超声联合载BDNF逆转录病毒微泡开放血脑屏障治疗阿尔茨海默病

Opening the blood-brain barrier by focused ultrasound and microbubbles loaded with BDNF-carrying retrovirus for treatment of Alzheimer’s disease

目的制备载脑源性神经营养因子(BDNF)逆转录病毒超声微泡(MpLXSN-BDNF),探讨其联合超声开放血脑屏障对阿尔茨海默病(AD)模型大鼠的治疗作用。方法成年雄性SD大鼠32只,随机分为4组:超声+载pLXSN-EGFP微泡组(U+MpLXSN-EGFP组)、超声+载pLXSN-BDNF微泡组(U+MpLXSN-BDNF组)、超声+微泡+pLXSN-BDNF病毒组(U+M+pLXSN-BDNF组)和对照组(Normal组),每组8只。脑内注射β-淀粉样蛋白1-40建立AD大鼠模型,U+MpLXSN-EGFP组、U+MpLXSN-BDNF组、U+M+pLXSN-BDNF组大鼠分别经尾静脉注射载pLXSN-EGFP的微泡、载pLXSN-BDNF的微泡、pLXSN-BDNF病毒和微泡;上述3组注射完成后采用磁共振引导的低频聚焦超声辐照大鼠左侧海马区开放血脑屏障,Normal组不进行处理。处理前、处理1个月后,各组大鼠采用Morris水迷宫实验评价大鼠行为学改变。采用免疫组化法和高效液相色谱法检测大鼠海马组织乙酰胆碱转移酶(ChAT)阳性细胞数目及乙酰胆碱(ACh)含量。结果Morris水迷宫实验显示,与处理前比较,处理1个月后U+MpLXSN-BDNF组大鼠穿越平台次数明显增多(P<0.05);其余3组处理前后穿越平台次数比较,差异均无统计学意义(均P>0.05)。免疫组化和高效液相色谱法检测显示,与U+MpLXSN-BDNF组比较,U+MpLXSN-EGFP组和U+M+pLXSN-BDNF组大鼠海马组织ChAT阳性神经元数目和Ach含量均明显减少(均P<0.05)。结论超声联合载BDNF病毒微泡可增加脑内神经细胞的转染效率,促进外源基因BDNF高表达,对AD模型大鼠起到治疗作用。

Objective To prepare ultrasound microbubbles loaded with retrovirus carrying brain-derived neurotrophic factor(BDNF)(MpLXSN-BDNF),and to investigate the therapeutic effect of MpLXSN-BDNF combined with ultrasound on Alzheimer's disease(AD)by opening the blood-brain barrier in rat models.Methods Thirty-two adult male SD rats were randomly divided into 4 groups:ultrasound+pLXSN-EGFP microbubble group(U+MpLXSN-EGFP group),ultrasound+pLXSN-BDNF microbubble group(U+MpLXSN-BDNF group),ultrasound+microbubbles+pLXSN-BDNF virus group(U+M+pLXSN-BDNF group)and control group(normal group)(n=8 each).The rat model of AD was established by intracerebral injection ofβ-amyloid 1-40.Rats in the U+MpLXSN-EGFP group,U+MpLXSN-BDNF group,U+M+pLXSN-BDNF group were injected with pLXSN-EGFP microbubbles,pLXSN-BDNF-loaded microbubbles,pLXSN-BDNF virus and microbubbles,respectively,via the caudal vein.After the injections in these three groups were completed,MR-guided low-frequency focused ultrasound was used to irradiate the left hippocampus of the rats to open the blood-brain barrier.Rats in the normal group did not receive any treatment.At baseline and at one month after treatment,the rats in each group were evaluated for behavioral changes using Morris water maze test.Immunohistochemistry and high performance liquid chromatography were used to detect the number of acetylcholine transferase(ChAT)positive cells and the content of acetylcholine(ACh)in rat hippocampus.Results Morris water maze test showed that,compared with baseline,the number of times crossing the platform was significantly increased in rats of the U+MpLXSN-BDNF group at one month after the treatment(P<0.05).There was no statistically significant difference in the number of crossing platforms before and after treatment in the remaining three groups(all P>0.05).Immunohistochemistry and high performance liquid chromatography showed that compared with the U+MpLXSN-BDNF group,the number of ChAT-positive neurons and the Ach content in the hippocampus of rats in the U+MpLXSN-EGFP group and U+M+pLXSN-BDNF group obviously decreased(both P<0.05).Conclusion Ultrasound combined with microbubbles loaded with BDNF-carrying virus can increase the transfection efficiency of nerve cells in the brain,promote high expression of exogenous gene BDNF,and thereby offer therapeutic effect on AD in rat models.