Effects of opioids on local anesthesia in the rat : a codeine and tramadol study

Opioids are central analgesics that act on the CNS (central nervous system) and PNS (peripheral nervous system). We investigated the effects of codeine (COD) and tramadol (TRAM) on local anesthesia of the sciatic nerve. Eighty Wistar male rats received the following SC injections in the popliteal fossa: local anesthetic with epinephrine (LA); local anesthetic without vasoconstrictor (LA WV); COD; TRAM; LA + COD; LA + TRAM; COD 20 minutes prior to LA (COD 20’ + LA) or TRAM 20 minutes prior to LA (TRAM 20’ + LA). As a nociceptive function, the blockade was considered the absence of a paw withdraw reflex. As a motor function, it was the absence of claudication. As a proprioceptive function, it was the absence of hopping and tactile responses. All data were compared using repeated-measures analysis of variance (ANOVA). Opioids showed a significant increase in the level of anesthesia, and the blockade duration of LA + COD was greater than that of the remaining groups (p < 0.05). The associated use of opioids improved anesthesia efficacy. This could lead to a new perspective in controlling dental pain. Descriptors: Codeine; Tramadol; Anesthesia, Local; Analgesics, Opioid. Introduction One of the most important factors for success in dental practice is the patient’s well-being. Currently, several methods are being used to control pain, and drug therapy is the most common and popular worldwide. However, the use of a single drug may not be sufficient to achieve the comfort level expected by the patient. Pain control is important because it allows the patient to leave the clinic in maximum comfort. LA is the surgeon’s first choice, because it precedes the procedure and is designed to offer comfort. However, administration of these drugs is stressful, and, in many cases, the drugs do not provide the expected comfort, especially in patients with psychological disorders, such as panic, and systemic and / or neurological disorders that hinder treatment. In this regard, some adjuvant drugs have been proposed to enhance anesthesia and analgesia, such as clonidine, diazepam, morphine, codeine and tramadol.1 Although modern LAs are effective and safe for most clinical practice, there are some patients who need more comfort. In these cases, an intense search for antinociceptive agents continues, aiming at improving nerve selectivity, promoting less motor block and lowering systemic toxDeclaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. Submitted: Feb 20, 2013 Accepted for publication: Jul 31, 2013 Last revision: Aug 13, 2013 http://dx.doi.org/10.1590/S1806-83242013000600003 Effects of opioids on local anesthesia in the rat: a codeine and tramadol study 456 Braz Oral Res., (São Paulo) 2013 Nov-Dec;27(6):455-62 icity incidences.2 In regard to pain control, the literature documents attempts to find the best therapy for each procedure. As reported in recent papers, it is evident that the association of more than one drug promotes better results than following the established conventional therapies.3 Considering the association therapy concept, COD and TRAM are centrally acting analgesics. When combined with other drugs, such as painkillers and LA, they reduce the occurrence of adverse effects, while producing an equivalent or greater degree of analgesia. Certain associations have been established in the medical field such as: • paracetamol + COD, • paracetamol + TRAM and • TRAM + LA. The products of these combinations of opioids with other drugs have been shown to produce analgesic and anesthetic synergism.4 The mechanism of opioid and LA association has not been elucidated. It is known that LAs act by blocking the influx of sodium in their channels, preventing increased permeability of the ion, needed for the potential action to occur.5 Opioids act by mechanisms different from those of LAs, i.e., through specific receptor opioid pathways, or through receptors coupled to ion channels, and these receptors cause changes in cellular mechanisms and balance.6 There is recent evidence of synergism in the association of COD with paracetamol in rats, demonstrated by the Hargreaves hypernociception model,7 and in the association of diclofenac sodium in the reduction of nociception in formalin-stimulated mice.8 However, no investigations regarding the synergism of action with LAs have been published. The possibility of synergism resulting in maximum antinociception with minimal doses of active therapy is considerably attractive and particularly valuable when increased efficacy occurs without increased side effects.9 Methodology Eighty healthy male Wistar rats, about 12 weeks old and weighing 300–350 g were selected and acclimatized at the Animal Facility of the Oral Biology Laboratory (Universidade de São Paulo USP, Dentistry School), in plastic cages at a temperature of 20°C–25°C, with 12-hour light / dark cycles, receiving food and water ad libitum. The blinded study was approved by the Research Ethics Committee of the Biomedical Science Institute (057ICB). All the experiments took place between 8:00 a.m. and 2:00 p.m. Solution preparations The following solutions were used to perform the sciatic nerve block: Local anesthetic solution • 2% lidocaine chlorhydrate with 1:100000 epinephrine (Alphacaine 100, DFL, Rio de Janeiro, Brazil): volume, 0.14 mL; • lidocaine chlorhydrate with no vasoconstrictor (Lidostesim, Dentsply Pharmaceutical, Catanduva, Brazil): volume, 0.14 mL. Codeine phosphate (Codein®, Cristália, São Paulo, Brazil) • 30  mg / mL, 2  mL / ampule. The recommended dose of injectable COD for rats, according to the manufacturer, is 5  mg / kg; thus, each rat was administered 0.05 mL of codeine phosphate + 0.10 mL of saline solution for a final solution volume of 0.15 mL. Tramadol chlorhydrate (Tramal®, Pfizer, Guarulhos, Brazil) • 50  mg / mL, 1  mL / ampule. The recommended dose of injectable tramadol chlorhydrate for rats, according to the manufacturer, is 20  mg / kg; thus, each rat was administered 0.12  mL of TRAM + 0.03 mL of saline solution, for a final solution volume of 0.15 mL. The gap time of the opioid in a LA injection was selected according to the pilot experiment. It was observed that 20 minutes was the time required for maximum decrease in the action potential amplitude for the peripheral nerve. Carnaval TG, Sampaio RM, Lanfredi CB, Borsatti MA, Adde CA 457 Braz Oral Res., (São Paulo) 2013 Nov-Dec;27(6):455-62 Thalhammer et al.11 All animals were analyzed before treatment (baseline) and at the end of the experiment, to observe if they recovered their normal function. Nociceptive response This was achieved through two paw withdrawal reflex (PWR) tests: 1. Withdraw reflex: applying pressure as a nociceptive stimulus using an analgesiometer (UgoBasile, Comerio, Italy) across the skin, over the lateral metatarsus, exerting and increasing linear force (16 g / s) of up to 250 g (cutoff) on the rat’s paw (Randall-Selitto method).12 2. Mechanical stimulation: a 2  mm forceps was used to exert pressure across the skin fold over the lateral metatarsus. The paw withdraw reflex (PWR) was assessed by scores (1–3),12 where the reactions were 1 = normal, 2 = weak (partial block), 3 = none (complete block). Motor function Normally, the extensor function is quantified when exerting the force required to reposition the metatarsus on the platform. The gait and spontaneous locomotive activity were evaluated by toe flexion (claudication); when the region received drugs, there was a deficit in motor function, which could Experimental procedures Two weeks before the experiments, the rats were randomly assigned to one of eight groups, and were exposed to preliminary experimental conditions to familiarize them with the experimenter. The experiment involved functional steps; the paradigm is shown in Figure 1. The nerve block performed was evaluated by sensitive, motor and proprioceptive testing, and the sciatic block was conducted by an operator with access to the hind leg popliteal fossa, by SC injection.10 Thus, the experiment involved an injection procedure and a neurological evaluation, with analysis at 5-minute intervals. Eighty rats were randomly divided into groups, and received different volumes of drugs. The abbreviations of each group represent drugs and volumes, respectively: 1. COD (0.05 mL); 2. TRAM (0.12 mL); 3. LA (0.14 mL); 4. LA WV (0.14 mL); 5. LA + TRAM (0.14 mL + 0.12 mL); 6. LA + COD (0.14 mL + 0.05 mL); 7. COD 20’ + LA (0.15 mL + 0.14 mL); 8. TRAM 20’ + LA (0.12 mL + 0.14 mL). Function evaluation The functions were evaluated according to popliteal Mixed Pressure Pressure stimulus test (forceps 2mm) stimulus test extensor Figure 1 Experimental paradigm.


Introduction
One of the most important factors for success in dental practice is the patient's well-being.Currently, several methods are being used to control pain, and drug therapy is the most common and popular worldwide.However, the use of a single drug may not be sufficient to achieve the comfort level expected by the patient.
Pain control is important because it allows the patient to leave the clinic in maximum comfort.LA is the surgeon's first choice, because it precedes the procedure and is designed to offer comfort.However, administration of these drugs is stressful, and, in many cases, the drugs do not provide the expected comfort, especially in patients with psychological disorders, such as panic, and systemic and / or neurological disorders that hinder treatment.In this regard, some adjuvant drugs have been proposed to enhance anesthesia and analgesia, such as clonidine, diazepam, morphine, codeine and tramadol. 1lthough modern LAs are effective and safe for most clinical practice, there are some patients who need more comfort.In these cases, an intense search for antinociceptive agents continues, aiming at improving nerve selectivity, promoting less motor block and lowering systemic tox-Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.icity incidences. 2 In regard to pain control, the literature documents attempts to find the best therapy for each procedure.As reported in recent papers, it is evident that the association of more than one drug promotes better results than following the established conventional therapies. 3onsidering the association therapy concept, COD and TRAM are centrally acting analgesics.When combined with other drugs, such as painkillers and LA, they reduce the occurrence of adverse effects, while producing an equivalent or greater degree of analgesia.Certain associations have been established in the medical field such as: The products of these combinations of opioids with other drugs have been shown to produce analgesic and anesthetic synergism. 4he mechanism of opioid and LA association has not been elucidated.It is known that LAs act by blocking the influx of sodium in their channels, preventing increased permeability of the ion, needed for the potential action to occur. 5Opioids act by mechanisms different from those of LAs, i.e., through specific receptor opioid pathways, or through receptors coupled to ion channels, and these receptors cause changes in cellular mechanisms and balance. 6here is recent evidence of synergism in the association of COD with paracetamol in rats, demonstrated by the Hargreaves hypernociception model, 7 and in the association of diclofenac sodium in the reduction of nociception in formalin-stimulated mice. 8owever, no investigations regarding the synergism of action with LAs have been published.The possibility of synergism resulting in maximum antinociception with minimal doses of active therapy is considerably attractive and particularly valuable when increased efficacy occurs without increased side effects. 9

Methodology
Eighty healthy male Wistar rats, about 12 weeks old and weighing 300-350 g were selected and ac-climatized at the Animal Facility of the Oral Biology Laboratory (Universidade de São Paulo -USP, Dentistry School), in plastic cages at a temperature of 20°C-25°C, with 12-hour light / dark cycles, receiving food and water ad libitum.The blinded study was approved by the Research Ethics Committee of the Biomedical Science Institute (057-ICB).All the experiments took place between 8:00 a.m. and 2:00 p.m.

Solution preparations
The following solutions were used to perform the sciatic nerve block:
Codeine phosphate (Codein ® , Cristália, São Paulo, Brazil) • 30 mg / mL, 2 mL / ampule.The recommended dose of injectable COD for rats, according to the manufacturer, is 5 mg / kg; thus, each rat was administered 0.05 mL of codeine phosphate + 0.10 mL of saline solution for a final solution volume of 0.15 mL.
Tramadol chlorhydrate (Tramal ® , Pfizer, Guarulhos, Brazil) • 50 mg / mL, 1 mL / ampule.The recommended dose of injectable tramadol chlorhydrate for rats, according to the manufacturer, is 20 mg / kg; thus, each rat was administered 0.12 mL of TRAM + 0.03 mL of saline solution, for a final solution volume of 0.15 mL.
The gap time of the opioid in a LA injection was selected according to the pilot experiment.It was observed that 20 minutes was the time required for maximum decrease in the action potential amplitude for the peripheral nerve.
Braz Oral Res., (São Paulo) 2013 Nov-Dec;27(6):455-62 Thalhammer et al. 11 All animals were analyzed before treatment (baseline) and at the end of the experiment, to observe if they recovered their normal function.

Nociceptive response
This was achieved through two paw withdrawal reflex (PWR) tests: 1. Withdraw reflex: applying pressure as a nociceptive stimulus using an analgesiometer (Ugo-Basile, Comerio, Italy) across the skin, over the lateral metatarsus, exerting and increasing linear force (16 g / s) of up to 250 g (cutoff) on the rat's paw (Randall-Selitto method). 122. Mechanical stimulation: a 2 mm forceps was used to exert pressure across the skin fold over the lateral metatarsus.

Motor function
Normally, the extensor function is quantified when exerting the force required to reposition the metatarsus on the platform.The gait and spontaneous locomotive activity were evaluated by toe flexion (claudication); when the region received drugs, there was a deficit in motor function, which could

Experimental procedures
Two weeks before the experiments, the rats were randomly assigned to one of eight groups, and were exposed to preliminary experimental conditions to familiarize them with the experimenter.
The experiment involved functional steps; the paradigm is shown in Figure 1.The nerve block performed was evaluated by sensitive, motor and proprioceptive testing, and the sciatic block was conducted by an operator with access to the hind leg popliteal fossa, by SC injection. 10Thus, the experiment involved an injection procedure and a neurological evaluation, with analysis at 5-minute intervals.

Function evaluation
The functions were evaluated according to

Proprioceptive function
This was assessed by the lack of hopping and tactile-placing response.In the hopping test, the rat was kept at normal posture, where the toes of one foot were flexed with their dorsa placed on a supporting surface, and their ability to reposition their toes was evaluated as normal or abnormal.
Rats normally hop with their weight put in the direction of the movement, to avoid falling over, but when the proprioception is blocked, this response is absent (score 3) or classified as normal (0), slightly impaired (1) or severely impaired (2).

Statistical methods
All time duration data are shown as means ± SD, and were compared using ANOVA and Tukey as complementary tests, because the distribution was normal and homogeneous.As for the scores, the nonparametric data were analyzed using Kruskal-Wallis and Student-Newman-Kels as complementary tests.The significance level was 5%.

Results
Local anesthetic associated with COD was effective in promoting a block in rats, and proved longerlasting than using COD alone, which was administered 20 minutes prior to LA (p <0.05), and which provided a greater period of pain control; at the end of the experiment, all animals recovered their normal function.
The potential was verified when it caused a mean sensitive blockade lasting 36 minutes compared with only 20.5 minutes for TRAM (Figure 2).Although no significant difference was determined between these two groups (p > 0.05), the only factor confirmed was the greater potency of COD (Table 1).
The results presented in Figure 2 suggest a groundbreaking development regarding the confirmation of synergisms, since they indicate that the LA + COD association increased the mean duration of the sensitive block 2.6-fold in comparison with LA alone (Figure 3).
Thus, although the results of combinations achieved similar mean values (p > 0.05), COD was more promising, since a dose of only 5 mg / kg was able to achieve effects similar to 20 mg / kg of TRAM.These data are consistent with the therapeutic aim and pursuit of synergism, by obtaining maximum antinociception with minimal active dos-    es of the drugs used, promoting increased therapeutic efficacy without major side effects. 9egarding the dosing interval between the associated drugs to achieve sensitive blockade, it was observed that both the COD20' + LA and TRAM20' + LA groups-opioid injection 20 minutes prior to the LA-proved to be a less effective blockade than the association of LA + COD or LA + TRAM.Although a period of 20 minutes is indicated for the maximum reduction of the action potential, better activity was not achieved when COD and TRAM were injected prior to LA.This suggests that opioids presented the best sensitive block activity when injected at the same time as LA. 16We also have to consider that TRAM peaked at about 10 minutes; therefore, it will be possible to obtain different results if we use 10 minutes as a gap time in future research.

Discussion
This is the first study to evaluate the synergic effect of COD and TRAM in the duration of local anesthesia, considering nociceptive, motor and proprioceptive functions.
It is very common in dental health practice for patients to associate oral procedures with pain, and, since their experience is perceived as painful, this can lead them to avoid or delay treatment. 17This highlights the growing need for drug development and technical associations that meet patients' expectations of comfort, so that they may continue to seek good oral healthcare.
The association of local anesthetic and opioids may improve the duration of the analgesia.This is important, since the use of long-acting anesthetics, such as bupivacaine, is controversial.It can trigger an inflammatory reaction and increase postsurgical pain at the time of maximal inflammatory reaction. 18pioid are adjuvants that prove successful in research and clinical use.Anesthetic adjuvants now represent some of the most important developments in current research in this area. 2They are considered well tolerated and have few systemic effects following peripheral administration; however, COD is also a potent opioid analgesic with a complex mechanism of action.Its performance as an analgesic on the PNS is excellent, i.e., up to twice as good as that of the CNS. 19his strong effect on the PNS corroborates evidence of synergism and demonstrates that COD could be a potent anesthetic adjuvant.In addition, considering its natural origin, low cost and availability, compared with TRAM and other relevant opioids, COD has the potential of becoming an important anesthetic adjuvant.
A study by Tsai et al. 20 stated that TRAM has some mechanism that provides a greater power differential for local anesthetic action and synergisms.
The blockade could be mediated by peripheral specific receptor or nonspecific opioid receptor actions on the cell membrane dependent on coupled receptors; however, the mechanisms of action remain unclear. 6The possibilities include stimulation of opioid-specific receptors (µ, δ, κ), which according to their cell expression, could generate direct effects on the intracellular mechanisms that regulate transmission inside the cell through exocytotic vesicles. 21nother possibility is the action of nonspecificopioid receptors that are usually attached to cell membrane channels, where their activation is able to change the modulation ion.In fact, Rhim and Miller 22 demonstrated that presynaptic membrane activation could inhibit Ca 2+ channels and induce K + . 23lternatively, nonspecific opioid activity could occur in Na + channels, through molecular mechanisms different from those occasioned by LA. 24 Regarding previous attempts at pharmacological associations, the combination of a centrally acting drug (COD) with a peripherally acting drug (LA) supports the idea that drugs with actions that involve uniting central and peripheral pathways generate onset times, activity durations and various sites of drug action that could increase the analgesic capacity; moreover, these additive and synergistic effects could occur at lower doses. 25oth COD and LA have hydroxyl groups on a molecular chain attached to the benzene ring.This is an important factor for the nervous system. 26In contrast, COD is considered very potent, unlike TRAM, which has no monoaminergic interference, and which could have great beneficial action, instead of being just another opioid. 19t is known that codeine opioid molecularity is considered different in the opioid group, since it promotes greater concentration of effects on ionic membrane mechanisms and less dependency.Thus, activity in K + , Ca 2+ and even Na + channels is possible, 26,23,22 thus implying a reduction in or blockage of potential cellular hyperpolarization. 22he influx of Ca 2+ and induction of K + in the nerve terminal is considered the trigger for release in synaptic cleft neurotransmitters. 22Considering the possible blockade of Ca 2+ and K + channels induced by opioid codeine and tramadol, together with the blockade of Na + channels by LAs, which keep the cell hyperpolarized, a synergistic effect would occur, resulting in nerve conduction impairment.
The stereospecificity of the properties of these opioids has been discussed for some time.Some of the separated isomers and active metabolites of COD have been determined.Despite understanding that L-codeine presents good nociceptive activity, studies have also clarified that the antitussive effect of opioids is not dependent on stereospecificity. 27oncerning TRAM toxicity in the CNS, risk of neuronal toxicity has not been reported, and in the PNS, there is also no evidence of damage.Nevertheless, the value of TRAM as an anesthetic adjunct is uncertain; therefore, caution is necessary. 1Concerning COD in the PNS, it can produce a dose-dependent and reversible decrement in motor function. 9ur results further support the concept that combinations of different analgesic modalities could reduce the incidence and severity of pain and patient anxiety. 10e also must remember that long-action solutions are available in dentistry.The use of bupivacaine, a long-lasting local anesthetic, has potential for providing anesthesia in dentistry. 27ven though the mechanisms of LA effects of opioid codeine have not been fully elucidated, an analysis of the results obtained in this study suggests that when this opioid is administered as an adjunct to the LA, it may offer many benefits to controlling pain in dental procedures.

Conclusion
The evidence of synergism and the strong effect of minimal doses of codeine emphasize its potential as an adjunct to local anesthetic.Codeine is a strong candidate for playing an adjunct role in pain control.However, studies are necessary to confirm its clinical use.

Figure 3 -
Figure 3 -Mean values of paw withdrawal reflex (PWR) intensity following analgesiometer nociceptive stimulus assessed over time.

Table 1 -
Description of significance.