Name: in vivo vascular permeability detection in mouse submandibular gland
Uploaded: 2022-08-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

This study was supported by the National Natural Science Foundation of China (grants 31972908, 81991500, 81991502, 81771093, and 81974151) and the Beijing Natural Science Foundation (grant 7202082).

All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Male wild-type (WT) mice in the age group of 8-10 weeks were used for the present study. The experimental animals were carefully treated to minimize their pain and discomfort.
1. Animal procedures
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	<li>Prepare and administer the anes...

<ol>
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The maintenance and regulation of endothelial barrier function are essential for vascular homeostasis. Endothelial cells and their intercellular junctions play a critical role in maintaining and controlling vascular integrity12. The shear force of blood flow, growth factors, and inflammatory factors can cause changes in vascular permeability and, thus, participate in the occurrence and development of systemic diseases such as hypertension, diabetes, and autoimmune diseases13</sup...

Various salivary glands produce saliva, which primarily acts as the first line of defense against infections and helps digestion, thereby playing an essential role in oral and overall health1. Blood supply is crucial for salivary gland secretion since it constantly provides water, electrolytes, and molecules that form the primary saliva. Endothelial barrier function, regulated by the tight junction (TJ) complex, strictly and delicately limits the permeation of capillaries, which are highly permeable to water, solutes, proteins, and even cells moving from the circulating blood vessels into the salivary gland tissues2,3. We have previously found that the opening of the endothelial TJs in response to a cholinergic stimulus facilitates saliva secretion, whereas the impairment of the endothelial barrier function is interlinked with hyposecretion and lymphocytic infiltration in the submandibular glands (SMGs) in Sj&#246;gren&#39;s syndrome4. These data suggest that the contribution of endothelial barrier function needs to be paid enough attention regarding a variety of salivary gland diseases.
A two-photon laser-scanning microscope is a powerful tool for observing the dynamics of cells in intact tissue in vivo. One of the advantages of this technique is that near-infrared light (NIR) has deeper tissue penetration than visible or ultraviolet light when specimens are excited by NIR and does not cause obvious light damage to tissues under appropriate conditions5,6. Indeed, the salivary glands are a very homogenous and superficial tissue, in which the surface acinar cells are only around 30 &#181;m away from the gland surface7,8. It has been shown that intravital confocal microscopy can study exocrine secretion and actin cytoskeleton in live mouse salivary glands at subcellular resolution8. Two-photon laser-scanning microscopy, nevertheless, not only has the advantage of conventional confocal microscopy but can also be used to detect deeper tissue and image more clearly. Here, fluorescence-labeled dextrans, which are frequently used as paracellular permeability tracers and have the advantage of different sizes, can be used to test the magnitude of TJ pore9. In the present study, an intravital real-time two-photon laser-scanning microscopy technique is established for in situ evaluation of endothelial barrier function in mouse SMGs. Each work step for in vivo vascular permeability detection in mouse SMGs is described in the current protocol. Here is an example of detecting endothelial barrier function in the mouse SMG duct ligation model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-photon microscope (TCS-SP8 DIVE)</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>70 kDa rhodamine B-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt tissue separation nickel</td>
			<td>Bejinghuabo Company</td>
			<td>NZW28</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile syringe</td>
			<td>Zhiyu Company</td>
			<td></td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>0253316</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 1x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Bejinghuabo Company</td>
			<td>M286-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>JITIAN Bio</td>
			<td>JT0781</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo vascular permeability detection in mouse submandibular gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

Following the protocol, the unilateral SMG was attached to a custom-made holder, and the gland was kept as far away from the mouse body as possible to prevent breathing from causing motion artifacts. The rapid flow of the red blood cells (black dots) in blood vessels was observed under the microscope. After finding the tissue field under an ocular lens, one must switch to manipulate the microscope software. In the control group, both the tracers existed in the blood vessels of the mouse SMG. In particular, due to its sma...

Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

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