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 anesthetics and tracers.
	<ol>
		<li>Maintain sterile conditions throughout the study and sterilize the instruments by autoclaving. Divide the mice into two groups. 
		NOTE: The control group was left untreated, and the other group with unilateral SMG duct ligation for 1 day served as the experimental group. When grouping, mice with similar body weight and age need to be selected to reduce the influence of weight and age on vascular permeability.</li>
		<li>Choose appropriate tracers such as fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight: 4 kDa; FD4) and rhodamine B-labeled dextran (molecular weight: 70 kDa; RD70) (see Table of Materials) with distinct excitation/emission spectra to minimize the interference between fluorescence signals (using the FITC or rhodamine B filter, respectively) for the permeability assay. 
		NOTE: The excitation wavelengths of FD4 and RD70 are 488 nm and 594 nm, respectively, and the selected emission wavelengths are 511-564 nm and 617-669 nm, respectively.</li>
		<li>Dilute the tracers in sterile phosphate-buffered saline (1x PBS) into 100 mg/mL stocks and store the aliquots protected from light at &#8722;20 &#176;C. 
		NOTE: Dextran with a molecular weight such as 4&#160;kDa will rapidly leak from the blood in mouse SMGs even without pathological conditions10.</li>
		<li>Intraperitoneally inject the tribromoethanol (the dose for a 25 g mouse was about 600 &#181;L) to anesthetize the mice. Provide analgesia as recommended by the local animal ethics committee. 
		NOTE: After the induction of anesthesia, use vet ointment on the eyes to prevent dryness. Take good care of the animals11. Wait until the mouse tolerates mechanical stimulation e.g. toe pinch without motor response.</li>
		<li>Inject a mixture of two paracellular permeability tracers with different molecular weights (FD4 and RD70) into the angular vein (0.5 mg/g body weight). Inject each mouse with a 100 &#181;L tracer solution mixture, with 50 &#181;L of each dye mixed in a 1:1 ratio.
		<ol>
			<li>After anesthesia, gently hold the head and neck of the mouse to make one side of the eyeball protrudes slightly. Insert an insulin syringe containing fluorescent tracers (be careful not to introduce any air into the syringe) along the corner of the eye at a right angle to the eye. 
			&#8203;NOTE: If the angular vein is inserted correctly, the dye solution will be easily injected into the vein, and there will be no resistance during the injection. Additionally, tail vein injection in mice might also be a candidate for intravenous tracer molecules.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform the SMG isolation following the steps below. 
	NOTE: Use sterile tools, including tissue scissors and separation nickels, for the surgery.
	<ol>
		<li>Separate a unilateral gland carefully so as not to damage the surrounding blood vessels and nerves under a stereoscopic microscope.
		<ol>
			<li>After the injection of paracellular permeability tracers, quickly put the mice on the cardboard and place them in a supine position with their limbs and heads taped. Apply hair removal cream to the neck skin to remove the mouse&#39;s hair. Use 75% ethanol to disinfect the skin before surgery.</li>
			<li>Then, under a stereomicroscope, cut the epidermis of the neck with general tissue scissors to expose both sides of the SMGs (the right gland was treated in this experiment). Next, use a blunt tissue separation nickel (see Table of Materials) to gently separate the capsule from the gland surface, taking care not to damage the gland tissue and blood vessels.</li>
			<li>Separate the capsule from the gland surface without damaging the glandular structure. In addition, ensure that the entire process of mouse restraint and gland separation is accomplished within 10 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Two-photon microscope set-up
<ol>
	<li>Connect the customized holder to the negative pressure device, and check whether the negative pressure effect is properly achieved in advance. 
	NOTE: The holder is shaped like a miniature magnifying glass with a flat piece of round glass in the center (diameter: 4.32 mm, Figure 1). The negative pressure device was designed in-house in such a way that the holder is connected to a rubber tube, which is linked with a drainage bottle, and the bottle is attached to the negative pressure controller through a short rubber tube. In this way, the negative pressure controller can adjust the pressure to ensure that the tissue is sucked up into the holder.
	<ol>
		<li>Link one side of the holder with the negative pressure device. To test whether the negative pressure device is intact and appropriate, turn the holder upside down, add a drop of water, and set the negative pressure value to 20 units. If the water is completely and quickly sucked away, the negative pressure device is successfully installed with the holder.</li>
	</ol>
	</li>
	<li>Gently place the exposed SMG of the mouse 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.</li>
	<li>Image the support material attached to the gland under a 25x/NA 1.0 water immersion objective (special for NIR). 
	&#8203;NOTE: Use an upright two-photon microscope (see Table of Materials) in this experiment. The blood vessels must be visible immediately with obvious fluorescence after the tracer injection.</li>
</ol>
3. Vessel imaging and permeability detection
<ol>
	<li>Commence imaging soon after the sight of the gland was chosen. 
	NOTE: Typically, time series of 45-50 images in 20 s or longer are generally captured depending on the experimental design.</li>
	<li>Acquire sequential images along the Z-axis, stepped 2 &#181;m apart, from the gland&#39;s surface up to 70-140 &#181;m into the tissue to generate a three-dimensional (3D) construction.</li>
	<li>Acquire time-lapse imaging of the vessels to measure vascular permeability in the SMG. 
	NOTE: Set the conditions of the program menu as follows after running the microscope software (see Table of Materials).
	<ol>
		<li>In the Explorer dialog box, click on Add New Folder and change the file name.</li>
		<li>Go back to the Acquisition column. In XY, the Format is 512 x 512, and the Speed is 400 Hz. Turn Bidirectional X on.</li>
		<li>Set the Zoom factor to 0.75 and 1.5 for Zoom.</li>
		<li>To acquire time-lapse imaging, select xyt under Acquisition Mode. Set Duration to 20 s, 30 min, or longer according to the experimental need.</li>
		<li>To make the 3D construction, set the Acquisition Mode to xyz. Z-Step size can be set according to the actual situation.</li>
		<li>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.</li>
		<li>Click on Start to start imaging. 
		&#8203;NOTE: The mouse must not be left unattended during the imaging process while under anesthesia.&#160;</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>To evaluate the vessel permeability, use Image J software to calculate the fluorescence intensity ratio of FD4 and RD70 between the outside and inside vessels and denote them as extra- and intravascular intensity4. 
	NOTE: The alteration of vascular permeability through tracing the transport of dextrans reflects the change in endothelial barrier function. Moreover, observe the vascular morphology and density by calculating the diameter and number of vessel changes.
	<ol>
		<li>Run Image J software (see Table of Materials).</li>
		<li>Click on File and select Open to open the blood vessel images captured under the two-photon microscope.</li>
		<li>Select Image and set Type to 16-bit to convert the image format.</li>
		<li>Select Analyze and select Area, Mean, IntDen under Set Measurements. Click on OK to confirm.</li>
		<li>Under Analyze, select Tools, open ROI Manger, and select Show All and Labels.</li>
		<li>Measure the intravascular fluorescence intensity value: click on the lasso tool on the main interface to sketch the outline of the blood vessel. Click on Add in ROI Manager. Follow this step to continue to select multiple vessels. Select all of the ROI Manager&#39;s objects and click on Measure.</li>
		<li>Measure the total fluorescence intensity value: outline the whole image, click Measure, and the result is the total fluorescence intensity value of the image.</li>
		<li>Calculate the extravascular fluorescence intensity value. Copy the data to Excel for calculation. Extravascular fluorescence intensity ratio = (1 &#8722; intravascular fluorescence intensity value/total fluorescence intensity value) &#215; 100%.</li>
	</ol>
	</li>
</ol>
5. Downstream applications
<ol>
	<li>During the experiment, calculate the blood flow rate by measuring the distance that a red cell (shown as a black dot under the two-photon microscope)4 undergoes in a time duration.</li>
	<li>To visualize the movement of blood cells across the endothelial cells, label the blood cells by fluorescence. Then, inject the fluorescence-labeled cells with FD4 or RD70 via the mouse tail vein. After circulating for 5 min, trace the interested cells for a period. 
	&#8203;NOTE: A total of 2 x 106 CFSE (carboxyfluorescein succinimidyl ester, a membrane-permeable fluorescent dye)-labeled lymphocytes derived from the spleen of donor mice were transferred into recipients by tail vein injection, and the infiltration of lymphocytes into SMGs was investigated in experimental animal models4.</li>
</ol>
6. Animal care and recovery
<ol>
	<li>Take good care of the animals throughout the experiment. 
	NOTE: Ensure that the animal that has undergone surgery is not returned to the company of other animals until fully recovered during the experiment.</li>
	<li>Do not leave the animal unattended until it regains enough consciousness. 
	NOTE: Observe the breathing status of the mice continuously and keep them warm by putting them on the animal heating pad. Provide postsurgical pain recovery housing and analgesia, as recommended by&#160;the local animal ethics committee.</li>
</ol>

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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. 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The authors have nothing to disclose.

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 diseases13,14,15. The vascular distribution and blood flow of the salivary gland is one of the most abundant in all organs and tissues, but research on the vascular system of the salivary gland has not been carried out widely. A deeper and more comprehensive understanding of salivary gland blood vessels, particularly the endothelial barrier function, will provide new clues to the mechanism of salivary secretion and promote vascular biology research.
The in vivo two-photon laser-scanning microscopy technique can quantify the vascular permeability by intravenous injection of fluorescent dye without sacrificing the animal. Fluorescence-labeled dextrans with microspheres of different molecular weights or sizes can better assess the extent of endothelial injury. Meanwhile, the dynamic kinetics of vascular permeability are determined in real-time by using the intravital evaluation system of vascular permeability. Another advantage is that it is easy to distinguish the types of blood vessels, such as arterioles, venules, and capillaries16. Furthermore, destruction of the vascular endothelial barrier and migration of immune cells are inevitably linked to inflammation, but there are many studies investigating a single factor, and only a few studies have focused on the connection between both aspects17,18. Uhl et al. recently established a research method to analyze the role of different pathogenic factors in salivary gland diseases by injecting fluorescence-labeled dextrans and immune cell-specific antibodies with different fluorescent markers simultaneously in vivo19. Here, fluorescence-labeled immune cells can also be traced through tail vein injection to explore pathogenesis in the current working model. Therefore, the present experimental method may provide a unique system for studying the interaction between leukocyte extravasation and endothelial barrier integrity in SMG disease animal models in vivo.
Nevertheless, it must not be ignored that the responsiveness of mice to anesthetics is different, and the longer the imaging time takes, the longer the anesthesia time needed, and the more difficult it is for mice to recover. Therefore, this experiment is better used to investigate when no subsequent in vivo experiments are required after the imaging. In addition, another limitation is that this technique is not suitable for experiments with a long time span. Dextrans are water-soluble and easy to metabolize, resulting in weaker fluorescence with a longer imaging time, and thus, it is better to maintain imaging time within 30 min.
Altogether, the study focuses on the penetration of paracellular permeability tracers and cells from blood vessels and microvessels into glandular tissues and has rigorously established contrast-enhanced intravital dynamic two-photon laser-scanning microscopy as an advanced method to measure vascular permeability to evaluate endothelial barrier function in the mouse SMG.

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><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 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 (as A and D point out in Figure 2A). By contrast, dextrans with higher molecular weights, such as 40 kDa and 70 kDa, could not differentiate the SMG morphology. Indeed, RD70 was dominantly distributed in large-sized blood vessels and microvessels. 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 then increase the permeability to large molecules. The semi-quantitative FD4 and RD70 fluorescence intensity results also confirmed the above phenomena (Figure 2B). Besides, the diameter of the blood vessels was increased, indicating that duct ligation for 1 day induced dilation of blood vessels. Furthermore, the 3D images showed much more obscured fluorescence of FD4 and RD70 around blood vessels in the ligation group (Figure 2C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64167/64167fig01.jpg" /> 
Figure 1: A schematic diagram showing the customized holder. The holder is shaped like a miniature magnifying glass with a flat piece of round glass in the center (diameter: 4.32 mm). One side of the holder is connected with the negative pressure device to suck up the tissues under the glass, while the other side of the holder is dead. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64167/64167fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64167/64167fig02.jpg" /> 
Figure 2: In vivo vascular permeability assay and 3D images of blood vessels in mice submandibular glands (SMGs). The mice were divided into control and duct ligation groups. The unilateral glands in both groups were exposed and observed. (A) The in vivo vascular permeability assay was performed by injecting 4 kDa FITC-labeled dextran (FD4) and 70 kDa rhodamine B-labeled dextran (RD70) into the angular vein. Arrowheads indicate the changes in the distribution of tracers in the SMG. A, acini. D, duct. Scale bar = 100 &#181;m. (B) For the semi-quantification analysis of the above images, the fluorescence intensity, including FD4 and RD70 within blood vessels (intravascular) and outside blood vessels (extravascular), was measured by the Image J software. (C) 3D images of blood vessels and microvessels in SMG. Arrowheads indicate the changes in the distribution of tracers in the SMG. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64167/64167fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.9K Views.  Ministry of Education, and Beijing Key Laboratory of Cardiovascular Receptors Research. Het huidige protocol beschrijft een in vivo paracellulaire permeabiliteitsdetectiemaat om de functie van strakke endotheelverbindingen in submandibulaire klieren van muizen te evalueren. Twee fotonenlaserscanmicroscopie heeft echter niet alleen het voordeel van conventionele confocale microscopie, maar kan ook worden gebruikt om dieper weefsel en beeld duidelijker te detecteren. Dit experiment biedt een goede methodemaat voor het beoordelen van de vasculaire permeabiliteit van verschillende w...

Video: In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Medicine

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Cancer Research

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Bioengineering

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, ...

Immunology and Infection

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Neuroscience

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

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