Undesirable provocation of the inflammatory response is a detrimental feature of numerous diseases such as diabetes, atherosclerosis, and asthma and thus impacts a significant population. Current strategies utilized to identify key markers of inflammatory disease at the cellular and molecular level have often been centered on in vitro cell culture studies or analyses of tissue excised from animal models of disease. In this proposal we describe the development of a platform for the real-time, in vivo analysis of multiple cellular and molecular mediators of inflammation in diseased animal models using quantum dot nanocrystals and retinal fluorescence microscopy. Our design differs from previous approaches to study inflammation in vivo in that it is enabled by the harnessing of unique and highly-desirable optical properties conferred to quantum dots. These properties include higher quantum efficiency relative to conventional dyes, a resistance to fading, narrow and size-tunable emission spectra all excitable by one wavelength, and amenability to surface engineering of proteins and polymers for optimum stability and targeting. The second enabling technology is our laboratory expertise in in vivo retinal fluorescence imaging which provides unique continuous optical accessibility to the in vivo vasculature. To validate the potential of our proposed technique, in Aim 1 we seek to optimize an imaging modality / nanoprobe design to simultaneously detect the therapeutically-significant biomarkers of inflammation ICAM-1, VCAM-1, PECAM-1, and E-selectin, and to observe their relative molecular expression levels in the retinal vasculature in a rat model of diabetes. Furthermore, we seek to adapt the probe/fluorescence imaging design established in Aim 1 to track multiple leukocyte subsets in real time in vivo in the same animal model. Our preliminary work demonstrates the potential of this technique for the specific labeling and in vivo detection of the endothelial surface markers ICAM-1, VCAM-1 and PECAM-1 in vivo, as well as moving, in vivo-labeled neutrophils in the retinal circulation, with high spatial and temporal resolution and high signal to background ratios. A highlight of our preliminary studies was the first in vivo validation of VCAM-1 and ICAM-1 upregulation in diabetes. This technology has the potential to elucidate complex, cellular and molecular events as they occur in real-time not only in inflammation, but in other diseases such as cancer and ocular disorders as well. ? ? ?
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