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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Abstract

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.

We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.

IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Introduction

Intravital microscopy (IVM) is a commonly applied tool in the field of leukocyte biology. Leukocyte recruitment follows a cascade of well-defined events initiated by leukocyte capture, rolling and adhesion to the endothelial wall, and finally transmigration and extravasation of leukocytes to the actual site of inflammation1. Each step is mediated and controlled by various chemokines (e.g., IL-8/CXCL8), receptors (e.g., LFA-1, Mac-1) and corresponding endothelial cell adhesion molecules (e.g., ICAM-1, VCAM-1 and E-Selectin)2,3. The interaction of different regulatory sites, controlling f....

Protocol

Animals were housed under controlled and specific pathogen-free conditions at the IBF (Interfakultäre Biomedizinische Forschungseinrichtung), Heidelberg. All the procedures described here were approved by the local IRB and the Regierungspraesidium Karlsruhe, Baden-Wuerttemberg, Germany.

1. Anesthesia administration

  1. Anesthetize the mouse by intraperitoneal (i.p.) bolus injection of 125 mg/kg ketamine and 12.5 mg/kg xylazine.
  2. Place and fix the mouse in a dorsal recumbe.......

Representative Results

IVM as per the provided protocol will yield unique insights into the cascade of leukocyte recruitment in skeletal muscle. The results section will focus on typical results obtained by IVM and highlight potential problems that may encounter.

The experimental setup for intravital microscopy is outlined in Figure 1. Preparation of the cremaster muscle and removal of connective tissue is crucial to obtain focused microscopic images with a uniform surface. Excess conne.......

Discussion

IVM as a method has been widely used to study different cell types in different organs and has been extensively described and discussed19. The main aim of this study is to provide an efficient approach to set up and perform IVM in the cremaster muscle. Practicing the method will produce reliable and reproducible results. Thus, planning and standardization are key factors to master the technique. Above all, the technique is very dependent on hemodynamic and microvascular parameters, which need to b.......

Acknowledgements

This study was supported by the German Federal Ministry of Education and Research (BMBF) 01GL1746E as part of the PRIMAL Consortium. The authors acknowledge Britta Heckmann and Silvia Pezer for skillful technical assistance.

....

Materials

NameCompanyCatalog NumberComments
Material
Ketanest SPfizer Pharma GmbHPZN: 08509909anesthesia. Generic / IUPAC Name: ketamine
XylazineCP-Pharma GmbHArticle-nr.: 1205 anesthesia. Generic / IUPAC Name: xylazine (as hidrochloride)
Saline SolutionB. Braun Melsungen PZN 02737756surgical preparation. Generic / IUPAC Name: sodium chloride
Syringe needle Omnican FB. Braun Melsungen REF 9161502surgical preparation 
Suture 6/0 USPResorbaREF 4217surgical preparation 
Polyethylene tube #10 BD GmbHSupplier No. 427401surgical preparation 
Polyethylene tube #90 BD GmbHSupplier No. 427421surgical preparation 
Rhodamine 6GSigma-Aldrich Chemie GmbHCAS Number 989-38-8 leukocyte staining. Generic / IUPAC Name: ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate
Setup Equipment
Upright microscope Olympus BX51W1microscopy
40-fold objective ZeissAchroplan 40 × /0.80 Wmicroscopy
ImSpector softwareLavision Biotec GmbHver. 4.0.469software
ImageJNational Institute of Health, USAver. 1.51j8software

References

  1. Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Reviews. Immunology. 7 (9), 678-689 (2007).
  2. Zanardo, R. C. O., et al.

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