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目的观察鞘内注射入β-内啡肽(β-EP)基因重组腺病毒(Ad-NEP)对慢性神经病理痛大鼠的镇痛作用。方法36只雄性SD大鼠,体重210-260 g,随机分为手术组(n=26)、假手术组(n= 5)、空白对照组(n=5),手术组分为三个亚组:Ad-NEP组(n=9)、绿色荧光蛋白基因重组腺病毒(Ad- GFP)组(n=9)、生理盐水组(n=8)。大鼠腹腔注射氯胺酮100 mg/kg和阿托品50 mg/kg麻醉后,手术组构建大鼠坐骨神经慢性捆扎(CCI)模型,假手术组进行同样手术,但不捆扎坐骨神经,空白对照组不进行手术;手术组经L5,6间隙蛛网膜下腔置入PE-10导管,7d后分别注射1×108pfu的Ad-NEP、Ad- GFP和40μl生理盐水。于手术后7 d(T0)、鞘内注射前当日(T1)、注射后1 d(T2)、1周(T3)、2周(T4)、3 周(T5)、4周(T6)、5周(T7)分别测定五组大鼠的左右足热痛阈;T3时取Ad-NEP、Ad-GFP组大鼠各1 只,取脊髓L3-6段作免疫组化检测;鞘内注射后10 d取Ad-NEP、Ad-GFP组大鼠,经腹腔内注射1 mg/kg 纳络酮,每间隔10min记录右足热痛阈(t0-9,共观察90min),并分别测定这两组T1-7时的脑脊液内β- EP浓度。结果Ad-GFP组和生理盐水组大鼠右足热痛阈明显低于假手术组和空白对照组(P< 0.01)。Ad-NEP组在T0,1,7时右足热痛阈明显低于假手术组和空白对照组(P<0.01),在T2-6时的右足热痛阈高于T0,也高于Ad-GFP组和生理盐水组(P<0.05或0.01),t1-6时右足热痛阈低于t0时(P< 0.01),与Ad-GFP组和生理盐水组比较差异无统计学意义(P>0.05)。Ad-NEP组脊髓外膜可见明显的橙黄色浓染带,后角区可见少量橙黄色细胞,T2-7时脑脊液内β-EP浓度均高于Ad-GFP组(P< 0.01。)结论鞘内注射重组腺病毒Ad-NEP对慢性神经病理痛大鼠有明显的镇痛效果,作用时间长达4周以上。
Objective To observe the analgesic effect of Intrathecal injection of β-EP gene recombinant adenovirus (Ad-NEP) on chronic neuropathic pain rats. Methods Thirty-six male Sprague-Dawley rats weighing 210-260 g were randomly divided into operation group (n = 26), sham operation group (n = 5) and blank control group (n = 5) Group: Ad-NEP group (n = 9), GFP group (n = 9) and saline group (n = 8). Rats were anesthetized with ketamine 100 mg / kg and atropine 50 mg / kg. Rats in the operation group were given chronic constriction injury (CCI) In the operation group, the PE-10 catheter was placed in the space subarachnoid space of L5,6 and injected with 1 × 108pfu of Ad-NEP, Ad-GFP and 40μl of saline respectively after 7 days. The rats were sacrificed at 7 days after operation (T0), 1 day before intrathecal injection (T1), 1 d after injection (T2), 1 week (T3), 2 weeks (T4), 3 weeks (T5), 4 weeks The left and right foot pain thresholds of five groups were determined at 5 weeks (T7). One group of Ad-NEP and one group of Ad-GFP were taken at T3 and the L3-6 of spinal cord was used for immunohistochemistry. The rats in Ad-NEP and Ad-GFP groups were injected intraperitoneally with 1 mg / kg naloxone 10 days later. The pain threshold of right foot was recorded every 10 minutes (t0-9, observed for 90 minutes) Β-EP concentration in cerebrospinal fluid in group T1-7. Results The pain threshold of right foot in Ad-GFP group and saline group was significantly lower than that in sham operation group and blank control group (P <0.01). The pain threshold of right foot in Ad-NEP group was significantly lower than that in sham operation group and blank control group at T0, 1, 7 (P <0.01), and the pain threshold of right foot was higher than T0 at T2-6, GFP group and saline group (P <0.05 or 0.01), and the right pain threshold at t1-6 was lower than that at t0 (P <0.01), but there was no significant difference between Ad-GFP group and saline group (P> 0.05). Ad-NEP group showed obvious orange-yellow staining zone in the adventitia, a small amount of orange-yellow cells in the posterior horn area, and β-EP concentration in cerebrospinal fluid in T2-7-7 group was higher than that in Ad-GFP group (P <0.01) Conclusion The sheath Intrathecal injection of recombinant adenovirus Ad-NEP on chronic neuropathic pain rats have significant analgesic effect, the role of time up to 4 weeks.