The ability to study the microcirculation in real time is key to elucidating how the immune system and the associated microvasculature interact and influence one another within the lymph node (LN). Here, we present a method for near in-situ imaging of the inguinal LN. In particular, this method is ideal for the assessment of overall vascular health that influences immune functions and for the evaluation of T-cell motility. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, quantification of vessel magnitude and vessel integrity. Modified intravital microscopy (M-IVM) of the LNs allows direct evaluation of microvascular functions as well as real-time imaging of the direct interface between immune cells, the LN, and the microcirculation. Importantly, M-IVM technique can be readily combined with many other vascular and immunological techniques such as fluorescent cell labeling and assessment of sticking and rolling time as descripted. Furthermore, it can be adapted to study vasculature of other than the inguinal LN. Overall, this chapter provides a dependable method for fundamental vascular immunological assessment of LNs that is decidedly useful in a diverse range of investigations.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by the Canadian Institutes of Health, Centre for Blood Research, Michael Smith Foundation and University of Northern British Columbia & University of British Columbia. SLS is supported by Michael Smith Foundation for Health Research/Centre for Heart Lung Innovation—Providence Health Care Research Institute—St. Paul’s Foundation Research Trainee Award.
Segal SS (2005) Regulation of blood flow in the microcirculation. Microcirculation 12:33–45CrossRefGoogle Scholar
Payne GW (2006) Effect of inflammation on the aging microcirculation: impact on blood flow control in skeletal muscle. Microcirculation 13:315–324CrossRefGoogle Scholar
Bearden SE, Payne GW, Chisty A, Segal SS (2004) Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle. J Physiol 561:535–545CrossRefGoogle Scholar
Soderberg KA, Payne GW, Sato A, Medzhitov R, Segal SS, Iwasaki A (2004) Innate control of adaptive immunity via remodeling of lymph node feed arteriole. Proc Natl Acad Sci U S A 102:16315–16320CrossRefGoogle Scholar
Sellers SL, Payne GW (2011) Intravital microscopy of the inguinal lymph node. J Vis Exp 4:50Google Scholar
Kumamoto Y, Mattei LM, Sellers S, Payne GW, Iwasaki A (2011) CD4+ T cells support cytotoxic T lymphocyte priming by controlling lymph node input. Proc Natl Acad Sci U S A 108:8749–8754CrossRefGoogle Scholar
Sellers SL, Iwasaki A, Payne GW (2013) Nitric oxide and TNFα are critical regulators of reversible lymph node vascular remodeling and adaptive immune response. PLoS One 8:e60741CrossRefGoogle Scholar
McDonald B, Kubes P (2015) Interactions between CD44 and hyaluronan in leukocytes trafficking. Front Immunol 17(6):68Google Scholar
Payne GW, Madri JA, Sessa WC, Segal SS (2003) Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS −/− mice. Am J Physiol Heart Circ Physiol 285:H493–H498CrossRefGoogle Scholar
Looft-Wison RC, Payne GW, Segal SS (2004) Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle. J Appl Physiol 97:1152–1158CrossRefGoogle Scholar
Murooka TT, Mempel TR (2012) Multiphoton intravital microscopy to study lymphocyte motility in lymph nodes. Methods Mol Biol 757:247–257CrossRefGoogle Scholar
Sammicheli S, Kuka M, Iannacone M (2018) Intravital imaging of B cell responses in lymph nodes. Methods Mol Biol 1763:63–74CrossRefGoogle Scholar
de With MC, de Vries AM, Kroese AB, van der Heijden EP, Bleys RL, Segal SS, Kon M (2009) Vascular anatomy of the hamster retractor muscle with regard to its microvascular transfer. Eur Surg Res 42:97–105CrossRefGoogle Scholar
Meijer EFJ, Jeong HS, Pereira ER, Ruggieri TA, Blatter C, Vakoc BJ, Padera TP (2017) Murine chronic lymph node window for longitudinal intravital lymph node imaging. Nat Protoc 12:1513–1520CrossRefGoogle Scholar
Sumen C, Mempel TR, Mazo IB, Andrian UH (2004) Intravital microscopy: visually immunity in context. Immunity 21:315–329PubMedGoogle Scholar
Mempel TR, Scimone ML, Mora JR, Von Andrain UH (2004) In vivo imaging of leukocytes trafficking in blood vessels and tissues. Curr Opin Immunol 16:406–417CrossRefGoogle Scholar
Germain RN, Miller MJ, Dustin ML, Nussenzweig MC (2006) Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol 6:497–507CrossRefGoogle Scholar
Progatzky F, Dallman MJ, Celso CL (2013) From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus 3:20130001CrossRefGoogle Scholar