Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Nat Protoc. 2011 Jun;6(6):817-26. doi: 10.1038/nprot.2011.359. Epub 2011 May 19.
This protocol describes a powerful in vivo method to quantitatively study the formation of new lymphatic vessels in the avascular cornea without interference of pre-existing lymphatics. Implantation of 100 ng of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, VEGF-C or fibroblast growth factor-2, together with slow-release polymers, into a surgically created micropocket in the mouse cornea elicits a robust lymphangiogenic response. Newly formed lymphatic vessels are detected by immunohistochemical staining of the flattened corneal tissue with lymphatic endothelial-specific markers such as lymphatic vessel endothelial hyaluronan receptor-1; less-specific markers such as vascular endothelial growth factor receptor 3 may also be used. Lymphatic vessel growth in relation to hemangiogenesis can be readily detected starting at day 5 or 6 after pellet implantation and persists for ∼14 d. This protocol offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function.
本方案描述了一种强大的活体方法，可在无血管角膜中定量研究新淋巴管的形成，而不会干扰先前存在的淋巴管。将 100ng 的血管内皮生长因子 (VEGF)-A、VEGF-C 或成纤维细胞生长因子-2 等淋巴管生成因子与缓释聚合物一起植入小鼠角膜中手术创建的微囊中，会引发强烈的淋巴管生成反应。通过用淋巴管内皮特异性标志物（如淋巴管内皮透明质酸受体-1）对扁平角膜组织进行免疫组织化学染色，可检测到新形成的淋巴管；也可以使用不太特异性的标志物，如血管内皮生长因子受体 3。在植入小丸后 5 或 6 天开始，很容易检测到与血管生成相关的淋巴管生长，并持续约 14 天。该方案为研究淋巴管形成、重塑和功能的机制提供了独特的机会。
