人硬脑膜淋巴管的免疫组织化学可视化：方法学视角。
Immunohistochemical visualization of lymphatic vessels in human dura mater: methodological perspectives.
机构信息
Department of Neurosurgery, Oslo University Hospital-Rikshospitalet, PB 4950 Nydalen, Oslo, 0424, Norway.
Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
出版信息
Fluids Barriers CNS. 2023 Mar 28;20(1):23. doi: 10.1186/s12987-023-00426-3.
BACKGROUND
Despite greatly renewed interest concerning meningeal lymphatic function over recent years, the lymphatic structures of human dura mater have been less characterized. The available information derives exclusively from autopsy specimens. This study addressed methodological aspects of immunohistochemistry for visualization and characterization of lymphatic vessels in the dura of patients.
METHODS
Dura biopsies were obtained from the right frontal region of the patients with idiopathic normal pressure hydrocephalus (iNPH) who underwent shunt surgery as part of treatment. The dura specimens were prepared using three different methods: Paraformaldehyde (PFA) 4% (Method #1), paraformaldehyde (PFA) 0.5% (Method #2), and freeze-fixation (Method #3). They were further examined with immunohistochemistry using the lymphatic cell marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and as validation marker we used podoplanin (PDPN).
RESULTS
The study included 30 iNPH patients who underwent shunt surgery. The dura specimens were obtained average 16.1 ± 4.5 mm lateral to the superior sagittal sinus in the right frontal region (about 12 cm posterior to glabella). While lymphatic structures were seen in 0/7 patients using Method #1, it was found in 4/6 subjects (67%) with Method #2, while in 16/17 subjects (94%) using Method #3. To this end, we characterized three types of meningeal lymphatic vessels: (1) Lymphatic vessels in intimate contact with blood vessels. (2) Lymphatic vessels without nearby blood vessels. (3) Clusters of LYVE-1-expressing cells interspersed with blood vessels. In general, highest density of lymphatic vessels were observed towards the arachnoid membrane rather than towards the skull.
CONCLUSIONS
The visualization of meningeal lymphatic vessels in humans seems to be highly sensitive to the tissue processing method. Our observations disclosed most abundant lymphatic vessels towards the arachnoid membrane, and were seen either in close association with blood vessels or remote from blood vessels.
背景
尽管近年来人们对脑膜淋巴功能重新产生了浓厚的兴趣，但脑膜的淋巴结构仍未得到充分描述。现有的信息仅来自尸检标本。本研究探讨了可视化和鉴定患者硬脑膜中淋巴管的免疫组织化学方法的一些方面。
方法
从接受分流手术治疗的特发性正常压力脑积水（iNPH）患者的右侧额区获得硬脑膜活检。硬脑膜标本分别采用 4%多聚甲醛（PFA）（方法 1）、0.5%多聚甲醛（PFA）（方法 2）和冷冻固定（方法 3）三种方法进行处理。然后，使用淋巴管内皮透明质酸受体 1（LYVE-1）作为淋巴细胞标志物和 Podoplanin（PDPN）作为验证标志物，对其进行免疫组织化学检查。
结果
本研究纳入了 30 例接受分流手术的 iNPH 患者。硬脑膜标本在右侧额区距上矢状窦平均 16.1±4.5mm 处获取（距眉间后约 12cm）。使用方法 1 在 0/7 例患者中观察到淋巴结构，而在 4/6 例（67%）使用方法 2 和 16/17 例（94%）使用方法 3 中观察到。为此，我们鉴定了三种脑膜淋巴管：（1）与血管密切接触的淋巴管。（2）无附近血管的淋巴管。（3）血管间散布的 LYVE-1 表达细胞簇。总体而言，在靠近蛛网膜的位置观察到淋巴管密度最高，而非靠近颅骨。
结论
在人类中观察脑膜淋巴管对组织处理方法高度敏感。我们的观察发现，蛛网膜处的淋巴管最丰富，并且与血管密切相关或远离血管。
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