A new chamber technique for intravital microscopic observations in the different soft tissue layers of mouse hindleg

Background: A newly developed observation chamber has been designed for com fortable limb immobilization during intravital microscopic analysis, which permits direct, repeated, long-lasting observations of the microcirculation in the various hindleg soft tissues. Methods: Experiments were performed under inhalation isoflurane/nitrous or; ide anesthesia. Intravenously injected fluorescein isothiocyanate (FITC)-de xtran (M-r 150,000) and Rhodamine 6G (Sigma, St, Louis, MO) allowed for vis ualization of both microcirculatory phenomena in arterioles, capillaries, a nd venules and macrocirculatory structures as superficial saphenous artery and vein. Skin microcirculation analysis was performed from the epidermal s ide (group A, n = 7), whereas observation of deeper situated tissues was pe rformed after oval skin excision on the medial surface of the tibial area ( group n, n = 7), FITC-dextran (M-r 150,000; group C, n = 8) injected into t he foot pad permitted visualization of venous, arterial, and accompanying l ymphatic vessels. With the aid of a computer-assisted microcirculation anal ysis system, functional capillary density, vessel diameter, edema formation , and leukocyte-endothelial cell interactions were evaluated. The ratio of rolling leukocytes, given as percentage of all leukocytes passing the vesse l segment during a 30-second observation interval, and the number of sticki ng; leukocytes per square millimeter of endothelial surface were determined . Results: This new model allows the analysis of the complex in vivo changes of macro- and microcirculatory parameters in the different (venous, arteria l, lymphatic) vessels of the covering tissues (skin and muscle) of the mous e hindleg. Conclusion: The potential applications of this technique include the study of mechanical trauma, ischemia-reperfusion injury, and tissue compression m imicking both acute and prolonged venous stasis on both the microcirculator y and macrocirculatory levels in the different tissue compartments.