Chronic pain as expression of neural substrates. Issues from the neuronal dynamics and mutual relations.

The Thalamo-Cortical somatosensory loop shows important synaptic re-organization in cases of chronic pain. Animal models exhibit severe functional distortions, potentially related to the anatomic rearrangements. Connectivity and information theoretic measurement represent important tools to quantify the functional disarrays. We performed electrophysiological experiments with multisite, multielectrode simultaneous recordings in the Thalamus and in the Somatosensory Cortex. The recurrent anomalies in the analytic estimates induce to hypothesize a potential neurodynamical explanation of the sensory context.

Repeated treatment with the synthetic cannabinoid WIN 55,212-2 reduces both hyperalgesia and production of pronociceptive mediators in a rat model of neuropathic pain.

The antinociceptive properties of cannabinoids in persistent pain are not fully elucidated. We investigated the effect of repeated treatment with the synthetic cannabinoid receptor agonist WIN 55,212-2 on the neuropathic pain induced in rats by chronic constriction of the sciatic nerve. WIN 55,212-2 administered daily throughout the development of neuropathy reversed the hyperalgesia, at a dose (0.1 mg x kg(-1), s.c.) that had no effect on the nociceptive responses of either paw contralateral to the sciatic ligation or of animals subjected to sham surgery. At 14 days after injury, the levels of mediators known to be involved in neuropathic pain, such as prostaglandin E2, NO and the neuronal NOS, were increased. Repeated treatment with WIN 55,212-2 abolished these increases. In the light of the current clinical need for neuropathic pain treatments, these findings indicate that cannabinoid agonists, at doses devoid of psychoactive effects, could constitute important compounds for the development of new analgesics.

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission.

Contralateral treatment with lidocaine reduces spinal neuronal activity in mononeuropathic rats.

In anaesthetised and paralysed rats with chronic constriction of the sciatic nerve, the effects of subcutaneous contralateral lidocaine (100 microl) on the activity of lumbar (L(4)-L(5)) wide dynamic range neurons ipsilateral to the constriction have been investigated. The results show reduction of the spontaneous hyperactivity for 60 min; suppression or reduction of the responses to contralateral noxious stimulation for 60 min; lack of effect on the responses to ipsilateral noxious stimulation, except for the afterdischarge duration, reduced for 60 min. The finding that the altered neuronal activity following peripheral nerve injury associated to behavioural signs of neuropathic pain, can be reduced by contralateral treatment, may provide further suggestions to neuropathic pain mechanisms and management.

Acupuncture produces central activations in pain regions.

Acupuncture is largely used for pain control in several pathological conditions. Its effects on the central nervous system are not well defined. We investigated the effect of the application of acupuncture to 13 normal subjects (males, 21-32 years). H2(15)O bolus PET scans were read before the application of the needles (Rest, R) and after 25 min of needle insertion. Data were acquired by scanning in 3-D mode. The acupuncture application, true acupuncture (TA), was alternated to a placebo needle application (PA) in two different sequences (seven and six subjects, respectively), either R,PA,R, TA or R,TA,R,PA, a period of 15 min being left after every first TA or PA to allow for the recovery of basal conditions. Here we show that classic acupuncture activates the left Anterior Cingulus, the Insulae bilaterally, the Cerebellum bilaterally, the left Superior Frontal Gyrus, and the right Medial and Inferior Frontal Gyri. Most of the activated areas are shared with areas activated in acute and chronic pain states as described in the literature. Thus acupuncture appears to act by activating areas also involved in pain. This indicates that acupuncture could relief pain by unbalancing the equilibrium of distributed pain-related central networks.

Effect of different concentrations of iontophoretic nociceptin on distinct classes of nociceptive neurons in rat spinal cord.

Iontophoretically applied nociceptin (NC) was tested at different concentrations on the activity of spinal nociceptive specific (NS) and wide dynamic range (WDR) neurons. Low NC dosages inhibited the noxious response of NS neurons, higher dosages inhibited the noxious responses of the WDR neurons but had little effect on the non-noxious response. Naloxone did not antagonize the NC effect. Thus, appropriate dosages of NC may be selective, both for neuronal classes and for sensory modalities.

Contribution of central sensitization to the pain-related abnormal activity in neuropathic rats.

The possibility of different contributions from peripheral and central sensitization to distinct neuropathic pain syndromes has been studied in rats with chronic constriction of the sciatic nerve (CCI), showing positive behavioral signs of neuropathic pain. In anesthetized, paralyzed rats extracellular recordings were performed in the spinal sciatic afferent territory (L5-L6), ipsilateral to the injured nerve, from wide dynamic range (WDR) neurons. The spontaneous activity and the responses to noxious stimuli applied to the proper area, i.e., the skin innervated by the constricted sciatic nerve, and to "inappropriate" areas, like the tail and the area of skin supplied by the contralateral sciatic and saphenous nerves, were analyzed before and after input from the constricted nerve was reversibly blocked at the ganglionic level by local anesthetic. The neurons discharged spontaneously with high frequencies, and responded to the stimulation of proper and "inappropriate" areas with high frequency discharge and prolonged afterdischarges During the ganglionic block, confirmed by the lack of responses to proper area stimulation, theWDR neuron background activity was significantly reduced; the responses to all "inappropriate" afferences were present, the frequency discharges being comparable to the preblock ones while the afterdischarges were significantly shorter. Since the efficacy of "inappropriate" inputs is related to neuronal sensitization, the persistence of these responses indicates that central neurons remain sensitized during peripheral block. In view of the relationship between the examined spontaneous and stimulated activities and neuropathic pain symptoms, the data suggest that central sensitization contributes with different drive strength to such symptoms, playing a crucial role in extraterritorial pain.

Differential effects of MK-801, a N-methyl-D-aspartate non-competitive antagonist, on the dorsal horn neuron hyperactivity and hyperexcitability in neuropathic rats.

N-methyl-D-aspartate (NMDA) involvement in altered spinal neuron activity following peripheral nerve injury has been investigated in rats with chronic constriction of the sciatic nerve. Extracellular single neuron recordings were performed, in anesthetized, paralyzed rats, from the sciatic spinal cord segments (lumbar, L5-L6) ipsilateral to the constriction, and the effect of iontophoresized MK-801, an NMDA receptor non-competitive antagonist, was tested on baseline hyperactivity and hyperresponsiveness to noxious stimulation. The results show that baseline activity was unaffected whereas the noxious evoked responses were significantly modified, there being amplitude reduction and after-discharges suppression. The different role of NMDA in the abnormal pain states related to the abnormal neuronal activities is discussed.

Multifractal analysis of wide dynamic range neuron discharge profiles in normal rats and in rats with sciatic nerve constriction.

Using normal rats and rats with a chronic constriction injury of the sciatic nerve (injured, model of Bennett-Xie) we investigated the possibility of classifying, by statistical tools, the temporal sequences of neuronal discharges during different noxious and non-noxious stimuli. An analysis was made of both the distribution of the inter-spike intervals and the temporal density of spike trains, the latter being studied within the framework of stochastic universal multifractals, to allow the identification of different random processes involved in the discharge distributions through the Lévy index alpha. The statistical analysis shows that the parametrization based on the Lévy index seems able to discriminate between different noxious stimuli (mechanical pinching and thermal), both in normal and injured animals. Furthermore, comparing normal and injured animals, although the spontaneous basal and non-noxious stimuli (brushing) evoked activities presented different frequencies, these seem to have the same multifractal structure, while the corresponding statistics of the inter-spike intervals are quite different. This information might be relevant to the understanding of a code of neuronal firing and to the modelling of temporal patterns in acute and chronic noxious signals.

Facilitation of spinal sciatic neuron responses to hindpaw thermal stimulation after formalin injection in rat tail.

A recent model of formalin injection in the tail induced a facilitation of the hindpaw withdrawal reflexes. In the present work we tried, after injecting formalin into the tail of the albino rat, to determine the spontaneous activity and response changes of lumbar sciatic wide-dynamic-range neurons to thermal stimulations of the paw at 45 degrees C and 48 degrees C (the respective thresholds for noxious and non-noxious thermal stimuli). The experiments were carried out with multiple recording electrodes placed in a comb array in the lumbar segments of the spinal cord at L4-L6 level in the sciatic projection field. A significant facilitation of the spontaneous activity was already evident 2 min after injection; at 5 min there were strong facilitations to the thermal stimuli. Stimuli at 45 degrees C, often ineffective prior to the formalin injection, became strongly excitatory. Stimuli at 48 degrees C evoked more conspicuous responses. This facilitatory effect on spontaneous and thermal responses followed a time-course comparable to that described for the excitations seen after paw formalin injection, but the duration was more prolonged, lasting more than 2 h. These data indicate a facilitatory role of the formalin effects on spinal sciatic neurons after injection in the tail. It is proposed that the mutual effects of spinal neurons in distant spinal segments could explain the facilitation and such a time-course, and that a role in the development of prolonged pain could be envisaged.

Central effect of ketorolac involving NMDA receptors activity.

Possible central mechanisms underlying the analgesic action of Ketorolac, a non-steroidal antiinflammatory drug (NSAID) have been investigated using an iontophoretic approach. We found that the excitation induced by N-methyl-d-aspartate (NMDA) on spinal wide dynamic range (WDR) neurons was prevented, or reduced, by Ketorolac applied before or after the start of the NMDA ejection. The data suggest that Ketorolac can achieve its central analgesic effect by interfering with the NMDA receptor activity on the spinal neurons.

Contralateral inhibitory control of spinal nociceptive transmission in rats with chronic peripheral nerve injury.

The effects of contralateral homotopic noxious stimulation on the spinal neurons nociceptive responses were examined in rats with chronic constriction of one sciatic nerve, in intact and in sham operated rats. Wide dynamic range (WDR) neurons activity was recorded in the spinal sciatic territory in anesthetized, paralyzed rats. Noxious stimulation of the contralateral homologous area strongly reduced the WDR nociceptive responses, the inhibitory effect outlasting the conditioning stimulus duration by 20-40 s. During a reversible blockade of descending pathways the inhibitory effect, except for the overlasting component, was still present, after a spinal cord longitudinal cut at the lumbar level the inhibitory effect was powerfully attenuated. The role of propriospinal connections in this phenomenon and how the contralateral input contributes to the control of nociceptive transmission is discussed.

Spinal neuron sensitization facilitates contralateral input in rats with peripheral mononeuropathy.

The effects of noxious stimulation of one hindpaw on the dorsal horn wide dynamic range (WDR) neurons activity of the opposite side were examined and compared in rats with chronic constriction of one sciatic nerve (CCI), in intact rats and in sham operated rats. Extracellular recordings, were performed in anesthetized, paralyzed rats in the sciatic spinal cord segments (L5-L6). Both the number and the magnitude of the responses were significantly larger in CCI than in intact and in sham rats. The effect was mainly excitatory, only in a few cases was inhibition observed. The relation of the contralateral increased efficacy with the level of excitability of the target neurons is discussed and a role of the potentiated cross transmission in some contralateral pain disorders is hypothesized.

Topical axonal transport blocker vincristine prevents nerve injury-induced spinal neuron sensitization in rats.

The effect of vincristine (Vin, a fast axonal transport blocker) to prevent any alteration in the excitability of dorsal horn neurons, following peripheral nerve injury, was investigated on 31 rats: 20 with chronic constriction injury (CCI) of the sciatic nerve and 11 sham preparations. In 15 of the 20 CCI rats, a small piece of gelfoam soaked with Vin was applied to the sciatic nerve before ligation (Vin+); in the remaining 5 rats the nerve was ligated without Vin (Vin-). The 11 sham rats were 7 Vin+ and 4 Vin-. The dorsal horn neuronal activity was recorded after 2-3 postoperative (PO) weeks. In the CCI Vin- rats, the neurons showed increased spontaneous activity and hyperresponsiveness to noxious stimulus with prolonged afterdischarges, events considered to signal central neuron sensitization. In the CCI Vin+ rats, the neuronal spontaneous and stimulated activity values were significantly lower (p < 0.001) than in the CCI Vin- rats being comparable to normal values. In sham Vin+ and Vin- rats, the neuronal activities had normal values. Given the crucial role attributed to central neuron sensitization for the development of neuropathic pain, the possibility that vincristine, by blocking the axonal transport, exerts a preventive action on this syndrome is discussed.

Rough annealing by two-step clustering, with application to neuronal signals.

To accomplish analyses on the properties of neuronal populations it is mandatory that each unit activity is identified within the overall noise background and the other unit signals merged in the same trace. The problem, addressed as a clustering one, is particularly difficult as no assumption can be made on the prior data distribution. We propose an algorithm that achieves this goal by a two-phase agglomerative hierarchical clustering. First, an inflated estimation (overly) of the number of clusters is cast down and, by a maximum entropy principle (MEP) approach, is made to collapse towards an arrangement near natural ones. In the second step consecutive partitions are created by merging, two at time previously aggregated partitions, according to similarity criteria, in order to reveal a cluster solution. The procedure makes no assumptions about data distributions and guarantees high robustness with respect to noise. An application on real data out of multiple unit recordings from spinal cord neurons of mixed gas-anaesthetized rats is presented.

Interaction between neurons in different laminae of the dorsal horn of the spinal cord. A correlation study in normal and neuropathic rats.

Simultaneous recordings of 135 pairs of units, located respectively in the superficial (I-IIo) and deep (V) laminae of the dorsal horn of the lumbar spinal cord of anaesthetized and paralysed animals, were performed both from normal (62 pairs) and from peripherally injured (chronically constricted sciatic nerve) rats (73 pairs). In each pair, one neuron was classified as nociceptive, responding only to noxious stimuli, and the other as a wide dynamic range neuron, responding to both non-noxious and noxious stimuli. To understand if some interaction was present between diverse neurons modulated by noxious inputs, we used cross-correlation techniques. The responses of simultaneously recorded pairs of units to suprathreshold (5 mA, 0.5 ms) electrical stimuli were used. A clearly delayed peak in the cross-correlograms of recordings from normal animals was present, indicating connectivity of superficial and deep-layer cells. This feature was not present in the cross-correlograms obtained from nerve-injured animals. Even if a specific pathway cannot be explicitly assigned to support these functional results, an overall connection between superficial and deep layers of the spinal cord is suggested. These connections are supposed to be either inactive or rearranged in the nerve-injured rats, thus suppressing a well timed coordinated connectivity. This anomaly could underlie a reduced degree of functional coherence in the modulation of nociceptive spinal inputs in cases of chronic pain.

Role of input from saphenous afferents in altered spinal processing of noxious signal that follows sciatic nerve constriction in rats.

An investigation was made into a possible contribution of saphenous nerve to altered central processing of noxious information following sciatic nerve chronic constriction (CCI) in rats. Spinal sciatic neuron spontaneous and noxious evoked activities were recorded in CCI rats, CCI rats with saphenous nerve sectioned just before the sciatic nerve constriction and sham operated rats. The results show (1) high values of spontaneous and noxious evoked activities with prolonged afterdischarges in CCI rats with intact saphenous, (2) comparable high values of spontaneous activity, but significantly reduced noxious evoked activity and afterdischarges in CCI rats with sectioned saphenous, (3) values in the normal range in the sham rats. Potential mechanisms underlying these results are discussed.

Injured nerve block alters adjacent nerves spinal interaction in neuropathic rats.

Baseline activity and responses to simultaneous saphenous stimulation of pairs of neurones recorded from sciatic (L5-6) and saphenous (L2) spinal cord segments, in rats with thermal hyperalgesia following sciatic constriction, were analysed before, during and after a sciatic nerve block with a local anaesthetic. In sciatic neurones, during the block, reductions of baseline activity (p < 0.001), increases in threshold of saphenous electrical stimulation (p < 0.01) and reductions of responses to electrical and to natural noxious saphenous stimuli (p < 0.001) were consistently found. The neuronal baseline and evoked activities remained unmodified in saphenous neurones. The contribution of input from injured periphery to central neurone circuitry mechanisms underlying the unmasking of improper afferents is discussed.

Peripheral mononeuropathy affects hypothalamic and splenocyte beta-endorphin levels but not immune function in the rat.

Beta-endorphin and substance P levels were measured in the hypothalamus of rats 14 days after chronic constriction injury of right sciatic nerve. Furthermore, beta-endorphin concentrations in splenocytes, phytoemoagglutinin-induced proliferation of splenocytes, and natural killer activity were assessed. We observed a significant increase of beta-endorphin and substance P hypothalamic levels, and a significant decrease of beta-endorphin concentrations in the immune cells. In contrast, the peripheral mononeuropathy did not affect the immune function. This study presents a picture of central and peripheral peptide changes consistent with a painful condition, but different from what previously observed in rats which underwent peripheral nerve deafferentation or stressful conditions.