| Pain is an unpleasant feeling that produced by different pathological state. Pain,especially for chronic pain, has already become a major threat to the quality of our humanlife globally. Therefore, the mechanism of pain generation and transmission, and effectiveanalgesic drugs and clinical targeted therapy method for pain have become a hot topic intoday’s life science research.Dorsal root ganglia and spinal dorsal horn are the primary portal for the transmissionand regulation of somatosensory signals. Glutamate localized in these two parts plays avery important role during the transmission of peripheral pain information. Previousstudies have confirmed that glutamate is one of the most important neurotransmitter forpain information transmission and regulation. Glutamate is synthesized in the soma ofneuron and transported into synaptic vesicles for exocytosis. Specific vesicular glutamatetransporters (VGLUTs) is indispensable to this transport process. In the recent decade,three kinds of VLGUTs have been cloned: VGLUT1, VGLUT2and VGLUT3. AndVGLUT1and VGLUT2, have been normally used as specific markers for glutamatergic neurons in central nervous system so far. Long sensory pathway is the structuralfoundation for glutamate participating the sensory information transmission and regulation.VGLUT2is found distrubuted in almost all the glutamatergic long pathway tracts.Moreover, except the long fiber tracts, many glutamatergic interneurons take part in thesensory information transmission and regulation as well, and these interneurons’regulatory effect also derives from VGLUT2. Resent research results further confirm thatin DRG and spinal dorsal horn it is VGLUT2mediated glutamatergic neurons thatcontribute to the peripheral pain signals transmission and regulation.For the lack of specific agonists and antagonists for VGLUT2, it was difficult toinvestigate the mechanism of VGLUT2mediated glutamatergic neurotransmission thatparticipate in the peripheral pain signals transmission and regulation. With thedevelopment of molecular biology, gene knock out technology solve the problem ideally,but it is unrealistic to develop more effective analgesic drugs by using gene knock outtechnology. with the development of gene therapy techniques, such as the RNAinterference (RNAi) technology, that provide new possibilities to investigate specific genefunction and related diseases.However, the mechanism of how the VGLUT2mediated glutamatergicneurotransmission in spinal interneurons that contribute to the generation and delivery ofpain is still unintelligible. Moreover, VGLUT2immunoreactive terminals in spinal doralhorn derive from two souses, one is the primary afferent neurons and the other is thespinal interneurons. Under chronic pain state, how the two kinds of VGLUT2immunoreactive terminals participate in the pain signals transmission and regulation is yetto be investigated. Therefore, to answer the two questions the following trails weredesigned by using RNA interference technology, antergrade tract tracing method,immunohistochemical technology, cell culture and electrophysiological techniques.Part IUsing the two selected lentiviral vectors that carried with VGLUT2-shRNA sequenceto transfect primary cultured spinal neurons in vitro, and to further select the most effective lentivirus that could down-regulate the expression of VGLUT2in spinalneurons.Object: To verify whether the two lentiviral vectors that could specifically interferethe expression of VGLUT2, which were selected by using the cultured cortex neurons,could effectively transfect primary cultured spinal neurons, and to further select the mosteffective lentiviral vectors. Methods: According to the different types of the vectors thatthe primary cultured spinal neurons from embryonic rats were transfected, the trials weredivided into4groups: Normal group, Negative control group, VGLUT2-shRNA-1groupand VGLUT2-shRNA-2group. Florescent microscope was used to observe the expressionof green fluorescent protein (GFP) directly and Western blot was used to investigate theexpression of VGLUT2in different groups. Results: Florescent results show that inNegative control group, VGLUT2-shRNA-1group and VGLUT2-shRNA-2group allcould be observed apparent GFP fluorescence, indicating that all the3groups neuronscould be infected by the lentiviral effectively. However, the expression of VGLUT2inVGLUT2-shRNA-1group and VGLUT2-shRNA-2group is significantly lower than theNormal group and Negative control group. The Western blot results also show the sametendency. The expression of VGLUT2in VGLUT2-shRNA-1group andVGLUT2-shRNA-2group accounting for the Normal group62.42±1.12%and66.07±1.33%respectively, but there’s no significantly difference between VGLUT2-shRNA-1group and VGLUT2-shRNA-2group (P>0.05), neither between the Normal group andNegative group (P>0.05). Conclusion: Both reconstructed lentiviral vectors used inVGLUT2-shRNA-1group and VGLUT2-shRNA-2group could effectively transfectprimary spinal neurons and could significantly down-regulate the expression of VGLUT2.Part IIBy stereotactic injecting the reconstructed lentiviral vectors that could effectivelydown-regulate the expression of VGLUT2in vitro into spinal dorsal horn of rats, toinfect spinal dorsal neurons and to investigate the effect of down-regulated VGLUT2 to the mechanical hyperalgesia in neuropathic pain rat model. Then with the help ofinfrared visual patch clamp system, to observe the change of electrophysiologicalproperties of the superficial dorsal horn neurons in rat spinal cord slices in vitroafter the down-regulation expression of VGLUT2.Object: To observe the change of rats’ pain behavior and the spinal dorsal hornneurons’ electrophysiological properties and synaptic plasticity after targeteddown-regulation of VGLUT2in spinal dorsal horn. Methods:(1) Construct theneuropathic pain model rats;(2) The reconstructed lentiviral vectors that could effectivelydown-regulate the expression of VGLUT2were stereotactic injected into spinal dorsalhorn of rats, and transfected the spinal neurons. Then, the effect of the vectors on the rats’pain behavior was observed.(3)The selected vectors were also used to infect the culturedspinal cord slices of rat in vitro, to study the change of electrophysiological properties ofthe superficial dorsal horn neurons. Results:(1) The mechanical and thermal hyperalgesiaof the model rats appeared just since the first day after the SNL operation, and thehyperalgesia was most serious on the3d.(2) The reconstructed lentiviral vectors couldefficiently infect the superficial dorsal horn neurons of rat, and down-regulate theexpression of VGLUT2. Compared with the Normal group the expression of VGLUT2inVGLUT2-shRNA-1group and VGLUT2-shRNA-2group only had20.71±5.52%and19.74±3.92%of the Normal group respectively. Meanwhile, the rat’s threshold tonociceptive heat and mechanical stimuli were obviously increased and pain behavior ofneuropathic pain rats released apparently.(3) In acutely isolated rat spinal cord slices invitro, after infected with the vectors, the amplitude of spontaneous excitatory postsynapticcurrents (sEPSC) of the spinal dorsal neurons decreased obviously and the frequency alsoreduced significantly. Conclusion: Down-regulating the expression of VGLUT2couldaffect the function of neuron and relieve pain behavior in neuropathic rats.Part IIIUsing neurobiotin-antergrade tract tracing method to distinguish different derives ofVGLUT2immunoreactice terminals in spinal dorsal horn, whether from DRG (the primary afferent neuron) or spinal dorsal neuron. Then to study the change ofVGLUT2terminals in the spinal dorsal horn that come from the primary afferentneurons at different time points under neuropathic pain state.Object: To observe the change of VGLUT2in the primary afferent terminal ofrats’spinal dorsal horn at different time points after spinal nerve ligation (SNL). Methods:All the afferents fibers that come from the DRG were labeled in the superficial dorsal hornby using neurobiotin-antergrade tract tracing method. Meanwhile, all the terminals thatcontain VGLUT2in the spinal cord were marked by immunofluorescence techniques withanti-VGLUT2antibody. Then, through the double labeled fluorescent the VGLUT2couldbe easily distinguished whether derive from DRG or the spinal dorsal horn. Via thismethod, the change of VGLUT2in primary terminals in the spinal dorsal horn at differenttime points after the operation of spinal nerve ligation was studied. Results: The primaryafferent fibers and terminals in spinal dorsal horn were clearly marked by neurobiotin, anddouble-labeled with VGLUT2. Moreover, the number of double-labeled neurons increasedapparently with the time prolongation (0d,3d,5d and7d). Conclusion: The resultsindicate that the expression of VGLUT2in the primary afferent terminals in the spinalcord dorsal horn would have an obviously increasement during the chronic painmaintenance stage. |
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