Summary

Неинвазивная оптических изображений из лимфатических сосудов от мыши

Published: March 08, 2013
doi:

Summary

В последнее время разработаны методы визуализации использованием ближней инфракрасной флуоресценции (NIRF) может помочь выяснение роли лимфатической системы играет в раковых метастаз, иммунный ответ, заживления ран и других лимфатических-ассоциированных заболеваний.

Abstract

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph “pumping” action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 μl) of ICG (645 μM) or cABD-IRDye800 (200 μM) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 μg/kg for ICG or 113-1,700 μg/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess “packets” of lymph moving through vessels.

Protocol

Все исследования на животных были проведены в соответствии со стандартами Техасского университета Научного центра здоровья (Хьюстон, Техас), кафедры сравнительной медицины и Центром молекулярной визуализации после рассмотрения и утверждения протокола их соответствующими Институци…

Representative Results

Пример NIRF лимфатической изображений в мышах Когда МКГ или Абд-IRDye800 вводится ID в основании хвоста нормальной мыши, лимфатических сосудов между инъекции в основание хвоста и паховых лимфатических узлов (ЛУ) должны быть немедленно визуализируются. Вскоре после инъекции (не?…

Discussion

Мы используем обычай, мелких животных NIRF системы визуализации для захвата изображений меченых лимфатических сосудов у мышей. Для построения фильмов движение лимфы, 300 или более изображений собираются. Для функционального анализа лимфатические из фильмов, два или более трансформирова…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Эта работа была поддержана следующими грантами Еве Sevick: NIH R01 CA128919 и NIH R01 HL092923.

Materials

Solutions, Reagents, and Equipment Company Catalog Number Comments
Indocyanine green (ICG) Patheon Italia S.P.A. NDC 25431-424-02 Reconstitute to 645 μM (5 μg/10 μL)
Cyclic Albumin Binding Domain(cABD) Bachem Custom Reconstitute to 200 μM (6.8 μg/10 μL)
IRDye800 Li-COR IRDye 800CW Reconstitute according to manufacture’s instructions; conjugate with cABD at equilmolar concentrations
Sterile Water Hospira, Inc., Lake Forest, IL NDC 0409-4887-10
NAIR Church & Dwight Co., Inc. Local Stores www.nairlikeneverbefore.com
Imaging System (components below) Center for Molecular Imaging N/A Custom-built in our laboratories.
Electron-multiplying charge-coupled device (EMCCD) camera Princeton Instruments, Trenton, NJ Photon Max 512
Nikon camera lens Nikon Inc., Melville, NY Model No. 1992, Nikkor 28mm
Optical filter Andover Corp., Salem,NH ANDV11333 Two 830.0/10.0 nm bandpass filters are used in front of lens
785-nm laser diode Intense Ltd, North Brunswick, NJ 1005-9MM-78503 500 mW of optical output
Collimating optics Thorlabs, Newton, NJ C240TME-B Collimates laser output prior to cleanup filter
Clean-up filter Semrock, Inc., Rochester, NY LD01-785/10-25 Removes laser emission in fluorescence band
Optical diffuser Thorlabs, Newton, NJ ED1-C20 Diffuses the laser over the animal
V++ Digital Optics, Browns Bay, Auckland, New Zealand Version 5.0 Software used to control camera system and save images to computer. http://digitaloptics.net/
Analytic Software Either of the following software packages can be used for image analysis
ImageJ National Institutes of Health, Bethesda, MD Most current version available Freeware available at http://rsbweb.nih.gov/ij/
MATLAB MathWorks, Natick, MA Version 2008a or later http://www.mathworks.com/

References

  1. Alitalo, K. The lymphatic vasculature in disease. Nat. Med. 17, 1371-1380 (2011).
  2. Rasmussen, J. C., Tan, I. C., Marshall, M. V., Fife, C. E., Sevick-Muraca, E. M. Lymphatic imaging in humans with near-infrared fluorescence. Curr. Opin. Biotechnol. 20, 74-82 (2009).
  3. Rasmussen, J. C., et al. Human Lymphatic Architecture and Dynamic Transport Imaged Using Near-infrared Fluorescence. Transl. Oncol. 3, 362-372 (2010).
  4. Sevick-Muraca, E. M. Translation of near-infrared fluorescence imaging technologies: emerging clinical applications. Annu. Rev. Med. 63, 217-231 (2012).
  5. Kwon, S., Sevick-Muraca, E. M. Noninvasive quantitative imaging of lymph function in mice. Lymphat. Res. Biol. 5, 219-231 (2007).
  6. Kwon, S., Sevick-Muraca, E. M. Mouse phenotyping with near-infrared fluorescence lymphatic imaging. Biomed Opt Express. 2, 1403-1411 (2011).
  7. Marshall, M. V., et al. Near-infrared fluorescence imaging in humans with indocyanine green: a review and update. The Open Surgical Oncology Journal. 2, 12-25 (2010).
  8. Davies-Venn, C. A., et al. Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging. Mol. Imaging Biol. , (2011).
  9. Sharma, R. Quantitative imaging of lymph function. Am. J. Physiol. Heart Circ. Physiol. 292, 3109-3118 (2007).
  10. Kwon, S., Sevick-Muraca, E. M. Functional lymphatic imaging in tumor-bearing mice. J. Immunol. Methods. 360, 167-172 (2010).
  11. Karlsen, T. V., McCormack, E., Mujic, M., Tenstad, O., Wiig, H. Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin. Am. J. Physiol. Heart Circ. Physiol. 302, 391-401 (2012).
  12. Zhou, Q., Wood, R., Schwarz, E. M., Wang, Y. J., Xing, L. Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice. Arthritis Rheum. 62, 1881-1889 (2010).
  13. Adams, K. E., et al. Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema. Biomed. Opt. Express. 1, 114-125 (2010).
  14. Tan, I. C., et al. Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging. Arch. Phys. Med. Rehabil. 92, 756-764 (2011).
  15. Lapinski, P. E., et al. RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice. J. Clin. Invest. 122, 733-747 (2012).
  16. Maus, E. A., et al. Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema. Head Neck. 34, 448-453 (2012).
  17. Galanzha, E. I., Tuchin, V. V., Zharov, V. P. Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening. World J. Gastroenterol. 13, 192-218 (2007).
  18. Schramm, R., et al. The cervical lymph node preparation: a novel approach to study lymphocyte homing by intravital microscopy. Inflammation research : official journal of the European Histamine Research Society. 55, 160-167 (2006).
  19. Hall, M. A., et al. Imaging prostate cancer lymph node metastases with a multimodality contrast agent. Prostate. 72, 129-146 (2012).
  20. Zhu, B., Sevick-Muraca, E. M. Minimizing excitation leakage and maximizing measurement sensitivity for molecular imaging with near-infrared fluorescence. J. Innovat. Opt. Health Sci. 4, 301-307 (2011).

Play Video

Cite This Article
Robinson, H. A., Kwon, S., Hall, M. A., Rasmussen, J. C., Aldrich, M. B., Sevick-Muraca, E. M. Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse. J. Vis. Exp. (73), e4326, doi:10.3791/4326 (2013).

View Video