Changes in the expression of large-conductance calcium-activated potassium channels in dorsal root ganglion neurons after electrical injury in rats′ sciatic nerves and its influence on sensory conduction function
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摘要: 目的 研究大鼠坐骨神经电损伤后脊髓背根神经节(DRG)中大电导钙激活钾(BKCa)通道表达变化及其对感觉传导功能的影响。 方法 取成年SD大鼠136只,按照随机数字表法分为正常对照组8只,假电伤组及75、100、125 V电伤组各32只。正常对照组大鼠不做处理,假电伤组大鼠仅行左侧坐骨神经主干钝性分离,75、100、125 V电伤组大鼠左侧坐骨神经主干造成相应电压电损伤。正常对照组大鼠于饲养1周时,假电伤组及各电伤组大鼠于伤后3 d及1、2、3周,每组各取8只测定大鼠机械缩足反射阈值(PWMT),其中100 V电伤组大鼠伤后1周行前述检测后于鞘内注射NS1619,注射后3 h内每隔30分钟测定1次PWMT。各组各时相点大鼠完成PWMT检测后处死,取脊髓L4~L6段DRG,采用免疫组织化学染色法观察BKCa通道分布,蛋白质印迹法、RT-PCR法分别检测BKCa通道蛋白、mRNA表达。对数据行单因素方差分析、析因设计方差分析、SNK检验。 结果 (1)与正常对照组的(11.2±2.0)g和假电伤组的(11.3±2.1)、(12.0±2.0)、(11.1±1.6)、(10.3±2.1)g比较,75、100 V电伤组大鼠伤后3 d及1、2、3周PWMT显著降低[(5.8±0.6)、(5.0±0.8)、(4.2±0.3)、(5.9±1.1)g,(5.3±1.3)、(5.9±2.0)、(4.5±2.7)、(4.3±1.3)g,
P 值均小于0.05]。125 V电伤组大鼠伤后3 d、1周PWMT[(6.1±1.6)、(5.7±1.7)g]较正常对照组和假电伤组显著降低,伤后2、3周PWMT[(26.7±3.3)、(21.7±3.4)g]明显高于其余4组,P 值均小于0.05。100 V电伤组大鼠伤后1周注射后3 h内PWMT呈先增高后降低趋势,其中注射后30、60、90、120 min时PWMT为(8.5±0.8)、(9.7±1.2)、(11.0±1.5)、(8.6±0.8)g,较干预前明显增高(P 值均小于0.05)。(2)正常对照组及各时相点假电伤组大鼠DRG神经元细胞质、细胞膜BKCa通道阳性表达均较多;各电伤组大鼠DRG神经元细胞质、细胞膜BKCa通道阳性表达随伤后时间延长而减少,以125 V电伤组最为明显。(3)伤后3 d,各组大鼠DRG中BKCa通道蛋白水平相近(P 值均大于0.05)。与正常对照组的0.477±0.027、0.521±0.034、0.475±0.022和假电伤组的0.511±0.025、0.489±0.025、0.483±0.032比较,75、100、125 V电伤组大鼠伤后1、2、3周DRG中BKCa通道蛋白水平明显下调(0.274±0.026、0.202±0.019、0.285±0.033,0.253±0.022、0.233±0.024、0.203±0.017,0.092±0.017、0.095±0.021、0.087±0.016,P 值均小于0.05)。125 V电伤组大鼠伤后1、2、3周DRG中BKCa通道蛋白水平较75、100 V电伤组明显降低(P 值均小于0.05)。(4)与正常对照组的0.581±0.051和假电伤组的0.603±0.045、0.586±0.032、0.614±0.045、0.572±0.038比较,75、100、125 V电伤组大鼠伤后3 d及1、2、3周BKCa通道mRNA水平显著下调(0.326±0.021、0.238±0.019、0.291±0.022、0.364±0.018,0.264±0.020、0.293±0.017、0.243±0.023、0.295±0.021,0.134±0.023、0.089±0.017、0.074±0.018、0.087±0.020,P 值均小于0.05)。125 V电伤组大鼠各时相点BKCa通道mRNA水平较75、100 V电伤组明显降低(P 值均小于0.05)。 结论 大鼠坐骨神经电损伤后,对应脊髓节段DRG中BKCa通道表达降低,其参与神经电损伤后触痛觉过敏的病理过程。Abstract: Objective To study the changes in the expression of large-conductance calcium-activated potassium (BKCa) channels in dorsal root ganglion (DRG) neurons after electrical injury in rats′ sciatic nerves and its influence on sensory conduction function. Methods One-hundred and thirty-six adult SD rats were divided into normal control group, sham electrical injury group, and 75, 100, 125 V electrical injury groups according to the random number table, with 8 rats in normal control group and 32 rats in each of the rest 4 groups. Rats in normal control group were routinely fed without any treatment. Blunt dissection of the sciatic nerves of left hind leg of rats was performed in sham electrical injury group, while sciatic nerves of left hind leg of rats in electrical injury groups were electrically injured with corresponding voltage. Eight rats of normal control group fed for one week, and 8 rats from each of the rest four groups on post injury day (PID) 3 and in post injury week (PIW) 1, 2, 3 respectively were collected to detect the paw withdrawal mechanical threshold (PWMT). In addition, rats of 100 V electrical injury group in PIW 1 were collected and intrathecally injected with NS1619 after former PWMT detection, and PWMT was detected per 30 minutes within three hours post injection. The rats in each group at each time point were sacrificed after PWMT detection. The DRG of L4 to L6 segments of spinal cord was sampled to observe the BKCa channels distribution with immunohistochemical staining and to detect the protein and mRNA expressions of BKCa channels with Western blotting and reverse transcription-polymerase chain reaction respectively. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test. Results (1) The PWMT values of rats in 75 and 100 V electrical injury groups on PID 3 and in PIW 1, 2, 3 were (5.8±0.6), (5.0±0.8), (4.2±0.3), (5.9±1.1) g; (5.3±1.3), (5.9±2.0), (4.5±2.7), (4.3±1.3) g, respectively, which were significantly lower than the value (s) in normal control group [(11.2±2.0) g] and sham electrical injury group [respectively (11.3±2.1), (12.0±2.0), (11.1±1.6), (10.3±2.1) g, withP values below 0.05]. The PWMT values of rats in 125 V electrical injury group decreased obviously on PID 3 and in PIW 1 [(6.1±1.6) and (5.7±1.7) g] as compared with the value (s) in normal control group and sham electrical injury group, and they were obviously increased in PIW 2 and 3 [(26.7±3.3) and (21.7±3.4) g] as compared with the value (s) of the rest 4 groups (withP values below 0.05). The PWMT of 100 V electrical injury group in PIW 1 firstly increased and then decreased within three hours post injection, which increased significantly at post injection minutes 30, 60, 90, 120 as compared with that before intervention [respectively (8.5±0.8), (9.7±1.2), (11.0±1.5), (8.6±0.8) g, withP values below 0.05]. (2) The positive expression of BKCa channels in large amount was observed in the cytoplasm and cytomembrane of neurons on the DRG of rats in normal control group and sham electrical injury group at each time point. The positive expression of BKCa channels in the cytoplasm and cytomembrane of neurons on the DRG of rats decreased over time in electrical injury groups, which was most obvious in 125 V electrical injury group. (3) There were no statistically significant differences in the protein expression of BKCa channels in DRG of rats among the five groups on PID 3 (withP values above 0.05). Compared with those in normal control group (0.477±0.027, 0.521±0.034, 0.475±0.022) and sham electrical injury group (0.511±0.025, 0.489±0.025, 0.483±0.032) in PIW 1, 2, 3, the protein expressions of BKCa channels in DRG of rats in 75, 100, 125 V electrical injury groups were decreased significantly (0.274±0.026, 0.202±0.019, 0.285±0.033; 0.253±0.022, 0.233±0.024, 0.203±0.017; 0.092±0.017, 0.095±0.021, 0.087±0.016, withP values below 0.05). The protein expressions of BKCa channels in DRG of rats in 125 V electrical injury group in PIW 1, 2, 3 were obviously lower than those in 75 and 100 V electrical injury groups (withP values below 0.05). (4) The mRNA expression levels of BKCa channels in DRG of rats in 75, 100, 125 V electrical injury groups on PID 3 and in PIW 1, 2, 3 were 0.326±0.021, 0.238±0.019, 0.291±0.022, 0.364±0.018; 0.264±0.020, 0.293±0.017, 0.243±0.023, 0.295±0.021; 0.134±0.023, 0.089±0.017, 0.074±0.018, 0.087±0.020, respectively, significantly decreased as compared with the level (s) in normal control group (0.581±0.051) and sham electrical injury group (0.603±0.045, 0.586±0.032, 0.614±0.045, 0.572±0.038), withP values below 0.05. The mRNA expression levels of BKCa channels in DRG of rats in 125 V electrical injury group at each time point were lower than those in 75 and 100 V electrical injury groups (withP values below 0.05). Conclusions The electrical injury in sciatic nerves results in reduction of the BKCa channels expression in rat′s DRG of corresponding spinal segments, which plays a role in the pathological process of sensory conduction dysfunction.
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