Veterinary Anaesthesia and Analgesia 2017, xxx, 1e9 https://doi.org/10.1016/j.vaa.2017.08.

009

RESEARCH PAPER
64
65
1 Lack of postexposure analgesic efficacy of low 66
2 67
3 Q21 concentrations of eugenol in zebrafish 68
4 69
5 70
6 Bernardo Baldisserottoa, Thaylise V Parodia & E Don Stevensb 71
Q1
Q20
7 Q2 a 72
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Santa Maria,
8 73
9 RS, Brazil
b 74
10 Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward
75
11 Island, Charlottetown, PEI, Canada
76
12 77
13 Correspondence: Bernardo Baldisserotto, Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, 97105- 78
14 Q3 900 Santa Maria, RS, Brazil. E-mail: bbaldisserotto@hotmail.com 79
15 80
16 81
17 82
18 Abstract Introduction 83
19 84
20 Objective To test the postexposure analgesic effi- Although there is a controversy regarding the ca- 85
21 cacy of low doses of eugenol in zebrafish. pacity of fish to experience pain (Braithwaite 2010; 86
22 Rose et al. 2014), essentially all persons involved in 87
23 Study design Prospective experimental study. 88
the controversy agree that fish are capable of noci-
24 89
25 Animals A total of 76 large adult zebrafish (Danio ception, the perception of a noxious stimulus
90
26 rerio). (Malafoglia et al. 2013; Curtright et al. 2015). Fish
91
27 have nociceptors with properties that are very similar 92
Methods Fish swimming behavior (median veloc-
28 to those found in mammals (Braithwaite 2010). After 93
29 ity, freeze time, high-speed swimming and distance
neural encoding of a noxious stimulus, fish respond to 94
30 moved in the vertical direction) was recorded in a
it in a variety of ways depending on the stimulus and 95
31 1.6 L video arena before and after exposure to 96
fish species. This response is referred to as a noci-
32 eugenol (0, 1, 2, 5, 10 and 20 mg L
1). In a 97
33 fensive response (Rose et al. 2014). Persons working
second experiment, fish were anesthetized with 2- 98
34 with fish generally agree that it is appropriate to try
phenoxy-ethanol and treated with an injection of 99
35 to minimize these nocifensive responses. 100
5% acetic acid (noxious stimulus), and then
36 A variety of anesthetic drugs have been investi- 101
37 exposed to 0, 1, 2 and 5 mg L
1 eugenol. The fish
gated for their properties to reduce the nocifensive 102
38 swimming behavior was also recorded.
responses, to reduce the stressful impacts of handling, 103
39
Results The higher doses (10 and 20 mg L
1) especially during routine procedures, such as 104
40 105
41 reduced the median velocity, high-speed swim- weighing, vaccination, blood sampling, tagging,
106
42 ming and distance moved in the vertical direction, experimental surgery and veterinary procedures
107
43 and increased the freeze time. Zebrafish behavior (Cunha et al. 2010a,b; Becker et al. 2012; Gressler 108
44 was not altered by eugenol (1, 2 and 5 mg L
1) et al. 2012; Parodi et al. 2014). Immersion anes- 109
45 after noxious stimulation. thesia in fish is analogous to gaseous inhalant anes- 110
46
thesia in terrestrial animals. The fish ventilates the 111
47 Conclusions and clinical relevance The change in 112
anesthetic dissolved in the water, which enters the
48 the behavior of zebrafish associated with a noxious 113
49 bloodstream mainly through the gills.
stimulus can be monitored and is a good model for 114
50 However, there are few studies investigating or
studying analgesia in fish. Eugenol (10 and 20 mg 115
51 demonstrating analgesic activity of drugs used as
L
1) induced zebrafish sedation. The response after 116
52 anesthetics in fish. Most studies focusing on anes- 117
53 a noxious stimulus was not affected by post-
thesia and analgesia measure and report the com- 118
54 exposure to lower doses, and thus we cannot
plete immobility of the animal. However, the 119
55 recommend its use as an analgesic. 120
distinction between an anesthetic with analgesic
56 121
57 properties and an immobilizing drug (anesthetic
Keywords fish, noxious stimulus, pain, sedation, 122
58 without analgesic properties) is not always clear
swimming behavior. 123
59 124
60 125
61 1 126
62 127
63 128
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
2 67
(Harms 2005). In addition, anesthetics do not always Chemicals and eugenol
3 68
4 provide analgesic effects, and can increase or have no 69
All chemicals were purchased from Sigma-Aldrich
5 effect on the stress response of fish (Weber et al. 70
(MO, USA). Solutions of acetic acid (5%) were dis-
6 2009) and/or on the nocifensive response of fish 71
solved in deionized water. Eugenol was purchased
7 (Rose et al. 2014). 72
8 commercially (Odontofarma, RS, Brazil), and the 73
Eugenol [2 methoxy-4-(2-propenyl) phenol] is the
9 analysis of this product by gas chromatography/mass 74
principal active ingredient of clove oil, which is
10 spectrometry (Varian Saturn 2200; Agilent Tech- Q4 75
derived from the leaves, buds and stems of the clove
11 nologies Inc., CA, USA) revealed 99.03 ± 0.15% 76
12 tree (Eugenia caryophyllata), and has wide use as an 77
eugenol (n ¼ 3) (Gomes et al. 2011).
13 anesthetic for aquatic organisms because of its low 78
14 price and ready availability (Roubach et al. 2005; 79
Behavioral variables
15 Hoseini et al. 2015). Several studies have evaluated 80
16 its use to reduce fish hypermobility, thus reducing Behavioral testing was performed between 1100 and 81
17 fish stress during handling (Palic et al. 2006; Cunha 1500 hours because the response of zebrafish to 82
18 83
et al. 2010a). Eugenol has a peripheral local anes- eugenol can change with the period of the day
19 84
20 thetic (antinociceptive) action on axons (Markowitz (S

anchez-V
azquez et al. 2011). Each fish was placed 85
21 et al. 1992), especially on specific voltage-gated so- individually in a 1.6 L video arena (17  8  12 cm 86
22 dium (Naþ) channels, at least in mammals (Wang deep) supplied with tank water at 25  C, pH 6.8e7.4 87
23 et al. 2015). The aim of the present study was to and constant aeration. The video arenas were 88
24 analyze the swimming behavior of zebrafish to test covered with a black lid and with blue Styrofoam on Q5 89
25 the efficacy of postexposure to subanesthetic and all sides but the front to diminish the influence of 90
26 91
subsedative doses of eugenol for analgesia in fish. Our external stimuli. After a 5 minute settling period, the
27 92
28 hypothesis was that a noxious stimulus decreases swimming activity was recorded for 35 seconds (time 93
29 zebrafish swimming activity and that subsedative pre) using a camera facing the front of the tank with a 94
30 doses of eugenol will prevent this behavioral change 17  12 cm view. Online records were stored on the 95
31 even after the fish are no longer exposed to eugenol. computer and later analyzed for activity (LoliTrack 96
32 Version 3.0.0 or 4.1; Loligo Systems, Denmark). 97
33 Materials and methods Distance was calculated as the change in position in 98
34 99
two dimensions, and velocity as this distance per
35 All experiments were performed in accordance with 100
36 the Canadian Council on Animal Care guidelines, and time. Q6 101
37 were approved by the local Animal Care Committee The variables used to analyze the analgesic effect 102
38 at the University of Prince Edward Island, Atlantic were based on those proposed by Cachat et al. 103
39 (2010), and measurement time was 30 seconds: 1) 104
Veterinary College (number 09-004).
40 median velocity during the trial (cm second
1); 2) 105
41 freeze time (time during the trial with no change in 106
42 Animals and housing 107
position): % of time that the fish moved with speed
43 108
44
Large adult zebrafish (Danio rerio) were obtained from <0.5 cm second
1; 3) high-speed swimming (time 109
45
a local supplier, and maintained in 40 L tanks at 28.3 spent swimming at high speed): % of the time that 110
46 ± 0.4  C (mean ± standard deviation). They were fed fish swam with speed 10 cm second
1; and 4) 111
47 flake food three times daily (Nutrafin Max; Hagen vertical distance (total distance moved in the vertical 112
48 Ltd., QC, Canada) with automatic feeders (model direction): total cm in a 30 second trial. 113
49 3581; EHEIM GmbH & Co. KG, Germany), and The difference between pre-exposure and post- 114
50 maintained on a 12/12 hour photoperiod. Illumina- 115
exposure was calculated for each variable, such that
51 116
tion was provided by fluorescent lights on a 24 hour zero would indicate no effect of eugenol or treatment.
52 117
53
cycle (on at 0600 hours/off at 1800 hours). Deion- 118
54 ized water was supplemented with Nutrafin Aqua Experiment 1: postexposure effects of low doses 119
55 Plus (5 mL in 40 L; Hagen Ltd.), EasyBalance (5 mL in of eugenol 120
56 40 L; Tetra Holding Inc., VA, USA) and sea salt (2 g in 121
57 40 L; D-D The Aquarium Solution Ltd., UK). The fish After recording the behavioral parameters in un- 122
58 were acclimated to fish housing for 2 months prior to treated fish (pre-exposure), each fish was transferred 123
59 to a 100 mL beaker containing eugenol in tank water 124
the experimental trials. Holding tank water was used
60
to make anesthetic and recovery tank water. (0, 1, 2, 5, 10 or 20 mg L
1, previously diluted in 125
61 126
62 2 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
2 67
ethanol 1:9; density of eugenol 1.07) where it was with an injection of 5% acetic acid (noxious stim-
3 68
4 exposed to the drug for 45 minutes (Fig. 1). After ulus). The injection was 5 mL into the face, between 69
5 exposure to eugenol, the fish was returned to the the nostrils, using a Hamilton syringe (33 gauge Q7 70
6 video arena and, after a rest time of 5 minutes, the needle) and an automatic injector. The maximum 71
7 swimming behavior was recorded for 35 seconds time for anesthesia, injection and recovery was 3 72
8 (time post1). The fish was then returned to aquarium minutes. After the completion of each experiment, 73
9 water without eugenol for 45 minutes, before being the fish were euthanized using the National Institutes 74
10 75
transferred to the video arena where, after a rest time of Health guidelines with an overdose of MS-222
11 76
12 of 5 minutes, the swimming behavior was recorded (300 mg L
1; Aqualife TMS; Syndel, BC, Canada). 77
13 for 35 seconds (time post2). This procedure was 78
14 repeated for a third recording of the swimming Statistical analysis 79
15 behavior after exposure to eugenol (time post3; 80
16 Different eugenol concentrations in experiment 1 (no 81
Fig. 1). Exactly 30 seconds of the approximate 35
17 noxious stimulus) were compared using the general 82
second video clip was analyzed.
18 linear model (GLM), followed by a post hoc Dunnett’s Q8 83
The eugenol concentrations and exposure times
19 test that was used to compare each dose with control 84
20 were chosen based on previous tests using concen- 85
(dose ¼ 0). This was further tested using a one-
21 trations of <40e60 mg L
1 that induce anesthesia in 86
sample t test to test if the variable differed from
22 fish. Five to seven zebrafish were studied for each 87
0 (i.e. no effect) with a Bonferroni correction for four
23 concentration tested, no fish was tested twice and 88
24 tests (i.e. there were four variables per trial; use 0.05/ 89
drug concentrations were randomized so that all doses
25 4 ¼ 0.0125 for 95% confidence). 90
were tested on each trial day, but the order of doses
26 The high doses (10 and 20 mg L
1) were pooled to 91
was determined using a table of random numbers.
27 test the duration of the effect. The data at time post2 92
28 and time post3 were tested to see if the mean was 93
29 Experiment 2: postexposure effects of low doses 94
significantly different from 0 using a one-sample t test
30 of eugenol on the response to a noxious stimulus 95
with a Bonferroni correction for eight tests (i.e. there
31 96
32 Based on the results from experiment 1, eugenol were four variables per trial and two times; use 0.05/ Q9 97
33 concentrations of 0, 1, 2 and 5 mg L
1 were tested 8 ¼ 0.0062 for 95% confidence). 98
34 and swimming behavior recorded before and at 5 Different eugenol concentrations in experiment 2 99
35 minutes after exposure (times pre and post1). Nine were compared using GLM and post hoc Dunnett’s 100
36 zebrafish were tested at each dose. The protocol was test. Data were expressed as mean ± standard error of 101
37 similar to experiment 1 except that, immediately the mean. The two experiments were carried out on 102
38 103
before the exposure to eugenol, each fish was different batches of fish at different times, and thus it
39 104
40 immediately anesthetized with 2-phenoxyethanol is inappropriate to compare them statistically. 105
41 (0.4 mL L
1 until loss of equilibrium and response The minimum significance level was set to p ¼ 106
42 to touching; usually about 1 minute) and treated 0.05. Non-normal data were transformed. All statis- 107
43 tical analyses were performed using the software 108
44 Statistica Academic Version 10.0 (TIBCO Software 109
45 Inc., CA, USA) and Minitab Version 17.3 (Minitab 110
46 111
Inc., PA, USA).
47 112
48 113
49
Results 114
50 There were no mortalities or adverse side effects 115
51 116
noted during any of the trials in either experiment.
52 117
53 118
54 Figure 1 Timeline for experiment 1. Arrows indicate
Experiment 1 119
55 recording of swimming behavior in the video arena after 120
The effect of eugenol immersion was only evident at
56 acclimatization for 5 minutes. After baseline recording 121
57 (pre), the fish was exposed to a eugenol concentration for
the first observation period 5 minutes after the end of 122
58 45 minutes. The swimming behavior was recorded 5 mi- exposure (time post1), and the behavior returned to 123
59 nutes after it was returned to the video arena (post1), and normal for the later observation periods. There was a 124
60 again at 55 and 105 minutes after exposure (post2 and significant dose effect for all four variables (median 125
61 post3). 126
62 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 3 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
2 67
velocity, p ¼ 0.001; vertical movement, p ¼ 0.009); two variables using GLM (median velocity, p ¼
3 68
4 freeze time, p ¼ 0.001; time at high velocity, p ¼ 0.005; vertical distance, p ¼ 0.015) and for freeze 69
5 Q10 0.009; Fig. 2). Post hoc tests comparing the results of time using KruskaleWallis (p ¼ 0.001), but not for 70
6 different doses with a dose of zero showed that the time at high-velocity swimming (p ¼ 0.058; Fig. 3 & Q11 71
7 higher doses (10 and 20 mg L
1) differed from the Table 2). Based on the results of this first experiment, 72
8 control value (dose of zero). However, lower doses (1, in experiment 2, only the lower doses (0, 1, 2 and 5 73
9 2 and 5 mg L
1) did not differ from a dose of zero for mg L
1) were studied, and only the post1 time point. 74
Q12
10 75
any of the four variables (Bonferroni p ¼ 0.5 for all
11 76
12 tests; Table 1). Experiment 2 77
13 The duration of the effect of eugenol was tested 78
None of the four behavior variables tested were altered
14 using the higher doses only (pooled values at 10 and 79
by eugenol after the noxious stimulation. For fish
15 20 mg L
1). There was a significant time effect for 80
16 subjected to a noxious stimulus, there were no 81
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57 Figure 2 Effect of eugenol dose on swimming behavioral variables of zebrafish (five to seven fish per dose): (a) median 122
58 velocity, (b) vertical distance, (c) freeze time and (d) high-speed swimming time. Pre-exposure (pre) values were compared 123
59 with values recorded 5 minutes after removal from the eugenol solution (post1). Data points were calculated as post1 minus 124
60 pre values, and presented as least-square means ± standard error of the mean. The reference line at zero denotes no difference 125
61 between pre and post. *Significantly different from dose 0 (p < 0.05). 126
62 4 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
2 67
Table 1 Effect of exposure to eugenol (pooled values from 10 and 20 mg L
1) for 45 minutes on zebrafish behavior
3 68
Q19 measured 5.3 minutes after removal from the eugenol solution (n ¼ 11)*
4 69
5 70
6 Variable Mean SEM py 71
7 72
Median velocity (cm second
1) e2.898 0.399 <0.001
8 73
Vertical distance (cm in 30 seconds) 134.9 28.7 0.001
9 74
Freeze (% total time) 76.36 9.91 <0.001
10 75
High-speed swimming (% total time) e15.84 4.10 0.003
11 76
12 SEM, standard error of the mean. *Data are the measured value minus the value recorded pre-exposure. yThe p value reflects the result of testing if the 77
13 variable was different from 0, where 0 indicates no effect. 78
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57 Figure 3 Duration of the effect of eugenol (pooled values from 10 and 20 mg Le1) on swimming behavioral variables of 14 122
58 zebrafish: (a) median velocity, (b) vertical distance, (c) freeze time and (d) high-speed swimming time. Time 0 is the end of 123
59 exposure to eugenol. Data points are the measured value minus the pre-exposure value presented as means ± standard error 124
60 of the means. The reference line at zero denotes no difference between pre and post. *Significantly different from zero, or the 125
61 reference line of no effect (p < 0.05). 126
62 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 5 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
2 67
Table 2 Effect of exposure to eugenol (pooled values from 10 and 20 mg L
1) for 45 minutes on zebrafish behavior
3 68
measured 55.9 and 106.5 minutes, time points post2 and post3, respectively, after removal from the eugenol solution
4 (n ¼ 11)* 69
5 70
6 71
7 Variable Mean SEM p 72
8 73
Time post2
9 74
Median velocity (cm second
1) e0.024 0.623 0.97
10 75
Vertical distance (cm in 30 seconds) 80.9 71.0 0.28
11 Freeze (% total time) 8.48 8.49 0.34
76
12 High-speed swimming (% total time) 0.4 6.07 0.95 77
13 Time post3 78
14 Median velocity (cm second
1) e0.680 0.692 0.35 79
15 Vertical distance (cm in 30 seconds) 29.4 77.4 0.71 80
16 Freeze (% total time) 6.30 12.5 0.62 81
17 High-speed swimming (% total time) 4.77 6.18 0.46 82
18 83
19 SEM, standard error of the mean. *Data are the measured value minus the value recorded pre-exposure. 84
20 85
21 86
22
significant differences between fish without eugenol eugenol in fish (Hikasa et al. 1986; Keene et al. 1998; 87
23 and fish administered with eugenol at 1, 2 or 5 mg L
1: Roubach et al. 2005; Cunha et al. 2010a; Gomes et al. 88
24 median velocity during the trial (p ¼ 0.72), vertical 2011; Filiciotto et al. 2012; Fabiani et al. 2013; 89
25 distance moved during the trial (p ¼ 0.95), freeze time Iversen et al. 2013; Hoseini et al. 2015), but those 90
26 (p ¼ 0.89) or time swimming at high speeds (p ¼ 0.79) that presented data regarding sedation analyzed only 91
27 (Fig. 4). When compared with the results of experi- the time to induce partial loss of response to external 92
28 93
ment 1, the noxious stimulus resulted in a decrease in stimuli (Hikasa et al. 1986; Roubach et al. 2005;
29 94
30
median velocity, vertical distance and high-speed Cunha et al. 2010a; Gomes et al. 2011; Filiciotto 95
31 swimming, and an increase in freeze time (Figs 2 & 4). et al. 2012). Sedation was also demonstrated in 96
32 zebrafish in the present study, because at the higher 97
33 Discussion doses tested (10 and 20 mg L
1), the median velocity 98
34 decreased and the freeze time increased at 5 minutes. 99
Analgesic protocols are available for a variety of ani-
35 Exposure to 45 mg L
1 clove oil (whose main com- 100
36 mals (Rang et al. 2012), and some information is 101
available about the use of analgesics in fish (Neiffer & pound is eugenol) for 1 minute in the light phase
37 102
38 Stamper 2009; Newby et al. 2009; Correia et al. decreased the swimming activity of zebrafish (S
anchez- 103
39 2011; Jones et al. 2012; Ward et al. 2012; Stevens V
azquez et al. 2011). The minimum eugenol dose to 104
40 2013; Sneddon 2015). Zebrafish is a good model for sedate silver catfish (Rhamdia quelen) in a study 105
41 analyzing the partial loss of response to external stimuli 106
the study of nociception (Curtright et al. 2015), and
42
shows neuroanatomical structures similar in embry- was 20 mg L
1 (Cunha et al. 2010a). Other studies 107
43 verified that the lowest eugenol doses tested induced 108
44 ological origin and organization to that of higher 109
vertebrates (Malafoglia et al. 2013). The behavioral sedation: 20 mg L
1 in common carp (Cyprinus carpio)
45 110
46 responses of zebrafish to a noxious stimulus of acetic- (Hikasa et al. 1986), 30 mg L
1 in sea bass (Dicen- 111
47 acid injection observed in the present study are similar trarchus labrax) (Filiciotto et al. 2012) and 35 mg L
1 112
48 to those of previous studies, that is, there is a decrease in tambaqui (Colossoma macropomum) (Roubach et al. 113
49 2005). Consequently, the analysis of the swimming 114
in activity (Reilly et al. 2008; Correia et al. 2011).
50 activity, as investigated in the present study, allows a 115
The first experiment of the present study was carried
51 more precise identification of the sedation stage in fish 116
52 out to establish the lowest dose of eugenol that pro- 117
duces neither anesthetic nor sedative effects, because if because minor alterations can be detected. Ethanol
53 118
54 fish are anesthetized or sedated, their behavior would was used to dilute eugenol. According to Readman 119
55 change, and it would not be possible to determine if et al. (2013), 330 mL L
1 ethanol is not aversive to 120
56 there is an analgesic effect. The results of these tests zebrafish. Moreover, experiments that test the effect of 121
57 ethanol typically expose zebrafish to 1% or 2% (Guo 122
also demonstrated that the duration of the effect of the
58 et al. 2015; Tran et al. 2016). The maximum con- 123
higher doses of eugenol was present at 5 minutes after
59 centration used in the present study was 180 mL L
1 or 124
60 exposure, but were gone by 55 minutes. Several 125
studies have demonstrated the anesthetic effect of 0.018%. Based on all studies of ethanol on zebrafish,
61 126
62 6 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
1 66
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41 Figure 4 Effect of 45 minutes of exposure to eugenol at 1, 2 and 5 mg Le1 followed by a noxious stimulus (acetic-acid 106
42 injection) on the 5 minute postexposure swimming behavior of zebrafish (nine fish per treatment): (a) median velocity, (b) 107
43 vertical distance, (c) freeze time and (d) high-speed swimming time. Data points are the measured value minus the pre- 108
44 exposure value presented as means ± standard error of the means. The reference line at zero denotes no difference be- 109
45 tween pre and post. 110
46 111
47 the probability of ethanol confounding our results is which it exerts its analgesic effects in mammals (Oz 112
48 extremely low. et al. 2015). The duration of zebrafish observation 113
49 114
Zebrafish brain has Naþ channels that are activated used in the present study would be sufficient for Q14
50 115
Q13 by extracellular Hþ (acid-sensing channels, zASIC), eugenol to produce an analgesic effect, because the
51 116
52 and zASIC1.1 may be expressed in nociceptors and act sedation effect occurs within 20e150 seconds in 117
53 as receptors for noxious stimuli (Paukert et al. 2004). some fish species (Hikasa et al. 1986; Roubach et al. 118
54 The activation of Naþ channels in peripheral termi- 2005; Cunha et al. 2010a; Gomes et al. 2011; 119
55 nals generates receptor potentials that can be sum- Filiciotto et al. 2012). Eugenol was probably still 120
56 med and effect an action potential (Ruiz & Kraus present in zebrafish at 5 minutes after withdrawal 121
57 122
2015). Eugenol inhibits Naþ currents in rat neurons from eugenol exposure (post1) when observation of
58 123
(Huang et al. 2012), apparently through its interac- the swimming activity was performed, because esti-
59 124
60 tion with both resting and inactivated Naþ channels, mates for elimination half-time are 26 minutes at 17 125
and it is considered as one of the mechanisms by  C in rainbow trout (Oncorhynchus mykiss) (Meinertz
61 126
62 © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 7 127
63 Elsevier Ltd. All rights reserved., ▪, 1e9 128
64 129
65 130
Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
 Eugenol for analgesia in fish B Baldisserotto et al.
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17
good model for studying analgesia in fish. The 82
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mento Científico e Tecnol
ogico (Brazil) and ED Ste- Rhamdia quelen. J Fish Biol 81, 1436e1445. 94
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Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009
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© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by 9
Elsevier Ltd. All rights reserved., ▪, 1e9

Please cite this article in press as: Baldisserotto B, Parodi TV, Stevens ED, Lack of postexposure analgesic efficacy of low
concentrations of eugenol in zebrafish, Veterinary Anaesthesia and Analgesia (2017), https://doi.org/10.1016/
j.vaa.2017.08.009