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Objective:To evaluate the relationship between the euchromatic histone-lysine N-methyltransferase (G9a) and sodium-dependent activation of potassium channel (Slack) in the dorsal root ganglia (DRG) of rats with neuropathic pain (NP).Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 1 month, weighing 100-120 g, were divided into 4 groups ( n=12 each) by a random number table method: sham operation group (S group), vector plus sham operation group (VS group), vector plus NP group (VN group), and G9a CRISPR/Cas9 knockout plus NP group (GN group). Sham operation was performed at the age of 2 months in group S. In group VS, AAV5 1 μl was microinjected into L 4 and L 5 DRG at the age of 1 month, and sham operation was performed at the age of 2 months.In VN group and GN group, AAV5 and G9a CRISPR/Cas9 knockout plasmid 1 μl were microinjected into L 4 and L 5 DRG at the age of 1 month, and NP model was established by spinal nerve ligation (SNL) at the age of 2 months.Six rats in each group were selected to measure the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) before microinjection (T 0), before SNL (T 1), and at 3, 5 and 7 days after SNL (T 2-4). The animals were sacrificed after the last behavioral testing, the DRGs of lumbar segment (L 4, 5) were removed for determination of the expression of G9a, dimethylation of histone H3 at lysine 9(H3K9me2) and Slack (by Western blot). At 7 days after establishing the model, 6 rats from each group were selected to culture the primary DRG neurons.The frequency and amplitude of Slack current in DRG neurons and miniature excitatory post-synaptic currents (mEPSCs) in the spinal dorsal horn were measured by whole-cell patch-clamp technique. Results:Compared with group S, the TWL was significantly shortened and the MWT was decreased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was up-regulated, the expression of Slack was down-regulated, the amplitude and frequency of Slack currents in DRG neurons were decreased, and the frequency of mEPSCs was increased in group VN ( P<0.05), and no significant change was found in the parameters mentioned above in group VS ( P>0.05). Compared with group VN, the TWL was significantly prolonged and the MWT was increased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was down-regulated, the expression of Slack was up-regulated, the amplitude and frequency of Slack currents in DRG neurons were increased, and the frequency of mEPSCs was decreased in group GN ( P<0.05). Conclusion:The mechanism of NP is related to up-regulating the expression of G9a in DRG, thus inhibiting the expression and opening of Slack channels in rats.
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Objective To evaluate the role of spinal nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in hydrogen-induced reduction of inflammatory pain in rats.Methods Sixty-four SPF healthy adult male Sprague-Dawley rats,weighing 200-250 g,were divided into 4 groups (n =16 each) using a random number table:control group (group C),inflammatory pain group (group IP),inflammatory pain plus hydrogen-rich saline group (group IP+H2) and inflammatory pain plus hydrogen-rich saline plus Nrf2 inhibitor all-trans retinoic acid (ATRA) group (group IP+H2+ATRA).Chronic inflammatory pain was induced by injecting complete Freund's adjuvant (CFA) 100 μl into the plantar surface of the left hind paw in IP group and IP+H2 group.The equal volume of normal saline was given instead in group C.Hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 7consecutive days starting from 1 day after injecting CFA in group IP+H2 and group IP+H2+ATRA,and the equal volume of normal saline was given instead in the other groups.ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days starting from 2 days before injecting CFA.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before establishing the model (T0) and 1,3 and 7 days after establishing the model (T1-3).Six rats were sacrificed after the last measurement of pain threshold on day 7 after establishing the model,and the L4-6 lumbar segments of the spinal cord were removed for determination of the expression of Nrf2,HO-1 and glial fibrillary acidic protein (GFAP) by Western blot.Results Compared with group C,the MWT was significantly decreased and the TWL was shortened at T1-3,and the expression of Nrf2,HO-1 and GFAP was up-regulated in IP and IP+H2 groups (P＜0.05).Compared with group IP,the MWT was significantly increased and the TWL was prolonged at T1-3,the expression of Nrf2 and HO-1 was up-regulated,and the expression of GFAP was down-regulated in group IP+H2 (P＜0.05),and no significant change was found in the parameters mentioned above in group IP+H2+ATRA (P＞0.05).Compared with group IP+H2,the MWT was significantly decreased and the TWL was shortened at T1-3,the expression of Nrf2 and HO-1 was down-regulated,and the expression of GFAP was up-regulated in group IP+H2+ATRA (P＜0.05).Conclusion Activation of spinal Nrf2/HO-1 signaling pathway is involved in hydrogen-induced reduction of infflammatory pain in rats.
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Objective To evaluate the influence of autophagy on pain behavioristics and astrocytic activation in rats with inflammatory pain.Methods Seventy-eight clean male Sprague-Dawley (SD) adult rats were randomly divided into four groups: control group (n = 12), model group (n = 42), autophagy inducer rapamycin (Rap) pretreatment group (n = 12) and autophagy inhibitor 3 methyladenine (3-MA) pretreatment group (n = 12). The inflammatory pain rat model was reproduced by subcutaneous injection of freund's complete adjuvant (CFA) 100μL at foot sole, whilein control group, the same volume 0.9% normal saline 100μL was injected at the same site. One hour before modeling, Rap 10 mg/kg and 3-MA 15 mg/kg were intraperitoneally injected in rats in Rap and 3-MA pretreatment groups respectively, and the same volume of 0.9% normal saline was injected intraperitoneally in rats of control and model groups. Before modeling and 6, 12, 24 hours and 3 days after modeling, the L4-L6 spinal cord tissue was harvested from 6 rats in model group, and autophagy protein membrane microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and autophagy related gene Beclin-1 expressions were detected by Western Blot in the tissue; the changes of pain behavioral indexes mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL,n = 6), were observed at6, 12, 24 hours and 3 days, 7 days after modeling in the 6 rats taken from each group; in another 6 rats in each group, 24 hours after modeling, L4-L6 spinal cord tissue was collected, immunofluorescence staining was used to observe the changes of astrocytes and the positive expression of glial fibrillary acidic protein (GFAP) under a confocal microscopy, and the protein expression quantity of GFAP was detected by Western Blot in the tissue.Results ① The inflammatory pain could induce the increase of rat autophagy protein LC3Ⅱ and Beclin-1 expressions in spinal cord tissue, reaching their peaks at 24 hours (A value: 0.59±0.07, 0.51±0.06, respectively), and then they were gradually decreased. ② With the prolongation of time, in the model group MWT was gradually decreased, TWL was gradually shortened, they reached their valley values at 24 hours after modeling [MWT (g): 17.8±1.9, TWL (s): 6.8±0.4], and from 12 hours they were significantly decreased compared with those in control group [12hours MWT (g): 21.5±2.4 vs. 43.4±5.1, TWL (s): 12.0±1.1 vs. 17.6±1.2, bothP < 0.05], after modeling for 3 days they were increased; Compared with model group, 12 hours after autophagy inducer Rap was given, MWT was significantly increased (g: 36.8±4.9 vs. 21.5±2.4,P < 0.05), TWL was significantly prolonged (s: 14.3±1.1 vs. 12.0±1.1,P < 0.05); from 12 hours after autophagy inhibitor 3-MA was given, MWT was further reduced (g: 18.6±1.9 vs. 21.5±2.4, P<0.05), TWL was further shortened (s: 8.4±0.6 vs. 12.0±1.1,P < 0.05). ③ Confocal microscopic findings showed, there was no significant acstrocytic change, and only litter GFAP expression was seen in control group. In model group, the inflammatory pain induced astrocyte activation, manifesting glial cell hypertrophy, hyperplasia, gelatinousnetwork deformation, and GFAP expression was obviously increased compared with that in the control group (A value: 0.54±0.09 vs. 0.16±0.02,P < 0.05). Since autophagy inducer Rap can decrease astrocyte activation and inhibit GFAP expression, there was statistical significant difference between Rap pretreatment and model groups (A value: 0.33±0.06 vs.0.54±0.09,P < 0.05); autophagy inhibitor 3-MA can further aggravate astrocytes activation and up-regulate GFAP expression in 3-MA pretreatment group (A value: 0.73±0.08 vs. 0.54±0.09,P < 0.05).Conclusion Autophagy participates in the process of astrocytic activation and pain behavioristics in rats with inflammatory pain.
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Objective To investigate the effects of HO/CO pathway on inflammation cytokines in a rat model of incisional pain. Methods Thirty-six rats were executed to collect ipsilateral spinal cord tissues for HO-1 detection by Western blot assay, and cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6 and high mobility group box (HMGB)1 were detected by ELISA before and at 1, 4, 8, 12 and 24 h after establishing incisional pain model. Additionally, 36 rats without establishment of incisional pain model were used as control group. A total of 144 model rats of incisional pain were divided into incisional pain (IP) group, IP+hemin group (100 mg/kg hemin was injected by i.p. before operation), IP+Znpp-IX group (45μmoL/kg Znpp-IX was injected by i.p. before operation) and IP+CORM-2 group (10 mg/kg CORM-2 was injected by i.p. before operation). Values of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were detected, and expressions of TNF-a, IL-1 b, IL-6 and HMGB1 were measured by ELISA before and at 1, 4, 8, 12 and 24 h after operation. Results Compared with pre-operation of incisional pain in rats, expression levels of HO-1 protein and cytokines TNF-a, IL-1 b, IL-6 and HMGB1 were increased at 1, 4, 8, 12 and 24 h after operation (P PWTL were decreased and cytokines TNF-α, IL-1β, IL-6 and HMGB1 were increased in IP+Znpp-IX group (P<0.05). Conclusion Incisional pain can increase the expression of HO-1, and HO-1/CO pathway exists the regulatory effect on inflammatory cytokines in the rat model of incisional pain.
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Objective To evaluate the significance of clinical grading methods in acute pulmonary embolism (APE). Methods Clinical data of 259 patients suspected APE were retrospectively analyzed. The clinical probability was classified into low, intermediate and high grade by the Geneva score, the Wells score and the SYSU score. The result was contrasted with gold standard. Results Through the three, methods, pa-tients were classified into low pmbability (43.9%-52.5%), intermediate probability (38.0%-42.1%) and high probability (9.5%-14.0%), and the actual frequencies of APE in each category were 6.2%-14.4% in low probability, 65.9%-76.2% in intermediate probability, 88.5%-90.5% in high probability. The SYSU score had the lowest rate of missed diagnosis in low probability (P<0.05 ).The Geneva score was the most accurate in predicting the intermediate probability (P<0.05). But in high probahility, three prediction rules had no significant difference (P>0.05). Combined with D--dimer test, the rote of missed diagnosis in low probability can be lowered. Conclusions The clinical grading methods can predict the clinical probability of APE. It exists similar accuracy, but has different scope of application. Clinical doctor should choose the ap-propriate grading methods in different patients.