| Study one Expression of acid-sensing ion channels (ASICs) on the ipsilateral dorsal root ganglia (DRG) in a rat model of bone cancer painObjective In attempt to investigate the underlying mechanisms of cancer-induced bone pain, we investigated the presence of ASICs in DRG neurons in an animal model of bone cancer pain.Method Forty-five female Sprague-Dawley rats were randomized into three groups: sham operation group (sham), cancer-bearing animals killed after 7 days (C7) and cancer-bearing animals killed after 14 days group (C14). After establishment of bone cancer pain model, pain-related behavioral tests were performed to determine the paw withdrawal threshold (PWT) of thermal hyperalgesia and mechanical allodynia, respectively. Series of tests including body weight, microCT were carried out at the final time point of each group to make sure the animal model is successfully established. RT-PCR, Western bolt and Immunofluorescence were used to determine mRNA and protein expression of ASICs in ipsilateral and contralateral lumbar4-5 DRG neurons.Results Compared to sham group, PWT of mechanical allodynia and thermal hyperalgesia in C14 group exhibited a significant decrease (P<0.01) from post-operation day (POD) 7 to the termination of the experiment. Among six ASIC subunits, only the ipsilateral ASIC3 mRNA expression was up-regulated in C14 group compared with Sham group. However, the ipsilateral protein expression of ASIC1, ASIC3, ASIC4 subunits were all increased in different extent except for ASIC2a. Double-labelled immunofluorescence revealed that ASIC3 and isolectin-B4 (IB4) co-localized small DRG neurons in C14 group are more than that in sham group. Furthermore, we also found that there are more ASIC3 and neurofilament 200 (NF200) co-localized DRG neurons in C14 group than that in sham group.Conclusion The up-regulation of mRNA and protein levels of ASIC3 suggested its potential involvement in the development and maintenance of cancer-induced bone pain.Study two Modulation of SDF-1 and its downstream signal on Nav1.8 sodium channel currentObjective Several chemokines, known as a new class of neuromodulators, participate in pain-sensing process at the spinal and supraspinal levels. Stromal cell-derived factor 1 (SDF-1)/chemokine CXC motif ligand 12 (CXCL12), a chemokine that is up-regulated in the dorsal root ganglion (DRG) in chronic pain models, has recently been found to play a central role in pain hypersensitivity. However, the intracellular mechanisms responsible for changes of tetrodotoxin-resistant (TTXR) Navl.8 sodium currents triggered by SDF-1 have not been well understood. The purpose of the present study was to investigate the functional impact of SDF-1 and its receptor, chemokine CXC motif receptor 4 (CXCR4), on Nav1.8 sodium channel in rat DRG neurons using electrophysiological techniques.Methods Double-labelled immunofluorescence was used to detect the distribution of Nav1.8 and CXCR4 in DRG sections. Acutely dissociated DRG neurons were incubated with various concentrations of SDF-1 for 1-2 hours. Whole-cell voltage-clamp recordings were performed to record Navl.8 currents from control or SDF-1 treated small DRG sensory neurons. The inhibition effects of CXCR4 antagonist AMD3100, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, pertussis toxin (PTX) and cholera toxin (CTX) on Navl.8 currents were also measured by whole-cell voltage-clamp recordings in the presence or absence of 50ng/ml SDF-1.Results Immunofluorescence analysis indicated that over 42% of CXCR4 positive DRG neurons colocalize with Navl.8 sodium channel. Preincubation with SDF-1 caused a concentration-dependent increase of the amplitudes of Navl.8 currents in acutely isolated small diameter DRG neurons in short-term culture. As to Navl.8, inhibitors (AMD3100, PTX, CTX, LY294002) used in present study all increased the amplitude of Nav1.8 current and shifted activation curve in a hyperpolarizing direction in the presence or absence of SDF-1 (50ng/ml).Conclusions Our data demonstrated that SDF-1 may excite primary nociceptive sensory neurons by acting on the biophysical properties of Navl.8 currents. However, the intracellular mechanisms that lead to the increase of Navl.8 current triggered by SDF-1 may not depend on CXCR4, G-protein, and PI3K pathway.Study three Modulation of SDF-1 and its downstream signal on Nav1.9 sodium channel currentObjective Several studies have identified the chemokines, a superfamily of small proteins with a crucial role in immune and neuromodulation functions. Stromal cell-derived factor 1 (SDF-1)/chemokine CXC motif ligand 12 (CXCL12), a chemokine that is up-regulated in the dorsal root ganglion (DRG) in chronic pain models, has recently been found to play a central role in pain hypersensitivity. However, the intracellular mechanisms responsible for changes of the tetrodotoxin-resistant (TTXR) sodium Navl.9 currents triggered by SDF-1 have not been well understood. The purpose of the present study was to investigate the functional impact of SDF-1 and its receptor, chemokine CXC motif receptor 4 (CXCR4), on Navl.9 sodium channel in rat DRG neurons using electrophysiological techniques.Methods Double-labelled immunofluorescence was used to detect the distribution of Nav1.9 and CXCR4 in DRG sections. Acutely dissociated DRG neurons were incubated with various concentrations of SDF-1 for 1-2 hours. Whole-cell voltage-clamp recordings were performed to record Nav1.9 currents from control or SDF-1 treated small DRG sensory neurons. The inhibition effects of CXCR4 antagonist AMD3100, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, pertussis toxin (PTX) and cholera toxin (CTX) on Navl.9 currents were also measured by whole-cell voltage-clamp recordings in the presence or absence of 50ng/ml SDF-1.Results Immunofluorescence analysis indicated that over 37% of CXCR4 positive DRG neurons colocalize with Navl.9 sodium channel. Preincubation with SDF-1 caused a concentration-dependent increase of the amplitudes of Nav1.9 currents in acutely isolated small diameter DRG neurons in short-term culture. As to Navl.9, changes in current density and kinetic properties of Nav1.9 current evoked by SDF-1 (50ng/ml) was eliminated by AMD3100. The increase in Navl.9 current was also blocked by PTX but not CTX, showing involvement of Gi/o but not Gs subunits. Furthermore, the enhancement in Navl.9 currents was also prevented by pretreatment with LY294002.Conclusions Our data demonstrated that SDF-1 may excite primary nociceptive sensory neurons by acting on the biophysical properties of Navl.9 currents. The present study also evidenced the role of SDF-1 as a positive modulator of Navl.9 current by triggering a cascade involving CXCR4 receptor, Gi/o protein, and PI3K. |
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