Changes In Expression And Function Of VGLUT2 During Neuropathic Pain And Their Influence On Nociceptive Processing

BackgroundGlutamate plays an important role in transmission and modulation of pain signals. Up-regulation of glutamate release has been reported and proposed as a crucial mechanism of neuropathic pain. Vesicular glutamate transporters(VGLUTs), which transport glutamate into vesicles to accomplish storage, isolation and release processes, play an important role in synaptic glutamate homeostasis. Among the three VGLUT isoforms, VGLUT2 was reported to be predominantly expressed in the spinal cord, thalamus and brainstem, and to be present in certain amygdaloid nuclei and cortical layers, suggesting that VGLUT2 may be involved in transmission and processing of pain signals in these brain structures. Several studies in heterozygous mice have demonstrated that VGLUT2 deficiency results in attenuation or deletion of some neuropathic pain symptoms. In addition, loss of VGLUT2 expression leads to reduction in EPSC frequency and amplitude in thalamic neurons, suggesting a possible correlativity between VGLUT2 and neuropathic pain.However, it is still not well understood whether VGLUT2 expression would change during neuropathic pain. Moreover, no study investigates how changes in VGLUT2 expression would affect glutamatergic transmission and whether inhibiting VGLUT function would have antinociceptive effects in neuropathic pain models. Previously, we have revealed that expression of VGLUT2 upregulated in SNI rat dorsal root ganglia and lumbar spinal cord. The present study investigated possible changes in VGLUT2 expression and function in mouse brain during neuropathic pain, and effects in the VGLUT inhibitors in glutamate release and pain behaviors. ObjectiveAnalyse VGLUT2 expression and function changes in mouse brain during neuropathic pain and the mechanisms in nociceptive signals transmission affected by VGLUT2 changes. Observe effects in VGLUT functional inhibitors in pain response and glutamate release. MethodsFirst, we established neuropathic pain models, and then examined VGLUT2 expression changes in pain-related brain areas at different time points during neuropathic pain, investigated influence in synaptic vesicles glutamate uptake and glutamate release. Including following experiments: 1) Establishment of SNI model: within C57 mice the three terminal branches of the sciatic nerve, both the common peroneal and tibial nerves were ligated and transected, leaving the sural nerve intact. After surgery, von Frey test was used to examine changes of mechanical allodynia in sural innervation domain. 2) Examination of VGLUT2 expression: Immunohistochemistry was used to examine changes in VGLUT2 expression in the thalamus, PAG, amygdala, and m PFC on postoperative days 0.5, 1, 1.5, 3, 7, 14 and 28. 3) Examination of VGLUT2 function: glutamate uptake into synaptic vesicles: Isolated synaptic vesicles on postoperative days 1 and 14 incubated with a series of glutamate, samples were collected by microdialysis probe and the concentrations of glutamate from the vesicular uptake assay were determined by HPLC with electrochemical detection; thalamic glutamate release: Isolating thalamic synaptosomes on postoperative days 1 and 14, incubated with exogenous glutamate dehydrogenase and NADP+. Fluorescence microplate reader measured the generation of fluorometrically detected NADPH resulting from the oxidative deamination of released glutamate by exogenous glutamate dehydrogenase to ensure depolarization-evoked glutamate release.Second, VGLUT inhibitors were used as pharmacological tools to examine whether inhibition of VGLUT activity would result in attenuation of thalamic pain behaviors and glutamate release, as well as the number of c-Fos positive neurons in pain-related brain areas; observed pharmacological effects of the VGLUT inhibitors in different pain models. Including following experiments: 1) Effects in inhibiting VGLUT function: VGLUT inhibitor(i.c.v.) during development or maintenance of SNI or Oxaliplatin-induced neuropathic pain, changes in mechanical allodynia was measured by von Frey test. 2) Effects in motor function and other pain models: an accelerating rotarod was used to measure motor function after i.c.v. administration of VGLUT inhibitor, the hot plate test, acetic acid writhing and formalin tests were used to observe effect of VGLUTs inhibitor. 3) Effects of VGLUT inhibitors on c-Fos expression: with the administration of VGLUT inhibitors, the expression of c-Fos in pain-related brain areas was tested by immunohistochemistry. 4) Effects of VGLUT inhibitors on glutamate release in neuropathic pain: isolated thalamic synaptosomes of SNI on postoperative day 1 preincubated with VGLUT inhibitor, effects of inhibitor on depolarization-evoked glutamate release measured by fluorescence microplate reader. Results 1) Changes in VGLUT2 expression and function after SNIVGLUT2 expression significantly increased in thalamus, PAG and amygdala in SNI groups on postoperative day 1. Then the upregulation of VGLUT2 expression was transit and returned to basal level from 1.5 days to 28 days; the rate of glutamate uptaked into thalamic synaptic vesicles which incubated with glutamate at 1000 M and 2000 M significantly increased in SNI groups on postoperative day 1, no changes on postoperative day 14; a significant increase in depolarization-evoked glutamate release in SNI mice relative to sham-operated mice on postoperative day 1, no changes on postoperative day 14 as well. 2) Effects of VGLUT inhibitors in on pain response in neuropathic painVGLUT inhibitor CSB6B(5 μg/per mouse), Rose Bengal(5 μg/per mouse), Xanthurenic Acid(10 μg/per mouse), Brilliant Yellow(5 μg/per mouse) i.c.v. administrations immediately after SNI surgery followed by repeated administrations at 12-h intervals, significantly inhibited in the development of mechanical allodynia on postoperative day 0.5 and 1, whereas did not affect mechanical allodynia in maintenance of SNI with CSB6 B and Rose Bengal(5 μg,i.c.v.) treatment; CSB6 B i.c.v. administrations repeated at 12-h intervals during oxaliplatin treatment significantly inhibited the development of oxaliplatin-induced mechanical allodynia from 2.0 to 3.5 days after the first Oxaliplatin injection. CSB6B(1 mg·kg-1,i.p.) during development or maintenance of SNI had no effect on mechanical allodynia. 3) Effects of VGLUT inhibitors on c-Fos expression and glutamate release inneuropathic painChanges in c-Fos expression indicated that the SNI-induced upregulation of c-Fos expression occurred in thalamus and PAG on postoperative day 1, and in PAG and m PFC(contra) on postoperative day 1.5. I.c.v. administration of CSB6B(5 μg) produced a tendency of reduction in c-Fos expression in all the analyzed areas and statistic significance was observed in thalamus on postoperative days 0.5 and in m PFC(ipsi) on postoperative days 1.5. Effects of CSB6 B on glutamate release indicated that preincubation with CSB6 B for 30 min significantly reduced the glutamate release from thalamic synaptosomes on postoperative day 1. The inhibitory effects of CSB6 B were concentration-dependent, with an IC50 value of 25.7 μM. Rotarod test showed that there was no significant difference of latency to fall between the mice i.c.v. treated with a CSF and CSB6B(5 μg). 4) Effects of VGLUT inhibitors on other pain modelsCSB6B(2.5 mg·kg-1, i.p.) did not affect the thermal pain responses in 50 or 55℃ hot plate test; CSB6B(2.5 mg·kg-1, i.p.) attenuated the acetic acid-induced writhing in a dose- and time-dependent manner. In addition, CSB6B(0.5 mg·kg-1, i.p.) reduced the licking behavior during the second phase, but did not affect the first phase, following intraplantar injection of formalin. ConclusionUp-regulation in VGLUT2 expression occurred in some brain areas at early stage of neuropathic pain, resulted in an increase in synaptic vesicles glutamate uptake and glutamate release in thalamic neurons, suggesting VGLUT2 plays a role in early stage of neuropathic pain. VGLUT inhibitors, such as CSB6 B, reduced early mechanical allodynia without affecting motor function, decreased c-Fos expression in brain. The effects of CSB6 B were related wtih reduced glutamate release in thalamus synaptosomes after SNI. CSB6 B did not affect motor function, and have antinociceptive effects in chronic pain induced by chemical stimulation. The findings of this study support the involvement of VGLUT2 in neuropathic pain.