Intrathecal Administration of an Anti-nociceptive Non-CpG Oligodeoxynucleotide Reduces Glial Activation and Central Sensitization.

Inflammatory pain associates with spinal glial activation and central sensitization. Systemic administration of IMT504, a non-CpG oligodeoxynucleotide originally designed as an immunomodulator, exerts remarkable anti-allodynic effects in rats with complete Freund´s adjuvant (CFA)-induced hindpaw inflammation. However, the anti-nociceptive mechanisms of IMT504 remain unknown. Here we evaluated whether IMT504 blocks inflammatory pain-like behavior by modulation of spinal glia and central sensitization. The study was performed in Sprague Dawley rats with intraplantar CFA, and a single lumbosacral intrathecal (i.t.) administration of IMT504 or vehicle was chosen to address if changes in glial activation and spinal sensitization relate to the pain-like behavior reducing effects of the ODN. Naïve rats were also included. Von Frey and Randall-Selitto tests, respectively, exposed significant reductions in allodynia and mechanical hypersensitivity, lasting at least 24 h after i.t. IMT504. Analysis of electromyographic responses to electrical stimulation of C fibers showed progressive reductions in wind-up responses. Accordingly, IMT504 significantly downregulated spinal glial activation, as shown by reductions in the protein expression of glial fibrillary acidic protein, CD11b/c, Toll-like receptor 4 (TLR4) and the phosphorylated p65 subunit of NFκB, evaluated by immunohistochemistry and western blot. In vitro experiments using early post-natal cortical glial cultures provided further support to in vivo data and demonstrated IMT504 internalization into microglia and astrocytes. Altogether, our study provides new evidence on the central mechanisms of anti-nociception by IMT504 upon intrathecal application, and further supports its value as a novel anti-inflammatory ODN with actions upon glial cells and the TLR4/NFκB pathway. Intrathecal administration of the non-CpG ODN IMT504 fully blocks CFA-induced mechanical allodynia and hypersensitivity, in association with reduced spinal sensitization. Administration of the ODN also results in downregulated gliosis and reduced TLR4-NF-κB pathway activation. IMT504 uptake into astrocytes and microglia support the concept of direct modulation of CFA-induced glial activation.

Role of the spinal TrkB-NMDA receptor link in the BDNF-induced long-lasting mechanical hyperalgesia in the rat: A behavioural study.

BACKGROUND: Intrathecal/intracisternal BDNF in rodents produces long-lasting hyperalgesia/allodynia, which implies BDNF plays a role in the establishment and maintenance of central sensitization. Both self-regeneration of endogenous BDNF and neuroplastic modifications of spinal NMDA receptors downstream TrkB signalling could be involved in such enduring hyperalgesia. We investigated to what extent BDNF by itself could participate in the generation and maintenance of mechanical hyperalgesia using pharmacological tools. METHODS: We studied sensitivity of mechanical hyperalgesia induced by a single intrathecal (i.t.) injection of BDNF (3 ng/10 µL i.t.) administered at time zero, for: (1) chronic NMDA receptor inhibition with subcutaneously implanted 7-day delivery osmotic pumps loaded with ketamine; (2) TrkB receptor inhibition with intraperitoneal (i.p.) cyclotraxine-B; and (3) chronic glial inhibition with repeated propentofylline i.t. injections. Nociceptive threshold to paw pressure, tested on days -3, 0, 3, 7, 10 and 14, was used as the index of central sensitization. Locomotor patterns and food and water consumption were assessed with LABORAS. RESULTS: Chronic ketamine prevented the mechanical hyperalgesia induced by BDNF, without affecting locomotion and food and water consumption. After pump depletion, a late hyperalgesic response to paw pressure stimulation emerged, which can be lastingly antagonized by cyclotraxine-B. Chronic propentofylline treatment irreversibly suppressed BDNF-induced hyperalgesia. CONCLUSION: Activation of NMDA receptors downstream to TrkB signalling is essential for behavioural expression of the mechanical hyperalgesia induced by intrathecal BDNF. However, maintenance of the hyperalgesia depends mainly from self-regenerating glial BDNF rather than from a NMDA receptor-dependent form of neuroplasticity. SIGNIFICANCE: Intrathecal BDNF induces long-lasting central sensitization via a glial-likely BDNF self-regenerating mechanism, whose behavioural expression depends on downstream activation of NMDA receptors. This knowledge suggests that TrkB antagonists could represent an interesting lead for the development of novel therapeutic strategies for some chronic pain conditions.

Interactions of pannexin 1 with NMDA and P2X7 receptors in central nervous system pathologies: Possible role on chronic pain.

Pannexin 1 (Panx1) is a glycoprotein that acts as a membrane channel in a wide variety of tissues in mammals. In the central nervous system (CNS) Panx1 is expressed in neurons, astrocytes and microglia, participating in the pathophysiology of some CNS diseases, such as epilepsy, anoxic depolarization after stroke and neuroinflammation. In these conditions Panx1 acts as an important modulator of the neuroinflammatory response, by secreting ATP, by interacting with the P2X7 receptor (P2X7R), and as an amplifier of NMDA receptor (NMDAR) currents, particularly in conditions of pathological neuronal hyperexcitability. Here, we briefly reviewed the current evidences that support the interaction of Panx1 with NMDAR and P2X7R in pathological contexts of the CNS, with special focus in recent data supporting that Panx1 is involved in chronic pain signaling by interacting with NMDAR in neurons and with P2X7R in glia. The participation of Panx1 in chronic pain constitutes a novel topic for research in the field of clinical neurosciences and a potential target for pharmacological interventions in chronic pain.

Comparative evolution history of SINEs in Arabidopsis thaliana and Brassica oleracea: evidence for a high rate of SINE loss.

Brassica oleracea and Arabidopsis thaliana belong to the Brassicaceae(Cruciferae) family and diverged 16 to 19 million years ago. Although the genome size of B. oleracea (approximately 600 million base pairs) is more than four times that of A. thaliana (approximately 130 million base pairs), their gene content is believed to be very similar with more than 85% sequence identity in the coding region. Therefore, this important difference in genome size is likely to reflect a different rate of non-coding DNA accumulation. Transposable elements (TEs) constitute a major fraction of non-coding DNA in plant species. A different rate in TE accumulation between two closely related species can result in significant genome size variations in a short evolutionary period. Short interspersed elements (SINEs) are non-autonomous retroposons that have invaded the genome of most eukaryote species. Several SINE families are present in B. oleracea and A. thaliana and we found that two of them (called RathE1 and RathE2) are present in both species. In this study, the tempo of evolution of RathE1 and RathE2 SINE families in both species was compared. We observed that most B. oleracea RathE2 SINEs are "young" (close to the consensus sequence) and abundant while elements from this family are more degenerated and much less abundant in A. thaliana. However, the situation is different for the RathE1 SINE family for which the youngest elements are found in A. thaliana. Surprisingly, no SINE was found to occupy the same (orthologous) genomic locus in both species suggesting that either these SINE families were not amplified at a significant rate in the common ancestor of the two species or that older elements were lost and only the recent (lineage-specific) insertions remain. To test this latter hypothesis, loci containing a recently inserted SINE in the A. thaliana col-0 ecotype were selected and characterized in several other A. thaliana ecotypes. In addition to the expected SINE containing allele and the pre-integrative allele (i.e. the "empty" allele), we observed in the different ecotypes, alleles with truncated portions of the SINE (up to the complete loss of the element) and of the immediate genomic flanking sequences. The absence of SINEs in orthologous positions between B. oleracea and A. thaliana and the presence in recently diverged A. thaliana ecotypes of alleles containing severely truncated SINEs suggest a very high rate of SINE loss in these species.

The evolutionary origin and genomic organization of SINEs in Arabidopsis thaliana.

We have characterized the two families of SINE retroposons present in Arabidopsis thaliana. The origin, distribution, organization, and evolutionary history of RAthE1 and RAthE2 elements were studied and compared to the well-characterized SINE S1 element from Brassica. Our studies show that RAthE1, RAthE2, and S1 retroposons were generated independently from three different tRNAs. The RAthE1 and RAthE2 families are older than the S1 family and are present in all tested Cruciferae species. The evolutionary history of the RAthE1 family is unusual for SINEs. The 144 RAthE1 elements of the Arabidopsis genome cannot be classified in distinct subfamilies of different evolutionary ages as is the case for S1, RAthE2, and mammalian SINEs. Instead, most RAthE1 elements were probably derived steadily from a single source gene that was maintained intact and active for at least 12-20 Myr, a result suggesting that the RAthE1 source gene was under selection. The distribution of RAthE1 and RAthE2 elements on the Arabidopsis physical map was studied. We observed that, in contrast to other Arabidopsis transposable elements, SINEs are not concentrated in the heterochromatic regions. Instead, SINEs are grouped in the euchromatic chromosome territories several hundred kilobase pairs long. In these territories, SINE elements are closely associated with genes. A retroposition partnership between Arabidopsis SINEs and LINEs is proposed.

Analysis of the SINE S1 Pol III promoter from Brassica; impact of methylation and influence of external sequences.

Transcription is an important control point in the transposable element mobilization process. To better understand the regulation of the plant SINE (Short Interspersed Elements) S1, its promoter sequence was studied using an in vitro pol III transcription system derived from tobacco cells. We show that the internal S1 promoter can be functional although upstream external sequences were found to enhance this basal level of transcription. For one putative 'master' locus (na7), three CAA triplets (in positions -12, -7 and -2) and two overlapping TATA motifs (in positions -54 to -43) were important to stimulate transcription. For this locus, two transcription initiation regions were characterized, one centered on position + 1 (first nucleotide of the S1 element) and one centered on position - 19 independently of the internal motifs. The CAA triplets only influence transcription in + 1 and work in association with the internal motifs. We show that methylation can inhibit transcription at the na7 locus. We also observe that S1 RNA is cleaved in a smaller Poly (A) minus product by a process analogous to the maturation of mammalian SINEs.

Effect of ketamine on spinal cord nociceptive transmission in normal and monoarthritic rats.

The effects of systemically and intrathecally administered ketamine on spinal wind-up of normal and monoarthritic rats were studied by using C-fiber reflex responses evoked by repetitive (0.6 Hz) electric stimulation. Both systemic and intrathecal ketamine induced dose-dependent depression of wind-up activity in normal rats, as revealed by the dose-related inhibitory effects of the drug. At the same intraperitoneal doses, ketamine produced a greater inhibitory effect on wind-up activity of monoarthritic rats, compared to normal animals. The intrathecal administration of ketamine also produced wind-up inhibition, the efficacy being higher in the monoarthritic rats. Results indicate that ketamine depresses spinal wind-up, specially in rats submitted to chronic pain, probably due to its antagonistic properties on dorsal horn NMDA receptors, which play a crucial role in the maintenance of chronic pain.

Antinociceptive effect of clomipramine in monoarthritic rats as revealed by the paw pressure test and the C-fiber-evoked reflex.

The antinociceptive effect of clomipramine was studied in monoarthritic rats by using the paw pressure test and the C-fiber-evoked reflex. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the tibio-tarsal joint. Joint circumference as well as vocalization threshold to graded paw pressure were evaluated weekly during a 14-week period after the intra-articular injection. At week 8, monoarthritic and vehicle-injected control rats were given either clomipramine or saline and both the paw pressure threshold and inhibition of the C-fiber-evoked reflex response were evaluated. Results showed that (i) 1.5, 3.0, and 6.0 mg/kg, i.v. of clomipramine induced significantly greater dose-dependent antinociception to paw pressure testing in the monoarthritic group, as compared to the control one; and (ii) 0.75, 1.5, 3.0, and 6.0 mg/kg, i.v. of clomipramine exerted significantly higher dose-dependent inhibition of the C-reflex activity in monoarthritic rats than in controls. Results suggest that the higher sensitivity to clomipramine in monoarthritic rats could be related to adaptive changes occurring in monoamine metabolism or in other neurotransmitter systems during chronic pain.

Lesion of the bulbospinal noradrenergic pathways blocks desipramine-induced inhibition of the C-fiber evoked nociceptive reflex in rats.

Desipramine-induced inhibition of spinal cord nociceptive transmission was studied in rats with or without lesion of the bulbospinal noradrenergic system by recording the C-fiber evoked nociceptive reflex from a hind limb. Bulbospinal noradrenergic projections were lesioned by injecting intrathecally 20 microg of 6-hydroxydopamine 2 weeks before the electrophysiological experiments. Results show that desipramine (5, 10 and 20 mg/kg intraperitoneally) produced dose-dependent inhibition of the C reflex response duration in rats having intact noradrenergic bulbospinal systems. The inhibitory effect of desipramine was reduced or even abolished in rats pre-treated with 6-hydroxydopamine. In addition, [3H]-noradrenaline uptake was significantly lower in spinal cord slices arising from 6-hydroxydopamine lesioned animals, as compared to that from intact rats. These observations support the notion that the antinociceptive activity of antidepressants with noradrenergic selectivity depends on a normal rate of endogenous noradrenaline released by bulbospinal neurons.

SINE retroposons can be used in vivo as nucleation centers for de novo methylation.

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences.

Differential effects of trigeminal tractotomy on Adelta- and C-fiber-mediated nociceptive responses.

In this study we have tested in the rat, whether trigeminal tractotomy, which deprives the spinal trigeminal nucleus caudalis (Sp5C) of its trigeminal inputs, affected differentially nociceptive responses mediated by C- vs. Adelta-nociceptors from oral and perioral regions. Tractotomy had no effect on the threshold of the jaw opening reflex, induced by incisive pulp stimulation (Adelta-fiber-mediated), but blocked the formalin response (mainly C-fiber-mediated). These results suggest that nociceptive responses mediated by trigeminal C-fibers completely depend on the integrity of the Sp5C, while intraoral sensations triggered Adelta-fibers (especially of dental origin) are primarily processed in the rostral part of the spinal trigeminal nucleus.

A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.

In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed.

Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation.

Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobacco plants has suggested that an RNA-DNA interaction could trigger de novo methylation of PSTVd transgene sequences. Using the genomic sequencing technique, the methylation pattern associated with the RNA-directed DNA methylation process has been characterized. Three different PSTVd transgene constructs all showed a similar pattern of methylation. Most of the cytosines at symmetrical as well as non-symmetrical positions appeared to be methylated in both DNA strands of the viroid sequences. Heavy methylation was mostly restricted to the viroid cDNA sequences. Flanking DNA regions immediately adjacent to the viroid cDNA displayed a lower but significant level of cytosine methylation. The observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act as a strong and highly specific signal for de novo DNA methylation. These data also confirmed that de novo methylation was not limited to canonical CpG and CpNpG sites, but can also involve all the cytosine residues located in the genomic region where the RNA-DNA interaction takes place.

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.

A model for RNA-mediated gene silencing in higher plants.

Homology-dependent gene silencing (HdGS) which is the generic term for transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS) and RNA-mediated virus-resistance (RmVR) has been shown to frequently occur in transgenic plants. The role of RNA as a target and initiator of PTGS and RmVR is more and more manifested. Because TGS is assumed to be induced by a DNA-DNA interaction-mediated promoter methylation, a possible involvement of RNA in TGS was not really considered up to now. In this review we attempt to demonstrate that all three types of HdGS could be triggered by one RNA-based mechanism. A model proposing TGS as a consequence of RNA-directed DNA methylation (RdDM) and a refined mRNA threshold mechanism are presented. In contrast to the view that high amounts of mRNA are required we assume that the concentration of RNAs that can serve as efficient templates for a plant-encoded RNA-directed RNA polymerase (RdRP) plays a key role in HdGS and possibly also in natural gene regulation of non-transformed cells. According to this idea a particular information must be encoded to render mRNA turn-over products a suitable RdRP substrate. It will be discussed that such a mechanism could account for the silencing phenomena of poorly transcribed transgenes. Finally, an explanation for the coherency between PTGS and DNA methylation is documented.

Interactions in the antinociceptive effect of tramadol in mice: an isobolographic analysis.

Tramadol is a widely-used analgesic for pre- and post-operative pain which has a different pharmacological profile to that of classical opioids, since it does not induce respiratory depression, constipation, sedation, tolerance or dependence. However, tramadol frequently produces nausea and vomiting as side-effects. In the present study, the interactions between tramadol and several adrenergic and serotonergic compounds with antinociceptive activity were studied by isobolographic analysis. Antinociceptive activity was evaluated using the acetic acid writhing test in mice. Dose-response curves for the antinociceptive effect of tramadol, prazosin, clonidine, xylamine, clomipramine and cyproheptadine were obtained, and ED(50)s were calculated for isobolographic analysis, which was performed by administration of fixed-ratios of tramadol with each of these drugs, given both systemically and intrathecally. The isobolograms of all combinations tested, either systemically or intrathecally showed superadditivity. The synergies observed with these combinations suggest a complex modulation of the descending noradrenergic and serotonergic systems that exert inhibitory influences on the transmission of nociceptive information, probably in addition to effects on receptors in the primary neurons of the spinal cord. The co-administration of analgesic drugs that produce superadditive effects constitutes a significant new avenue for the treatment of pain, since a similar level of antinociception can be obtained with considerable reductions in the dose of each analgesic. Copyright 1998 European Federation of Chapters of the International Association for the Study of Pain.

Effects of clomipramine and desipramine on a C-fiber reflex in rats.

A C-fiber nociceptive reflex evoked by electrical stimulation within the territory of the sural nerve, was recorded from the ipsilateral biceps femoris muscle in urethane anesthetized rats. Intravenously administered clomipramine and desipramine produced a dose-dependent depression of the C-fiber reflex. High doses of intrathecal desipramine also inhibited the C-fiber reflex, while similar intrathecal doses of clomipramine produced only a modest inhibition of the response. Intracerebroventricular administration of clomipramine decreased dose-dependently the C-fiber reflex whereas intracerebroventricular desipramine increased this reflex. These findings suggest that tricyclic antidepressants with noradrenergic selectivity, as desipramine, inhibit the spinal processing of C inputs by acting directly at the spinal cord level, while those with serotonergic spectra, as clomipramine, depress the C-fiber-evoked spinal reflex by acting at a supraspinal modulatory site.

Paracetamol exerts a spinal antinociceptive effect involving an indirect interaction with 5-hydroxytryptamine3 receptors: in vivo and in vitro evidence.

Rats (Sprague-Dawley), submitted to a mechanical noxious stimulus (paw pressure), were tested to determine 1) the antinociceptive effects of p.o. (200, 400 and 800 mg/kg), i.v. (50, 100, 200 and 300 mg/kg) and intrathecal (i.t.) (100 and 200 micrograms/rat) administrations of paracetamol; 2) the influence of i.t. administered tropisetron, a 5-hydroxytryptamine3 (5-HT3) receptor antagonist (0.5, 1 or 10 micrograms/rat) on paracetamol-induced antinociception; 3) the influence of indomethacin (25 mg/kg s.c.), naloxone (10 micrograms/rat i.t.) and yohimbine (1 mg/kg i.v.) on the effect of paracetamol (200 mg/kg i.v.) to determine the involvement of prostaglandins, opioids and alpha-2 adrenoceptors. The displacement by paracetamol of radioligand binding to various receptors was also investigated. Paracetamol induced a significant antinociceptive effect after p.o., i.v. and i.t. administration. A total inhibition of the effect of paracetamol, administered p.o. or i.t., occurred at the dose of 0.5 microgram/rat of tropisetron, whereas 10 micrograms/rat of this antagonist was needed to totally inhibit the action of i.v. administered paracetamol. Indomethacin, naloxone and yohimbine failed to modify paracetamol antinociceptive action. In vitro studies failed to show any binding of paracetamol to 5-HT3 and several other receptors and to 5-HT uptake sites. It is concluded that paracetamol has a central antinociceptive effect, based on an indirect involvement of spinal 5-HT3 receptors.

DNA regions flanking the major Arabidopsis thaliana satellite are principally enriched in Athila retroelement sequences.

An analysis of Arabidopsis thaliana heterochromatic regions revealed that genomic sequences immediately flanking the major 180 bp satellite are essentially made of middle repetitive sequences and that most of these sequences correspond to defective Athila retroelements. Using YAC and lambda clones, we evaluated the distribution of Athila elements in the Arabidopsis genome and showed that, despite the presence of numerous euchromatic copies, these elements are especially concentrated in or near heterochromatic regions. Sequencing of the various DNA transitions between satellite and Athila repeats provides strong evidence that most of the heterochromatic elements retrotransposed directly into 180 bp satellite clusters.