To elucidate the role of RAGE and ICAM-1 in the context of PC-induced inhibition of leukocyte recruitment, leukocyte adhesion in postcapillary venules of surgically prepared cremaster muscles of wild-type (WT), [RAGE.sup.-/-], and [Icam-1.sup.-/-] mice was observed using intravital microscopy.
For this purpose, Giemsa staining of whole mounts of fMLP-stimulated cremaster muscles, obtained after the respective intravital microscopic experiment, was performed (Figure 1(c)).
Interestingly, in vivo experiments using the models of trauma- and TNF[alpha]-induced inflammation of murine cremaster muscles revealed that RAGE and ICAM-1 act together in mediating leukocyte adhesion in a stimulus-dependent manner [5, 6].
To this end, superfusion of murine cremaster muscles with SB203580 (100 nM) for 30 min prior to and for 1 h following the placement of KC gel did not significantly modify the rolling flux and rolling velocity of neutrophils as compared to the control saline-superfused cremaster muscle (P > 0.05; Figures 1(a) and 1(b)).
To analyse the subsequent steps of neutrophil recruitment in the same cremaster muscles, we further quantitatively-visualized and determined the number of adherent and emigrated neutrophils.
The mouse cremaster muscle preparation was used to study neutrophil behaviour in microcirculation and tissue as described previously [24-26].