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The Feasibility of Using Stored Mouse Blood Vessels for Mechanical Testing

The Feasibility of Using Stored Mouse Blood Vessels for Mechanical Testing. Amber Kunkel Advisor: Jessica Wagenseil , D.Sc. Department of Biomedical Engineering Saint Louis University. Radial cut. Opening angle (OA). What Are Blood Vessel Mechanics?.

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The Feasibility of Using Stored Mouse Blood Vessels for Mechanical Testing

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

  2. Radial cut Opening angle (OA) 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

  3. 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

  4. 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

  5. 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

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

  7. Mechanical Testing Ctd. • Inflation protocols • At 1, 1.1, and 1.2x in vivo length • 3 cycles, 0-175 mmHg, steps of 25, 12 sec/step • Stretch protocols • At 50, 100, and 150 mmHg • 3 cycles, 1-1.2x in vivo length

  8. 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

  9. 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)

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

  11. 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

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

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

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

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

  16. 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

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

  18. 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

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

  20. Opening Angle • No significant difference between vessels

  21. 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?

  22. 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

  23. 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)

  24. Vessel Bath Translation Stage Microscope Force Transducer Pressure Transducer Pressure Controlled Pump Desktop Computer 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

  25. 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

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

  27. 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, 1217-1224. • Wagenseil, J. E., Nerurkar, N. L., Knutsen, R. H., Okamoto, R.J., Li, D. Y., Mecham, R.P. (2005). Effects of elastinhaploinsufficiency 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, 1610-1619. • 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, 664-672. • Fung, Y.C., & Liu, S.Q. (1989). Change of residual strains in arteries due to hypertrophy caused by aortic constriction. Circulation Research, 65, 1340-1349. • 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, 512-522. • 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), 349-357. • 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, 3213-3218.

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