UNASSIGNED: The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology.
UNASSIGNED: Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram.
UNASSIGNED: Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR\'s key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.
UNASSIGNED:该研究包括体外和体内方法。体外,我们用大鼠B-35细胞进行研究,在温度和兴奋性毒性损伤下,AAV-shRNA-HuR递送是否会影响存活和氧化应激标志物。体内方法由两种不同的设置组成。在第一个,35只8周龄大鼠接受AAV-shRNA-HuR或AAV-shRNA扰乱对照的玻璃体内注射。动物接受视网膜电图测试,并在注射后2、4或6个月处死。收集视网膜和视神经并进行免疫染色处理,电子显微镜和体视学。对于第二种方法,动物接受了类似的基因构建体。诱发慢性青光眼,AAV注射后8周,进行单侧巩膜上静脉烧灼术.每组动物接受玻璃体内注射金属硫蛋白II。动物进行视网膜电图测试并在8周后处死。收集视网膜和视神经并进行免疫染色处理,电子显微镜和体视学。
UNASSIGNED:沉默hur诱导B-35细胞凋亡和氧化应激标志物增加。此外,shRNA处理损害了细胞对温度和兴奋性毒性损伤的应激反应。在体内,注射后6个月,shRNA-HuR组RGC计数减少39%,与shRNA扰乱对照组相比。在神经保护研究中,在用金属硫蛋白和shRNA-HuR治疗的青光眼动物中,RGC的平均损失为35%,在用金属硫蛋白和scramble对照shRNA治疗的青光眼动物中,RGC的平均损失为11.4%.HuR细胞含量的改变导致视网膜电图中的明视负反应减少。
未经评估:根据我们的发现,我们得出的结论是,HuR对于RGC的存活和有效的神经保护至关重要,并且诱导的HuR含量改变加速了年龄相关和青光眼诱导的RGC数量和功能下降,进一步证实了HuR在维持细胞稳态中的关键作用及其可能参与青光眼的发病机制。