| BackgroundComplex regional pain syndrome(CRPS)is a chronic pain syndrome.The ability of CRPS patients to participate in daily activities is reduced,which is seriously affected.Antagonists of N-methyl-D-aspartic acid receptors(NMDARs)have been shown to have relief effects on hyperalgesia,but their distribution and role in the periphery are unclear.NR2B receptor(N-methyl-d-aspartate receptor subunit 2B receptor)is a regulatory subunit in NRMDRs,and its palmitoylation may play a role in hyperalgesia as an important mechanism for regulating the membrane transport of this receptor.This thesis is consisted of two parts of animal experiments,using a chronic post-ischemia pain(CPIP)model to mimic CRPS and further explore its hyperalgesia mechanism and use palmitoylation inhibitors in the periphery to verify the whether palmitoylation of NR2B plays a role in enhancing peripheral inflammation and hyperalgesia.MethodsIn the first part of the experiment,male SD rats were randomly divided into sham operation(Sham)group and chronic post-ischemic pain(CPIP)group.In the CPIP group,an O-ring(inner diameter 0.56 cm)was inserted into the ankle joint of the right hind limb of the rat to cause ischemia of the right hind limb,after 3 hours,the O-ring was cut off and the limb was reperfused,while the rats of the Sham group placed a cut-off O-ring.Parameters such as paw mechanical withdrawal threshold(PMWT),paw withdrawal thermal latency(PWTL),skin temperature of the feet,and thickness of the palms of the feet were recorded at various time points after ischemia reperfusion(IR),and the skin and tibial nerves were obtained and stored at 1 and 3 days after ischemia.HE staining was used to observe the pathological changes of the skin after IR,protein immunoblotting was used to detect the expression of NR2B and inflammatory factors IL-1β,TNF-α,and immunohistochemistry was used to detect the distribution and expression level of nerve fiber PGP9.5 in the skin,transmission electron microscope scanning was used to detect tibial nerve.In the second part of the experiment,male SD rats were randomly divided into a simple vehicle(Veh)group,a vehicle(CPIP+Veh)group,a palmitoylation inhibitor(CPIP+2-BP)group,and a simple palmitoylation inhibitor(2-BP)group.Three days before the right foot ischemia,subcutaneous injection of 50 μl of vehicle(0.1%ethanol solution)or 50μL palmitoylation inhibitor(100 mM 2-BP)was started daily.Rats in the Veh group and 2-BP group were anesthetized for only 3 hours on the day of ischemia,and the right hind limb was not treated for ischemia;CPIP+Veh group and CPIP+2-BP group were fitted with an O-ring at the ankle joint of the right hind limb of the rat,after 3 hours of ischemia of the right hind limb,the O-ring was cut off and the limb was reperfused.After IR,the PMWT,PWTL,skin temperature of the feet,and thickness of the palms of the feet were recorded at various time points,and the skin was harvested 3 days after IR.Western blotting was used to detect the expression of NR2B,immunofluorescence was used to detect the distribution and expression of nerve fibers PGP9.5 and mast cells in the skin,and immunoprecipitation and acyl-biotin exchange were used to detect palmitoylation of NR2B in skin.ResultsThe first part of the experiment showed that compared with the Sham group,the PMWT and PWTL of the CPIP group decreased to the lowest point at 6 hours after ischemia(P<0.05).Both the temperature and the thickness of the foot increased at 6 hours(P<0.05).The PMWT and PWLT gradually increased with the extension of the reperfusion time,but it was not restored to the baseline after 14 days of reperfusion(P<0.05),the phenomenon of increased skin temperature recovered after 12 hours of reperfusion(P>0.05),and the thickness of the foot palm recovered after 2 days of reperfusion(P>0.05).HE staining showed an increase of dermal fibers and infiltration of inflammatory cells in the skin of the CPIP group.The expression of NR2B increased significantly after IR(P<0.05).At the same time,the distribution of nerve fibers PGP9.5 in the skin of the CPIP group decreased,and the tibial nerve electron microscope showed demyelination phenomenon.The second part of the experimental results showed that compared with the Veh or 2-BP group,the PMWT and PWTL of the CPIP+Veh and CPIP+2-BP groups fell to the lowest point 6 hours after ischemia(P<0.05),the skin temperature and the thickness of the foot of the ischemic side foot compared with the healthy side foot in CPIP+Veh group and CPIP+2-BP group increased at 6 hours(P<0.05),with the extension of the reperfusion time,the PMWT and PWTL were gradually increased,but their level did not return to the baseline level after 14 days of reperfusion(P<0.05),and the increased skin temperature recovered after 12 hours of reperfusion(P>0.05),thickness of the foot palm recovered after 1 day of reperfusion(P>0.05);compared with the CPIP+Veh group,the PMWT and PWTL of the CPIP+2-BP group increased after 24 hours and 2 days(P<0.05),respectively.But the skin temperature and the thickness of the operation foot were not significantly changed at each time point(P>0.05).Compared with Veh or 2-BP group,the expression of NR2B in CPIP+Veh group and CPIP+2-BP group increased significantly after ischemia(P<0.05);compared with CPIP+Veh group,the expression of NR2B in CPIP+ 2-BP group decreased.Compared with the CPIP+Veh group,CPIP+2-BP group immunofluorescence showed that the reduction of nerve fiber PGP9.5 in the skin was relieved and the ratio of mast cells close to nerve fibers was greater(P<0.05),palmitoylation of NR2B was decreased in skin.ConclusionsAfter chronic ischemia in the hind feet of rats,local inflammation increased significantly in a short period of time,and the pain thereshold was decreased most.NR2B activation in the skin,denervation of the skin,and demyelination of the tibial nerve played a role in hyperalgesia.Inhibition of NR2B palmitoylation has no obvious improvement effect on local skin inflammation and PMWT and PWTL in a short time after ischemia,but it can improve the denervation of the skin to improve hyperalgesia from 1-2 days after ischemia. |
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