preproenkephalin and Inflammation

Expression of the opioid peptide genes proopiomelanocortin (Pomc), proenkephalin (Penk), and prodynorphin (Pdyn), in immune cells plays a key role in endogenous pain control. In a rat model of painful unilateral paw inflammation, we isolated cells from popliteal lymph nodes and evaluated the role of CpG island C5-methylation on the transcriptional activation of those genes. Using methylated DNA immunoprecipitation, we sorted gDNA into methylated (me) and non-me fractions and then determined the CpG island methylation status of each fraction via quantitative Real Time-PCR (qRT-PCR). In silico analysis by MethPrimer software identified one CpG island in Pdyn and three each in Pomc and Penk. No substantial changes in C5-methylation of any gene were observed. In conclusion, the CpG island methylation status does not seem to be a key regulator of opioid gene activation in immune cells during peripheral tissue inflammation.

Topics: Animals; CpG Islands; Disease Models, Animal; DNA Methylation; Enkephalins; Gene Expression Regulation; Inflammation; Male; Pain; Pro-Opiomelanocortin; Protein Precursors; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction

Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice.. The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis.. Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice.. These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.

Topics: Animals; Behavior, Animal; Brain; Brain Chemistry; Dynorphins; Enkephalins; Freund's Adjuvant; Immunohistochemistry; Inflammation; Injections, Spinal; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pain; Protein Precursors; Receptors, N-Methyl-D-Aspartate; Reverse Transcriptase Polymerase Chain Reaction; RNA; Spinal Cord; Synaptic Transmission

Neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. We investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behaviour at the physiological and molecular levels after peripheral reinflammation in adulthood.. A decrease in paw withdrawal latency (PWL) after a heat stimulus was documented in rats that received inflammatory injections in their left hind paws on postnatal day one (P1) and a reinflammation stimulus at postnatal 6-8 weeks of age, compared with normal rats. An increase in the expression of the prodynorphin (proDYN) gene was noted after reinflammation in the spinal cord ipsilateral to the afferents of the neonatally treated hind paw. The involvement of the activation of extracellular signal-regulated kinases (ERK) in peripheral inflammatory pain hypersensitivity was evidenced evident by the increase in phospho-ERK (pERK) activity after reinflammation.. Our results indicate that peripheral inflammation in neonates can permanently alter the pain processing pathway during the subsequent sensory stimulation of the region. Elucidation of the mechanism underlying the developing pain circuitry will provide new insights into the understanding of the early pain behaviours and the subsequent adaptation to pain.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Enkephalins; Gene Expression Regulation; Hindlimb; Hot Temperature; Hyperalgesia; Inflammation; Physical Stimulation; Protein Precursors; Rats; Rats, Sprague-Dawley; Spinal Cord; Up-Regulation

A chronic pancreatitis model was developed in young male Lewis rats fed a high-fat and alcohol liquid diet beginning at three weeks. The model was used to assess time course and efficacy of a replication defective herpes simplex virus type 1 vector construct delivering human cDNA encoding preproenkephalin (HSV-ENK).. Most surprising was the relative lack of inflammation and tissue disruption after HSV-ENK treatment compared to the histopathology consistent with pancreatitis (inflammatory cell infiltration, edema, acinar cell hypertrophy, fibrosis) present as a result of the high-fat and alcohol diet in controls. The HSV-ENK vector delivered to the pancreatic surface at week 3 reversed pancreatitis-associated hotplate hypersensitive responses for 4-6 weeks, while control virus encoding beta-galactosidase cDNA (HSV-beta-gal) had no effect. Increased Fos expression seen bilaterally in pain processing regions in control animals with pancreatitis was absent in HSV-ENK-treated animals. Increased met-enkephalin staining was evident in pancreas and lower thoracic spinal cord laminae I-II in the HSV-ENK-treated rats.. Thus, clear evidence is provided that site specific HSV-mediated transgene delivery of human cDNA encoding preproenkephalin ameliorates pancreatic inflammation and significantly reduces hypersensitive hotplate responses for an extended time consistent with HSV mediated overexpression, without tolerance or evidence of other opiate related side effects.

Topics: Animals; Behavior, Animal; DNA, Complementary; Enkephalins; Genetic Vectors; Herpesvirus 1, Human; Inflammation; Male; Models, Animal; Pancreatitis, Chronic; Protein Precursors; Rats; Rats, Inbred Lew; Receptors, Opioid, mu; Spinal Cord; Time Factors

Cyclooxygenase-2 (COX-2) is a major contributor to the elevation of spinal prostaglandin E2, which augments the processing of nociceptive stimuli following peripheral inflammation, and dynorphin has been shown to have an important role in acute and chronic pain states. Moreover, the transcription factor, nuclear factor-kappa B (NF-kB), regulates the expressions of both COX-2 and dynorphin. To elucidate the role of spinal NF-kB in the induction of inflammatory pain hypersensitivity, we examined whether activated NF-kB affects pain behavior and the expressions of the mRNAs of COX-2 and prodynorphin following peripheral inflammation. Intrathecal pretreatment with different NF-kB inhibitors, namely, NF-kB decoy or pyrrolidine dithiocarbamate, significantly reduced mechanical allodynia and thermal hyperalgesia following unilateral hindpaw inflammation evoked by complete Freund's adjuvant (CFA). These NF-kB inhibitors also suppressed the activation of spinal NF-kB and the subsequent remarkable elevation of spinal COX-2 mRNA, but not that of prodynorphin mRNA. In addition, the activation of spinal NF-kB following CFA injection was inhibited by intrathecal pretreatments with interleukin-1 beta receptor antagonist or caspase-1 inhibitor. In view of the fact that interleukin-1 beta (IL-1 beta) is the major inducer of spinal COX-2 upregulation following CFA injection, our results suggest that IL-1 beta-induced spinal COX-2 upregulation and pain hypersensitivity following peripheral inflammation are mediated through the activation of the NF-kB-associated pathways.

Topics: Animals; Cyclooxygenase 2; Enkephalins; Enzyme Inhibitors; Freund's Adjuvant; Gene Expression; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Isoenzymes; Male; NF-kappa B; Pain; Prostaglandin-Endoperoxide Synthases; Protein Precursors; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Spinal Cord; Up-Regulation

Activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase in dorsal horn neurons of the spinal cord by peripheral noxious stimulation contributes to short-term pain hypersensitivity. We investigated ERK activation by peripheral inflammation and its involvement in regulating gene expression in the spinal cord and in contributing to inflammatory pain hypersensitivity. Injection of complete Freund's adjuvant (CFA) into a hindpaw produced a persistent inflammation and a sustained ERK activation in neurons in the superficial layers (laminae I-IIo) of the dorsal horn. CFA also induced an upregulation of prodynorphin and neurokinin-1 (NK-1) in dorsal horn neurons, which was suppressed by intrathecal delivery of the MEK (MAP kinase kinase) inhibitor U0126. CFA-induced phospho-ERK primarily colocalized with prodynorphin and NK-1 in superficial dorsal horn neurons. Although intrathecal injection of U0126 did not affect basal pain sensitivity, it did attenuate both the establishment and maintenance of persistent inflammatory heat and mechanical hypersensitivity. Activation of the ERK pathway in a subset of nociceptive spinal neurons contributes, therefore, to persistent pain hypersensitivity, possibly via transcriptional regulation of genes, such as prodynorphin and NK-1.

Topics: Animals; Butadienes; Disease Models, Animal; Enkephalins; Enzyme Activation; Enzyme Inhibitors; Freund's Adjuvant; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Pain; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Substance P; Up-Regulation

Control and treatment of chronic pain remain major clinical challenges. Progress may be facilitated by a greater understanding of the mechanisms underlying pain processing. Here we show that the calcium-sensing protein DREAM is a transcriptional repressor involved in modulating pain. dream(-/-) mice displayed markedly reduced responses in models of acute thermal, mechanical, and visceral pain. dream(-/-) mice also exhibited reduced pain behaviors in models of chronic neuropathic and inflammatory pain. However, dream(-/-) mice showed no major defects in motor function or learning and memory. Mice lacking DREAM had elevated levels of prodynorphin mRNA and dynorphin A peptides in the spinal cord, and the reduction of pain behaviors in dream(-/-) mice was mediated through dynorphin-selective kappa (kappa)-opiate receptors. Thus, DREAM appears to be a critical transcriptional repressor in pain processing.

Topics: Animals; Base Sequence; Behavior, Animal; Calcium-Binding Proteins; Cells, Cultured; Consensus Sequence; Down-Regulation; Enkephalins; Heart; Hyperalgesia; Inflammation; Kv Channel-Interacting Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Neurons; Physical Stimulation; Presenilin-1; Presenilin-2; Protein Precursors; Proto-Oncogene Proteins c-fos; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, kappa; Repressor Proteins; Spinal Cord; Stimulation, Chemical; Transcription, Genetic

By using a dual-labeling immunohistochemical/in situ hybridization technique we examined if enkephalin-expressing neurons in the pontine parabrachial nucleus, a major brain stem relay for ascending visceral and homeostatic information, were activated by systemic immune challenge. While rats subjected to intravenous injection of bacterial wall lipopolysaccharide expressed dense labeling for the immediate-early gene product FOS in parts of the parabrachial nucleus that also demonstrated dense preproenkephalin expression, only a small proportion of the enkephalin-positive neurons were FOS-positive. These data indicate that enkephalins, although implicated in a variety of autonomic responses, are not primarily involved in the transmission of immune-related information from the parabrachial nucleus to its different forebrain and brain stem targets.

Topics: Animals; Dose-Response Relationship, Drug; Enkephalins; Immune System; Immunohistochemistry; Inflammation; Lipopolysaccharides; Male; Neurons; Pons; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Visceral Afferents

Both c-Fos and prodynorphin mRNA and peptide increase unilaterally in nociceptive-specific neurons in the lumbar rat spinal cord during chronic hindpaw inflammation. To study the mechanisms underlying prodynorphin gene expression, we examined transcription factors and their interactions at the CRE/AP-1-like site, DYNCRE3, found in the prodynorphin gene promoter. CREB repressed while c-Fos and c-Jun activated transcription through the DYNCRE3 site in transient co-transfections in PC12 cells. Following inflammation of the rat hindpaw, immunostaining demonstrated a bilateral increase in phosphorylated CREB (P-CREB)-positive neurons in the spinal cord. Gel supershift studies showed that spinal cord extracts contained CREB, P-CREB, and phosphorylated c-Jun (P-c-Jun) proteins that bound to the DYNCRE3 site. We propose a model in which inflammation-induced phosphorylation of CREB relieves CREB repression at the DYNCRE3 site, P-CREB binds to the c-Fos promoter, and Fos/Fra, P-CREB, and P-c-Jun interact at the DYNCRE3 site to activate prodynorphin gene transcription.

Topics: Animals; Binding Sites; Cell Nucleus; Cyclic AMP Response Element-Binding Protein; Enkephalins; Gene Expression Regulation; Hindlimb; Inflammation; Male; Models, Neurological; Neurons; PC12 Cells; Phosphorylation; Promoter Regions, Genetic; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Spinal Cord; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transfection

The expression of preproenkephalin (PPE) mRNA has previously been shown to be regulated by steroid hormones in the ventromedial nucleus of the hypothalamus (VMH) and to be regulated by noxious stimuli in the dorsal horn of the spinal cord (DH). The present in situ hybridization study in ovariectomized rats showed that PPE mRNA expression in both the VMH and the lumbar DH, responds to the interaction between a noxious peripheral stimulus and ovarian steroid hormones. In the VMH, either estradiol or estradiol + progesterone increased the mean PPE mRNA content per cell by 100% compared with vehicle-treated rats. Unilateral hindpaw injection of 5% formalin, as compared to saline, significantly increased mean PPE mRNA content per VMH cell in rats treated with vehicle or estradiol but not those treated with estradiol + progesterone. Regression analysis for mean PPE mRNA content per VMH cell as a function of intensity of hindpaw inflammation showed a significant positive correlation coefficient after vehicle and estradiol treatment (P < 0.02) but a strong trend towards a negative correlation coefficient after estradiol + progesterone treatment (P < 0.06). ANOVA for homogeneity of regression coefficients showed a significant difference across hormone groups (P < 0.01). In the lumbar DH, mean PPE mRNA content per cell was greater in rats injected with formalin than with saline and was greatest in rats given steroids + formalin. Mean PPE mRNA content per DH cell was greater ipsilateral than contralateral to the formalin injection in estradiol-treated rats, but no laterality difference was seen in the other hormone groups. No significant differences in mean PPE mRNA levels per DH cell were found among the rats treated with saline + hormone, saline + vehicle, formalin + vehicle, or uninjected rats. For all hormone groups combined, mean PPE mRNA per DH cell showed a significant positive regression on intensity of hindpaw inflammation (P < 0.05). Taken together these data are consistent with reports of increased pain threshold during pregnancy, descending control of antinociception from the basomedial hypothalamus and positive correlations between VMH levels of PPE mRNA and lordosis, a behavior evoked by somatosensory stimulation below nociceptive threshold.

Topics: Animals; Enkephalins; Female; Hindlimb; In Situ Hybridization; Inflammation; Nociceptors; Ovariectomy; Pain; Protein Precursors; Rats; RNA, Messenger; Spinal Cord; Steroids; Ventromedial Hypothalamic Nucleus

An animal model of nociception involving unilateral hindpaw inflammation has been used to examine behavioral, molecular, and biochemical aspects of well-characterized spinal cord neural circuits involved in pain transmission. The neurotoxin capsaicin administered neonatally was used to modify this neuronal system by producing a selective destruction of most small, unmyelinated primary afferent axons. Capsaicin had minimal effects on the behavioral hyperalgesia and edema associated with the hindpaw inflammation and on the constitutive expression of preprodynorphin (PPD) mRNA and preproenkephalin mRNA in the spinal cord. However, the inflammation-induced increases in Fos-like immunoreactivity (Fos-LI) and in PPD mRNA were greatly attenuated by neonatal capsaicin treatment. The data indicate that input from small-diameter unmyelinated primary afferents is important for the stimulus-induced increase in Fos-LI and PPD mRNA. Our finding that neonatal capsaicin reduces the levels of Fos-LI and PPD mRNA in a related fashion in the spinal dorsal horn provides further evidence for a relationship between the protein product of the c-fos protooncogene and regulation of dynorphin gene transcription.

Topics: Animals; Animals, Newborn; Capsaicin; Dynorphins; Enkephalins; Female; Foot Diseases; Gene Expression; Hyperalgesia; Inflammation; Male; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; Spinal Cord; Substance P

Increased levels of preproenkephalin mRNA in spinal cord neurons induced by peripheral tissue inflammation were examined using in situ hybridization histochemistry. In addition, in situ hybridization histochemistry was combined with immunocytochemistry to determine whether increases in preproenkephalin mRNA were co-localized in spinal cord neurons with increases in immunoreactivity for Fos and Fos-related proteins coded by the immediate-early proto-oncogene, c-fos, and related genes. Dorsal horn laminae I-II, V-VI and VII showed a greater than 200% increase in preproenkephalin mRNA-labeled neurons on the inflamed side as compared to the contralateral control. Inflammation also induced Fos-like immunoreactivity in cell nuclei, mainly in the superficial laminae I-II and the neck of the dorsal horn (laminae V-VI). Few labeled nuclei were detected on the contralateral side. Inflammation resulted in double-labeling of neurons ipsilateral to the inflamed limb whereas they were almost completely absent on the contralateral side. Double-labeled neurons were most frequently found in laminae V-VI. Double-labeled laminae I-II neurons were concentrated in the medial two-thirds of the dorsal horn, the site that receives innervation from the inflamed limb. There were also many double-labeled neurons in laminae VII. Over 90%, 82% and 69% of all neurons expressing preproenkephalin mRNA co-localized Fos immunoreactivity in laminae V-VI, I-II, and VII, respectively. However, the number of neurons expressing increased Fos immunoreactivity was substantially greater than the subpopulation of double-labeled neurons. Our findings indicated that peripheral inflammation induces an increase in preproenkephalin mRNA levels in spinal cord neurons and that most neurons exhibiting preproenkephalin mRNA labeling also co-localized Fos and Fos-related immunoreactivity. These data are consistent with evidence supporting the role of Fos and Fos-related proteins in the regulation of transcription of the preproenkephalin gene in spinal neurons.

Topics: Animals; Cell Nucleus; Enkephalins; Gene Expression Regulation; Genes, fos; Inflammation; Male; Neurons; Nucleic Acid Hybridization; Oncogene Proteins v-fos; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord

Peripheral inflammation produces a rapid elevation (within 4 h) in preprodynorphin mRNA in neurons of the dorsal spinal cord and an even more rapid elevation in c-fos proto-oncogene mRNA (within 30 min). During this period a relatively modest increase is also observed in spinal cord preproenkephalin mRNA. Previous anatomical studies have shown that the neurons in which these transcripts increase have overlapping distributions. Assuming that these events occur in the same cells, it suggests the possibility that newly synthesized c-fos protein may participate in transcriptional regulation of opioid genes in spinal cord.

Topics: Animals; Dynorphins; Enkephalins; Gene Expression Regulation; Hyperalgesia; Inflammation; Male; Protein Precursors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord