Journal of Visualized Experiments www.jove.com Copyright © 2020 Journal of Visualized Experiments May 2020 | 159 | e61178 | Page 1 of 5 Video Article Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice Yujia Lin 1,2 , Jingna Xue 2 , Shan Liao 2 1 Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin Medical University 2 Inflammation Research Network, Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary Correspondence to: Yujia Lin at kly1319@163.com, Shan Liao at liaos@ucalgary.ca URL: https://www.jove.com/video/61178 DOI: doi:10.3791/61178 Keywords: Immunology and Infection, Issue 159, Lymphatic vessel, Lymph node, Lymph flow, Antigen delivery, High endothelial venules Date Published: 5/14/2020 Citation: Lin, Y., Xue, J., Liao, S. Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice. J. Vis. Exp. (159), e61178, doi:10.3791/61178 (2020). Abstract Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses. Introduction The spatial organization of the lymphatic system provides structural and functional support to efficiently remove extracellular fluid and transport antigens and antigen-presenting cells (APCs) to the draining LNs. The initial lymphatic vessels (also named lymphatic capillaries) are highly permeable due to their discontinuous intercellular junctions, which facilitate the effective collection of fluids, cells, and other materials from surrounding extracellular spaces 1 . The initial lymphatic vessels merge into collecting lymphatic vessels, which have tight intercellular junctions, a continuous basement membrane, and lymphatic muscle coverage. Collecting lymphatic vessels are responsible for transporting collected lymph to the draining LNs and eventually returning lymph to the circulation 2,3 . The collecting lymphatic vessels that propel lymph into the draining LN are the afferent lymphatic vessels 4,5,6,7 . Obstruction of afferent lymphatic vessels can block lymph flow into the LNs, which is a useful technique when studying the function of lymph flow. Previous studies have shown that lymph flow plays a significant role in transporting antigens and APCs, as well as maintaining LN homeostasis. It is well understood that tissue-derived APCs, typically activated migrating dendritic cells (DCs), travel through the afferent lymphatic vessels to the LN to activate T cells 8 . The idea that free-form antigens, such as microbes or soluble antigens, passively flow with lymph to the LN to activate LN-resident APCs has been gaining acceptance in the past decade 9,10,11,12 . Free-form antigens traveling with lymph take minutes after the infection to travel to the LN, and the LN-resident cell activation may occur within 20 min after the stimulation. This is much faster than the activation of migrating DCs, which takes more than 8 h to enter the draining LN 9 . Besides transporting antigens to initiate immune protection, lymph also carries cytokines and DCs to the LN to maintain its microenvironment, and to support immune cell homeostasis 13,14 . Previously, blocking lymph flow by suturing the afferent lymphatic vessels demonstrated that lymph is required to maintain the HEV phenotype required for supporting homeostatic T cell and B cell homing to the LN 15,16,17 . CCL21 is a critical chemokine that directs DC and T cell positioning in the LN 8,18 . Blocking lymph flow interrupts CCL21 expression in the LN and potentially interrupts DC and T cell positioning and/or interaction in the LN 19 . Thus, blocking lymph flow can directly or indirectly abrogate antigen/DC access to the draining LN by disrupting the LN microenvironment that regulates immune responses in the LN. To better investigate the function of lymph flow, an experimental protocol is presented (Figure 1) to block lymph flow in mice by suturing the afferent lymphatic vessels from the footpad to the pLN. This method can be an important technique for future studies on lymphatic function in healthy and diseased conditions. Protocol All animal work needs to be approved by institutional and governmental ethics and animal handling committee. 1. Preparation of materials 1. Prepare 100 mL of 70% ethanol by mixing 70 mL of 100% ethanol with 30 mL of sterile water. Autoclave all surgical tools before surgery and keep the tools in 70% ethanol before and during the surgery to maintain sterilization. 2. Prepare an injection apparatus.