Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats

Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats

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F. Ceccherelli 1, 2, G. Gagliardi 1, 2, A. Roveri 2, 3, M.R. Meneghetti 1,2, C. Ori 1

Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats Der Einfluss von Metoclopramid auf Akupunkturund Elektroakupunkturanalgesie – eine einfachblinde, kontrollierte Studie an Ratten Abstract

Zusammenfassung

Background: Metoclopramide is an antagonist that is selective of dopamine DA2-receptors. In high doses it appears to have antagonist properties also towards DA1 receptors. It has been demonstrated that the dopaminergic system is closely correlated to opioid actions; in particular it has been observed that the destruction of DA2 receptors in rats increases analgesia by μ and k1 agonists. The role of dopamine on acupuncture-induced analgesia is not fully defined; the data available is scarce and contrasting.

Hintergrund: Metoclopramid ist ein DA2-Rezeptor-selektiver Dopaminantagonist. In hohen Dosierungen scheint es seine antagonistischen Eigenschaften auch auf DA1-Rezeptoren auszudehnen. Es wurde gezeigt, dass das dopaminerge System eng mit den opioid-vermittelten Wirkungen verbunden ist; speziell wurde beobachtet, dass die Zerstörung von DA2-Rezeptoren im Rattenmodell die durch μ- und k1-Agonisten vermittelte Analgesie augmentiert. Die Rolle des Dopamins im Rahmen der akupunkturinduzierten Analgesie ist noch nicht vollständig geklärt; zu diesem Thema sind nur wenige und widersprüchliche Daten verfügbar. Ziel: Ziel ist die Klärung der Frage, ob Metoclopramid die analgetische Wirksamkeit von traditioneller und Elektroakupunktur an Ratten verstärken kann. Methode: Es wurden 140 Sprangue-Dawley-Ratten mit einem Gewicht zwischen 180 und 220 g verwendet, verteilt auf sieben Behandlungsgruppen zu je 20 Ratten. Kontrollgruppe 1 erhielt keinerlei Manipulation; Kontrollgruppe 2 erhielt Manipulation ohne Behandlung (weder Akupunktur noch Metoclopramid); Gruppe 3 wurde mit Metoclopramid behandelt; Gruppe 4 mit Akupunktur; Gruppe 5 wurde mit Akupunktur und Metoclopramid behandelt; Gruppe 6 mit Elektroakupunktur und Metoclopramid; Gruppe 7 erhielt ausschließlich Elektroakupunktur. Die Schmerzschwelle wurde mit dem Plantartest ermittelt. Ergebnis: Im gesamten Experiment zeigten sich keine signifikanten Unterschiede in der Schmerzgrenze der beiden Kontrollgruppen. Ratten, die Metoclopramid erhielten, hatten eine erhöhte Schmerzschwelle. Die Gruppen, die Akupunktur bzw. Elektroakupunktur erhielten, zeigten eine erhöhte Schmerzschwelle im Plantartest. Vorbehandlung mit Metoclopramid erhöhte den analgetischen Effekt der Stimulation. Schlussfolgerung: Es konnte gezeigt werden, dass Metoclopramid analgetische Eigenschaften besitzt und die analgetische Wirkung von Akupunktur und Elektroakupunktur verstärken kann.

Aim: The aim of the present study was to verify whether the administration of metoclopramide potentiates the analgesic efficacy of manual and electro-acupuncture in the rats. Methods: In this study we used 140 Sprangue-Dawley rats weighing between 180–220 g, divided into 7 groups of 20 on the basis of type of treatment. Control group 1: no manipulation; control group 2: subjected to all the manipulations without treatment (acupuncture and/or metoclopramide); group 3: treated with metoclopramide; group 4: treated with acupuncture; group 5: treated with metoclopramide and acupuncture; group 6: treated with metoclopramide and electro-acupuncture; group 7: treated with electro-acupuncture. The pain threshold was measured using the plantar test. Results: Throughout the experiment there were no significant variations in the pain thresholds of either of the two control groups. Rats treated with metoclopramide showed an increased pain threshold. Both groups treated with acupuncture and electro-acupuncture showed an increased pain thresholds in the plantar test. Pre-treatment with metoclopramide increased the analgesic effect of the stimulation. Conclusion: It can be demonstrated that metoclopramide possesses analgesic properties and can potentiate the effect of acupuncture and electro-acupuncture.

Keywords

Schlüsselwörter

Acupuncture, metoclopramide, RCT, pain, analgesia, plantar test

Akupunktur, Metoclopramid, RCT, Schmerz, Analgesie, Plantartest

FRANCESCO CECCHERELLI, MD., Dept. of Pharmacology and Anesthesiology, University of Padova, Via Cesare Battisti N°267, 35121 I-Padova

1

Tel.: +39 (0) 49 / 8 21 30 92 Fax: +39 (0) 49 / 87 54 25 6 [email protected]

Department of Pharmacology and Anesthesiology, University of Padova, Italy. A.I.R.A.S. (the Italian Association of Scientific Research and Development) Padova, Italy 3 Department of Biological Chemistry, University of Padova 2

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Introduction The use of drugs that influence acupuncture analgesia has a double purpose: it helps to understand certain mechanisms of acupuncture which have previously not been perfectly clear, as well as enhances its analgesic effects in clinical practice. Metoclopramide is a drug belonging to the benzamide derivatives and is a structural analog of procainamide. Metoclopramide is a drug normally used to control nausea and vomiting and to facilitate bowel transit. However, in reality its properties are numerous and involve different mechanisms and different areas of activity at both the central and peripheral levels of the nervous system. With regard to the mechanism of action, its properties may be summarized as follows [1]: 1. Central nervous system: a) block of DA2-receptors, particularly in the brainstem; b) control of gastrointestinal motility through central mechanisms; 2. Peripheral nervous system: a) increase in cholinergic response and in the release of acetylcholine from the postganglionic nerve endings. b) sensitization of the muscarinic receptors in the smooth muscle; c) decrease of the serotonergic activity d) decrease of the dopaminergic activity. e) decrease of the activity of the noradrenergic inhibitory terminals. Metoclopramide is a selective DA2 receptor antagonist and as such belongs to the class of antidopaminergic drugs; in high doses it appears to possess antagonist properties also towards DA1 receptors for dopamine [2]. There exists data assigning analgesic properties to metoclopramide [3] on pain induced by intravenous injection of propofol (2,6-diisopropylfenol), as well as on pain induced by ureterolithiasis [4]. Acupuncture in association with metoclopramide has been studied in rabbits. The drug was administered intravenously in three groups of rabbits 30 minutes before acupuncture in the following doses: 2 mg/kg, 4 mg/kg e 6 mg/kg, respectively. The dose administered for 6 mg/kg lead to a rise in the pain threshold [5]. Comparing the effects of the association with metoclopramide vs. procaine 0,1 % and procaine 1 % in patients anesthesized with acupuncture for thyreoidectomy, it was observed that the best results were obtained in the group treated with metoclopramide compared to the group treated with procaine 0,1 %, while there was no difference with the group treated with procaine 1 %. In conclusion, Xu and Coll. were able to affirm that metoclopramide may be used to assist acupuncture-induced analgesia [5]. The study by Junilla [6] carried out on patients affected for at least six months by chronic pain contrasts with these results; 10 mg of metoclopramide were administered orally 1 hour before the acupuncture session (the control group

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received 10 mg of placebo). A decrease in pain was observed in both groups starting 1 week after the second session of acupuncture and was maintained for the whole period of acupuncture treatment throughout the following 6 months. However, in no phase of the study, any differences were observed between the 2 groups (metoclopramide and placebo) [6].

Aim The aim of the present study was to verify whether the administration of metoclopramide potentiates the analgesic efficacy of manual and electro-acupuncture in rats.

Materials and Methods Animals In this study we used 140 Sprangue-Dawley rats weighing between 180–220 g, divided into 7 groups of 20 on the basis of type of treatment: • Control group 1: animals without any manipulation; • Control group 2: subjected to all the manipulations without treatment (manual acupuncture or electro-acupuncture and/or metoclopramide); • group 3: treated with metoclopramide; • group 4: treated by acupuncture; • group 5: treated with metoclopramide and acupuncture; • group 6: treated with metoclopramide and electro-acupuncture; • group 7: treated with electro-acupuncture. Experimental design The experiment was always carried out in the morning, between 9.00 a.m. and 1.00 p.m. The basal pain threshold of the right paw of all the animals was determined by the plantar test before they underwent any type of treatment [7]. Subsequently, after an interval of 30 minutes, all animals underwent light anaesthesia with ether. All groups with the exception of group 1 were then immobilized: they were placed stomach-down on a wooden board and retained with a strip of sticking-plaster on each of the four paws. Group 1 was purely a no-manipulations control group; 30 minutes after the first measurement these rats underwent very light anaesthesia with ether, and after an interval of 20 minutes they underwent the further measurements. Group 2 was also a control group, but unlike group 1 it underwent the immobilization, light anaesthesia and injection procedures (a intraperitoneal needle without injection of any substance) and without undergoing any type of treatment of stimulation; after 20 minutes they underwent further measurements. Group 2 was submitted only to the stress of experimental procedures.

Originalia | original articles F. C e c c h e r e l l i , G . G a g l i a r d i , A. Roveri, M.R. Meneghet ti, C. Ori

Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats

Group 3 was treated with metoclopramide. The rats in this group, after measurement of the basal threshold, were injected with metoclopramide intraperitoneally (6 mg/kg). Subsequently, after 30 minutes they underwent further measurements. Group 4, after the initial 30 minutes, was immobilized, anesthetized with ether, and then treated with acupuncture for 20 minutes. Four points then were stimulated: St 36 (Zusanli) and LI 4 (Hegu), bilaterally. After the puncture the needles were stimulated with a right-left turn movement for 30 seconds and the stimulation was repeated after 10 minutes. Also in this case, at the end of the treatment which lasted 20 minutes, further measurements took place. Group 5 underwent both treatments. After measurement of the basal painthreshold, the rats in this group were injected with metoclopramide intraperitoneally (6 mg/kg). Subsequently, after 30 minutes the animals were anesthetized with ether and then underwent acupuncture treatment for 20 minutes. Also, in this case, at the end of treatment further measurements took place. Group 6 underwent both electro-acupuncture and treatment with metoclopramide. After measurement of the basal threshold, the rats in this group were injected with metoclopramide intraperitoneally (6 mg/kg). Subsequently, after 30 minutes the animals were anesthetized with ether and treated with electro-acupuncture (15 Hz) for 20 minutes. The points stimulated were GB 30 (Huantiao) and St 36 (Zusanli), bilaterally. Also in this case, at the end of treatment further measurements took place.

After the first 30 minutes group 7 was immobilized, anesthetized with ether, and then treated with electro-acupuncture (15 Hz) in the same way as the preceding group. Also, in this case, at the end of treatment further measurements took place.

For electro-acupuncture the technique of inserting the needles was the same as that used for conventional acupuncture. After insertion, the needles were connected to an electric stimulator (Sedatelec® mod. Agistim Duo) with a pulsating current of 15 Hz and an intensity of 5 mA. The choice of stimulation frequency was due to the efficacy on the central production and release of opioids [8]. The current intensity used in the present work was chosen on the basis of previous experimental evidences of efficacy in rats [9–10]. The impulses were represented by a positive square wave and a negative spike; the mean current was zero. The amplitude of the impulses was maintained through a device with a constant current and was checked continuously by an oscilloscope (Hameg mod. HM 408).

Figure 1: Time course of pain threshold values of groups treated with manual acupuncture. Data are compared to those of both control groups and metoclopramide treated group. Controls: control rats without manipulations; contr+manip: control rats with all manipulation; acupuncture: rats with manual acupuncture stimulation; meto+acup: rats with acupuncture and metoclopramide; metoclopr: rats with administration of metoclopramide.

Figure 2: Time course of pain threshold values of groups treated with electro-acupuncture. Data are compared to those of both control groups and metoclopramide treated group. Controls: control rats without manipulations; contr+manip: control rats with all manipulation; metoclopr: rats with administration of metoclopramide; meto+electroa: rats with metoclopramide and electro-acupuncture; electroacupun: rats with electro-acupuncture.

Needles Steel needles by the French manufacturer Sedatelec® were used; they were 0.16 mm thick and shortened to about 7 mm in length; the needles were inserted into the muscle at a depth of 4–5 mm from the surface of the skin. Electrical stimulation

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Measurement of the pain threshold The pain threshold of each rat in all seven groups was measured six times with the same time intervals for each animal: • T0 (before treatment began); • T1 (after treatment with acupuncture, or 50 minutes after the first measurement): • T2 (after 30 minutes from T1); • T3 (after 60 minutes from T1); • T4 (after 90 minutes from T1); • T5 (after 120 minutes from T1); In the time intervals between the various measurements, the rats were left quietly inside the special cages. The pain threshold was measured by the plantar test (Ugo Basile) [7]. This consisted of a plate in plexiglas with 3 boxes fixed to it in which the rats were placed. There was an infrared lamp underneath the transparent base with a slit at the top. The lamp was placed correspondingly beneath the rat’s paw, heating it. When the heat reached the pain threshold, the rat moved its paw, a photo-electric cell fixed the time of irradiation. The irradiation times obtained in the different groups were compared with the values observed in the control rats. Statistical Analysis Averages and Standard Deviations were calculated for the results obtained; the ANOVA test was applied for repeated measurements to evaluate the statistical significance of the differences between the groups.

Results The results are summarized in Table 1. Neither of the control groups showed significant variations of the pain threshold for the entire duration of the experiment. This indicates that the methodology used does not put the animals under stress from manipulation at a level that would induce analgesia. The group of rats treated with metoclopramide showed an increase in the pain threshold at T1; 50 minutes after the first measurement the increase was 51.76 %; T2 and T3 times registered a further small increase that was not statistically significant. Statistical analysis showed a significance of p < 0.05 only at T1 time versus both the control groups. The others times showed no statistically significant variations. In the groups treated with acupuncture, the analgesic effect of the technique was confirmed: conventional acupuncture increased the pain threshold by a value of 55.4 % at T1 time which was a statistically significant value compared to both control groups. The administration of metoclopramide increased the analgesic effect of acupuncture; in fact at the T1 time the group of rats thus treated showed an increase of 71.24 % with statistical significance of p < 0.05, compared with all the

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other groups in the study with the exception of the group treated with EAP and metoclopramide. Electro-acupuncture In the group that underwent electro-acupuncture, an important analgesic effect was observed: the increase of the pain threshold was 57.12 % at T1 time compared to the basal value, and was statistically significant compared with the control groups; there was no statistical significance, however, towards the group treated with metoclopramide and acupuncture. The administration of metoclopramide associated with electro-acupuncture potentiated the analgesic effect; in fact the T1 time of the group of rats thus treated showed an increase of 83.69 %. The statistical comparison with the group treated solely with electro-acupuncture did not however show any significance (value of p = 0.064); neither did it show any significance for the group treated with metoclopramide.

Discussion and Conclusions More recent data relegates an important role in antinociception to the dopaminergic system, in particular referring to chronic pain. In fact, it seems that the inhibition of chronic pain is mediated also through the activation of dopaminergic mesolimbic neurons arising from cell bodies of the ventral tegmental area and projecting to the nucleus accumbens. This control system is activated by acute stress and is mediated by the release of opioids and substance P within the ventral tegmental area [11]. Other experimental evidence shows an increase in the D2 receptors and a reduced D1/D2 ratio in the substantia nigra system to be associated with conditions of chronic orofacial pain [12] In any case, it has been demonstrated that the dopaminergic system is closeley associated to the actions of opioids. In particular, it has been observed that the destruction of D2 receptors in rats increases analgesia induced by μ− and k1 agonists in a dose-dependant way, while it does not influence analgesia by δ agonists [13]. The role of dopamine in acupuncture-induced analgesia has not been perfectly defined; acupuncture increases brain dopamine and homovanillic acid levels [14]. The administration of chlorpromazine decreases the analgesic effect of acupuncture [14]. Kho [15] reports there were no alterations in the brain dopamine levels after a single treatment of the Zusanli point, but an increase of it was seen in the caudate nucleus. Four dopamine agonists were tested regarding their effects on acupuncture analgesia and on the L-tetrahydropalmatine (L-THP)-induced analgesia in rabbits. The results showed that the analgesic efficacy of both the techniques is notably reduced by intracerebroventricular (ICV) administration of dopamine agonists like apomorphine. Similar results have been obtained by administering SKF-38393, a D1 selective agonist. On the contrary, quinpirole hydrochloride, a selective D2 agonist, appears to increase EA analgesia [16].

Originalia | original articles F. C e c c h e r e l l i , G . G a g l i a r d i , A. Roveri, M.R. Meneghet ti, C. Ori

Table 1

Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats

Values of the means and SD of the pain threshold expressed in seconds registered in the groups of rats treated at the various times; # significance for p < 0.05 vs controls; § significance for p < 0.05 vs metoclopramide; * significance for p < 0.05 vs manual acupuncture; $ significance for p < 0.05 vs electro-acupuncture. T0

T1

T2

T3

T4

T5

Controls

14,6 ± 3,12

14,78 ± 3,74

14,54 ± 3,57

14,29 ± 3,17

13,97 ± 3,69

13,15 ± 2,8

Controls with Manipulation

15,59 ± 3,77

15,28 ± 4,03

14,92 ± 3,14

15,06 ± 3,66

15,49 ± 5,29

13,68 ± 3,18

Metoclopramide

15,28 ± 2,32

23,19# ± 4,51

15,86 ± 3,61

16,13 ± 3,37

13,43 ± 3,3

13,71 ± 2,89

Manual Acupuncture

14,8 ± 2,15

23,0# ± 5,34

16,6 ± 3,41

15,0 ± 3,04

14,4 ± 4,07

12,8 ± 3,01

Metoclopramide and Acupuncture

16,1 ± 1,8

27,6#§*$± 4,05

19,15# ± 5,14

16,86 ± 4,3

14,51 ± 3,69

14,27 ± 3,1

Metoclopramide and Electro-Acupuncture

13,74 ± 1,91

25,2# ± 6,36

15,53 ± 4,29

12,47 ± 4,14

10,38 ± 3,0

11,92 ± 3,9

Electro-Acupuncture

13,62 ± 2,3

21,4# ± 6,23

15,38 ± 4,98

13,19 ± 4,06

14,13 ± 3,99

13,44 ± 5,58

In another work, two selective agonists of D1 or D2 receptors, SKF-38393 and quinpirole (Qui), were injected into the nucleus accumbens of rats to investigate the roles of D1 and D2 receptors in the potentiation of EA analgesia induced by L-THP. The injection of D1 agonist SKF-38393 (5 μg or 10 μg) attenuated the potentiation of EA analgesia induced by L-THP, while the injection of D2 agonist Qui (10 μg or 20 μg) had no effect on EA analgesia and the potentiation of EA analgesia induced by l-THP. The results indicate that the D1,but not the D2, receptor in N. accumbens plays an important role in EA analgesia [17]. Our results have confirmed the analgesic effect of acupuncture, as well as a fair analgesic effect of metoclopramide when used alone. We cannot confirm the hypothesis suggested by Ranaswamy and Coll. [18] that the analgesic activity of metoclopramide is mediated by opioids, since a opioid receptor antagonist appears to be able to decrease the analgesic effect of the drug which seems to be linked also to the action of prolactin; bromocriptine, a dopaminergic agonist inhibitor of prolactin release, seems to lessen the analgesic response to metoclopramide. Appadu and Lambert do not seem to agree with this hypothesis [19]. In the “metoclopramide plus acupuncture” group, an increase in the pain threshold was registered at time T1 which was significantly higher than that in the “metoclopramide-“ and acupuncture-only” groups. The “metoclopramide plus acupuncture” group was the only group to maintain a significantly higher threshold compared to the controls, also in time T2. Our results agree with the study by Xu and Coll. [20] who demonstrated an increase in acupuncture-induced analgesia in rabbits. In our study, the group that received electroacupuncture plus metoclopramide showed an increase in the threshold at time T1 greater than that of the group which received only electro-acupuncture, but this difference was not statistically significant.

The results obtained confirm that acupuncture, metoclopramide, and electro-acupuncture each have, singularly, an analgesic effect; but they also show that this property is potentiated when the above are used in combination. It may be hypothesized that the effect obtained from the combination of metoclopramide with the acupunctural techniques we used is due to the antidopaminergic property of the drug and to its augmentation of opioid activity. Further studies are necessary to confirm this hypothesis. Role of Authors Prof. F. Ceccherelli: compilation of the protocol, and final version of the work. Dott. G. Gagliardi: execution of animal experimentation and statistical elaboration Dott.ssa A. Roveri: English translation and collaboration on the discussion Dott.ssa M.R. Meneghetti: execution of animal experimentation. Prof. C. Ori: supervision Financial Support This study was supported financially by A.I.R.A.S. (Associazione Italiana per la Ricerca e l’Aggiornamento Scientifico). Conflict of Interests Prof. F. Ceccherelli and Dott. G. Gagliardi are teaching to the AIRAS course of medical acupuncture and reflexotherapy. Acknowledgements The Author wishes to thank A.I.R.A.S. for financial support and collaboration.

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