Cooperative N-methyl-D-aspartate (NMDA) receptor antagonism and mu-opioid receptor agonism mediate the methadone inhibition of the spinal neuron pain-related hyperactivity in a rat model of neuropathic pain.

Methadone (Racemic methadone) exerts its antinociceptive effect by activation of mu-opioid receptors and/or blockade of NMDA receptors. The aim of this study is to determine whether the methadone analgesic effect on neuropathic pain is achieved only by the agonism of the mu-opioid receptors or cooperatively with the antagonism of the NMDA receptors. To this purpose, in rats with neuropathic pain model of chronic constriction of one sciatic nerve (CCI rats), we administered methadone before or after opioid receptor blockade with naloxone and checked its effects on the spinal Wide Dynamic Range (WDR) neuron dynamics in three experimental conditions: on the spontaneous and noxious evoked neuronal activities in control rats (sham operated and naïve); on iontophoretic NMDA induced neuronal hyperactivity in intact rats; on pain-related spontaneous and noxious evoked hyperactivities in CCI rats. The results, as from the spike-frequency analysis, show that: (i) in control rats, methadone inhibits the noxious evoked neuronal activity and naloxone prevents or reverses about 94% of methadone inhibitory effect; (ii) in intact rats, pretreated with naloxone, methadone reduces the NMDA induced neuronal hyperactivity; (iii) in CCI rats, methadone inhibits the neuronal spontaneous and noxious evoked hyperactivities, and naloxone prevents or reverses about 60% of methadone inhibitory effect. These findings allow to conclude that methadone inhibition of the noxious evoked activity in normal rats is achieved predominantly through the agonism of the mu-opioid receptors, while the inhibition of the pain-related hyperactivity in rats with signs of neuropathic pain (CCI rats), involves also the NMDA receptors antagonism.

Contribution by DRt descending facilitatory pathways to maintenance of spinal neuron sensitization in rats.

We investigated in different experimental rat models the potential facilitatory contribution of the medullary dorsal reticular nucleus (DRt) descending pathway to the expressions of the sensory spinal neuron sensitization such as increased spontaneous and noxious evoked activities, responsivity to heterotopic afferences stimulation and long lasting afterdischarges (ADs). We carried out experiments by recording from ipsilateral lumbar Wide Dynamic Range (WDR) neurons and by simultaneously monitoring the DRt neuron activity in neuropathic pain rats with chronic constriction injury of one sciatic nerve (CCI), in sham-operated and in "intact" rats. In particular, we recorded the spinal neuron spontaneous activities and the activities evoked by noxious stimulations of ipsi- and contralateral sciatic supplied areas before and during DRt activity blockade. Additionally, in "intact rats" we modulated WDR activity by iontophoretic NMDA to mimic CCI WDR hyperactivity without peripheral damage. We found that during DRt activity blockade in CCI rat neurons and in "intact" rat NMDA-treated neurons, the spontaneous activity was significantly reduced, the responses to contralateral sciatic area stimulation were reduced or suppressed, the responses to ipsilateral sciatic area were poorly affected (slightly reduced or unaffected), except for the poststimulus afterdischarges that were mostly suppressed. In sham-operated rats, the neuronal activity was not affected by DRt blockade. The finding that during the DRt nucleus blockade some expressions of spinal neurons sensitization, seemingly associated to sensory disorders in neuropathic pain, fade or extinguish designates a likely facilitatory role of DRt in the maintenance of neuronal sensitization and thus a contribution to neuropathic pain state.

Effects of the bisphosphonate ibandronate on hyperalgesia, substance P, and cytokine levels in a rat model of persistent inflammatory pain.

The anti-inflammatory and analgesic properties of different bisphosphonates have been demonstrated in both animal and human studies. Ibandronate is a third-generation bisphosphonate effective in managing different types of bone pain. In this study we investigated its effects in a standard pre-clinical model of inflammatory pain. We evaluated the effects of a single injection of different doses (0.5, 1.0, and 2.0 mg/kg i.p.) of ibandronate on inflammatory oedema and cutaneous hyperalgesia produced by the intraplantar injection of complete Freund's adjuvant (CFA) in the rat hind-paw. In addition, we measured the effects of this drug (1.0 mg/kg i.p.) on hind-paw levels of different pro-inflammatory mediators (PGE-2, SP, TNF-alpha, and IL-1beta). We also measured the levels of SP protein and of its mRNA in the ipsilateral dorsal root ganglia (DRG). Ibandronate proved able to reduce the inflammatory oedema, the hyperalgesia to mechanical stimulation, and the levels of SP in the inflamed tissue as measured 3 and 7 days following CFA-injection. This drug significantly reduced the levels of TNF-alpha and IL-1beta only on day 7. On the other hand, the levels of PGE-2 in the inflamed hind-paw were unaffected by the administration of this bisphosphonate. Finally, ibandronate blocked the overexpression of SP mRNA in DRG induced by CFA-injection in the hind-paw. These data help to complete the pharmacodynamic profile of ibandronate, while also suggesting an involvement of several inflammatory mediators, with special reference to substance P, in the analgesic action of this bisphosphonate.

Heterotopic inputs facilitate poststimulus afterdischarges of spinal WDR neurons in rats with chronic nerve constriction.

Heterotopic inputs activate spinal wide dynamic range (WDR) neurons in rats with chronic constriction of one sciatic nerve (CCI rats). A possible contribution from these inputs, to long-lasting afterdischarges (ADs) of noxious evoked responses, was investigated during reversible input blockade from adjacent saphenous nerve and contralateral peripheral nerve territories. The results show significant AD reduction or suppression, indicating that heterotopic afferences contribute to mechanisms underlying prolonged ADs.

AM404, an inhibitor of anandamide uptake, prevents pain behaviour and modulates cytokine and apoptotic pathways in a rat model of neuropathic pain.

An attractive alternative to the use of direct agonists at the cannabinoid receptor type 1 (CB1) in the control of neuropathic pain may be to potentiate the actions of endogenous cannabinoids. Thus, the effects of AM404, an inhibitor of anandamide uptake, were assessed in an experimental model of neuropathic pain in rats. Daily treatment with AM404 prevented, time- and dose-dependently, the development of thermal hyperalgesia and mechanical allodynia in neuropathic rats. Antagonists at cannabinoid CB1 or CB2 receptors, or at the transient receptor potential vanilloid type 1 receptor, each partially reversed effects induced by AM404. A complete reversal was obtained when the three antagonists were given together, suggesting that all three receptors are involved. AM404 treatment affected two pathways involved in the generation and maintenance of neuropathic pain, one mediated by nitric oxide (NO) and the other by cytokines. AM404 completely prevented the overproduction of NO and the overexpression of nNOS, inhibited the increase in tumour necrosis factor alpha (TNFalpha) and enhanced the production of interleukin-10. Both NO and TNFalpha are known to contribute to the apoptotic process, which plays an important role in the establishment of chronic pain states. AM404 treatment prevented the increase in the ratio between pro- and anti-apoptotic gene bax/bcl-2 expression observed in the spinal cord of neuropathic rats. Taken together, these findings suggest that inhibition of endocannabinoid uptake, by blocking the putative anandamide carrier, results in the relief of neuropathic pain and may represent a novel strategy for treating chronic pain.

Efficacy of nociceptin inhibition on WDR neuron activity is enhanced in mononeuropathic rats.

Nociceptin (NC), administered microiontophoretically at different concentrations, significantly reduced the spontaneous and stimulus-evoked activity on WDR neuron in rats with chronic constriction of one sciatic nerve and showing signs of neuropathic pain. The effect was not antagonized by Naloxone. The same concentrations of NC were ineffective on the noxious stimulus evoked responses of WDR neurons in sham and intact rats. This result indicates a facilitated inhibitory action of NC on nociceptive transmission in this pain model.

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization.

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5-L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.

Neuronal sensitization and its behavioral correlates in a rat model of neuropathy are prevented by a cyclic analog of orphenadrine.

N-methyl-D-aspartic acid (NMDA) is an agonist at the homonymous receptor implicated in the development of neuronal sensitization and its behavioral correlates. An effective modulation of the NMDA effects, achieved also by uncompetitive antagonists, could contribute to controlling pain symptoms in several neuropathic syndromes. Because nefopam is a known analgesic derivative of orphenadrine and of its congener diphenhydramine, both uncompetitive NMDA receptor antagonists, we tested the effect of nefopam on the developing pain and neuronal anomalies in an animal model of chronic pain with NMDA receptor involvement. A single intraperitoneal injection of nefopam was administered twenty minutes prior to the chronic constriction injury of the sciatic nerve (CCI rats). In the first 10 days, nefopam (30 mg/kg) significantly decreased behavioral signs of neuropathic pain and the stimulus-evoked electrophysiological anomalies in recordings at 14 days, with only slight manifestation afterwards. The dose of 20 mg/kg was ineffective. Nefopam injected after constriction was ineffective. In normal non-operated rats, Nefopam had no effect on the electrophysiological and behavioral parameters. Iontophoretic nefopam (1 mM, 50-80 nA, positive current) in normal rats did not change the spontaneous neuronal activity, but reduced the mean response to noxious stimuli and the concurrent iontophoretic NMDA evoked activity. In CCI rats, iontophoretic nefopam did not significantly modify the spontaneous hyperactivity but reduced significantly both the frequency of the responses to noxious stimuli, and the duration of the afterdischarge. We propose that nefopam exerts a preventive analgesic effect, with a possible role in modulating NMDA receptor-mediated effects in central sensitization.

The mGluR5 selective antagonist 6-methyl-2-(phenylethynyl)-pyridine reduces the spinal neuron pain-related activity in mononeuropathic rats.

In rats with chronic constriction of one sciatic nerve (CCI rats), showing behavioural signs of neuropathic pain, 6-methyl-2-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, was intraperitoneally administered at 0.75, 1.0 and 1.5 mg/kg or spinally microejected and the effects on the lumbar wide dynamic range neurons activity were investigated. In CCI rats MPEP at 1.0 and 1.5 (but not at 0.75) mg/kg, or spinally microejected induced a significant reduction of the spontaneous (SA) and noxious evoked activity (NEA), and a significant decrease of the suppression of the afterdischarge duration. In sham rats SA was unaffected and NEA was significantly reduced by 1.0 and 1.5 mg/kg MPEP dosages. These findings indicate that the metabotropic GluR5 receptor plays a role in the spinal cord processes underlying neuropathic pain and represents a potential target for new therapeutic approaches.

NMDA and strychnine diversely modulate spinal dorsal horn noxious responsiveness in normal rats: potential significance to sensory disorders in neuropathic pain.

Changes in neuronal excitability due to increase in excitatory transmitters and/or removal of local inhibition underlie central neuron sensitization and altered responsiveness related to painful sensory disorders. To distinguish the contribution of each of the two mechanisms, they have been mimicked separately in intact rats, by iontophoretically applying excitatory (NMDA) and disinhibitory (the glycine antagonist strychnine) substances during dorsal horn neuron recording. Wide dynamic range (WDR) neurons were extracellularly recorded at the L5-L6 lumbar level in anesthetized and paralyzed rats and an analysis was made, before and during the substance application, of the characteristics of the response to noxious stimuli applied to areas supplied by the ipsilateral sciatic nerve and the contralateral sciatic and saphenous nerves ("inappropriate" areas). The results show that the neuronal response properties were modified differently during the NMDA-induced hyperexcitability and strychnine-induced release of inhibition. Both manipulations brought about the unmasking of responses to previously ineffective, noxious stimuli applied to the contralateral sciatic and saphenous nerve areas, and the enhancement of the responses to noxious stimulation of the ipsilateral sciatic nerve area. However, it was only during the increased excitation induced by NMDA that the neurons exhibited hyperresponsiveness, with long-lasting afterdischarge, to noxious stimulation of the ipsi- and contralateral areas. Such response features resemble those described in sensitized neurons in neuropathic rats and associated with behavioral signs of hyperalgesia. This suggests, by inference, a crucial contribution of the NMDA-induced increased excitability to the expression of neuronal sensitization related to this painful sensory disorder.

Pre-injury lidocaine treatment prevents thermal hyperalgesia and cutaneous thermal abnormalities in a rat model of peripheral neuropathy.

The effect of lidocaine pretreatment on thermal hyperalgesia and thermal skin asymmetries provoked by experimental mononeuropathy was investigated in rats. Forty anesthetized rats were given sciatic nerve ligatures according to the technique of Bennett and Xie. Rats were divided into 3 groups: 16 were ligated without lidocaine, 16 were ligated after lidocaine bathing of the nerve, and 8 were ligated after systemic lidocaine (6-8 mg/kg). Six sham-operated rats for each group were also prepared. From the first postoperative day the responses to the hot-plate test were assessed daily for 4 weeks by tracking the paw-licking latency (PLL) for both hindpaws. Shorter or longer latencies on the operated side were respectively considered sign of hyperalgesia and hypoalgesia. Infrared thermographic images of plantar hindpaws were taken in 22 operated rats in the 2nd postoperative week. Thermographic images of 8 non-operated rats were used as control. Animals operated without lidocaine exhibited shorter PLL (P < 0.001) and a decreased skin temperature on the operated side (P < 0.001). In the lidocaine-pretreated rats, no paw-licking reflex was present for a variable postoperative period (1 week or more) and afterwards there was a trend toward recovery of normal PLL values at the 4th week; the hindpaw skin temperature was symmetrical and normal. Sham-operated rats had normal tests. It is postulated here that lidocaine prevents behavioral and thermal manifestation of mononeuropathy by blocking early afferent injury barrage.

Lidocaine test in neuralgia.

Ten patients with organic nerve injury causing chronic neuropathic pain were tested for the effects of intravenous lidocaine versus saline upon psychophysical somatosensory variables. The variables assessed were the subjective magnitude of pain, area of mechanical hyperalgesia and presence and magnitude of thermal heat/cold hyperalgesia. The study methods applied to evaluate these conditions were the conventional testing of somatosensory submodalities with area mapping and the subjective magnitude estimation of spontaneous pain. It was found that spontaneous pain and mechanical hyperalgesia were consistently improved, transiently, by intravenous administration of lidocaine in all 10 patients; areas of hyperalgesia which extended beyond the territory of the nerve also improved transiently. Spontaneous pain and mechanical hyperalgesia, but not hypoesthesia, were transiently improved by injection of saline in only 1 of the 10 patients. This outcome is probably due to a placebo effect. This improvement is in keeping with the inhibition of anomalous neural impulses which can be generated anywhere along the sensory channels responsible for generating spontaneous pain and hyperalgesia. Thus, intravenous lidocaine is proposed as a diagnostic aid in the examination of patients complaining of complex sensory disorders associated with nerve injury. The transient pain relief may allow a fuller identification of the area of sensory loss.

Inhibitory effect of the lateral reticular nucleus on neurons of the gigantocellularis nucleus which respond to noxious stimuli.

The effect was assessed of electrical stimulation of 'analgesic sites' in the lateral reticular nucleus (LRN) on the activity (spontaneous and evoked by noxious stimuli) of neurons in the nucleus gigantocellularis (NGC) in rabbits. The neurons (n = 145) were classified as 'nociceptive' (n = 98) and 'non-nociceptive' (n = 47) according to their responsiveness to a peripherally applied noxious heat stimulus (HS). The response to HS consisted in all cases of an increase in firing rate of up to 5 times the resting values. LRN stimulation was ineffective on the background discharge of the 'non-nociceptive' neurons, whereas it induced an inhibition lasting 40-120 msec on both spontaneous and noxious evoked activity in 66% (n = 68) of the 'nociceptive' neurons. Furthermore, prolonged LRN stimulation could induce a 'tonic' inhibition evaluable by the response to HS. This effect, quantified in 24 neurons, significantly decreased the noxious evoked activity to the baseline value. After hemilesion of the spinal cord, the inhibitory effects induced by LRN stimulation on the spontaneous and on the noxious evoked activity of NGC neurons were still present. The possibility that inhibition of the NGC neurons is a supraspinal component of the 'analgesic effects' resulting from LRN stimulation is discussed.