The Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China.
State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, China.
Invest Ophthalmol Vis Sci. 2024 Nov 4;65(13):25. doi: 10.1167/iovs.65.13.25.
White matter (WM) abnormalities are associated with various central nervous system (CNS) disorders, and the optic nerve provides a unique opportunity to study WM pathology. Large animal models offer a more suitable platform for preclinical testing of novel therapeutic strategies for optic neuropathy due to their similarities to humans in size and relevant anatomy. Transcriptomic analyses of optic nerve tissue are essential for understanding the underlying pathological mechanisms. However, extracting high-quality RNA from the optic nerve in large animals remains challenging.
We utilized in situ hybridization and single-nucleus RNA sequencing (snRNA-seq) to examine mRNA expression in WM cells and gray matter (GM) cells.
We discovered that mRNA expression levels in WM cells were only 15% to 66% of those in GM neurons. To overcome the low mRNA yield, we developed a specialized RNA extraction protocol for the intra-canalicular optic nerve in large animal models, achieving an RNA integrity number (RIN) of 6.8 ± 0.06. For single-cell transcriptomics (scRNA-seq), we obtained a cell density of 1.0 × 105 cells/µL, cell viability of 93% ± 1.84%, and an agglomeration rate of 5.37% ± 0.75%. This approach is also applicable for postmortem human optic nerve with a RIN of 8.3 ± 0.3 using snRNA-seq.
We first discovered that the mRNA expression in the WM was significantly lower than that in the GM. Our RNA extraction protocol from large animal models enhances transparency and reproducibility in transcriptomic studies of optic nerve and other WM tissues.
白质（WM）异常与各种中枢神经系统（CNS）疾病有关，而视神经为研究 WM 病理学提供了一个独特的机会。由于大型动物模型在大小和相关解剖结构上与人类相似，因此为视神经病变的新型治疗策略的临床前测试提供了更合适的平台。视神经组织的转录组分析对于理解潜在的病理机制至关重要。然而，从大型动物的视神经中提取高质量的 RNA 仍然具有挑战性。
我们利用原位杂交和单核 RNA 测序（snRNA-seq）来检测 WM 细胞和灰质（GM）细胞中的 mRNA 表达。
我们发现 WM 细胞中的 mRNA 表达水平仅为 GM 神经元的 15%至 66%。为了克服低 mRNA 产量的问题，我们针对大型动物模型的管内视神经开发了一种专门的 RNA 提取方案，得到的 RNA 完整性数（RIN）为 6.8±0.06。对于单细胞转录组学（scRNA-seq），我们获得了 1.0×105 个细胞/µL 的细胞密度、93%±1.84%的细胞活力和 5.37%±0.75%的聚集率。使用 snRNA-seq 对死后人类视神经进行该方法也适用，其 RIN 为 8.3±0.3。
我们首次发现 WM 中的 mRNA 表达明显低于 GM。我们从大型动物模型中提取 RNA 的方案增强了视神经和其他 WM 组织转录组研究的透明度和可重复性。
