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Revista Brasileira de Anestesiologia

Print version ISSN 0034-7094On-line version ISSN 1806-907X

Rev. Bras. Anestesiol. vol.58 no.4 Campinas July/Aug. 2008 



Local effect of tramadol on formalin evoked flinching behavior in rats*


Efecto analgésico local del tramadol en modelo de dolor provocado por formalina en ratones



Angela Maria Sousa, TSAI; Paula Andrea Baptista FrancoII; Hazem Adel Ashmawi, TSAIII; Irimar de Paula Posso, TSAIV

IMestre em Farmacologia pela Faculdade de Medicina de Ribeirão Preto da USP; Médica Assistente da Equipe de Controle de Dor da Divisão de Anestesia do Instituto Central do Hospital das Clínicas da FM/USP e do Laboratório de Investigação Médica - LIM 08, FM/USP
IIAnestesiologista; Médica Assistente do Serviço de Anestesia do Instituto do Coração do Hospital das Clínicas da FM/USP
IIIDoutor em Medicina; Professor Adjunto do Departamento de Medicina do Centro de Ciências Biológicas e da Saúde da Universidade Federal de São Carlos; Professor Colaborador do Departamento de Cirurgia FM/USP e Laboratório de Investigação Médica - LIM-08 da FM/USP
IVLivre-Docente pela FM/USP; Professor-Associado do Departamento de Cirurgia da FM/USP; Supervisor da Equipe de Controle de Dor da Divisão de Anestesia do Instituto Central do Hospital das Clínicas da FM/USP

Correspondence to




BACKGROUND AND OBJECTIVES: Tramadol hydrochloride is known as a centrally acting analgesic drug, used for the treatment of moderate to severe pain. A local analgesic effect has been demonstrated, but its mechanism of action remains unclear.
METHODS: In this study, we examined the effect of local, systemic and nerve block tramadol on the nociceptive flinching behavior elicited by injection of 50 mL of 1% formalin into the dorsal region of hind paw of rats. Nociceptive flinching behavior was observed for 60 minutes.
RESULTS: Local tramadol in higher concentrations (2.5 and 5mg) almost eliminated flinching behavior during the entire test. Systemic and neural block tramadol did not affect flinching behavior in phase I and partially decreased it in phase II.
CONCLUSIONS: Tramadol presented a local analgesic effect in formalin nociceptive flinching behavior that is different from its central analgesic effect. This analgesic effect, in this model, seems not to be linked to a local anesthetic like effect. 

Key Words: ANALGESICS: tramadol; ANIMALS: rats; PAIN: Experimental: formalin; PHARMACOLOGY, Pharmacodynamic: action mecanisms.


JUSTIFICATIVA Y OBJETIVOS: El clorhidrato de tramadol es un fármaco analgésico de acción central, utilizado en el tratamiento de dolores moderados e intensos. Quedó demostrado un efecto analgésico local del tramadol, pero su mecanismo de acción no fue establecido.
MÉTODO: Se estudió el efecto de la administración local, sistémica y por bloqueo de nervio periférico del tramadol sobre el comportamiento de elevación de la pata generado por la inyección de 50 µL de formalina a 1% en la región dorsal de la pata de los ratones. El número de elevaciones de la pata fue observado durante 60 minutos.
RESULTADOS: La administración local de tramadol en concentraciones mayores (2,5 y 5 mg) conllevó al bloqueo prácticamente completo del comportamiento de elevación de la pata durante todo el test. La administración sistémica y por bloqueo de nervio periférico, no afectó el comportamiento de elevación de la pata en la fase I y se redujo parcialmente en la fase II.
CONCLUSIONES: El tramadol presentó un efecto analgésico local en el modelo de comportamiento de elevaciones de la pata con formalina, que es diferente de su acción central. Ese efecto, en ese modelo, no parece estar vinculado al efecto anestésico local.




Tramadol hydrochloride (1RS, 2RS)-2-[(dimethylamino)-methyl]-1(3-methoxyphenyl)-cyclohexanol hydrochloride is a centrally acting analgesic drug used mainly for treatment of moderate to severe, as well as acute and chronic pain 1,2. A synthetic analog of codeine, tramadol is a 1:1 racemic mixture of two enantiomers [(+)- and (-)-tramadol] with different pharmacological properties, mainly in receptor affinities, which have synergic and complementary actions that result in the analgesic effect of tramadol 1-4. Two mechanisms of action are known for tramadol. Tramadol and its active metabolite M1 exhibit a low and selective affinity for µ opioid receptors 1,2,5. Tramadol presents indirect action on monoaminergic receptors through noradrenaline and serotonin reuptake inhibition, blocking nociceptive inputs at the medular level 1,4,6-10. Pre-treatment with a2adrenergic receptor antagonists, yohimbine and idazoxan, and µ opioid receptor antagonist, naloxone, reduced analgesic effects of tramadol 1,11,12. Its direct action on µ opioid receptors and indirect action in inhibition of monoaminergic reuptake are synergic for analgesic effect 4.

It has been observed in clinical studies that tramadol has a local analgesic effect when injected intradermally 9, reducing propofol pain injection 13, prolonging brachial plexus blockade when added to mepivacaine 11,14 and reducing consumption of lidocaine in intravenous regional anesthesia 12. Tramadol analgesic effects in experimental pain models were shown in thermal hyperalgesia model 15, intraplantar carrageenan inflammation 16, not being related to a direct inhibitory action on prostaglandin endoperoxidase synthases 17, and in spinal somatosensory evoked potential measurements 18.

The formalin test is widely used to evaluate analgesic drugs, being considered a reliable model of tonic inflammatory pain 19-21. Subcutaneous injection of formalin into a rat hind paw evokes an array of stereotyped behaviors. Among these behaviors, flinching (elevating and withdrawing the injected paw) is a reliable parameter of pain behavior 22. Nociceptive response to formalin occurs in a biphasic pattern: there is an initial acute period (phase I, duration of 5 ± 10 min), and then after a short period of remission, phase II begins, consisting of a longer period (20 ± 40 min) of sustained activity 23-25. The initial response is usually attributed to a direct activation of nociceptors 26,27, whereas phase II is associated with the release of local endogenous mediators, leading to local inflammation responsible for sensitisation of primary and spinal sensory neurons and subsequent activation of nociceptors 23,28.

This study aimed to verify whether there is a local analgesic effect for tramadol in a formalin pain model and to investigate a possible mechanism of action.



All experiments were performed on Wistar male rats (280 - 360 g), which had free access to food and water and were supplied by own breeding facilities of the University of São Paulo Faculty of Medicine. The experiments were approved by the Bioethics Committee of the Hospital das Clínicas of the University of São Paulo Faculty of Medicine in accordance with the Committee for Research and Ethical Issues of IASP (29). All behavioral experiments were conducted between 9:00 a.m. and 3:00 p.m. and under the same temperature.

Formalin-evoked nociceptive flinching behavior test

Pain was induced with formalin 1% (50 µL) in the dorsal region of a right hind paw. Before receiving the injection, the rats were placed in an open plexiglass observation chamber for 20 minutes to acclimate them to the surroundings, then removed for the drug administration and returned to the observation chamber. A mirror was placed behind the chamber to enable unhindered observation of the formalin-injected paw. The rats were observed for nociceptive behavior immediately after formalin injection, every 5 minutes until 60 minutes after injection. Nociceptive flinching behavior was quantified as the number of flinches of the injected paw during the observation period. Such behavior varied from a simple lifting of the paw (not associated with locomotion) to a vigorous shaking of the limb, or a rippling of the back muscles associated with movement. The flinches were discrete and easily quantifiable 23,24,30,31-33.

In the first part of the study, twenty five animals were used, divided into five groups, containing five animals each: FORM (formalin 1%, 50 µL in hind paw), TRA1.25 (50 µL containing 1.25 mg of tramadol in hind paw and 10 minutes later, 50ìL of 1% formalin solution in hind paw), TRA2.5 (50 µL containing 2.5 mg of tramadol in hind paw and 10 minutes later, 50 µL of 1% formalin solution in hind paw) TRA5 (50 µL containing 5mg of tramadol in hind paw and 10 minutes later, 50 µL of 1% formalin solution in hind paw). A group with tramadol via intraperitoneal route, TRA-IP (100 µL containing 5mg of tramadol intraperitoneally, and 10 minutes later 50 µL of 1% formalin solution in hind paw), was used to show possible systemic analgesic effect. In the second part of the study a group of five animals was used for neural blockade of the dorsal region of hind paw with tramadol, TRA-BLOC (50 µL containing 10mg of tramadol at the anterior region of ankle, to block nervus fibularis profundus and superficialis and 10 minutes later, 50 µL of 1% formalin).

The mean number of flinches was compared by ANOVA. When a significant difference was observed in phase I, Dunnet multiple comparisons procedure was used to identify the noteworthy groups. Tukey multiple comparisons procedure was used in phase II. A value of p < 0.05 was considered as statistically significant.



Effects on nociceptive flinching behavior.

The number of flinches were grouped in two phases, the first 10 minutes (1-10 minutes) and the following 50 minutes (11-60 minutes), recorded in two phases, first (0-10 minutes) and second (11-60 minutes). Nociceptive flinching behavior scores of all groups throughout the test are shown in figure 1. FORM displayed the typical biphasic behavior of formalin. TRA-BLOC and TRA-IP also presented biphasic behavior, not present in TRA1.25, TRA2.5 and TRA5 (Figure 1). In phase I, tramadol, when used locally, blocked almost completely flinching response with the three doses used, significant differences occurred among group means of TRA1.25, TRA2.5 and TRA5 and means of the control group (FORM) and systemic tramadol (TRA-IP), as shown in figure 2. In phase II, the number of flinches in local injected tramadol groups decreased with the increase of amount of drug, with significant difference between TRA1.25 group mean compared to TRA2.5 and TRA5 means. The blockade of flinching response was almost complete in higher doses (Fig. 3). TRA-BLOC in phase I was not different from FORM and TRA-IP, but it presented significant differences when compared to local injected groups (Figure 2). In phase II TRA-BLOC resulted in similar mean to TRA-IP, but different from FORM and local injected groups (Figure 3).



In the present investigation, flinches were used to quantify formalin evoked pain behaviors since they correlate well with formalin evoked cardiovascular responses and provide a reliable correlation of pain in the conscious and freely moving rat 22. Among various stereotyped behaviors observed in the formalin test, the frequency of flinches is simple to assess as well as directly proportional to formalin concentration 23-25,30-32.

A local analgesic effect of tramadol has been shown in this pain model with formalin in rats. Analgesia was found in both phases of the nociceptive flinching behavior test. Some authors have attributed the analgesic effect as part of local anesthetic effect 18,34, similar to clonidine 12 or anti-inflammatory effects 16. In this study the possibility of this analgesic effect being due to a local anesthetic effect was tested. This was done through the use of a neural blockade of the area where the nociceptive stimuli was applied (nervus fibularis profundus and superficialis). A higher dose of tramadol was used in this group to avoid an insufficient block of the nerves. When tramadol was used to block neural transmission, nociceptive flinching behavior did not change in phase I and showed a small decrease in phase II. These results support the possibility that the analgesic action of tramadol observed is not linked to a local anesthetic effect in the flinching behavior elicited by formalin in rats hind paw.

In previous study, TSAI et al (2001) have found that direct application of tramadol in the sciatic nerve of rats led to a dose-dependent reduction in both amplitude and conduction speed of somatosensory evoked potentials when compared to pre-treatment measures. This inhibition was attributed to a local anesthetic effect of tramadol. The doses of tramadol used were the same in our and their study. The discrepancy between the results may be explained by the local anesthetic effect of tramadol (electrophysiological data) observed in their study, being of no clinical relevance as observed in ours. The small decrease observed in phase II seems to be consequence of a systemic effect of tramadol since it did not occur in phase I. The same phenomenon was observed when tramadol was used through the intraperitoneal route. However, systemic tramadol analgesia only occurred in phase II, and was less than that observed in local injected tramadol groups. It is expected that systemic effect occurs after tramadol reaches peak in CNS. These results may represent analgesia provided by tramadol central action through classical mechanisms, a decrease in serotonin and noradrenaline reuptake, similar to the findings observed in the same model using tramadol enantiomers via intraperitoneal route and serotonin and noradrenalin antagonists 35. Tramadol effect on serotonin receptors present on sensory afferent nerve endings is questionable. Serotonin receptors present, like 5-HT1A, 5-HT3 and 5-HT4, have pronociceptive action on sensory afferent nerve endings on formalin pain model while 5-HT2A seems not to affect pain behavior 30,32. To explain local tramadol analgesia through action on serotonin receptors, it would be necessary to conceive that tramadol acts in different modes at central level and primary afferent neuron ending, decreasing reuptake of serotonin and noradrenalin in the former and acting as serotonin antagonist in the latter. A possible anti-inflammatory effect for tramadol in the formalin pain model to explain the analgesia observed may be present. Although phase I is believed to be linked to direct stimulation of nociceptors 26,27, some authors believe that phase I is also a consequence of local inflammation32. Phase II is known to be inflammatory 20 and tramadol presented anti-inflammatory action after yeast injection in paw, reducing paw edema, though this reduction was also small 16. However, it is not probable that only the anti-inflammatory effect of tramadol may explain local analgesia obtained in evoked flinching behavior, since the analgesia observed was complete, and anti-inflammatory effect seems to be partial.



Tramadol hydrochloride was kindly provided by Cristália Prod. Quim. Farm., Brazil.



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Correspondence to: 
Dr. Hazem Adel Ashmawi
LIM-08, Anestesiologia Experimental
Faculdade de Medicina da Universidade de São Paulo
Av. Dr. Arnaldo, 455, 2º andar
01246-903 São Paulo, SP

Submitted em 13 de novembro de 2007
Accepted para publicação em 14 de abril de 2008



* Received from Laboratório de Investigação Médica LIM-08 da Faculdade de Medicina da Universidade de São Paulo (FM/USP), São Paulo, SP

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