In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Summary

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.

Abstract

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.

Introduction

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 tissues^2....

Protocol

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

1. Prepare and administer the anesthetics and tracers.
   1. Maintain sterile conditions throughout the study and sterilize the instruments by autoclaving. Divide the mice into t....

Results

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.......

Discussion

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

Materials

Name	Company	Catalog Number	Comments
2-photon microscope (TCS-SP8 DIVE)	Leica, Germany
4 kDa FITC-labeled dextran	Sigma Aldrich	46944
70 kDa rhodamine B-labeled dextran	Sigma Aldrich	R9379
Blunt tissue separation nickel	Bejinghuabo Company	NZW28
Depilatory cream	Veet
Disposable sterile syringe	Zhiyu Company		1 mL
Image J software	National Institutes of Health
Insulin syringe	Becton, Dickinson and Company	0253316	1 mL
Leica Application Suite X software	Leica Microsystems
Microtubes	Axygen	MCT-150-C	1.5 mL
Phosphate buffered saline 1x	Servicebio	G4207-500
Tissue scissors	Bejinghuabo Company	M286-05
Tribromoethanol	JITIAN Bio	JT0781