Comparative effects of levobupivacaine and racemic bupivacaine on excitotoxic neuronal death in culture and N-methyl-d-aspartate-induced seizures in mice

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Abstract

We compared the neurotoxic profile of racemic bupivacaine and levobupivacaine in: (i) a mouse model of N-methyl-d-aspartate (NMDA)-induced seizures and (ii) in an in vitro model of excitotoxic cell death. When used at high doses (36 mg/kg) both bupivacaine and levobupivacaine reduced the latency to NMDA-induced seizures and increased seizure severity. However, levobupivacaine-treated animals underwent less severe seizures as compared with bupivacaine-treated animals. Lower doses of levobupivacaine and bupivacaine had opposite effects on NMDA-induced seizures. At doses of 5 mg/kg, levobupivacaine increased the latency to partial seizures and prevented the occurrence of generalized seizures, whereas bupivacaine decreased the latency to partial seizures and did not influence the development of generalized seizures. In in vitro experiments, we exposed primary cultures of mouse cortical cells, containing both neurons and astrocytes, to 100 μM NMDA for 10 min for the induction of excitotoxic neuronal death. This treatment killed 70–80% of the neuronal population, as assessed 24 h after the excitotoxic pulse. In this particular model, both levobupivacaine and bupivacaine were neuroprotective against NMDA toxicity. However, neuroprotection by levobupivacaine was seen at lower concentrations (with respect to bupivacaine) and was maintained at concentrations of 3 mM, which are much higher than the plasma security threshold for the drug in vivo. In contrast, no protection against NMDA toxicity was detected when 3 mM concentrations of bupivacaine were applied to the cultures. Our data show a better neurotoxic profile of levobupivacaine as compared to racemic bupivacaine, and are indicative of a safer profile of levobupivacaine in clinical practice.

Introduction

Bupivacaine is a local anaesthetic commonly used in dental, ophthalmologic, and simple surgical procedures. Its widespread use is derived from its potency, long half-life and its tendency to provide more sensory than motor block. The clinical use of bupivacaine is hampered by the occurrence of severe adverse effects ensuing from the systemic absorption of the drug, such as cardiotoxic and neurotoxic effects (Graf et al., 2002, Mather and Chang, 2001, Simon et al., 2002, Gristwood, 2002, Heavner, 2002). Bupivacaine toxicity results from the inhibition of voltage-gated sodium channels and several types of potassium channels (Clarkson and Hondeghem, 1985, Olschewski et al., 1999, Longobardo et al., 2001, Friederich et al., 2004). Neurotoxicity depends on the impact of the drug on excitatory and inhibitory processes in the central nervous system (CNS). Bupivacaine induces generalized seizures by depressing the activity of inhibitory neurons and by inhibiting potassium channels in excitatory neurons (Graf et al., 2002). The neurotoxic and cardiodotoxic (increased QTc interval, arrhythmias, and hypotension) effects of bupivacaine have been ascribed to the racemic nature of the drug, and, in particular, to the presence of the d (+) isomer in the commercial preparation. Electrophysiological studies show that the d (+) isomer of bupivacaine is a more potent and faster blocker of voltage-gated Na+ channels, and inactivates several subtypes of ATP-dependent and voltage-dependent K+ channels (Kv) with higher affinity than the l (−) isomer (Arias, 2002, Franqueza et al., 1997, Kawano et al., 2004). The recent development of the l(−)-enantiomer of bupivacaine, levobupivacaine, holds the potential of a better safety profile. However, while several studies show that levobupivacaine treatment is associated with a lower risk of cardiovascular toxicity (Mather and Chang, 2001, Morrison et al., 2000, Gristwood, 2002), there are only a few studies that compare the neurotoxic profile of levobupivacaine and bupivacaine. In sheeps, levobupivacane induces less CNS toxicity than racemic bupivacaine (Ladd and Mather, 2000, Santos and DeArmas, 2001), and higher doses of levobupivacaine are required for the induction of motor seizures, as compared with racemic bupivacaine (Huang et al., 1998); in addition, levobupivacaine induces lesser electroencephalography (EEG) abnormalities than bupivacaine in human volunteers (Arias, 2002) and causes less apnea and death when infused in rats (Srinivasa et al., 2003). How these drugs affect mechanisms of neuronal excitation under physiological and pathological conditions is unknown. Here, we have examined the comparative effects of racemic bupivacaine and levobupivacaine in an in vitro model of excitotoxic death (mixed cultures of mouse cortical cells challenged with NMDA) and in an in vivo model of chemically induced seizures, i.e. NMDA-induced motor seizures in mice.

Section snippets

Materials

Levobupivacaine was bought from Chirocaine; Purdue Pharma LP (Stamford, CT, USA), bupivacaine and all other drugs were bought from Sigma (Milano, Italy).

In vivo studies

Animal studies were performed according to the European Community guidelines for the use of experimental animals. CD1 mice, 30–35 g body weight, were used. We induced epileptic seizures by injecting mice with NMDA. This model is widely used to test the activity of anticonvulsant drugs (Sofia et al., 1994, Velisek and Mares, 1995, Villetti et

In vivo experiments

We compared the pro-convulsant effect of levobupivacaine and racemic bupivacaine in mice co-injected with NMDA. All control mice injected with NMDA (100 mg/kg i.p.) showed generalized tonic–clonic convulsions, but none of them reached scores 5 or 6 of seizure severity (generalized tonic–clonic convlusions, status epilepsicus, and death) during the 2-h observation period (see Table 1, Table 2). Bupivacaine and leobupivacaine were combined with NMDA either at low or high doses (5 or 36 mg/kg,

Discussion

Bupivacaine, a local anaesthetic, possesses steroisomeric properties that have been related to the cardiovascular and neurological toxicity of the drug. Within the last few years, the l (−) isomer of bupivacaine, levobupivacaine, has been introduced into clinical practice in the attempt of reducing the side effects of the racemic formulation. Induction of seizures is one of the major concerns related to the neurotoxic adverse effects of local anaesthetics. This apparent paradoxical effect

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