Effects And Molecular Mechanism Of Tramadol Combined With Acetaminophen On A Rat Model Of Neuropathic Pain

Effects and molecular mechanism of tramadol combined with acetaminophen on a rat model of neuropathic painNeuropathic pain is a kind of pain syndrome induced by injuries or diseases in central or peripheral nervous system.It is usually chronic pain,last several days or months,which is different from the acute pain caused by tissue injury,and conventional single drugs couldn't cause evident effects to neuropathic pain.The clinical causes of neuropathic pain are extensive,and the pathomechanism and symptoms are complicated and variform.The pathophysiologic characteristic mainly appears as hyperreactivity to algesthesia,such as hyperalgesia,allodynia and spontaneous pain,et al.The clinical traditional drugs usually target at neurons,while more and more recent literatures indicate that glial cells(astrocytes and microglia) and pro-inflammatory cytokines(tumor necrosis factor-α(TNFα), interleukin-1(IL-1),interleukin-6(IL-6),etc.) released from the glial cells play significant roles in the initiation and maintenance of neuropathic pain,providing a new viewpoint on drug discovery for clinical pain prevention and control.The sequential treatment of conventional single drugs in present clinical work usually could't produce satisfactory effect due to the complexity of the mechanism of neuropathic pain.The combination of different paregoric drugs with different machanism to treat neuropathic pain can be more effective,also can reduce the paregoric drugs' side effects on account of the reduction of dosages.Tramadol and acetaminophen are both paregoric drugs commonly used in clinic,and usually used to relieve lower grade pain and medium pain,but seldom applied in the treatment ofneuropathic pain.Their machanism and the combination of the two drugs in the treatment of neuropathic pain have been less intensely studuied.Using the rat model of L₅ spinal nerve transection,we investigated the effects and mechanism of tramadol combined with acetaminophen on mechanical allodynia and thermal hyperalgesia by studing the relationship between pain behavior and expression of microglia and pro-/anti-inflammatory cytokines.Partâ… ,Establishment of neuropathie pain model and investigation of the related mechanismâ… .Establishment of neuropathic pain rats model and alteration of mechanical and thermal pain thresholdsThe rats with normal pain sensation were sieved from male Sprague-Dawley rats(230-280 g) between the age of 10-15 weeks.Using a nerve injury model of L₅ spinal nerve ligation and transection(modified from Kim and Chung),we investigated the general ethology after operation,and measured the hind paw mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) on the 1^st,3^rd,7^th,14^th and 21^st day after operation with von Frey hairs and Hargreaves tests,respectively.Results:conspicuous mechanical allodynia and thermal hyperalgesia appeared and reached the peak on the 3^rd day in the rats after spinal nerve injury which were still with good general state of health,suggesting that all rats have successfully developed neuropathic pain syndromes.â…¡.The investigation of mechanism underlying rat model of neuropathic pain(â… ) Myelonie expression of mieroglia in neuropathie pain ratsAfter the measurement of mechanical and thermal pain thresholds,the rats were sacrificed with paraform.The lumbar spinal cord containing the L₄-L₅ segments was immunostained for microglial activation on the 1^st,3^rd,7^th,11^th,14^th and 21^st day after spinal nerve injury.Results:OX-42 immunoreactive microglial activation was less expressed in normal spinal cord dorsal horns,with a smaller soma beating small processus spinosus and slender branches;while after operation,prominent microglial proliferation was observed at each time points,their branches coarsened and lengthened,and they changed their morphology into amoeboid shape.By counting the number of cells,we found that the expression of microglial activation reached the peak on the 3^rd and the 7^th day,and then gradually decreased to normal level on the 21^st day after operation,which was similar to the changes of pain thresholds of rats.(â…¡) Expression of pro-/anti-inflammatory eytokines in the CNS of neuropathie pain ratsAfter the measurement of mechanical and thermal pain thresholds on the 1^st,3^rd,7^th,11^th, 14^th and the 21^st day after spinal nerve injury,the rats were sacrificed.The brain and all the spinal cord tissues(from cervical cord to sacral cord) were removed to liquid nitrogen for assays.Expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),prostaglandin E₂(PGE₂) and interleukin-10(IL-10) in the CNS was quantified using enzyme-linked immunosorbent assay(ELISA).Results:compared with normal rats,the expression of pro-inflammatory cytokines TNF-α,IL-1βand PGE₂ in the CNS were significantly elevated after operation,all being maximum on the 3^rd day(PGE₂ on the 7^th day),and coincident with the development of pain threshold,while the expression of anti-inflammatory cytokine IL-10 undergo a progressive increase after operation,reaching the maximum on the 21^th day.(â…¢) Expression of nuclear factor-κB(NF-κB) in the CNS of neuropathic pain ratsAnimals were allowed to survive for 1,3,7,11,14,and 21 days after spinal nerve injuries,respectively.EMSA(electrophoretic mobility shift assay) experiments were performed to examine the activation of NF-κB.Results:Spinal cord and cerebral expression of NF-κB was markedly increased after operation compared with normal rats,and both peaked on the 3^rd day,suggesting that spinal nerve injury might induce the activation of NF-κB in the CNS,and enable the level of NF-κB keep higher during the whole lab process.Partâ…¡,The synergistic effects of tramadol combined with aeetaminophen on neuropathic pain rats modelâ… .Synergistic effects of the intraperitoneal injection of tramadol combined with acetaminophen on neuropathic pain rats modelThe rats with L₅ spinal nerve injury were randomly assigned into ten groups according to different drugs injected through the way of abdominal cavity on the 7^th day after operation: group 1,acetaminophen 40 mg/kg;group 2,acetaminophen 80 mg/kg;group 3, acetaminophen 160 mg/kg;group 4,tramadol 5 mg/kg;group 5,tramadol 10 mg/kg;group 6, tramadol 20 mg/kg;group 7,acetaminophen 40 mg/kg+tramadol 5 mg/kg;group 8, acetaminophen 80 mg/kg+tramadol 10 mg/kg;group 9,acetaminophen 160 mg/kg+ tramadol 20 mg/kg;group 10,the saline group(control group).Results:compared with the saline group,pronounced elevated mechanical and thermal pain threshold and conspicuous reduced mechanical allodynia and thermal hyperalgesia occurred at each time points in all the experimental groups except the acetaminophen-treated groups and the tramadol 5 mg/kg-treated group;compared with the groups which used tramadol alone,the intraperitoneal injection of tramadol combined with acetaminophen could bring synergistic antinociptive effect to mechanical allodynia and thermal hyperalgesia in spinal nerve ligated rats.â…¡.Synergistic effects of the intrathecal injection of tramadol combined with acetaminophen on neuropathic pain rats modelThe rats with L5 spinal nerve injury were randomly assigned into ten groups according to different drugs intrathecal injected on the 7^th day after operation:group 1,acetaminophen 8μg;group 2,acetaminophen 40μg;group 3,acetaminophen 80μg;group 4,tramadol 1μg; group 5,tramadol 5μg;group 6,tramadol 10μg;group 7,acetaminophen 8μg+tramadol 1μg;group 8,acetaminophen 40μg+tramadol 5μg;group 9,acetaminophen 80μg+ tramadol 10μg;group 10,the saline group(control group).Results:compared with the saline group,pronounced elevated mechanical and thermal pain threshold and conspicuous reduced mechanical allodynia and thermal hyperalgesia occurred in all the experimental groups except the acetaminophen-treated groups and the tramadol 1μg-treated group; compared with the groups which used tramadol alone,the intrathecal injection of tramadol combined with acetaminophen could bring synergistic antinociptive effect to mechanical allodynia and thermal hyperalgesia in spinal nerve ligated rats.Partâ…¢,The effects and underlying machanism of tramadoi combined with aeetaminophen on neuropathic pain rats modelâ… .Effects of tramadoi combined with acetaminopnen on the ethology on the formation and after the establishment of neuropathic pain rats model(â… ) Effects of tramadol combined with acetaminopnen on the formation of neuropathic pain rats modelThe rats with L₅ spinal nerve injury were randomly assigned into four groups according to different drugs injected through the way of abdominal cavity which were administered twice daily(10:00 and 20:00) and lasted seven days from the day after operation:group 1, tramadol 20 mg/kg+acetaminophen 160 mg/kg;group 2,tramadol 20 mg/kg;group 3, acetaminophen 160 mg/kg;group 4,saline.Results:compared with the saline group,the tramadol-treated group and the tramadol combined with acetaminophen-treated group could both restrain the formation of mechanical allodynia and thermal hyperalgesia in neuropathic pain rats,and the depressant effect was the most conspicuous from the 3^rd to the 7^th day, moreover the depressant effect in the tramadol combined with acetaminophen-treated group could last to the 21^st day.(â…¡) Effects of tramadol combined with acetaminopnen after the establishment of neuropathic pain rats modelThe rats with L₅ spinal nerve injury were randomly assigned into four groups according to different drugs injected through the way of abdominal cavity which were administered twice daily(10:00 and 20:00) and lasted seven days from the 7^th day after operation:group 1, tramadol 20 mg/kg+acetaminophen 160 mg/kg;group 2,tramadol 20 mg/kg;group 3, acetaminophen 160 mg/kg;group 4,saline.Results:compared with the saline group, tramadol treatment and tramadol combined with acetaminophen treatment could both lessen the degree of mechanical allodynia and thermal hyperalgesia in neuropathic pain rats,and the effect of lessening mechanical allodynia and thermal hyperalgesia in neuropathic pain rats in the tramadol combined with acetaminophen-treated group could last to the 14^th day after operation,but compared with sham group,there is no significant difference on the variance of MWT and TWL,and return to the baseline after stopping injection.â…¡Effects of tramadol combined with aeetaminophen on spinal cord mieroglial activation on the formation and after the establishment of neuropathie pain rats modelThe expression of microglia was investigated by immunohistochemistry of lumbar spinal cord containing the L₄-L₅ segments in different groups.Results:OX-42 immunoreactive microglia in each group significantly proliferated with hypertrophic soma and manifolded branches which was like a crown of a tree after spinal nerve injury,and mainly spotted in the spinal cord dorsal horns cinerea on the operation side.Compared with the saline group, pre-injection with the combination of tramadol and acetaminophen significantly inhibited the activation of microglia after operation,with a peak of activation on the 7^th day,and then gradually decreased to normal level by the 21^st day.While the diffference of the effect on the expression of microglia was unconspicuous in the tramadol or acetaminophen single used groups compared with the saline group,but compared with the saline group,there is no significant difference on the expression ofmicroglia after neuropathic pain is established.â…¢Effects of tramadol combined with aeetaminophen on the expression of pro-/anti-inflammatory cytokines in the CNS on the formation and after the establishment of neuropathic pain rats modelELISA was used to measure the expression of TNF-α,IL-1β,PGE₂ and IL-10 in the CNS of the rats in different groups on the 1^st,3^rd,7^th,11^th,14^th and 21^st day after spinal nerve injury.Results:Compared with the saline group,the acetaminophen-pretreated group and tramadol combined with acetaminophen-pretreated group could both inhibit the level of pro-inflammatory cytokines TNFα,IL-1βand PGE₂ after operation at each time points,while significantly induce the expression of anti-inflammatory cytokines IL-10,which peaked on the 11^th day after operation,while tramadol combined with acetaminophen-treated group after neuropathic pain is established also could inhibit the level of pro-inflammatory cytokines TNFα,IL-1βand PGE₂.â…£Effects of tramadol combined with aeetaminophen on NF-κB expression in the CNS on the formation and after the establishment of neuropathie pain rats modelActivation of NF-κB in the CNS was quantified on the 1^st,3^rd,7^th,11^th,14^th and 21^st day after spinal nerve injury in different groups.Results:tramadol combined with acetaminophen-pretreated group could significantly inhibit the activation of NF-κB in the CNS after operation at each time points compared with the saline group,coincident with the changes of pain thresholds in rats,but compared with the saline group,there is no significant difference on the inhibition of the activation of NF-κB after neuropathic pain is established.Conclusion In the present study,we established a rat model of neuropathic pain by L₅ spinal nerve injury,investigated the effects of tramadol combined with acetaminophen on mechanical and thermal hyperalgesia in rats,detected the expression of NF-κB,pro-/anti-inflammatory cytokines and microglia coherent with pain,and investigated the mechanism.We found that microglia and the inflammatory neurogens released from microglia were involved in the formation and development of allodynia and hyperalgesia,neural inflammation and neural immune reaction in the CNS participated in the adjusting process of neuropathic pain.The intraperitoneal injection of tramadol combined with acetaminophen could prevent the formation of neuropathic pain in rats,which might act by inhibiting the expression of microglia,the activity of NF-κB,the content of pro-inflammatory cytokines and up-regulating the level of anti-inflammatory cytokines.The results observed may provide an experimental basis of clinical medication for pathological pain control.