TITLE: Modeling acute visceral pain in adult zebrafish
AUTHORS: Fabiano Costa, Denis Rosemberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a reliable procedure for assessing acute pain responses in adult zebrafish (</span><span style = "font-style:italic;">Danio rerio</span><span>) based on the abdominal writhing-like phenotype following a single intraperitoneal injection of acetic acid. The method is an inexpensive, fast, and easy-to-use protocol for measuring pain-like responses in adult zebrafish. The protocol involves five steps: analyses of baseline behavior, drug injection, post-injection recordings of behavior, euthanasia, and data analyses/interpretation. Intraperitoneal injection of 2.5–5.0% acetic acid elicits a robust pain-like behavior by changing zebrafish body curvature, which can be easily quantified using freely available imaging software. This response is sensitive to pharmacological manipulations, as morphine prevents altered body curvature, while naloxone blocks these effects. Pretreatment with diclofenac sodium (a non-steroidal anti-inflammatory drug commonly used as an analgesic) also prevents writhing-like behavior. Demonstrating high predictive and face validity, this unbiased protocol can be performed over the course of ~2 days, enabling a reliable assessment of acute pain-like responses in zebrafish, thus fostering in-depth analyses of complex pain-related mechanisms and anti-pain drug discovery.</span></div></div>

[STEPS]
?. [ACCLIMATIZATION AND BASELINE BEHAVIORAL RECORDINGS ●TIMING ~ 1 h for acclimatization; 6 min per animal]
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).
?. [ACCLIMATIZATION AND BASELINE BEHAVIORAL RECORDINGS ●TIMING ~ 1 h for acclimatization; 6 min per animal]
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
? TROUBLESHOOTING Problems: (1) Inappropriate video lighting.Possible reasons: (1) Lack or excess of brightness, heterogeneous background.Solutions: (1) Measure the light intensity above the experimental tank with a lux meter or open Android or IOS application (e.g., Lux meter, Crunchy ByteBox, Germany, or similar) to ensure adequate illumination. Use a styrofoam background to improve contrast.
?. [ACCLIMATIZATION AND BASELINE BEHAVIORAL RECORDINGS ●TIMING ~ 1 h for acclimatization; 6 min per animal]
Prepare the acetic acid solutions in 1.5 mL microcentrifuge tubes (2.5% or 5.0% diluted in PBS or 0.9% NaCl).
?. [ACCLIMATIZATION AND BASELINE BEHAVIORAL RECORDINGS ●TIMING ~ 1 h for acclimatization; 6 min per animal]
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
? TROUBLESHOOTING Problem: (1) Pretreatment alters the normal zebrafish behavior.Possible reasons: (1) The drug used affects the swimming activity of fish and/or elicits abnormal phenotypes. Incorrect injection. Solutions: (1) Run a pilot experiment using different concentrations of the drug. Analyze zebrafish behavior at different time intervals after pretreatment. Use highly-trained researchers to perform the injections.
?. [INTRAPERITONEAL INJECTIONS ●TIMING ~ 3 min per animal]
Before i.p. injections, video recordings must be stopped.
?. [INTRAPERITONEAL INJECTIONS ●TIMING ~ 3 min per animal]
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 (e.g., depth preference, immobility, and locomotion).?TROUBLESHOOTING
? TROUBLESHOOTING Problem: (1) Unconventional responses (e.g., fish do not exhibit curvature, fish die).Possible reasons: (1) Incorrect injection, incorrect acetic acid concentration.Solutions: (1) Use highly-trained researchers to perform the injections. Ensure the appropriate acetic acid concentration is used.
?. [FINAL BEHAVIOR RECORDING ●TIMING 6 min per animal]
Relocate the fish into the experimental tank to record the behavioral phenotypes following i.p. injections for 6 minutes.
?. [CLEANUP ●TIMING 1–2 min per animal]
Stop the behavioral recording and remove the fish from the experimental tank. Animals must be euthanized following national guidelines and standard protocols.
?. [CLEANUP ●TIMING 1–2 min per animal]
Discard the water used previously, wash the tank thoroughly with tap water and refill it with non-chlorinated water before testing another fish.
?. [CLEANUP ●TIMING 1–2 min per animal]
Repeat steps 4–9 for the next subject tested.
?. [VIDEO ANALYSES ●TIMING 20–30 min per animal]
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.
?. [VIDEO ANALYSES ●TIMING 20–30 min per animal]
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
?TROUBLESHOOTINGProblem: (1) Fish do not swim at the sagittal plane. (2) The image is not appropriate to measure the body curvature.Possible reasons: (1) Fish freeze during the entire test period due to increased stress responses; (2) The quality of video and/or image acquisition makes the analysis difficult. High-speed swimming in the experimental tank due to increased erratic movements.Solutions: (1) Ensure adequate manipulation to minimize handling and/or other stressful conditions. Use a narrow experimental tank to restrict the lateral swimming activity. Alternatively, record videos from frontal- and top-view positions to generate 3D traces; (2) Ensure a proper manipulation and/or injection to minimize stress. Improve video/image resolution. Use a high-resolution camera to record the behaviors. Select a more appropriate image to quantify the writhing-like response at the respective time interval by using the Prt Sc" (print screen) Windows tool (to obtain high definition screenshots).
?. [STATISTICAL ANALYSES ●TIMING 20–30 min, depending on amount of data collected]
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).
?. [ANTICIPATED RESULTS]
The anticipated results described below show robust pain-like responses in zebrafish as reported elsewhere.
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.