dynorphins and Inflammation

A rat model has been developed to characterize the responses of brainstem trigeminal neurons to orofacial deep and cutaneous tissue inflammation and hyperalgesia. Complete Freund's adjuvant (CFA) was injected unilaterally into the rat temporomandibular joint (TMJ) or perioral (PO) skin to produce inflammation in deep or cutaneous tissues, respectively. The TMJ and PO inflammation resulted in orofacial behavioral hyperalgesia and allodynia that peaked within 4-24 h and persisted for at least 2 weeks. Compared to cutaneous CFA injection, the injection of CFA into the TMJ produced a significantly stronger inflammation associated with a selective upregulation of preprodynorphin mRNA in the trigeminal spinal complex, an enhanced medullary dorsal horn hyperexcitability, and a greater trigeminal Fos protein expression, a marker of neuronal activation. The Fos-LI induced by TMJ inflammation persisted longer, was more intense, particularly in the superficial laminae, and more widespread rostrocaudally. Thus, the inflammatory irritant produces a stronger effect in deep than in cutaneous orofacial tissue. As there is heavy innervation of the TMJ by unmyelinated nerve endings, a strong nociceptive primary afferent barrage is expected following inflammation. An increase in TMJ C-fiber input after inflammation and strong central neuronal activation may initiate central hyperexcitability and contribute to persistent pain associated with temporomandibular disorders. Since deep inputs may be more effective in inducing central neuronal excitation than cutaneous inputs, greater sensory disturbances may occur in pain conditions involving deep tissues than in those involving cutaneous tissues.

Topics: Animals; Behavior, Animal; Dynorphins; Facial Pain; Inflammation; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; RNA, Messenger; Skin; Temporomandibular Joint Disorders; Trigeminal Nerve; Up-Regulation

Negative affective states affect quality of life for patients suffering from pain. These maladaptive emotional states can lead to involuntary opioid overdose and many neuropsychiatric comorbidities. Uncovering the mechanisms responsible for pain-induced negative affect is critical in addressing these comorbid outcomes. The nucleus accumbens (NAc) shell, which integrates the aversive and rewarding valence of stimuli, exhibits plastic adaptations in the presence of pain. In discrete regions of the NAc, activation of the kappa opioid receptor (KOR) decreases the reinforcing properties of rewards and induces aversive behaviors. Using complementary techniques, we report that in vivo recruitment of NAc shell dynorphin neurons, acting through KOR, is necessary and sufficient to drive pain-induced negative affect. Taken together, our results provide evidence that pain-induced adaptations in the kappa opioid system within the NAc shell represent a functional target for therapeutic intervention that could circumvent pain-induced affective disorders. VIDEO ABSTRACT.

Topics: Affect; Animals; Dynorphins; Inflammation; Mice; Mood Disorders; Neural Inhibition; Neuronal Plasticity; Neurons; Nucleus Accumbens; Pain; Rats; Receptors, Opioid, kappa

Dynorphin 1-17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1-17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, μ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1-17 and fragments (1-6, 1-7 and 1-9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1-7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund's Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1-7, 1-17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1-7 and 1-9 displayed the greatest activity across all receptor subtypes, while dynorphin 1-7, 1-9 and 1-17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1-7 and U50488H, but not dynorphin 1-17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1-7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1-7 mediates analgesia.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesia; Animals; Disease Models, Animal; Dynorphins; HEK293 Cells; Humans; Inflammation; Pain; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Transfection

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™

Topics: Animals; Cell Line; Dynorphins; Humans; Immunomodulation; Inflammation; Interleukin-1beta; Lipopolysaccharides; NF-kappa B; Protein Transport; Signal Transduction; Toll-Like Receptor 4; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Processed Chuanwu (PCW), the mother root of Aconitum carmichaelii Debeauxv, has been widely used as a classic Traditional Chinese Medicine for pain relieve for over two millennia clinically. However, its action on chronic inflammatory pain has not been clarified. Here, we investigated the antinociceptive effect of PCW in complete freund's adjuvant (CFA)-induced mice and its possible mechanisms associated with opioid system and TRPV1 ion channel.. Male ICR mice were intraplantarly injected with CFA. PCW (0.34, 0.68 and 1.35 g/kg) was orally given to mice once a day for 7 days. Von frey hairs and planter test were assessed to evaluate the antinociceptive effect of PCW. To investigate the participation of dynorphin/opioid system in PCW antinociception, subtype-specific opioid receptor antagonists or anti-dynorphin A antiserum were used. To eliminate other central mechanisms that contribute to PCW antinociception, hot plate (50 °C) test were performed. Further, involvements of TRPV1 in PCW antinociception were evaluated in CFA-induced TRPV1(-/-) and TRPV1(+/+) C57BL/6 male mice, and in capsaicin-induced nociception ICR naive mice pretreated with nor-BNI. Meanwhile, calcium imaging was performed in HEK293T-TRPV1 cells. Finally, rotarod, open-field tests and body temperature measurement were carried out to assess side effects of PCW.. PCW dose-dependently attenuated mechanical and heat hypersensitivities with no tolerance, which could be partially attenuated by coadministration of k-opioid receptor antagonist nor-binaltorphimine (nor-BNI) or anti-dynorphin A (1-13) antiserum. And PCW antinociception was totally erased by pretreatment with nor-BNI in the hot plate test. In addition, PCW antinociception was decreased in TRPV1(-/-) mice compared to TRPV1(+/+) group. And PCW still manifested inhibitory effects in capsaicin-induced nociception with nor-BNI pretreatment. PCW significantly inhibited capsaicin-induced calcium influx in HEK293T-TRPV1 cells. Finally, no detectable side effects were found in naive mice treated with PCW.. This study shows PCW's potent antinociceptive effect in inflammatory conditions without obvious side effects. This effect may result from the activation of κ-opioid receptor via dynorpin release and the inhibition of TRPV1. These findings indicate that PCW might be a potential agent for the management of chronic inflammatory pain.

Topics: Aconitum; Administration, Oral; Analgesics; Analgesics, Opioid; Animals; Body Temperature; Calibration; Cell Survival; Chromatography, High Pressure Liquid; Dynorphins; Freund's Adjuvant; HEK293 Cells; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Plant Extracts; Plant Roots; Receptors, Opioid, kappa; TRPV Cation Channels

Microglial activation worsens neuronal loss and contributes to progressive neurological diseases like Parkinson's disease (PD). This inflammatory progression is countered by dynorphin (Dyn), the endogenous ligand of the kappa-opioid receptor (KOR). We show that microglial β-arrestin mediates the ability of Dyn/KOR to limit endotoxin-elicited production of pro-inflammatory effectors and cytokines, subsequently protecting neurons from inflammation-induced neurotoxicity. Agonist-activated KOR enhances the interaction of β-arrestin2 with transforming growth factor-beta-activated kinase 1 (TAK1)-binding protein 1 (TAB1), disrupting TAK1-TAB1 mediated pro-inflammatory gene expression. We reveal a new physiological role for β-arrestin in neuroprotection via receptor internalization-triggered blockade of signal effectors of microglial inflammatory neurotoxicity. This result offers novel drug targets in the convergent KOR/β-arrestin2 and inflammatory pathways for treating microglial inflammatory neuropathologies like PD.

Topics: Adaptor Proteins, Signal Transducing; Animals; Arrestins; beta-Arrestins; Cell Death; Cytokines; Dynorphins; Female; Inflammation; Male; MAP Kinase Kinase Kinases; Mice; Mice, Inbred C57BL; Microglia; Neurons; Opioid Peptides; Parkinson Disease; Pregnancy; Receptors, Opioid, kappa

Interleukin-17 (IL-17) is involved in a wide range of inflammatory disorders and in recruitment of inflammatory cells to injury sites. A recent study of IL-17 knock-out mice revealed that IL-17 contributes to neuroinflammation and neuropathic pain after peripheral nerve injury. Surprisingly, little is known of micro-environment modulation by IL-17 in injured sites and in pathologically related neuroinflammation and chronic neuropathic pain. Therefore, we investigated nociceptive sensitization, immune cell infiltration, myeloperoxidase (MPO) activity, and expression of multiple cytokines and opioid peptides in damaged nerves of wild-type (IL-17(+/+)) and IL-17 knock-out (IL-17(-/-)) mice after partial sciatic nerve ligation. Our results demonstrated that the IL-17(-/-) mice had less behavioral hypersensitivity after partial sciatic nerve ligation, and inflammatory cell infiltration and pro-inflammatory cytokine (tumor necrosis factor-α, IL-6, and interferon-γ) levels in damaged nerves were significantly decreased, with the levels of anti-inflammatory cytokines IL-10 and IL-13, and expressions of enkephalin, β-endorphin, and dynorphin were also decreased compared to those in wild-type control mice. In conclusion, we provided evidence that IL-17 modulates the micro-environment at the level of the peripheral injured nerve site and regulates progression of behavioral hypersensitivity in a murine chronic neuropathic pain model. The attenuated behavioral hypersensitivity in IL-17(-/-) mice could be a result of decreased inflammatory cell infiltration to the injured site, resulting in modulation of the pro- and anti-inflammatory cytokine milieu within the injured nerve. Therefore, IL-17 may be a critical component for neuropathic pain pathogenesis and a novel target for therapeutic intervention for this and other chronic pain states.

Topics: Animals; Behavior, Animal; beta-Endorphin; Central Nervous System Sensitization; Cytokines; Disease Models, Animal; Dynorphins; Enkephalins; Hyperalgesia; Inflammation; Interleukin-10; Interleukin-13; Interleukin-17; Interleukin-1beta; Interleukin-2; Interleukin-6; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Neutrophils; Nociception; Peripheral Nerve Injuries; Peroxidase; Sciatic Nerve; T-Lymphocytes; Tumor Necrosis Factor-alpha

The striato-nigral projecting pathway contains the highest concentrations of dynorphin in the brain. The functional role of this opioid peptide in the regulation of mesencephalic dopaminergic (DAergic) neurons is not clear. We reported previously that exogenous dynorphin exerts potent neuroprotective effects against inflammation-induced dopaminergic neurodegeneration in vitro. The present study was performed to investigate whether endogenous dynorphin has neuroprotective roles in vivo.. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (MA), two commonly used neurotoxins in rodent models of Parkinson's disease, were administered to wild-type (Dyn⁺/⁺) and prodynorphin-deficient mice (Dyn⁻/⁻). We examined dopaminergic neurotoxicity by using an automated video tracking system, HPLC, immunocytochemistry, and reverse transcription and polymerase chain reaction (RT-PCR).. Treatment with MPTP resulted in behavioral impairments in both strains. However, these impairments were more pronounced in Dyn-l- than in Dyn⁺/⁺. Dyn⁻/⁻ showed more severe MPTP-induced dopaminergic neuronal loss in the substantia nigra and striatum than Dyn⁺/⁺. Similarly, the levels of dopamine and its metabolites in the striatum were depleted to a greater extent in Dyn⁻/⁻ than in Dyn⁺/⁺. Additional mechanistic studies revealed that MPTP treatment caused a higher degree of microglial activation and M1 phenotype differentiation in Dyn⁻/⁻ than in Dyn⁺/⁺. Consistent with these observations, prodynorphin deficiency also exacerbated neurotoxic effects induced by MA, although this effect was less pronounced than that of MPTP.. The in vivo results presented here extend our previous in vitro findings and further indicate that endogenous dynorphin plays a critical role in protecting dopaminergic neurons through its anti-inflammatory effects.

Topics: Animals; Corpus Striatum; Dopaminergic Neurons; Dynorphins; Inflammation; Methamphetamine; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Motor Skills Disorders; MPTP Poisoning; Neurotoxins; Substantia Nigra

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective μ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 μM)/thiorphan (1-100 μM) combinations or by P8B (1-100 μM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

Topics: Alanine; Amino Acid Sequence; Animals; Antibodies; CD13 Antigens; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enzyme Inhibitors; Flow Cytometry; Hindlimb; Inflammation; Leucine; Leukocytes; Male; Narcotic Antagonists; Neprilysin; Neurons; Opioid Peptides; Pain; Pain Threshold; Phosphinic Acids; Rats; Rats, Wistar; Receptors, Opioid; Thiorphan

Dynorphin A 1-17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Topics: Animals; Chromatography, Liquid; Dynorphins; Hindlimb; Inflammation; Rats; Serum; Swine; Tandem Mass Spectrometry; Trypsin

Postoperative pain management is a critical aspect of patient care. The inflammatory state of the post-sternotomy surgical wound sensitizes nerve endings, causing pain. Unrelieved or improperly managed pain compromises wound healing. Peripheral opioid receptors play a major role in analgesia, particularly under inflammatory conditions where both opioid receptor expression and efficacy are increased. Leukocytic opioid peptides include β-endorphin (END), met-enkephalin (ENK), and dynorphin-A (DYN), with END and ENK being predominant.. This work represents the first study of inflammatory cells collected from post-sternotomy wounds of patients undergoing cardiac surgery including coronary artery bypass grafting (CABG). Wound fluid (WF) and cells were collected from sternal wounds using a JP Blake drain at 24, 48, and 72 hours post sternum closure. Anti-CD15 staining and flow cytometry revealed that polymorphonuclear neutrophils (PMN) are the predominant cells present in wound fluid collected post-surgery. Compared to peripheral blood (PB) derived PMN, significant increases in CD177+/CD66b+ PMN were observed suggesting activation of wound-site PMN. Such activation was associated with higher levels of opioid peptide expression in PMN derived from WF. Indeed, increased level of opioid peptides in sternal wound environment was noted 72 h post-surgery. We demonstrate that WF contains factors that can significantly induce POMC transcription in human PMNs. IL-10 and IL-4 were abundant in WF and both cytokines significantly induced POMC gene expression suggesting that WF factors such as IL-10 and IL-4 contribute towards increased opioid peptide expression in wound-site PMN.. This approach provided a unique opportunity to study the cross-talk between inflammation and opioid peptides in PMN at a sternotomy wound-site. Wound-site PMN exhibited induction of END and ENK. In addition, sternal wound fluid significantly induced END expression in PMN. Taken together, these data constitute first clinical evidence that human wound-site PMNs are direct contributors of opioids at the sternal wound-site.

Topics: Aged; beta-Endorphin; Dynorphins; Enkephalin, Methionine; Female; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation; Interleukin-10; Interleukin-4; Male; Middle Aged; Neutrophils; Pain Management; Postoperative Period; Sternotomy; Wound Healing

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

An upregulation of the endogenous opioid, dynorphin A, in the spinal cord is seen in multiple experimental models of chronic pain. Recent findings implicate a direct excitatory action of dynorphin A at bradykinin receptors to promote hyperalgesia in nerve injured rats, and its upregulation may promote, rather than counteract, enhanced nociceptive input due to injury. Here we examined a model of inflammatory pain by unilateral injection of complete Freund's adjuvant (CFA) into the rat hind paw. Rats exhibited tactile hypersensitivity and thermal hyperalgesia in the inflamed paw by 6 hours after CFA injection, whereas a significant elevation of prodynorphin transcripts in the lumbar spinal cord was seen at day 3 but not at 6 hours. Thermal hyperalgesia at day 3, but not at 6 hours, after CFA injection was blocked by intrathecal administration of anti-dynorphin antiserum or by bradykinin receptor antagonists. The antihyperalgesic effect of the latter was not due to de novo production of bradykinin or upregulation of spinal bradykinin receptors. These data suggest that elevated spinal dynorphin on peripheral inflammation mediates chronic inflammatory hyperalgesia. The antihyperalgesic effect of bradykinin receptor antagonists requires the presence of upregulated spinal dynorphin but not of de novo production of bradykinin, supporting our hypothesis that pathological levels of dynorphin may activate spinal bradykinin receptors to mediate inflammatory hyperalgesia.. This study shows that chronic peripheral inflammation induces a significant upregulation of the endogenous opioid peptide dynorphin. Elevated levels of spinal dynorphin and activation of spinal bradykinin receptors are essential to maintain inflammatory hyperalgesia. The results suggest that blockade of spinal bradykinin receptors may have therapeutic potential in chronic inflammatory pain.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Bradykinin; Dynorphins; Freund's Adjuvant; Hyperalgesia; Inflammation; Injections, Spinal; Kallidin; Kininogens; Male; Pain; Pain Measurement; Pain Threshold; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Bradykinin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Tritium; Up-Regulation

Our previous results indicated that dynorphin in the spinal dorsal horn mediates the analgesic effect of high frequency electroacupuncture stimulation (EAS). Here we report that the transcriptional repressor downstream regulatory element antagonist modulator (DREAM) of dynorphin precursor-preprodynorphin (PPD) may participate in this process. In normal rats, 100 Hz, but not 2 Hz EAS triggered the nuclear export and membrane translocation of DREAM concomitantly with the upregulation of PPD mRNA in the dorsal horn. In inflammatory rats, both 2 and 100 Hz EAS alleviated thermal and mechanical hypersensitivity and caused the nuclear export and membrane translocation of DREAM, but only 100 Hz EAS enhanced the mRNA level of PPD and DREAM. These results suggest the role of DREAM in the dorsal horn in the regulation of PPD gene expression by EAS is frequency dependent, and DREAM may exert different roles in different frequency EAS under physiological and inflammatory conditions.

Topics: Animals; Dynorphins; Electroacupuncture; Freund's Adjuvant; Inflammation; Kv Channel-Interacting Proteins; Male; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; Repressor Proteins; Up-Regulation

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

Topics: Animals; beta-Endorphin; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Freund's Adjuvant; Hyperalgesia; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Protein Precursors; Reaction Time; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Somatostatin

Recent clinical studies have demonstrated that when morphine is used to control pain in cancer patients, psychological dependence is not a major concern. The present study was undertaken to ascertain the modulation of psychological dependence on morphine under a chronic pain-like state in rats. The prototypical mu-opioid receptor agonist morphine (8 mg/kg, i.p.) induced a dose-dependent place preference. In the present study, we found that an inflammatory pain-like state following formalin injection significantly suppressed the morphine-induced rewarding effect. This effect was almost reversed by s.c. pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI, 5 mg/kg). Furthermore, the morphine-induced increase in dopamine (DA) turnover in the limbic forebrain was significantly inhibited by treatment with formalin. This inhibition was also suppressed by pretreatment with nor-BNI. In addition, in vivo microdialysis studies clearly showed that the morphine-induced increase in the extracellular levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the nucleus accumbens (N.Acc.) was significantly decreased in rats that had been pretreated with formalin. This effect was in turn reversed by the microinjection of a specific dynorphin A antibody into the N.Acc. These findings suggest that the inflammatory pain-like state induced by formalin injection may have caused a sustained activation of the kappa-opioidergic system within the N.Acc., resulting in suppression of the morphine-induced rewarding effect in rats. The present study provides further evidence of the clinical usefulness of morphine in patients suffering from severe pain.

Topics: Animals; Brain Chemistry; Conditioning, Operant; Dopamine; Dynorphins; Edema; Foot; Formaldehyde; Inflammation; Limbic System; Male; Microdialysis; Microinjections; Morphine; Naltrexone; Narcotic Antagonists; Narcotics; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reward

Spinal nociceptive neural circuits undergo considerable changes during the postnatal period. This study showed that neonatal rats exhibited earlier upregulation and faster recovery of spinal preprodynorphin (PPD) mRNA than did the adults during complete Freund's adjuvant (CFA)-induced peripheral inflammation. These data suggest that the central nervous systems of neonates and adults respond differently to peripheral noxious inputs, a fact that should be considered when selecting pain treatment strategies for neonate populations.

Topics: Aging; Animals; Animals, Newborn; Behavior, Animal; Blotting, Northern; Dynorphins; Electrophoresis, Polyacrylamide Gel; Inflammation; Protein Precursors; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Up-Regulation

The aging process is associated with various morphological and biochemical changes in the nervous system that may affect the processing of noxious inputs. This study showed greater hyperalgesia and up-regulation of spinal dynorphin (DYN) expression in aging than in young adult rats during CFA-induced peripheral inflammation. These data indicate that nociception is regulated differently in aging individuals, a fact that should be considered when selecting treatment strategies for aging populations with persistent pain.

Topics: Aging; Animals; Chronic Disease; Disease Models, Animal; Dynorphins; Freund's Adjuvant; Functional Laterality; Hyperalgesia; Immunohistochemistry; Inflammation; Male; Peripheral Nervous System Diseases; Physical Stimulation; Posterior Horn Cells; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

Glucocorticoids (GC) play important roles in response to stressful stimuli, including pain. This study examined the effects of bilateral adrenalectomy (ADX) and dexamethasone (DEX) replacement on the hyperalgesia and spinal preprodynorphin (PPD) mRNA expression induced by injecting complete Freund's adjuvant (CFA) into rats' hind paws. The results demonstrated that CFA induced more intense hyperalgesia and up-expression of spinal PPD mRNA in ADX rats than in control rats, while both of these intensified reactions could be significantly suppressed by subcutaneous pretreatment with DEX. This leads to the conclusion that both exogenous (pharmacological) and endogenous (physiological) GC suppresses the behavioral hyperalgesia and the up-regulation of spinal PPD mRNA induced by sustained peripheral inflammation. The results also suggest that spinal PPD mRNA suppression may partially underlie the inhibition of behavioral hyperalgesia.

Topics: Animals; Dynorphins; Freund's Adjuvant; Glucocorticoids; Hyperalgesia; Inflammation; Male; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Up-Regulation

Previous studies suggest that cholecystokinin (CCK) is implicated in the modulation of pain sensitivity and the development of neuropathic pain. We used CCK(2) receptor deficient (CCK(2) (-/-)) mice and assessed their mechanical sensitivity using Von Frey filaments, as well as the development and time course of mechanical hyperalgesia in a model of neuropathic pain. We found that CCK(2) (-/-) mice displayed mechanical hyposensitivity, which was reversed to the level of wild-type animals after administration of naloxone (0.1-10 mg/kg). On the other hand, injection of L-365260 (0.01-1 mg/kg), an antagonist of CCK(2) receptors, decreased dose-dependently, mechanical sensitivity in wild-type mice. The mechanism of reduced mechanical sensitivity in CCK(2) (-/-) mice may be explained by changes in interactions between CCK and opioid systems. Indeed, CCK(2) (-/-) mice natively expressed higher levels of lumbar CCK(1), opioid delta and kappa receptors. Next, we found that CCK(2) (-/-) mice did not develop mechanical hyperalgesia in the Bennett's neuropathic pain model. Induction of neuropathy resulted in decrease of lumbar pro-opiomelanocortin (POMC) gene expression in wild-type mice, but increase of POMC expression in CCK(2) (-/-) mice. In addition, induction of neuropathy resulted in further increase of opioid delta receptor in CCK(2) (-/-) mice. Gene expression results indicate up-regulation of opioid system in CCK(2) (-/-) mice, which apparently result in decreased neuropathy score. Our study suggests that not only pain sensitivity, but also mechanical sensitivity and the development of neuropathic pain are regulated by antagonistic interactions between CCK and opioid systems.

Topics: Animals; Benzodiazepinones; Disease Models, Animal; Dose-Response Relationship, Drug; Dynorphins; Enkephalins; Gene Expression; Hyperalgesia; Inflammation; Ligation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naloxone; Narcotic Antagonists; Pain Measurement; Pain Threshold; Phenylurea Compounds; Pro-Opiomelanocortin; Protein Precursors; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Receptors, Opioid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sciatic Neuropathy; Time Factors

Neuroimmune interactions control pain through activation of opioid receptors on sensory nerves by immune-derived opioid peptides. Here we evaluate mechanisms of intrinsic pain inhibition at different stages of Freund's adjuvant-induced inflammation of the rat paw. We use immunohistochemistry and paw pressure testing. Our data show that in early (6 h) inflammation leukocyte-derived beta-endorphin, met-enkephalin and dynorphin A activate peripheral mu-, delta- and kappa-receptors to inhibit nociception. In addition, central opioid mechanisms seem to contribute significantly to this effect. At later stages (4 days), antinociception is exclusively produced by leukocyte-derived beta-endorphin acting at peripheral mu and delta receptors. Corticotropin-releasing hormone (CRH) is an endogenous trigger of these effects at both stages. These findings indicate that peripheral opioid mechanisms of pain inhibition gain functional relevance with the chronicity of inflammation.

Topics: Animals; Corticotropin-Releasing Hormone; Dynorphins; Edema; Endorphins; Enkephalin, Methionine; Freund's Adjuvant; Hindlimb; Inflammation; Injections, Subcutaneous; Leukocytes; Male; Naloxone; Pain; Pain Threshold; Rats; Rats, Wistar; Stress, Physiological; Time Factors

We previously reported that partial sciatic nerve ligation (PSNL) dramatically up-regulates cyclooxygenase 2 (COX2) in injured sciatic nerve, and local injection of the COX inhibitor, ketorolac, reverses tactile allodynia and suppresses increased phosphorylation of the transcription factor cAMP responsive element binding protein [Eur J Neurosci 15 (2002) 1037]. These findings suggest that peripheral prostaglandins (PGs) are over-produced and contribute to the central plasticity and the maintenance of neuropathic pain after nerve injury. PGs, particularly PGE2, are well known to facilitate the release of the pro-nociceptive neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) from primary sensory afferents. Thus, suppressing peripheral PG over-production may inhibit the release of these two neuropeptides from primary afferents and thereby increase the content of these neuropeptides remaining in afferent terminals in the dorsal horn. In this study we tested this hypothesis by examining the immunoreactivities of SP and CGRP in the dorsal horn of PSNL rats intraplantarly injected with saline and ketorolac. Four weeks after PSNL, SP- and CGRP-immunoreactivities (IR) in the ipsilateral dorsal horn were not significantly different from the contralateral side. Five days following intraplantar injection of ketorolac, CGRP- and SP-IR in the ipsilateral and contralateral dorsal horn were dramatically reduced compared with saline-injected PSNL rats. Local ketorolac also suppressed PSNL-induced increase in dynorphin-IR in dorsal horn neurons. Since abundant production of PGs during inflammation is well documented, we further examined the effect of intraplantar ketorolac on neuropeptide expression in the dorsal horn following carrageenan inflammation. We observed that co-administration of ketorolac with carrageenan in the hindpaw also reduced SP- and dynorphin-IR in the ipsilateral and contralateral dorsal horn. These findings are in contrast to our hypothesis, suggesting that peripherally over-produced PGs following nerve injury and inflammation possibly contribute to the production of SP and CGRP in primary sensory neurons, to the up-regulation of dynorphin in the dorsal horn neurons, and finally to the mechanisms of neuropathic and inflammation pain.

Topics: Animals; Behavior, Animal; Calcitonin Gene-Related Peptide; Carrageenan; Cell Count; Cyclooxygenase Inhibitors; Drug Interactions; Dynorphins; Functional Laterality; Hyperalgesia; Immunohistochemistry; Inflammation; Injections, Spinal; Ketorolac; Male; Pain Measurement; Physical Stimulation; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Spinal Cord; Substance P; Time Factors

The experience of pain is highly variable among individuals, which may be due in part to the effects of genetic factors on the central transmission and modulation of noxious inputs. This study examined behavioral responses and the expression of preprodynorphin (PPD) mRNA at the spinal level during complete Freund's adjuvant-induced inflammation of the unilateral hind paw in male Fischer 344 (F344), Sprague-Dawley (SD), and Lewis (LEW) rats. Experiments showed that F344 rats exhibited stronger hind paw hyperalgesia and greater spinal PPD mRNA induction than SD or LEW rats. These results indicate that genetic factors that determine the spinal PPD mRNA and dynorphin production underlie strain-dependent differences in pain perception.

Topics: Animals; Behavior, Animal; Blotting, Northern; Dynorphins; Freund's Adjuvant; Inflammation; Male; Pain Threshold; Peripheral Nervous System Diseases; Protein Precursors; Rats; Rats, Inbred F344; Rats, Inbred Lew; Rats, Sprague-Dawley; RNA, Messenger; Species Specificity; Spinal Cord; Time Factors

The influence of chronic arthritic pain on two endogenous opioid peptides, dynorphin B and [Met5]enkephalin-Arg6-Phe7, and multiple opioid receptors in discrete brain, lumbar spinal cord and pituitary pools was investigated. Using radioimmunoassay and receptor binding assay, we examined the changes in regional opioid peptide levels and opioid receptor activity due to chronic inflammation in adjuvant arthritic rats. At 4 weeks post-inoculation, increased levels of immunoreactive dynorphin B and [Met5]enkephalin-Arg6-Phe7 were measured in tissues of arthritic rats compared with controls. No significant changes in mu-, delta- or kappa-opioid receptors were seen after chronic inflammation. Taken together, these results indicate that in chronic arthritis, opioid receptor changes do not follow the peptide alterations of pro-dynorphin and pro-enkephalin systems. Thus, dynamic modification and modulation of nociceptive information takes place during chronic inflammation. This supports the key role of the central nervous system in chronic inflammatory pain conditions.

Topics: Animals; Brain; Chronic Disease; Disease Models, Animal; Dynorphins; Endorphins; Enkephalin, Methionine; Female; Inflammation; Opioid Peptides; Pain; Pain Measurement; Rats; Rats, Inbred Lew; Receptors, Opioid; Spinal Cord

We have previously shown that beta-endorphin (END) is contained and released from memory-type T-cells within inflamed tissue and that it is capable to control pain (J Clin Invest 100(1) (1997) 142). Methionine-enkephalin (MET) and Dynorphin-A (DYN) are endogenous opioids with preference for delta- and kappa-opioid receptors, respectively. Both MET and DYN are produced and contained within immune cells. The goal of this study was to determine the release characteristics of MET and DYN in a rat model of localized hindpaw inflammation and to examine the antinociceptive role of MET and DYN in a Freund's adjuvant induced model of inflammatory pain. We found that corticotropin-releasing factor (CRF) can stimulate the release of both MET and DYN from lymphocytes. This release is dose-dependent and reversible by the selective CRF antagonist alpha-helical-CRF. Furthermore, CRF (1.5 ng) produces analgesia when injected into the inflamed paw, which is reversible by direct co-administration of antibodies to MET. Lymphocyte content of MET was 7.0+/-1.4 ng/million cells, whilst DYN content was ~30-fold lower. Both END and DYN, but not MET, were released by IL-1. Consistently, IL-1 produced peripheral analgesic effects which were not reversed by antibodies to MET. These results indicate that both MET and DYN play a role in peripheral analgesia but have different characteristics of release. These studies further support a role of the immune system in the control of inflammatory pain. This may be particularly important in patients suffering from compromised immune systems as with cancer and AIDS.

Topics: Animals; Antibodies; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Methionine; Humans; Inflammation; Interleukin-2; Lymphocytes; Male; Pain; Rats; Rats, Wistar

The neuromodulatory interactions of sex steroids with the opioid system may result in sex differences in pain and analgesia. Dynorphin is an endogenous kappa-opioid peptide that is upregulated in an animal model of peripheral inflammation and hyperalgesia and is possibly regulated by circulating levels of sex steroids. The present study compared behavioral responses of male, cycling female, and gonadectomized Sprague-Dawley rats in a model of persistent pain. Cycling female rats were behaviorally tested over a 14-day period, and their estrous cycles were monitored by daily vaginal smears. Thermal hyperalgesia was measured by paw withdrawal latencies taken prior to and 24-72 h after rats received a unilateral hindpaw injection of complete Freund's adjuvant (CFA). Prior to CFA administration, there was no significant difference in paw withdrawal latencies between male rats, cycling female rats, and ovariectomized female rats. Following CFA administration, female rats in proestrus exhibited significantly increased hyperalgesia compared with male rats, ovariectomized female rats, and female rats in other estrous stages (P

Topics: Animals; Autoradiography; Behavior, Animal; Blotting, Northern; Dynorphins; Estrus; Female; Freund's Adjuvant; Hyperalgesia; Inflammation; Male; Neurons; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sex Characteristics; Spinal Cord

The newly identified neuropeptide nociceptin/orphanin FQ (NOC) was measured in different rat brain areas related to the descending anti-nociceptive pathways and compared to two opioid peptides, dynorphin B (DYN B) and Met-enkephalinArgPhe (MEAP). Two experimental models of chronic nociception, one neurogenic and one inflammatory, used in this study, reveal how different pathological conditions may influence these endogenous systems. Nerve injury is induced by ligation of the sciatic nerve and inflammation by a carrageenan injection in the gluteal muscle, 2 weeks prior to decapitation. Selected brain areas were dissected out and frozen. NOC-, DYN B- and MEAP-like immunoreactivity (LI) is determined by radioimmunoassay. Nerve injury increased the NOC-LI levels in the cortex cinguli, DYN B-LI levels in the dorsal and the ventral part of the spinal cord, whereas a decrease in the MEAP-LI levels is seen in the dorsal part of the periaqueductal grey (PAG). After inflammation, the NOC-LI levels increased in cortex cinguli, hypothalamus and in the dorsal spinal cord, whereas DYN B-LI levels increased in the dorsal part of the PAG. A general increase in MEAP-LI levels is found after inflammation in all analyzed brain areas except in hippocampus. In conclusion, increased levels of NOC-LI were found in cortex cinguli in both treatment groups and in hypothalamus and spinal cord following carrageenan treatment. The changes in the NOC-LI concentrations were not parallelled by changes in DYN B-LI and MEAP-LI, suggesting that NOC and opioid peptides elicit different reactions in the systems of nociception/antinociception.

Topics: Animals; Brain; Dynorphins; Efferent Pathways; Endorphins; Enkephalin, Methionine; Inflammation; Male; Nerve Crush; Nociceptin; Nociceptors; Opioid Peptides; Pain; Rats; Rats, Sprague-Dawley

Inflammation of the temporomandibular joint (TMJ) produces an increase in preprodynorphin (PPD) mRNA expression in the spinal trigeminal nucleus (Vsp) and paratrigeminal nucleus (Pa5) of the rat. In this study, we further demonstrated that a portion of the TMJ inflammation-induced PPD mRNA positive neurons in the Vsp and Pa5 projected to the parabrachial nucleus (PB). In inflamed rats, the percentage of trigemino- and paratrigeminoparabrachial neurons with up-regulation of PPD mRNA was significantly increased in the ipsilateral Vsp (5.7+/-1.8%) and Pa5 (22.8+/-7.4%, n = 3) when compared with the contralateral side and with saline-treated controls (p < 0.05). These results suggest that the selective up-regulation of PPD mRNA in the Vsp and Pa5 following TMJ inflammation involves ascending trigeminal nociceptive pathways.

Topics: Animals; Dynorphins; Inflammation; Male; Neural Pathways; Neurons; Pons; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Temporomandibular Joint; Trigeminal Nucleus, Spinal

We have analyzed central Fos immunoreactivity (Fos-IR) brainstems of adult rats after three clinically relevant dental injuries: filled dentin (DF) cavities that cause mild pulp injury and heal within 1-2 weeks; open pulp exposures (PX) that cause gradual pulp loss and subsequent periodontal lesions; and filled pulp exposures (PXF). By 1 week after DF cavities, no Fos-IR remained except for sites such as lateral-ventral periolivary nucleus (LVPO) that had Fos-IR in all rats including controls. PX injury induced (1) a delayed transient expression of Fos at 1-2 weeks at three loci (ipsilateral neurons in dorsomedial nucleus oralis, paratrigeminal nucleus, and trigeminal tract), (2) persistent ipsilateral Fos for at least 4 weeks after injury in dynorphin (Dyn)-rich regions (rostral lateral solitary nucleus, periobex dorsal nucleus caudalis), and (3) late Fos-IR at 2-4 weeks (bilateral superficial cervical dorsal horn, contralateral dorsal nucleus caudalis, contralateral rostral lateral solitary nucleus). Rats with PXF injury were examined at 2 weeks, and they had greater numbers and more extensive rostro-caudal distribution of Fos neurons than the PX group. One week after PX injury, Fos-IR neurons were found in regions with strong Dyn-IR central fibers. Co-expression of Dyn and Fos was found in some unusually large neurons of the ipsilateral rostral lateral solitary nucleus, trigeminal tract, and dorsal nucleus caudalis. Immunocytochemistry for the p75 low affinity neurotrophin receptor (p75NTR) or for calcitonin gene-related peptide (CGRP) showed no consistent change in trigeminal central endings in any Fos-reactive brainstem areas, despite the extensive structural and cytochemical reorganization of the peripheral endings of the dental neurons. The Fos responses of central neurons to tooth injury have some unusual temporal and spatial patterns in adult rats compared to other trigeminal injury models.

Topics: Animals; Brain Stem; Calcitonin Gene-Related Peptide; Dental Pulp Cavity; Dynorphins; Female; Inflammation; Male; Molar; Pilot Projects; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley

Inflammation and hyperalgesia induce a dramatic up-regulation of opioid messenger RNA and peptide levels in nociceptive neurons of the spinal dorsal horn. Descending axons modulate nociceptive transmission at the spinal level during inflammatory pain, and may play a role in the development of persistent pain. The role of descending bulbospinal pathways in opioid-containing nociceptive neurons was examined. Removal of descending inputs to the spinal cord was performed by complete spinal transection at the midthoracic level. Seven days after spinal transection, rats received a unilateral hindpaw injection of complete Freund's adjuvant, a noxious stimulus that produces inflammation and hyperalgesia. Tissues from the L4 and L5 segments of the spinal cord were removed and analysed by northern blotting and immunocytochemistry. Spinal transection resulted in a further increase in both dynorphin and enkephalin messenger RNA content following complete Freund's adjuvant injection. There was a similar distribution and number of dynorphin-immunoreactive cells in transected rats compared to rats which received sham surgery. These data suggest that increased dynorphin messenger RNA ipsilateral to inflammation, in rats without descending axons, was due to increased expression within the same cells and not to recruitment of additional dynorphin-expressing cells. This reflects a greater dynamic response of nociceptive neurons to noxious stimuli in the absence of descending modulation. Therefore, the net effect of descending afferents on spinal nociceptive circuits may be to reduce the response of opioid-containing neurons to noxious stimulation from the periphery.

Topics: Animals; Blotting, Northern; Calcitonin Gene-Related Peptide; Colchicine; Denervation; Dynorphins; Enkephalins; Freund's Adjuvant; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Male; Neurons, Afferent; Nociceptors; Protein Precursors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Tachykinins

The effects of i.v. dynorphin A(1-17) and its main nonopioid biotransformation fragment, dynorphin A(2-17), were compared in rhesus monkeys with those of the selective kappa-opioid agonist, U69, 593, in assays of operant behavior, thermal antinociception, and neuroendocrine function (prolactin release). Dynorphin A(1-17) (0. 1-3.2 mg/kg i.v.) and U69,593 (0.001-0.032 mg/kg s.c.) decreased rates of schedule-controlled (fixed ratio 20) food-reinforced responding, whereas dynorphin A(2-17) (1-3.2 mg/kg i.v.) was ineffective. Pretreatment studies with the opioid antagonist quadazocine (0.32 mg/kg s.c.) revealed that the operant effects of dynorphin A(1-17) were not mediated by kappa- or micro-opioid receptors. A different profile was observed in the warm water tail withdrawal assay of thermal antinociception, where both dynorphin A(1-17) and A(2-17) (0.032-3.2 mg/kg i.v., n = 4) were modestly effective in 50 degrees C water, and both were ineffective in 55 degrees C water. By comparison, U69,593 (0.032-0.18 mg/kg s.c.) was maximally effective in 50 degrees C water and partially effective in 55 degrees C. kappa-opioid agonists increase serum levels of prolactin in animals and humans. Dynorphin A(1-17) (ED(50) = 0.0011 mg/kg i.v.), similar to U69,593 (ED(50) = 0.0030 mg/kg i.v.), was very potent in increasing serum prolactin levels in follicular phase female rhesus monkeys, whereas dynorphin A(2-17) (0.32 mg/kg i.v.) was ineffective. The effects of dynorphin A(1-17) and U69,593 on serum prolactin were both antagonized by quadazocine (0.32 mg/kg s.c.) in a surmountable manner, consistent with opioid receptor mediation. The present studies show that serum prolactin levels are a sensitive quantitative endpoint to study the systemic effects of the endogenous opioid peptide, dynorphin A(1-17), in primates.

Topics: Analgesics, Opioid; Animals; Azocines; Behavior, Animal; Conditioning, Operant; Cytokines; Dose-Response Relationship, Drug; Dynorphins; Female; Inflammation; Injections, Intravenous; Macaca mulatta; Male; Narcotic Antagonists; Pain Measurement; Prolactin; Reinforcement Schedule

The effects of N-methyl-D-aspartate(NMDA) receptor antagonist, Mk-801, on the expression of spinal dynorphin (DYN) mRNA and the hyperalgesia induced by peripheral inflammation were studied by Northern analysis and behavioral test. Following an unilateral injection of complete Freund's adjuvant (CFA) into the rat hindpaw, there appeared a significant hyperalgesia of inflamed hindpaw and up-regulation of ipsilateral spinal DYN mRNA; while the pre-emptive and continuous intrathecal administration of Mk-801 (10 microg/microl per h) could significantly suppress both the hyperalgesia and the up-regulation of spinal DYN mRNA induced by peripheral inflammation. The results suggest that NMDA receptor activation may contribute to the development and maintenance of the thermal hyperalgesia that is associated with the up-regulation of DYN expression in spinal dorsal horn.

Topics: Animals; Binding, Competitive; Chronic Disease; Disease Models, Animal; Dizocilpine Maleate; Dynorphins; Freund's Adjuvant; Hindlimb; Hyperalgesia; Inflammation; Infusion Pumps, Implantable; Injections, Spinal; Male; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Spinal Cord; Up-Regulation

Proto-oncogenes of the fos and jun family are rapidly expressed in the central nervous system following various stimuli. Proto-oncogene encoded nuclear proteins such as c-Fos or c-Jun act as transcription factors that may link neuronal excitation to changes in target gene expression. However, the precise in vivo functions of proto-oncogenes in neuroplasticity are still poorly understood. In the present study the effect of systemically administered c-fos antisense oligodeoxynucleotides (ODNs) on c-Fos and dynorphin protein levels in rat L4 spinal cord has been investigated by immunohistochemistry during carrageenan-induced hindpaw inflammation. Continuous infusion of terminal-phosphorothioated c-fos antisense ODNs by subcutaneously implanted miniosmotic pumps for 3 days sequence-specifically suppressed c-Fos protein expression in dorsal horn neurons by about 50%, while the increase in c-Jun immunopositive nuclei was not affected. Digital image analysis revealed a concomitant decrease in spinal dynorphin immunoreactivity. Moreover, 48 hr after carrageenan injection into one hindpaw plasma protein extravasation was observed in numerous blood vessels in the ipsilateral dorsal horn using intravenously administered Evans Blue. Our results provide further evidence that c-Fos may contribute to the regulation of spinal dynorphin gene expression following noxious stimulation. The local increase in blood-spinal cord barrier permeability during sustained peripheral inflammation may permit penetration of hydrophilic antisense ODNs into the central nervous system.

Topics: Animals; Base Sequence; Capillary Permeability; Carrageenan; Dynorphins; Gene Expression Regulation; Genes, fos; Inflammation; Infusions, Parenteral; Mice; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-fos; Rats; Spinal Cord; Thionucleotides

Dynorphin A (DYN) peptides, administered into the central nervous system, have produced inconsistent analgesic actions in tests using thermal stimuli. This study examined antinociceptive effects of intravenous and intraplantar DYN-(2-17) against noxious pressure in rats with Freund's adjuvant-induced unilateral hindpaw inflammation. The effects of DYN-(2-17) were compared to those of the opioid agonists morphine. (D-Ala2,N-Methyl-Phe4,Gly-ol5)-enkephalin (DAMGO) and DYN-(1-17). Intravenous DYN-(2-17) (0.188-10 mg/kg) produced dose-dependent elevations of paw pressure thresholds in inflamed and in non-inflamed paws. These effects were similar in magnitude to those of subcutaneous morphine (2 mg/kg), at doses of 0.375-1.5 mg/kg they were significantly greater on the inflamed (right) than on the non-inflamed (left) paw, and they were not reversible by intravenous naloxone (1-10 mg/kg). Intraplantar Dyn-(2-17)(0.001-0.3 mg) was ineffective, whereas both intraplantar DYN-(1-17)(0.15-0.3 mg) and DAMGO (0.008-0.016 mg) produced dose-dependent and naloxone-reversible elevations of paw pressure thresholds. The intraplantar injection of both Dyn peptides produced a transient increase in the volume of non-inflamed paws. These findings suggest that intravenous DYN-(2-17) produces possibly centrally mediated, non-opioid antinociceptive effects against noxious pressure. At certain doses these effects are more potent in inflamed than in non-inflamed paws. In contrast to the opioid peptides DYN-(1-17) and DAMGO, DYN-(2-17) does not appear to have no peripheral antinociceptive actions.

Topics: Analgesics; Analgesics, Opioid; Animals; Dynorphins; Edema; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Foot; Inflammation; Male; Nociceptors; Peptide Fragments; Rats; Rats, Wistar

Microprobes bearing immobilised antibodies to dynorphin A(1-8) were used to study the basal and evoked release of this prodynorphin derived peptide in the spinal cord of urethane anaesthetised normal rats and those with a peripheral inflammation. In the absence of any active peripheral stimulus the antibody microprobes detected immunoreactive (ir)-dynorphin A(1-8) in two areas (lamina I and laminae IV-V) in the dorsal horn of the spinal cord of normal rats. With the development of unilateral ankle inflammation over 3 to 5 days following subcutaneous injections of Freund's complete adjuvant, a basal presence of ir-dynorphin A(1-8) was found in both the dorsal and ventral horn regions of both sides of the spinal cord. Lateral compression of the ankles of the normal animals did not release ir-dynorphin A(1-8) during the period of stimulation, but this neuropeptide was detected in increased amounts in the ventral horn following the stimulus. By contrast, compression of inflamed ankles produced elevated levels of ir-dynorphin A(1-8) during the period of stimulus application at three major sites in the ipsilateral spinal grey matter. The largest peak was in the deep dorsal horn/upper ventral horn (laminae VI-VII), with further sites of significant release in the mid dorsal horn (laminae II-V) and the lower ventral horn. The observation that ir-dynorphin A(1-8) is physiologically released in the ventral and deep dorsal in addition to the superficial dorsal horn of the rat suggests an involvement of dynorphins in several aspects of spinal function.

Topics: Animals; Dynorphins; Functional Laterality; Immunologic Techniques; Inflammation; Male; Peptide Fragments; Physical Stimulation; Pressure; Rats; Rats, Wistar; Reference Values; Spinal Cord; Tarsus, Animal

Dynorphin, an opioid peptide, is thought to play an important role in the modulation of nociceptive neural networks at the level of the spinal cord. Fos protein is involved in the transcriptional regulation of the dynorphin gene. Although several studies have been carried out on dynorphin gene expression by noxious somatic stimuli, few have evaluated the effect of noxious visceral stimuli on the expression of dynorphin gene. In the present studies we analysed the expression of the dynorphin gene mediated by a noxious visceral stimulus in a rat model by exposure of abdominal tissue to carrageenan. Expression of preprodynorphin and c-fos mRNAs in the spinal cord neuron was examined using ribonuclease protection assays. After inflammation, a rapid increase in the levels of c-fos mRNA in the thoracic spinal cord was observed. c-fos mRNA levels rose within 30 minutes after injection, and remained elevated for 1 hour, subsequently falling to control levels. In contrast, preprodynorphin mRNA began to increase from 30 minutes after injection and remained elevated for at least 2 days. In situ hybridization with alpha 35S-labeled cRNA probe demonstrated that in the lower thoracic spinal cord preprodynorphin mRNA was expressed in dorsal horn neurons. In celiac ganglia, both preprodynorphin and c-fos mRNAs were not detected. In the peripancreatic abdominal tissue, there was acute severe inflammation consisting of necrosis and marked polymorphonuclear leucocytic infiltration. These data demonstrate that after abdominal tissue inflammation, activation of dynorphin biosynthesis occurred in thoracic spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abdomen; Animals; Carrageenan; Dynorphins; Gene Expression; Genes, fos; In Situ Hybridization; Inflammation; Male; Pain; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord; Stimulation, Chemical

Local analgesic effects of exogenous opioid agonists are particularly prominent in painful inflammatory conditions and are mediated by opioid receptors on peripheral sensory nerves. The endogenous ligands of these receptors, opioid peptides, have been demonstrated in resident immune cells within inflamed tissue of animals and humans. Here we examine in vivo and in vitro whether interleukin 1 beta (IL-1) or corticotropin-releasing factor (CRF) is capable of releasing these endogenous opioids and inhibiting pain. When injected into inflamed rat paws (but not intravenously), IL-1 and CRF produce antinociception, which is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively, and by the immunosuppressant cyclosporine A. In vivo administration of antibodies against opioid peptides indicates that the effects of IL-1 and CRF are mediated by beta-endorphin and, in addition, by dynorphin A and [Met]enkephalin, respectively. Correspondingly, IL-1 effects are inhibited by mu-, delta-, and kappa-opioid antagonists, whereas CRF effects are attenuated by all except a kappa-antagonist. Finally, IL-1 and CRF produce acute release of immunoreactive beta-endorphin in cell suspensions freshly prepared from inflamed lymph nodes. This effect is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively. These findings suggest that IL-1 and CRF activate their receptors on immune cells to release opioids that subsequently occupy multiple opioid receptors on sensory nerves and result in antinociception. beta-Endorphin, mu- and delta-opioid receptors play a major role, but IL-1 and CRF appear to differentially release additional opioid peptides.

Topics: Analysis of Variance; Animals; Antibodies; beta-Endorphin; Corticotropin-Releasing Hormone; Cyclosporine; Dose-Response Relationship, Drug; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Humans; Inflammation; Injections; Interleukin-1; Male; Naloxone; Pain; Rats; Rats, Wistar; Recombinant Proteins; Regression Analysis; Somatostatin

This electrophysiological study uses the mixed peptidase inhibitor kelatorphan and the selective kappa-antagonist nor-binaltorphimine (nor-BNI) to investigate whether there is altered modulation of spinal nociceptive transmission by endogenous opioids 3 h after injection of carrageenan into the ipsilateral paw. Intrathecal kelatorphan (5-250 micrograms) inhibited the C-fibre evoked response of dorsal horn neurones in both normal and carrageenan animals, with no difference in this inhibitory effect found between the 2 groups of animals. In both groups of animals, this inhibition reached a plateau at 50%. Thus there was no change in the effects exerted by the spinal enkephalins at this point in the inflammatory state. Nor-BNI (10 and 100 micrograms) produced a bidirectional change in the C-fibre evoked response of dorsal horn neurones in both normal and carrageenan animals, facilitating the evoked response of some neurones whilst inhibiting others. The magnitude of the change in the neuronal response induced by nor-BNI in carrageenan animals was significantly greater than that seen in normal animals, suggesting a greater release of spinal dynorphin in the inflammatory state. Dorsal horn neurones showed a bidirectional change in response as carrageenan-induced inflammation developed, although the direction of this change did not correlate with the subsequent direction of effect of nor-BNI. There was, however, a significant correlation between the magnitude of the change in the C-fibre evoked response after the injection of carrageenan and the magnitude of change produced in the same cells by nor-BNI.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Carrageenan; Dipeptides; Dynorphins; Electrophysiology; Endorphins; Inflammation; Injections, Spinal; Male; Microelectrodes; Naltrexone; Nerve Fibers; Neurons; Nociceptors; Rats; Rats, Sprague-Dawley; Spinal Cord; Synaptic Transmission

Exogenous kappa-opioid agonists have been shown to produce peripheral antinociceptive effects in inflamed tissue. This study sought to determine whether endogenous kappa-receptor ligands are present at the site of inflammation. In Freund's adjuvant-induced hindpaw inflammation in the rat, we show, by immunohistochemistry, that dynorphin is detectable within inflammatory cells and in the cutaneous nerves in a similar distribution as calcitonin gene-related peptide, a specific marker for sensory neurons. These findings extend our previous observations in that not only beta-endorphin and Met-enkephalin (mu- and delta-receptor ligands), but also a preferential kappa-ligand is present within inflamed subcutaneous tissue.

Topics: Animals; Calcitonin Gene-Related Peptide; Dynorphins; Freund's Adjuvant; Inflammation; Male; Nerve Fibers; Pain; Rats; Rats, Wistar; Receptors, Opioid, kappa; Skin

Using a double-labeling technique, we evaluated the input of afferents immunoreactive for dynorphin peptide onto a population of lumbar spinal neurons contributing to the spinoparabrachial tract in rats with 1 inflamed hind paw. We found that the frequency and distribution with which dynorphin immunoreactive varicosities were in apposition to projection neurons varied according to neuron location. In particular, neurons in the superficial dorsal horn and neck of the dorsal horn receive a high degree of dynorphin input. We also determine that unilateral peripheral inflammation is associated with both an increase in the number of projection neurons receiving detectable DYN input and in the frequency of this input onto a given neuron, with the largest increase seen in the superficial dorsal horn. Since almost all superficial dorsal horn neurons contributing to the spinoparabrachial tract respond either exclusively or maximally to noxious stimulation, our data supports dynorphin's involvement in nociception.

Topics: Animals; Axons; Dendrites; Dynorphins; Immunohistochemistry; Inflammation; Male; Nerve Endings; Neurons; Pain; Peptide Fragments; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Spinal Cord

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

Spinal cord application of the kappa-opioid receptor agonists dynorphin (50 nmol) or (1S,2S)U-50,488H (0.19-1.9 mumol) produced changes in the excitability of some superficial dorsal horn nociceptive neurons. One-third of the cells exhibited expansion of their receptive fields as defined using mechanical stimuli following a spinal kappa agonist (dynorphin or U-50,488H); receptive field expansions were of the same order as those observed immediately after a conditioning electrical stimulus applied to a peripheral nerve. In addition, spinal U-50,488H produced changes in mechanical and thermal thresholds of the majority of superficial dorsal horn neurons. These changes were dose-dependent. Facilitation of responses occurred at lower doses and inhibition occurred primarily at higher doses, but these effects were not reversed by subsequent administration of naloxone. The data are consistent with the hypothesis that one action of increases in spinal dynorphin levels due to peripheral inflammation, tissue injury or nerve damage, is to contribute to enhanced neuronal excitability in superficial dorsal horn neurons.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Dynorphins; Electric Stimulation; Inflammation; Neurons; Nociceptors; Physical Stimulation; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, kappa; Spinal Cord

Fos and Fos-related proteins are increased in spinal dorsal horn neurons following noxious stimulation. The laminar location of neurons that exhibit this increase is coincident with those that exhibit an increase in dynorphin in a rat model of peripheral inflammation and hyperalgesia. In order to determine whether the increase in Fos or related proteins and dynorphin occurs in the same dorsal horn neurons, two kinds of double-labeling methods were used: in situ hybridization histochemistry to label dynorphin mRNA autoradiographically, and immunocytochemistry to label Fos and Fos-related proteins, or a double immunocytochemical method that labeled Fos and Fos-related proteins and dynorphin peptide with distinct chromagens. With both methods more than 80% of the neurons in laminae I, II, V and VI exhibiting an increase in either dynorphin mRNA or peptide following peripheral inflammation also colocalized increased nuclear Fos-like immunoreactivity. However, the number of neurons displaying increased Fos-like immunoreactivity was substantially greater than the number of neurons colocalizing increased dynorphin. These data suggest that the activation of nuclear Fos and Fos-related proteins may be related to the induction of dynorphin gene expression in a subpopulation of spinal cord neurons following peripheral inflammation and hyperalgesia.

Topics: Animals; Autoradiography; Dynorphins; Hyperalgesia; Inflammation; Male; Neurons; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord; Sulfur Radioisotopes

We have examined the frequency and distribution of neuron profiles immunoreactive for glutamic acid decarboxylase, a biosynthetic enzyme for the putative inhibitory neurotransmitter, gamma aminobutyric acid, in the lumbar spinal cord of colchicine-treated rats with unilateral inflammation of a hindpaw. Ipsilateral to the inflamed hindpaw, there was an apparent increase in the levels of glutamic acid decarboxylase, as indicated by significant increases in the number of visible glutamic acid decarboxylase-like immunoreactive profiles within the superficial dorsal horn, neck of the dorsal horn and the deep gray matter at L4. An increase limited to the deep gray matter at L6 was also seen. No alteration was identified at L2. These results are the first to demonstrate that peripheral inflammation is associated with altered levels of glutamic acid decarboxylase-like immunoreactivity.

Topics: Animals; Dynorphins; Foot; Glutamate Decarboxylase; Inflammation; Male; Rats; Rats, Inbred Strains; Spinal Cord; Synaptic Transmission

Opioid systems modulate nociceptive input at several levels of the CNS. At the spinal cord level neurons are present that express the genes coding for the precursors of the dynorphin and enkephalin opioid peptide families. We found that two conditions in rats, a chronic constriction injury to the sciatic nerve and peripheral inflammation, have a common consequence centrally: they evoke a large, rapid and sustained up-regulation of preprodynorphin mRNA. Both are also characterized by signs of hyperalgesia and increased primary afferent input. In contrast, there is little or no up-regulation of preprodynorphin mRNA following complete transection of the sciatic nerve or sciatic nerve crush. Furthermore, only minor alterations in the levels of preproenkephalin mRNA occur in any of the conditions, except for inflammation where the elevation is relatively small compared to that of preprodynorphin mRNA. These data imply that specific regulatory processes that include stimulation of opioid gene expression are strongly engaged in the spinal cord in certain types of peripheral nerve injuries and inflammation, but not in others. Marked and sustained up-regulation of the spinal cord dynorphin system distinguishes the chronic constriction injury model from other nerve injury models of pain.

Topics: Animals; Carrageenan; Constriction, Pathologic; Denervation; Densitometry; Dynorphins; Endorphins; Gene Expression; Hindlimb; Inflammation; Male; Nerve Crush; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger; Sciatic Nerve; Spinal Cord; Up-Regulation

The influence of prolonged pain upon hypothalamic opioid peptide release in vitro was examined in rats subjected to Freund's adjuvant (FA)-induced unilateral inflammation of the hindlimb. Basal release of enkephalin (ENK) but not beta-endorphin (END) or dynorphin (DYN) was increased 10 days following FA treatment. Superfusion of corticotropin-releasing factor (CRF; 10(-8) M) stimulated the release of opioid peptides in control hypothalami. CRF, however, failed to modify beta-END and DYN release in hypothalami of FA-treated rats, whereas ENK release was markedly reduced. In contrast, KCl-stimulated opioid peptide release did not differ between FA and control hypothalami. These data demonstrate that prolonged inflammatory pain alters the responsiveness of hypothalamic opioid systems to CRF. It is suggested that this effect is mediated at the level of the CRF neuron or its receptor.

Topics: Animals; Arthritis, Experimental; Behavior, Animal; beta-Endorphin; Corticotropin-Releasing Hormone; Dynorphins; Electroshock; Endorphins; Enkephalin, Methionine; Hypothalamus; In Vitro Techniques; Inflammation; Male; Pain; Potassium Chloride; Radioimmunoassay; Rats; Rats, Inbred Strains; Restraint, Physical; Stress, Psychological

Exogenous opioids can produce localized opioid receptor-mediated antinociception in peripheral inflamed tissue. Previous studies show that activation of endogenous opioids by a cold water swim in rats with hind paw inflammation results in a similar local antinociceptive effect but suggest that pituitary-adrenal opioid pools are not directly involved in producing this effect. Here we show increased amounts of opioid peptides in immune cells infiltrating the inflamed tissue. Furthermore, we demonstrate immunoreactive opioid receptors on peripheral terminals of sensory neurons. The local administration of antibodies against opioid peptides or receptors or systemic pretreatment with the immunosuppressant cyclosporine blocks cold water swim-induced antinociception. These findings suggest that antinociception in inflammation can be brought about by endogenous opioids from immune cells interacting with opioid receptors on peripheral sensory nerves.

Topics: Animals; Antibodies, Monoclonal; beta-Endorphin; Dynorphins; Enkephalin, Methionine; Freund's Adjuvant; Immunoenzyme Techniques; Inflammation; Male; Neurons, Afferent; Pain; Physical Exertion; Radioimmunoassay; Rats; Rats, Inbred Strains; Receptors, Opioid; Reference Values; Skin

The present study combined the retrograde transport of fluorescent tracers with the immunocytochemical identification of dynorphin A(1-8) in superficial dorsal horn neurons to examine whether peripheral inflammation-induced dynorphin increases are found in local circuit neurons only or also in neurons projecting at least to the caudal mesencephalon. Evidence is presented that complete Freund's adjuvant-induced inflammation produces a large increase in the number of lamina I dynorphin-containing projection and non-projection neurons, and in the number of lamina II dynorphin local circuit neurons.

Topics: Animals; Cell Count; Dynorphins; Fluorescent Dyes; Immunohistochemistry; Inflammation; Male; Peptide Fragments; Peripheral Nervous System Diseases; Rats; Rats, Inbred Strains; 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

Dynorphin, an opioid peptide, is thought to play an important role in the modulation of nociceptive neural circuits at the level of the spinal cord. In a model of peripheral inflammation and hyperalgesia, an oligodeoxyribonucleotide probe complementary to a portion of preprodynorphin mRNA and antisera to dynorphin A-(1-8) were used to localize changes in dynorphin mRNA and peptide to individual spinal cord neurons. Intraplantar injection in rats of complete Freund's adjuvant resulted in edema and hyperalgesia to radiant heat stimulation of the injected hind paw that reached a peak at 4 days. At the same time, in situ hybridization histochemistry and immunocytochemistry identified an increase in transcription of preprodynorphin mRNA that was paralleled by an increase in dynorphin peptide. These changes were seen in spinal neurons in the medial two-thirds of laminae I and II and in laminae V and VI of lumbar segments receiving innervation from the inflamed paw. Since neurons demonstrating the increase in dynorphin biosynthesis are located in both the superficial and deep dorsal horn laminae, our data provide evidence for opioid modulation of nociceptive neural circuits in these two distinct spinal locations.

Topics: Animals; Disease Models, Animal; Dynorphins; Hyperalgesia; Hyperesthesia; Inflammation; Male; Neurons; Nucleic Acid Hybridization; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord

In a rat model of peripheral inflammation and hyperalgesia, dynorphin A(1-8)-like immunoreactive (DYN-LIr) spinal neurons were examined for contacts from calcitonin gene-related peptide-like immunoreactive (CGRP-LIr) varicosities using a double-label PAP method. Ipsilateral to the inflammation, CGRP-LIr varicosities contacted both dendrites and somata of DYN-LIr neurons in lumbar laminae I, II and V. Few such contacts were found on the contralateral side. The results suggest that opioid neurons which exhibit a dynamic change in dynorphin associated with inflammation, represent a subpopulation of neurons that receive contacts from presumptive nociceptive primary afferents.

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Count; Dynorphins; Hyperalgesia; Hyperesthesia; Immunohistochemistry; Inflammation; Male; Neuropeptides; Peptide Fragments; Peripheral Nervous System Diseases; Rats; Rats, Inbred Strains; Spinal Cord; Synapses

Inoculation of the right hind paw with Mycobacterium butyricum rapidly led to swelling and inflammation. The afflicted limb showed an enhanced sensitivity to noxious pressure (hyperalgesia) and a reduced sensitivity to noxious heat 24 h following treatment. Both naloxone and MR 2266 (which has greater activity at kappa-opioid receptors) further increased the sensitivity to pressure (that is, potentiated the hyperalgesia) but did not affect the response to heat. They did not affect the response of the uninflamed paw. At 1 week, only MR 2266 was effective. At both 24 h and 1 week, the inflamed paw showed pronounced supersensitivity to the antinociceptive action of morphine against noxious pressure. At both 24 h and (to a greater extent) 1 week, a rise in levels of immunoreactive (ir)-dynorphin (DYN) was seen in the ipsilateral dorsal horn of the lumbar spinal cord. There was no alteration in the contralateral dorsal horn or in either ventral horn. Furthermore, levels of ir-met-enkephalin (ME) and ir-leu-enkephalin (LE) were unaffected. There was no difference in the density of mu-, delta- or kappa-binding sites in any part of the lumbar cord, at either 24 h or 1 week, between ipsilateral and contralateral tissue. By 3 and 5 weeks postinoculation, the symptoms had spread to the contralateral hind limb and ir-DYN was elevated in the contralateral dorsal horn and the ipsilateral ventral horn. At 5 weeks, levels of ir-ME and ir-LE also were increased in the ipsilateral and contralateral dorsal horns, but not in the contralateral ventral horn. Furthermore, levels of ir-DYN were increased in the cervico-thoracic spinal cord, and rats displayed adrenal hypertrophy and a rise in plasma levels of ir-beta-endorphin (beta-EP). These data indicate: (1) Peripheral inflammation localized to a single limb selectively modifies levels of ir-DYN in ipsilateral dorsal horn. The effect is specific to DYN as compared to ME and LE. The density of mu-, delta-, or kappa-receptors in the lumbar spinal cord is unmodified. (2) The altered response to opioid agonists and antagonists shown by rats with an inflamed limb may be selective to the injured tissue. (3) Alterations in opioid systems associated with unilateral hind limb inflammation may not be exclusively chronic in nature: they appear very rapidly (within 24 h) of the induction of pain. With time, the contralateral limb becomes affected and, eventually, the effects resemble those seen with generalized polyarthritis.

Topics: Animals; Benzomorphans; Disease Models, Animal; Dynorphins; Endorphins; Hindlimb; Inflammation; Male; Naloxone; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Spinal Cord

The stimulus specificity for enhancement of dynorphin gene expression in rat spinal cord was studied by combined measurements of the peptide dynorphin A 1-8 and preprodynorphin mRNA levels during peripheral inflammation induced by several agents. The density of kappa receptors, the putative receptor for dynorphin peptides, was examined using receptor binding with autoradiographic visualization. Mu and delta receptor classes were also studied. All inflammatory agents tested (carrageenan, phorbol ester, yeast and Freund's adjuvant) rapidly induced edema and thermal hyperalgesia. All agents also induced a rapid (within 8 h) elevation in dynorphin mRNA and, in comparison, a delayed (within 2 days) elevation of dynorphin A 1-8 peptide; peak peptide levels were reached at 4 days. No alteration of kappa, mu or delta receptor binding was observed at 4 h or 4 days post inflammation. The rapid development of thermal hyperalgesia and elevation of dynorphin mRNA and peptide content indicates that the involvement of dynorphin-containing neurons in nociceptive processing does not require a chronic abnormality and a dynamic picture of opioid modulation of sensory processing emerges. These data also demonstrate that activation of dynorphin biosynthesis in spinal cord is a feature common to hyperalgesia and peripheral inflammation and is not restricted to any one type of inflammatory agent. The lack of alteration in receptors suggests that the physiological effects of an increased biosynthesis are not accompanied by a concurrent down-regulation of opiate receptors.

Topics: Animals; Behavior, Animal; Body Weight; Dynorphins; Endorphins; Gene Expression Regulation; Hot Temperature; Hyperalgesia; Inflammation; Male; Peptide Fragments; Protein Precursors; Rats; Rats, Inbred Strains; Receptors, Opioid; Spinal Cord

The plasma extravasation response to dynorphin-(1-13) was investigated using the Evans blue dye leakage technique. Dynorphin induced plasma extravasation in rat and guinea-pig abdominal skin with a similar potency to substance P. In rat skin dynorphin, unlike substance P, produced its action entirely by release of histamine and 5-hydroxytryptamine since the response was abolished by pretreatment of rats with mepyramine and methysergide. Pretreatment of rats with capsaicin or the tachykinin antagonist, spantide, reduced but did not abolish the response to dynorphin, indicating that its action was not mediated primarily by a neurogenic mechanism. Since the response was not significantly reduced by naloxone it was concluded that the plasma extravasation response to dynorphin was mediated by receptors other than mu opiate receptors. Thus dynorphin, if released from sensory nerves, might play a role in neurogenic inflammation.

Topics: Analgesics; Animals; Dynorphins; Female; Inflammation; Male; Mast Cells; Naloxone; Peptide Fragments; Plasma; Rats; Rats, Inbred Strains; Skin; Substance P

Injection of a complete Freunds adjuvant-saline emulsion into the hindpaw of the rat induces a peripheral inflammatory process accompanied by pronounced increase (3 fold) in spinal cord dynorphin content. A marked increase (approximately 9 fold) in the mRNA coding for the preprodynorphin precursor accompanies the increase in peptide. The parallel elevations of the mRNA and peptide product suggest that an increased activity of dynorphin neurons occurs in spinal cord in response to altered afferent input due to the peripheral inflammatory process.

Topics: Animals; Arthritis, Experimental; Dynorphins; Inflammation; Male; Nucleic Acid Hybridization; Rats; Rats, Inbred Strains; RNA, Messenger; Spinal Cord