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
<ol>
	<li>Prepare and administer the anes...

<ol>
	<li>Carpenter, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretion,+components,+and+properties+of+saliva.">The secretion, components, and properties of saliva.</a> Annual Review of Food Science and Technology. 4, 267-276 (2013).</li><li>Garrett, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proper+role+of+nerves+in+salivary+secretion:+A+review.">The proper role of nerves in salivary secretion: A review.</a> Journal of Dental Research. 66 (2), 387-397 (1987).</li><li>Berndt, P., 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=Tight+junction+proteins+at+the+blood-brain+barrier:+Far+more+than+claudin-5.">Tight junction proteins at the blood-brain barrier: Far more than claudin-5.</a> Cellular and Molecular Life Sciences. 76 (10), 1987-2002 (2019).</li><li>Cong, X., 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=Disruption+of+endothelial+barrier+function+is+linked+with+hyposecretion+and+lymphocytic+infiltration+in+salivary+glands+of+Sj&#246;gren's+syndrome.">Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sj&#246;gren's syndrome.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (10), 3154-3163 (2018).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+Multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: Multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21 (11), 1369-1377 (2003).</li><li>Masedunskas, A., Sramkova, M., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostasis+of+the+apical+plasma+membrane+during+regulated+exocytosis+in+the+salivary+glands+of+live+rodents.">Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents.</a> Bioarchitecture. 1 (5), 225-229 (2011).</li><li>Masedunskas, 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=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13552-13557 (2011).</li><li>Balda, M. S., 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=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of Cell Biology. 134 (4), 1031-1049 (1996).</li><li>Enis, D. R., 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=Induction,+differentiation,+and+remodeling+of+blood+vessels+after+transplantation+of+Bcl-2-transduced+endothelial+cells.">Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (2), 425-430 (2005).</li><li>Wang, X., 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=Application+of+digital+subtraction+angiography+in+canine+hindlimb+arteriography.">Application of digital subtraction angiography in canine hindlimb arteriography.</a> Vascular. 30 (3), 474-480 (2022).</li><li>Trani, M., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+control+of+vascular+permeability:+vascular+endothelial-cadherin+and+other+players.">New insights in the control of vascular permeability: vascular endothelial-cadherin and other players.</a> Current Opinion in Hematology. 22 (3), 267-272 (2015).</li><li>Viazzi, F., 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertension Research. 31 (5), 873-879 (2008).</li><li>Scheppke, L., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> The Journal of Clinical Investigation. 118 (6), 2337-2346 (2008).</li><li>Blanchet, M. R., 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=Loss+of+CD34+leads+to+exacerbated+autoimmune+arthritis+through+increased+vascular+permeability.">Loss of CD34 leads to exacerbated autoimmune arthritis through increased vascular permeability.</a> Journal of Immunology. 184 (3), 1292-1299 (2010).</li><li>Egawa, G., Ono, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+of+vascular+permeability+by+two-photon+microscopy.">Intravital Imaging of vascular permeability by two-photon microscopy.</a> Methods in Molecular Biology. 2223, 151-157 (2021).</li><li>Vestweber, D., Wessel, F., Nottebaum, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+the+regulation+of+leukocyte+extravasation+and+vascular+permeability.">Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.</a> Seminars in Immunopathology. 36 (2), 177-192 (2014).</li><li>Schulte, 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=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> The EMBO Journal. 30 (20), 4157-4170 (2011).</li><li>Uhl, B., 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+novel+experimental+approach+for+in+vivo+analyses+of+the+salivary+gland+microvasculature.">A novel experimental approach for in vivo analyses of the salivary gland microvasculature.</a> Frontiers in Immunology. 11, 604470 (2020).</li></ol>

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 ...

The present protocol describes an in vivo paracellular permeability detection measure to evaluate the function of tight endothelial junctions in mouse submandibular glands. 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. This experiment provides a good method measure for assessing the vascular permeability of different tissues especially the surface tissues and organs of animals.Begin by choosing appropriate tracers such as fluorescein, isothiocyanate, labeled dextran and Rhodamine B, labeled dextran with distinct excitation emissions spectra, to minimize the interference between fluorescence signals for the permeability assay. Dilute the traces into sterile phosphate buffered saline into 100 milligrams per milliliter stocks and store the aliquots protected from light at minus 20 degrees Celsius. After anesthetizing the 8 to 10 weeks old male wild type mouse, gently hold the head and neck of the mouse to make one side of the eyeball protrude slightly.Insert an insulin syringe containing 100 microliters of fluorescent tracer solution mixture along the corner of the eye at a right angle to the eye. After the injection, quickly place the mouse on the cardboard in a supine position with its limbs and head taped. For submandibular gland or SMG isolation, under a stereoscopic microscope, cut the epidermis of the neck with general tissue scissors to expose both sides of the SMGs.Next, use a blunt tissue separation needle to gently separate the capsule from the gland surface without damaging the gland tissue, blood vessels, and glandular structure. Connect the customized holder to the negative pressure device and check whether the negative pressure effect is properly achieved in advance. Gently place the exposed SMG on the customized holder and suck up the tissue by negative pressure suction, as far away from the mouse body as possible, to reduce motion artifacts caused by respiration and other conditions.Commence imaging soon after the site of the gland is chosen. Acquire sequential images along the Z axis stepped two micron apart from the gland's surface up to 70 to 140 microns into the tissue to generate a three-dimensional construction. Next, acquire time lapse imaging of the vessels to measure vascular permeability in the SMG.In the Explorer dialogue box, click on Add New Folder"and change the file name. Then, go back to the acquisition column. In XY, the format is 512 by 512, and the speed is 400 hertz.Turn Bidirectional X"on, set the zoom factor to 0.75 and 1.5 for zoom. Next, select XYT under Acquisition Mode"to acquire time lapse imaging. Set duration to 20 seconds, 30 minutes or longer according to the experimental need.After setting the conditions, click on Live"to observe the vascular imaging of two channels and overlapping channels in the photo forming frame and choose the areas of interest. Click on Start"to begin imaging. In vivo vascular permeability assay, and three dimensional images of blood vessels in mice.Submandibular glands are shown here. In the control group, both the tracers existed in the blood vessels of the SMG. Due to its small molecular weight, FD4 was able to leak out of the blood vessels to the basal sides of acini and ducts, thereby clearly depicting the shape of the acini and ducts.The RD70 was distributed in large sized blood vessels and micro vessels. In the duct ligation group, both FD4 and RD70 were extravasated to the basal sides of acini, which indicated that duct ligation could disrupt the endothelial barrier function and increase the permeability to large molecules. Further, the semi-quantitative FD4 and RD70 fluorescence intensity results confirmed these observations.The three dimensional images showed much more obscured fluorescence of FD4 and RD70 around blood vessels in the ligation group compared to the control group. Careful SMG isolation and appropriate placement and the microscope are essential prerequisites for successful detection. Following the procedures, the blood flow rate can be calculated by marrying the movement of red blood cells and the infiltration of the fluorescence labeled blood cells can be chased.

in-vivo-vascular-permeability-detection-in-mouse-submandibular-gland

Research

JoVE Journal

Biology

1.8K visualizações.  Ministry of Education, and Beijing Key Laboratory of Cardiovascular Receptors Research. O presente protocolo descreve uma medida de detecção de permeabilidade paracelular in vivo para avaliar a função de junções endoteliais apertadas em glândulas submandibulares de camundongos. A microscopia de varredura a laser de dois fótons, no entanto, não só tem a vantagem da microscopia confocal convencional, mas também pode ser usada para detectar tecidos e imagens mais profundos com mais clareza. Este experimento fornece uma boa medida de método para avaliar a permeabilidade ...