A Dual-Tracer Method for Differentiating Transendothelial Transport from Paracellular Leakage in Vivo and in Vitro

Inflammation-induced impaired function of vascular endothelium may cause leakage of plasma proteins that can lead to edema. Proteins may leave the vascular lumen through two main paracellular and transcellular pathways. As the first involves endothelial cell (EC) junction proteins and the second caveolae formation, these two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during pathology that causes inflammation. Here we present a newly developed dual-tracer probing method that allows differentiation of transcellular from paracellular transport during pathology. This fluorescence-based method can be used in vitro to test changes in EC layer permeability and in vivo in various animal vascular preparations. The method is based on comparison of low molecular weight molecule (LMWM) transport to that of high molecular weight molecule (HMWM) transport through the EC layer or the vascular wall during physiological and pathological conditions. Since the LMWM will leak through mainly the paracellular and HMWM will move through paracellular (when gaps between the ECs are wide enough) and transcellular pathways, the difference in transport rate (during normal conditions and pathology) of these molecules will indicate the prevailing transport pathway involved in overall protein crossing of vascular wall. Thus, the novel approach of assessing the transport kinetics of different size tracers in vivo by intravital microscopy can clarify questions related to identification of target pathways for drug delivery during various pathologies associated with elevated microvascular permeability

Role of Fibrinogen in Vascular Cognitive Impairment in Traumatic Brain Injury

Fibrinogen (Fg) is one of the biomarkers of inflammation and a high risk factor for many cardiovascular and cerebrovascular diseases. Elevated levels of Fg (hyperfibrinogenemia, HFg) are also associated with traumatic brain injury (TBI). HFg in blood alters vascular reactivity and compromises integrity of endothelial cell layer that ultimately can result in extravasation of Fg and other plasma proteins. Proteins deposited in extravascular space may form plaques which can lead to neurodegeneration. Among these plasma proteins are amyloid beta (Aβ) and/or cellular prion protein (PrPC) that can form degradation resistant complexes with Fg and are known to be involved in memory impairment. The purpose of this chapter is to propose and discuss some possible mechanisms involved in HFg-mediated cerebrovascular dysfunction leading to neuronal degeneration during TBI

Localization of Fibrinogen in the Vasculo-Astrocyte Interface after Cortical Contusion Injury in Mice

Besides causing neuronal damage, traumatic brain injury (TBI) is involved in memory reduction, which can be a result of alterations in vasculo-neuronal interactions. Inflammation following TBI is involved in elevation of blood content of fibrinogen (Fg), which is known to enhance cerebrovascular permeability, and thus, enhance its deposition in extravascular space. However, the localization of Fg in the extravascular space and its possible interaction with nonvascular cells are not clear. The localization of Fg deposition in the extravascular space was defined in brain samples of mice after cortical contusion injury (CCI) and sham-operation (control) using immunohistochemistry and laser-scanning confocal microscopy. Memory changes were assessed with new object recognition and Y-maze tests. Data showed a greater deposition of Fg in the vascular and astrocyte endfeet interface in mice with CCI than in control animals. This effect was accompanied by enhanced neuronal degeneration and reduction in short-term memory in mice with CCI. Thus, our results suggest that CCI induces increased deposition of Fg in the vasculo-astrocyte interface, and is accompanied by neuronal degeneration, which may result in reduction of short-term memory