Modeling acute visceral pain in adult zebrafish

Transport animals from their holding facility to the experimental room for acclimation 1 h prior to the experiments. Avoid low- or high density of fish to prevent social isolation or crowding stress (tanks must have 1–2 fish per 1 L of water). Fish must acclimate to the facility before testing, and the water used must have similar physicochemical characteristics to those of housing tanks. The use of home tank or holding tank water (with properly adjusted temperature and salinity) is required. !CAUTION: Ensure that the experimental tank is filled with non-chlorinated water before use set at optimal temperature (27-28°C).

Mount a camera on the frontal side of tank test and connect video recorder to the power in a switch-on mode. Provide adequate illumination (fluorescent light bulbs), to ensure that the enlightenment is proper to differentiate the subject from the apparatus, allowing a precise detection of fish. However, avoid excessive brightly lit environments (stressful for zebrafish) and dark areas (poor fish recognition and video-tracking detection of locomotion if desirable). ? TROUBLESHOOTING

Prepare the acetic acid solutions in 1.5 mL microcentrifuge tubes (2.5% or 5.0% diluted in PBS or 0.9% NaCl).

Transfer the fish from the holding tank to the experimental tank and start recording for 6 minutes to measure the baseline behavioral phenotypes. Animals should be transferred individually to the experimental tank. Ensure adequate transport to minimize handling stress. !CAUTION: If more than a single injection is applied, the baseline behavior should be recorded after the first injection to avoid false positive/negative results. ?TROUBLESHOOTING

Before i.p. injections, video recordings must be stopped.

Remove the fish from the tank with a fishing net and proceed with the i.p. injection using a BD Ultra-fine™ 30U syringe (needle size 6 x 0.25 mm) with a volume of 10 μL (which does not impair normal zebrafish behaviors). Animals must be gently handled, briefly anesthetized, and later immobilized using a small wet fishing net (< 5 s). The i.p. injections should be quickly performed into the midline between the pelvic fins. To minimize potential interference of drugs in the behavioral endpoints measured and to allow a fast evaluation of the swimming activity, avoid using anesthetics (tricaine or other similar drugs). Injections can be quickly performed through the net in fish previously anesthetized in cold water without affecting complex behaviors ( e.g. , depth preference, immobility, and locomotion). ?TROUBLESHOOTING

Relocate the fish into the experimental tank to record the behavioral phenotypes following i.p. injections for 6 minutes.

Stop the behavioral recording and remove the fish from the experimental tank. Animals must be euthanized following national guidelines and standard protocols.

Discard the water used previously, wash the tank thoroughly with tap water and refill it with non-chlorinated water before testing another fish.

Repeat steps 4–9 for the next subject tested.

Digital pictures of fish at sagittal plane (Fig. 2) must be taken every 30 s, totaling 24 photos per fish. We strongly recommend the use of "Prt Sc" ( print screen ) Windows tool.

With ImageJ software (available for download at https://imagej.nih.gov/ij/download.html), open the digital pictures of fish sagittal plane that represent the first 30 s (1-2) , and using the angle tool (3) , select three positions to estimate the fish body angulation: a frontal (in the front of the head), a central (in the middle of the animal’s body – between the anal and dorsal fins), and a posterior one (at the caudal fin) (4) . Then, analyze and measure the fish angular value (5-7) . !CAUTION: Results must be subtracted from 180° to calculate a value representing the body curvature index (Fig. 3) . ?TROUBLESHOOTING

Several options exist for analyzing the data, based on the specific experimental design. Use the nonparametric Wilcoxon-Mann-Whitney U-test or parametric Student’s t -test (if data are homoscedastic or normally distributed) for comparing two groups. For more than two groups, use analysis of variance (ANOVA), followed by appropriate post-hoc comparison ( e.g. , Tukey, Dunn, Student-Newman-Keuls or Dunnet tests). Depending on the study design, an n -way ANOVA can generally be applied to assess various factors ( e.g. , drug/treatment, dose, sex, strain, time, trial, age). Even if a small number of cohorts is used ( n = 5), robust differences can be detected in acetic acid-treated fish vs. PBS (control group) ( e.g. , effect size calculated using Cohen's d = 3.646). Thus, the use of adult zebrafish to assess pain-like responses following a single i.p. acetic acid injection brings direct “3R's” benefits (refinement, replacement, reduction) of animal experimentation. Use ANOVA with repeated measures to assess time-dependent modulation of the observed phenotypes, if necessary. To facilitate data analysis, export data from every 30 s into separate Excel spreadsheets (one spreadsheet per fish tested in a 6-min trial). The area under curve (AUC) from each individual can be further calculated to express the specific behavioral endpoint using specific statistical packages ( e.g. , GraphPad Prism).

The anticipated results described below show robust pain-like responses in zebrafish as reported elsewhere.

Citation

Intraperitoneal injection of 5.0% acetic acid (AA) induces a typical, well-defined abdominal constriction writhing-like phenotype (measured as body curvature index) in zebrafish, analogous to writhing response observed in rodents. Fig. 4 illustrates typical results obtained using the present protocol. Notably, these results are robust and replicable even with a small number of subjects per cohort (e.g. , n = 5), obtained by trained researchers blinded to the experimental condition with inter-rater reliability > 0.85–0.90. Acetic acid-treated fish show a prominent increase in the body curvature index when compared to control (PBS). Interestingly, intraperitoneal injections do not affect the baseline curvature since no differences are observed in PBS-treated group.

Our protocol also enables studying the involvement of the opioid system in zebrafish pain-like behaviors, as well as the effects of potential pro- and anti-pain drugs. For example, co-administration of 2.5 mg/kg morphine (MOR), an opioid agonist classically used as an analgesic drug, inhibits the effects of acetic acid on the body curvature index, while a common ‘reference’ non-specific opioid antagonist naloxone (NAL, 5.0 mg/kg) predictably antagonizes morphine-evoked analgesic responses (Fig. 5) .

Pretreatment with the non-steroidal anti-inflammatory drug, diclofenac (40 mg/kg, for 15 min), another commonly used analgesic agent that does not target the opioidergic system, prevents acetic acid-induced changes in the body curvature index (Fig. 6) . These findings suggest that assessing writhing-like responses in the present protocol may be a sensitive and specific approach to evaluate pain-related phenotypes in adult zebrafish.

Overall, these robust behavioral and pharmacological responses show high predictive and face validity of the present zebrafish-based pain model. The protocol can be run using animals selected randomly from their home tanks since no differences in the body curvature index was observed when different genders are tested (Fig. 7) . Collectively, these data show that the protocol described here enables measuring specific acute pain responses in adult zebrafish.