Nuclear Length-to-Width ratio USE OF ENDOTHELIAL MORPHOMETRY TO DETERMINE BLOOD FLOW PATTERNS NEAR RABBIT AORTIC BRANCHES A.R. Bond & P.D. Weinberg Physiological Flow Studies Group, Department of Bioengineering, Imperial College London, UK Introduction Lipid deposition (precursor to atherosclerosis) varies around branch points with age (Fig.1) Blood flow / haemodynamics may be a crucial factor Difficult to determine flow in vivo under physiological conditions Endothelial cells (EC) and nuclei (ECn) are dynamic and can be used as in situ biological flow sensors EC and ECn elongate in regions of higher shear stress, and align with the direction of flow Results Immature Mature Nuclear Length-to-Width ratio Immature Mature Figure 2. Pair of rabbit intercostal branch ostia. En face preparation stained with propidium iodide. 200x Magnification. Mean blood flow is from top to bottom (arrow). Scale bar = 500µm. A B Nuclear Orientation Figure 1. Lipid deposition (red) surrounding intercostal branch ostia of immature and mature rabbits. Mean blood flow is from top to bottom (arrow) (Barnes & Weinberg, 1999). Methods Male NZW rabbit (immature n=4 ,mature n=4) thoracic aortas fixed in vivo at physiological pressure and then excised Tissue dehydrated in graded alcohol series Pairs of branches pressed, endothelium down, onto double-sided adhesive tape attached to glass slide Adventitia and media peeled away leaving endothelium fixed to slide ECn stained with propidium iodide before viewing with fluorescence microscope Montages produced of region surrounding branch ostia (Fig. 2) ECn analysed for length-to-width (LW) ratio and angle of orientation using image processing software (ImageTool, UTHSCSA) Nuclear LW ratios (Fig. 3a,b) and angles (Fig. 3c,d) averaged per 100µm*100µm regions and colour maps produced C D Figure 3. Colour maps representing average nuclear length-to-width ratios (a,b), and angle of orientation (c,d) surrounding immature (a,c: n=4 rabbits) and mature (b,d: n=4 rabbits) aortic branch ostia. L/W Ratio – Significant differences between regions (p<0.01) and ages (p<0.01), and interaction between age and region (p<0.01) (Repeated measures ANOVA). Orientation – Significant differences between regions (p<0.01). No significant differences between ages (p>0.1). No significant interaction between age and region (p>0.1) (Repeated measures ANOVA). Discussion Pattern of nuclear elongation very different between immature and mature rabbits Overall LW ratios lower in immature rabbits, suggesting lower shear stresses, with an elevated shear stress region downstream of ostia Mature rabbits have higher LW ratios with elevated regions particularly in lateral regions The data are consistent with previous findings (Al-Musawi et al, 2004) and suggest an age-related change in blood flow that correlates with age-related lipid deposition and atherosclerotic lesion development Orientations suggest flow from local regions taken into branch, whilst remainder of blood continues down aorta producing secondary flows Technique has great potential as could enable visualisation of the distribution of flow-controlled mediators of atherosclerosis within the vasculature References Barnes, SE & Weinberg, PD (1999) Arterioscler Thromb Vasc Biol 19(10): 2376-86 Al-Musawi et al (2004) Atherosclerosis 172(1): 79-84 This study is funded by the British Heart Foundation