Intravital analysis of vascular permeability in mice using two-photon microscopy (1)
A number of groups have used an in-vitro assay system that measures the flux of variety sizes of molecules that traverse endothelial cells cultured in transwell chambers.
Dextran is a polysaccharide that consists of glucose chains and its molecular mass is variable from 3 to 2000 kDa.
Arterioles were delineated immediately after dextran injection and visualization of capillaries and postcapillary venules followed, thus allowing us to distinguish blood vessel types. The black-and-white images obtained were then converted to rainbow color-scale images according to fluorescent intensity. To monitor tracer dynamics, we separately circumscribed the blood vessel area and interstitial space.
20-kDa FITC-dextran readily leaked into the interstitial space and the MFI of the interstitium peaked around 10 min after injection. 40-kDa dextran exhibited mild but sustained leakage, as indicated by a persistent elevation of the MFI in the interstitial space. 70-kDa dextran and FITC-albumin were retained in the blood 10 min after injection. However, a mild elevation of the interstitial space MFI was observed after 60 min.
70-kDa dextran shifted into the interstitial space 1–2 mins after the histamine injection. 150-kDa and even 2000- kDa dextran shifted into the interstitial space within 2 mins.
As the passive anaphylaxis model as a classical type I allergic reaction, IgE antibody against TNP was injected intraperitoneally, followed by intravenous injection of TNP-conjugated OVA. This hyper-permeability was not suppressed with bepotastine besilate treatment but was suppressed in WBB6F1-W/Wv mice in which mast cells were absent, suggesting that mast cells but not histamine were indispensable in this model.
A classical type IV allergic reaction. T cells from DNFB-sensitized mice were transferred into non-sensitized mice and DNFB was painted on the ear skin. Twenty hours later, injected dextran readily leaked from postcapillary venules.
Just after the histamine injection, leakages of tracer were detected around postcapillary venules, but not around arterioles or capillaries.
Dextran larger than 70 kDa was retained in the blood for hours under homeostatic con- ditions, indicating that a traffic restriction exists for plasma contents around 70-kDa. This observation is physiologically convincing because not only albumin (molecular weight (MW) 66 kDa) but also other carrier proteins in plasma, such as transferrin (MW 80 kDa), transthyretin-tetramer (MW 56 kDa), and thyroxine- binding globulin (MW 54 KDa) represents similar molecular sizes.
Under inflammatory conditions, such as type I and type IV allergy, even 2000-kDa dextran promptly shifted into the interstitial space. Since the molecular sizes of immunoglobulins (Igs) range from 146- to 990-kDa all subclasses of Igs, including IgM, should promptly traverse the vascular endothelium at sites of inflammation.
Extravasation of albumin, a major osmotic protein, results in the expansion of interstitial fluid contributing to wash out of harmful pathogens. Permits the efflux of plasma proteins, particularly immunoglobulins. This should be an essential system that enables selective accumulation of immunoglobulins into sites of inflammation for the clearance of pathogens.
