| Background and objective Postoperative pain involves a series of physiological,psychological and behavioral reactions that occur after the patient is stressed by the operation.About 86% of postoperative patients reported moderate to severe postoperative pain.Postoperative pain causes patients to suffer a great deal of pain and may lead to a series of organ function damaga.The severe pan may even affect the postoperative rehabilitation and quality of life,greatly increasing the burden of patients and society.With the improvement of people’s living standards,the demands for comfortable medical treatment has become higher in recent years,the use of painless technology to eliminate people in the hospital teeatment process of pain fear and confusion has become a real need to take seriously.Postoperative pain management is one of the most difficult problem.Effective pain management can speed up the recovery of patients,reduce the length of hospital stay,reduce the financial burden of patients and the country,improve patient satisfaction,and ultimately promote harmonious doctor-patient relationship.It is consistent with the concept of Enhanced Recovery After Surgery(ERAS)proposed in recent years.Although there are many postoperative analgesia models in use,the fact that clinical research showa that: patients with effective postoperative acute pain treatment satisfaction is still less than 47%~75%.Therefore,it is of great significance to explore the mechanism of acute pain in order to find specific therapeutic targets and deveop drugs to management postoperative acute pain.It is now believed that the astrocyte and microglia of the spinal cord play a key role in the development of pain due to inflammation and injury and in the development now believed that the astrocyte and microglia of the spinal cord play an important role in the development of pain due to inflammation and injury and in the maintenance of chronic pain.In the rat model of postoperative pain,the activity of glial cells was significantly increased after the rat incision surgery.Interestingly,the paper suggests that the microglia inhibitor Minocyline acts no effect on postoperative pain behavior hypersensitivity.It seems that astrocytes play a major role in the development of allodynia and hyperalgesia in acute pain.However,exact cellular and molecular mechanisms underlying postoperative pain remain unclear in astrocyte.In central nervous system,astrocytes are interconnected by gap junctions to construct the functional syncytium organization.Gap junction(GJ),a direct channel connecting two neighboring cells,provides convenient pathway for rapid intercytoplasmic transfer of hydrophilic metabolites and messenger molecules between adjacent cells.Gap junction is composed of two hemichannels,and each one of which locates in one of two opposing cells.Every hemichannel is composed of six homogenous or heterogenous connexins(Cxs).More than 21 subtypes of Cx family,which are named with its molecular weight,have been identified in humans.The predominant Cx protein in astrocytes is Cx43.In a variety of neuropathic pain conditions,including the chronic constriction of sciatic nerve,complete Freund’s adjuvant and partial sciatic nerve ligation-induced pain,Cx43 is increased in spinal astrocytes and blockade of gap junctions or gene deletion of Cx43 has been showed to reduce peripheral nerve injury-induced central sensitization in dorsal horn.Importantly,whether the expression of Cx43 is also increased in acute pain and whether the involvement of astrocytes in acute incision pain may engage a gap junction mechanism needs to be further elucidated Plantar hind paw incision is an established model of postoperative pain.In present study,we determined the astrocyte-specific gap junction protein Cx43 and the activation of spinal astrocytes,as well as gap junction proteins in neurons and oligodendrocytes in model of acute incision pain.Moreover,we investigated the effect of carbenoxolone,a gap junction decoupler,and INI-0602,a hemichannel inhibitor on mechanical and thermal hypersensitivity and spinal astrocyte activation in rats with planter incision surgery.Methods 1.The experiments were performed on male Sprague-Dawley rat(200-220g)obtain from the Animal Experimental Center,Sun Yat-sen University,P.R.China.The method for intrathecal administration of gap junction channel inhibitors and hemichannel blocker and saline was described by Hylden and Wilcox(1980).The rat model of post-operative pain was performed plantar surgery as previously described by Schuwaller et al(2015).2.Sensitivity to mechanical stimuli was measured with an electronic von Frey esthesiometer equipped with a rigid cylindrical tip(diameter0.8mm)(Electronic von Frey esthesiometer,IITC Inc.Life Science,USA).Heat sensitivity was recorded as paw withdrawal latency time(PWL).3.The expressions of Cx32,Cx36,Cx43 and GFAP were detected by Western Blot.4.Immunofluorescence demonstrated the expressions of GFAP and Cx43.Results 1.In acute incisional pain model group,the activity of astrocyte increased,and the expression of GFAP increased.In rats following planter incision.A significant increase in GFAP expression was observed at 30 min and peaked at 2 h.It gradually reduced to the control level at 12 h post-incision.Immunochemical analysis also showed an increase of GFAP expression in ipsilateral spinal dorsal horn at 2h following incision surgery.2.Planter incision increased astrocyte Cx43 expression in spinal dorsal horn,but had no effect on Cx expression in neurons and oligodendrocytes Cx43 level in ipsilateral spinal dorsal horn was apparently increased from 30 min and peaked at 2h after planter incision,where the density was 1 fold higher than the rats without incision at 2 h post-incision.Then it declined from 2h and was comparable to control at 24 h post-incision.Cx32,which is mainly expressed in oligodendrocyte,and Cx36,which is mainly expressed in neuron after incision.The level of Cx32 and Cx36 in spinal dorsal horn were unchanged at all analyzed time points.Although the expression level of Cx36 seems lightly elevated at 30 min after incision,no significant difference was observed.these results suggest that astrocyte Cx43 may play an important role in acute incisional pain model.3.Gap junction inhibitor reduced postoperative pain response in rats after incision surgery Gap junction inhibitor carbenoxolone significantly induced the hypersensitivity to thermal and mechanical stimulus,with the reduction of paw withdrawal threshold and latency from 30 min and saturated at 2 h after incision surgery.Carbenoxolone by itself induced no alteration on ipsilateral withdrawal threshold and latency,while it significantly reversed ipsilateral mechanical and thermal pain hypersensitivity from 30 min following surgery.After 4 h post-incision,no obvious difference was observed between rats with or without pretreatment with carbenoxolone before paw incision.These results indicated that gap junctions composed of Cx43 in spinal dorsal horn is associated with the early phase of acute pain response in rats with planter incision.4.Block of hemichannel attenuate ipsilateral pain response of the rats at early phase and enhance the pain response at late phase after incision surgery Both withdrawal threshold for mechanical stimuli and withdrawal latency for thermal stimuli in contralateral paw were not altered by INI-0602 in rats with or without incision surgery,while it significantly attenuated the ipsilateral thermal and mechanical hypersensitivity in surgery rats before 4 h post-incision.After 4 h post-incision,INI-0602 showed no effect on ipsilateral paw withdrawal threshold and withdrawal latency of rats receiving incision surgery.That suggest the inhibitory effect of INI-0602 on incision-induced hypersensitivity at the initial stage of post-incision. 5.The effects of gap junction and hemichannel inhibitor on GFAP expression in spinal dorsal horn Paw incision induced the increase in GFAP expression in ipsilateral spinal dorsal horn at 2h after paw incision.Pretreatment of carbenoxolone reduced GFAP expression at 2h post-incision.Intrathecally injection with hemichannel blocker INI-0602 reduced GFAP expression at 2 h following incision surgery.Immunofluorescence analysis also showed the change of GFAP staining intensity in ipsilateral spinal dorsal horn at 2 h following incision surgery.GFAP staining intensity at 2h post-incision was significantly increased when compared with control rats.Intrathecally injection with carbenoxolone or INI-0602 reduced the GFAP staining intensity at 2h post-incision surgery,accompanied with the decreased expression of Cx43.Conclusion The astrocyte Cx43 in spinal dorsal horn is involved in the initiation of the paw incision induced acute pain. |
