Effect of the analgesics dipyrone, tramadol, and meloxicam on the behavior of laboratory rats
Introduction
The research community has an ethical responsibility to prevent or minimize pain and suffering in laboratory animals by refining experimental procedures and administrating appropriate analgesics. Moreover, the management of pain has important scientific and methodological implications for experimental design since stress profoundly affects the welfare and behavior of laboratory animals with a consequential effect on the quality of data obtained (Jirkof, 2017).
Nevertheless, it has been widely reported that less than half of researchers describe pain management in their publications (Stokes et al., 2009; Coulter et al., 2017; Alemán-Laporte et al., 2019). Among the reasons that may explain the lack of analgesia in painful procedures with laboratory animals are: (i) the possible clinical side effects of analgesics, (ii) the potential of analgesics to confound the results from experimental studies, and (iii) an apparent uncertainty about when and how to administer analgesics (Richardson and Flecknell, 2005).
The main analgesics employed in experimental procedures involving rodents are nonsteroidal anti-inflammatory drugs (NSAID) and opioids. Dipyrone (DIP) and meloxicam (MEL) are NSAIDs that relieve pain by suppressing the cyclooxygenases that catalyze the first step in the synthesis of prostaglandins (Nunamaker et al., 2018), while the synthetic opioid tramadol (TRA) acts on the central nervous system by activating μ-opioid receptors and inhibiting the reuptake of serotonin and norepinephrine (Taylor et al., 2016). These drugs are widely used in veterinary field, especially in Europe and Latin America (Escobar et al., 2011). They are usually used alone in procedures that inflict minimal pain. However, in procedures that generate moderate or more severe pain, multimodal analgesia (the combination of 2 or more analgesics) is recommended because their different mechanisms of action can potentiate pain relief (Foley et al., 2019).
In behavioral neuroscience research, an understanding of how each external variable affects animal behavior is crucial, and awareness of the advantages, disadvantages, and complications associated with commonly used analgesics is clearly essential (Gaertner et al., 2008). A wide array of tools are available to evaluate behavior in laboratory rodents, ranging from tests that measure basic motor and sensory functions u to those that assess more complex behavior patterns such as cognition and emotionality. The Open Field (OF) and the Elevated Plus Maze (PM) are the most common tests used to evaluate murine exploratory and anxiety-like behavior and have been employed routinely to screen drugs for psychopharmacological potential (Schmitt and Hiemke, 1998). The more recently developed Grooming Transfer Test (GTT; Oliver et al., 2018) allows indirect assessment of grooming behavior by monitoring the elimination of fluorescent gel applied to the fur of an experimental animal. This test can be used to evaluate the long-term pharmacological effects of analgesics on grooming behavior as a function of time after drug administration.
It is known that NSAIDs may provoke various adverse effects on animal metabolism, including renal, cardiovascular and gastrointestinal toxicity (Jirkof, 2017), while some studies have demonstrated that high doses of TRA can produce sedation in rats (Wolfe et al., 2015). However, alterations in the behavior of laboratory rats following administration of DI and MEL, either alone or in combination with TRA, have been poorly studied. For this reason, the aim of the present investigation was to evaluate the changes in behavior induced by these analgesics and to emphasize the importance of considering such alterations in studies involving the evaluation of rodent behavior.
Section snippets
Animals
Forty-eight male Wistar-Han rats (8-12 weeks old) were obtained from the Institute of Biomedical Sciences of the University of São Paulo (USP) and housed in the animal facility of the Department of Pathology of the School of Veterinary Medicine and Animal Science at USP. Animals were free of endo- and ecto-parasites, Mycoplasma pulmonis, Pasteurella pneumotropica, P. multocida, Bordetella bronchiseptica, Klebsiella oxytoca, K. pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus,
Open Field Test
Minute-by-minute analysis of locomotion revealed a main effect of Analgesic [F(5,42) = 9.16, < 0.001, η2 = 0.52], Minute [F(5.30,222.73) = 77.94, < 0.001, η2 = 0.65], and Analgesic*Minute interaction [F(26.52,222.73) = 1.60, < 0.05, η2 = 0.16]. Independent analyses revealed that animals treated with DIP, MEL, DIP+TRA, or SAL showed progressive reductions in locomotion over the 10 minutes period (all < 0.001, η2 = 0.72 - 0.78) with a substantial decrease from 1 to 5 minutes (Bonferroni: < 0.001
Discussion
The purpose of analgesic therapy is to minimize pain without compromising the well-being of an experimental animal (National Research Council, 2009). However, analgesic drugs generally give rise to diverse effects on the metabolism of the animal and these may provoke changes in physiological and behavioral parameters (Jirkof, 2017). Therefore, the information obtained only using analgesic drugs should eliminate the effect created by scientific procedures and point out the most appropriate
Conclusions
Administration of DIP, either alone or in combination with TRA and in the specific doses and route employed in the present study, depressed general activity in rats. Moreover, treatments that included TRA decreased grooming behavior while those that involved MEL increased locomotor activity in the experimental animals. These behavioral changes were observed in naïve rats, and so they should be considered when certain experimental procedures, including surgical procedures, are applied to
Authorship Statement
The idea for the study was conceived by J.A-L. and C.M.C.M. The experiments were designed by J.A-L., G.A., A.T.F.B.A., M.R-C. and C.M.C.M. and performed by J.A-L., G.A. and C.M.C.M. The data were analyzed by J.A-L., G.A., M.R-C. and C.M.C.M. and the manuscript was written by J.A-L., G.A., A.T.F.B.A., J.F.T., M.R-C. and C.M.C.M. All authors reviewed and approved the final version of the manuscript.
Conflict of Interest
The authors declare no conflicts of interest.
Ethical Statement
All procedures were approved by, and conducted following the guidelines of, the Ethics Committee of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil (protocol number 3611111119).
Acknowledgments
This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Vice Rector for Research, University of Costa Rica (projects 723-B9-197, 837-B7-603 and 837-B8-123).
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