The Effection Of Astrocytes Gap Junctions In Mirror-image Pain And The Mechanism Of Its Function Regulated By Glutamic Acid

BackgroundAfter a certain part of the body injury, in addition to the injury part will have spontaneous pain or hyperalgesia, the corresponding contralateral parts of the body also appeare spontaneous pain or hyperalgesia phenomenon, known as the Mirror image of Pain(MIP). It is a special phenomenon of neuropathic pain. The mechanism of MIP is unclear, more and more evidence indicate that gap junctions between astrocytes in the spinal cord may play an important role in the formation of Mirror image of Pain.Gap junctions between cells is a way to communicate information directly. Gap junction is composed of two half-channels formed by a special connexin protein. Gap junctions allows ions and other small molecules, such as adenosine triphosphate (ATP), inositol triphosphate (IP3), cyclic adenosine monophosphate (cMAP) and the free passage of small molecules of amino acids. In the central nervous system, connexin43(CX43) is mainly expressed in astrocytes and constitutes astrocytes gap junction channels and half channels. In some models of neuropathic pain, CX43is highly expressed in astrocytes of spinal cord.Spared nerve injury (SNI) is a common model of neuropathic pain, widely used in studies of chronic neuropathic pain.There have no research report about SNI model induced MIP phenomenon and The role of CX43in astrocytes. So we want to observe the changes of GFAP and CX43protein expression in the bilateral spinal dorsal horn,explore the effects of the CX43on bilateral mechanical hyperalgesia and its possible mechanism by intrathecal injection of carbenoxolone (CBX). In addition,the expression of connexin43proteins and its phosphorylated form has an important role on the function of gap junctional intercellular communication. The half-life of CX43protein is shorter(1-5h), and its phosphorylated form is dynamic and can be regulatived by different kinase. Its phosphorylated form can affect the CX43protein half-life, and the opening and closing of the channel state. There have reports show that the astrocyte gap intercellular communication may be regulatived by protein kinase C (PKC), and its role is achieved by phosphorylation of the phosphorylation carboxyl terminal368sites of CX43protein. Glutamate is an important neurotransmitter and an excitatory amino acid. After nerve injury, the central presynaptic neuron will release more glutamate to enhance the conduction of postsynaptic neuron.Besides, glutamate is also acting on astrocytes and active the astrocytes which will release more material and these material will regulating neuronal activity. However,there have been not yet reported that whether glutamate dose can active astrocytes by regulating CX43protein phosphorylation and the function of gap junctional communication.So we want to observe how does the glutamate regulate the gap junctional intercellular communication and active of astrocytes via cultured astrocytes., Glutamate in the regulation of gap junctional communication function in what role, and how it works provide an experimental basis to further studying the body in a state of neuropathic pain.This study will provide new information for elucidating the role and mechanism of CX43in neuropathic pain, provide an important experimental basis for prevention targets and drug exploring of neuropathic pain.Part Ⅰ:Intrathecal injection of carbenoxolone decreased the expression of cx43and its effects on mirror-image pain in ratsObjective: To investigate the expression of gap junction connexin43(Cx43) and the effective the mirror image pain.Methods:96males SD rats were randomly divided into4groups (n=24):the sham operation group, the spared nerve injury(SNI) group,SNI group with carbenoxoxone(CBX) and SNI group with normal saline (NS). Mechanical allodynia was assessed by measuring hind paw withdrawal thresholds bilaterally using von Frey filaments atld preoperative and1st,5th,7th,10th,14th,21th day postoperative. Then,on the14th postoperative day the sham group and sni group animals were sacrificed and the spinal cord was removed for detection of the expression of connexin43(Cx43)and glial fibrillary acid protein(GFAP) in the spinal dorsal horn by immunohistochemistry. Other groups of rats received either intrathecal infusion of saline or carbenoxo lone (20ng/d) for5days on days5-9postoperative. After completing the intrathecal infusion, lumbar spinal cord sections were assessed for immunostaining of astrocytic GFAP and detecting of CX43protein expression with the method of westernblot on the10th,14th and21th postoperative day.Results:The mechnical withdraw threshold (MWT) of SNI group t is reduced,the expression of CX43and GFAP is increased compared with the Sham group. Intrathecal injection of carbenoxoo lone decreased the expression of CX43and GFAP in spinal cord dorsal horn compared with the NS group,and increased the MWT of bilateral foot on the14th and21th day after surgery(P<0.05).Conclusion:Intrathecal injection of carbenoxolone decreased the expression of CX43, inhibits the activation of astrocytes and relieves the Mirror-image pain of rats. Part II:Glutamic acid enhances gap junction intercellular communication and its effects on the astrocyte activationObjective:To explore the functional modulation of gap junction intercellular communication and the mechanism of astrocytes activation by glutamic acid in vitro culture astrocytes of spinal cord.Methods:Cultured neonatal SD rat astrocytes were divided into normal control group, Glutamic acid stimulation group(treated with10μmol/Glu for24h) PKC inhibitor group and CBX blocker group (treated with20nmol/L CBX for24h).’Western blotting assay were used to detect p-Cx43, p-PKC, GFAP, p-ERK protein expression, Cell immunofluorescence assay were used to detect the p-Cx43expression of astrocytes membrane proteins and GFAP protein expression, Scrape-loading and dye transfer assay were used to detect the functional changes of gap junction intercellular communication of the astrocytes.Results:Compared with the normal control cells, Glutamic acid treatment resulted in a significantly increased the expression of p-Cx43、p-PKC、GFAP protein in the astrocytes and enhanced gap junction intercellular communication of the astrocytes (P<0.01); On the basis of glutamic acid stimulation,PKC inhibitor reduced the expression of p-Cx43and GFAP protein and gap junction communication function; After given gap junction blocker CBX, astrocytes reduced GFAP protein expression.(P<0.01).Conclusion:Glutamic acid can enhance gap junction intercellular communication of the astrocytes possibly by regulating the expression of p-Cx43protein through PKC pathway and further activity the astrocytes.