nonsteroidal anti - inflammatory drugs ( nsaids ) have been commonly used but are effective only for treating inflammatory pain by inhibiting prostaglandin production . understanding endogenous pain modulatory systems that alter nociceptive transmission in the nervous system may lead to the development of pain treatment . the opioid receptor ( or ) and monoamine systems are the main inhibitory transmission mechanisms against pain sensation . endogenous opioid receptor agonists are very powerful analgesic mechanisms . at the spinal cord level , clinically , or agonists , such as morphine , are commonly used for treating rather serious pain , such as postoperative pain . monoamines , such as noradrenalin   and serotonin , are also strong endogenous pain - relieving agents . increasing monoamines with reuptake inhibitors has been used for pain relief in patients with several neuropathic pains . we previously reported that serotonin reuptake inhibitors ameliorated neuropathic pain induced by spinal cord injury . in the study reported here , we focused on adenosine receptors , another endogenous nerve transmission system . adenosine is an endogenous neuromodulator that inhibits synaptic transmission in both the peripheral and central nervous systems . adenosine a1 receptor activation can produce postsynaptic inhibition by activating potassium ( k ) channels . the working mechanism of adenosine a1 receptors on pain signals is believed to inhibit pain signals by hyperpolarization of the neuronal membrane . these reports indicate that adenosine a1 receptor signal activation may become a new therapeutic method for treating several kinds of pain . we previously reported that selective adenosine a1 receptor agonists [ r - pia : r(-)n6-(2phenylisopropyl ) adenosine ] inhibited thermal hyperalgesia after spinal cord injuries in rats . to establish a clinical use for adenosine , we examined which kinds of pain are the ideal targets for adenosine treatment and the best timing for adenosine application . in the study reported here , to clarify the effects of adenosine on pain signals , we tested intrathecal adenosine injections in several kinds of animal pain models . in addition , change of adenosine receptors in the spinal cord was assessed by real - time polymerase chain reaction ( pcr ) and immunohistochemistry . one hundred and one female wistar rats ( 250  g , purchased from japan clea co. , japan ) were used for this study . experiments were performed according to the ethical recommendations of the committee for research and ethical issues of the international association for the study of pain . the research protocol was accepted by the ethical committee for animal experiments of ehime university ( ehime , japan ) . a plantar incision ( pi ) a 1-cm longitudinal incision through skin , fascia , and muscle was made in the left hind paw . the spinal cord compression model was produced according to our previous reports [ 6 , 11 ] . under general anesthesia with halothane , the rat spinal cord was carefully exposed by removing the vertebral lamina at the 11th vertebra . direct compression was performed using a 20-g weight , of which the point of contact to the dura consisted of very soft and rounded silicone . the weight was gently placed on the thoracic spinal cord extradurally for 20  min ( sci ) . we observed no serious damage , such as hyperextension , paresis of the hind limbs , or histological hemorrhage with tissue destruction at the point of compression . in some experiments , our cci model was produced according to our previous report . under general anesthesia with halothane , a 27-gauge needle was placed along the sciatic nerve and the nerve and needle were ligated using polyglycolic acid strings ( 4 - 0 , nesco company , osaka , japan ) at four points . too much constriction produces irreversible nerve damage ; therefore , careful constriction is required to produce the appropriate level of damage . usually , stable hypersensitivity , such as allodynia and hyperalgesia , was observed 3  days after operation . cl - adenosine ( 10  nmol in 10  l ; research biochemicals inc . ma , usa ) , a nonselective adenosine receptor agonist , was intrathecally injected through the intervertebral foramen between l3 and l4 at 24 and 72  h after surgery . in each model , animals injected with 10  l of saline instead of cl - adenosine solution were defined as vehicles . to evaluate the withdrawal threshold of thermal paw stimulation , we used the hargreaves plantar test apparatus ( ugo basile , varese , italy ) . rats were placed on a 2-mm - thick glass floor . a mobile infrared heat generator with an aperture of 10  mm diameter was aimed at the rat s hind paw from under the floor . when the rats felt pain and withdrew their paw , the power was shut off and the reaction time ( paw withdrawal latency ) was recorded automatically . the temperature of the glass floor was kept at 22.523.5  c .   in the cci and the pi models , l4 and l5 lumbar spinal segments 1  cm of the cord at the center of the compressed part was taken . spinal cord tissue was dissected under rnase - free conditions , and samples were stored at 80  c before use . total rna was extracted by tissue homogenization in trizol reagent , quantified by absorbance at 260  nm , normalized , and reverse - transcribed into first - strand complementary dna ( cdna ) using an rneasy mini kit ( qiagen , md , usa ) . for the a1r gene ( takara . shiga , japan ) , the forward primer was 5-atcgatacctccgagtcaagatcc-3 and the reverse primer was 5-tccagtcttgctctaccacactcag-3. for the gapdh gene ( takara ) , the forward primer was 5-ggcacagtcaaggctgagaatg-3 and the reverse primer was 5-atggtggtgaagacgccagta-3. two - step real - time pcr denaturing , annealing , and extension reactions were performed for 40 cycles of 15  s at 95  c and then 1  min at 60  c ( for a1r and gapdh ) . increasing curves of reporter dye fluorescence emission were recorded and analyzed with the sybr premix ex taq ( takara ) to determine the threshold cycle ( ct ) value . each sample was run and analyzed in triplicate , and ct values for a1r were subtracted from ct values of gapdh to yield ct values . the average ct was calculated for the control group , and this value was subtracted from the ct of all other samples ( including the control group ) . this resulted in a ct value for all samples , which was then used to calculate the fold induction of the mrna levels of a1r using the formula  2 , as recommended by the manufacture ( bio - rad , hercules , ca , usa ) [ 15 , 16 ] . the spinal cord at the 11th vertebral level ( sci model ) and l4l5 lumbar segments ( lumbar enlargement ; cci and pi models ) were immediately removed , and axial freezing microtome sections of 10-m thickness were prepared . the sections were fixed on glass slides with 4  % paraformaldehyde in phosphate - buffered saline ( pbs ) for 5  min . then , after washing twice with pbs , slices on the slides were exposed to an anti - adenosine a1r antibody ( abcam , inc . cambridge , uk : 1  g / ml in pbs ) overnight at 4  c . slices were then washed twice with pbs and exposed to a fluorescein isothiocyanate ( fitc)-conjugated anti - rabbit immunoglobulin g ( igg ) antibody ( sigma , mo , usa : 20  g / ml in pbs ) for 30  min . analysis of variance ( anova ) , followed by fisher s protected least significant difference ( plsd ) , was used . one hundred and one female wistar rats ( 250  g , purchased from japan clea co. , japan ) were used for this study . experiments were performed according to the ethical recommendations of the committee for research and ethical issues of the international association for the study of pain . the research protocol was accepted by the ethical committee for animal experiments of ehime university ( ehime , japan ) . a plantar incision ( pi ) was made similar to that described by brennan et al . . a 1-cm longitudinal incision through skin , fascia , and muscle was made in the left hind paw . the spinal cord compression model was produced according to our previous reports [ 6 , 11 ] . under general anesthesia with halothane , the rat spinal cord was carefully exposed by removing the vertebral lamina at the 11th vertebra . direct compression was performed using a 20-g weight , of which the point of contact to the dura consisted of very soft and rounded silicone . the weight was gently placed on the thoracic spinal cord extradurally for 20  min ( sci ) . we observed no serious damage , such as hyperextension , paresis of the hind limbs , or histological hemorrhage with tissue destruction at the point of compression . in some experiments , our cci model was produced according to our previous report . under general anesthesia with halothane a 27-gauge needle was placed along the sciatic nerve and the nerve and needle were ligated using polyglycolic acid strings ( 4 - 0 , nesco company , osaka , japan ) at four points . too much constriction produces irreversible nerve damage ; therefore , careful constriction is required to produce the appropriate level of damage . after the strings were inserted , usually , stable hypersensitivity , such as allodynia and hyperalgesia , was observed 3  days after operation . cl - adenosine ( 10  nmol in 10  l ; research biochemicals inc . ma , usa ) , a nonselective adenosine receptor agonist , was intrathecally injected through the intervertebral foramen between l3 and l4 at 24 and 72  h after surgery . in each model , animals injected with 10  l of saline instead of cl - adenosine solution were defined as vehicles . to evaluate the withdrawal threshold of thermal paw stimulation , we used the hargreaves plantar test apparatus ( ugo basile , varese , italy ) . rats were placed on a 2-mm - thick glass floor . a mobile infrared heat generator with an aperture of 10  mm diameter was aimed at the rat s hind paw from under the floor . when the rats felt pain and withdrew their paw , the power was shut off and the reaction time ( paw withdrawal latency ) was recorded automatically . shortening withdrawal latency indicated thermal hyperalgesia . in the cci and the pi models , l4 and l5 lumbar spinal segments were taken for messenger rna ( mrna ) measurement . in the sci model , 1  cm of the cord at the center of the compressed part was taken . spinal cord tissue was dissected under rnase - free conditions , and samples were stored at 80  c before use . total rna was extracted by tissue homogenization in trizol reagent , quantified by absorbance at 260  nm , normalized , and reverse - transcribed into first - strand complementary dna ( cdna ) using an rneasy mini kit ( qiagen , md , usa ) . for the a1r gene ( takara . shiga , japan ) , the forward primer was 5-atcgatacctccgagtcaagatcc-3 and the reverse primer was 5-tccagtcttgctctaccacactcag-3. for the gapdh gene ( takara ) , the forward primer was 5-ggcacagtcaaggctgagaatg-3 and the reverse primer was 5-atggtggtgaagacgccagta-3. two - step real - time pcr denaturing , annealing , and extension reactions were performed for 40 cycles of 15  s at 95  c and then 1  min at 60  c ( for a1r and gapdh ) . increasing curves of reporter dye fluorescence emission were recorded and analyzed with the sybr premix ex taq ( takara ) to determine the threshold cycle ( ct ) value . each sample was run and analyzed in triplicate , and ct values for a1r were subtracted from ct values of gapdh to yield ct values . the average ct was calculated for the control group , and this value was subtracted from the ct of all other samples ( including the control group ) . this resulted in a ct value for all samples , which was then used to calculate the fold induction of the mrna levels of a1r using the formula  2 , as recommended by the manufacture ( bio - rad , hercules , ca , usa ) [ 15 , 16 ] . the spinal cord at the 11th vertebral level ( sci model ) and l4l5 lumbar segments ( lumbar enlargement ; cci and pi models ) were immediately removed , and axial freezing microtome sections of 10-m thickness were prepared . the sections were fixed on glass slides with 4  % paraformaldehyde in phosphate - buffered saline ( pbs ) for 5  min . then , after washing twice with pbs , slices on the slides were exposed to an anti - adenosine a1r antibody ( abcam , inc . cambridge , uk : 1  g / ml in pbs ) overnight at 4  c . slices were then washed twice with pbs and exposed to a fluorescein isothiocyanate ( fitc)-conjugated anti - rabbit immunoglobulin g ( igg ) antibody ( sigma , mo , usa : 20  g / ml in pbs ) for 30  min . for statistical analysis , analysis of variance ( anova ) , followed by fisher s protected least significant difference ( plsd ) , was used . in all three kinds of pain models , significant shortening of withdrawal latencies were detected from 24  h to 1  week after the surgery ( fig .  1 ) . hyperalgesia was seen only ipsilaterally to the surgery side in the pi and cci models . in the foot contralateral to the operation , withdrawal latencies against thermal stimulation was identical to that in normal animals ( 11.1    0.24  s).fig . plantar incision ( pi ; n  =  6 ) , chronic constriction injury ( cci ; n  =  6 ) , and spinal cord injury ( sci ; n  =  15 ) were performed . statistical significance compared with preoperation levels for each time point is represented with an asterisk ( * p  <  0.05 ) time course of withdrawal latency by thermal stimulation following three kinds of pain models . plantar incision ( pi ; n  =  6 ) , chronic constriction injury ( cci ; n  =  6 ) , and spinal cord injury ( sci ; n  =  15 ) were performed . measurements were taken every 24  h until the 7th day following operations . data are the mean    standard error of mean ( sem ) . statistical significance compared with preoperation levels for each time point is represented with an asterisk ( * p  <  0.05 ) in the postsurgical model ( pi model ; fig . 2 ) , apparent thermal hyperalgesia was seen in the foot ipsilateral to the pi in the vehicle animal both at 24 and 72  h after the operation . when cl - adenosine ( 10  nmol in 10  l ) was applied 24  h after surgery , pain threshold returned to normal ( p  <  0.05 , 11.37    0.97  s ) . however , the antihyperalgesic action of cl - adenosine was not seen when the injection was done 72  h after the operation . in the cci model ( fig . 3 ) , hyperalgesia was also seen in the foot ipsilateral to sciatic nerve constriction in the vehicle animal both at 24 and 72  h after the operation . intrathecal injection of cl - adenosine normalized withdrawal threshold against thermal stimulation at 24 ( 11.36    0.69  s ) and 72  h ( 12.07    0.19  s ) after the operation . the difference of the cl - adenosine effect in the pi and cci models was seen at 72  h after the operation . in the sci model ( fig .  4 ) , thermal threshold decreased both at 24 and 72  h after the operation compared with that in the sham - operated animals . intrathecal injection of cl - adenosine significantly ( p  <  0.05 ) increased withdrawal latency both at 24 ( 11.12    0.55  s ) and 72  h ( 10.47    0.67  s ) after the operation.fig . 2effects of cl - adenosine on thermal stimulation in the plantar incision model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time point ) . asterisk statistical significance ( * p  <  0.05)fig . 3effects of cl - adenosine on thermal stimulation in the chronic constriction injury model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time point ) . 4effects of cl - adenosine on thermal stimulation in the spinal cord injury model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time points ) . asterisk , statistical significance compared vehicle animal ( * p  <  0.05 ) effects of cl - adenosine on thermal stimulation in the plantar incision model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time point ) . asterisk statistical significance ( * p  <  0.05 ) effects of cl - adenosine on thermal stimulation in the chronic constriction injury model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time point ) . asterisk statistical significance ( * p  <  0.05 ) effects of cl - adenosine on thermal stimulation in the spinal cord injury model at 24  h ( a ) and 72  h ( b ) after surgery . data are mean    standard error of mean ( sem ) ( n  =  6 in each time points ) . asterisk , statistical significance compared vehicle animal ( * p  <  0.05 ) real - time pcr analysis was represented as folds to normal mrna content ( fig . twenty - four hours after operation , adenosine a1r mrna expression significantly ( p  <  0.05 ) decreased in the pi model ( 0.2 folds compared with the normal level ) . in the sci model , adenosine a1r mrna level decreased to 0.7 folds . on the other hand , in the cci model it remarkably increased ( 2.5 folds ) , at 72  h after operation in all three models it moved toward the normal level . however , in the pi model , it was still lower than normal ( 0.7 folds).fig . 5comparison of adenosine a1r messenger rna ( mrna ) expression by real - time polymerase chain reaction ( pcr ) analysis in the normal group and the three pain models . data are mean    standard error of mean ( sem ) ( n  =  3 in each column ) . asterisk statistical significance compared with normal group ( * p  <  0.05 ) comparison of adenosine a1r messenger rna ( mrna ) expression by real - time polymerase chain reaction ( pcr ) analysis in the normal group and the three pain models . data are mean    standard error of mean ( sem ) ( n  =  3 in each column ) . asterisk statistical significance compared with normal group ( * p  <  0.05 ) adenosine a1r protein expression was evaluated immunohistochemically . in the normal animals , twenty - four hours after operation , a1 receptors were maintained in all three models ( data not shown ) . three days after operation , a1 receptors were rather increased in the dorsal horn of the cci model . a1 receptor levels in the sci model were similar to that in the normal animal . on the other hand , a1 receptor proteins remarkably decreased in pi models 3  days after operation compared to the other two models and normal control ( fig . seventy - two hours after operation , spinal cord sections from the 11th vertebra of the spinal cord mild - compression ( sci ) model and l4l5 lumbar segment [ lumbar enlargement ; plantar incision ( pi ) and chronic constriction injury of sciatic nerve model ( cci ) models and normal rat ] were stained by anti - adenosine a1r antibody . normal rat ( a ) , pi model ( b ) , cci model ( c ) , sci model ( d ) . apparent decrease in adenosine a1r expression was observed in pi model adenosine a1r protein expression in spinal cord dorsal horn . seventy - two hours after operation , spinal cord sections from the 11th vertebra of the spinal cord mild - compression ( sci ) model and l4l5 lumbar segment [ lumbar enlargement ; plantar incision ( pi ) and chronic constriction injury of sciatic nerve model ( cci ) models and normal rat ] were stained by anti - adenosine a1r antibody . normal rat ( a ) , pi model ( b ) , cci model ( c ) , sci model ( d ) . the dorsal horn of the spinal cord is believed to be one of the most important areas for pain signal transmission . in the spinal cord , adenosine a1 receptors are mainly distributed in postsynaptic neuronal - cells bodies and processes in dorsal superficial layers ( lamina ii ) . we also found that dense adenosine a1 receptor proteins existed in lamina ii of the dorsal horn ( fig . this area is not only for adenosine signal transmission but is also the main area of adenosine production . ecto-5-nucleotidase is a membrane - anchored protein that hydrolyzes extracellular adenosine 5-monophosphate ( amp ) to adenosine . ecto-5-nucleotidase is located on nociceptive neurons in dorsal root ganglia and on axon terminals in lamina ii ( substantia gelatinosa ) of the spinal cord . thus , this area should be a main spot of endogenous pain relief by adenosine signaling . we found the adenosine a1 receptor mrna remarkably decreased in the spinal cord after pi but increased after sciatic nerve constriction ( fig . this suggests that differential working mechanisms of adenosine in pain modulation existed in postoperative and neuropathic pain conditions . it is not clear why adenosine a1 receptor expression in the spinal cord dorsal horn was enhanced in our cci models . a remarkable difference between the cci and pi models is microglia activation in the spinal cord . microglia proliferation and activation occurs in the spinal cord in the cci model . on the other hand , the activated microglia in the spinal cord may enhance the pain signal by releasing nitric oxide or cytokines . therefore , it is possible that adenosine a1 receptor mrna expression enhanced to counteract the effect of activated microglia in our cci model . the change in adenosine a1 receptor mrna expression of the sci model was not remarkable compared with that in the cci model ( fig . although activated microglia also enhanced adenosine a1 receptor production in sci model , direct ischemic damage might scale back the amount of a1 receptors . the pathological pain mechanism of the sci model is different from that in the cci model . sci - induced pain is reported to induce inhibition of tonic descending inhibitory mechanisms , such as serotonergic and noradrenergic signaling , in the spinal cord . therefore , the working mechanisms of injected cl - adenosine were different between cci and sci models . in the sci model , injected cl - adenosine may compensate damaged descending inhibitory signaling from brain to spinal cord . in the cci model , in contrast , sensitivity to adenosine was increased in the dorsal horn by augmented adenosine a1 receptor expression . the reason for decreased adenosine a1 receptor expression in our pi model also remains unclear . hippocampal a1 receptor immunoreactivity in rats with cortex - kindled seizures remarkably decreased compared with that in normal rats . the number of a1 receptors in the hippocampus was also decreased in aged rats compared with young adult rats . in aged rats , in addition , the amounts of adenosine in the synaptic cleft further increased due to the decrease in adenosine transporter activity . it is possible that repetitive overstimulation of increased extracellular adenosine may induce adenosine a1 receptor expression inhibition in these models . in the pi pain model , stimulation from the hind paw may accelerate adenosine triphosphate ( atp ) release from the afferent nerve terminals in the dorsal horn . then , adenosine a1 receptor mrna expression may be depressed by overstimulation of adenosine in the same manner as in aged or kindled rat . released atp , which is the source of extracellular adenosine , may be depleted with time ; however , adenosine a1 receptor expression inhibition continued for several days after pi . as adenosine is a common substance existing in the entire nervous system , it inhibits nerve transduction at the baseline level . when the adenosine a1 receptor was blocked at the spinal cord level , the threshold of thermal pain decreased ( heat hyperalgesia ) . therefore , when adenosine a1 signal is reduced by decreasing adenosine a1 receptors , pain signals should be enhanced . if this phenomenon is a rational control of the self - protection system , a1 receptor inhibition may induce hyperalgesia as cautionary notices to avoid further nociception . the striking decrease of a1 receptor mrna occurred 24  h after pi ( fig . 5 ) , mrna level recovered within another 2  days , and change in a1 receptor protein occurred later than that in mrna level . decreased a1 receptor protein was apparent 3  days after pi ( fig .  6 ) . this indicates that desensitizing signals from adenosine are not effective for several days after incision . on the other hand , the desensitizing mechanism of adenosine signals was maintained after nerve injury . therefore , although adenosine is a potential pain - relief agent , application of adenosine may not be effective in postoperative pain compared with that in neuropathic pain . or agonists have been used to treat several types of neuropathic or postoperative pain . however , several studies suggest that or agonists , like morphine , show decreased analgesic potency against neuropathic pain . after peripheral nerve injuries , these reports suggest that or agonist application for treating neuropathic pain after a peripheral injury is not rational . relatively large amounts of the agonist may be required to achieve a large enough analgesic effect , which may produce considerable side effects such as constipation or nausea / vomiting . these results suggest that adenosine may be a reasonable choice for treating neuropathic pain that has not responded well to the opioids . clinically , chronic neuropathic pain is one of the most serious pathological conditions in the field of orthopedic surgery . as a result of this study , we propose a new possible therapeutic module for treating neuropathic pain after peripheral nerve injuries . brain barrier permeability , intrathecal injection of adenosine a1 receptor agonists , such as cl - adenosine or r - pia using a continuous infusion pump may effectively inhibit chronic neuropathic pain . alternative ideas to increase the adenosine effect are to increase extracellular adenosine concentrations by amplifying production by ecto-5-nucleotidase or to inhibit adenosine uptake or degradation . propentofylline , an adenosine reuptake inhibitor with a blood  brain barrier permeability , is reported to inhibit pain behavior after peripheral nerve injury in the rat . adenosine reuptake , kinase , and deaminase inhibitors may provide an avenue for the development of novel therapeutic methods against neuropathic pain . our study indicates that intrathecally administered adenosine may be an effective pain - relief therapy for neuropathic pain but is not effective on postoperation pain . we also found a decrease in adenosine a1 receptors in the spinal cord dorsal horn of the pi model , which may explain the variation of adenosine effects among the three pain models . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .