Intravital microscopy

The mesenteric microcirculation was observed using intravital microscopy, as described previously [25]. Briefly, mice were anesthetized with isoflurane (2–3%) in 100% oxygen at 2 L/min flow rate and placed in the supine position on a heated platform (37 °C). Subsequently, the intestines were mobilized, gently exteriorized, and placed into a warm isotonic 0.9% saline bath (37 °C). Intravital microscopy was performed with a CapiScope handheld video capillary microscope (KK Technology, Honiton, UK) to view the mesenteric microcirculation. The intravital microscope uses a sidestream dark field (SDF) camera that uses green light–emitting diodes. The green light is primarily absorbed by hemoglobin in RBCs in the microcirculation that allows RBCs to be viewed in contrast to the background. Glycocalyx thickness, as well as measurements of microvascular structure and hemodynamics (i.e., perfused boundary region, microvascular density, RBC fraction, and capillary blood volume [CBV] _absolute), was analyzed using an automated capture and analysis system (GlycoCheck, MicroVascular Health Solutions LLC, Alpine, UT), as described previously [11]. Briefly, the perfused boundary region (PBR) represents the depth of RBC penetration into the endothelial glycocalyx and is taken as a marker of glycocalyx barrier function, perfused microvascular density represents the cumulative length of perfused microvessel segments in a given area of tissue, RBC fraction represents an estimate of the longitudinal tube hematocrit in perfused microvessel segments, and CBV absolute represents the capillary perfusion and is calculated as density of perfused capillary segments (4–6 μm diameter) multiplied by their cross-sectional areas. We also derived an estimate of glycocalyx thickness by manually measuring the change in width of flowing RBCs before and after the passage of a spontaneous leukocyte in individual capillaries, as described and validated previously [26]. Glycocalyx thickness was calculated using the following equation: