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-3 protein 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.
Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adenoassociated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo.Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A,B,C,D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 mu;l phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 mu;l recombinant adenoassociated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5 mu;l rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects.Results Green fluorescence can be observed exactly in labeled RGC in B，C，and D groups. The AOD and transfection rate in group D was （95.02plusmn;7.25）% and（20.10plusmn;0.74）% , respectively; which were higher than those in group B and C (F=25.970，25.799；P＜0.01). The difference of the number of RGC among the four groups was not significant(F=0.877,P＞0.05). Conclusion Under the condition of low frequency and with certain energy, ultrasoundmediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.
Objective To observe the protection effects of ultrasonic microbubbles combined with memantine on rat retinal ganglion cells (RGCs) after optic nerve injury. Methods Forty Sprague-Dawley adult male rats were randomly divided into normal control group (group A), sham operation group (group B), blank control group (group C), memantine group (group D) and memantine and ultrasonic microbubbles group (group E), 8 rats in each group. Then A - E groups were randomly divided into 1 week subgroup and 2 weeks subgroup after the optic nerve injury, 4 rats in each subgroup. Group A had no interference treatment. The optic nerves in group B eyes were exposed but not clamped. Normal saline was injected into the vitreous, and those eyes were immediately radiated with ultrasound. The optic nerves in Group C - E were exposed and clamped to establish the optic nerve clamped models. Normal saline was injected into the vitreous of group C eyes; memantine was injected into the vitreous of group D eyes; ultrasonic microbubble and memantine was orderly injected into the vitreous of group E eyes and those eyes were immediately radiated with ultrasound. One week and 2 weeks after the optic nerve injury, RGC was labeled by retrograde fluorogold to count the RGC number; flash visual evoked potential (F-VEP) was used to record the incubation period and amplitude of P100 wave; fluorescence microscopy was used to observe the pathological morphology change of retinal cell. Results There were goldlabeled RGCs on the retina of group A-E. The difference of RGC count was not statistically significant between group A and B (q=0.018, 0.011; P=0.986, 0.873). Compared to group A, the RGC count in group C-E were decreased significantly (F=85.944, P=0.012). The RGC count in group D was significantly higher than that in group C (q=1.721, 1.924; P=0.043, 0.037). The RGC count in group E was significantly higher than that in group C and D (q=1.128, 1.482, P=0.027, 0.008; q=1.453, 1.855, P=0.031, 0.010).F-VEP showed that there was no statistically significant difference of incubation period and amplitude of P100 wave between group A and B (q=0.008, 0.019,P= 0.981, 0.946; q=0.072, 0.052, P=0.737, 0.851). Compared to group A, the incubation period were lengthened and the amplitude were decreased in group C- E with statistically significant (F=134.312, 106.312; P=0.017, 0.009). Observed under the electron microscope, the retinal structure of group A, B eyes was normal, but there were varying degrees of edema and thickening, RGC loss in group C-E eyes. Conclusions Memantine and ultrasonic microbubble can inhibit the rat RGC loss after optic nerve injury, and improve the visual function.