Protective effect of PNQX on motor units and muscle property after sciatic nerve crush in neonatal rats

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Abstract

Sciatic nerve injury in neonatal rats results in significant reduction in the number of surviving motoneurons and impairs muscle development. We examined the possible neuroprotective effects of daily in vivo administration of 1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3,4-f]quinoxaline-2,3-dione (PNQX), an AMPA/kainate receptor antagonist, on sciatic nerve injured rats, during the period of plasticity of the rat nervous system. Furthermore, we investigated the effect of PNQX on muscle properties impaired by nerve crush.

At the second postnatal day, the sciatic nerve of the rat left hind limb was crushed. Twenty-four rats were subsequently treated with PNQX and an equal number of rats, as control group, were treated with saline. PNQX was injected subcutaneously once daily (14 mg kg−1 body weight). Treatment continued until the rats were 14 days old. Measurements were then carried out to assess the contractile properties of the extensor digitorum longus (EDL), a fast-contracting muscle, and of the soleus muscle, a slow-contracting muscle, in four “age groups” of rats, each consisting of six PNQX-treated and six control animals: (a) postnatal day (P) 14, (b) postnatal day (P) 21, (c) postnatal day (P) 28 and (d) adult rats. The following parameters were recorded: number of motor units, muscle weight, maximal tetanic tension (TET 100), time to peak (TTP), half relaxation time (HRT), and fatigue index (FI). Improvement in motor unit survival after nerve injury was observed in all age groups administering PNQX. Also axotomy-induced impairment of muscle properties such as muscle weight, tension development, contraction and relaxation velocity was counteracted by injection of PNQX. The fatigue index was altered by axotomy and mostly normalized by treatment with this compound.

AMPA/kainate receptor antagonists, with low toxicity, may serve in the future, as possible neuroprotective agents after acute neural injury or even as therapeutic agents in neurodegenerative diseases.

Introduction

Motoneuron survival and development in the early postnatal period is strongly dependent upon interaction with the target-muscle [1], [2]. Acute injury of a peripheral nerve during this “critical” developmental stage causes massive loss of motoneurons [1], [2], [3]. In the limb muscles, such injury is reflected as a reduction in the number of motor units and alterations in the muscle properties [4]. A possible mechanism involved in axotomy-induced neuronal death is excessive activation of ionotropic glutamate receptors (NMDA and AMPA/kainate) and subsequent Ca2+ influx into the cell (excitotoxicity) [5]. Intracellular elevation of calcium concentration induces the activation of a cell death cascade (activation of proteases, lipases and other enzymes leading to cell lysis) [5], [6], [7].

Glutamate is an excitatory neurotransmitter in the CNS [8]. Ionotropic glutamate receptors (NMDA and AMPA/kainate), which have been identified throughout the brain and spinal cord [9], form an ion-channel through which Ca2+-ions enter the cell, when activated [10]. Function and properties of glutamate receptors undergo changes throughout CNS development [11]. It has also been shown that excitatory amino acids, in addition to their involvement in the mechanisms of pain transmission [12], participate in the pathogenesis of numerous nervous system diseases such as anxiety [13], [14], acute brain ischemia [15], injury of the central [16] and peripheral nervous system [17], Parkinson's [18], Alzheimer's [19], Huntington's [20] disease and Amyotrophic lateral sclerosis (ALS) [21]. Improving our understanding of the role of glutamate and its receptors could thus lead to the development of novel therapeutic agents for a variety of malfunctions of the nervous system.

Sciatic nerve injury by axotomy in neonatal rats results in significant reduction in the number of surviving motoneurons in the ventral horn of the lumbar segments. The reduction of motoneurons occurs due to retrograde cell death [22], [23] and impaired survival and development of motor units and muscles [24], [25], [26], [27], [28]. Nerve lesion on the fifth postnatal day, however, does not affect motoneuron survival [27].

Moreover, axotomy in the early postnatal period does not only reduce the number of surviving motoneurons and motor units, but also induces changes in the contractile properties of limb muscles [1], [24], [25].

Administration of an NMDA or an AMPA/kainate receptor antagonist such as dizocilpine maleate (MK-801) [29] or CNQX [30], [31] but also magnesium ions [32] within this critical period of development is thought to reverse the neurotoxic effects of axotomy and result in increased survival of motoneurons [29], [33], [34], [35]. On the other hand, it has been described that AMPA antagonist treatment may result in hypothermia, which itself is cytoprotective [36].

In the present study, we examined, for the first time, the possible effect of systemic administration of PNQX, an AMPA/kainate receptor antagonist [37], on peripheral nerve injury-induced loss of motor units and impairment of muscle properties. More precisely the efficacy of PNQX is investigated on motor unit survival and muscle properties (muscle weight, tension development, contraction and relaxation velocity and fatigue index) of a fast-contracting muscle (EDL) and a slow-contracting muscle (soleus muscle) after sciatic nerve crush in neonatal rats.

Section snippets

Materials and methods

Attention was paid to minimize pain and discomfort to the animals. Experiments have been conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) and were approved by the Ethical Committee of the School of Medicine of Aristotle University of Thessaloniki.

Forty-eight Wistar rats of both sexes were used in these experiments. The animals were housed in an

Results

In order to avoid unnecessary sacrifice of experimental animals the results regarding control and axotomy side of rats given saline are identical with the results obtained in experiments on influence on axotomy and Mg2+ administration, which were proceeded in parallel but was published previously [32].

Weight gain and physical condition of the PNQX-treated animals were similar to those of the control rats.

Physical examination findings on the operated leg locomotor recovery were similar to those

Discussion

The role of glutamate receptors in the pathogenesis of traumatic or ischemic damage to the nervous system has been well established [30], [43], [44]. There is evidence that the density of NMDA receptors on motoneurons is increased after neonatal nerve injury and that the binding affinity changes [43]. Blockade of NMDA receptor-mediated channels by treatment with magnesium sulphate [32], [35] or dizocilpine maleate (MK-801), an NMDA receptor antagonist [29], [33], [34], results in improved

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