There are numerous leukocyte-mediated acute and chronic pathologic conditions that affect the skin and result in severe suffering and considerable health care expenses. A central pathogenetic event in these conditions is the accumulation of leukocytes in dermal microvessels. This process is thought to require highly specific adhesive interactions between circulating leukocyte subsets and inflamed endothelial cells (EC). Intravital microscopy studies in nondermal vascular beds have demonstrated that intravascular adhesion follows a three (or more)-step scenario that is characterized by (l) initial loose attachment leading to leukocyte rolling, (2) an activation event mediated by locally expressed chemoattractants, and (3) activation-induced engagement of secondary adhesion molecules that promote firm adhesion. Additional molecular events are probably required for subsequent transendothelial migration. Although there is indirect evidence that a similar scenario may be required for leukocyte accumulation in the skin, there are currently no published reports that have tested the multi-step paradigm in this organ. This is due in part to the lack of a simple and versatile in vivo model that permits direct visualization of intravascular adhesion events in normal and diseased skin. Clearly, a reasonably simple model for intravital microscopy in the skin that possesses good optical properties, is accessible for local pro- and/or anti-inflammatory interventions, and permits intravital analysis of acute and chronic inflammatory conditions would be a valuable tool for research on inflammatory skin disease. This proposal is aimed at developing such a model by modifying the classic air pouch preparation in mice. Air pouches are generated by subcutaneous injection of sterile air in the back or abdomen of an animal resulting within six days in formation of a chronic bubble. This model has been used previously in investigations on acute and chronic reactions to bacteria and proinflammatory mediators, mast cell migration, granuloma formation, and cytokine production, to name a few. Adjuvant-induced granulomas in air pouches of mice have been observed for up to 12 weeks indicating that the model permits repeated long-term observations of chronic events. To make this model accessible for intravital microscopy, a transillumination device made from flexible fiberoptic cables will be inserted into the pouch cavity. A glass cover slip will be applied to the apex of the pouch to flatten the skin for microscopic observation of the dermal microvasculature. Using established fluorescence and bright field videomicroscopy and image analysis techniques, physiologic leukocyte behavior, microvascular anatomy, and hemodynamics will be characterized. Tissue samples will be obtained for histopathology and immunohistology. If feasible, this model will offer a novel and unique approach for high resolution studies on cutaneous physiology and pathophysiologic mechanisms of skin disease.
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