Involvement Of Peripheral And Spinal Group I MGluRs In The Formalin Induced Long-term Hyperalgesia And Spinal Microglia Activation And Potential Mechanisms Underlying

Within just the past decades,there has been increasing recognition that glia are far more than simply "housekeepers" for neurons,providing neurochemical precursors and energy sources to neurons,regulating extracellular ion concentrations,removing debris,and so on.It is now known that glia is also involved in the fever,alterations in sleep,disruption of learning and memory,as well as to neuroinflammatory/neurodegenerative conditions such as ischemia/stroke, Alzheimer's disease,and Parkinson's disease(Linda et al.,2007).In addition,recently glia is recognized to participate in creating and maintaining pain facilitation(Linda et al.,2007). Traditionally,the experimental model employed to use to demonstrate nociception or pain mechanisms focused exclusively on neurons,without pay attention to glia,as glial cells were not thought of in terms of neuronal signaling.Despite many decades of research and drug development,pain facilitation remains unresolved,and clinical trial pain is poorly controlled by currently available therapeutics,since all the trial were designed to target neurons.Increasing evidences suggest that glia cells(including microglia and astrocyte)are activated in almost all of chronic pain model.Subcutaneous formalin injection,peripheral nerve or spinal cord exposure to human immunodeficiency virus-1 viral envelope glycoprotein(gp120),and peripheral nerve and dorsal root injury all produce mechanical allodynia and thermal hyperalgesia,and meanwhile there are activation of microglias and astrocytes in the spinal cord dorsal horn(Colburn et al., 1999;Coyle et al.,1998;Herzberg et al.,2001;Milligan et al.,2000).Microglia are exquisitely sensitive to signals of "not self" or "not normal",leading them to be far more reactive in responding to CNS challenges than other candidate cell types(Kreutzberg,1996).Similarly, spinal cord microglia is the earliest glial cell type activated in response to peripheral inflammation and injury.Intrathecal injection of minocycline,microglia inhibitor,can prevent the onset of enhanced pain and the rapid upregulation of microglial activation markers relative to those of astrocytes.The formalin test has been widely used as an acute inflammatory model,characterized by biphasic behavioral responses(lasting for 1 to 2 hours)namedacute and tonic phase,which could be quantitively measured by accounting the time for lifting and licking towards the injected place or by other behavioral responses.However,recent evidence shows that subcutaneous injection of formalin could also produce dramatically a long-term hyperalgesia (LTH)even lasting for several weeks,meanwhile a strong activation of spinal microglia was observed.Previous study in our lab demonstrated that peripheral and spinal groupⅠmGluRs mediated the acute nociceptive process induced by formalin injection.The present study was designed to investigate if peripheral and spinal groupⅠmGluRs were involved in the formalin-induced LTH and the underlying mechanisms.The experiment includes three parts:1) observing the effects of peripheral application of the mGluR1-antagonist CPCCOEt or the mGluR5-antagonist MPEP,respectively,on the long-term hyperalgesia and spinal microglia activation;2)observing the effects of intrathecal application of CPCCOEt or MPEP,respectively, on the long-term hyperalgesia and spinal microglia activation;3)observing whether the p38 MAPK pathway mediated nociceptive facilitation of groupⅠmGluRs in the long-term hyperalgesia and spinal microglia activation induced by formalin injection.PartⅠPeripheral GroupⅠmGluRs are Involved in the Formalin Induced Long-Term Hyperalgesia and Activation of Spinal MicrogliaPrevious studies in our lab showed that peripheral groupⅠmGluRs are involved in the acute biphasic nociception.The present study was designed to observe the effects of peripheral application groupⅠmGluRs antagonist on the formalin-induced long-term hyperalgesia and the activation of spinal microglia.Experiments were performed on adult male Sprague-Dawley rats weighing 100-120g.Rats were divided into the following 3 groups(n=30 per group),CPCCOEt(100 nmol,25μl),MPEP (50 nmol,25μl)or HEPES(100 mM,pH 7.4)as control,was injected into the dorsal surface of the left hind paw 15 min prior to formalin injection and again 1,2,3 and 4 day after formalin injection.Paw withdrawal latency(PWL)in response to thermal stimuli was tested 1d before formalin injection as the baseline and 2h,1d,3d,7d,10d,2w,3w and 4w after formalin injection (including the ipsilateral and contralateral hindpaw),and immunohistochemistry observation was performed 2h,1d,7d,2w,3w and 4w after formalin injection.Each rat which join into immunohistochemistry observations were carried out to test its baseline behavioral response and behavioral response just before it was killed.The immunohistochemical intensity of microglia was compared by image and statistical analysis.Pain behavioral can be observed immediately after formalin treatment of the hindpaw of rats,which displayed the significant decrease of PWL and peaked in 2h lasting more than 10 d after formalin injection,and then fell to baseline at 14 d,21 d after formalin injection PWL increased again,and fell to baseline at 28 d.In CPCCOEt or MPEP treated group,PWL in ipsilateral hindpaw were increased,the differences were significant at 2h,1day and 3dayImmnohistochemical results showed that in CPCCOEt or MPEP treated group,OX-42 positive expressing in ipsilateral side was attenuated significantly compared with control (P＜0.05)at 2 h,7 d with significant differences respectively.These results suggested that:1)peripheral administration of formalin induced thermal hyperalgesia with peak at 2 h and lasted for more than 10 d,meanwhile spinal microglia activation were observed.The activation of spinal microglia may be one of the key factors mediating the long-term hyperalgesia;2)Peripheral administration of CPCCOEt,an antgonist of mGluR1,or MPEP,an antgonist of mGluR5,inhibited both long-term thermal hyperalgesia and spinal microglia activation.PartⅡSpinal GroupⅠmGluRs are Involved in the Formalin Induced Long-Term Hyperalgesia and Activation of Spinal MicrogliaThe first part of the present study showed that peripheral groupⅠmGluRs was involved in the long-term thermal hyperalgesia and spinal microglia activation.The present study try to determine whether spinal groupⅠmGluRs involved in the generation of formalin-induced long-term hyperalgesia and the activation of spinal microglia.Experiments were performed on adult male Sprague-Dawley rats weighing 100-120g.Rats were divided into the following 3 groups(n=18 per group):CPCCOEt(100 nmol,5μl),MPEP (50 nmol,5μl)or HEPES(100 mM,pH7.4,5μl)as control was intrathecally injected into the the lumbar enlargement of the spinal cord 15 min prior to formalin peripheral injection and again 1,2,3 and 4 day after formalin injection.Paw withdrawal latency in response to thermal stimuli was tested 1d before formalin injection as the baseline and 2h,1d,3d,7d,2w,3w and 4w after formalin injection(including the ipsilateral and contralateral hindpaw),and immunohistochemistry observation was performed 2h,7d,and 4w after formalin injection.The immunohistochemical intensity of microglia was compared by image and statistical analysis.Pain behavioral in control group can be observed immediately after formalin treatment of the hindpaw of rats,which displayed the significant decrease of PWL and peaked in 2h lasting more than 7 d after formalin injection,and then fell to baseline.In CPCCOEt group,PWL in ipsilateral hindpaw were increased,the differences were significant at 2h,1d,3d and 7d(P＜0.05). In MPEP group,PWL in ipsilateral hindpaw were increased,the differences were significant at 2 h and 1d(P＜0.05).Immnohistochemical results showed that in CPCCOEt or MPEP treated group,OX-42 positive expressing in ipsilateral side was attenuated significantly compared with control (P＜0.05)at 2 h and 7 d with significant differences respectively.These results suggested that:1)Spinal administration of CPCCOEt,or MPEP inhibited the long-term thermal hyperalgesia and spinal microglia activation;2)Spinal groupⅠmGluRs was involved in the long-term thermal hyperalgesia and spinal microglia activation.PartⅢMechanisms Underlying the Involvement of Peripheral and Spinal GroupⅠmGluRs in the Formalin Induced Long-term Hyperalgesia and Activation of Spinal Microglia—Analysis of p38 MAPK PathwayMitogen-activated protein kinases(MAPKs)are a family of important intracellular signaling molecules that transduce extracellular stimuli into intracellular responses in a wide variety of circumstances and controls cell function and survival in eukaryotes(Robinson and Cobb 1997;Widmann et al.1999;Johnson and Lapadat 2002).Three subfamilies of MAPKs have been identified in mammals:extracellular signal-regulated kinase(ERK),c-Jun NH2-terminal kinase(JNK),and p38 MAPK.As a member of the MAPK family p38 is known to regulate events associated with cellular stress such as inflammatory cytokines,heat shock, ultraviolet light,and ischemia(Widmann et al,1999).Accumulating evidences demonstrate that p38 MAPK is also invovled in the generation and maintenance of pain hypersentivity and pain facilitation and the activation of p38 mitogen-activated protein kinase(p38)in the spinal cord has been seen primarily in microglia(Kim et al.,2002;Milligan et al.,2003;Svensson et al., 2003a,b;Tsuda et al.,2004).Take together,evidences indicate that microglia might participate in the nociceptive process,especially pain facilitation or hyperalgesia;p38 MAPK(probably in microglia)also involved in nociception,and at least one of the cellular signal pathway underlying the pain facilitation in spinal level.Based on the preliminary experiments,in the present study,we tested the hypothesis that the p38 MAPK pathway mediated nociceptive facilitation of groupⅠmGluRs in the long-term hyperalgesia and spinal microglia activation induced by formalin injection.Experiments were performed on adult male Sprague-Dawley rats weighing 100-120g. Firstly we observed the level of phosphorylated p38(P-p38)expression in normal condition and the change of expression of P-p38 at different time point,5,15,60min,1,3,7,14,28 day after formalin injection assessed by western blotting.15mins before formalin injection,CPCCOEt or MPEP was given by peripheral or intrathecal injection.The lumbar enlargement of spinal cord was removed 5 minute or 1 day after formalin injection to test the effects of peripheral and spinal treatment of groupⅠmGluRs antagonists on the level of P-p38 in spinal cord.Results showed that if the baseline level of P-p38 was considered as 100%,the data of each time point treated with formalin was 303.78%,182.56%,101.68%,273.84%,101.22%,174.78%, 104.08%,108.97%,respectively.5 min after formalin injection into the plantar surface of the hind paw,a threefold increase in P-p38 immunoreactivity was detected in homogenates of lumbar ipsilateral dorsal horn by western blotting.P-p38 level returned towards baseline by 1 h. 1 d after formalin injection P-p38 level increased again and in turn returned towards baseline.Peripheral or intrathecal treatment of CPCCOEt or MPEP decreased the P-p38 level of 5 mins post formalin injection.Meanwhile the increased P-p38 level 1 day after formalin injection was inhibited only by peripheral treatment of CPCCOEt.The present results suggested that:1)Peripheral formalin injection induced p38 MAP kinase activation in spinal cord,which mediated the generation of long-term inflammatory pain; 2)Peripheral treatment of the antagonists of groupⅠmGluRs suppressed the increase of P-p38 5 minute after formalin injection;3)Intrathecal treatment of the antagonists of groupⅠmGluRs suppressed the phosphorylation of p38 MAPK 5 minute after formalin injection.4)These indicated that the involvement of peripheral and spinal groupⅠmGluRs in the pain process were mediated by p38 MAPK pathway. Conclusion:1.peripheral administration of formalin induced thermal hyperalgesia,lasting for more than 10 days,and meanwhile spinal microglia was activated;2.peripheral groupⅠmGluRs were involved in the generation of long-term hyperalgesia and the activation of spinal microglia;3.spinal groupⅠmGluRs mediated the long-term hyperalgesia and the activation of spinal microglia;4.spinal p38 MAPK pathway(probably in microglia)was the possible signal pathway to mediate the pain facilitation of groupⅠmGluRs;5.combining the founding that groupⅠmGluRs participate in the acute formalin pain response in our previous studies,we can draw a conclusion that peripheral and spinal groupⅠmGluRs play key roles in the generation and transmission of pain signals including the acute inflammation pain and long-term hyperalgesia.