超声微泡造影剂介导脑源性神经营养因子联合转染视网膜和视皮质对视神经损伤后视网膜神经节细胞的保护作用

　　目的　观察超声微泡造影剂介导脑源性神经营养因子（BDNF）联合转染大鼠视网膜和视皮质区细胞对视神经损伤后视网膜神经节细胞（RGC）的保护作用。方法　雄性Sprague-Dawley（SD）大鼠88只随机分为正常组（A组）、假手术组（B组）、空白对照组（C组）、单纯眼转染组（D组）、单纯脑转染组（E组）、联合转染组（F组）;A组8只大鼠,B～F组每组16只大鼠。建立钳夹视神经损伤模型,将B～F组大鼠随机分为视神经损伤1、2周亚组,各亚组8只大鼠。B、C组玻璃体腔和视皮质区分别注射磷酸盐缓冲液（PBS）,D、E组玻璃体腔和视皮质区分别注射BDNF质粒（pBDNF）微泡造影剂悬液,F组玻璃体腔和视皮质区同时注射pBDNF微泡造影剂悬液。D～F组注射pBDNF微泡微泡造影剂悬液后,立即用超声辐照相应转染部位。视神经损伤后1、2周,各组行逆行荧光金标记RGC计数;半胱氨酸蛋白酶-3(caspase-3）蛋白免疫组织化学染色,观察其阳性表达情况;图形视网膜电流图（PERG）检测,记录N95振幅。结果　荧光金标记RGC结果显示,各组均可见金黄色荧光散布于视网膜定向铺片上。A~F组间RGC计数差异有统计学意义（F=256.30,P＜0.01）;B~F组视神经损伤1、2周亚组间RGC计数差异也有统计学意义（F=6518,P＜0.01）。光学显微镜观察发现,A、B组大鼠视网膜均未见caspase-3蛋白阳性表达;C~F组均可见主要位于神经节细胞层的caspase-3蛋白阳性表达。PERG检测发现,A~F组间N₉₅振幅差异有统计学意义（F=121.56,P＜0.01）;B~F组视神经损伤1、2周亚组间N95振幅差异也有统计学意义（F=8238,P＜0.01）。结论　超声微泡造影剂介导BDNF联合转染视网膜和视皮质区细胞能抑制视神经损伤后RGC凋亡,提高RGC存活数,保护其视功能。

Protection of retinal ganglion cells from optic nerve injury by brain-derived neurotrophic factor transfected through microbubble contrast agent into the retina and visual cortex

Objective To observe the protective effect of ultrasound microbubble contrast agentmediated transfection of brain-derived neurotrophic factor(BDNF) into the retina and visual cortex on retinal ganglion cells (RGC) after optic nerve injury. Methods A total of 88 male Sprague-Dawley (SD) rats were randomly divided into normal group (group A, eight rats), sham operation group (group B, 16 rats),control group (group C, 16 rats), eyes transfection group (group D, 16 rats), brain transfection group (group E, 16 rats), combined transfection group (group F, 16 rats). The optic nerve crush injury was induced, and then the groups B to F were divided into one-week and two-week after optic nerve injury subgroup with eight rats each, respectively. The rats in group B and C underwent intravitreal and visual cortex injection with phosphate buffered solution respectively. The rats in group D and E underwent intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids respectively. The rats in group F underwent both intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids at the same time. The ultrasound exposure was performed on the rats in group D to F after injection with the mixture solution of microbubbles and BDNF plasmids. One and two weeks after optic nerve injury, RGC were retrogradely labeled with Fluorogold;active caspase-3protein was observed by immunohistochemistry and the N95 amplitude was detected by pattern electroretinogram (PERG). Results Golden fluorescence can be observed exactly in labeled RGC in all groups,the difference of the number of RGC between the six groups and ten subgroups were significant (F=256.30,65. 18;P＜0. 01). Active caspase-3 in ganglion cell layer was detected in group C to F, but not in group A and B. The difference of the N95 amplitude between the six groups and ten subgroups were significant (F= 121.56, 82. 38;P＜ 0. 01 ). Conclusion Ultrasound microbubble contrast agent-mediated BDNF transfection to the rat retina and visual cortex can inhibit the RGC apoptosis after optic nerve injury and protect the visual function.