Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 µm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6±0.06 compared to 0.23±0.05 h−1, P<0.05, thereby reducing the imaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.
Microvascular permeability is a serious complication of systemic inflammation in critically ill patients; yet, no direct techniques exist to quantify this in vivo. To overcome this limitation, we investigated the use of multiphoton microscopy to evaluate fluorescent macromolecular gradients in the eye. Following the induction of systemic inflammation in a CD1 mouse, a bolus of high (250 KD FITC-dextran) and low (70 KD rhodamine-dextran) molecular weight fluorescent macromolecules was injected via the tail vein. The anesthetized mouse was positioned in such a way that different microvessels in the eye could be imaged directly using an upright microscope. The fluorophores were simultaneously excited at 840nm and a series of images including a spectral scan (480 to 680nm), an xt line scan (96 lines) and an x,y,z image stack were collected from the iris, cornea and limbal plexus at one hour intervals for four hours. A simple fluorescent gradient across the vessel wall was used as an index of microvascular permeability. In all microvessels, the LMW dye was more permeable. We found that the fluorescent gradient increased dramatically in the limbal plexus up to three hours then declined. This may indicate that circulating fluid pooled near the limbal plexus. Consistent with the thick walls and tight junctions of the iris microvessels, no significant fluorescent gradients were detected in this area. The cornea, containing a collagen filled stroma layer, was found to have both lateral and perpendicular fluorescent gradients. This work demonstrates that inflammation causes differential microvascular permeability in the mouse eye.
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