Abstract
In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1-weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast. In addition, we introduce methods for optimal immunostaining of lymphatic and blood vessel markers in human dura mater ex-vivo.
Keywords: Lymphatic vessels, Brain, Meninges, MRI, Histopathology, Immunohistochemistry
Background
Among the causes of immune privilege in the brain is the absence of parenchymal lymphatic vessels. However, recent studies have uncovered an extensive lymphatic circulating system in the dura mater of rodents (Aspelund et al., 2015; Louveau et al., 2015), providing possible routes for the elimination of the brain’s waste products and for immune cells to access the deep cervical lymph nodes. In this protocol, we describe a way to: (1) visualize the lymphatic vessels in-vivo in the dura mater using MRI of the head, and (2) assess the local presence of lymphatic vessels using optimized immunostaining methods (Absinta et al., 2017). In-vivo imaging of lymphatics may enable more detailed studies of mechanisms of waste removal and immune function and their potential abnormalities in various diseases and aging.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Scan the entire slide and stitch it together by greater than 10x magnification using Zeiss Microscope, camera, and Zeiss Zen Blue software. On slides double-stained for lymphatic and vascular endothelial markers (D2-40/CD31 and PROX1/CD31), identify lymphatic structures and mark them on the screen under the microscope using the following criteria: (a) structures of endothelial cell-lined vessel; (b) vessel with thin endothelial cells, the nuclei of cell bulge into the lumen; (c) semi-collapsed thin vessel wall with poor basal lamina; and (d) no or only a few red blood cells in the lumen of the vessel (Killer et al., 2008). Lymphatic vessels are counted, and their dimensions are measured. If samples vary in disease type or treatment status, simple comparative statistics may be computed on the count and diameter data (Figure 5). Figure 5. Neuropathology of human dural lymphatic vessels, coronal section. A, B and C. Within the dura mater, lymphatic and blood vessels can be differentiated using double staining for PROX1 (a transcription factor involved in lymphangiogenesis, nuclear staining) and CD31 (a vascular endothelial cell marker). E, F and G. Similarly, lymphatic and blood vessels can be differentiated using double staining for D2-40 (endothelial membrane staining) and CD31. Red blood cells are seen within blood vessels, but not within lymphatic vessels. D and H. Using Zeiss Zen Blue software, lymphatic structures are marked on the digitalized slide. Insets (B, C, F, G) were rotated relative to the original Figures in A and E. Scale bars: 1 mm (A, G), 100 μm (B, C, F, G). Abbreviations: LV–lymphatic vessels; BV–blood vessels. (Modified from Figure 3 in Absinta et al. [2017]. Creative Commons Attribution License)
Notes
Recipes
Acknowledgments
The Intramural Research Program of NINDS supported this study. This protocol was adapted from procedures published in Absinta et al. (2017). Figures 2, 3, and 5 were modified and reproduced with permission from Absinta et al. (2017). The authors declare no conflicts of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Thanks for your question about our protocol for immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded (FFPE) tissue. Antigen retrieval is a critical point. Although formalin-based fixatives yield great preservation of morphology and tissue architecture, formalin fixation reduces the sensitivity of the IHC technique. Formaldehyde covalently binds to tissue protein and acts to crosslink adjoining proteins to form large accumulations of protein. The cross-linking of proteins to the antigen is supposed to “mask” the epitope and thus interfere with the binding of the antibody. So, antigen retrieval, such as heating (HIER) or enzyme processing, is necessary for FFPE tissue. Although the mechanisms by which HIER acts are unknown, many pathologists have thought it turns back the formaldehyde-mediated chemical modifications of the antigen. Since the initial description of HIER, a wide range of buffered solutions (even “universal buffers”) have been developed. At present, HIER solutions can be divided two categories based on pH and buffer compositions, as low pH citrate buffer VS high pH EDTA buffer.The classical theory is the knowledge that the heated efficiency of the citrate buffer-mediated HIER process breaks the cross-links that bind surrounding proteins to the antigen and open the epitope. On the other hand, EDTA buffer-mediated HIER is believed to work by removing bound calcium ions from the sites of cross-links. In this second theory, the chelation of calcium ions bound to proteins during fixation could be a critical step in HIER. Interestingly, EDTA buffers are particularly effective on over-fixed specimens and for the recovery of hard-to-detect antigens. In our experience in long-fixed brain samples, high-pH EDTA-based solutions work well for some antigens (CD3, CD4, CD8, PD-1, PD-L1, LYVE-1, Prox1, D2-40) that are difficult to retrieve with citrate. Additionally, most phospho-tyrosine-specific antibodies appear to require the EDTA buffer (reference: Cell Signal Technology brochure). In our study of the dural lymphatic system, the EDTA buffer showed very specific signal with less background than citrate, so we recommend EDTA for LYVE-1 IHC in FFPE tissue.
