José Manuel Castro-Lopes

Complex changes of GABAA and GABAB receptor binding in the spinal cord dorsal horn following peripheral inflammation or neurectomy.

Chronic peripheral inflammation or peripheral neurectomy cause changes in GABA levels and GABA immunoreactivity in the spinal cord dorsal horn. The present study aimed to investigate if such changes are accompanied by alterations in GABA receptor binding. Neurectomy of the sciatic nerve caused an ipsilateral down-regulation of GABAB receptor binding in lamina II of the spinal cord 2-4 weeks after the nerve injury. Since approximately 50% of GABAB receptor binding in that region is located on primary afferent endings, degenerative changes of such endings caused by the nerve lesion can explain the observed reduction. In contrast, GABAA binding was substantially enhanced following neurectomy, which may be due to an up-regulation of the receptors issued by the concomitant decrease of endogenous GABA. In rats bearing unilateral chronic peripheral inflammation induced by intraarticular injection of complete Freunds adjuvant we found a reduction of GABAB binding in the superficial dorsal horn. This effect, which was maximal at 3-4 weeks after adjuvant injection, was attributed to an enhanced release of GABA by spinal interneurons. GABAA receptor binding was not changed in this experimental model. Together, these results suggest that the two receptor types may be located at different loci and are differently affected by variations in sensory input.

Opiates modify induction of c-fos proto-oncogene in the spinal cord of the rat following noxious stimulation

The expression of the proto-oncogene c-fos in neurons of the spinal cord dorsal horn of the rat following noxious thermal stimulation was compared in morphine- and ketamine-treated animals. Intravenous injection of morphine reduced the number of c-fos-positive neurons by up to 85% in laminae III-VI and X. This effect was dose dependent and naloxone reversible. The non-competitive N-methyl-D-aspartate (NMDA) antagonist ketamine had no effect. The present data show that morphine suppresses the induction of c-fos. A block of Ca2+ influx through voltage- and ligand (NMDA)-gated channels does not influence c-fos protein synthesis in the dorsal horn of the spinal cord in vivo.

Increase in GABAergic Cells and GABA Levels in the Spinal Cord in Unilateral Inflammation of the Hindlimb in the Rat

The effects of chronic peripheral inflammation on spinal cord γ‐aminobutyric acid (GABA) were examined in the rat. Following the injection of complete Freunds adjuvant in the left hindlimb footpad an increased number of immunoreactive cells occurred in ipsilateral laminae I‐III of the dorsal horn from L3 to L5. GABA‐immunoreactive cells were more numerous than contralaterally 1 week after the onset of the inflammation, reached maximal numbers after 3–4 weeks, and declined thereafter. Differences from control sides were statistically significant except at week 6. GABA levels in homogenates of the ipsilateral lumbar enlargement were increased significantly at 4 weeks. Since increases in GABA occurred in the spinal cord zone of projection of the nerves supplying the inflamed foot, the central response is surmised to result from the increased nociceptive input arriving from the periphery. However, the transmission from primary axons to GABA interneurons is not likely to be monosynaptic since profiles containing glutamate decarboxylase or GABA immunoreactivity are known to be predominantly presynaptic, and rarely postsynaptic, to primary afferent endings in electron micrographs in the rat. The findings support the function attributed to spinal GABA in modulating nociceptive input at segmental level.

Ultrastructural changes of the central scalloped (C1) primary afferent endings of synaptic glomeruli in the substantia gelatinosa Rolandi of the rat after peripheral neurotomy

SummaryFine structural changes were observed in the dark scalloped central C1 terminals of type I synaptic glomeruli in spinal cord segments C6–C7 of the rat 3 days after cutting the three main forelimb nerves. Twenty-six per cent of the C1 terminals occurring on the ipsilateral side showed a lighter appearance due to a decrease in the number of synaptic vesicles. The number of synaptic vesicles per unit section area was only 42% of that present in normal C1 terminals on the contralateral side. The number of synaptic contacts of C1 terminals with the profiles surrounding them in each glomerulus was diminished and glial envelopment was increased to 15% of C1 terminal contour. Up to day 12, vesicle and synaptic losses were gradually aggravated and glial apposition was increased, but no obvious signs of glial engulfment were observed. From day 3 to day 12, altered C1 terminals increased in number, while those that appeared normal decreased. The latter had disappeared at day 12 and the altered ones at day 15, and from this stage type I glomeruli were no longer present on the treated side. The lack of electron-dense degenerative bouton changes characteristic of Wallerian degeneration offers an explanation for the lack of or minimal amount of argyrophilic structures which has been found consistently in the substantia gelatinosa during transganglionic degeneration. The gradual decay of the C1 terminals raises the question of their fate. Future studies with the use of a stable marker might provide an answer.

Effects of Kelatorphan and morphine before and after noxious stimulation on immediate-early gene expression in rat spinal cord neurons.

&NA; Expression of the immediate‐early genes (IEG) c‐FOS, NGF1‐A and c‐JUN was induced hy noxious thermal stimulation in neurons of the rat spinal cord dorsal horn. Intravenous injection of Kelatorphan (5, 10 and 20 mg/kg), an inhibitor of multiple enkephalin‐degrading enzymes, 20 min before noxious stimulation reduced the overall number of dorsal horn neurons expressing c‐FOS and NGF1‐A by up to 20–30%. While c‐FOS expression was suppressed in superficial and deep laminae of the spinal cord, NGF1‐A and c‐JUN was only suppressed in superficial laminae. Morphine (5, 7.5 and 10 mg/kg) produced a dose‐dependent reduction of c‐FOS expression by up to 70% only when injected before noxious stimulation. Morphine injected K) min after the noxious treatment was virtually ineffective. The depressant effect of Kelatorphan and morphine could be prevented by prior application of the opioid antagonist naloxone. Naloxone itself slightly increased the overall number of c‐FOS‐positive neurons in all laminae of the spinal cord. The present data support the existence of a tonic release of endogenous opioid peptides at the spinal level and show that inhibition of their peptidase‐induced degradation modulates IEG expression in dorsal horn neurons of the rat. The finding that opioid agonists were ineffective when applied after stimulation underline the necessity of pre‐emptive analgesia to prevent long‐term activity‐dependent changes in spinal cord neurons.

Epidemiology of Chronic Pain: A Population-Based Nationwide Study on Its Prevalence, Characteristics and Associated Disability in Portugal

UNLABELLED A cross-sectional nationwide epidemiological study was performed in a random sample of the Portuguese adult population, aiming to describe the prevalence and impact of chronic pain (CP). The 5,094 participants were selected by random digit dialing, between January 2007 and March 2008, and estimates were adequately weighted for the population. Prevalence of CP was 36.7% (95% confidence interval [CI] [35.3-38.2]), based on the definition of the International Association for the Study of Pain. Recurrent or continuous pain was present in 85% of those with CP, and moderate-to-severe intensity and disability were present in 68 and 35%, respectively. Highest CP prevalence was observed among the elderly, retired, unemployed, and less educated. Highest disability was found in relation with family/home responsibilities, recreational activities, occupation/work, and sleep/rest; 13% reported a diagnosis of depression and 49% reported interference in their job. The main factors associated with disability were sex, pain intensity, and depression or depressive symptoms. CP is highly prevalent, causes high personal and social burden, and affects particularly the most vulnerable subgroups. Portugal, depending on CP definition, could be placed in the lower prevalence group in Europe. Improvement in pain intensity management and special attention to affective components of CP are recommended. PERSPECTIVE In this cross-sectional nationwide epidemiological study, we showed that chronic pain is a significant problem that is present in 37% of the Portuguese adult general population, is associated with high personal, family, and social burden, and affects in particular the most vulnerable subgroups of the population.

Carrageenan-induced inflammation of the hind foot provokes a rise of GABA-immunoreactive cells in the rat spinal cord that is prevented by peripheral neurectomy or neonatal capsaicin treatment

&NA; An increase in the number of &ggr;‐aminobutyric acid (GABA)‐immunorcactive cells is reported in the superficial dorsal horn of the rat spinal cord upon unilateral inflammation of the hind foot caused by subcutaneous carrageenan injection. The rise of GABAergic cells was restricted to the ipsilateral dorsal horn, reaching a peak value of 23.4% over the contralateral side 4 days after carrageenan injection. Sciatic neurectomy or neonatal capsaicin treatment prevented this effect. These findings suggest that dorsal horn GABA is up‐regulated by the increase of noxious inflow conveyed by unmyelinated C fibers from the inflamed tissues.

Differential distribution of metabotropic glutamate receptor subtype mRNAs in the thalamus of the rat

L-Glutamate (L-Glu) is present in most excitatory synapses of the mammalian brain, acting on several receptor subtypes. Height different genes encoding metabotropic glutamate receptors (mGluRs) subtypes have been described (mGluR1-8), having a distinct distribution in the brain. In the present study, the distribution of mGluR1, 3, 4, 5 and 7 mRNAs was determined in 20 thalamic nuclei of adult rats by performing in situ hybridisation with subtype-specific 35S-labelled oligonucleotide probes. High expression of mGluR1 mRNA mainly occurred in midline nuclei such as the centromedial/centrolateral (CM/CL) nuclei, parafascicular and submedius nuclei, and in the ventroposteromedial (VPM) and posterior (Po) nuclei. In contrast, mGluR5 mRNA was more uniformly distributed at weak to moderate levels, except in the reuniens nucleus where a strong signal was detected. The mGluR3 mRNA was highly expressed in the reticular thalamic nucleus and almost not detectable in any other thalamic region. Additionally, mGluR3 mRNA was found not only in neurones but also in putative glial cells. The mGluR4 mRNA was abundant in most thalamic nuclei, with prominent expression in the CM/CL, Po and ventrobasal complex (VPM and ventroposterolateral, VPL). Finally, mGluR7 transcripts were found evenly distributed throughout the thalamus at moderate levels, the highest signal being detected in the paraventricular thalamic nucleus, VPM, VPL and Po. This differential distribution of mGluR subtypes in the rat thalamus may contribute to the heterogeneity of glutamate effects on thalamic neurones. The mGluR1, mGluR4 and mGluR7 receptors may be involved in the processing of somatosensory information because they are expressed in nuclei that receive direct sensory input.

Inhibition of ERK phosphorylation decreases nociceptive behaviour in monoarthritic rats.

&NA; In this study we investigated the role of the activation of the extracellular signal‐regulated kinases 1 and 2 (ERK) in chronic inflammatory articular nociception. Monoarthritis was induced in the left ankle of Wistar rats by injection of complete Freunds adjuvant (CFA). Movement of the inflamed joint increased ERK phosphorylation in neurones of the superficial and deep ipislateral dorsal horn laminae of L3‐L5 spinal cord segments. Spinal immunoreactivity to phosphoERK was more intense in animals in which the inflammation lasted longer, 7 days or more, than in rats with less time of inflammation. PhosphoERK levels were transient, since 2 h after ankle stimulation spinal immunoreaction had almost disappeared. PhosphoERK immunoreactivity was not induced by movement of ankles from non‐arthritic control animals, neither in monoarthritic rats in which the inflamed ankle was not stimulated. Intrathecal administration of PD 98059, an inhibitor of ERK phosphorylation, reduced nociceptive behaviour induced by the ankle bend test in monoarthritic rats. The anti‐nociceptive effect of PD 98059 was more prominent and in animals with short lasting (4 days) than in animals with longer (14 days) monoarthritis. Taken together, these findings suggest that ERK phosphorylation in spinal cord neurones plays an important role in chronic inflammatory articular pain and that its inhibition may provide significant anti‐nociception.

Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.

UNLABELLED Although there are several reports on pain behavioral tests in rat models of knee osteoarthritis (OA), most of them focus on the paw. The aim of this study was to investigate pain-related behaviors on the affected knee joint, the primary source of nociception, in animals with mono-iodoacetate-induced OA, using the knee-bend (which provides information on movement pain) and pin-prick tests, and to evaluate nociception elicited by walking using the CatWalk test. The von Frey and Randall-Selitto tests applied to the paw allowed us to compare our study results with previous studies. A further aim was to compare the behavioral nociceptive responses of the most used doses of mono-iodoacetate, 2 and 3 mg. Knee-bend score of OA animals was higher than those of control animals throughout the study (P < .05). At every time point, the ipsilateral hind-paw load of OA rats, as measured by the CatWalk test, was lower than that of control rats (P < .05), and paw withdraw threshold to von Frey filaments was also decreased (P < .01). No changes were observed in pin-prick and Randall-Selitto tests. Results obtained with the 2 doses of mono-iodoacetate were similar. The knee-bend and CatWalk tests are effective for evaluating movement-related nociception, a hallmark of clinical OA, which was present throughout the experimental period. PERSPECTIVE Behavioral characterization of models of OA pain is important and useful for use in future studies to test pharmacological treatments. Furthermore, it is important to find methods that correlate better with the human symptoms of OA.