The meninges not only surround the brain and the spinal cord but also the optic nerve. Meningeal-derived extracellular matrix (ECM) is a crucial component of the pial basement membrane, glia limitans and important for maintenance of optic nerve axon integrity, homeostasis and retinal ganglion cell health. To get closer insight into optic nerve meningeal-derived ECM composition, we performed proteomic analysis of the sheep optic nerve subarachnoid space (SAS). Candidate components were confirmed in cultures of primary human meningothelial cells (phMECs) and human optic nerve samples. Sheep optic nerve SAS samples were analysed by LC-MS, identified proteins were matched to their human orthologs and filtered using gene lists representing all major ECM components. To validate these findings digital droplet PCR (ddPCR) to evaluate mRNA expression of all candidate components identified was performed on cultures of phMECs. In addition, one protein per major ECM group was stained on human optic nerve sections and on phMEC cultures. Employing LC-MS, 1273 proteins were identified and subjected to bioinformatic analysis. Gene ontology analysis revealed six out of forty-four collagen types (1A1, 1A2, 3A1, 6A2, 6A3 and 14A1), three out of eleven laminin subunits (A4, B2, C1) and six out of twenty-seven hyaluronan binding proteins (CD44, versican (VCAN), C1q binding protein, neurocan (NCAN), brevican (BCAN) and hyalaluronan proteoglycan link protein 2 (HAPLN2)) were present in our cohort. DdPCR in phMEC cell culture confirmed presence of all candidate components except NCAN, BCAN and HAPLN2. Immunohistochemistry (IHC) staining on human optic nerve sections and immunofluorescence (IF) staining on in vitro cultured phMECs showed strong immunopositivity for collagen-typeI-α1 (COL1A1), lamininγ1 (LAMC1), and VCAN. Fibronectin (FN1) was exclusively present in cultures of phMECs. Using a combined bioinformatics and immunohistological approach, we describe the ECM composition of the optic nerve subarachnoid space. As this space plays an important role in maintaining optic nerve function, a better understanding of ECM composition in this delicate environment might be key to further pathophysiological insight into optic nerve degeneration and associated disorders.

脑膜不仅围绕大脑和脊髓，还围绕视神经。脑膜来源的细胞外基质（ECM）是膜基底膜，神经胶质细胞的重要组成部分，对于维持视神经轴突的完整性，体内稳态和视网膜神经节细胞的健康至关重要。为了更深入地了解视神经脑膜衍生的ECM组成，我们对绵羊视神经蛛网膜下腔（SAS）进行了蛋白质组分析。在原代人脑膜内皮细胞（phMEC）和人视神经样品的培养物中确认了候选成分。绵羊视神经SAS样品通过LC-MS分析，鉴定出的蛋白质与其人类直系同源物匹配，并使用代表所有主要ECM成分的基因列表进行过滤。为了验证这些发现，在phMEC的培养物中进行了数字液滴PCR（ddPCR）以评估鉴定出的所有候选组分的mRNA表达。此外，每个主要ECM组的一种蛋白质在人的视神经切片和phMEC培养物上染色。使用LC-MS，鉴定了1273种蛋白质并进行了生物信息学分析。基因本体分析显示了四十四种胶原蛋白类型中的六种（1A1、1A2、3A1、6A2、6A3和14A1），十一种层粘连蛋白亚基中的三种（A4，B2，C1）和二十七种透明质酸结合蛋白中的六种（ CD44，versican（VCAN），C1q结合蛋白，neurocan（NCAN），brevican（BCAN）和透明质酸蛋白聚糖连接蛋白2（HAPLN2））存在于我们的队列中。phMEC细胞培养物中的DdPCR证实除了NCAN，BCAN和HAPLN2之外，所有候选成分均存在。人体视神经切片的免疫组织化学（IHC）染色和体外培养的phMECs的免疫荧光（IF）染色显示对胶原I-α1（COL1A1），层粘连蛋白γ1（LAMC1）和VCAN的强免疫阳性。纤连蛋白（FN1）仅存在于phMEC的培养物中。使用生物信息学和免疫组织学相结合的方法，我们描述了视神经蛛网膜下腔的ECM组成。由于该空间在维持视神经功能中起着重要作用，因此在这种微妙的环境中更好地理解ECM组成可能是进一步了解视神经变性和相关疾病的病理生理学的关键。层粘连蛋白γ1（LAMC1）和VCAN。纤连蛋白（FN1）仅存在于phMECs培养物中。使用生物信息学和免疫组织学相结合的方法，我们描述了视神经蛛网膜下腔的ECM组成。由于该空间在维持视神经功能中起着重要作用，因此在这种微妙的环境中更好地理解ECM组成可能是进一步了解视神经变性和相关疾病的病理生理学的关键。层粘连蛋白γ1（LAMC1）和VCAN。纤连蛋白（FN1）仅存在于phMEC的培养物中。使用生物信息学和免疫组织学相结合的方法，我们描述了视神经蛛网膜下腔的ECM组成。由于该空间在维持视神经功能中起着重要作用，因此在这种微妙的环境中更好地理解ECM组成可能是进一步了解视神经变性和相关疾病的病理生理学的关键。