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Comprehensive confocal imaging and 3D computer analysis of blood and lymphatic vascular channels across entire lymph nodes

Inken Kelch1, 2*, Gib Bogle1, 3, Anthony Phillips1, 2, 4, Ian J. LeGrice3, 5, Gregory B. Sands3, Dane A. Gerneke3, Claudia J. Mansell1, 2 and P. Rod Dunbar1, 2
  • 1 Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand
  • 2 The University of Auckland, School of Biological Sciences, New Zealand
  • 3 The University of Auckland, Auckland Bioengineering Institute, New Zealand
  • 4 The University of Auckland, Department of Surgery, New Zealand
  • 5 The University of Auckland, Department of Physiology, New Zealand

Lymph nodes (LNs) are highly organized organs of the adaptive immune system (1,2). An intricate network of blood and lymphatic vascular channels serves as entry and exit routes for lymphocytes but its overall structure and dynamism during LN activation remains poorly understood (3,4,5). By using a new automated imaging system which enables extended tissue imaging of whole lymph nodes at confocal resolution (6), we aim to gain new spatial information on the LN topology and make structural changes measurable through computer analysis. Fluorescently labeled vascular systems of murine LNs were imaged at 2 µm pixel resolution in their entirety of up to 8 mm3 and analyzed using in-house designed software tools. High-resolution volume images of the blood and lymphatic networks alone provided new insights into their labyrinthine arrangement in LN subcompartments, while additional computer reconstruction allowed sophisticated measurements in 3D. As a first step, we calculated the blood vessel network volume, individual segment diameters, and the distribution of distance to the nearest blood vessel. Current experimentation is employing measurements of the vessel diameters to revisualize the blood vessel network, allowing functional elements such as the feed arteriole and putative high endothelial venules to be extracted and analyzed in detail. Subsequently, we plan to investigate LNs at different stages of the immune response to gather more information on the remodeling processes of vascular networks in response to increased lymphocyte traffic. Ultimately, we wish to elucidate the topological relationship and dynamism of structural elements in LNs and draw a comprehensive map of LN anatomy.

Acknowledgements

The authors wish to thank the Maurice Wilkins Centre for Molecular Biodiscovery and the University of Auckland for funding this project.

References

1. Willard-Mack C. Normal Structure, Function, and Histology of Lymph Nodes. Toxicol Pathol. (2006) 34(5):409-24.

2. Ohtani O, and Ohtani Y. Structure and function of rat lymph nodes. Arch Histol Cytol (2008) 71(2):69-76.

3. von Andrian UH, and Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol (2003) 3(11):867-78.

4. Bajenoff M, Egen JG, Qi H, Huang AY, Castellino F, Germain RN. Highways, byways and breadcrumbs: directing lymphocyte traffic in the lymph node. Trends Immunol (2007) 28(8):346-52.

5. Mueller SN, and Germain RN. Stromal cell contributions to the homeostasis and functionality of the immune system. Nat Rev Immunol (2009) 9(9):618-29.

6. Sands GB, Gerneke DA, Hooks DA, Green CR, Smaill BH, Legrice IJ. Automated imaging of extended tissue volumes using confocal microscopy. Microsc Res Tech (2005) 67(5):227-39.

Keywords: Lymph Node, lymph node stromal cells, Blood Vessels, lymph vessels, 3D imaging, computational analysis, 3D visualization

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Adaptive Immunity

Citation: Kelch I, Bogle G, Phillips A, LeGrice IJ, Sands GB, Gerneke DA, Mansell CJ and Dunbar P (2013). Comprehensive confocal imaging and 3D computer analysis of blood and lymphatic vascular channels across entire lymph nodes. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00568

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Received: 23 May 2013; Published Online: 22 Aug 2013.

* Correspondence: Ms. Inken Kelch, Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand, i.kelch@auckland.ac.nz