Name: leukocyte infiltration of cremaster muscle in mice assessed by intravital microscopy
Uploaded: 2020-04-15T13:00:00
Description: Watch this Scientific Journal Video about Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy at JoVE.com

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.

Animals were housed under controlled and specific pathogen-free conditions at the IBF (Interfakult&#228;re Biomedizinische Forschungseinrichtung), Heidelberg. All the procedures described here were approved by the local IRB and the Regierungspraesidium Karlsruhe, Baden-Wuerttemberg, Germany.
1. Anesthesia administration
<ol>
	<li>Anesthetize the mouse by intraperitoneal (i.p.) bolus injection of 125 mg/kg ketamine and 12.5 mg/kg xylazine.</li>
	<li>Place and fix the mouse in a dorsal recumbe...

<ol>
	<li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nature Reviews. Immunology. 7 (9), 678-689 (2007).</li><li>Zanardo, R. C. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+down-regulatable+E-selectin+ligand+is+functionally+important+for+PSGL-1-independent+leukocyte-endothelial+cell+interactions.">A down-regulatable E-selectin ligand is functionally important for PSGL-1-independent leukocyte-endothelial cell interactions.</a> Blood. 104 (12), 3766-3773 (2004).</li><li>Woodfin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICAM-1-expressing+neutrophils+exhibit+enhanced+effector+functions+in+murine+models+of+endotoxemia.">ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.</a> Blood. 127 (7), 898-907 (2016).</li><li>Frommhold, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAGE+and+ICAM-1+cooperate+in+mediating+leukocyte+recruitment+during+acute+inflammation+in+vivo.">RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo.</a> Blood. 116 (5), 841-849 (2010).</li><li>Braach, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAGE+controls+activation+and+anti-inflammatory+signalling+of+protein+C.">RAGE controls activation and anti-inflammatory signalling of protein C.</a> PloS One. 9 (2), 89422 (2014).</li><li>Frommhold, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAGE+and+ICAM-1+differentially+control+leukocyte+recruitment+during+acute+inflammation+in+a+stimulus-dependent+manner.">RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner.</a> BMC Immunology. 12 (1), 56 (2011).</li><li>Braach, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+functions+of+protein+C+require+RAGE+and+ICAM-1+in+a+stimulus-dependent+manner.">Anti-inflammatory functions of protein C require RAGE and ICAM-1 in a stimulus-dependent manner.</a> Mediators of Inflammation. 2014, 743678 (2014).</li><li>Girbl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Compartmentalization+of+the+Chemokines+CXCL1+and+CXCL2+and+the+Atypical+Receptor+ACKR1+Determine+Discrete+Stages+of+Neutrophil+Diapedesis.">Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.</a> Immunity. 49 (6), 1062-1076 (2018).</li><li>Smith, M. L., Olson, T. S., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR2-+and+E-selectin-induced+neutrophil+arrest+during+inflammation+in+vivo.">CXCR2- and E-selectin-induced neutrophil arrest during inflammation in vivo.</a> The Journal of Experimental Medicine. 200 (7), 935-939 (2004).</li><li>Emre, Y., Jemelin, S., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neutrophils+and+Monocytes+in+Mesenteric+Veins+by+Intravital+Microscopy+on+Anaesthetized+Mice+in+Real+Time.">Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time.</a> Journal of Visualized Experiments. (105), (2015).</li><li>Eriksson, E., Boykin, J. V., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+in+vivo+microscopy+of+the+cutaneous+microcirculation+of+the+hairless+mouse+ear.">Method for in vivo microscopy of the cutaneous microcirculation of the hairless mouse ear.</a> Microvascular Research. 19 (3), 374-379 (1980).</li><li>von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+peripheral+lymph+node+microcirculation+in+mice.">Intravital microscopy of the peripheral lymph node microcirculation in mice.</a> Microcirculation. 3 (3), 287-300 (1996).</li><li>Hudalla, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS-induced+maternal+inflammation+promotes+fetal+leukocyte+recruitment+and+prenatal+organ+infiltration+in+mice.">LPS-induced maternal inflammation promotes fetal leukocyte recruitment and prenatal organ infiltration in mice.</a> Pediatric Research. 84 (5), 757-764 (2018).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+ok+skeletal+muscle+blood+vessels+(rat+cremaster).">Direct observation ok skeletal muscle blood vessels (rat cremaster).</a> The Journal of Physiology. 172 (1), 123-137 (1964).</li><li>Thiele, J. R., Goerendt, K., Stark, G. B., Eisenhardt, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+digital+imaging+of+leukocyte-endothelial+interaction+in+ischemia-reperfusion+injury+(IRI)+of+the+rat+cremaster+muscle.">Real-time digital imaging of leukocyte-endothelial interaction in ischemia-reperfusion injury (IRI) of the rat cremaster muscle.</a> Journal of Visualized Experiments. (66), e3973 (2012).</li><li>Kranig, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+deficiency+promotes+leukocyte+recruitment+in+mdx+mice.">Dystrophin deficiency promotes leukocyte recruitment in mdx mice.</a> Pediatric Research. 11, 4457 (2019).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> Journal of Visualized Experiments. (52), e2874 (2011).</li><li>Reichenbach, Z. W., Li, H., Gaughan, J. P., Elliott, M., Tuma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IV+and+IP+administration+of+rhodamine+in+visualization+of+WBC-BBB+interactions+in+cerebral+vessels.">IV and IP administration of rhodamine in visualization of WBC-BBB interactions in cerebral vessels.</a> Microscopy Research and Technique. 78 (10), 894-899 (2015).</li><li>Secklehner, J., Lo Celso, C., Carlin, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+in+historic+and+contemporary+immunology.">Intravital microscopy in historic and contemporary immunology.</a> Immunology and Cell Biology. 95 (6), 506-513 (2017).</li><li>Nussbaum, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+and+endothelial+adhesive+function+during+human+fetal+ontogeny.">Neutrophil and endothelial adhesive function during human fetal ontogeny.</a> Journal of Leukocyte Biology. 93 (2), 175-184 (2013).</li></ol>

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...

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 factors and mediators of the leukocyte recruitment cascade like receptor of advanced glycation end products (RAGE), intercellular adhesion molecule 1 (ICAM-1), C-X-C motif ligand (CXCL)1/2 and their receptor CXCR2 were uncovered using IVM4,5,6,7,8,9.
The method of IVM has been described for many different organs and tissues such as the intestine10, skin11, lymph nodes12, the embryonic yolk sack13 and others. However, the most widely studied method of IVM is the cremaster model, first described in rats14. Whilst still used in rats15, the method is nowadays mainly applied in mice due to the high abundance of different transgenic lines. Our group has recently highlighted the potential role of cremaster IVM in the field of inflammatory muscolopathies like Duchenne Muscular Dystrophy (DMD) studying dystrophin-deficient mdx mice16. Due to its thin interwoven and easily accessible fiber composition, the cremaster muscle represents the ideal candidate muscle to be studied as a whole mount using light or fluorescent microscopy. Leukocyte recruitment and extravasation mainly take place in post-capillary venules, which can readily be identified on a continuous muscular layer in the cremaster muscle.
The advantage of in vivo imaging compared to other in vitro assays is its biological context in a living organism. At the same time, delineating cell-specific contributions to altered leukocyte recruitment may require additional in vitro models like flow chambers or endothelial assays. The combination of multiple methods will yield most convincing data. Scientists should be aware of the limitations of the cremaster model as any surgical manipulation will lead to increased leukocyte trafficking and recruitment. Hence, baseline recruitment is difficult to estimate with this method. Despite its broad application, IVM of the cremaster can be challenging and a novel setup may take time and resources to establish. We now provide an easy protocol which will help to avoid some of the common mistakes in IVM. Also, limitations will be discussed and complimentary methods will be highlighted where applicable.
IVM of the cremaster represents an ideal approach to be implemented in the field of inflammatory and infectious studies. More specifically, the cremaster model may be of high interest to scientists studying skeletal muscle biology in the context of inflammatory disease.

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			<td height="20" style="height:20px;width:279px;">Material</td>
			<td style="width:204px;"></td>
			<td style="width:175px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ketanest S</td>
			<td style="width:204px;">Pfizer Pharma GmbH</td>
			<td style="width:175px;">PZN: 08509909</td>
			<td>anesthesia. Generic / IUPAC Name: ketamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Xylazine</td>
			<td style="width:204px;">CP-Pharma GmbH</td>
			<td style="width:175px;">Article-nr.: 1205&#160;</td>
			<td>anesthesia. Generic / IUPAC Name: xylazine (as hidrochloride)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Saline Solution</td>
			<td>B. Braun Melsungen&#160;</td>
			<td>PZN 02737756</td>
			<td>surgical preparation. Generic / IUPAC Name: sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe needle Omnican F</td>
			<td>B. Braun Melsungen&#160;</td>
			<td style="width:175px;">REF 9161502</td>
			<td>surgical preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Suture 6/0 USP</td>
			<td style="width:204px;">Resorba</td>
			<td style="width:175px;">REF 4217</td>
			<td>surgical preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Polyethylene tube #10&#160;</td>
			<td>BD GmbH</td>
			<td style="width:175px;">Supplier No. 427401</td>
			<td>surgical preparation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:279px;">Polyethylene tube #90&#160;</td>
			<td>BD GmbH</td>
			<td style="width:175px;">Supplier No. 427421</td>
			<td>surgical preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Rhodamine 6G</td>
			<td style="width:204px;">Sigma-Aldrich Chemie GmbH</td>
			<td style="width:175px;">CAS Number 989-38-8&#160;</td>
			<td>leukocyte staining. Generic / IUPAC Name: ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate</td>
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			<td height="21" style="height:21px;width:279px;">Setup Equipment</td>
			<td style="width:204px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Upright microscope&#160;</td>
			<td style="width:204px;">Olympus&#160;</td>
			<td style="width:175px;">BX51W1</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">40-fold objective&#160;</td>
			<td style="width:204px;">Zeiss</td>
			<td style="width:175px;">Achroplan 40 &#215; /0.80 W</td>
			<td>microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ImSpector software</td>
			<td style="width:204px;">Lavision Biotec GmbH</td>
			<td style="width:175px;">ver. 4.0.469</td>
			<td>software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">ImageJ</td>
			<td style="width:204px;">National Institute of Health, USA</td>
			<td style="width:175px;">ver. 1.51j8</td>
			<td>software</td>
		</tr>
	</tbody>
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leukocyte infiltration of cremaster muscle in mice assessed by intravital microscopy

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.

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...

Watch this Scientific Journal Video about Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy at JoVE.com

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

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.

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

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