The feasibility of using stored mouse blood vessels for mechanical testing
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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

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


What are blood vessel mechanics

Radial cut Mechanical Testing

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


Why blood vessel mechanics
Why Blood Vessel Mechanics? Mechanical Testing

  • 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
Why Storage? Mechanical Testing

  • 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
Study Overview Mechanical Testing

  • 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

Vessel Bath Mechanical Testing

Translation

Stage

Microscope

Force

Transducer

Pressure

Transducer

D

Pressure

Controlled

Pump

L

Desktop

Computer

Stretch

z

d

Inflate

Mechanical Testing

  • Setup

  • Preconditioning


Mechanical testing ctd
Mechanical Testing Mechanical TestingCtd.

  • 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


Dimensions
Dimensions Mechanical Testing

  • 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
Data Analysis Mechanical Testing

  • 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
Dimensions: Left Carotid Diameters Mechanical Testing

  • No significant difference between time points

  • But there is a slight trend to each…


Left carotid thickness
Left Carotid Thickness Mechanical Testing

  • 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
Ascending Aorta Diameters Mechanical Testing

  • 1 day OD is significantly different from 7; 1 day ID is significantly different from 3, 7, and 28 days


Ascending aorta thickness
Ascending Aorta Thickness Mechanical Testing

  • This time, changes in diameter actually cancel each other out

  • 1 day is only significantly different from 28


Pressure diameter
Pressure-Diameter Mechanical Testing

  • At all pressures except 25, no difference between dates

  • At 25, only 7 day is different from 1 day


Pressure compliance
Pressure-Compliance Mechanical Testing

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


Pressure force
Pressure-Force Mechanical Testing

  • 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
Pressure- Circumferential Stretch Ratio Mechanical Testing

  • No significant differences between time points

  • Circumferential stretch ratio changes less the longer the vessels are stored


Pressure circumferential stress
Pressure-Circumferential Stress Mechanical Testing

  • 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
Pressure- Axial Stress Mechanical Testing

  • Significant difference between 1 day and 14 day for P0-P100

  • Still lacks a clear pattern


Opening angle
Opening Angle Mechanical Testing

  • No significant difference between vessels


Discussion
Discussion Mechanical Testing

  • 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
Vessel Degradation? Mechanical Testing

  • 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
Other Explanations: Dimensions Mechanical Testing

  • 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

Vessel Bath Mechanical Testing

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


Future work improvements
Future Work/ Improvements Mechanical Testing

  • 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
Acknowledgements Mechanical Testing

  • NSF

  • SLU BME

  • Dr. Wagenseil

  • Victoria Le

  • Dr. Willits

  • Neva Gillan


References
References Mechanical Testing

  • 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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