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 (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
<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 recumbent position on a heating pad to maintain body temperature of the mouse (36.5-38 °C). Use a non-absorbable sterile suture (6/0) to wind a simple loop around the frontal teeth and tape the joining ends of the suture to the heating pad. This fixation aims to keep the mouth open in order to avoid disturbance in breathing.</li>
	<li>Check the mouse for appropriate depth of anesthesia by interdigital toe pinch (withdrawal should not be seen).</li>
	<li>Once sufficient anesthesia is reached proceed to surgical preparation. Repeatedly confirm anesthesia along the ongoing experiment every 30 min. Re-administer drugs as described above if needed.</li>
	<li>Equilibrate physiological salt solution (PSS, composition: 132 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgS04, 2.0 mmol/L CaCl2, 18 mmol/L NaHCO3) with 5% CO2/95% N2 for 15 minutes. Continuously bubble PSS with 5% CO2/95% N2 throughout the experiment and keep at 37 °C.</li>
</ol>
2. Surgical preparation of the trachea and carotid artery (optional)
<ol>
	<li>Dissect the skin from the neck area by gently pulling the skin with tweezers in the midline. Use scissors to apply a circular cut of 1-2 cm in diameter to give enough space for surgical preparation.</li>
	<li>Carefully dissect the surrounding muscle, fat and connective tissues using tweezers.</li>
	<li>Make a transverse cut (~ 1.3 mm) into the trachea using small surgical scissors and introduce a polyethylene tube (I.D x O.D. 0.034" x 0.050") inside the caudal end of the trachea to secure the upper airways.</li>
	<li>Fix the tube located in the trachea by a single circular knot suture (6/0 USP).</li>
	<li>Locate the carotid artery along the right side of trachea and dissect the surrounding tissue from the carotid artery wall. Alternatively, the jugular or also the tail vein could be used for i.v. access. Try to avoid injury to the vagal nerve, as it is located closely.</li>
	<li>Pass two pieces of suture (6/0) underneath the carotid artery. Place the first cranial suture proximal, close to the bifurcation of carotid arteries and tie it permanently. The second suture will be located about 5-8 mm distal from the first one and later be used to secure the tube in the carotid artery. Do not tie it yet.</li>
	<li>Prepare a polyethylene tube (I.D x O.D. 0.011" x 0.024") with a length of 30 cm by bending the end part to a 1 mL syringe needle filled with saline (0.9% NaCl). Rigorous flushing is important to prevent air embolization during preparation.</li>
	<li>Use a 7 mm vessel clip to clamp the carotid artery distally of the second suture.</li>
	<li>Perform a small transverse cut (~0.5 mm) in the carotid artery and introduce the sterile polyethylene tube. Secure the tube in the artery using the second suture prepared before.</li>
	<li>Remove the vessel clip and gently apply a little tension to the syringe connected to the polyethylene tube. This carotid catheter can now be used to administer drugs or take blood samples if required, even monitoring of blood pressure or pulse rate is possible with respective devices.</li>
</ol>
3. Surgical preparation of the cremaster muscle
<ol>
	<li>Transfer the mouse on the heating pad (along with the heating pad) to the custom-made plastic frame holding the stage for later microscopy. Orient the mouse with its scrotum facing the microscope stage.
	<ol>
		<li>Reassess depth of anesthesia before proceeding; re-administer a quarter to half dose of anesthesia if required.</li>
	</ol>
	</li>
	<li>Localize the scrotum, holding it at its most distal part using tweezers, pull it gently and cut off a circular section of scrotal skin with about 5 mm in diameter. Make sure not to harm the underlying structures such as the cremaster muscle. Make sure to keep the open tissue well hydrated with saline solution.</li>
	<li>Dissect loose connective tissue using two tweezers and localize both testes.</li>
	<li>Hold one testis distally and gently start pulling it out, removing surrounding connective tissue step by step.</li>
	<li>Once the testis is exteriorized, pin its distal end to the rubber ring surrounding the stage. Upon exteriorization, hydrate the tissue with saline solution during the whole process.</li>
	<li>Critical step: Carefully remove connective tissue without harming the underlying cremaster muscle. Excess connective tissue may obstruct vision in later microscopy and may produce blurry images.</li>
	<li>Pin the distal part of the testis down and open the cremaster muscle by a small transverse cut (about 1 mm), followed by longitudinal incision from the very distal to the proximal end. As a result the cremaster muscle should open spherically. Macroscopically visible vessel should not be severed, as this may impact hemodynamics.</li>
	<li>Carefully spread the muscle over the glass stage and pin it to the rubber ring. Make sure not to touch or harm the central region, as microscopy will be performed here. 
	Caution: Excess stretch may obstruct blood flow.</li>
	<li>Pin the remaining testis aside to give access to the region of interest. Make sure to moisten with PSS repeatedly to prevent drying. The mounted muscle is now ready for microscopy.</li>
</ol>
4. Intravital microscopy
<ol>
	<li>Place the mounted cremaster muscle in the upright microscope and perform microscopy using a 40x objective.</li>
	<li>Acquire data and export using high throughput imaging spectrography.</li>
	<li>Perform recordings under continuous superfusion with preheated (35-37 °C) PSS17. Apply superfusion by a small tubing that has been taped to the upright objective of the microscope to allow continuous dripping of PSS alongside the objective down onto the muscle tissue.</li>
	<li>Identify post-capillary venules (confluence of two smaller vessels) and measure the microcirculation on venules with a diameter of 20-40 μm.</li>
	<li>Record high-resolution images of the microcirculation from the cremaster muscle in a time range of 30 s. As there might be slight contraction and relaxation of the muscle tissue during recording, make sure to continuously adjust the focus. Generally, the mouse tends to exhibit stable circulation for about 2 hours. It is possible to acquire several recordings of different vessels from different regions. One or both testis can be used subsequently. 
	NOTE: As this is a model of inflammation, common ways of induction are: systemic application or local scrotal injection of pro inflammatory mediators as well as superfusion with for example N-Formylmethionyl-leucyl-phenylalanine (fMLP). Local TNF-α stimulation is commonly used to trigger leukocyte recruitment; LPS-induced endotoxemia is a model of SIRS severe systemic inflammation.</li>
</ol>
5. Leukocyte visualization
NOTE: Adherent and rolling leukocytes can easily be seen without further visualization. To determine the center line velocity of freely moving leukocytes, perform differential staining.
<ol>
	<li>If eGFP labeled mice are used, directly analyze them by fluorescence microscopy.</li>
	<li>For non-eGFP labeled mouse strains, use rhodamine staining.
	<ol>
		<li>Administer rhodamine 6G at 0.2 mg/kg body weight. Repeated doses may be required to achieve sufficient staining intensities. Make sure to keep small volumes, as larger volumes may affect hemodynamic parameters.</li>
		<li>For instant staining of leukocytes, administer the stain via the carotid artery. If carotid artery dissection has not been performed, sufficient staining may be achieved by i.p. application using the same dose18. As an alternative to carotid artery catheterization, any other venous catheterization can be performed. 
		NOTE: Keep in mind that rhodamine staining, unlike eGFP labeling, shows time decay of fluorescence intensity as well as an overall staining maximum of about 80% of leukocytes at higher concentrations.</li>
		<li>Visualize freely moving rhodamine stained leukocytes by fluorescence microscopy.</li>
	</ol>
	</li>
</ol>
6. End of experiment
NOTE: The overall estimated time to perform this protocol should be 90 – 150 minutes.
<ol>
	<li>End the experiment with euthanasia by cervical dislocation.</li>
	<li>For further analyses, such as transmigration, fix the cremaster muscle in PFA whilst still stretched on the stage and stained for histology as required.</li>
</ol>
7. Offline analysis
<ol>
	<li>Analyze the following parameters as simple counts during video playback.
	<ol>
		<li>Calculate rolling flux [number/min]: number of rolling cells passing an imaginary line/min</li>
		<li>Calculate adhesion [number/mm2]: number of cells adhesive to the vessel wall within the field of view ≥ 30 s/vascular surface [mm2].</li>
	</ol>
	</li>
	<li>Measure the following hemodynamic and microvascular parameters are measured using ImageJ.
	<ol>
		<li>Calculate vessel diameter [µm] as the inner wall diameter.</li>
		<li>Calculate vessel length [µm] as the center line length of the vessel.</li>
		<li>Calculate center line velocity <img alt="Micrometers per second symbol, measuring velocity, scientific notation, concise unit definition." src="/files/ftp_upload/60509/60509eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> obtained from frame-to-frame video analysis of freely moving leukocytes in center line.</li>
	</ol>
	</li>
	<li>Use the following equations as an estimation.
	<ol>
		<li>Calculate vascular surface in [mm2]: vessel length [µm] x vessel diameter [µm] x π x 10-6.</li>
		<li>Calculate wall shear rate <img alt="Time-inverse symbol [1/s], mathematical notation, unit representation in formulas." src="/files/ftp_upload/60509/60509eq2.jpg" data-altupdatedat="1747911167000" data-alt="Equation 2">: 4.9 x (8 x center line velocity <img alt="Micrometers per second symbol, measuring velocity, scientific notation, concise unit definition." src="/files/ftp_upload/60509/60509eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> x 0.625/vessel diameter [µm]).</li>
		<li>Calculate mean blood flow velocity <img alt="Micrometers per second symbol, measuring velocity, scientific notation, concise unit definition." src="/files/ftp_upload/60509/60509eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1">: center line velocity <img alt="Micrometers per second symbol, measuring velocity, scientific notation, concise unit definition." src="/files/ftp_upload/60509/60509eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> x 0.625.</li>
		<li>Calculate total leukocyte flux <img alt="Kinetic measurement symbol, [number/min], used in scientific calculations; formula for reaction rates." src="/files/ftp_upload/60509/60509eq4.jpg" data-altupdatedat="1747911167000" data-alt="Equation 4">: systemic leukocyte count <img alt="Concentration formula [number/µL] with unit notation for quantitative analysis." src="/files/ftp_upload/60509/60509eq5.jpg" data-altupdatedat="1747911167000" data-alt="Equation 5"> x (mean blood flow velocity <img alt="Micrometers per second symbol, measuring velocity, scientific notation, concise unit definition." src="/files/ftp_upload/60509/60509eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> x 60/1000) x π x <img alt="Vascular diameter calculation formula, \((\text{vascular diameter [μm]}/{2 \times 1000})^2\)." src="/files/ftp_upload/60509/60509eq6.jpg" data-altupdatedat="1747911167000" data-alt="Equation 6">.</li>
		<li>Calculate rolling flux fraction [%]: rolling flux/total leukocyte flux x 100.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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 be closely monitored and controlled for. As such, different hemodynamics between groups will yield misleading results.
Despite its role in studying physiological and pathological conditions, IVM alone may not fully unravel individual contribution of specific cell types to leukocyte recruitment in inflammation. To do so, other complementary methods may be combined with IVM. For example, ex vivo methods like flow chamber experiments may delineate between effects of the endothelium or the leukocyte. Also, flow chambers are excellent setups to translate rodent findings to human leukocytes, for example in newborn infants20. Further, in vitro methods like FACS or immunohistochemistry should complement IVM experiments. Each method may add specific information acquired from a controlled environment with little confounding factors.
The traumatic cremaster preparation presented here resembles physiological baseline conditions of inflammation as closely as possible. Yet, the mandatory surgical preparation itself is a stimulus of inflammation, which must be considered for interpretation. Pharmacological TNF stimulation of the cremaster tissue may provide an additional mode to boost inflammatory recruitment beyond the traumatic surgical stimulus16. Further, different mouse strains may show altered responses towards standardized stimuli. We have demonstrated that even common wild type strains may exhibit different baseline states of leukocyte recruitment. This shows the importance of assigning correct genetic controls, and establishing baseline data for new experimental groups when planning experiments. As transgenic strains are highly abundant these days, it is likely that even same strains may differ over time depending on breeding.
Taken together, IVM is a powerful tool to monitor leukocyte recruitment in the biological context of the mouse and should be accompanied with ex vivo and in vitro techniques. Further, cremaster IVM allows a variety of different modes including specific cell staining, inflammatory stimulation or pharmacological manipulation and pretreatment. As such it may serve as a platform to evaluate different pharmacological mediators and their biological effects in preclinical studies especially in the field of inflammation and more specifically in inflammatory musculopathies.

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.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketanest S</td><td>Pfizer Pharma GmbH</td><td>PZN: 08509909</td><td>anesthesia. Generic / IUPAC Name: ketamine</td></tr><tr><td>Xylazine</td><td>CP-Pharma GmbH</td><td>Article-nr.: 1205&amp;nbsp;</td><td>anesthesia. Generic / IUPAC Name: xylazine (as hidrochloride)</td></tr><tr><td>Saline Solution</td><td>B. Braun Melsungen&amp;nbsp;</td><td>PZN 02737756</td><td>surgical preparation. Generic / IUPAC Name: sodium chloride</td></tr><tr><td>Syringe needle Omnican F</td><td>B. Braun Melsungen&amp;nbsp;</td><td>REF 9161502</td><td>surgical preparation&amp;nbsp;</td></tr><tr><td>Suture 6/0 USP</td><td>Resorba</td><td>REF 4217</td><td>surgical preparation&amp;nbsp;</td></tr><tr><td>Polyethylene tube #10&amp;nbsp;</td><td>BD GmbH</td><td>Supplier No. 427401</td><td>surgical preparation&amp;nbsp;</td></tr><tr><td>Polyethylene tube #90&amp;nbsp;</td><td>BD GmbH</td><td>Supplier No. 427421</td><td>surgical preparation&amp;nbsp;</td></tr><tr><td>Rhodamine 6G</td><td>Sigma-Aldrich Chemie GmbH</td><td>CAS Number 989-38-8&amp;nbsp;</td><td>leukocyte staining. Generic / IUPAC Name: ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate</td></tr><tr><td>&lt;strong&gt;Setup Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Upright microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>BX51W1</td><td>microscopy</td></tr><tr><td>40-fold objective&amp;nbsp;</td><td>Zeiss</td><td>Achroplan 40 &amp;times; /0.80 W</td><td>microscopy</td></tr><tr><td>ImSpector software</td><td>Lavision Biotec GmbH</td><td>ver. 4.0.469</td><td>software</td></tr><tr><td>ImageJ</td><td>National Institute of Health, USA</td><td>ver. 1.51j8</td><td>software</td></tr></tbody></table>

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 connective tissue eventually leads to blurry microscopic images (Supplemental Video 1) when performing IVM. Figure 2 shows representative microscopic images that can be obtained using IVM. First, post-capillary venules, which should be between 20-40 µm in diameter, are identified by confluence of two smaller vessels. Only adherent and rolling leukocytes can be visualized, circulating leukocytes may only be tracked in slow motion replay of recorded videos. The number of adherent leukocytes is defined as stationary leukocytes over a time of 30 seconds and expressed per mm2 of vessel surface (Figure 2). The number of rolling leukocytes passing a previously defined cross-section of the vessel is counted; rolling velocities may be obtained in offline analysis. Rolling may be expressed as rolling flux (RF = number of rolling cells over a predefined line/min) or as rolling flux fraction (RFF in % = RF/total leukocytes flux passing the vessel). RF is highly dependent on the number of circulating cells, whereas RFF is less affected by changes in white blood cell counts (WBCs). Note that in many cases leukocyte adherence and rolling are inversely related; a high number of adherent leukocytes may reduce the pool of freely circulating cells and thus dampen leukocyte rolling.
Technical challenges and potential pitfalls 
The cremaster muscle encloses the testis and assembles a spherical structure. To mount the muscle onto the recording stage, a longitudinal incision is required to open the sphere. This may affect the overall microvasculature as small vessels might be severed. To avoid bias to altered blood flow, it is advisable to choose the central area for reordering. This area should neither be touched nor manipulated. Additionally, microcautery may prevent bleeding and changes in microvascular hemodynamics. The blood flow of surrounding capillaries generally provides a valuable hint if circulation is affected due to preparation within the field of interest. In general, more proximal vessels (towards the body of the mouse) tend to show better microcirculation, however image capture may be complicated by increased movement and disturbances that result from breathing of the animal.
To prevent the cremaster muscle from drying out during microscopic recording, permanent superfusion of the tissue is eminent. Insufficient or interrupted moistening will result in drying of the tissue and failure of the experiment.
Pinning of the cremaster tissue is necessary to keep it stretched and in place. To flatten the striated muscle, gentle stretching is required. Too little stretch results in uneven tissue, which complicates microscopy, excessive stretching will restrict blood flow and harm the tissue. Again capillary blood flow provides a reliable status of the overall circulatory condition of the tissue. Experience and practice is required to determine the degree of stretch when mounting the tissue.
Long preparation times will affect the overall inflammatory state of the organism and bias data. Therefore, make sure to focus on the question of interest while designing the experiment and keep preparation times as short as necessary. If no drug administration, monitor or blood tests are required a singular preparation of cremaster tissue may be sufficient.
In most settings, different experimental groups will be compared to wild type animals. It is of pivotal importance to make sure that hemodynamic and microvascular parameters are similar between experimental groups (Table 1). A multitude of parameters may affect leukocyte recruitment in vivo, including cardiac output, vessel diameter, center line velocity, wall shear rate and the number of circulating leukocytes. Table 1 displays data from two different transgenic mouse lines, the dystrophin deficient mdx mouse and the lys-eGFP mouse (expressing eGFP under the lys promoter in neutrophils and macrophages). Whereas parameters such as vessel diameter can be controlled by the investigator, centerline velocity and shear rate depend on the hemodynamics of the individual experimental group or the mouse strain. Clearly, the centerline velocity and wall shear rate are significantly different between mdx and lys-eGFP mice (Table 1) making meaningful comparison of IVM data impossible (statistical analysis by t-test with Welch’s correction, significance was set at P &lt; 0.05). The centerline velocity is influenced by cardiac output, peripheral vascular resistance and intravasal volume. For example, a large volume of rhodamine or any other experimental substance given via the carotid catheter increases post capillary centerline velocity and inhibits leukocyte capture and rolling (Supplemental Video 2). On the contrary, cardiac failure, deep anesthesia or hypothermia may decrease post-capillary flow. Before approaching offline analysis of IVM data, requirements of data quality (for example hemodynamic parameters within physiological ranges) should be defined, in order to draw meaningful conclusions and reduce bias.
To determine the centerline velocity, freely circulating leukocytes need to be visualized. Either, fluorescent leukocytes (like in lys-eGFP mice) may be used or leukocytes must be dyed with fluorescent reagents like rhodamine (Figure 3). Rhodamine can be applied via the carotid catheter or as an i.p. injection. Since rhodamine is gradually taken up by other cell types as well, a titration of dosage and time is essential. Excess dosing and long intervals will produce significant background during microscopy.
Finally, we like to point out biological diversity of different mouse strains. Besides genetically altered strains, also generally accepted wild type strains may show physiological variance. Figure 4 compares commonly employed black 6 (C57BL/6) to black 10 (C57BL/10ScSn) mice. Black 6 mice clearly show enhanced rolling, adhesion and transmigration of leucocytes compared to black 10 mice (statistical analysis by one-way ANOVA with Tukey’s post-hoc test; significance was set at P &lt; 0.05), even though both strains would be referred to as wild type. Therefore, correct genetic background should be considered, especially when utilizing transgenic mice. In our experimental setting, a comparison of black 6 mice and transgenic mdx mice (black 10 background) would largely conceal the enhanced inflammatory state of dystrophin deficient mdx mice over their correct black 10 wild type background (Figure 4).
<img alt="Rodent muscle surgery setup, microscopy analysis for physiological study; labeled anatomy diagram." class="xfigimg" src="/files/ftp_upload/60509/60509fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Schematic experimental setup of IVM on cremaster muscle. After anesthesia, the trachea is cannulated and a catheter is placed in the carotid artery. The cremaster muscle is exposed and prepared for intravital microscopy on an upright microscope using either light microscopy or fluorescent microscopy. Videos are obtained for later offline analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/60509fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Leukocyte rolling dynamics; time-lapse microscopy images; adhesion process diagram; 40 μm scale." class="xfigimg" src="/files/ftp_upload/60509/60509fig2a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Representative post-capillary venule segment from intravital microscopy with sequential frames. The left column shows light microscopic images, for better visualization the vessel is outlined with dotted lines. The venule is identified by confluent vessels (marked by red asterisks) and a vessel diameter of &lt;40 µm. The right column depicts a schematic representation of the corresponding left image, showing leukocytes in red. The first four rows show sequential images of the same vessel over time. The bottom row indicates adherent leukocytes. In the schematic representation, individual leukocytes and their rolling distance are marked. In offline analysis adherence per square millimeter, rolling flux as well as rolling velocity may be calculated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/60509fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy of eGFP, rhodamine binding in cellular structures, experimental comparison." class="xfigimg" src="/files/ftp_upload/60509/60509fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Rolling leukocytes may be visualized by transgenic expression of fluorescent markers or by secondary staining with rhodamine. Representative microscopic images of leukocyte recruitment of eGFP leukocytes and rhodamine labeled leukocytes from n = 3 animals each. Rolling neutrophils expressing eGFP under the lys promoter from lys-eGFP transgenic mice can be directly detected by immunofluorescent microscopy (left column). Leukocytes without fluorescent makers may be stained by intravenous or intraperitoneal injection of rhodamine (right column). Notably, rhodamine is not exclusively taken up by neutrophils, giving substantially more background. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/60509fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar charts of RFF, adhesion, transmigration metrics in mouse models; statistical analysis indicated." class="xfigimg" src="/files/ftp_upload/60509/60509fig4a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Comparison of leukocyte recruitment in different transgenic and wild type mouse strains. (A) Rolling flux fraction, (B) adhesion and (C) transmigration of leucocytes is compared between black 6 (C57BL/6J), black 10 (C57BL/10ScSnJ) and mdx mice (C57BL/10ScSn-DmdmdxJ), shown as mean + SEM. Black 6 mice show enhanced leukocyte recruitment compared to black 10 mice. Similarly, mdx mice have greater recruitment compared to black 10, but not compared to black 6 wild type mice. This data demonstrates the importance of allocating correct genetic controls. Data from at least n = 3 animals per group. Statistical analysis by one-way ANOVA with Tukey’s post-hoc analysis. Significance was set at P &lt; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/60509fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Mouse strain</td>
			<td>No. of mice</td>
			<td>No. of venules</td>
			<td>Vessel diameter</td>
			<td>Centerline velocity</td>
			<td>Wall shear rate</td>
			<td>Systemic WBC</td>
		</tr>
		<tr>
			<td></td>
			<td>N</td>
			<td>n</td>
			<td>µm</td>
			<td>µm/s</td>
			<td>s-1</td>
			<td>/µl</td>
		</tr>
		<tr>
			<td>mdx</td>
			<td>3</td>
			<td>12</td>
			<td>28 ± 4</td>
			<td>1150 ± 50</td>
			<td>1010 ± 100</td>
			<td>5200 ± 300</td>
		</tr>
		<tr>
			<td>lys-eGFP</td>
			<td>3</td>
			<td>15</td>
			<td>27 ± 2</td>
			<td>2220 ± 90</td>
			<td>2030 ± 90</td>
			<td>5800 ± 100</td>
		</tr>
		<tr>
			<td>P</td>
			<td></td>
			<td></td>
			<td>0.83</td>
			<td>0.0005</td>
			<td>0.0016</td>
			<td>0.13</td>
		</tr>
	</tbody>
</table>
Table 1: Relevant hemodynamic and microvascular parameters from two different transgenic mouse lines. Vessel diameter, center line velocity, wall shear rate and systemic WBCs are displayed for mdx mice lacking dystrophin and lys-eGFP mice. In this example mdx mice (C57BL/10J, black 10 background) and lys-eGFP mice (C57BL/6J, black 6 background) show statistically different centerline velocity and wall shear rate and should not be compared using IVM. Exemplary data from at least n = 3 animals per group are presented as mean ± SEM. Statistical analysis by unpaired t-test with Welch’s correction. Significance was set at P &lt; 0.05.
Supplemental Video 1: Microscopic effect of insufficient removal of connective tissue on cremaster muscle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/Supplement_Video_1.mp4" target="_blank">Please click here to view this video (Right-click to download).</a>
Supplemental Video 2: Altered blood flow by either hypervolemia or hypotension. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60509/Supplement_Video_2.mp4" target="_blank">Please click here to view this video (Right-click to download).</a>

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 ...

leukocyte-infiltration-cremaster-muscle-mice-assessed-intravital

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Heidelberg University Children's Hospital. Deze praktische en reproduceerbare methode kan worden gebruikt om de endotheel leukocyten interacties die leiden tot leukocyte recruitment binnen een intacte muis te visualiseren. Deze techniek presenteert ook een krachtig instrument voor het monitoren van fysiologische en pathofysiologische processen binnen de leukocyte recruitment cascade in vivo. De methode kan worden toegepast in verschillende ontstekings- en septische modellen.Zo hebben we onlangs gewezen op de relevantie ervan in...

Video: Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Medicine

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Biology

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video