1. 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

2. 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)

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

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. 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

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

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