Comparative Medicine Copyright 2017 by the American Association for Laboratory Animal Science

Vol 67, No 2 April 2017 Pages 106–111

Original Research

Biochemical, Histopathologic, Physiologic, and Behavioral Effects of Nonsteroidal Antiinflammatory Drugs in Rainbow Trout (Oncorhynchus mykiss) Amy L Rizzo,1,* Gregory A Wooster,2 Luce E Guanzini,1 Christine M Peterson,1 Karryssa S Fenderson,4 Hollis N Erb,3 Paul R Bowser,2 and Mary E Martin1 Because the number of fish being used in research is increasing rapidly, evaluating the analgesic and pathologic effects of NSAID in fish is essential. To determine the biochemical, histopathologic, physiologic and behavioral effects of 3 NSAID, 48 rainbow trout underwent anesthesia with tricaine methanesulfonate and exploratory celiotomy and were randomly assigned to receive flunixin (0.5 mg/kg IM), ketorolac (0.5 mg/kg IM), ketoprofen (2 mg/kg IM), or saline. Clinical pathologic variables were assessed 1 wk before surgery and 48 h after surgery. Histopathology was performed to evaluate the healing of the incision, tissue reaction at the injection site, and potential organ toxicity. Physiologic and behavioral parameters, including weight, feeding, opercular rate, and vertical position in the water, were measured to establish parameters for identifying pain in fish. The difference between the preand postoperative phosphorus concentrations was greater in the flunixin group than the saline group and was the only pathologic difference between treatment groups. Histopathology of incision site, injection site, and internal organs appeared normal, and healing did not appear to be inhibited by the drugs used. The physiologic parameters of opercular rate and weight were consistent and may be helpful in identifying pain in fish in future studies, whereas feeding and vertical position in the water were unhelpful as indicators of pain in this rainbow trout surgical model. Overall, according to clinical pathology and histopathology, the use of ketoprofen, ketorolac, and flunixin at the dosages used in this study lack negative effects in rainbow trout undergoing surgery. Abbreviation: COX, cyclooxygenase

The rapid increase in the number of fish used as research animals has led to an urgent need to understand pain perception in these species as well as safe and effective alleviation of pain. Although multiple publications have evaluated whether fish are capable of experiencing pain after a noxious stimulus,5,13,18,19,20,21 few studies document safe and effective alleviation of pain after a surgical procedure.1 One study6 investigated the use of a κ opioid agonist, butorphanol, and an NSAID, ketoprofen, in koi carp (Cyprinus carpio) after exploratory celiotomy and revealed behavioral and physiologic changes at 48 h postoperatively. In the current study, we sought to extend the previous study6 by testing additional NSAID and by assessing clinical pathology and histopathology to focus on the safety of the administered analgesics. Because our study involved celiotomy, we also evaluated physiologic and behavioral parameters after surgery, to identify parameters that can be consistently and reliably used to identify pain in fish. Received: 28 Apr 2016. Revision requested: 08 Jun 2016. Accepted: 07 Sep 2016. 1 Center for Animal Resources and Education, Departments of 2Microbiology and Immunology and 3Population Medicine and Diagnostic Sciences, and 4College of Veterinary Medicine, Cornell University, Ithaca, New York *Corresponding author. Email: [email protected]

Rainbow trout (Oncorhynchus mykiss) are a commonly used laboratory animal species and are sufficiently robust to undergo surgical procedures. Implanting telemetry devices in trout is a common invasive procedure performed to facilitate studying physiologic parameters. Telemetry might also be applied in aquaculture studies and in the field to track the movement of wild fish populations.12 In the current study, we performed celiotomies on rainbow trout to model the conditions necessary for implantation of a telemetry unit. Because NSAID are known to be relatively safe and effective analgesics in other species undergoing surgical procedure and given the paucity of studies evaluating NSAID in fish, we evaluated 3 NSAID which were administered prior to celiotomy: ketoprofen (2 mg/kg IM), ketorolac (0.5 mg/kg IM), and flunixin (0.5 mg/kg IM). Ketorolac and flunixin help to alleviate both somatic and visceral pain, features that would be beneficial when performing surgical procedures (particularly in a field setting) that produced visceral pain and where opioids were not an option, due to their requirements regarding licensing and security. Ketoprofen is another common NSAID in laboratory animals and has been used at a dose of 2 mg/kg in fish.6 Furthermore, all of the NSAID we evaluated (unlike many other NSAID) are available in an injectable form, thus simplifying administration in an aquatic species.

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Effects of NSAID in rainbow trout

Because NSAID can cause harmful side effects, such as gastrointestinal ulceration and nephrotoxicity, in other species and because some NSAID, such as diclofenac, cause nephrotoxicity and hepatotoxicity after chronic administration to rainbow trout during ecotoxicology studies,17,25 the main objective of our current study was to assess the clinical pathology and histopathology of rainbow trout after surgery to ensure the safety of these drugs. An additional objective was to assess the physiologic and behavioral changes in rainbow trout after surgery to identify consistent and reliable parameters of identifying pain to optimize fish wellbeing during research. We hypothesized that at the dosages used in this study, the analgesics ketoprofen, ketorolac, and flunixin are safe to use in rainbow trout undergoing celiotomy, as determined by clinical pathology and histopathology.

Materials and Methods

Animals. Juvenile rainbow trout (Oncorhynchus mykiss; n = 48) of approximately equal numbers of males and females and a median weight of 159 g (minimum, 104 g; maximum, 252 g) were obtained from the New York State Department of Environmental Conservation Bath Fish Hatchery (Bath, NY). The trout were group-housed in a 350-gal flow-through stock tank until they entered the study. At 7 days prior to surgery, individual trout were transferred to 10-gal glass tanks, each with an air stone and flowthrough water supply. Two individual aquaria were positioned over a 140-gal tank (Living Stream, Frigid Units, Toledo, OH) that served as a water bath and effluent discharge basin. A total of 8 tanks, each containing 2 individual aquaria, were available, providing 16 aquaria per surgical trial. For both the stock tank and surgery trial tanks, water temperature was maintained at 14 to 16 °C; water-quality parameters were maintained at acceptable levels for rainbow trout (pH, 7.3 to 7.5; unionized ammonia, 0 mg/L; nitrite, 0 mg/L);22 and a 12:12-h light dark cycle was provided. Trout were fed a commercially available pelleted food (Zeigler, Gardners, PA) at 1% of body weight daily. All housing and experimental procedures were conducted in an AAALACaccredited facility under a protocol approved by the IACUC at Cornell University. Baseline bloodwork and behavior analysis. Seven days prior to surgery, trout were lightly anesthetized with tricaine methanesulfonate prepared as an 80-mg/L solution that was then buffered with sodium bicarbonate until the pH reached 7.0 to 7.5. Fish were weighed by being placed in a bucket of water on a calibrated scale that was tared between fish. Blood (approximately 0.6 mL) was collected from the caudal hemal arch by using a 1-mL syringe and 25-gauge needle and placed in a lithium heparin tube, and fish were placed individually in single-housing tanks for acclimation. At 24 h before surgery, baseline behavior was recorded. Surgery. On the day of surgery, fish were fasted for 12 to 16 h. Anesthesia was then induced by placing a fish in a 38-L bucket containing a 80-mg/L solution of buffered tricaine methanesulfonate. Once the righting reflex was lost, each fish was transferred from the induction bucket to the surgery table. Each anesthetized fish was received an analgesic drug or saline (equivalent volumes) as assigned by using a computer-generated list (random. org), with 12 fish per group. Physiologic saline (0.9% NaCl), flunixin (0.5 mg/kg), ketorolac (0.5 mg/kg), or ketoprofen (2 mg/ kg) was injected into the dorsal musculature just ventral to the dorsal fin of each fish at approximately 5 min prior to surgery. The fish was then placed in dorsal recumbency and underwent

exploratory celiotomy while maintained on a recirculating delivery system containing 80 mg/L buffered tricaine methanesulfonate. Chlorhexidine solution was used to disinfect the surgical site prior to incision; because surgical scrub solutions can be caustic to fish skin, the chlorhexidine solution was diluted by an additional factor of 10, and the area was rinsed with sterile saline after application. A clear sterile drape with a precut oval opening was placed over the fish. A no. 10 scalpel blade was used to incise the body wall lateral from ventral midline. The length of the incision was determined by measuring the distance from the pectoral fin to the pelvic fin and then incising the skin for half of this length; this method was used to account for the different sizes of the fish. Once the body wall was opened, the coelomic cavity was examined and then the incision was closed in a single layer by using 4-0 polytrimethylene carbonate suture (Maxon, Medtronic, Dublin, Ireland) in a horizontal mattress pattern. All surgeries were performed by the same, experienced surgeon. Fish recovered in their individual home aquaria. Physiologic and behavioral data. The behavioral and physiologic parameters were assessed at 24 h before surgery (baseline), 2 h after surgery (when we expected them to be fully recovered from anesthesia), and 24 h after surgery (when we anticipated the medication would be fully absorbed and active). Observers were blinded to the treatment group, and fish were examined for opercular rate, vertical position in the tank, and response to food pellets. To determine vertical position in the tank, the tank was equally divided into 3 sections by using a ruler, and lines were drawn on the tanks to identify the top, middle, and bottom of the tank. Three feed pellets were presented to the fish, which was observed for 60 s; each fish was scored regarding the number of pellets ingested: score of 0, none of the pellets eaten; 1, fish investigated but did not eat pellets; 2, ate some of the pellets; and 3, ate all of the pellets. Clinical pathology. At 48 h after surgery, blood was collected as described previously. Clinical pathology parameters measured at both collection time points were Hct, total protein, AST, creatine kinase, cortisol, glucose, calcium, sodium, phosphorus, and potassium. Total protein, calcium, phosphorus, sodium, potassium, AST, and glucose were analyzed automatically by using the manufacturer-provided profile for avians and reptiles (VetScan VS2, Abaxis, Union City, CA). Creatine kinase was analyzed at Cornell University Animal Health Diagnostic Center (Ithaca, NY). Plasma cortisol was measured by chemiluminescent immunoassay (Immulite analyzer, Cornell University Animal Health Diagnostic Center Endocrinology Lab, Ithaca, NY). Tissue collection. At 14 d after surgery, fish were euthanized with an overdose (250 mg/L) of buffered tricaine methanesulfonate. Excess water was blotted from the skin, and fish were weighed on a calibrated scale. Internal organs (kidney, liver, spleen, and gastrointestinal tract), the incision site, and the injection site were collected and fixed in 10% neutral buffered formalin for histopathology. Internal organs were evaluated for adverse effects, such as inflammation, hemorrhage, and ulceration, due to treatment with NSAID. The incision site and injection site were evaluated for inflammation and healing. Scoring systems were established to evaluate the healing of the incision site. All incision sites were evaluated grossly and histologically in a blinded fashion by 2 veterinarians (AR and MM). The gross inflammatory reaction was graded according to the following scale: 0, no inflammation, edema, necrosis, or erythema

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and suture tract completely healed (Figure 1 A); 1, mild inflammation, edema, and erythema but no necrosis and suture tract completely healed; 2, moderate inflammation, edema, and erythema, with slight necrosis and moderately healed suture tract; 3, severe inflammation, severe edema of wound edges and erythema, prominent necrosis, and dehiscence of suture tract (Figure 1 B). The histologic inflammatory reaction was graded as: 0, no apparent changes from normal skin and underlying subcutis; 1, mild changes with minimal inflammatory cells and edema, minimal fibrosis of the suture tract with well-organized granulation tissue (Figure 2 A); 2, mild changes, with mild inflammatory cells and edema and mild fibrosis of the suture tract with well-organized granulation tissue; 3, moderate changes with moderate mixed inflammatory infiltrates; 4, moderately severe changes; and 5, severe changes, marked inflammation and edema, with and large areas of coagulative necrosis of muscle bundles and poorly organized granulation of the suture tract (Figure 2 B). Data analysis. The sample size of 12 per group was based on a credible rule-out of lesion prevalence (that is, caused by the analgesics tested) of greater than 30%. Statistical analyses were performed by a quantitative epidemiologist (HNE) who used the commercial software program Statistix 10 (Analytical Software, Tallahassee, FL). Because data were asymmetrically distributed, sample size per treatment group was small, some data are scores, and Shapiro–Wilk tests indicated nonnormal distribution of some data, nonparametric statistics were used. We calculated the changes in respiratory rate and feeding score from baseline to the 2- and 24-h time points after surgery and the difference in weight between baseline and day 14. The Kruskal–Wallis test was used to compare pre- and postsurgery respiratory rate, weight, feeding scores, and clinicopathologic parameters among the 4 treatment groups. The Kruskal–Wallis test was also used to compare the median gross and histologic incision-site scores from the 2 observers for each fish by treatment group. Statistical significance was accepted at Bonferroni-adjusted P value less than 0.05 (to account for multiple comparisons of similar questions) for respiratory rate and feeding scores and at an unadjusted P value of 0.05 for potential harms (pathologic and clinicopathologic results) and for weight. We also evaluated the interobserver agreement regarding incision scores by calculating the weighted κ statistic (MedCalc Statistical Software version 13.1.2, MedCalc Software, Ostend, Belgium; http://www.medcalc.org).

Results

All fish survived general anesthesia and surgery (duration [mean ± SEM]), 7.2 ± 1.5 min) One postoperative complication occurred, in which a fish developed a fungal infection of the incision. This fish was treated by adding formalin (25 mg/L) to the tank until the condition resolved and was included in the analysis and data presentation. Pathologic parameters. Between treatment groups, the change in phosphorus concentration from before to after surgery differed between the saline (median decrease, 0.9 mg/dL) and flunixin (median decrease, 3.4 mg/dL; P = 0.015) groups. In contrast, phosphorus concentration changes in the ketorolac and ketoprofen groups did not differ from each other or from those of the saline and flunixin groups. No changes in the other 9 clinicopathologic analytes differed between groups (all P ≥ 0.07). In all fish groups, Hct, cortisol, glucose, and calcium were decreased (P < 0.0001) decreased and AST and CK were increased (P < 0.0001) postoperatively (Table 1).

Figure 1. (A) Example of a grade 0 gross incision, showing no inflammation, edema, or necrosis, and with complete healing of the suture tract. (B) Example of a grade 3 gross incision, with severe inflammation, erythema, and edema of wound edges as well as dehiscence of the suture tract.

Agreement between the 2 observers regarding the score for the gross incision site was only fair (weighted κ, 0.40; SE, 0.097). However, there was perfect agreement for 23 of 48 fish, and all disagreements were of only 1 score unit. No significant difference (P = 0.80) in gross incision scores was noted among the 4 treatment groups. The median (minimum, maximum) incision scores were: saline, 1.5 (0.5, 3); ketorolac, 1.75 (1, 3); flunixin, 1.5 (0.5, 3); and ketoprofen, 1.5 (0.5, 3). On necropsy examination, no gross lesions of major organs were observed (therefore, no formal statistical test was run). A common incidental finding (38 of 48 fish) was adhesion at the surgical site. There was no significant difference (P = 0.46) in the histologic score of the incision site among the 4 treatment groups. Median (minimum, maximum) scores were: saline, 2 (1, 5); ketorolac, 3 (2, 5); flunixin, 2.5 (1, 5); and ketoprofen, 3 (1, 5). Internal organs (including liver, kidney, gastrointestinal tract, and spleen) were histologically normal in all fish. In addition, the NSAID injection site in the dorsal musculature was histologically normal in all fish. Physiologic parameters. The median (minimum, maximum) opercular rate for all 48 fish before surgery was 66 (48, 96) operculations per minute. At both times after surgery (2 h and 24 h), some fish in each group had lower opercular rates than before surgery (as many as 24 fewer breaths per minute in the NSAID treatment groups), the median rates (by group, for all 4 groups) were 9 to 12 breaths per minute higher at 2 h after surgery than before (Figure 3). By 24 h after surgery, the median difference from before surgery was 0 in all 4 groups. Overall, the change in respiratory rate from baseline did not differ between the 4 treatment groups at either 2 h (P = 0.91) or 24 h (P = 0.75).

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Effects of NSAID in rainbow trout

Table 1. Preoperative (day –7) and postoperative (2 d) clinical pathology values (mean ± SEM) in rainbow trout (n = 48) Hct (%)

Day –7

Day 2

P

49.1 ± 1.2

39.3 ± 1.1

Biochemical, Histopathologic, Physiologic, and Behavioral Effects of Nonsteroidal Antiinflammatory Drugs in Rainbow Trout (Oncorhynchus mykiss).

Because the number of fish being used in research is increasing rapidly, evaluating the analgesic and pathologic effects of NSAID in fish is essential...
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