| [Background] Neuropathic pain is a kind of intractable chronic pain caused by the damage of sensory nervous system. The mechanisms of neuropathic pain are multifaceted, and clinical curative effect is poor, which affect the life quality of patients severely. Several studies have demonstrated that neurons, glial cells and the immune cells are related to the induction and maintenance of the neuropathic pain. After nerve injury, spinal astrocytes can be activated, the overexpression of glial fibrillary acidic proteins (GFAP), a marker of activated astrocytes, and followed by release of interleukin1β (IL-1(3).Previous studies have demonstrated that, IL-1β, combined with interleukin1receptor (IL-1R) on presynaptic neurons, induces the increasing of algogenic substance such as glutamate. While combined with IL-1R on postsynaptic neuron, activates the N-methyl-D-aspartate receptor (NMDAR) subunits NR1and phosphorylates serine896. The phosphorylation of NR1subunits change molecular conformation of NMDA receptor and enhance the reactivity of the NMDA receptor to its ligand glutamate, which makes the depolarization of the postsynaptic neuron cell membrane more obviously. The phosphorylation of NR1subunits take part in the induction and maintenance of the neuropathic Pain. In a word, IL-1β, combined with IL-1R on presynaptic and postsynaptic neuron, induces hyperalgesia.However, whether the down-regulation of phosphorylation of NR1via IL-1β allevates neuropathic pain is not clear, and it is necessary to have a further study. In this study, we construct IL-1β short hairpin RNA mediated by recombinant adenovirus (rAd/shRNA-IL-1β) expression vector, which selectively down-regulated the expression of IL-1β and blocked the phosphorylation of NMDA receptor NR1subunit serine-896(pNRlserine-896). Then we observed the effects of behaviors, paw withdrawal latency and protein expression on neuropathic pain in rats.These may provide a new therapeutic methods for neuropathic pain.[Objective](1) Construction of the rat models of neuropathic pain, chronic compression of dorsal root ganglion (CCD) model.(2) Intrathecal injection of rAd/shRNA-IL-1β expression vector, to observe the effects of behaviors, paw withdrawal latency and protein expression on neuropathic pain in rats.[Methods] Section one:Construction of the rat CCD models of neuropathic pain.60male SD rats weighing200-250g were randomly divided into3groups (n=20each):CCD group, sham operation group (group Sham) and control group (group Control). In group CCD, a fine steel needle was put into the intervertebral foramen between L4and L5of each rat for preparation of CCD model. In group Sham, the intervertebral foramen was only exposed without inserting the fine steel needle to the rats. The PWL were measured at1day before and1w,2w and4w after operation.4rats in each group were randomly selected after the measurement for PWL and sacrificed. The lumbar enlargement of spinal cord was removed for observing the change of GFAP, IL-1β and pNRlserine-896expression by immunohistochemistry and Western blot.Section two:Intrathecal injection of rAd/shRNA-IL-1β expression vector, to observe the effects of behaviors, paw withdrawal latency and protein expression on neuropathic pain in rats.100male SD rats weighing200-250g were randomly divided into5groups (n=20each):CCD group, rAd/IL-1β group, rAd+CCD group, rAd/IL-1β+rAd/shRNA-IL-1β+CCD group, rAd/shRNA-IL-1β+CCD group.In group rAd/IL-1β, intrathecal injection of5ul rAd/IL-1β to normal rats, additional intrathecal injection at2w. In group rAd+CCD, intrathecal injection of5ul rAd to CCD rats, additional intrathecal injection at2w. In group rAd/IL-1β+rAd/shRNA-IL-1β+CCD, intrathecal injection of5ul rAd/IL-1β and5ul rAd/shRNA-IL-1β to CCD rats, additional intrathecal injection at2w. In group rAd/shRNA-IL-1β+CCD, intrathecal injection of5ul rAd/shRNA-IL-1β to CCD rats, additional intrathecal injection at2w. The PWL were measured at lday before and lw,2w and4w after operation.4rats in each group were randomly selected after the measurement for PWL and sacrificed. The lumbar enlargement of spinal cord was removed for observing the change of GFAP, IL-1β and pNRlserine-896expression by immunohistochemistry and Western blot.[Results] Section one: Construction of the rat CCD models of neuropathic pain.1. Behavioral test. Protective behavior after operation:lifting and licking feet.2. PWL.①Compared with group Control, the PWL was significantly decreased, at each time point after operation in group CCD (P<0.05); the PWL was significantly decreased at1w after operation in group Sham(P<0.05), and there was no significant difference in the PWL, at other time points in group Sham(P>0.05).②Compared with Id before operation, the PWL was significantly decreased at other time points (P <0.05), and the PWL at2w after operation was decrease to the lowest and last to4w after operation in group CCD; the PWL was significantly decreased at1w after operation in group Sham(P<0.05), and there was no significant difference in the PWL, at other time points in group Sham(P>0.05).3. Immunohistochemistry. Compared with group Control, the astrocyte in ipsilateral become hypertrophy, hyperplasia and protuberances increased significantly in group CCD(P<0.05), IL-1β and pNRlserine-896was significantly increased in group CCD(P<0.05); there was no significant difference in group Sham(P>0.05).4. Western blot. Compared with group Control, the expression of IL-1β and pNRlserine-896were up-regulated in group CCD(P<0.05); there was no significant difference in group Sham(P>0.05).Section two:Intrathecal injection of rAd/shRNA-IL-1β expression vector, to observe the effects of behaviors, paw withdrawal latency and protein expression on neuropathic pain in rats. 1. Behavioral test. In group CCD, rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD, rats lift and lick feet after operation.2. PWL.①Compared with group CCD, there was no significant difference in the PWL, at each time points in group rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD (P>0.05); the PWL was significantly increased at1w,2w and4w after operation in group rAd/shRNA-IL-1β+CCD (P<0.05).②Compared with Id before operation, the PWL was significantly decreased, at each time point after operation in group CCD, rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD (P>0.05), and the PWL at2w after operation was decrease to the lowest and last to4w after operation; there was no significant differencein the PWL, at each time points in group rAd/shRNA-IL-1β+CCD (P>0.05).3. Immunohistochemistry.①Compared with group CCD, GFAP, IL-1β and pNRlserine-896was significantly decreased in group rAd/shRNA-IL-1β+CCD (P<0.05); there was no significant difference in group rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD (P>0.05).②Compared with group rAd/shRNA-IL-1β+CCD, GFAP, IL-1β and pNRlserine-896was significantly increased in group CCD, rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD(P<0.05).4.Western blot.①Compared with group CCD, the expression of IL-1β and pNRlserine-896were down-regulated in group in group rAd/shRNA-IL-1β+CCD (P<0.05); there was no significant difference in group rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD (P>0.05).②Compared with group rAd/shRNA-IL-1β+CCD, IL-1β and pNRlserine-896was significantly increased in group CCD, rAd/IL-1β, rAd+CCD and rAd/IL-1β+rAd/shRNA-IL-1β+CCD (P<0.05).[Conclusions](1) Construction of the rat CCD models of neuropathic pain was successful. In group CCD, PWL was significantly decreased. GFAP, IL-1β and pNRlserine-896was significantly increased. These deta demonstrated that the CCD model is successful.(2) Observed the effects of intrathecal injection of rAd/shRNA-IL-1β on beiaviors, paw withdrawal latency and protein expression of rats with neuropathic pain. Intrathecal injection of rAd/shRNA-IL-1β to CCD rats, the decrease of PWL was reversed, the change of astrocytes also was reversed. High expression of GFAP, IL-1β and pNRlserine-896were inhibited. These deta demonstrated that it maybe the role of rAd/shRNA-IL-1β. These date indicate that rAd/shRNA-IL-1β could effectively relieve the symptoms of neuropathic pain. |
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