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Objective: To investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation. Methods: Fifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention. Results: Compared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn. Conclusion: Tuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.
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Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty SPF healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),NL1-shRNA plasmid group (group NL),incisional pain plus remifentanil group (group I+R),incisional pain plus remifentanil plus blank vector group (group I+R+B),and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+R+NL).Negative lentivirus was intrathecally injected in group I+R+B.In NL and I+R+NL groups,10 μl NL-1-shRNA lentivirus at 1×10s IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+R groups.After transfection was stable,normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+R,I+R+B and I+R+NL groups,0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals,and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3,6,24 and 48 h after the end of administration (T1-4).The animals were sacrificed after measurement of pain threshold at T4,and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors,and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group C,the MWT was significantly decreased,and TFL was shortened at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated,and m/t ratio was increased in I+R and I+R+B groups (P＜0.05).Compared with I+R and I+R+B groups,the MWT was significantly increased and TFL was prolonged at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated,and m/t ratio was decreased in group I+R+NL (P＜0.05).Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane,which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
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Objective@#To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.@*Methods@#Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.@*Results@#Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05).@*Conclusion@#NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
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Objective To observe variation of Caveolin-1 expression in dorsal root ganglia and spinal dorsal horn in chronic neuropathic pain of rats.Methods Experimental animals of 56 SD rats were used for this study,and 28 rats for control.They were divided into 7 groups and their L5 nerves were ligated to produce the neuropathic pain model.After 1,3,5,7,10,14,21d of surgery,the mechanical shrinkage foot threshold and hot shrinkage foot incubation period were detected.Neuropathic pain models were confirmed to make successfully compared with the control group.Rats in the surgery group and the control group rats were all put to death on the 22d.The total protein of L5 spinal cord and dorsal root ganglia was extracted from the same side or the opposite side.The Caveolin-1 expression of different groups and sides was detected by the Western blot method in dorsal root ganglia and spinal dorsal horn in chronic neuropathic pain of rats.Results After 5,7,10,14,21d of L5 ligation,the expressions of Caveolin-1 were increased by 40.4％ ± 3.67％,126.9％ ± 5.46％,159.7％ ±4.89％,119.1％ ±5.77％ and 91.4％ ±5.31％ in the same sides.The difference was significant on 10d(P ＜0.01)after the surgery.After 5d,7 d,10d,14d,21 d of L5 ligation,the expressions of Caveolin-1 were increased by 33.3％ ± 4.89％,152.8％ ±3.56％,142.1％ ±5.43％,103.2％ ± 6.21％,175.6％ ±4.81％ in the opposite sides.It had statistic difference on 10d,14d,21d (P ＜ 0.05).There were no statistic differences among control groups.Conclusion The Neuropathic chronic pain can increase the Caveolin-1 expressions on the same side of dorsal root ganglia and spinal dorsal horn.
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Aim To investigate whether the pain modi-fication by group I metabotropic glutamate receptors (mGluRs)required the involvement of Src homology-2 domain-containing phosphatase-2 (SHP-2 ).Methods Co-immunoprecipitation was performed to examine the possible interaction between SHP-2 and group I mGluRs in spinal cord dorsal horn of mice.By measur-ing the paw withdrawal thresholds,the effects of SHP-2 inhibitor NSC-87877 or its catalytically inactive SHP-2 (C459S ) mutant on allodynia induced by group I mGluRs agonist DHPG (50 nmol)were observed.Re-sults Anti-mGluR5 antibody was able to co-immuno-precipitate SHP-2 from spinal dorsal horn of mice, while no SHP-2 was precipitated by anti-mGluR1 anti-body.Inactivation of SHP-2 by NSC-87877 (6 nmol) or SHP-2 (C459S ) effectively attenuated allodynia caused by DHPG.Conclusion SHP-2 can physically interact with mGluR5.The activation of SHP-2 may be necessary for group I mGluRs to process the nocicep-tive information.
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Aim Toinvestigatetheantagonisticeffect of intrathecal injection of carbenoxolone (CBX ) on neuropathic pain and its underlying mechanism.Meth-ods SixtymaleSprague-Dawleyratswererandomly divided into five groups (n =12 ):group I received sham surgery then treated with saline;group Ⅱ re-ceived SNT then treated with saline;groupⅢreceived SNT then treated with 0. 05 μg CBX;group Ⅳ re-ceived SNT then treated with 0. 5 μg CBX;group Ⅴreceived SNT then treated with 5 μg CBX.Treatment was undertaken with 10 μl volume as a single intrathe-cal injection on postoperative day 10.Mechanical with-drawl thresholds were measured 1 d before operation, 1,3,5,7 and 10 d after surgery,1 h before intrathe-cal administration,and 1 ,2,4,6 h after intrathecal administration.Lumbar spinal cord was obtained 2 h after intrathecal administration to determine the expres-sions of GFAP by immunohistology and TNF-α,IL-1βby ELISA in bilateral spinal dorsal horns.Results Comparedwiththeshamgroup,thebilateralMWTin group Ⅱ ~Ⅴ was significantly decreased.Compared with the MWT 1 h before intrathecal administration on day 10,the values at 1 ,2,4,6 h after administration of group Ⅱ and Ⅲ had no marked difference.The ip-silateral MWT in groupⅣhad no significant difference at 1,2,4 h after administration,the contralateral MWT was significantly increased,whereas GFAP and TNF-α,IL-1βwas significantly decreased in the spinal cord .In group Ⅴthe bilateral MWT was significantly improved at 1 ,2,4 h after administration,whereas GFAP and TNF-α,IL-1βwere significantly decreased inthespinalcord.Conclusions IntrathecalCBXcan inhibit the development of bilateral MWT.The analge-sic effect of CBX is implemented partly via suppressing the actation of GFAP and the realease of TNF-α,IL-1βin the spinal doral horn.
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Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%+/-12% and 76%+/-14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.
Subject(s)
Animals , Male , Rats , Acid Sensing Ion Channels/genetics , Amiloride/analogs & derivatives , Analgesics/pharmacology , Disease Models, Animal , Neuralgia/drug therapy , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/metabolism , Transcription, Genetic/drug effectsABSTRACT
The relative quantitative [~3H]-2-deoxyglucose (2DG)method was used to study the laminary projection of acupuncture and noxious information in the rat spinal cord dorsal horn. The noxious information projected to lamina Ⅰ、Ⅱ、Ⅳ、Ⅴ、Ⅵ and Ⅹ, and the acupuncture information projected to lamina Ⅰ、Ⅱ、Ⅳ and Ⅴ in the spinal cord dorsal horn. The noxious and acupuncture information had overlapping projection areas in the spinal cord. It is in such areas that the two types of information may be interacted on each other and consequently the nociception is modulated at the spinal level.