Department of Ophthalmology, Norfolk and Norwich University Hospital, Norwich, UK.
Br J Ophthalmol. 2011 May;95(5):720-6. doi: 10.1136/bjo.2010.181404. Epub 2010 Dec 17.
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.
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).
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 μM) 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.
HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes.
建立人类器官型视网膜培养物（HORC）以研究对缺血性和兴奋性毒性损伤的视网膜神经节细胞（RGC）死亡，已知这两种损伤都会导致 RGC 丧失，并被认为与青光眼视网膜神经退行性变有关。
人死后 24 小时内获得供体眼。视网膜通过两种技术进行分离和培养。通过实时定量 PCR 评估 THY-1 mRNA（作为 RGC 的标志物）和神经元核（NeuN）（通过免疫组织化学评估）。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）评估细胞凋亡。
在人类视网膜中，THY-1 mRNA 和 NeuN 标记的分布与 RGC 的预期分布一致。在 96 小时的培养期间，大体形态和视网膜结构保持稳定。THY-1 mRNA 和 NeuN 标记的 RGC 层细胞在培养期间减少，随着时间的推移 TUNEL 标记增加，但在无血清 DMEM/HamF12 培养基中培养的 HORC 可在培养中使用长达 48 小时。N-甲基-D-天冬氨酸（10 μM）在 24 小时内导致 THY-1 mRNA 减少，并在 48 小时内减少 NeuN 标记的 RGC 层神经元的数量，这表明 THY-1 mRNA 的丢失是 RGC 死亡前应激的标志物。模拟缺血（60 分钟缺氧/葡萄糖剥夺）导致 24 小时时 HORC 的 THY-1 mRNA 和 NeuN 标记神经元数量减少。
HORC 为研究可能导致人类青光眼的神经退行性机制对 RGC 损伤提供了有用的模型。
