TNFR1/2in The Dorsal Horn Of The Spinal Cord Regulate NMDA Receptor Phosphorylation Process Involved In Research Hyperalgesia

The traditional theory that pain transmission, pain is peripherally, passing the spinal cord and brain levels of neurons, ultimately perceived by the body, usually presents unpleasant feeling or emotional experience. When persistent inflammatory pain occurs in the nervous system to produce a large number of inflammatory mediators, such as IL-1β, TNF-α, these inflammatory mediators play an important role in the development and maintenance process in hyperalgesia. TNF-αis composed of neurons and glial cells secrete inflammatory cytokines, in the increased expression of the pain signal passed, and participate in hyperalgesia and allodynia development and maintenance, but it is the means by which this process is mediated It is not clear. Previous studies in our laboratory showed that, TNF-α can increase NMDA receptor NR1subunit phosphorylation levels increased hyperalgesia, but there are two kinds of TNFR subtypes, namely TNFR1(55KD) and TNFR2(75KD), in the end what kind of subtype-mediated phosphorylation of NR1is still controversial. The study will utilize siRNA technology were silent expression in rat spinal dorsal horn neurons TNFR1/TNFR2, the mechanism for the increase in the expression of TNFR mediated hyperalgesia occurred pNR1and conduct more in-depth study.Research Methods1. Preparation and heat-induced pain behavior observed pain modelMale SD rats (250-270g), Freund’s adjuvant injection side feet (CFA,0.08ml,40μg) or saline (NS). Sole injection time is set to0; intrathecal administration time was before the injection sole-5d; animals were placed on a hot plate to measure foot retractable lift latencies (PWL) were the sole time after injection1d,3d and7d. Grouping designed according to2×2two-factor.2. Western-blot detectionAfter the hot-plate test measure PWL, the animals were sacrificed quickly isolated spinal cord tissue, enzymatic ice bath environment using buffer solution after centrifugation ultrasonic homogenizer, SDS-PAGE protein electrophoresis, proteins were transferred to PVDF membrane. After the closure, with an anti-incubated with the appropriate secondary antibody incubation, detection immunoblotting. Measured by this method indicators:pNR1.3. siRNA TechnologyInterference TNFR1/TNFR2mRNA for small molecular fragments and adenovirus from reagent companies to obtain, respectively, after intrathecal injection of dissolved mixed lumbar enlargement of the spinal cord. The pre-treatment process. The method used to block the expression of gene silencing TNFR1/TNFR2:the use of siRNA technology, through adenovirus packaging transfection, respectively, will be targeted mRNA TNFR1/TNFR2for intrathecal injection of siRNA to advance the lumbar enlargement of the rat spinal cord, respectively inhibited the expression of TNFR1, TNFR2, or the TNFR1/2.4. Double immunofluorescence detectionWith NR1and TNFR1/2immunofluorescence of labeled neurons in the spinal cord. Anesthetized rats were fixed with paraformaldehyde (4%) perfusion. Remove the spinal lumbar enlargement, dehydration embedded with bone forceps, use frozen microtome cut after spinal cord tissue sections30μm, with0.01M phosphate buffer (PBS, NaCl137mmol/L, KC12.7mmo/L, Na2HPO44.3mmol/L, KH2PO41.4mmol/L, pH7.2～7.4) rinsed three times, each time3min. A5%BSA for1h. An anti-anti-NRl. antibody mouse anti-rat (millipore, USA1:250PBS diluted), a second anti-anti-TNFRl/2antibody rabbit anti-rat, and incubated at room temperature overnight. Rinsed three times, and incubated at room temperature for shutting light AlexFluo488labeled donkey anti-mouse IgG (Invitrogen, USA) AlexFluo594labeled donkey anti-rabbit IgG (Invitrogen, USA,1:1000PBS diluted)2h. Rinsed three times. Patch, after being set were mounted with an Olympus fluorescence microscope to observe and take pictures.ResearchA total of two partsPart Ⅰ:morphological examination.1. It will be marked with a green fluorescent protein virus injected into the lumbar enlargement visible green fluorescence, indicating that the virus is indeed injected cells.2. Light immunofluorescence showed co-expression of TNFR1and TNFR2and NR1.Part Ⅱ:Behavioral and Western blot analysis.1. Viral load intrathecal injection of the control groupThe viral load (GFP) was injected into the rat lumbar enlargement, foot and saline were injected with CFA.Results:Behavioral results showed<GFP+CFA> group, compared with<GFP+saline> group, the left foot under the skin of adult male rats after inj ection of inflammatory pain caused by substances carried CFA paw reaction on thermal pain behavior observed, PWL at1d,3d,7d decrease compared with the control group injected soles after (P<0.05), heat pain model successfully explained.2intrathecal injection of TNFR1-siRNA groupWill contain TNFR1-siRNA virus injected into the rat lumbar enlargement, plantar injection of CFA or saline.Results:Behavioral results showed that compared with<GFP+CFA> group,<TNFR1-siRNA+CFA> group, rat lumbar enlargement was given TNFR1-siRNA after CFA injection after animal feet thermal pain behavior observation, PWL injection in the foot after1d,3d,7d were significantly longer (P<0.05),, Description TNFRl-siRNA attenuated the CFA-induced thermal pain response.Western Blot results showed that, compared<GFP+CFA> group, after intrathecal administration of TNFRl-siRNA, reducing spinal dorsal horn TNFR1and expression of p-NRl.3intrathecal injection TNFR2-siRNA groupWill contain TNFR2-siRNA adenovirus was injected into the rat lumbar enlargement, plantar injection of CFA or saline.Results:Behavioral Analysis,<GFP+CFA> compared with the control group,<TNFR2-siRNA+CFA> rats given TNFR2-siRNA, the soles of the feet caused by the injection of CFA PWL on day1was significantly longer (p<0.05), but there was no significant difference between the third and seventh days PWL with the control groupWestern Blot results showed that, compared<GFP+CFA> group, intrathecal administration inhibits TNFR2-siRNA, expressing neurons TNFR2and p-NRl reduction in spinal dorsal horn.4.groups of intrathecal TNFR1&2-siRNAWill contain TNFR1&2-siRNA injected into rat lumbar enlargement, foot or saline were injected with CFA.Behavioral results showed:<TNFR1&2-siRNA+CFA> group, compared with<GFP+CFA>, after injection of CFA PWL value feet at1d,3d and7d seen significantly longer (P<0.05), which gives rats and did not give TNFR1&2-siRNA significantly increased TNFR1&2-siRNA PWL’s.Western Blot results showed that, compared<GFP+CFA> group, intrathecal TNFR1&2-siRNA, decreased expression of spinal p-NRl.In addition, compared with<TNFRl-siRNA+CFA>, PWL<TNFRl&2-siRNA+CFA> rats at1d,3d,7d did not show significant differences; compared with the<TNFR2-siRNA+CFA> ratio, PWL value<TNFRl&2-siRNA+CFA> group in the first3d,7d showed significant differences (p <0.05).ConclusionsIntraplantar CFA-induced inflammatory injury sustained activation of glial cells by stimulation stimulate and promote the release of TNF-a in the spinal cord, which is activated by receptors TNFR1/2, mainly TNFR1, raised dorsal horn NR1subunit phosphorylation technology, facilitated the transmission of pain signals in the spinal cord and the occurrence of hyperalgesia. Our experiments also more clearly this process, TNFR1is mediated major aspects of this process, TNFR1-mediated spinal hyperalgesia in the occurrence and development process played an important role, TNFR2may occur mainly in the early days some of the pain from effect.