Quantitative Assessment of Capillary Permeability in Deep Intracardiac Capillaries Using Fluorescent Dextran

Background

Blood vessels extend throughout the body to circulate blood and supply cells in all tissues with sufficient oxygen and nutrients, as well as to collect and discharge cellular waste products. In particular, capillaries in peripheral tissues are considered sites of material exchange, where small molecular substances pass through the endothelial cell layer under colloid osmotic pressure (COP). Each tissue cannot function without nutrition and oxygen; normal physiological tissue function depends on the regulation of vascular permeability, an important function of capillaries [1].

Capillaries are composed of a single endothelial cell layer. Leakage from blood vessels into tissues is the source of interstitial fluid and reflects the underlying biological function of each organ. The homeostasis of organ-specific function lies in the diversity of vascular permeability. Hence, vascular permeability dysfunction is a major cause of disease. In recent years, abnormalities in microvascular function have been reported to be closely related to tissue function and disease development [2]. In fact, dysfunction of endothelial cells due to inflammation, aging, and viral and bacterial infections leads to organ damage. Endothelial dysfunction is critically involved in many chronic human diseases, such as diabetes, arthritis, and dementia [3–5]. A large cohort of human studies has shown that dysfunction of the tissue microvasculature, a nutritional supply channel, is associated with a wide range of subjective symptoms such as headache, palpitations, fatigue, and chest pain and increases the risk of more severe cardiovascular events [6,7]. Previous studies have noted that blood flow disturbance in cerebral blood vessels, especially in small blood vessels, caused by microplastic particles, could lead to neurological and cognitive disorders [8,9]. In addition, maintaining tissue microvascular function regulates individual lifespan in mice [10].

Permeability in microvascular vessels in superficial layers, such as tumors [11,12], skin, and retina [13–15], has been evaluated using fluorescent dextran or Evans blue. Several in vivo evaluations of cardiac permeability have also been reported [16–18]. These reports have evaluated capillaries visible under a microscope—such as those in transparent tissues at early developmental stages and on organ surfaces—and have demonstrated that capillary permeability increases in response to inflammation, cancer, and other diseases. Here, because most capillaries lie deep within tissues, our overall understanding remains incomplete. Therefore, substance exchange between capillaries and tissues is thought to occur via interstitial fluid, which permeates and circulates throughout the tissue, enveloping the cells. However, the mechanism underlying this process and its relationship to organ function remain unclear, and it is not yet known whether there are differences between healthy states and states of functional decline or disease.

In in vitro experiments, the selection and replacement of culture media are critical determinants of cellular status (such as proliferation, differentiation, and cell death) and of functional assessment. In contrast, in three-dimensional culture, cells (or cell clusters) have a high degree of freedom of movement, and the contact surface with the culture medium is constantly changing. Furthermore, in vivo, changes in the composition of cell populations alter intercellular interactions, and the interstitial fluid flowing between cells also changes daily in response to the environment. These changes in mechanical sensitivity affect cell dynamics [19,20]. Therefore, in drug discovery aimed at organ-specific efficacy, there is a need for novel validation methods that integrate imaging endpoints with functional measurements in translational toxicology and mechanobiology [21,22].

To date, no method is available to detect the exchange of substances in cardiac capillaries deep within the heart tissue or to evaluate what is detected. In this study, we develop a new method to detect and evaluate molecules leaking from cardiac capillaries. By administering fluorescent dextran to mice via the tail vein, followed by rapid processing of the heart tissue, we have detected leaking fluorescent material from intracardiac microvessels [23]. In addition, we calculated the diffusion area of the fluorescent substance and used it as an indicator of capillary permeability. Using this new method, we compared the diffusion area of molecules leaking from blood circulation into cardiac tissue in young and aged mice and found that it was significantly reduced in the latter [23]. In this way, we are now able to assess the presence of interstitial fluid that leaks from capillaries deep within tissues—a phenomenon that was previously impossible to observe. As a result, it has become clear that the flow of interstitial fluid within the heart changes with age, suggesting that this may be a contributing factor to age-related cardiac dysfunction and to the increased risk of disease.