Amber Kunkel Advisor: Jessica Wagenseil, D.Sc. Department of Biomedical Engineering Saint Louis University The Feasibility of Using Stored Mouse Blood Vessels for Mechanical Testing

What Are Blood Vessel Mechanics? How longitudinal force, diameter, compliance respond to inflation and longitudinal stretching Unloaded dimensions Opening angle (residual strain) Stress and strain in response to stretches or inflations Radial cut Opening angle (OA)

Why Blood Vessel Mechanics? Model in vivo conditions Understand normal functions and any abnormalities Mechanics of mouse aorta and carotid used to study: Elastin +/- mice and Supravalvular Aortic Stenosis (Wagenseil lab) Smoking Aortic development Muscular dystrophy and Marfan syndrome

Why Storage? Vessels usually tested within 1 day of dissection But the carotid and aorta are large elastic arteries, could theoretically be stored longer Applications of longer storage time: Improved collaboration Easier scheduling Insurance against equipment failure or other unexpected circumstances

Study Overview 30 mice (8 week old) sacrificed over 1 month Ascending aorta, left common carotid, and right common carotid removed Five time points: refrigerated in physiologic saline for 1, 3, 7, 14, or 28 days

Mechanical Testing Setup Preconditioning Vessel Bath Pressure Controlled Pump Force Transducer Microscope Pressure Transducer Desktop Computer Translation Stage L D z d Stretch Inflate

Mechanical Testing Ctd. Inflation protocols At 1, 1.1, and 1.2x in vivo length 3 cycles, mmHg, steps of 25, 12 sec/step Stretch protocols At 50, 100, and 150 mmHg 3 cycles, 1-1.2x in vivo length

Dimensions Three rings cut from left carotid and ascending aorta Image under microscope Measure inner diameter, outer diameter, thickness Ascending aortas cut radially, used for opening angle measurements

Data Analysis Pressure-force, pressure-diameter, and pressure-compliance curves from 1 cycle of first inflation protocol Image J to compute thickness, inner and outer diameters Circumferential stretch, circ stress, and axial stress Matlab for opening angle ANOVA and Scheffe post hoc test (P<.05)

Dimensions: Left Carotid Diameters No significant difference between time points But there is a slight trend to each…

Left Carotid Thickness Slightly decreasing outer diameter and increasing inner diameter leads to steadily decreasing thickness 1 day vessels are significantly different from 7 and 28 days

Ascending Aorta Diameters 1 day OD is significantly different from 7; 1 day ID is significantly different from 3, 7, and 28 days

Ascending Aorta Thickness This time, changes in diameter actually cancel each other out 1 day is only significantly different from 28

Pressure-Diameter At all pressures except 25, no difference between dates At 25, only 7 day is different from 1 day

Pressure-Compliance At 50, 75, and 100 mmHg, the 1 day compliance is significantly different from 14 and 28 days

Pressure-Force 1 day is significantly different from 14 at all but the highest pressures However, there is no clear pattern to the force differences

Pressure- Circumferential Stretch Ratio No significant differences between time points Circumferential stretch ratio changes less the longer the vessels are stored

Pressure-Circumferential Stress 1 day is significantly different from 7 and 28 at P25 and P50, and from 28 at P75 Again, no clear pattern to differences between days

Pressure- Axial Stress Significant difference between 1 day and 14 day for P0-P100 Still lacks a clear pattern

Opening Angle No significant difference between vessels

Discussion 1 and 3 day vessels the same except for ASC inner diameter Every other time point shows several differences, so 3 days could be our limit Where are these differences coming from?

Vessel Degradation? Supported by decreases in LCC thickness and ASC inner diameter Could explain why compliance decreases with storage time But this doesn’t fit some of the other trends we’ve seen

Other Explanations: Dimensions Measurements done by hand, not blindly Could be influenced by order measured Rings cut by 2 different people Lighting, angle, and height of rings can also influence readings 1 day (6/9 lcc1) 28 day (7/6 lcc1 )

Other Explanations: Mechanical Testing Some variation between days is normal More could be explained by abnormal myograph functions Outflow pressure often lagging Temporary solutions could have interfered with readings Vessel Bath Pressure Controlled Pump Force Transduc er Microscope Pressure Transduc er Desktop Computer Translation Stage

Future Work/ Improvements Test more vessels Protein content analysis and histology to understand changes in dimensions Use data from additional protocols for modeling Consistency in cutting rings, maybe blind measurements for dimension analysis Fix leak in myograph

Acknowledgements NSF SLU BME Dr. Wagenseil Victoria Le Dr. Willits Neva Gillan

References Huang, Y., Guo, X., & Kassab, G. S. (2005). Axial nonuniformity of geometric and mechanical properties of mouse aorta is increased during postnatal growth. American Journal of Physiology: Heart and Circulatory Physiology, 290, H657-H664. Wagenseil, J. E., Ciliberto, C. H., Knutsen, R. H., Levy, M. A., Kovacs, A., & Mecham, R. P. (2009). Reduced vessel elasticity alters cardiovascular structure and function in newborn mice. Circulation Research, 104, Wagenseil, J. E., Nerurkar, N. L., Knutsen, R. H., Okamoto, R.J., Li, D. Y., Mecham, R.P. (2005). Effects of elastin haploinsufficiency on the mechanical behavior of mouse arteries. American Journal of Physiology: Heart and Circulatory Physiology, 289, H1209-H1217. Shifren, A., Durmowicz, A.G., Knusten, R.H., Faury, G., & Mecham, R.P. (2008). Elastin insufficiency prediscposes to elevated pumonary circulatory pressures through changes in elastic artery structure. Journal of Applied Physiology, 105, Guo, X., Oldham, M. J., Kleinman, M.T., Phalen, R.F. & Kassab, G.S. (2006). Effects of cigarette smoking on nitric oxide, strucutral, and mechanical properties of mouse arteries. American Journal of Physiology: Heart and Circulatory Physiology, 291, H2354-H2361. Dye, W.W., Gleason, R.L., Wilson, E., & Humphrey, J.D. (2007). Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy. Journal of Applied Physiology, 103, Fung, Y.C., & Liu, S.Q. (1989). Change of residual strains in arteries due to hypertrophy caused by aortic constriction. Circulation Research, 65, Chung, A.W.Y., Au Yeung, K., Sandor, G.G.S., Judge, D.P., Dietz, H.C., & van Breemen, C. (2007). Loss of elastic fiber integrity and reduction of vascular smooth muscle contraction resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfan Syndrome. Circulation Research, 101, Guo, X., Lanir, Y., & Kassab, G.S. (2007). Effect of osmolarity on the zero-stress state and mechanical properties of aorta. American Journal of Physiology: Heart and Circulatory Physiology, 293, H2328-H2334. Mercier, N., Osborne-Pellegrin, M., El Hadri, K., Kakou, A., Labat, C., Loufrani, L., Henrion, D., Challande, P., Jalkanen, S., Feve, B., & Lacolley, P. (2006). Carotid arterial stiffness, elastic fibre network and vasoreactivity in semicarbazide-sensitive amine-oxidase null mouse. Cardiovascular Research, 72(2), Gleason, R.L., Dye, W.W., Wilson, E., & Humphrey, J.D. (2008). Quantification of the mechanical behavior of carotid arteries from wild- type, dystrophin-deficient, and sarcoglycan-delta knockout mice. Journal of Biomechanics, 41,