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Published on: May 22, 2020
Immunohistochemical visualization of lymphatic vessels in human dura mater: methodological perspectives
César Luis Vera Quesada1,2, Shreyas Balachandra Rao3, Reidun Torp3
1Department of Neurosurgery, Oslo University Hospital-Rikshospitalet, PB 4950 Nydalen, Oslo, 0424, Norway.
Insights
Optimizing tissue processing is key to visualizing human meningeal lymphatic vessels. Freeze-fixation and a 0.5% paraformaldehyde method significantly improve detection of these lymphatic structures in the dura mater.
Area of Science:
- Neuroscience
- Anatomy
- Immunology
Background:
- Human meningeal lymphatic vessels remain poorly characterized, with existing data solely from autopsy specimens.
- Recent years have seen renewed interest in meningeal lymphatic function, highlighting the need for better visualization techniques.
Purpose of the Study:
- To address methodological aspects of immunohistochemistry for visualizing and characterizing lymphatic vessels in the human dura mater.
- To compare different tissue preparation methods for optimal detection of meningeal lymphatics.
Main Methods:
- Dura biopsies were obtained from 30 patients with idiopathic normal pressure hydrocephalus (iNPH).
- Specimens were processed using three methods: 4% PFA, 0.5% PFA, and freeze-fixation.
- Immunohistochemistry was performed using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and podoplanin (PDPN) markers.
Main Results:
- Lymphatic structures were detected in 0/7 patients with 4% PFA, 67% (4/6) with 0.5% PFA, and 94% (16/17) with freeze-fixation.
- Three types of meningeal lymphatic vessels were characterized: associated with blood vessels, remote from blood vessels, and LYVE-1-expressing cell clusters.
- Lymphatic vessels were most abundant towards the arachnoid membrane compared to the skull.
Conclusions:
- Human meningeal lymphatic vessel visualization is highly sensitive to tissue processing methods.
- Freeze-fixation and 0.5% PFA are superior methods for detecting meningeal lymphatics.
- Meningeal lymphatics are predominantly located towards the arachnoid membrane, associated with or independent of blood vessels.
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.
