ABSTRACT
Our laboratory has previously characterized age-dependent changes in nociception upon acute morphine withdrawal. This study characterizes changes in mechanical and thermal nociception following acute, intermittent, or continuous morphine administration in infant (postnatal days 5-8) and young (postnatal days 19-21) rats. Morphine was given as a single acute administration (AM), intermittently twice a day for 3 days (IM), or continuously for 72 h via pump (CM). AM did not produce long-term changes in mechanical or thermal nociception in either infant or young rats. CM produced changes in mechanical nociception that included the development of tolerance, opioid-induced mechanical allodynia and withdrawal-associated mechanical allodynia in young rats, but only tolerance and a prolonged withdrawal-associated mechanical allodynia in infant rats. IM produced withdrawal-associated mechanical allodynia in both infant and young rats. Measuring paw withdrawal responses to thermal stimuli, infant and young rats showed tolerance without opioid-induced thermal hyperalgesia or withdrawal-associated thermal hyperalgesia following CM. In contrast to CM, withdrawal-associated thermal hyperalgesia was seen in both ages following IM. In conclusion, CM versus IM differentially modified mechanical and thermal nociception, suggesting that opioid-dependent thermal hyperalgesia and mechanical allodynia can be dissociated from each other in infant and young rats. Furthermore, tolerance, opioid-induced hypersensitivity, and withdrawal-associated hypersensitivity are age-specific and may be mediated by distinct mechanisms.
Subject(s)
Aging/psychology , Analgesics, Opioid/adverse effects , Analgesics, Opioid/pharmacology , Morphine/adverse effects , Morphine/pharmacology , Pain/chemically induced , Pain/etiology , Substance Withdrawal Syndrome/psychology , Analgesics, Opioid/administration & dosage , Animals , Animals, Newborn , Behavior, Animal/drug effects , Drug Implants , Drug Tolerance , Female , Hot Temperature , Hyperalgesia/chemically induced , Hyperalgesia/psychology , Infusion Pumps, Implantable , Injections, Subcutaneous , Male , Morphine/administration & dosage , Pain Measurement/drug effects , Pain Threshold/drug effects , Physical Stimulation , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects , Weight Gain/drug effectsABSTRACT
The development of painful peripheral neuropathy is a dose-limiting side effect of numerous cancer chemotherapeutic agents. The present study utilized a rodent model of vincristine-induced neuropathy to determine whether a glial modulating agent, propentofylline, could attenuate vincristine-induced mechanical allodynia. Intravenous vincristine administered on days 1 through 5 and days 8 through 11 produced mechanical allodynia using 2 and 12 g von Frey filaments. Lumbar spinal cord from animals on day 15 expressed mild bilateral microglial and astrocytic activation as compared to saline-treated animals. Daily intraperitoneal propentofylline at 10 mg/kg attenuated mechanical allodynia induced by vincristine administration. In addition, propentofylline was found to decrease spinal microglial and astrocytic activation on day 15. These data suggest that central glial cells may play an important role in the development of painful neuropathy following vincristine administration.
Subject(s)
Antineoplastic Agents/adverse effects , Hyperalgesia/chemically induced , Hyperalgesia/prevention & control , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/prevention & control , Vincristine/adverse effects , Xanthines/administration & dosage , Animals , Drug Combinations , Hyperalgesia/diagnosis , Male , Neuroprotective Agents/administration & dosage , Pain Measurement/drug effects , Pain Threshold/drug effects , Peripheral Nervous System Diseases/pathology , Rats , Rats, Sprague-Dawley , Treatment OutcomeABSTRACT
Mononeuropathy was induced by placing an ameroid ring around the sciatic nerve and was compared with chronic constriction injury (CCI) of the sciatic nerve [Pain 33 (1988) 87] in rats. Mechanical allodynia was assessed and the role of sciatic and saphenous afferents (Adelta and C) in thermal hyperalgesia investigated. A shorter duration of mechanical allodynia in ameroid rats as compared to CCI rats was observed. Thermal hyperalgesia was observed in the saphenous innervated skin of the hindpaw for Adelta and C nociceptors in ameroid and for Adelta nociceptors only in CCI rats, respectively. The sciatic innervated skin showed a thermal hypoalgesia with a fast onset for Adelta afferents and a slower onset for C afferents in CCI and ameroid rats. The duration of both thermal hypo- and hyperalgesia was longer in ameroid rats. We conclude that ameroid rings are a useful tool for the investigation of long-duration hyperalgesic effects of nerve injury, as the effects were more stable and seen for a longer time (>8 weeks) as compared to the CCI model. The uninjured saphenous afferents, in particular C fibers, mediate thermal hyperalgesia after chronic constriction of the sciatic nerve using an ameroid ring.
Subject(s)
Biocompatible Materials/adverse effects , Hydrogels/adverse effects , Mononeuropathies/complications , Neuralgia/etiology , Sciatic Nerve/physiopathology , Analysis of Variance , Animals , Caseins , Constriction, Pathologic , Hyperalgesia/physiopathology , Male , Pain Measurement , Physical Stimulation , Rats , Rats, Sprague-Dawley , Reaction Time , Reproducibility of Results , Sciatic Nerve/injuries , Skin/innervation , Skin/physiopathology , Time FactorsABSTRACT
The stress-activated mitogen-activated protein kinase (MAPK) p38 is emerging as an important mediator of pain. The present study examined the possible involvement of peripheral and spinal p38 MAPK in capsaicin-induced thermal hyperalgesia. Topical capsaicin produced phosphorylation of p38 MAPK in the skin from the affected hindpaw as well as the corresponding lumbar spinal cord in a time dependent manner. Topical capsaicin produced robust C-fiber mediated thermal hyperalgesia that was inhibited by systemic, local peripheral, or central intrathecal pre-treatment with the p38 MAPK inhibitor, SD-282. Intraperitoneal SD-282 (10-60 mg/kg) significantly and dose-dependently attenuated capsaicin-induced C-fiber mediated thermal hyperalgesia. Similarly, 0.1-5mg/kg subcutaneous SD-282 in the hindpaw dose-dependently attenuated capsaicin-induced thermal hyperalgesia. Intrathecal administration of 1microg SD-282 was also anti-hyperalgesic in this model. Functionally, SD-282 decreased capsaicin-induced release of calcitonin gene related peptide in an in vitro skin release assay, consistent with a role for p38 MAPK in peripheral nerve function. These results suggest that p38 MAPK plays a role in the development of hyperalgesic states, exerting effects both centrally in the spinal cord and peripherally in sensory C fibers.
Subject(s)
Capsaicin/toxicity , Hyperalgesia/enzymology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Male , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Rats , Rats, Sprague-Dawley , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
The present study was designed to determine the role of central expression of immunoregulatory molecules in the development and maintenance of allodynia following a peripheral inflammatory insult or nerve transection. Differential spinal expression of major histocompatibility complex (MHC) class II, platelet-endothelial cellular adhesion molecule (PECAM), intercellular adhesion molecule (ICAM) and CD4 was observed in the two injury models. Intraplantar zymosan produced transient allodynia and only PECAM and ICAM immunoreactivity. In contrast, persistent mechanical allodynia and enhanced spinal PECAM, ICAM, MHC class II and CD4 immunoreactivity was observed following peripheral nerve transection. MHC class II knockout mice exhibited attenuated allodynia following spinal nerve transection as compared to wild-type control mice. These findings suggest that central neuroimmune activation may contribute to the maintenance of neuropathic pain following peripheral L5 spinal nerve transection but not following a peripheral inflammatory insult.
Subject(s)
Histocompatibility Antigens Class II/immunology , Intercellular Adhesion Molecule-1/immunology , Neuralgia/immunology , Neuritis/immunology , Spinal Cord/immunology , Animals , CD4 Antigens/analysis , Denervation , Disease Models, Animal , Histocompatibility Antigens Class II/genetics , Intercellular Adhesion Molecule-1/analysis , Lumbar Vertebrae , Male , Mice , Mice, Transgenic , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Rats , Rats, Sprague-Dawley , Spinal Cord/chemistry , Spinal Nerves/physiologyABSTRACT
The specific mechanisms by which nervous system injury becomes a chronic pain state remain undetermined. Historically, it has been believed that injuries proximal or distal to the dorsal root ganglion (DRG) produce distinct pathologies that manifest in different severity of symptoms. This study investigated the role of injury site relative to the DRG in (1) eliciting behavioral responses, (2) inducing spinal neuroimmune activation, and (3) responding to pharmacologic interventions. Rats received either an L5 spinal nerve transection distal to the DRG or an L5 nerve root injury proximal to the DRG. Comparative studies assessed behavioral nociceptive responses, spinal cytokine mRNA and protein expression, and glial activation after injury. In separate studies, intrathecal pharmacologic interventions by using selective cytokine antagonists (interleukin-1 [IL-1] receptor antagonist and soluble tumor necrosis factor [TNF] receptor) and a global immunosuppressant (leflunomide) were performed to determine their relative effectiveness in these injury paradigms. Behavioral responses assessed by mechanical allodynia and thermal hyperalgesia were almost identical in the two models of persistent pain, suggesting that behavioral testing may not be a sensitive measure of injury. Spinal IL-1beta, IL-6, IL-10, and TNF mRNA and IL-6 protein were significantly elevated in both injuries. The overall magnitude of expression and temporal patterns were similar in both models of injury. The degree of microglial and astrocytic activation in the L5 spinal cord was also similar for both injuries. In contrast, the pharmacologic treatments were more effective in alleviating mechanical allodynia for peripheral nerve injury than nerve root injury, suggesting that nerve root injury elicits a more robust, centrally mediated response than peripheral nerve injury. Overall, these data implicate alternate nociceptive mechanisms in these anatomically different injuries that are not distinguished by behavioral testing or the neuroimmune markers used in this study.
Subject(s)
Antigens, CD , Antigens, Neoplasm , Antigens, Surface , Avian Proteins , Blood Proteins , Cytokines/metabolism , Ganglia, Spinal/injuries , Gliosis/metabolism , Neuroglia/metabolism , Pain/physiopathology , Peripheral Nervous System Diseases/metabolism , Spinal Cord/metabolism , Animals , Basigin , Cytokines/antagonists & inhibitors , Cytokines/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Ganglia, Spinal/pathology , Ganglia, Spinal/physiopathology , Glial Fibrillary Acidic Protein/metabolism , Gliosis/etiology , Gliosis/physiopathology , Hyperalgesia/etiology , Hyperalgesia/metabolism , Hyperalgesia/physiopathology , Immunohistochemistry , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Lumbar Vertebrae , Male , Membrane Glycoproteins/metabolism , Neuroglia/drug effects , Neuroglia/pathology , Pain/drug therapy , Peripheral Nervous System Diseases/pathology , Peripheral Nervous System Diseases/physiopathology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Tumor Necrosis Factor/therapeutic use , Sialoglycoproteins/pharmacology , Spinal Cord/drug effects , Spinal Cord/pathology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The present study was undertaken to determine whether propentofylline, a glial modulating agent, could both prevent the induction of mechanical allodynia and attenuate existing mechanical allodynia in a rodent L5 spinal nerve transection model of neuropathic pain. In a preventative paradigm, propentofylline (1 and 10 mg/kg intraperitoneally) was administered systemically daily, beginning 1 day prior to nerve transection. This regimen produced a dose-dependent decrease in mechanical allodynia (p < 0.01). In another preventative paradigm, propentofylline (0.1, 1, or 10 microg) was administered daily intrathecally via direct lumbar puncture. Intrathecal administration of propentofylline was more effective than systemic administration at dose dependently reducing mechanical allodynia (p < 0.01). The effect of systemic propentofylline on existing allodynia was examined with 0.1-, 1-, and 10-mg/kg intraperitoneal administration initiated on day 4 post L5 spinal nerve transection. Systemic propentofylline was found to be equally effective in the attenuation of existing allodynia (p < 0.01) as in the prevention of allodynia in this rodent model of neuropathic pain. Spinal cords (L4-L6 segments) were removed for immunohistochemical analysis on day 10 or 20 post-transection. Microglial and astrocytic activation was decreased by both peripheral and central administration of propentofylline in both preventative and existing allodynia paradigms. This research supports a growing body of literature highlighting the importance of glial activation in the development of persistent neuropathic pain states, and the potential to therapeutically modulate glial activation in the treatment of neuropathic pain.
Subject(s)
Hyperalgesia/drug therapy , Neuroglia/drug effects , Somatoform Disorders/drug therapy , Xanthines/administration & dosage , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Hyperalgesia/complications , Hyperalgesia/prevention & control , Immunohistochemistry , Injections, Intraperitoneal , Injections, Spinal , Male , Neuroglia/cytology , Neuroglia/metabolism , Pain Measurement/drug effects , Physical Stimulation , Rats , Rats, Sprague-Dawley , Somatoform Disorders/complications , Spinal Cord/cytology , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Nerves/physiology , Treatment OutcomeABSTRACT
In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.
Subject(s)
Bilirubin/metabolism , Cytochrome P-450 CYP1A2/metabolism , Jaundice/metabolism , Animals , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/deficiency , Cytochrome P-450 CYP1A2/genetics , Disease Models, Animal , Iron-Dextran Complex/pharmacology , Jaundice/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Polychlorinated Biphenyls/pharmacology , Polychlorinated Dibenzodioxins/toxicity , Rats , Rats, Gunn , Rats, Wistar , Teratogens/toxicityABSTRACT
Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.
Subject(s)
Alzheimer Disease/enzymology , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Glycoproteins/metabolism , Oligosaccharides/chemistry , Polysaccharides/metabolism , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases/genetics , Brain/enzymology , CHO Cells , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line , Cricetinae , Endopeptidases , Glycoproteins/chemistry , Glycoproteins/genetics , Glycosylation , Humans , Molecular Sequence Data , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spodoptera , TransfectionABSTRACT
The expression of interleukin-1beta and tumor necrosis factor has previously been shown to be up-regulated in the spinal cord of several rat mononeuropathy models. This present study was undertaken to determine whether blocking the action of central interleukin-1beta and tumor necrosis factor attenuates mechanical allodynia in a gender-specific manner in a rodent L5 spinal nerve transection model of neuropathic pain, and whether this inhibition occurs via down-regulation of the central cytokine cascade or blockade of glial activation. Interleukin-1 receptor antagonist or soluble tumor necrosis factor receptor was administered intrathecally via lumbar puncture to male Holtzman rats in a preventative pain strategy, in which therapy was initiated 1h prior to surgery. Administration of soluble tumor necrosis factor receptor attenuated mechanical allodynia, while interleukin-1 receptor antagonist alone was unable to decrease allodynia. Interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor, administered to both male and female rats in a preventative pain strategy, significantly reduced mechanical allodynia in a dose-dependent manner (P<0.01). The magnitude of attenuation in allodynia was similar in both males and females. Immunohistochemistry on L5 spinal cord revealed similar astrocytic and microglial activation regardless of treatment. At days 3 and 7 post-transection, animals receiving daily interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibited significantly less interleukin-6, but not interleukin-1beta, in the L5 spinal cord compared to vehicle-treated animals. In an existing pain paradigm, in which treatment was initiated on day 7 post-transection, interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor attenuated mechanical allodynia (P<0.05) in male rats. These findings further support a role for central interleukin-1beta and tumor necrosis factor in the development and maintenance of neuropathic pain through induction of a proinflammatory cytokine cascade.
Subject(s)
Immunoglobulin G/pharmacology , Neuralgia/drug therapy , Neuralgia/metabolism , Sialoglycoproteins/pharmacology , Animals , Disease Models, Animal , Drug Therapy, Combination , Etanercept , Female , Injections, Spinal , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/metabolism , Interleukin-6/metabolism , Male , Neuralgia/immunology , Neuroglia/immunology , Neuroglia/metabolism , Neurons/immunology , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Tumor Necrosis Factor , Sex Factors , Spinal Cord/cytology , Spinal Cord/metabolism , Spinal Cord/physiology , Spinal Nerves/physiopathology , Spinal Nerves/surgery , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Interleukin-6 (IL-6) is a pleiotrophic cytokine with a diverse range of actions including the modulation of the peripheral and central nervous system. We have previously shown significant IL-6 protein and messenger RNA elevation in rat spinal cord following peripheral nerve injury that results in pain behaviors suggestive of neuropathic pain. These spinal IL-6 levels correlated directly with the mechanical allodynia intensity following nerve injury. In the current study, we sought to determine whether it is possible to attenuate mechanical allodynia and/or alter spinal glial activation resulting from peripheral nerve injury by specific manipulation of IL-6 with neutralizing antibodies or by global immune modulation utilizing immunogamma-globulin (IgG). Effects of peripheral administration of normal goat IgG and intrathecal (i.t.) administration of IL-6 neutralizing antibody, normal goat or normal rat IgG on mechanical allodynia associated with L5 spinal nerve transection were compared. Spinal glial activation was assessed at day 10 post surgery by immunohistochemistry. Low dose (0.01-0.001 microg) goat anti-rat IL-6 i.t. administration (P=0.025) significantly decreased allodynia and trended towards significance at the higher dose (0.08 microg to 0.008 microg, P=0.062). Low doses (0.01-0.001 microg) i.t. normal goat and rat IgG significantly attenuated mechanical allodynia, but not at higher doses (0.08-0.008 microg; P=0.001 for both goat and rat IgG). Peripherally administered normal goat IgG (30 or 100 mg/kg) did not attenuate mechanical allodynia. Spinal glial activation was unaltered by any treatment. These data provide further evidence for the role of central IL-6 and neuroimmune modulation in the etiology of mechanical allodynia following peripheral nerve injury.
Subject(s)
Antibodies/pharmacology , Immunoglobulin G/pharmacology , Interleukin-6/physiology , Pain/physiopathology , Spinal Cord/physiopathology , Spinal Nerves/physiology , Animals , Antibodies/administration & dosage , Immunoglobulin G/administration & dosage , Injections, Spinal , Interleukin-6/immunology , Male , Microglia/physiology , Neuralgia/physiopathology , Neuroglia/physiology , Physical Stimulation , Rats , Rats, Sprague-Dawley , Spinal Cord/physiology , Spinal Nerves/injuriesABSTRACT
The purpose of this study was to investigate the role of peripheral macrophages in the generation of mechanical allodynia utilizing a modification of the Chung rat model of neuropathy. Three distinct methods were used: (1) systemic and perineural macrophage inhibition utilizing CNI-1493; (2) depletion of the peripheral macrophage population by liposome-encapsulated clodronate; and (3) perineural administration of activated or inactivated bone marrow-derived macrophages (BMDM) in sham-surgery rats. Mechanical allodynia was tested on days 1, 3, 5, 7, and 10 post-intervention or surgery using von Frey monofilaments. In order to assess the role of spinal glia following these interventions, microglial (CNS macrophages) and astrocytic activation was assessed using immunohistochemistry. CNI-1493 did not attenuate mechanical allodynia, or spinal glial expression as compared to the saline control group. Similarly, the clodronate depletion of peripheral macrophages prior to nerve injury did not have any effect on the resultant mechanical allodynia or spinal glial activation. Perineural administration of activated or inactivated BMDM did not evoke mechanical allodynia in sham surgery rats. Of interest, we observed an ipsilateral, dorsal horn increase in microglial expression following perineural administration of activated macrophages. In summary, these data suggest a limited role of activated macrophages in the onset of mechanical allodynia in an animal model of neuropathy.
Subject(s)
Hyperesthesia/etiology , Macrophages/physiology , Spinal Nerves/injuries , Wounds, Penetrating/complications , Analgesics, Non-Narcotic/administration & dosage , Analgesics, Non-Narcotic/pharmacology , Animals , Bone Marrow Transplantation , Cell Count , Clodronic Acid/administration & dosage , Clodronic Acid/pharmacology , Hydrazones/pharmacology , Hyperesthesia/physiopathology , Immunohistochemistry , Liposomes , Macrophages/drug effects , Macrophages/transplantation , Male , Neuroglia/pathology , Pain Measurement , Physical Stimulation , Rats , Rats, Sprague-Dawley , Spinal Cord/pathology , Wounds, Penetrating/pathology , Wounds, Penetrating/psychology , Wounds, Penetrating/surgeryABSTRACT
Our laboratory has previously shown that glial activation and increased proinflammatory cytokine expression are observed in the rat spinal cord following peripheral nerve injuries that result in neuropathic pain behaviors. In the present study, we sought to determine whether acute peripheral inflammation induces changes in central glial and cytokine (Interleukin-1beta) expression similar to those seen following peripheral spinal nerve transection. Two models of peripheral inflammation were used in this study: formalin (5% solution) or zymosan (25 mg/ml) injected subcutaneously into the plantar portion of the left hind paw of male Holtzman-strain Sprague-Dawley rats. The rats were euthanized at 1 h, 6 h, and 1, 3, 7 days post-injection (n=4 or 5/group/time point). As expected, the animals treated with formalin showed a spontaneous pain response and mechanical allodynia that persisted for approximately 60 min following injection. The animals treated with zymosan exhibited mild spontaneous pain responses during the first hour and mechanical allodynia at 6 h and 1 day following injection. Immunohistochemistry for glial activation and cytokine expression was performed on L4-L5 spinal levels in all rats. Spinal sections from both formalin and zymosan treated animals exhibited microglial and astrocytic activation and increased Interleukin-1beta immunoreactivity at 1 and 6 h, respectively. Spinal glial activation and upregulation of Interleukin-1beta appear to parallel the development and maintenance of zymosan and formalin-induced mechanical allodynia. These findings support a unifying theory that glial activation and cytokine expression have a similar, if not related, role in producing hyperalgesia following either peripheral inflammation or peripheral nerve injury.
Subject(s)
Interleukin-1/biosynthesis , Neuritis/physiopathology , Neuroglia/physiology , Peripheral Nervous System Diseases/physiopathology , Spinal Nerves/physiopathology , Acute Disease , Animals , Astrocytes/drug effects , Immunohistochemistry , Male , Microglia/drug effects , Neuritis/chemically induced , Neuritis/metabolism , Neuroglia/metabolism , Pain Measurement , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/metabolism , Rats , Rats, Sprague-Dawley , Spinal Nerves/drug effects , Stress, Mechanical , ZymosanABSTRACT
The dynamin family of GTPases is essential for receptor-mediated endocytosis and synaptic vesicle recycling, and it has recently been shown to play a role in vesicle formation from the trans-Golgi network. Dynamin is believed to assemble around the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane. This role would make dynamin unique among GTPases in its ability to act as a mechanochemical enzyme. Data presented here demonstrate that purified recombinant dynamin binds to a lipid bilayer in a regular pattern to form helical tubes that constrict and vesiculate upon GTP addition. This suggests that dynamin alone is sufficient for the formation of constricted necks of coated pits and supports the hypothesis that dynamin is the force-generating molecule responsible for membrane fission.
Subject(s)
GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/ultrastructure , Guanosine Triphosphate/physiology , Lipid Bilayers/metabolism , Dynamins , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/isolation & purification , Hydrolysis , Light , Liposomes , Phosphatidylserines , Phosphorylation , Proline , Protein Binding , Protein Conformation , Protein Kinase C/metabolism , Recombinant Fusion Proteins/isolation & purification , Scattering, Radiation , SubtilisinsABSTRACT
It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.
Subject(s)
DNA-Binding Proteins/metabolism , Growth Hormone/pharmacology , Milk Proteins , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Receptors, Somatotropin/metabolism , Signal Transduction , Trans-Activators/metabolism , Animals , Humans , Intracellular Signaling Peptides and Proteins , Janus Kinase 2 , Liver/metabolism , Mice , Mice, Mutant Strains , Phosphorylation , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Receptors, Somatotropin/genetics , STAT5 Transcription Factor , Sequence Deletion , Subcellular Fractions/metabolism , Tumor Suppressor ProteinsABSTRACT
Poxviruses encode a large number of proteins that attenuate the inflammatory and immune responses to infection. In this report we demonstrate that a number of orthopoxviruses express a type I interferon (IFN)-binding protein, which is encoded by the B18R open reading frame in the WR strain of vaccinia virus. The B18R protein has significant regions of homology with the alpha subunits of the mouse, human, and bovine type I IFN receptors, bound human IFN alpha 2 with high affinity, and inhibited transmembrane signaling as demonstrated by inhibition of Fc receptor factor gamma 1/gamma 2 and interferon-stimulated gene factor-3 formation as well as inhibition of the IFN alpha antiviral response. Among viral host response modifiers, the B18R protein is unique inasmuch as it exists as a soluble extracellular as well as a cell surface protein and thus should effectively block both autocrine and paracrine functions of IFN.
Subject(s)
Interferon-alpha/antagonists & inhibitors , Orthopoxvirus/genetics , Receptors, Interferon/genetics , Signal Transduction , Viral Proteins/genetics , Amino Acid Sequence , DNA-Binding Proteins/metabolism , Genes, Viral/genetics , Molecular Sequence Data , Receptors, Interferon/metabolism , STAT1 Transcription Factor , STAT2 Transcription Factor , Sequence Homology, Amino Acid , Trans-Activators/metabolism , Vaccinia virus/genetics , Viral Proteins/metabolismABSTRACT
The cytokine signaling pathways that activate the Janus family of tyrosine kinases (Jaks) and the "signal transducers and activators of transcription" (Stats) have been well characterized in mammalian systems. Work shown here provides evidence that an analogous signaling pathway exists in Drosophila melanogaster. Because many of the ligand-receptor pairs in Drosophila have not been fully characterized, it was necessary to bypass the receptor stimulation event that normally triggers intracellular Jak/Stat activation. This was done by treating Drosophila Schneider 2 cells with vanadate/peroxide, which has been shown to closely mimic some signaling events triggered by interferon gamma, including the activation of Jak1, Jak2, and the Stat1 alpha protein. Evidence presented here demonstrates that vanadate/peroxide can induce a gamma response region binding complex in Drosophila Schneider 2 cells. This complex contains two phosphoproteins of 100 and 150 kDa, respectively, and shares many features with the vanadate/peroxide-stimulated binding complex in the mammalian system. Southern blot analysis of genomic DNA using the src homology domain 2 (SH2) of Stat1 alpha confirms the presence of a related gene in the Drosophila genome.
Subject(s)
DNA-Binding Proteins/analysis , Drosophila melanogaster/chemistry , Trans-Activators/analysis , Animals , Base Sequence , Blotting, Southern , Drosophila melanogaster/genetics , Humans , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic , Tyrosine/metabolism , Vanadates/pharmacologyABSTRACT
Interferon-alpha (IFN-alpha) and IFN-gamma regulate gene expression by tyrosine phosphorylation of several transcription factors that have the 91-kilodalton (p91) protein of interferon-stimulated gene factor-3 (ISGF-3) as a common component. Interferon-activated protein complexes bind enhancers present in the promoters of early response genes such as the high-affinity Fc gamma receptor gene (Fc gamma RI). Treatment of human peripheral blood monocytes or basophils with interleukin-3 (IL-3), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-gamma response region (GRR) located in the promoter of the Fc gamma RI gene. Although tyrosine phosphorylation was required for the assembly of each of these GRR binding complexes, only those formed as a result of treatment with IFN-gamma or IL-10 contained p91. Instead, complexes activated by IL-3 or GM-CSF contained a tyrosine-phosphorylated protein of 80 kilodaltons. Induction of Fc gamma RI RNA occurred only with IFN-gamma and IL-10, whereas pretreatment of cells with GM-CSF or IL-3 inhibited IFN-gamma induction of Fc gamma RI RNA. Thus, several cytokines other than interferons can activate putative transcription factors by tyrosine phosphorylation.
Subject(s)
Cytokines/pharmacology , DNA-Binding Proteins/metabolism , Monocytes/metabolism , Trans-Activators , Transcription Factors/metabolism , Tyrosine/metabolism , Base Sequence , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interferon-gamma/pharmacology , Interleukin-10/pharmacology , Interleukin-3/pharmacology , Interleukins/pharmacology , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic , Receptors, IgG/genetics , Receptors, IgG/metabolism , STAT1 Transcription FactorABSTRACT
The records of 1,029 consecutive patients undergoing bilateral vasectomy were reviewed. Of the 1,029 patients 375 (36%) never returned postoperatively and the partners of 2 of these 375 later became pregnant. A total of 560 patients (54%) was followed until the recommended demonstration of azoospermia occurred on 2 consecutive occasions. There were 84 patients (8%) who did not return after the first demonstration of azoospermia, while 7 (1%) still had sperm in the semen at the last examination. Three other patients had persistent nonmotile sperm postoperatively; 2 of them underwent repeat vasectomy followed by azoospermia and 1 was placed in a special clearance category of sterility as described. We discuss practical and legal considerations regarding the management of noncompliant patients.
PIP: Urologists reviewed the medical charts of 1029 consecutive patients who had experienced a bilateral vasectomy (cauterization of the mucosa at each end and of the divided vas deferens) at the University of Louisville's School of Medicine in Kentucky between February and December 1986 to examine the medical and legal ramifications of vasectomized men not returning to have physicians examine their semen. Only 54.4% of the men returned for the recommended 2 semen examinations. 8.2% returned only once. 7 men (1% of the 654 who returned for semen examination) had sperm in their last semen sample. 36.4% (375) did not return at all after the vasectomy. Partners of 2 of these men conceived. 1 pregnancy occurred 29 months postoperatively. The men did not want to have his semen examined, however, for fear of learning he was sterile. The other pregnancy occurred 67 months after the vasectomy and the man's sperm had many motile sperm. Late recanalization of the vas deferens most likely occurred in this case. Other than recommending to patients before and during the vasectomy to submit 2 semen samples after the vasectomy, the urologists had no mechanisms in place to assure compliance. The high noncompliance rate in this study made the researchers consider their new legal responsibilities. Court cases for somewhat related medical situations have resulted in the jury finding the physician negligent because he/she did not fully inform the patient of the risk of noncompliance. Thus physicians, even though they may believe they are not responsible for noncompliance, should prudently inform patients of their risk of not complying by perhaps sending a punctually written notice. These warnings may protect them from potential litigation. Nevertheless the courts may appropriate more responsibility of patient actions to the physicians than is either morally justified or reasonable.
Subject(s)
General Surgery/legislation & jurisprudence , Paternity , Patient Compliance , Pregnancy, Unwanted , Pregnancy , Semen/analysis , Vasectomy , Female , Humans , Informed Consent/legislation & jurisprudence , Kentucky , Male , Malpractice/legislation & jurisprudence , Statistics as TopicABSTRACT
Our experience with children who require supravesical diversion in the face of a compromised urinary tract indicates that in selected cases transureteroureterostomy with cutaneous ureterostomy will lead to a stabilized urinary tract that will be free from infection and postoperative complications.