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Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time
Published on: November 16, 2015
In vivo imaging of leukocyte recruitment to glomeruli in mice using intravital microscopy
A Richard Kitching1, Michael P Kuligowski, Michael J Hickey
1Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, Melbourne, Victoria, Australia.
Insights
Leukocyte recruitment in severe glomerulonephritis is hard to study. A new hydronephrosis model in mice allows direct visualization of leukocyte adhesion in glomerular capillaries, revealing inflammatory mechanisms.
Area of Science:
- Nephrology
- Immunology
- Microcirculation
Background:
- Leukocytes are implicated in severe glomerulonephritis.
- Leukocyte recruitment in glomerular capillaries is poorly understood due to visualization challenges.
Purpose of the Study:
- To develop and validate a method for visualizing leukocyte recruitment in glomerular capillaries.
- To investigate the mechanisms of leukocyte adhesion in the glomerulus during inflammation.
Main Methods:
- A murine hydronephrosis model was created by ureter ligation.
- Intravital microscopy was used to visualize leukocyte adhesion in glomerular capillaries during anti-GBM antibody-induced inflammation.
- Epifluorescence was used sparingly to avoid phototoxicity.
Main Results:
- Leukocyte adhesion in glomerular capillaries was successfully visualized and quantified.
- The hydronephrosis model did not impair the kidney's inflammatory response to anti-GBM antibody.
- The method allows detailed investigation of leukocyte recruitment mechanisms within glomeruli.
Conclusions:
- The murine hydronephrosis model provides a viable approach to study leukocyte recruitment in glomerular capillaries.
- This technique facilitates detailed investigation into the mechanisms underlying leukocyte adhesion in the glomerulus during inflammatory conditions.
Abstract:
Leukocytes mediate some forms of glomerulonephritis, particularly severe proliferative and crescentic forms. The renal glomerulus is one of the few sites within the microvasculature in which leukocyte recruitment occurs in capillaries. However, due to the difficulty of directly visualising the glomerulus, the mechanisms of leukocyte recruitment to glomerular capillaries are poorly understood. To overcome this, a murine kidney can be rendered hydronephrotic, by ligating one ureter, and allowing the mouse to rest for 12 weeks. This allows the visualisation of the glomerular microvasculature during inflammatory responses. In inflammation, in this example induced by anti-glomerular basement membrane (GBM) antibody, leukocytes can be observed undergoing adhesion in glomerular capillaries using intravital microscopy. Leukocyte adhesion can be quantitated using this approach. An observation protocol involving few, limited periods of epifluorescence avoids phototoxicity-induced leukocyte recruitment. The process of hydronephrosis does not alter the ability of anti-GBM-antibody to induce a glomerular inflammatory response. This approach allows detailed investigation of the mechanisms of leukocyte recruitment within glomeruli.
