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 recumbent position on a heating pad to maintain body temperature of the mouse (36.5-38 &#176;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 &#176;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&#34; x 0.050&#34;) 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&#34; x 0.024&#34;) 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 &#176;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 &#956;m.</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-&#945; 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 &#8211; 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 &#8805; 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 [&#181;m] as the inner wall diameter.</li>
		<li>Calculate vessel length [&#181;m] as the center line length of the vessel.</li>
		<li>Calculate center line velocity <img alt="Equation 1" src="/files/ftp_upload/60509/60509eq1.jpg" /> 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 [&#181;m] x vessel diameter [&#181;m] x &#960; x 10-6.</li>
		<li>Calculate wall shear rate <img alt="Equation 2" src="/files/ftp_upload/60509/60509eq2.jpg" />: 4.9 x (8 x center line velocity <img alt="Equation 1" src="/files/ftp_upload/60509/60509eq1.jpg" /> x 0.625/vessel diameter [&#181;m]).</li>
		<li>Calculate mean blood flow velocity <img alt="Equation 1" src="/files/ftp_upload/60509/60509eq1.jpg" />: center line velocity <img alt="Equation 1" src="/files/ftp_upload/60509/60509eq1.jpg" /> x 0.625.</li>
		<li>Calculate total leukocyte flux <img alt="Equation 4" src="/files/ftp_upload/60509/60509eq4.jpg" />: systemic leukocyte count <img alt="Equation 5" src="/files/ftp_upload/60509/60509eq5.jpg" /> x (mean blood flow velocity <img alt="Equation 1" src="/files/ftp_upload/60509/60509eq1.jpg" /> x 60/1000) x &#960; x <img alt="Equation 6" src="/files/ftp_upload/60509/60509eq6.jpg" />.</li>
		<li>Calculate rolling flux fraction [%]: rolling flux/total leukocyte flux x 100.</li>
	</ol>
	</li>
</ol>

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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 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:875px;" width="874">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<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>
		</tr>
		<tr height="21">
			<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...

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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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Research

JoVE Journal

Immunology and Infection

7.7K Views.  Heidelberg University Children's Hospital. 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.

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

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 ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

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 ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...