This work was supported by the Deutsche Forschungsgemeinschaft (DU 1190/3-1).

All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA (www.felasa.eu/guidelines.php) and the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html). The animal welfare committee of the University of Freiburg as well as the local authorities (Regierungspr&#228;sidium Freiburg) approved all animal experiments.
1. Animal Handling, Preparation and ...

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Herein we describe the preparation and performance of intravital microscopy of mesentery veins of mice in vivo. This method gives us the opportunity to observe leukocyte-endothelial and platelet-endothelial interactions11 directly in the living organism.
As an early response to inflammation the endothelium gets activated and interacts with leukocytes and platelets resulting in rolling, adhesion and transmigration12. But leukocytes also interact with platelets for...

The purpose of this protocol is to describe a simplified technique of intravital microscopy of mesentery veins in living mice for direct observation of leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions.
Intravital microscopy was developed to study leukocyte-endothelial interactions in vivo under inflammatory conditions1,2, which is not trivial but important for understanding inflammatory leukocyte recruitment and function. The method we describe in this protocol was developed based on previously published publications. Similar to visualizing platelet incorporation in a growing thrombus3 and in parallel to what has been published earlier4, an exteriorized mesentery vein is examined by transmitted light microscopy. There are various other models of intra-vital microscopy such as the cremaster muscle of rats5 or liver of mouse and rats6.
Intravital microscopy of mesentery vein has been developed and applied in previous studies by several groups. We have used this technique to observe differences in leukocyte recruitment between wild type mice and tryptophane hydroxylase1 (Tph1) -/- mice, which are deficient of non-neuronal serotonin and compared the findings to mice whose platelet serotonin was pharmacologically depleted by long-term application the a selective serotonin reuptake inhibitor fluoxetine7. We have also examined leukocyte-endothelial interactions after acute fluoxetine treatment8.
In this protocol we focus on intravital microscopy of mesentery veins, because this model can be performed quickly, and allows valid measurements of leukocyte-endothelial and platelet-leukocyte interactions. This is much more challenging and time-consuming in intravital microscopy of other organs, such as bone, liver, skin, or cremaster muscle. The model described here is ideal for reproducible evaluation of inflammatory cell-cell interaction after challengeing it with an inflammatory stimulus, such as intraperitoneal injection of lipopolysaccharide.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;989-38-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli 055:B5</td>
			<td>Sigma-Aldrich</td>
			<td>L2637&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

An overview of the experimental setting described in this protocol is shown in Figure 1. It shows a mouse with an exteriorized ileum-loop and its vessels, which can be observed in intravital microscopy. Figure 2 shows certain degree of activation in untreated animals due to the procedure itself. But there is almost no slow rolling or firm adhesion of leukocytes compared to LPS-treated mice. Figure 2 also shows the different results of intravital microscopy in wildtype mi...

Watch this Scientific Journal Video about Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice at JoVE.com

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

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 ...

intravital-microscopy-leukocyte-endothelial-platelet-leukocyte

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JoVE Journal

Immunology and Infection

12.0K Aufrufe.  Heart Center, University of Freiburg. 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.