Acetyl-L-Carnitine Induces Muscarinic Antinocieption in Mice and Rats

FLORE Repository istituzionale dell'Università degli Studi di Firenze ACETYL-L-CARNITINE INDUCES MUSCARINIC ANTINOCICEPTION IN MICE AND RATS Questa è la Versione finale referata (Post print/Accepted manuscript) della seguente pubblicazione: Original Citation: ACETYL-L-CARNITINE INDUCES MUSCARINIC ANTINOCICEPTION IN MICE AND RATS / C. GHELARDINI; N. GALEOTTI; M. CALVANI; L. MOSCONI; R. NICOLAI; A. BARTOLINI;. - In: NEUROPHARMACOLOGY. - ISSN 0028- 3908. - STAMPA. - 43(2002), pp. 1180-1187. [10.1016/S0028-3908(02)00225-3] Availability: This version is available at: 2158/311633 since: 2016-11-09T12:32:15Z Published version: DOI: 10.1016/S0028-3908(02)00225-3 Terms of use: Open Access La pubblicazione è resa disponibile sotto le norme e i termini della licenza di deposito, secondo quanto stabilito dalla Policy per l'accesso aperto dell'Università degli Studi di Firenze (https://www.sba.unifi.it/upload/policy-oa-2016-1.pdf) Publisher copyright claim: (Article begins on next page) 27 September 2021 Neuropharmacology 43 (2002) 1180–1187 www.elsevier.com/locate/neuropharm Acetyl-l-carnitine induces muscarinic antinocieption in mice and rats Carla Ghelardini a,∗, Nicoletta Galeotti a, Menotti Calvani b, Luigi Mosconi b, Raffaella Nicolai b, Alessandro Bartolini a a Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini 6, I-50139 Florence, Italy b Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina km 30,400, I-00040 Pomezia, Rome, Italy Received 25 January 2002; received in revised form 24 July 2002; accepted 13 August 2002 Abstract The analgesic activity of acetyl-L-carnitine (ALCAR) in neuropathic pain is well established. By contrast, its potential efficacy in the relief of acute pain has not been reported. The antinociceptive effect of ALCAR was, therefore, examined in the mouse hot- plate and abdominal constriction tests, and in the rat paw-pressure test. ALCAR (100 mg kgϪ1 s.c. twice daily for seven days) produced an increase of the pain threshold in both mice and rats. ALCAR was also able to reverse hyperalgesia induced by kainic acid and NMDA administration in the mouse hot-plate test. The antinociception produced by ALCAR was prevented by the unselec- tive muscarinic antagonist atropine, the M1 selective antagonists pirenzepine and S-(-)-ET126, and by the choline uptake inhibitor hemicholinium-3 (HC-3). By contrast the analgesic effect of ALCAR was not prevented by the opioid antagonist naloxone, the GABAB antagonist CGP 35348, the monoamine synthesis inhibitor (a)-methyl-p-tyrosine, and the Gi-protein inactivator pertussis toxin. Moreover, ALCAR antinociception was abolished by pretreament with an antisense oligonucleotide (aODN) against the M1 receptor subtype, administered at the dose of 2 nmol per single i.c.v injection. On the basis of the above data, it can be postulated that ALCAR exerted an antinociceptive effect mediated by a central indirect cholinergic mechanism. In the antinociceptive dose- range, ALCAR did not impair mouse performance evaluated by the rota-rod and hole-board tests. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acetyl-L-carnitine; Acetylcholine; M1 muscarinic receptor subtype; Analgesia; Pain; Hyperalgesia 1. Introduction been demonstrated to improve cognitive deficits in some experimental paradigms (Pettegrew et al., 1995; Acetyl-L-carnitine (γ-trimethyl-β-acetylbutyrobetaine; Dell’Anna et al., 1997), to modify the cerebral electrical ALCAR), the acetyl ester of carnitine, is a small water- activity (Mancia et al., 1990); and to induce an antide- soluble molecule naturally present in the central nervous pressant-like effect (Pulvirenti et al., 1990). Furthermore, system. ALCAR exerts a neuroprotective effect by ALCAR modulates the release of various neurotransmit- increasing glutathione and by decreasing the concen- ters such as dopamine (Harsing et al., 1992), excitatory tration of malondialdehyde (Fariello et al., 1987), pro- amino acids, taurine (Toth et al., 1993), acetylcholine moting nerve growth factor receptor synthesis and (Imperato et al., 1989), and GABA (Fariello et al., 1988). increasing its levels in the hippocampus (Taglialatela et ALCAR has also been demonstrated to be signifi- al., 1991, 1992). cantly effective in reducing neuropathic pain, a condition Several studies have indicated that ALCAR is characterised by spontaneous pain, allodynia and hyper- involved in different aspects of neuronal activity. It has algesia caused by traumatic injury, diabetes, and viral infections. Intramuscular chronic treatment with ALCAR significantly improves the outcome of painful ∗ Corresponding author. Tel.: +39 055 4271312; fax: +39 055 neuropathies or radiculopathies (Onofrj et al., 1995). A 4271280. beneficial effect of ALCAR has been reported in the E-mail address: carla.ghelardini@unifi.it (C. Ghelardini). treatment of symptomatic diabetic neuropathy (Quatraro 0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(02)00225-3 C. Ghelardini et al. / Neuropharmacology 43 (2002) 1180–1187 1181 et al., 1995) and in the treatment of pain in distal sym- scopically after sectioning. The accuracy of the injection metrical polyneuropathy related to HIV infection technique was evaluated and the percentage of correct (Scarpini et al., 1997). However, the mechanism under- injections was 95. lying the analgesic effect of ALCAR is still obscure. By contrast, its potential efficacy in the relief of acute 2.3. Intrathecal injection technique pain has not been reported. On these bases, the aim of the present study was to first investigate the analgesic Intrathecal injections were performed under ether properties of ALCAR in animal models of acute pain anaesthesia as described by Hylden and Wilcox (1980) by using different nociceptive stimuli, such as thermal, in which the mouse was gently restrained and a 30- chemical and mechanical. We also investigated the gauge, 1/2-inch needle mated to a 50 µl Hamilton syr- receptor subtypes involved in the increase of pain thres- inge was inserted between L5 and L6 of the mouse spi- hold induced by ALCAR to elucidate its mechanism nal column. A volume of 5 µl was used for i.t. injection. of action. In order to exclude that the effects produced by 2.4. Hot-plate test ALCAR treatment were due to the induction of side effects, some additional behavioural tests (rota rod, hole The method adopted was described by O’Callaghan board) were performed. and Holzman (1975). Mice were placed inside a stainless steel container, thermostatically set at 52.5 ± 0.1°Cina precision water-bath from KW Mechanical Workshop, 2. Methods Sienna, Italy. Reaction times (s), were measured with a stop-watch before and at regular intervals up to a 2.1. Animals maximum of 120 min after treatment. The endpoint used was the licking of the fore or hind paws. Before pretreat- Maple Swiss albino mice (23–25 g) and Wistar rats ing animals with ALCAR, a pretest was performed: (150–200 g) from the Morini (San Polo d’Enza, Italy) those mice scoring below 12 and over 18 s in the pretest breeding farm were used. Fifteen mice or five rats were were rejected (30%). An arbitrary cut-off time of 45 s housed per cage (26 × 41 cm). The cages were placed was adopted. in the experimental room 24 h before the test for acclimatisation. The animals were fed a standard labora- 2.5. Abdominal constriction test tory diet and tap water ad libitum and kept at 23 ± 1°C with a 12 h light/dark cycle, light on at 7 a.m. Each Mice were injected i.p. with a 0.6% solution of acetic animal was used only once and was killed immediately acid (10 ml kgϪ1), according to Koster et al. (1959). The after the end of the experiment by exposure to diethyl number of stretching movements was counted for 10 ether. All experiments were carried out in accordance min, starting 5 min after acetic acid injection. with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for experimental animal 2.6. Paw pressure test care. All efforts were made to minimise animal suffer- ing, and to reduce the number of animals used. The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy), 2.2. Intracerebroventricular injection technique according to the method described by Leighton et al. (1988). Threshold pressure was measured before and 15, Intracerebroventricular (i.c.v.) administration was per- 30, 45 and 60 min after treatment. Before pretreating formed under ether anaesthesia, according to the method animals with ALCAR a pretest was performed: rats scor- described by Haley and McCormick (1957). Briefly, dur- ing below 30 g or over 85 g were rejected (25%). An ing anaesthesia, mice were grasped firmly by the loose arbitrary cut-off value of 250 g was adopted. skin behind the head. A 0.4 mm external diameter, hypo- dermic needle attached to a 10 µl syringe was inserted 2.7. Hole-board test perpendicularly through the skull and no more than 2 mm into the brain of the mouse, where 5 µl were then The hole board test consisted of a 40 cm square plane administered. The injection site was 1 mm to the right with 16 flush mounted cylindrical holes (3 cm diameter) or left from the midpoint on a line drawn through to the distributed 4 × 4 in an equidistant, grid-like manner. anterior base of the ears. Injections were performed into Mice were placed on the centre of the board one by one the right or left ventricle randomly. To ascertain that the and allowed to move about freely for a period of 10 min drugs were administered exactly into the cerebral ven- each. Two electric eyes, crossing the plane from mid- tricle, some mice (20%) were injected with 5 µlof point to mid-point of opposite sides, thus dividing the diluted 1:10 India ink and their brains examined macro- plane into four equal quadrants, automatically signalled 1182 C.

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