Michal Korostynski

Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain.

ABSTRACT We investigated the efficacy of local intraplantar (i.pl.) injection of peptide and non‐peptide μ‐, δ‐ and κ‐opioid receptor agonists in rat models of inflammatory and neuropathic pain. Locally applied agonists dose‐dependently reduced formalin‐induced flinching of the inflamed paw and induced antiallodynic and antihyperalgesic effects in sciatic nerve ligation‐induced neuropathic pain. These effects were mediated by peripheral opioid receptors localized at the side of tissue/nerve injury, as was demonstrated by selective and non‐selective opioid receptors antagonists. The ED50 dose range of μ‐ and κ‐agonists required to induce analgesia in neuropathy was much higher than the ED50 for inflammation; moreover, only δ‐agonists were effective in the same dose range in both pain models. Additionally, effective antinociception was achieved at a lower dose of peptide, compared to non‐peptide, opioids. Such findings support the use of the peripheral administration of opioid peptides, especially δ‐agonists, in treating chronic pain. Furthermore, in order to assess whether adaptations in the expression of opioid genes could underlie the clinical observation of reduced opioid effectiveness in neuropathic pain, we analyzed the abundance of opioid transcripts in the spinal cord and dorsal root ganglia (DRG) during the neuropathy and inflammation. Nerve injury down‐regulated mRNA for all types of opioid receptors in the DRG, which is predicted to decrease in the synthesis of opioid receptors to possibly account for the reduced effectiveness of locally administered opioids in neuropathy. The obtained results differentiate inflammatory and neuropathic pain and provide a novel insight into the peripheral effectiveness of opioids in both types of pain.

Neuropsin cleaves EphB2 in the amygdala to control anxiety

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron–matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2–NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2–NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2–NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain.

The pharmacological attenuation of glial activation represents a novel approach for controlling neuropathic pain, but the role of microglial and astroglial cells is not well established. To better understand the potential role of two types of glial cells, microglia and astrocytes, in the pathogenesis of neuropathic pain, we examined markers associated with them by quantitative RT-PCR, western blot and immunohistochemical analyses in the dorsal horn of the lumbar spinal cord 7days after chronic constriction injury (CCI) to the sciatic nerve in mice. The mRNA and protein of microglial cells were labeled with C1q and OX42(CD11b/c), respectively. The mRNA and protein of astrocytes were labeled with GFAP. The RT-PCR results indicated an increase in C1q mRNA that was more pronounced than the increased expression of GFAP mRNA ipsilateral to the injury in the dorsal spinal cord. Similarly, western blot and immunohistochemical analyses demonstrated an ipsilateral upregulation of OX42-positive cells (72 and 20%, respectively) and no or little (8% upregulation) change in GFAP-positive cells in the ipsilateral dorsal lumbar spinal cord. We also found that chronic intraperitoneal injection of the minocycline (microglial inhibitor) and pentoxifylline (cytokine inhibitor) attenuated CCI-induced activation of microglia, and both, but not fluorocitrate (astroglial inhibitor), diminished neuropathic pain symptoms and tactile and cold sensitivity. Our findings indicate that spinal microglia are more activated than astrocytes in peripheral injury-induced neuropathic pain. These findings implicate a glial regulation of the pain response and suggest that pharmacologically targeting microglia could effectively prevent clinical pain syndromes in programmed and/or anticipated injury.

The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum

BackgroundVarious drugs of abuse activate intracellular pathways in the brain reward system. These pathways regulate the expression of genes that are essential to the development of addiction. To reveal genes common and distinct for different classes of drugs of abuse, we compared the effects of nicotine, ethanol, cocaine, morphine, heroin and methamphetamine on gene expression profiles in the mouse striatum.ResultsWe applied whole-genome microarray profiling to evaluate detailed time-courses (1, 2, 4 and 8 hours) of transcriptome alterations following acute drug administration in mice. We identified 42 drug-responsive genes that were segregated into two main transcriptional modules. The first module consisted of activity-dependent transcripts (including Fos and Npas4), which are induced by psychostimulants and opioids. The second group of genes (including Fkbp5 and S3-12), which are controlled, in part, by the release of steroid hormones, was strongly activated by ethanol and opioids. Using pharmacological tools, we were able to inhibit the induction of particular modules of drug-related genomic profiles. We selected a subset of genes for validation by in situ hybridization and quantitative PCR. We also showed that knockdown of the drug-responsive genes Sgk1 and Tsc22d3 resulted in alterations to dendritic spines in mice, possibly reflecting an altered potential for plastic changes.ConclusionsOur study identified modules of drug-induced genes that share functional relationships. These genes may play a critical role in the early stages of addiction.

Interleukin-1alpha has antiallodynic and antihyperalgesic activities in a rat neuropathic pain model

&NA; Nerve injury and the consequent release of interleukins (ILs) are processes implicated in pain transmission. To study the potential role of IL‐1 in the pathogenesis of allodynia and hyperalgesia, IL‐1alpha and comparative IL‐1beta, IL‐6, and IL‐10 mRNA levels were quantified using competitive RT‐PCR of the lumbar spinal cord and dorsal root ganglia (DRG; L5–L6) three and seven days after chronic constriction injury (CCI) in rats. Microglial and astroglial activation in the ipsilateral spinal cord and DRG were observed after injury. In naive and CCI‐exposed rats, IL‐1alpha mRNA and protein were not detected in the spinal cord. IL‐1beta and IL‐6 mRNAs were strongly ipsilaterally elevated on day seven after CCI. In the ipsilateral DRG, IL‐1alpha, IL‐6, and IL‐10 mRNA levels were increased on days three and seven; IL‐1beta was elevated only on day seven. Western blot analysis revealed both the presence of IL‐1alpha proteins (45 and 31 kDa) in the DRG and the down‐regulation of these proteins after CCI. Intrathecal administration of IL‐1alpha (50–500 ng) in naive rats did not influence nociceptive transmission, but IL‐1beta (50–500 ng) induced hyperalgesia. In rats exposed to CCI, an IL‐1alpha or IL‐1 receptor antagonist dose‐dependently attenuated symptoms of neuropathic pain; however, no effect of IL‐1beta was observed. In sum, the first days after CCI showed a high abundance of IL‐1alpha in the DRG. Together with the antiallodynic and antihyperalgesic effects observed after IL‐1alpha administration, this finding indicates an important role for IL‐1alpha in the development of neuropathic pain symptoms.

Gene Expression Profiles in Human Ruptured and Unruptured Intracranial Aneurysms: What Is the Role of Inflammation?

Background and Purpose— Mechanisms underlying development and rupture of intracranial aneurysms (IA) are poorly recognized. The majority of studies on human tissue have focused on predefined pathways. We sought to analyze global gene expression patterns of ruptured IA, unruptured IA, and control vessels. Methods— Transcription profiles were studied in human ruptured (n=8) and unruptured (n=6) IA, as well as in control intracranial arteries (n=5), using oligonucleotide microarrays. Real-time reverse-transcription polymerase chain reaction was used for confirmation. Functional analysis for determination of over-represented ontological groups among gene expression profiles was also performed. Results— The expression of 159 genes differed among the studied groups. Compared to the controls, 131 genes showed common directions of change in both IA groups. The most impacted biological processes for IA are: (1) the muscle system; (2) cell adhesion (downregulation); and (3) the immune system and inflammatory response (upregulation). Ruptured and unruptured IA differed in genes involved in immune/inflammatory processes; expression was reduced in ruptured IA. Conclusions— Decreased expression of genes related to muscle system and cell adhesion is important for the development of IA. The role of immune/inflammatory processes is unclear. Inflammation may participate in the healing process within IA while playing a protective role against IA rupture.

Morphine effects on striatal transcriptome in mice

BackgroundChronic opiate use produces molecular and cellular adaptations in the nervous system that lead to tolerance, physical dependence, and addiction. Genome-wide comparison of morphine-induced changes in brain transcription of mouse strains with different opioid-related phenotypes provides an opportunity to discover the relationship between gene expression and behavioral response to the drug.ResultsHere, we analyzed the effects of single and repeated morphine administrations in selected inbred mouse strains (129P3/J, DBA/2J, C57BL/6J, and SWR/J). Using microarray-based gene expression profiling in striatum, we found 618 (false discovery rate < 1%) morphine-responsive transcripts. Through ontologic classification, we linked particular sets of genes to biologic functions, including metabolism, transmission of nerve impulse, and cell-cell signaling. We identified numerous novel morphine-regulated genes (for instance, Olig2 and Camk1g), and a number of transcripts with strain-specific changes in expression (for instance, Hspa1a and Fzd2). Moreover, transcriptional activation of a pattern of co-expressed genes (for instance, Tsc22d3 and Nfkbia) was identified as being mediated via the glucocorticoid receptor (GR). Further studies revealed that blockade of the GR altered morphine-induced locomotor activity and development of physical dependence.ConclusionOur results indicate that there are differences between strains in the magnitude of transcriptional response to acute morphine treatment and in the degree of tolerance in gene expression observed after chronic morphine treatment. Using whole-genome transcriptional analysis of morphine effects in the striatum, we were able to reveal multiple physiological factors that may influence opioid-related phenotypes and to relate particular gene networks to this complex trait. The results also suggest the possible involvement of GR-regulated genes in mediating behavioral response to morphine.

Full inhibition of spinal FAAH leads to TRPV1-mediated analgesic effects in neuropathic rats and possible lipoxygenase-mediated remodeling of anandamide metabolism.

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

Gene expression profiling in the striatum of inbred mouse strains with distinct opioid-related phenotypes

BackgroundMouse strains with a contrasting response to morphine provide a unique model for studying the genetically determined diversity of sensitivity to opioid reward, tolerance and dependence. Four inbred strains selected for this study exhibit the most distinct opioid-related phenotypes. C57BL/6J and DBA/2J mice show remarkable differences in morphine-induced antinociception, self-administration and locomotor activity. 129P3/J mice display low morphine tolerance and dependence in contrast to high sensitivity to precipitated withdrawal observed in SWR/J and C57BL/6J strains. In this study, we attempted to investigate the relationships between genetic background and basal gene expression profile in the striatum, a brain region involved in the mechanism of opioid action.ResultsGene expression was studied by Affymetrix Mouse Genome 430v2.0 arrays with probes for over 39.000 transcripts. Analysis of variance with the control for false discovery rate (q < 0.01) revealed inter-strain variation in the expression of ~3% of the analyzed transcripts. A combination of three methods of array pre-processing was used to compile a list of ranked transcripts covered by 1528 probe-sets significantly different between the mouse strains under comparison. Using Gene Ontology analysis, over-represented patterns of genes associated with cytoskeleton and involved in synaptic transmission were identified. Differential expression of several genes with relevant neurobiological function (e.g. GABA-A receptor alpha subunits) was validated by quantitative RT-PCR. Analysis of correlations between gene expression and behavioural data revealed connection between the level of mRNA for K homology domain containing, RNA binding, signal transduction associated 1 (Khdrbs1) and ATPase Na+/K+ alpha2 subunit (Atp1a2) with morphine self-administration and analgesic effects, respectively. Finally, the examination of transcript structure demonstrated a possible inter-strain variability of expressed mRNA forms as for example the catechol-O-methyltransferase (Comt) gene.ConclusionThe presented study led to the recognition of differences in the gene expression that may account for distinct phenotypes. Moreover, results indicate strong contribution of genetic background to differences in gene transcription in the mouse striatum. The genes identified in this work constitute promising candidates for further animal studies and for translational genetic studies in the field of addictive and analgesic properties of opioids.

Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.