Neuropathic Animals Research Articles

5-HT 1A receptors in endogenous regulation of neuropathic hypersensitivity in the rat

The role of medullary and spinal 5-HT 1A receptors in endogenous regulation of neuropathic hypersensitivity was studied. When administered in the rostroventromedial medulla or subcutaneously, WAY-100635, a 5-HT 1A receptor antagonist, attenuated mechanical hypersensitivity in rats with a spinal nerve injury. Thermal or mechanical nociception outside of the injured area was not influenced by medial medullary or subcutaneous administration of WAY-100635. Intrathecal administration of WAY-100635 had no significant effect on pain-related behavior. Suppression of mechanical hypersensitivity induced by medial medullary administration of WAY-100635 was reversed by intrathecal administration of WAY-100635 or atipamezole, an α 2-adrenoceptor antagonist, but not by naloxone, an opioid receptor antagonist. The results indicate that endogenous release of 5-HT, via action on medial medullary 5-HT 1A receptors, tonically suppresses descending inhibition in neuropathic animals. Following medial medullary administration of a 5-HT 1A receptor antagonist, descending pain regulatory pathways are disinhibited. This leads to selective attenuation of neuropathic hypersensitivity, due to action on spinal 5-HT 1A receptors and α 2-adrenoceptors.

Antihyperalgesic effects of local injections of anandamide, ibuprofen, rofecoxib and their combinations in a model of neuropathic pain

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit fatty acid amidohydrolase (FAAH), the enzyme responsible for the metabolism of anandamide, an endocannabinoid. The analgesic interactions between anandamide (0.01 μg), ibuprofen (0.1 μg) and rofecoxib (0.1 μg) or their combinations administered locally in the hind paw of neuropathic rats were investigated together with the effects of specific antagonists for the cannabinoid CB 1 (AM251; 80 μg) and CB 2 (AM630; 25 μg) receptors. Mechanical allodynia and thermal hyperalgesia were evaluated in 108 Wistar rats allocated to: (1–4) NaCl 0.9%; anandamide; ibuprofen; rofecoxib; (5–6) anandamide + ibuprofen or rofecoxib; (7–8) AM251 or AM630; (9–10) anandamide + AM251 or AM630; (11–12) ibuprofen + AM251 or AM630; (13–14) rofecoxib + AM251 or AM630; (15–16) anandamide + ibuprofen + AM251 or AM630; (17–18) anandamide + rofecoxib + AM251 or AM630. Drugs were given subcutaneously in the hind paw 15 min before pain tests. Anandamide, ibuprofen, rofecoxib and their combinations significantly decreased mechanical allodynia and thermal hyperalgesia with an ED 50 of 1.6 ± 0.68 ng and 1.1 ± 1.09 ng for anandamide, respectively. The effects of NSAIDs were not antagonized by AM251 or AM630 but those of anandamide were inhibited by AM251 but not by AM630. In conclusion, locally injected anandamide, ibuprofen, rofecoxib and their combinations decreased pain behavior in neuropathic animals. Local use of endocannabinoids to treat neuropathic pain may be an interesting way to treat this condition without having the deleterious central effects of systemic cannabinoids.

Inflammatory and Neuropathic Pain Animals Exhibit Distinct Responses to Innocuous Thermal and Motoric Challenges

Most current methods for assessing pain in animals are based on reflexive measures and require constant interaction between the observer and the animal. Here we explore two new fully automated methods to quantify the impact of pain on the overall behavior of the organism. Both methods take advantage of the animals' natural preference for a dark environment. We used a box divided into two compartments: dark and bright. In the motoric operant task, "AngleTrack", one end of the box was raised so that the animals had to climb uphill to go from the light to the dark compartment. In the thermal operant task, "ThermalTrack", the floor of the dark compartment was heated to a given temperature, while the light compartment remained at 25°C. Rats were individually placed in the light box and their crossing between chambers monitored automatically for 30 minutes. The angle of the box, or the temperature of the dark compartment, was altered to challenge the animals' natural preference. We test the hypothesis that different models of pain (inflammatory or neuropathic) can be differentiated based on performance on these devices. Three groups of rats were tested at five different challenge levels on both tasks: 1) normal, 2) neuropathic injury pain (Spared Nerve Injury), and 3) inflammatory pain (intraplantar injection of Carrageenan). We monitored the position of the animals as well as their rate of switching between compartments. We find significant differences between the three groups and between the challenge levels both in their average position with respect to time, and in their switching rates. This suggests that the angle-track and thermal-track may be useful in assessing automatically the global impact of different types of pain on behavior.

Differential pharmacological modulation of the spontaneous stimulus-independent activity in the rat spinal cord following peripheral nerve injury

Peripheral nerve injury is a significant clinical problem that is often difficult to treat. The major clinical symptoms are numbness, tactile and cooling allodynia, hyperalgesias as well as ongoing pain. In animal models of neuropathy, abnormal responses to applied (or evoked) stimuli can be gauged, but spontaneous pain, a major clinical issue, has proved very difficult to assess. In neuropathic animals, spinal neuronal hyperexcitability indicative of peripheral and central changes with high levels of spontaneous neuronal firing has been reported. This latter stimulus-independent firing of sensory neurones may be a measure related to ongoing pain. Two weeks after L5/6 spinal nerve ligation, deep dorsal horn neurones were recorded in halothane-anesthetized rats. The majority of neurones in neuropathic rats showed increased levels of spontaneous firing with irregular firing patterns. We examined and compared the effects of 5 centrally acting pharmacological agents: morphine (i.t. or i.v.), gabapentin, ketamine, memantine and mepyramine on stimulus-independent neuronal firing. This ongoing activity showed high sensitivity to gabapentin (s.c.) and morphine (i.t.) administration, being significantly reduced in a dose-dependent manner. Morphine administered via the systemic route produced modest but non-significant reductions of spontaneous activity. The two NMDA receptor antagonists, ketamine and memantine, and the histamine H 1 receptor antagonist, mepyramine, produced minor effects at doses known to be effective on stimulus evoked measures of deep dorsal horn neurones. This may form an electrophysiological basis for the efficacy of gabapentin and spinal morphine on ongoing pain in patients with peripheral neuropathy.

RFamide-related peptides signal through the neuropeptide FF receptor and regulate pain-related responses in the rat

The mammalian RFamide-related peptide RFRP1 was found to signal through the neuropeptide FF 2 receptor expressed in Xenopus oocytes. The peptide induced a dose-dependent outward current, which was dependent on the simultaneous expression of GIRK1 and GIRK4 potassium channels. In neuropathic rats, RFRP1 administered intrathecally induced tactile antiallodynia and thermal antinociception, whereas in the solitary tract nucleus it produced only mechanical antihyperalgesia. Expression of the RFamide-related peptide mRNA in the rat CNS was distinctly different from that of neuropeptide FF. Most notably, the gene was not expressed in the hindbrain or spinal cord at detectable levels. However, there was a prominent group of RFamide-related peptide mRNA-expressing neurons in the central hypothalamus, in the area in and between the dorsomedial and ventromedial nuclei. The results suggest that RFamide-related peptides are potentially involved in pain regulation through a hypothalamo-medullary projection system, and possibly via action on neuropeptide FF 2 receptors. In neuropathic animals, the pain suppressive effect of RFamide-related peptide varies depending on the submodality of noxious test stimulation and the site of RFamide-related peptide administration.

Nerve growth factor-induced hyperexcitability of rat sensory neuron in culture

Abnormal spontaneous firing of primary sensory neurons is considered to be a cause of neuropathic pain. However, pathogenic mechanisms of hyperexcitable sensory neurons in neuropathic model animals are unclear. We examined effects of chronic treatment of nerve growth factor (NGF), one of candidate mediators for the pathogenesis, on excitability of sensory neurons by voltage-clamped recording in a cell-attached configuration. From rat dorsal root ganglion (DRG) neurons cultured without NGF, only stable holding currents without spontaneous firing activity were recorded. On the other hand, more than 20% neurons cultured in the presence of NGF for more than 3 days showed spontaneous current spikes at frequencies between 0.1 and 5 Hz. Each spikes had an initial inward phase followed by the outward phase, resulted from spontaneous transient depolarization followed by transient hyperpolarization. These spontaneous spikes were abolished by tetrodotoxin, lidocaine and reduction of extracellular concentration of Na+ from 154 mM to 100 mM, in all-or-none fashion, suggesting that spontaneous current spikes reflected spontaneous action potentials. From these results, it became evident that DRG neurons of adult rats had a nature to respond to NGF and obtained the abnormal hyperexcitability to fire spontaneously.

Increased WDR spontaneous activity and receptive field size in rats following a neuropathic or inflammatory injury: implications for mechanical sensitivity

Spontaneous activity and receptive field size for spinal wide dynamic range (WDR) neurons were measured and related to the mechanical allodynia in both neuropathic (L5–L6 ligation, 14 days post-injury) and complete Freund's adjuvant-inflamed rats (CFA, 2 days post-injury). The size of the WDR receptive field located on the hindpaw expanded significantly ( p < 0.01) following both modes of injury, with no difference between CFA and neuropathic animals. Likewise, the spontaneous firing of WDR neurons was significantly elevated following both the CFA (4.4 ± 0.6 spikes/s, p < 0.01) and neuropathic (3.2 ± 0.3 spikes/s, p < 0.05) injuries compared to naïve (2.1 ± 0.2 spikes/s) and sham-neuropathic (1.9 ± 0.3 spikes/s) rats. Furthermore, the spontaneous WDR activity recorded from CFA rats was also significantly greater ( p < 0.05) than neuropathic rats. Mechanical allodynia, as measured by application of a von Frey hair stimulus, was observed from both CFA and neuropathic rats, however, the degree of sensitivity was significantly greater ( p < 0.01) for the CFA animals. These data suggest that the differences in mechanical sensitivity between CFA and neuropathic rats may be related to their respective changes in WDR spontaneous activity, but not to the changes in receptive field size, and is further demonstration of the importance of spontaneous WDR activity in determining mechanical sensitivity following injury.

The effect of a kainate GluR5 receptor antagonist on responses of spinothalamic tract neurons in a model of peripheral neuropathy in primates

The responses of antidromically identified spinothalamic tract (STT) neurons to mechanical and thermal stimuli were compared in anesthetized normal and neuropathic monkeys before and after administration of a GluR5 kainate receptor antagonist (LY382884) into the spinal cord dorsal horn through a microdialysis fiber. Peripheral neuropathy was induced by tight ligation of the L7 spinal nerve 13–15 days prior to the experiment. STT neurons recorded in the animals with neuropathy showed increased responsiveness to weak mechanical stimuli and to heating and cooling of the skin compared to STT cells in normal animals. In both normal and the neuropathic monkeys the responses of the STT neurons to mechanical and thermal stimuli were attenuated by LY382884 application in a concentration-dependent manner. Intraspinal application of LY382884 in the neuropathic animals led to a potent reduction of those responses of the STT neurons that were aggravated by the peripheral neuropathy (weak mechanical, heat and innocuous cooling stimuli). These results suggest that kainate receptors are involved in synaptic activation of STT cells in the normal state and may also play an important role in pathological pain states such as peripheral neuropathy in primates. Kainate receptor antagonists could thus be useful for the treatment of certain forms of allodynia and hyperalgesia.

Endogenous and exogenous melanocortin antagonists induce anti-allodynic effects in a model of rat neuropathic pain

A number of studies suggest melanocortin (MC) system involvement in nociceptive modulation. Although the mechanism through which this occurs is still unknown, experimental evidence would suggest a primary role of MC4 receptors. To further investigate the implication of this MC receptor subtype in chronic pain, we have studied the effects of several MC antagonists on spinal nerve ligation-induced nociceptive behavior in rats. The intrathecal injection of synthetic antagonists with different selectivity to MC4 receptor and of an endogenous antagonist (Agouti related protein; AgRP) reduced mechanical allodynia in neuropathic rats, as measured by von Frey hair test. Treatments produced an anti-allodynic effect at the dose of 1.5 nmol (25–30% maximum possible effect, MPE, P < 0.05). To further investigate the possible physiological role of AgRP in pain modulation we studied its expression in both sham and neuropathic rat spinal cord and dorsal root ganglia (DRG) by quantitative real time PCR and immunohistochemistry. AgRP was present in both spinal cord and DRG, and its expression, was unchanged in neuropathic animals. In conclusion MC4 receptor antagonists with different selectivity profile, induce anti-allodynic effects in one of the most relevant neuropathic pain model. In addition the expression of AgRP in spinal cord and DRG suggests an endogenous tonic inhibitory control on MC system activity. In pathological conditions this steady control could be insufficient to cope with an over activated MC system leading to increase in nociception. These data suggest that targeting MC4 with synthetic antagonists could restore the balance and hence reduce nociception.

Melanocortin 4 receptor is expressed in the dorsal root ganglions and down-regulated in neuropathic rats

Recent reports have demonstrated effectiveness of melanocortin antagonists as potent analgesics, and have suggested that the spinal melanocortin 4 receptor (MC4-R) mediates their effects on pain transmission. These findings prompted us to investigate the changes in MC4-R mRNA level in the spinal cord and dorsal root ganglia (DRG) of neuropathic animals at different time points after sciatic nerve injury by quantitative real-time PCR. The spinal MC4-R mRNA level was not affected by sciatic nerve injury. In contrast, down-regulation of MC4-R mRNA in DRG developed 2 weeks after the injury and was parallel with the attenuated effectiveness of MC4-R ligands in neuropathic animals. The MC4-R adaptation in DRG observed in neuropathic rats indicates their important role in presynaptic modulation of activity of the primary afferents in neuropathic pain.

Plasticity in action of intrathecal clonidine to mechanical but not thermal nociception after peripheral nerve injury.

Intrathecal clonidine reduces tactile allodynia in animal models of neuropathic pain, and this effect is blocked by atropine. However, the role of tonic spinal cholinergic activity and its interaction with alpha2-adrenergic systems in normal and neuropathic conditions and to different sensory methods has not been systematically examined. The authors examined cholinergic receptor involvement in thermal and mechanical sensitivity in normal and neuropathic animals and its interaction with intrathecal clonidine. Normal rats and rats that received L5/L6 spinal nerve ligation were tested with acute radiant heat, paw pressure, and punctate mechanical stimulation before and after the intrathecal administration of saline, the muscarinic receptor antagonist, atropine, or a toxin to destroy cholinergic neurons, and then after intrathecal clonidine. Atropine, the cholinergic neuronal toxin, and saline did not alter baseline withdrawal thresholds. In nerve-injured rats, neither saline nor atropine altered antinociception from clonidine to a thermal stimulus, but atropine reduced the effect of clonidine to von Frey filament withdrawal threshold (34 +/- 5.6 vs. 14 +/- 5.8 g [mean +/- SEM], saline vs. atropine; P < 0.05) and to withdrawal threshold to paw pressure after clonidine (174 +/- 18 g vs. 137 +/- 16 g, saline vs. atropine; P < 0.05). These data suggest that after nerve injury, mechanical but not thermal antinociception from intrathecal clonidine relies on a muscarinic interaction, because only mechanical antinociception was antagonized by atropine. These results do not favor a regulation of nociceptive transmission by a tonic release of acetylcholine in nerve-injured rats.

Long-term effects of decreased noradrenergic central nervous system innervation on pain behavior and opioid antinociception.

Here we examine whether a permanent reduction in the noradrenergic (NA) innervation of the spinal cord leads to a chronic decreased nociceptive threshold. NA denervation of rats was achieved by intrathecal injection of dopamine beta-hydroxylase antibodies conjugated to the toxin saporin. A subset of animals also underwent unilateral L5 spinal nerve ligature to induce sustained neuropathic pain behavior. NA fibers and terminals were lost throughout the spinal cord 2 weeks after toxin application and were still absent 12 months later, indicating that regeneration did not occur. There was also a widespread loss of NA terminals in the cerebral cortex, whereas innervation of the hypothalamus and amygdala were close to normal and NA innervation of the brainstem was moderately reduced. There was extensive loss of NA cells in the locus coeruleus and A5 and A7 cell groups. Dopaminergic and serotoninergic innervation was normal. Intracerebroventricular injection of the toxin resulted in additional NA reduction in the hypothalamus, amygdala, and A1 and A2 cell groups. Long-term removal of NA afferents did not affect nociceptive thresholds. Neuropathic animals showed greater mechanical hyperalgesia in the affected hindpaw only during the first 60 days after toxin. Rats lacking NA spinal afferents were less responsive to the antinociceptive effects of morphine, especially in the neuropathic hindpaw, and did not display opioid-dependent stress analgesia. Finally, in the spinal cord of toxin-treated rats, immunoreactivity for substance P was decreased, whereas that of its receptor (NK1) was increased. These animals exhibited antinociception to a low dose of an NK1 receptor antagonist. Our results suggest that NA contributes only modestly to determining the nociceptive threshold and that its antinociceptive effects are closely linked to opioidergic and tachykinergic neurotransmission.

A dissociative change in the efficacy of supraspinal versus spinal morphine in the neuropathic rat

The efficacy of spinally versus supraspinally administered morphine was studied in rats with a spinal nerve ligation-induced neuropathy. Behavioural assessment indicated that the effect of intrathecally administered morphine on pain-related responses was attenuated when compared with unoperated controls. The decreased efficacy of spinal morphine was associated with neuropathic symptoms, since sham ligation or nerve ligation without accompanying tactile allodynia did not lead to spinal inefficacy of morphine. In contrast, the pain attenuating effect of morphine in the periaqueductal gray (PAG) was enhanced in neuropathic animals. The effect of systemically administered morphine on pain-related behavior of neuropathic rats was in the same range as in controls or decreased, depending on the test. Coadministration of lidocaine or MK-801, a N-methyl- d-aspartate (NMDA) receptor antagonist, into the rostroventromedial medulla enhanced the tactile antiallodynic but not the thermal antinociceptive effect of intrathecally administered morphine in neuropathic animals. Supraspinal administration of MK-801 or lidocaine did not influence efficacy of spinal morphine in sham-operated animals. Electrophysiological recordings of nociceptive wide-dynamic range (WDR) neurons in the deep spinal dorsal horn of pentobarbitone-anesthetized animals corresponded to a large extent with behavioral results. The inhibitory effect of spinally and systemically administered morphine on WDR neuron responses was attenuated whereas that induced by morphine in the PAG was enhanced in neuropathic animals. The results indicate that in spinal nerve ligation-induced neuropathy the efficacy of spinal morphine is decreased whereas that of supraspinal morphine is increased. Descending influence from brainstem-spinal pathways, involving NMDA receptors in the rostroventromedial medulla, may contribute to the selective reduction in tactile antiallodynic efficacy of spinal morphine.

The influence of chemical sympathectomy on pain responsivity and α 2-adrenergic antinociception in neuropathic animals

We studied the effect of chemical sympathectomy by 6-hydroxydopamine (6-OHDA) on pain behavior and α 2-adrenergic antinociception in rats with a spinal nerve ligation-induced neuropathy. For assessment of α 2-adrenergic antinociception, the rats were treated systemically with two α 2-adrenoceptor agonists, one of which only poorly (MPV-2426) and the other very well (dexmedetomidine) penetrates the blood–brain barrier. Moreover, the effect of MPV-2426 on spontaneous activity of dorsal root nerve fibers proximal to the nerve injury was determined. Systemic treatment with 6-OHDA produced a marked decrease in immunocytochemical labeling of sympathetic nerve fibers in the skin but it produced no marked change in basal pain sensitivity to mechanical stimulation either in neuropathic or sham-operated animals. Systemic administration of MPV-2426 and dexmedetomidine produced a dose-dependent tactile antiallodynic effect in neuropathic animals. Intraplantar injection of MPV-2426 had an identical antiallodynic effect independent of whether it was injected into the neuropathic or contralateral hindpaw. In a test of mechanical nociception and hyperalgesia, dexmedetomidine markedly attenuated pain responses in all experimental groups, whereas MPV-2426 had a weak but significant pain attenuating effect only in neuropathic animals. In the tail flick test, both α 2-adrenoceptor agonists had a significant antinociceptive effect. The pain attenuating effect of MPV-2426 was enhanced by pretreatment with 6-OHDA, except in a test of tactile allodynia. MPV-2426-induced modulation of spontaneous activity was not a general property of dorsal root fibers proximal to the injury. The results indicate that a chemical destruction of sympathetic postganglionic nerve fibers innervating the skin does not markedly influence cutaneous pain sensitivity nor is it critical for the α 2-adrenoceptor agonist-induced attenuation of pain behavior in neuropathic or non-neuropathic animals. Chemical sympathectomy, independent of neuropathy, enhanced the pain attenuating effect by MPV-2426, probably due to a peripheral action, whereas in non-sympathectomized control and neuropathic animals peripheral mechanisms have only a minor, if any, role in the α 2-adrenoceptor agonist-induced antinociception.

A Role of the Ubiquitin–Proteasome System in Neuropathic Pain

Neuropathic pain (characterized by hyperalgesia and allodynia to mechanical and thermal stimuli) causes cellular changes in spinal dorsal horn neurons, some of which parallel those in synaptic plasticity associated with learning. Ubiquitin C-terminal hydrolase (UCH) appears to play a key role in long-term facilitation in Aplysia. The cooperation of UCH with the proteolytic enzyme complex known as the proteasome is required for the degradation of a number of signaling molecules within the cell that may remove normal restraints on synaptic plasticity. We have used electrophysiology, in situ hybridization histochemistry, semiquantitative RT-PCR, Western blotting, and in vivo behavioral reflex analysis to investigate the ubiquitin-proteasome system in a model of neuropathic pain. In neuropathic animals, ionophoretic application of selective proteasome inhibitors attenuated dorsal horn neuron firing evoked by normally innocuous brush or cold stimuli and by noxious mustard oil stimuli. In control animals, only mustard oil-evoked responses were inhibited. Intrathecal administration of proteasome inhibitors attenuated hyperalgesia and allodynia in neuropathic rats. Expression of UCH-L1 (a rat homolog of Aplysia neuronal UCH and of the human UCH-L1, also known as PGP 9.5) and its mRNA were selectively increased within the ipsilateral dorsal horn of neuropathic rats, supporting the idea of a role for the ubiquitin-proteasome system in nociceptive processing. Proteasome inhibitors selectively attenuate allodynic and hyperalgesic responses in neuropathic pain, with some reduction in normal nociceptive, but not non-nociceptive responses, and potentially represent a novel therapeutic strategy for neuropathic pain.

Response properties of neurons in the rostroventromedial medulla of neuropathic rats: attempted modulation of responses by [1DMe]NPYF, a neuropeptide FF analogue

We determined whether chronic neuropathy changes response properties of neurons in the rostroventromedial medulla of rats, and whether (d-Tyr)L(Me-Phe)QPQRF-amide, a neuropeptide FF analogue, in the periaqueductal gray produces changes in responses of rostroventromedial medullary neurons that might underlie its antiallodynic effect described earlier. Single unit recordings of medullary neurons were performed in lightly anesthetized neuropathic and control animals. Spontaneous activity and the responses to noxious thermal and mechanical stimulation of the hind paw were determined with and without administration of (d-Tyr)L(Me-Phe)QPQRF-amide. The neurons were classified into three groups: ON-neurons increased, OFF-neurons decreased, and NEUTRAL-neurons did not change their discharge rate prior to a limb withdrawal induced by noxious stimulation of the skin. Spontaneous activity and heat-evoked responses of ON-neurons were not different between neuropathic and control animals, whereas their mechanically evoked responses were reduced in neuropathy. Response properties of OFF-neurons were not different between neuropathic and control animals. Spontaneous activity of NEUTRAL-neurons was not different between neuropathic and control animals. (d-Tyr)L(Me-Phe)QPQRF-amide in the periaqueductal gray had no significant effect on evoked responses or spontaneous activity of ON- or OFF-neurons, independent of the experimental group. However, (d-Tyr)L(Me-Phe)QPQRF-amide produced a significant attenuation of spontaneous activity of NEUTRAL-neurons in neuropathic animals. In a behavioral study performed in unanesthetized animals it was found that intrathecal administration of methysergide, a serotonin antagonist, selectively attenuated neuropathic symptoms. Also, light pentobarbitone anesthesia markedly attenuated, but did not abolish, behaviorally determined neuropathic symptoms. From these results we suggest that NEUTRAL-neurons of the rostroventromedial medulla may have a role in neuropathy and they may be involved in attenuation of mechanical hypersensitivity by (d-Tyr)L(Me-Phe)QPQRF-amide in the periaqueductal gray. It is proposed that in neuropathy the synaptic effects of descending impulses from medullary NEUTRAL-neurons on their axonal targets in the spinal cord are changed so that this contributes to mechanical hypersensitivity, due to mechanisms that are at least partly serotoninergic.

Modulation of pain by [1DMe]NPYF, a stable analogue of neuropeptide FF, in neuropathic rats

The pain modulatory effects of ( d-Tyr) l(Me-Phe)QPQRF-amide ([1DMe]NPYF), a stable analogue of neuropeptide FF were studied in rats with a chronic neuropathy induced by unilateral ligation of two spinal nerves. According to behavioral assessments, intrathecal (i.t.) administration of [1DMe]NPYF induced mechanical antiallodynic and thermal antinociceptive effects in a parallel and dose-dependent fashion, whereas following administration in the periaqueductal gray (PAG) it produced only mechanical antiallodynia. I.t. or PAG administration of FLFQPQRF, a non-amidated form of NPFF, or intraplantar injection of [1DMe]NPYF into the neuropathic paw had no effects. Electrophysiological results indicated that administration of [1DMe]NPYF suppressed responses of nociceptive spinal dorsal horn neurons in a submodality selective way and without an effect on their spontaneous activity; PAG administration predominantly suppressed brush-evoked responses and i.t. administration heat-evoked responses. The descending inhibitory effect by conditioning electrical stimulation of the PAG was enhanced by i.t. administration of [1DMe]NPYF. The reversibility of [1DMe]NPYF-induced effects by naloxone (1 mg/kg subcutaneously) depended on the submodality of test stimulation and the route of drug administration. The amplitude of the innocuous H-reflex was not changed by [1DMe]NPYF administered i.t. in control rats. The present results indicate that [1DMe]NPYF produces a selective attenuation of pain in neuropathic animals due to naloxone-sensitive or -insensitive central mechanisms depending on the submodality of pain and route of drug administration. The amide-group is essential for the [1DMe]NPYF-induced attenuation of pain.

Non-opioid actions of lamotrigine within the rat dorsal horn after inflammation and neuropathic nerve damage.

Some opioid-resistant pain conditions can be alleviated by voltage-dependent Na(+) channel blockers such as lamotrigine. The mu-opioid-receptor agonist morphine can modulate cation entry into cells to affect overall cellular excitability, an effect which can in turn be endogenously antagonised by the neuropeptide cholecystokinin (CCK). However, lamotrigine may also modulate cellular excitability by non-specifically blocking voltage-dependent ion channels. We have looked for interactions of lamotrigine with the opioid/CCK pathway within the spinal dorsal horn, to rule out the possibility that lamotrigine may attenuate nociceptive responses via actions on this pathway. Both lamotrigine and the mu-opioid agonist DAMGO inhibited mustard oil-evoked cell firing by approximately 50% compared with control levels. Co-application of CCK8S reversed DAMGO-, but not lamotrigine-induced inhibition of cell firing and this reversal was prevented with the selective CCK(B) receptor antagonist PD 135158. Although lamotrigine inhibited both brush- and cold-evoked cell firing in neuropathic animals, lamotrigine inhibition of mustard oil-evoked cell firing in the same animals was not significantly greater than that observed in controls. These results suggest that the antinociceptive properties of lamotrigine within the spinal dorsal horn are unlikely to be mediated via interactions with the opioid/CCK pathway.

Spinal and supraspinal components of opioid antinociception in streptozotocin induced diabetic neuropathy in rats

This study investigated the antinociceptive effect of opioids given via intraperitoneal and intrathecal routes in a diabetes-induced neuropathic pain model in rats. Streptozotocin induced diabetes in 91% of juvenile male Wistar rats at the dose of 150 mg/kg (75 mg/kg intraperitoneal on 2 successive days). When compared with younger weight-matched saline treated rats, the diabetic rats developed hyperalgesia assessed by the paw pressure nociceptive test. Nociceptive thresholds and responses to fentanyl in all nociceptive tests in these younger normal rats were the same as those described previously for older normal rats. Fentanyl (10–100 μg/kg, i.p.) produced a dose-related antinociceptive effect in both neuropathic ( n=6–8) and non-neuropathic ( n=6–8) rats in electrical current, paw pressure and tail flick nociceptive tests. Higher doses of fentanyl were needed in neuropathic animals to achieve similar antinociceptive effects to those in non-neuropathic animals. Intrathecal injections of fentanyl (0.05–0.5 μg) in non-neuropathic rats, produced a spinally-mediated, dose-related antinociceptive effect assessed by all tests. In contrast, intrathecal administration of fentanyl that confined the drug action to the spinal cord produced little antinociceptive effect in neuropathic rats in all three tests. These experiments suggest that supraspinal μ opioid receptors are responsible for the antinociceptive effect of opioids in this model of neuropathic pain and that spinal cord opioid systems are in some way rendered ineffective for antinociception assessed with noxious heat, electrical and pressure stimuli.

EFFECTS OF MIDAZOLAM IN THE SPINAL NERVE LIGATION MODEL OF NEUROPATHIC PAIN IN RATS

Potential changes in the spinal GABAergic activity after nerve injury were studied by comparing the effects of systemic administration of the benzodiazepine midazolam on the noxious evoked responses of dorsal horn in rats with spinal nerve ligation of neuropathy and control animals. The tight ligation of the L5 and L6 spinal nerves was performed in adult male Sprague‐Dawley rats and resulting mechanical and cold allodynia were assessed with von Frey hairs and the acetone drop test. Single unit extracellular recordings of dorsal horn neurones were performed 15‐18 days after the surgery under halothane anaesthesia using transcutaneous electrical stimulation of the receptive field at three times the C‐fibre threshold. The rats in the spinal nerve ligation group, but not in the sham‐operated control group developed mechanical and cold allodynia. Subcutaneous administration of midazolam 0.1–3.0 mg/kg reduced the A delta‐fibre evoked activity in a dose‐related manner in all study groups, but the C‐fibre evoked activity was significantly reduced only in the spinal nerve ligation group. The inhibitory effects of s.c. midazolam were significantly reversed by i.t. administration of flumazenil, suggesting a spinal site of action. Midazolam reduced C‐fibre evoked firing significantly more in the spinal nerve ligation model than in the non‐operated or sham controls. These results indicate changes in the spinal GABAergic system in the neuropathic animals and could be of importance in the development of new treatments for neuropathic pain.