Effect Of Extracellular Adenosine Diphosphate On Neuropathic Pain Via AMPK In Spinal Astrocytes

Glial cells activation plays important role in the maintenance and promotion of neuropathic pain (NPP). Extracellular ATP is a powerful stimulator to activate glial cells, but its origins and the producing mechanisms are unclear.Early experimental results suggested P2Y receptors have effects on the modulation of the intracellular ATP level, and now need to detect their effects on extracellular ATP level and find out the mechanisms. AMPK is the key regulator of ATP synthesis and expenditure, its upstream signals is sometimes consistency with the downstream signals of G-protein-coupled P2Y receptors. Whether AMPK is a modulator of the signals from P2Y receptors to mitochondrion need to be studied.This topic aims to detect the effects of AMPK on the rat spinal dorsal horn astrocytes. Experimental techniques such as morphology, MTT assay, Western blot and high performance liquid chromatography were employed to study the effects of extracellular ADP on ATP synthesis, release and AMPK expression in astrocytes in vitro. Effects of AMPK inhibitor(Compound C) on the behavior of pain and GFAP expression were observed in chronic contracted sciatic nerve injury(CCI) rats to study the relationship about AMPK, astrocyte activation and chronic pain occur. Research results will be helpful for understanding the mechanisms of ATP-induced pain and finding a new way to inhibit NPP.Methods:Primary dissociated spinal dorsal horn astrocytes were prepared from Sprague–Dawley rats for studying the effects of ADP on cells in vitro. Cellular ATP levels were measured using a firefly luciferase ATP assay kit and mitochondrial membrane potential(MMP) was assessed by using 5,5,6,6-tetrachloro-1,1'3,3'- tetraethylbenzamidazol- carboncyanine(JC-1) in both a direct and an indirect manner. Expression of AMPK in the astrocytes were determined by muti-labeling immunofluorescence and western blot methods. Effect of Compound C on ADP and ADPβS-evoked glutamate release were detected by using HPLC. The rats with intrathecal catheter intubation received sciatic nerve ligation for study the behavior of pain. Paw-withdrawal thermal latency and mechanical threshold were tested in 0, 3,7,14 and 21 days after operation and immunofluorescence was used to determine the expression of GFAP in the ipsilateral dorsal horn at 14th day. Results:Part One:1. The immunohistochemical staining of primary cultured rat spinal astrocytes after two generations showed that the purity of the astrocytes was more than 95%.2. ADP and ADPβS can dose-and time-dependently increase the number of cultured rat dorsal horn astrocytes. 20μM GDPβS, an G protein-coupled inhibitor, inhibited such a cell proliferation effectively. But MRS2179, a P2Y1-specific antagonist, can dose-dependently promote the astrocyte proliferation induced by 100μM ADP or ADPβs for 24 h.3. ADP and ADPβs induced a significant increase both of the intracellular and extracellular ATP concentration, and which could be fully blocked by GDPβs. Pretreatment with MRS2179 did not inhibit ATP content increase, but augmented it to a much higher level.4. ADP or ADPβs significantly improved MMP after 24 h, for which MRS2179 had a significant enhancing effect. The results showed that the red fluorescence in the astrocytes turned stronger than normal. In the presence of 100μM MRS2179, red fluorescence in the astrocytes was strengthened further and green fluorescence turned weaker. The red:green ratio indicated the degree of MMP. It was found that 100μM MRS2179 significantly enhanced the MMP increase induced by ADP or ADPβs.Part Two:1. The AMPK expression in the astrocytes was initially detected with multi-channel immunofluorescence, which indicated AMPK was in a good concordance with the astrocyte specific glial fibrillary acidic protein (GFAP) (red) and DAPI (blue, stained for nuclei) in the normal cultured spinal astrocytes.2. After ADP treatment, the expression of AMPK was much stronger than that in the untreated cells. On co-incubation with MRS2179 and ADP, the immunodetection of AMPK in the astrocytes was significantly increased. Similar changes were also found following ADPβs treatment. The amount of AMPK protein in the astrocytes measured by Western Blotting revealed that 100μM MRS2179 increased the AMPK protein expression induced by ADP and ADPβs.3. To determine the direct effect of AMPK, 20μM Compound C was used to directly block the AMPK activation and investigated its effect on the ADP-induced ATP accumulation and astrocyte proliferation. The results revealed that the extra- and intracellular ATP concentrations were decreased significantly regardless of the presence of MRS2179. JC-1 assay suggested that MMP increase induced by ADP was inhibited by the Compound C. Moreover, the MTT assay showed that the time-dependent astrocyte proliferation induced by ADP was also inhibited by 20μM Compound C.4. After 24h treatment by ADP, ADPβs and Compound C, little changes were found about the contents of glutamate in cell culture medium.Part Three:1. The rats implanted with intrathecal catheter and subjected to CCI in right leg have lower disability and mortality and are favorable to study the behavior of pain.2. Intrathecal injections of Compound C reversed CCI-induced thermal and mechanical hypersensitivity. Following ligation, rats developed significant thermal and mechanical hypersensitivity by 3 d, which was maintained until 21 d. Na?ve control and sham-CCI rats completely failed to produce thermal and mechanical hypersensitivity. After Compound C intrathecal injections, hypersensitivity induced by CCI was reversed.3. Immunocytochemistry revealed that expression of GFAP in the ipsilateral spinal cord was markedly increased after nerve injury. Up-regulation of GFAP expression was significantly higher in ipsilateral dorsal horn of spinal cord after injury, peaked at 14 d and persisted for at least 21 d, but was occasionally observed in na?ve control and sham-CCI groups.4. Compound C intrathecal injection could significantly inhibit the activiation of astrocytes in the ipsilateral dorsal horn of spinal cord after injury. Conclusion:1. Extracellular ADP modulated ATP metabolism through increasing MMP (ATP biosynthesis) and accumulating ATP in the extra- and intracellular space of the astrocyte.2. Decrease of AMPK expression resulted from the activation of the P2Y1 receptor may contribute to the long-term inhibition of ATP accumulation in astrocytes.3. Significant inhibition effects of Compound C on the time-dependent astrocyte proliferation and ATP accumulation induced by extracellular ADP indicating that AMPK is also an adjustable target in inhibiting astrocyte activation induced by extracellular ADP.4. Inhibition effect of Compound C on the CCI-induced thermal and mechanical hypersensitivity and activation of astrocytes in the spinal dorsal horn of CCI rats indicated a new way to suppress the NPP.The primary findings of this study show that activation of the P2Y receptors in astrocytes induced the ATP accumulation through AMPK modulation. This report provides additional insight into extracellular ADP pathophysiology in the spinal astrocyte activation and highlights the importance of the P2Y/AMPK pathway in the ATP metabolism and the cell proliferation, which could potentially lead to a novel understanding of the extracellular ATP-induced neuropathic pain and other related diseases.