Pain management in zebrafish

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe.

Reliability: A reliable assessment tool is one that yields similar results when applied to the same individual experiencing the same level of pain at different times (test-retest reliability or intra-observer reliability) and when applied by different raters (inter-rater or inter-observer reliability).Reliability measures the ratio of true variance in the test to total variance.In a perfect test, true and total variance are the same, and the ratio will be 1. 1 Validity: Validity assesses a tool's capacity to quantify the construct it is designed to measure.Validity can be divided broadly into three concepts: • face and content validity -a judgement (not assessed empirically) that the tool is assessing what it purports to; assessed subjectively • criterion-related/concurrent validity -correlation of results of the tool compared with an existing gold standard test; without a gold standard, this form of validity cannot be established • construct validity -a tool's capacity to objectively assess the construct that it sets out to assess.This can be established by determining whether the tool can distinguish between a group known to have pain and one that does not.It can also be established by examining whether the tool yields results that vary appropriately according to changes in pain intensity, for example as a result of treatment. 2

Pain Assessment in Larval Zebrafish
Larval (typically less than 5 days post fertilisation (dpf) but see below) have been used in the study of nociception and pain.The types of parameters assessed are listed:

•
Reduction of activity 0-6 dpf (number of movements) 3,4 • Reduction in speed of swimming (5 dpf, mm/sec) 4 • Increased distance travelled, 5 dpf (measured as cm or mm/10sec) 5,6 , effect reduced after use of medicine (4 dpf) 5 • Reduction of time being active (5 dpf, %), seems to be a more sensitive parameter, this alteration was prevented with the use of analgesics 4 • Altered swimming speed (5dpf, up or down depending on stimulus) 7 • Increased thigmotaxis (5 dpf) 8,9 • Increased heart rate (5 and 7 dpf, not 4 dpf, normoxia at 25ºC =180 beats/min 2-14 dpf) 10,11 • Ventilation rate is altered but the noxious stimulus was lack of oxygen or hypoxia (3-5 dpf) 12 A range of drugs have been tested within the above cited studies in larval zebrafish to determine if these reduce behavioural responses to potentially painful treatment and these are listed in a simple summary below (Table 1).

Drugs with analgesic properties
Table 2 provides a simple summary of drugs with pain-relieving properties for adult zebrafish -see the main text for references.For injection a maximum of 10 µl is proposed and for immersion where the drug is dissolved in the holding water.Deakin et al. immersed for 70 minutes and demonstrated that the effective drugs and doses prevented behavioural changes in response to painful treatment. 14low we highlight a range of drugs with analgesic properties that were demonstrated in zebrafish or other fishes.The following drugs are used in mammals to provide analgesia and it is possible with further testing they could be administered to zebrafish.

Medetomidine
The (alpha-2 adrenoceptor agonist) medetomidine is used as an anaesthetic pre-med with strong sedative and analgesic properties Medetomidine injected intramuscularly (IM) at 0.025 mg/kg into goldfish reduced the amount of additional anaesthetic needed and thus demonstrated an anaestheticsparing effect where lower concentrations of MS222 could be used in combination with medetomidine. 15However, rainbow trout exposed to 6mg/l medetomidine became sedated rather than anesthetised. 16Medetomidine combined with ketamine (100 μg/mL ketamine + 1.25 μg/mL medetomidine) induced a rapid surgical anaesthesia in zebrafish but recovery was more prolonged than MS222. 17Further studies are required to understand the analgesic properties of medetomidine and its use in combination with other drugs.

Clonidine
In an assay exploring the use of small molecules with known analgesic properties as fish analgesics, 5 dpf zebrafish larvae sensitised with mustard oil were subjected to Clonidine immersion (5μM) and displayed an inhibition of acute and sensitized temperature aversion. 13

Amitriptyline
Amitriptyline is a tricyclic antidepressant that is commonly used to treat chronic pain in humans. 17mersion with this drug (0.5μM, dissolved in DMSO), successfully mitigated thermal aversion in 5 dpf zebrafish. 13

Feprosidnine
Feprosidnine is a stimulant drug which was developed in the Soviet Union in the 1970s; injected intraperitoneally in cod at 15-100 mg/kg this compound showed a substantial and strongly dosedependent reduction in nocifensive behaviour after challenge with electric shocks. 19

Anaesthesia in zebrafish
The following are recommended as suitable agents for inducing general anaesthesia in fish and some drugs have analgesic properties.See Table 3 for anaesthetics not described in detail and Table 4 for anaesthesia indicators.

Tricaine/MS222 and benzocaine
Tricaine (MS222) and benzocaine are members of the amine family of voltage-gated sodium channel blockers and, in other species, are more commonly used as local anaesthetic agents. 20Tricaine is easily soluble in water or embryo medium, but the resulting aqueous solution has low pH and therefore requires buffering with the same amount of sodium bicarbonate. 213][24] However, since tricaine has a history of safe use in zebrafish, and that its efficacy, safety and recovery profile is superior to the other commonly used anaesthetic agents, it remains the preferred anaesthetic agents for most researchers and in some countries, is one of the only approved anaesthetics for use in fish. 25Researchers must check the regulations and availability of drugs in their own country since this can vary widely.Since tricaine and benzocaine are used at sufficiently high concentration to induce general anaesthesia, there would seem to be little pharmacological rationale for using drugs of the same class (e.g.lidocaine and novocaine) for peri-operative analgesia.Indeed, there is the possibility that the administration of such agents of the same pharmacological class may be more likely to result in over-dosing if used in combination, but this has been done for zebrafish where benzocaine was used as anaesthetic and a low dose of lidocaine (5 mg/L) provided effective pain-relief. 14,26However, there is a case for using combinations of agents, where the drugs in question act via different receptors.Such an approach is commonly used in mammals (see main text).One such study in zebrafish demonstrated propofol/lidocaine combined use induced a quicker loss of equilibrium, and loss of response to light and painful stimuli compared with MS222. 27Since propofol acts via GABA receptors whereas lidocaine works via voltage-gated sodium channels, it is possible that these drugs could be used at lower concentrations than when used singly.It is also necessary to consider the half-life of anaesthetic drugs with analgesic properties since it is possible that the analgesic effects may persist after recovery from anaesthesia if the agent used has a long half-life.Studies on the pharmacokinetics of these drugs are limited in fish.Further environmental (e.g.temperature, salinity) and biological factors (e.g.developmental stage, body condition) will affect both the action and half-life of anaesthetics (review 28 ).Although there is little information about half-life, adult zebrafish recover equilibrium within ~90-140 seconds after anaesthesia with tricaine, suggesting that it may be rapidly metabolised or excreted. 25,29Although pain reflexes are abolished during the period of anaesthesia, this rapid recovery indicates that additional peri-and post-operative analgesia should be considered. 25The number of times a fish is anaesthetised during one experiment is an important factor to consider in case repetitive anaesthesia and handling impairs the welfare of the fish.However, this may be beneficial if it results in a reduction in the number of animals used. 30Caution should be applied since it has been reported that daily MS222 exposure can cause mortality. 31her anaesthetic agents, such as clove oil (eugenol and isoeugenol), etomidate and metomidate, quinaldine sulphate, ketamine, alphaxalone, have been reported as suitable for use in fish.For many of these agents there is very little data on their efficacy or safety in zebrafish and are therefore less relevant for consideration here but are included in the table below as potential alternatives.
Table 3. Overview of other agents used as anaesthetics for fishes, with notes on their use, aversiveness and reference.Drugs in bold have been used in zebrafish.

Table 2 .
Effective pain relieving drugs used in adult zebrafish administer via immersion, injection intraperitoneally (IP) or injected intramuscularly (IM).For references, please see Table3of the main publication.