Atherosclerotic lesion size in mice is routinely evaluated by morphometrical measurements on serial sections in order to obtain volume measurements. The technique of confocal scanning laser microscopy (CSLM) makes it possible to optically scan and thereby evaluate a tissue sample. We here describe a method for measuring lesion volume in ApoE/LDLr deficient mice at 20 and 30 weeks of age using the non-destructive procedure of CSLM. Whole mount preparations of opened aorta with the lumen side facing the cover slip were analysed under 10x magnification in a CSLM (Leica). The autofluorescence of the elastic fibres of the lamina interna as opposed to the non-fluorescing lesion was used to define the bottom and top of the lesion during scanning. Ten to forty images were collected 2.4 microm apart, depending on the size of the lesion, and the stack of images was then analysed using Imaris (Bitplane). After the CSLM evaluation, the aortas were de-mounted, embedded in paraffin, sectioned, stained in hematoxylin and eosin and the volume re-evaluated with conventional morphometry. Statistical evaluation showed that the results obtained with CSLM and the results of morphometry were positively correlated. Area measurements of the plaques using en face preparations of aorta showed that the plaque area was generally larger at the left side and a significant increase of plaque area along the length of the thoracic aorta. Our results showed that atherosclerotic lesions in mice can be quantitatively evaluated by CSLM.
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