The Analgesic Effect Of Progesterone On Neuropathic Pain And Its Possible Mechanisms In Rats

Objective Progesterone is a female gonadal steroid hormone synthesized in the ovary and placenta that exerts a wide range of actions,it is mainly used in obstetrics and gynecology.The research shown that in recent years,Progesterone as a neurosteroid is also synthesized within the nervous system,such as neurons and glial cells,including oligodendroglia cell and astrocyte.In the nervous system,progesterone exerts pleiotropic effects by regulating gene transcription,intracellular signaling pathways and neurotransmission.Recently,a few researches have shown that progesterone was able to alleviate CCI-induced nociceptive behaviors and to restore the electrophysiological changes in both motor(tibial)and sensory(sural)peripheral nerves,indicating that this steroid may have a therapeutic effect against neuropathic pain,But there is no clear relationship between progesterone and the activation of glial cells in the development of Neuropathic pain.So,this study used spinal nerve ligation rat model to investigate the relationship between the expression of microglia and astrocyte and the protein expression of COX-2 and i NOS in the lumbosacral enlargement and L5 ganglion.We used behavioral methods and immunofluorescence staining technique and ELISA kit for the experiment.MethodsExperiment 1: Making SNL neuropathic pain models and measuring the mechanical paw withdrawal threshold of rats after spinal nerve ligation24 male SD rats were randomly divided into three groups,normal group、sham group and the surgery group.SNL neuropathic pain model were made according the following procedure.first,anesthetized the rats with 10% chloral hydrate(300mg/kg,ip);Second,slited the waist iliac crest and exposed L5 spinal processus transverses then ligated the L5 spinal nerve with the 6th silk tightly;Finally,rats in model group were given intraperitoneal injection of gentamicin to prevent infection.Mechanical paw withdrawal threshold were determined with von Frey Filament everyday.Experiment 2: Effect of progesterone on the mechanical withdrawal threshold in SNL rats32 healthy adult male SD rats were randomly divided into four groups.The groups were as follows:(1)normal group:without any treatment;(2)control group: treatment with subcutaneous injection of 22.5 % Hydroxypropyl-beta-cyclodextrin(3ml/kg),(3)progesterone low and high dose groups(8 mg/kg,16 mg/kg),treatment with subcutaneous injection of progesterone.The mechanical withdrawal threshold of the both hindpaws of every rats were measured with von Frey Filament everyday from day 6 before SNL to day 21 postoperation.The results were expressed as 50% mechanical paw withdrawal threshold(50% PWT).The control group were subcutaneously injected with 22.5%Hydroxypropyl-beta-cyclodextrin(3ml/kg)daily from day 1 to day 21 postoperation.The treatment groups were administered subcutaneously with low(8 mg/kg)and high(16 mg/kg)dose of progesterone respectively,the treatment method was the same as the control group.Experiment 3: Effect of progesterone on the activation of glia cells in SNL ratsAfter drug treatment 1 hour,On day 3、day 7、day 14 and day 21 postoperation,some of the rats in each group were deeply anaesthetized and perfused with fixative,the lumbar spinal enlargment was removed,embedded and cut transversally at 20 μm thickness in a cryostat.The slices were stained immunofluorescencly and the activation of spinal microglia and astrocyte were observed under confocal microscope.Experiment 4: Effects of continuous subcutaneous injections of progesterone for 21 days on the expression of COX-2 and i NOS.24 male SD rats were randomly divided into three groups,normal group、control group and the pro-high((16 mg/kg)group.normal group without any treatment;The control group were subcutaneously injected with 22.5 % Hydroxypropyl–beta-cyclodextrin(3ml/kg)daily from day 1 to day 21 postoperation;The treatment groups were administered subcutaneously with high(16 mg/kg)dose of progesterone from day 1 to day 21 postoperation;On day3、day7、day14 and day21 postoperation,some of the rats in each group were deeply anaesthetized and perfused with fixative,the lumbar spinal enlargments and L5 ganglion was removed,then detect protein expression for lumbosacral enlargement in the spinal dorsal horn and L5 ganglion of COX-2 and i NOS with ELISA kit.Results1.Mechanical withdrawal threshold after spinal nerve ligation over timeThe mechanical paw withdrawal threshold(PWT)reduced significantly after spinal nerve ligation in rats,the PWT of ipsilateral side remaining below 5g from day 3 to day 13.Mechanical paw withdrawal threshold was significantly decreased(P <0.01),compared with sham-operated and contralateral side.The PWT of ipsilateral side from day 14 to day 21 was also significantly decreased(P <0.05).2.The effects of continuous subcutaneous injections of progesterone for 21 days on mechanical withdrawal thresholdProgesterone significantly improved the mechanical paw withdrawal thresholds of the ipisilateral hindpaws.The 50% PWT of the the ipisilateral hindpaws of the normal group,control group,progesterone 8mg/kg and 16mg/kg groups were 14.29±1.63、14.41±1.01g、14.27±1.02 g and 14.49±0.95 g respectively before the operation,and 14.74±0.73、3.84±1.09g、4.47±0.96 g and 4.59±1.47 g respectively on 3 days post operation,and 14.49±0.95、3.47±1.78g、4.76±1.72 g and 5.41±0.76 g respectively on day 7 postoperation,and 14.32±1.30、4.39±0.61g、5.48±0.54 g and 7.20±0.81 g respectively on day 14 postoperation,14.33±0.93、5.37±0.98、8.55±1.10、11.65±1.83 g respectively on day 21 postoperation.On day 14 and day 21 postoperation,the 50% PWT of the ipisilateral hindpaws of the low and high-dose group of progesterone increased significantly(P<0.05,P<0.01 respectively)as compared with those of the control group;the 50% PWT of the ipisilateral hindpaws of the progesterone high-dose group increased significantly(P <0.01)as compared with those of the low-dose group.Progesterone had no significant effects on the mechanical paw withdrawal thresholds of the contralateral hindpaws.3.The effects of continuous subcutaneous injections of progesterone for 21 days on the activation of OX-42.Immunofluorescence staining showed that SNL induced the activation of spinal microglia,and continuously subcutaneous injection of progesterone could inhibit the activation of microglia.After microglia cells activated,resting state microglia with many thin processes extending away from their soma turned into a rounded amoeboid-like appearance.The immunofluorescence staining intensity of the optical density(IOD)of OX-42 in the ipisilateral sides of the spinal cords was not significantly lowered on day 3 and day 7 postoperation(P>0.05),but the IOD of OX-42 was significantly lowered on day 14 and day 21 postoperation(P<0.01)as compared with those of the control group;Progesterone had no significant effects on the activation of microglia and astrocyte of the contralateral side of the spinal cords.4.The effects of continuous subcutaneous injections of progesterone for 21 days on the activation of GFAP.Immunofluorescence staining showed that SNL induced the activation of spinal astrocyte,and continuously subcutaneous injection of progesterone could inhibit the activation of astrocyte.After astrocyte cells activated,a series of changes have been produced,their performance is hyperplasia and hypertrophy.the IOD of GFAP in the normal group at baseline levels,the IOD of GFAP in the ipisilateral sides of the spinal cords was not significantly lowered on day 3 and day 7 postoperation(P>0.05),but the IOD of GFAP was significantly lowered on day 14 and day 21 postoperation(P<0.01)as compared with those of the control group.Progesterone had no significant effects on the activation of microglia and astrocyte of the contralateral side of the spinal cords.5.The effects of continuous subcutaneous injections of progesterone for 21 days on the expression of COX-2 and i NOS.The expression of COX-2 and iNOS for lumbosacral enlargement in the spinal dorsal horn and L5 ganglion was significantly lowered.The expression of COX-2 and i NOS at baseline levels of the normal group,the average level is 11.24±1.71ng/L、7.71±1.29 ng/L and 0.80±0.09μmol/L、0.58±0.11μmol/L.The concentration of two proteins dramatic increase after SNL operation of the control group.The average level of COX-2 and i NOS for lumbosacral enlargement in the spinal dorsal horn and L5 ganglion is 84.10±8.62 ng/L、88.85±6.91 ng/L and 7.40±0.59μmol/L、7.58±0.60μmol/L.In the pro-high((16 mg/kg)group,the protein expression of COX-2 and i NOS was not significantly lowered on day 3 and day 7 postoperation(P>0.05).On day 3 the average level of COX-2 and i NOS in the spinal dorsal horn and L5 ganglion is 84.59±9.23 ng/L、87.56±8.26 ng/L and 7.45±0.87μmol/L、7.52±1.25μmol/L.On day 7 the average level of COX-2 and i NOS in the spinal dorsal horn and L5 ganglion is 80.36±5.96 ng/L、85.29±8.41 ng/L and 7.14±0.47μmol/L、7.20±0.91μmol/L.but the expression was significantly lowered on day 14 and day 21 postoperation(P<0.01)as compared with those of the control group.On day 14 the average level of COX-2 and i NOS in the spinal dorsal horn and L5 ganglion is 58.84±5.44 ng/L、61.21±5.43 ng/L and 5.17±0.82μmol/L、5.23±0.58μmol/L.On day 21 the average level of COX-2 and i NOS in the spinal dorsal horn and L5 ganglion is 32.77±5.07 ng/L、31.78±8.37 ng/L and 2.42±0.29μmol/L、2.40±0.36μmol/L.Conclusions Progesterone dose-dependently inhibited the mechanical pain induced by SNL through the inhibition of the activation of glial cells and the protein expression of COX-2 and i NOS.