2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and Disease-Models--Animal

Our previous study together with other investigations have reported that neonatal hypoxia or ischemia induces long-term cognitive impairment, at least in part through brain inflammation and hypomyelination. However, the detailed mechanisms are not fully understood. Here, we used a rodent model of neonatal hypoxia by subjecting postnatal day 0 (P0) rat pups to systemic hypoxia (3.5 h). We found that neonatal hypoxia increased the glutamate content and initiated inflammatory responses at 4 h and 1 day after hypoxia, caused hypomyelination in the corpus callosum, and impaired hippocampus-dependent learning and memory when assessed 30-60 days after hypoxia. Interestingly, much of the hypoxia-induced brain damage was ameliorated by treatment with the ATP analogue 2',3'-0-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP; blocks all ionotropic P2X1-7 receptors), whereas treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; inhibits P2X1-3 and P2X5-7 receptors) was less neuroprotective. Our data indicated that activation of ionotropic ATP receptors might be partially, if not fully, involved in glutamate deregulation, neuroinflammation, hypomyelination, and cognitive dysfunction after neonatal hypoxia.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Brain; Cognition Disorders; Disease Models, Animal; Female; Glutamic Acid; Hypoxia-Ischemia, Brain; Immunoblotting; Male; Maze Learning; Myelin Sheath; Neurogenesis; Neuroprotective Agents; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley

The role of peripheral neurosteroids and their related mechanisms on nociception have not been thoroughly investigated. Based on emerging evidence in the literature indicating that neurosteroids and their main target receptors, i.e., sigma-1, GABAA and NMDA, affect P2X-induced changes in neuronal activity, this study was designed to investigate the effect of peripherally injected dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulfate (PREGS) on P2X receptor-mediated mechanical allodynia in rats. Intraplantar injection of either neurosteroids alone did not produced any detectable changes in paw withdrawal frequency to the innocuous mechanical stimulation in naïve rats. However, When DHEAS or PREGS were co-injected with a sub-effective dose of αβmeATP, mechanical allodynia was developed and this was dose dependently blocked by pre-injection of the P2X antagonist, TNP-ATP. These results demonstrates that DHEAS and PREGS potentiate the activity of P2X receptors which results in the enhancement of αβmeATP-induced mechanical allodynia. In order to investigate the potential role of peripheral sigma-1, GABAA and NMDA receptors in this facilitatory action, we pretreated animals with BD-1047 (a sigma-1 antagonist), muscimol (a GABAA agonist) or MK-801 (a NMDA antagonist) prior to DHEAS or PREGS+αβmeATP injection. Only BD-1047 effectively prevented the facilitatory effects induced by neurosteroids on αβmeATP-induced mechanical allodynia. Collectively, we have shown that peripheral neurosteroids potentiate P2X-induced mechanical allodynia and that this action is mediated by sigma-1, but not by GABAA nor NMDA, receptors.

Topics: Adenosine Triphosphate; Animals; Dehydroepiandrosterone Sulfate; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Ethylenediamines; Hyperalgesia; Male; Pain Measurement; Pregnenolone; Purinergic P2X Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X; Receptors, sigma; Time Factors

Previously we described the drop of the noxious heat threshold in response to mild heat injury or plantar incision. While mild heat injury elicits an immediate and short-lasting thermal hyperalgesia, surgical incision leads to a delayed and sustained heat hyperalgesia. Only very few peripheral mediators of these phenomena have been identified. Therefore the present study aimed at comparing the peripheral mediator background of heat hyperalgesia evoked by mild heat injury or surgical incision.. Heat hyperalgesia was assessed by measuring the behavioural noxious heat threshold in conscious rats employing an increasing-temperature water bath.. The heat threshold drop evoked by a mild heat injury and measured 10min afterwards was reduced by intraplantarly applied HOE 140, a bradykinin B(2) receptor antagonist, NDGA, a non-selective lipoxygenase inhibitor, L-NOARG, a non-selective nitric oxide synthase inhibitor, TNP-ATP, a P2X purinoceptor antagonist and AMG9810, an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The heat threshold drop evoked by plantar incision and measured 18h later was reduced by intraplantarly applied HOE 140, [des-Arg(10)]-HOE 140, a bradykinin B(1) receptor antagonist, L-NOARG, TNP-ATP and the TRPV1 receptor antagonist SB-366791.. Only small differences have been revealed between the examined peripheral mediators of the acute heat hyperalgesia evoked by mild heat injury and the sustained increase in heat responsiveness induced by surgical incision. The B(2) and B(1) bradykinin receptor, P2X purinoceptors, TRPV1 receptor, nitric oxide synthase and lipoxygenase(s) are involved in at least one of these hyperalgesia models.

Topics: Acrylamides; Adenosine Triphosphate; Adrenergic beta-Antagonists; Animals; Bradykinin; Bradykinin B1 Receptor Antagonists; Bradykinin B2 Receptor Antagonists; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Enzyme Inhibitors; Female; Fluorescent Dyes; Heat Stress Disorders; Hyperalgesia; Lipoxygenase; Nitric Oxide Synthase; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Receptors, Purinergic P2; Receptors, Purinergic P2X; TRPV Cation Channels

This study explored purinergic signaling in lumbosacral (LS) and thoracolumbar (TL) dorsal root ganglion neurons innervating the urinary bladder. In naïve mice, a greater proportion of LS (93%) than that of TL (77%) bladder neurons responded to purinergic agonists. Three types of purinergic currents were identified: 'sustained' (homomeric P2X2) currents were detected only in LS neurons, rapidly activating, 'slow' deactivating (heteromeric P2X2/3) currents predominated in both LS and TL neurons, and 'fast' activating/de-activating (homomeric P2X3) currents were detected only in TL neurons. Relative to TL bladder neurons, slow current density was greater in LS neurons, which also had a more negative action potential threshold and generated more action potentials in response to purinergic agonists (suggesting greater excitability of LS neurons). Single cell nested PCR documented P2X2 and P2X3 subunit expression in both TL and LS bladder neurons. Relative to saline treatment, bladder wall thickness and weight increased after cyclophosphamide (CYP) treatment. Both LS and TL neuron excitability increased (rheobase was decreased and responses to purinergic agonists increased) after CYP treatment. The proportion of sustained currents in LS bladder neurons increased fourfold after CYP bladder inflammation. Although proportions of slow and fast purinergic currents in TL neurons were unchanged by CYP treatment, the fast current density was greater than in saline-treated mice. These results in mouse, as previously described in rat, reveal differential purinergic signaling in TL and LS bladder neurons. The predominant currents and significant changes after inflammation, however, occur in different ganglia/sensory pathways in mouse and rat.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Cyclophosphamide; Disease Models, Animal; Dogs; Ganglia, Spinal; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Patch-Clamp Techniques; Peroxidase; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Sensory Receptor Cells; Signal Transduction

Activation of P2X3,2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia. Given the clinical importance of mechanical hyperalgesia in inflammatory states, we hypothesized that the activation of P2X3,2/3 receptors by endogenous ATP contributes to carrageenan-induced mechanical hyperalgesia and that this contribution is mediated by an indirect and/or a direct sensitization of the primary afferent nociceptors. Co-administration of the selective P2X3,2/3 receptors antagonist A-317491, or the non-selective P2X3 receptor antagonist, TNP-ATP, with carrageenan blocked the mechanical hyperalgesia induced by carrageenan, and significantly reduced the increased concentration of tumor necrosis factor alpha (TNF-alpha) and chemokine-induced chemoattractant-1 (CINC-1) but not of interleukin-1 beta (IL-1 beta) induced by carrageenan. Co-administration of the selective P2X3,2/3 receptors antagonist A-317491 with carrageenan did not affect the neutrophil migration induced by carrageenan. Intrathecal administration of oligonucleotides antisense against P2X3 receptors for seven days significantly reduced the expression of P2X3 receptors in the saphenous nerve and significantly reduced the mechanical hyperalgesia induced by carrageenan. We concluded that the activation of P2X3,2/3 receptors by endogenous ATP is essential to the development of the mechanical hyperalgesia induced by carrageenan. Furthermore, we showed that this essential role of P2X3,2/3 receptors in the development of carrageenan-induced mechanical hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of TNF-alpha and by a direct sensitization of the primary afferent nociceptors.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Carrageenan; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Enzyme-Linked Immunosorbent Assay; Hyperalgesia; Inflammation; Male; Oligodeoxyribonucleotides, Antisense; Pain Measurement; Pain Threshold; Peroxidase; Phenols; Polycyclic Compounds; Polysaccharides; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Time Factors

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

Topics: Adenosine Triphosphate; Animals; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Male; Mice; Nociceptors; Pain Measurement; Physical Stimulation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; RNA, Messenger; Sensory Receptor Cells; Up-Regulation

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterised by abdominal pain and bloating in association with altered bowel movements. Its pathogenesis and the underlying molecular mechanisms of visceral hyperalgesia remain elusive. Recent studies of somatic and other visceral pain models suggest a role for purinergic signalling mediated by the P2X receptor (P2XR) family.. To examine the role of P2XR signalling in the pathogenesis in a rat model of IBS-like visceral hyperalgesia.. Visceral hypersensitivity was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were conducted at 8 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labelled by injection of DiI (1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate) fluorescence into the colon wall.. Visceral hypersensitivity was reversed by TNP-ATP (2'-(or-3')-O-(trinitrophenyl) ATP), a potent P2X1, P2X3 and P2X2/3 receptor antagonist. Rapid application of ATP (20 microM) induced a fast inactivating current in colon-specific DRG neurons from both control and AA-treated rats. There was a twofold increase in the peak ATP responses in neurons from AA-treated rats. These currents were sensitive to TNP-ATP (100 nM). Under current-clamped conditions, ATP evoked a larger membrane depolarisation in neurons from neonatal AA-treated rats than in controls. P2X3R protein expression was significantly enhanced in colon-specific DRGs 8 weeks after neonatal AA treatment.. These data suggest that the large enhancement of P2XR expression and function may contribute to the maintenance of visceral hypersensitivity, thus identifying a specific neurobiological target for the treatment of chronic visceral hyperalgesia.

Topics: Acetic Acid; Adenosine Triphosphate; Animals; Chronic Disease; Colon; Disease Models, Animal; Hyperalgesia; Irritable Bowel Syndrome; Membrane Potentials; Neurons; Patch-Clamp Techniques; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X; Signal Transduction; Up-Regulation

The present study was undertaken to determine the role of P2X3 receptor (P2X3R) on heat hyperalgesia in a newly developed rat model of trigeminal neuropathic pain. The unilateral infraorbital nerve (IoN) was partially ligated by 6-0 silk. To assess heat sensitivity, a vibrissal pad (VP) was placed on a hot plate and the latency until the rats withdrew their head was measured. Mechanical sensitivity of VP was also assessed by the use of von Frey filament. Both heat and mechanical hyperalgesia were observed at the VP ipsilateral to the IoN ligation. The latency to heat stimuli was prolonged after subcutaneous administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X1,2,3,5,7,1/5,2/3R antagonist) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP, P2X1,3,2/3,1/5R antagonist). The latency was shortened after administration of alpha,beta-methylene ATP (alpha,beta-meATP, P2X1,3,2/3R agonist), although no changes appeared after administration of beta,gamma-methylene-L-ATP (beta,gamma-me-L-ATP, P2X1R agonist). The protein gene product-9.5 and calcitonin gene-related peptide immunoreactive nerve fibers significantly decreased in the VP skin of ipsilateral to the IoN ligation. In the ipsilateral trigeminal ganglion, the number of P2X3-immunoreactive neurons significantly increased in the small cell group. In this study, we developed an experimental model of trigeminal neuropathic pain by partial ligation of IoN, which produced heat and mechanical hyperalgesia in the VP. Pharmacological and immunohistochemical studies revealed that the P2X3R plays an important role in the heat hyperalgesia observed in this model.. The study describes the development of a novel model of trigeminal neuropathic pain. Heat hyperalgesia in this model was inhibited by peripheral injection of P2XR antagonists. The results suggest that P2X3R is a potential target for development of a novel therapy for trigeminal neuropathic pain.

Topics: Adenosine Triphosphate; Animals; Calcitonin Gene-Related Peptide; Disease Models, Animal; Hyperalgesia; Immunohistochemistry; Ligation; Male; Neurons, Afferent; Nociceptors; Pain Measurement; Pain Threshold; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Inbred Lew; Reaction Time; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Trigeminal Ganglion; Trigeminal Nerve; Trigeminal Neuralgia; Ubiquitin Thiolesterase

Pain during inflammatory joint diseases is enhanced by the generation of hypersensitivity in nociceptive neurons in the peripheral nervous system. To explore the signaling mechanisms of mechanical hypersensitivity during joint inflammation, experimental arthritis was induced by injection of complete Freund's adjuvant (CFA) into the synovial cavity of rat knee joints. As a pain index, the struggle threshold of the knee extension angle was measured. In rats with arthritis, the phosphorylation of extracellular signal-regulated kinase (ERK), induced by passive joint movement, increased significantly in dorsal root ganglion (DRG) neurons innervating the knee joint compared to the naïve rats that received the same movement. The intrathecal injection of a MEK inhibitor, U0126, reduced the phosphorylation of ERK in DRG neurons and alleviated the struggle behavior elicited by the passive movement of the joint. In addition, the injection of U0126 into the joint also reduced the struggle behavior. These findings indicate that the ERK signaling is activated in both cell bodies in DRG neurons and peripheral nerve fibers and may be involved in the mechanical sensitivity of the inflamed joint. Furthermore, the phosphorylated ERK-positive neurons co-expressed the P2X3 receptor, and the injection of TNP-ATP, which antagonizes P2X receptors, into the inflamed joint reduced the phosphorylated ERK and the struggle behavior. Thus, it is suggested that the activation of the P2X3 receptor is involved in the phosphorylation of ERK in DRG neurons and the mechanical hypersensitivity of the inflamed knee joint.

Topics: Adenosine Triphosphate; Animals; Arthritis, Experimental; Axonal Transport; Butadienes; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Freund's Adjuvant; Ganglia, Spinal; Hyperalgesia; Injections, Intra-Articular; Injections, Spinal; Male; Neurons, Afferent; Nitriles; Osteoarthritis, Knee; Pain; Phosphorylation; Protein Processing, Post-Translational; Purinergic P2 Receptor Antagonists; Range of Motion, Articular; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Signal Transduction; Stifle; Stress, Mechanical

The mechanism of mechanical hyperalgesia in inflammation might involve a 'mechanochemical' process whereby stretch evokes the release of adenosine 5'-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (alphabetame-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after alphabetame-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or 2'- (or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.

Topics: Adenosine Triphosphate; Animals; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Immunohistochemistry; Inflammation; Male; Mitogen-Activated Protein Kinases; Neurofilament Proteins; Neurons; Phosphorylation; Physical Stimulation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X

Pain after nerve damage is an expression of pathological operation of the nervous system, one hallmark of which is tactile allodynia-pain hypersensitivity evoked by innocuous stimuli. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. Here we report that pharmacological blockade of spinal P2X4 receptors (P2X4Rs), a subtype of ionotropic ATP receptor, reversed tactile allodynia caused by peripheral nerve injury without affecting acute pain behaviours in naive animals. After nerve injury, P2X4R expression increased strikingly in the ipsilateral spinal cord, and P2X4Rs were induced in hyperactive microglia but not in neurons or astrocytes. Intraspinal administration of P2X4R antisense oligodeoxynucleotide decreased the induction of P2X4Rs and suppressed tactile allodynia after nerve injury. Conversely, intraspinal administration of microglia in which P2X4Rs had been induced and stimulated, produced tactile allodynia in naive rats. Taken together, our results demonstrate that activation of P2X4Rs in hyperactive microglia is necessary for tactile allodynia after nerve injury and is sufficient to produce tactile allodynia in normal animals. Thus, blocking P2X4Rs in microglia might be a new therapeutic strategy for pain induced by nerve injury.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Cells, Cultured; Disease Models, Animal; Male; Microglia; Neurons; Pain; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X4; Spinal Cord; Spinal Nerves; Touch