The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration
The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration
复制标题
DOI：
10.1136/bjo.2010.181404
复制
发表时间：
2011-05-01
影响因子：
4.1
通讯作者：
Sanderson, Julie
中科院分区：
文献类型：
--
作者：
Niyadurupola, Nuwan;Sidaway, Peter;Sanderson, Julie
目的开发人器官型视网膜培养物（HORC），研究视网膜神经节细胞（RGC）的死亡，缺血性和兴奋性毒性的侮辱，都已知会导致损失的RGC和提出的机制参与昏迷性视网膜neurodegeneration.Methods人供体眼死后24小时内获得。分离视网膜并使用两种技术培养外植体。THY-1 mRNA（通过实时定量PCR评估）和神经元核（NeuN）（通过免疫组织化学评估）被用作RGC的标志物。结果THY-1 mRNA和NeuN标记在人视网膜中的分布与预期的RGCs分布一致。大体形态和视网膜结构在96小时的培养期内保持稳定。THY-1 mRNA和NeuN标记的RGC层细胞在培养期间减少，并且TUNEL标记随时间增加，但在无血清DMEM/HamF 12培养基中培养的HORC在培养中长达48 h是有用的。N-甲基-D-天冬氨酸（10 μ M）引起减少THY-1 mRNA的24小时，减少NeuN标记的RGC层神经元的数量48小时，这表明THY-1 mRNA的损失是RGC应激死亡前的标志。模拟缺血（60 min氧/糖剥夺）24 h后HORC的THY-1 mRNA和NeuN标记神经元数量均减少。结论HORC为研究RGC损伤导致青光眼的神经退行性机制提供了一个有用的模型。
Aims To develop human organotypic retinal cultures (HORCs) to study retinal ganglion cell (RGC) death in response to ischaemic and excitotoxic insults, both known to cause loss of RGCs and proposed as mechanisms involved in glaucomatous retinal neurodegeneration.Methods Human donor eyes were obtained within 24 h post mortem. The retina was isolated and explants cultured using two techniques. THY-1 mRNA (assessed by real-time quantitative PCR) and neuronal nuclei (NeuN) (assessed by immunohistochemistry) were used as markers of RGCs. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL).Results The distribution of THY-1 mRNA and NeuN-labelling within the human retina was consistent with the expected distribution of RGCs. Gross morphology and retinal architecture remained stable over a 96 h culture period. THY-1 mRNA and NeuN-labelled RGC layer cells decreased over the culture period, and there was an increase in TUNEL-labelling with time, but HORCs cultured in serum-free DMEM/HamF12 medium were useful for up to 48 h in culture. N-methyl-D-aspartate (10 mu M) caused a reduction in THY-1 mRNA by 24 h and decreased the numbers of NeuN-labelled RGC layer neurons by 48 h, suggesting that the loss of THY-1 mRNA was a marker of RGC stress prior to death. Simulated ischaemia (60 min oxygen/glucose deprivation) caused a reduction at 24 h in both THY-1 mRNA and the numbers of NeuN-labelled neurons of HORCs.Conclusion HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes.