal,

8

but also supraspinal pathways.

9

Dennis and Melzack first described an essential role for the dopamine system in

10

mediating antinociception (Dennis and Melzack, 1983). Basal ganglia, limbic areas, thalamus,

11

periaqueductal gray matter (PAG), and other brain areas modulated by the dopaminergic

12

system play a critical role in the processing of pain (Chudler and Dong, 1995; Hagelberg et

13

al., 2002). The dopaminergic circuitry could also directly influence nociceptive behavior

14

acting in the dorsal horn of spinal cord through hypothalamic A11 projections to spinal D2

15

receptors (Kim and Abdi, 2014; Taniguchi et al., 2011). Recent findings have shown a

16

dysregulation of GABAergic neurotransmission in the PAG ventrolateral (the major

17

component of the descending inhibitory pain pathway) induced by 6-OHDA nigrostriatal

18

lesions, suggesting an impairment of descending analgesic system and stimulation of the

19

descending pain facilitatory system (Domenici et al., 2019). This unbalance promotes

20

downregulation of the spinal opioidergic modulation and, consequently, leads to a nociceptive

21

sensitization. Based on these findings, we speculate that dysfunctions in limbic areas and at

22

the spinal level could be responsible for the decreased nociceptive threshold observed in the

23

6-OHDA-induced PD model in the present work.

24

In patients, decreased function of the nigrostriatal pathway is associated with pain,

25

(Hagelberg et al., 2004) while persistent and chronic pain reduces intracranial self-stimulation

26

of the medial forebrain bundle (Leitl et al., 2014; Pereira Do Carmo et al., 2009). Since these

27

pieces of evidence indicate that chronic pain leads to a significant impairment of dopamine

28

activity (Chudler and Dong, 1995; Magnusson and Fisher, 2000; Taylor et al., 2016; Wood,

29

2008), the loss of dopaminergic nigrostriatal activation after injection of 6-OHDA into the

30

striatum can directly contribute to the decreased nociceptive threshold observed in the current

31

work.

32

The involvement of the endocannabinoid system in dopamine signaling within reward

33

circuits affected by chronic pain is few explored (Mlost et al., 2019). Despite the

34

antinociceptive potential of cannabinoids (Jensen et al., 2015), CBD effect on nociception

15 1

induced by striatal lesions in mice has never been investigated. Our results show that a CB1

2

receptor antagonist blunted CBD-induced antinociception while a CB2 receptor antagonist did

3

not affect CBD response, suggesting that CB1 receptors play an important role in CBD

4

antinociception. Although CBD has a low affinity for cannabinoid receptors, CBD can act

5

directly, at micromolar concentrations, as a partial agonist of CB1 and CB2 receptors

6

(McPartland et al., 2015; Pertwee, 2008a). CB2 receptors are mainly expressed in cells of the

7

immune system (Cabral and Griffin-Thomas, 2009) and mediated many anti-inflammatory

8

properties of cannabinoids (Turcotte et al., 2016), and albeit celecoxib has produced

9

antinociception in our experimental model of PD, CB2 receptors are unlikely to mediate CBD

10

effects in our studysince the selective CB2 antagonist did not change the CBD

11

antinociception.

12

Conversely, a TRPV1 antagonist and a FAAH inhibitor increased the CBD-induced

13

analgesia, suggesting that the TRPV1 have a different role from CB1 increasing nociception

14

and that CBD actions might be depending on AEA. CBD can inhibit FAAH, increasing

15

endogenous AEA concentration (Leweke et al., 2012; McPartland et al., 2015). AEA is an

16

agonist of both CB1 and TRPV1 receptors (Di Marzo and De Petrocellis, 2012), as well as

17

can bind to CB2 receptor, but with less efficacy, behaving like a CB2 antagonist in some in

18

vitro bioassays (McPartland et al., 2015; Pertwee, 2008b). CB1 and TRPV1 have a distinct

19

mechanism of action: CB1 is a metabotropic receptor coupled to a Gi-protein while TRPV1 is

20

a channel permeable to several cations such as calcium (Di Marzo and De Petrocellis, 2012).

21

Therefore, CBD or AEA can produce opposite effects acting on these receptors (Di Marzo

22

and De Petrocellis, 2012).

23

Although the activation of CB1 receptors exert an anti-nociceptive and TRPV1

24

activation, a pro-nociceptive effect, the anandamide (AEA) has low intrinsic efficacy at

25

TRPV1 as compared to CB1 ((Ross, 2003; Toth et al., 2009; McPartland et al., 2015), and this

26

could explain the preference of the CB1, instead TRPV1, modulation by increased levels of

27

anandamide. In addition, the efficacy of AEA on TRPV1 is influenced by many factors and

28

physiological and pathological states, including the fact that AEA action on CB1 receptors

29

can change the TRPV1-mediated AEA response. In this sense, it is possible to suggest that the

30

smaller dose of CBD increase AEA, by inhibiting the FAAH, evoking a CB1-mediated

31

response while intermediate doses of CBD increase AEA to levels sufficient to activate both

32

CB1 and TRPV1 receptors eliciting lesser CBD-mediated antinociception, thus explaining the

33

U-shaped curve for the CBD in our experimental model. Another work using the FAAH

34

inhibitor URB597 injected into the PAG also found a dual effect for AEA in nociception. A

16 1

low dose of URB597 into the ventrolateral PAG evoked a hyperalgesic effect that was

2

converted to antinociceptive when injected along with the CB1 receptor inverse agonist

3

AM251. However, a TRPV1 antagonist converted the antinociceptive effect of the high dose

4

of URB597 to a hyperalgesic effect (Maione, 2006).

5

In vivo microdialysis showed that intraplantar injection of formalin increase AEA

6

release within the PAG (Walker et al., 2002) while electrical stimulation of dorsal or lateral

7

columns of the PAG evokes CB1-mediated antinociception along with a rise in AEA levels in

8

the PAG (Martin et al., 1999). Moreover, mice lacking FAAH presents increased thermal

9

analgesia and reduced nociceptive behavior in the formalin and carrageenan models (Carey et

10

al., 2016). In the same experimental pain model, intra-spinal injection of AEA also evokes

11

analgesia while CB1 receptor blockade inhibits these AEA effects (Carey et al., 2016). In the

12

same way, the acute administration of URB597 decrease allodynia in a neuropathic pain

13

model, and the pretreatment with a CB1 antagonist blunts this effect (Kinsey et al., 2009).

14

Altogether, these results suggest that AEA has antinociceptive effects acting via CB1

15

receptors in different experimental pain models.

16

The CB1 receptor is present in several brain regions involved in pain processing such

17

as PAG, dorsal root ganglion, superficial laminae of the spinal cord, cortical areas, and

18

amygdala (Di Marzo and De Petrocellis, 2012; Starowicz and Finn, 2017). Administration of

19

WIN55,212-2, a synthetic cannabinoid agonist, into the amygdala increases tail-flick latency

20

in rats, while the pharmacological blockade of CB1 receptors within the basolateral amygdala

21

attenuates the stress-induced analgesia in the same pain mode (Connell et al., 2006). In

22

nonhuman primates, the amygdala is essential to cannabinoid-induced antinociception

23

(Manning et al., 2018), thus suggesting that amygdala may be a target for AEA-induced

24

analgesia after peripheral CBD administration.

25

Amygdala and prefrontal cortex are areas that play a major role in mood disorders

26

(Price and Drevets, 2010), and both are involved in the neurocircuitry of pain (Taylor, 2018;

27

Xiao and Zhang, 2018). Neuroimaging studies show that these areas are affected in PD

28

patients (Chagas et al., 2017; Yoshimura et al., 2005), thus contributing to the increased risk

29

of anxiety and depression in these patients. Mood disorders can promote hypersensitivity to

30

pain (Bushnell et al., 2013; Taylor, 2018), and a study showed a correlation between

31

depression and pain in PD (Rana et al., 2017). Therefore, since several works demonstrate

32

that CBD is useful to treat affective disorders such as depression (Blessing et al., 2015; Crippa

33

et al., 2018; (Campos et al., 2016), the modulation of the emotional component of pain can be

34

part of CBD evoked-antinociception in our experimental model.

17 1

5.

Conclusion

2

In summary, our results suggest that CBD decreases the enhanced nociception of

3

animals with selective loss of dopaminergic nigrostriatal pathway. URB (selective FAAH

4

inhibitor) treatment potentialized the CBD antinociception effect. On the other hand, the

5

blockage of the CB1 receptors with the selective antagonist AM251 inhibited the CBD effect

6

while TRPV1 receptor antagonism increased the antinociception effect of CBD. Altogether,

7

these results suggest that CBD decrease the nociception inhibiting FAAH and consequently

8

increasing AEA. In turns, AEA can bind to CB1 and TRPV1 receptors. These receptors have

9

opposite effects on pain modulation. Thus, AEA binds to CB1 receptors promoting analgesia

10

while decreases the nociception via TRPV1 receptors.

11 12 13 14

Acknowledgments We thank Célia Aparecida da Silva for technical assistance and Mauricio dos Santos

15

Pereira for drug offering.

16

Author contributions

17

G.C.N., D.P.F, M.B. and N.C.F.J. performed experiments. F.S.G. and E.A.D.B.

18

provided feedback on project and participated in writing the manuscript. G.C.N., D.P.F,

19

M.B. and N.C.F.J. designed study, analyzed data, interpreted results, and wrote the

20

manuscript.

21

Financial Support

22 23 24

This study was supported by grants from CNPq, CAPES, and FAPESP. Conflict interests There is no conflict of interests to declare.

25 26 27

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28 29 30 31

Figure legends Figure 1. Time-course of 6-OHDA-induced hyperalgesia and allodynia responses. (A)

28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Photomicrographs of coronal sections from brains at different positions relative to Bregma illustrating the loss of TH + fibers in the rostral (A’), medial (A’’) and caudal (A’’’) striatum. 10x, Bar=50um. (B-D) Two-way analysis of variance (repeated measure) with Bonferroni’s post-hoc test for Basal measures (1 day before surgery: -1) and for 6-OHDA-treated rats 1, 7, 14 and 21 days after surgery. Decrease in thermal (Hot Plate - B, Tail Flick - C, Acetoneinduced cold stimulus - D) and mechanical (Von Frey - E) withdrawal thresholds in 6-OHDAtreated rats have distinct temporal profiles. Data represent the mean ± SEM. *p < 0.05 from Basal (-1) measure, n = 8/test. Figure 2. Antinociceptive effects of CBD on experimental parkinsonism. Antinociceptive effects induced by CBD (10, 30, and 100 mg/kg), in the Hot Plate (A), Tail Flick (B), acetone-induced cold stimulus (C), and Von Frey tests (D). Data represent the mean ± SEM. *p < 0.05 from VEH group, #p < 0.05 from CBD100 group; +p < 0.05 from MOR groups n = 8/group. Figure 3. Antinociceptive effects of chronic therapy with CBD on experimental parkinsonism. Antinociceptive effects induced by chronic injection of CBD (10 mg/kg), in the Hot Plate (A), Tail Flick (B), acetone-induced cold stimulus (C), and Von Frey tests (D). Data represent the mean ± SEM. *p < 0.05 from VEH group, #p < 0.05 from CBD100 group; +p < 0.05 from MOR groups n = 8/group. Figure 4. Antinociceptive effects of URB597 (URB), AM251 (AM), capsazepine (CPZ) and SCH336 (SCH) on experimental parkinsonism. Antinociceptive effects induced by URB (0.5 mg/kg), AM251 (1 mg/kg), CPZ (5 mg/kg) and SCH (2 mg/kg), in the Hot Plate (A), Tail Flick (B), acetone-induced cold stimulus (C), and Von Frey tests (D). Data represent the mean ± SEM. n = 8/group. Figure 5. Involvement of CB1, CB2, FAAH and TRPV1 in the antinociceptive effects induced by CBD on experimental parkinsonism: proposal mechanisms. Effects of the pretreatment with FAAH inhibitor URB (0.5 mg/kg); CB1 receptor antagonist AM251 (1 mg/kg) and TRPV1 antagonist CPZ (5 mg/kg) followed by CBD (10 mg/kg) in the Hot Plate (A), Tail Flick (B), acetone-induced cold stimulus (C) and Von Frey tests (D). Data represent the mean ± SEM. *p < 0.05 from VEH group, #p < 0.05 from CBD10 group n = 5-7/group.

1

Highlights •

The CBD treatment decreases hyperalgesia and allodynia in experimental parkinsonism;

•

The inverse agonist of the CB1 receptor prevents the antinociceptive effect of CBD;

•

FAAH inhibitor or TRPV1 antagonist potentiates the CBD antinociceptive effect