Slide 1Saint 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
Radial cut
Mechanics of mouse aorta and carotid used to study:
Elastin +/- mice and Supravalvular Aortic Stenosis (Wagenseil lab)
Smoking
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
Study Overview
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
3 cycles, 0-175 mmHg, steps of 25, 12 sec/step
Stretch protocols
Dimensions
Image under microscope
Ascending aortas cut radially, used for opening angle measurements
Data Analysis
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
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 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
ASC Opening Angle v. Days Stored
168.6733333333333 133.1359 101.9362333333333 136.8606666666667 146.5883 131.4934666666666 146.0199666666667 150.0785333333333 122.6741333333333 129.1080666666667 120.7046333333333 130.5290666666666 149.8543 153.7153666666667 162.1733666666667 121.6126 142.1938333333333 144.3939333333333 124.9123666666667 113.0233666666667 119.9171666666667 137.4893 150.0012666666667 117.4649 120.8451666666667 146.6872333333333 126.6370333333333 88.7879 100.1814666666667 104.7001333333333
Days Stored
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
Lighting, angle, and height of rings can also influence readings
1 day (6/9 lcc1)
28 day (7/6 lcc1)
Other Explanations: Mechanical Testing
More could be explained by abnormal myograph functions
Outflow pressure often lagging
Vessel Bath
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
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 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, 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.
LCC Outer Diameter v. Days Stored
340
360
380
400
420
440
460
480
0
5
10
15
20
25
30
200
220
240
260
280
300
320
340
0
5
10
15
20
25
30
y = -1.1192x + 80.146
82.2703465732
70.9769275701
88.4326323988
88.2518496885
81.0796339564
79.3143496885
81.6237344237
80.2806658879
72.2630451713
72.4480140187
77.3065615265
76.4631035826
83.4377920561
62.0133372274
67.906055296
62.0602609034
55.0263823988
58.644665109
77.3759735202
77.1770833333
72.2093068536
65.2406542056
71.738123053
59.7335475078
48.6127336449
44.5149922118
53.3370327103
43.6554711838
50.9013823988
44.1852609034
Sheet1
3
443.1074766355
329.5560747664
362.6168224299
250.8177570093
386.3317757009
306.7172897196
39.8072429907
od
id
thick
od
id
thick
od
id
thick
date tested
lcc thickness
order tested
Days stored
LCC thickness
lcc thickness by order tested (no frozen)
asc thick by age
lcc id by age no frozen
lcc id by order inc frozen (similar trend w/o)
asc od by order
asc od by storage
asc id by order, no frozen (same trend inc frozen)
try this one
-4.03E+00
-4.03E+00
6.64E+02
6.61E+02
-2.79E+00
6.72E+02
1.53E+00
8.60E-01
sample size is too small for iqr to really work…
1.66E+00
14
-2.5
14
-0.5
14
2.5
14
-1
14
n/a
7
3
0.6
7
2.5
7
-2
7
1
7
-1.5
7
n/a
28
1
-0.4
28
0.5
28
-2
28
-2.5
28
1
Stored
800
850
900
950
1000
1050
1100
1150
1200
0
5
10
15
20
25
30
Stored
500
550
600
650
700
750
800
850
900
950
0
10
20
30
80
90
100
110
120
130
140
150
160
170
180
0
5
10
15
20
25
30
6/3/09
2
25
JW
0
6
6
4.5
4
1.33
1.50
6/3/09
3
24.3
JW
0
6
6
4
4
1.50
1.50
Good pressure reading, didn't leave alone very long before trying to get in LV
6/4/09
1
23.2
JW
0
6
6
4.5
4.5
1.33
1.33
6/10/09
6/11/09
1
2
22.8
VL
0
6
6
4.5
4
1.33
1.50
6/11/09
2
23.1
VL
0
6
6
4.4
4
1.36
1.50
6/12/09
1
24.6
JW
0
5.5
5.5
3.77
3.56
1.46
1.54
6/15/09
2
27.9
VL
0
6
6
3.84
3.3
1.56
1.82
6/15/09
3
24.7
VL
0
6
6
3.67
4.23
1.63
1.42
6/16/09
1
27.9
VL
0
6
6
4.21
3.8
1.43
1.58
6/16/09
2
28.3
VL
0
6
6
4.05
3.95
1.48
1.52
6/16/09
3
24.5
VL
0
6
6
3.89
3.68
1.54
1.63
6/22/09
1
26.9
VL
0
5.5
6
3.54
4.09
1.55
1.47
6/22/09
2
27.1
VL
0
6
6
4.05
3.81
1.48
1.57
Practice- introduced catheter, but could not advance. Deduced RCC in vivo
6/22/09
3
29.5
VL
0
6
3.5
4.08
2.11
1.47
1.66
6/23/09
1
33.6
VL
97
75
82
586
6
3.5
4.05
2.14
1.48
1.64
6/23/09
2
29.9
VL
112
83
92
489
6
6
4.46
4.06
1.35
1.48
6/24/09
3
28.3
VL
100
68
79
423
6
6
4.05
4.13
1.48
1.45
7/6/09
7/6/09
7/6/09
7/7/09
7/7/09
7/8/09
-1
405
745
Ran 2 additional cycles b/c having trouble tracking diameter, tubing interfering w/ length
3
8.76
1.35
5
7.3-8.76
100
5
550
650
6
7.3-8.76
150
7
650
750
6/8/09
6/9/09
1
2
LCC
1
5.8
7.18
1.24
** Fixed tubing, but MAX STRETCH POSSIBLE = 8.5 mm
** Make sure vessel starts short enough to make this work - Test micro ~ 7.1, unl = ~5.45
6/8/09
6/9/09
1
1
LCC
1
5.2
6.05
1.16
5
100
6
150
6/9/09
6/12/09
3
1
LCC
1
5.35
6.08
1.14
2
6.77
1.26
-2
400
610
for all 6/25 vessels: set on no flow because outflow pressure could not go above 150. Target outflow pressure may stay 0 for all protocols.
2
6.82
1.25
6/11/09
6/25/09
14
2
LCC
1
5.45
6.20
1.14
-2.5
350
610
no flow method may make force change stepwise instead of smooth b/c of pressure directly on the force transducer
2
7.32
1.33
outflow lagging for this vessel but still reaching pressures eventually
2
6.16
1.26
5
100
6
150
6/12/09
6/26/09
14
2
LCC
1
5.5
5.70
1.04
2
6.27
1.14
2
n/a
n/a
n/a
n/a
n/a
4
50
5
100
6
150
Had a leak, maybe from us trying to increase the outflow pressure
6/15/09
6/29/09
2
6.49
1.1589285714
6
325
585
4
50
5
100
6
150
don't use 1st cycle- had trouble setting focus. Stopped too soon going down from 3rd cycle
6/16/09
6/30/09
-3
420
700
closed off left side b/c outflow was lagging, not reaching 175. Also forgot to open new log file- added to end of 1st vessel
2
6.49
1.1589285714
0 - 175
has a hole- there originally or I could have blown it while trying to fix pressure
3
7.08
1.2642857143
2
6.05
1.1747572816
manually closed outflow valve and it seemed to perform better
3
6.6
1.2815533981
6
290
460
4
50
Stopped to move a piece of suture. pressures were acting funky so I turned off no flow. Rechecked for hole after testing- there was one. Not sure when it appeared...
5
100
*also having a problem w/ sutures breaking when I tighten them… how do I avoid that?
6
150
6/23/09
6/30/09
7
4
50
5
100
6
150
6/23/09
6/30/09
7
-2
385
outflow lagging 5 below regardless of flow. Doesn't appear to be a leak…
2
7.095
1.3911764706
-3.5
430
changed 150 to 155 and 175 to 185 on sequence because neither inflow nor outflow performing
2
6.82
1.1559322034
5
100
6
150
set target to 155 for 1st, 160 for rest; pressures jumping around a lot
6/24/09
7/1/09
7
2
LCC
1
5.6
5.9
1.0535714286
-2
410
640
same problem as 1st vessel- pressures not going all the way up, so changed sequence
2
6.49
1.1589285714
1
365
600
I think it might be slightly twisted.. Doesn't seem to be a problem though
3
7.08
1.2642857143
4.5
320
575
forgot to make a new file- added to end of 2nd one
4
50
5
100
6
150
6/24/09
7/1/09
7
3
LCC
1
5.6
6.6
1.1785714286
-3.5
400
710
150 changed to 155, 175 to 185 but still jumping around, having troubling reaching 175
2
7.26
1.2964285714
3
7.92
1.4142857143
3
360
680
4
50
5
100
6
150
set target to 160, mean should be around 150 but pressure chancing a lot
6/4/09
7/2/09
28
1
LCC
1
5.25
5.5
1.0476190476
0-175
-3
400
620
*forgot to note unloaded length before testing… I think it was around 5.25 though
2
6.05
1.1523809524
0-175
1
360
600
3
6.6
1.2571428571
0-175
5
320
540
4
50
5
100
had trouble getting the focus right…think it's ok at the end
6
150
6/4/09
7/2/09
28
2
LCC
1
5.35
5.75
1.0747663551
0-175
-3
425
660
trouble w/ focus on 1st 2 cycle, last should be fine
2
6.325
1.1822429907
0-175
0.5
380
650
3
6.9
1.2897196262
0-175
5
340
600
4
50
had to move pretty close to cannula to avoid fat
5
100
6
150
6/4/09
7/2/09
28
3
LCC
1
0-175
suture not tight enough, vessel slipped off and got a hole
2
0-175
3
0-175
4
50
5
100
6
150
6/5/05
7/6/05
31
1
LCC
1
4.15
4.85
1.1686746988
0-175
-3
410
690
2
5.335
1.2855421687
0-175
-2
380
680
brief stop after 1st cycle because of IP address error
3
5.82
1.4024096386
0-175
1
360
660
started leaking on 3rd cycle. Pulled sutures on further. New length = 5.18
4
4.32-5.18
50
5
100
6
150
6/5/05
7/6/05
31
2
LCC
1
4
4.4
1.1
0-175
-3.5
365
630
2
4.84
1.21
0-175
-2.5
340
610
3
5.28
1.32
0-175
-0.5
320
600
4
50
5
100
6
150
6/5/05
7/6/05
31
3
LCC
1
4.25
4.5
1.0588235294
0-175
-2.5
360
620
2
4.95
1.1647058824
0-175
1
330
560
3
5.4
1.2705882353
0-175
4
290
485
4
50
5
100
6
150
comp_summ
* highlighted vessels have at least 2 points > 3SD from mean and are not included in means or statistics for any of the data
Pressure
Pressure
Force
Compliance (%)
4.16E+02
4.42E+02
4.90E+02
5.46E+02
5.94E+02
6.21E+02
6.32E+02
6.37E+02
2.21E+02
8.47E-01
3.52E-01
-1.09E-01
-4.77E-01
-6.79E-01
*these are all around the same (-3) as they should be
1.1470801102
1.1008119708
1.0042174372
0.9513050977
0.9851571998
1.1103598863
1.3226197573
1.4263296904
0-25
25-50
50-75
75-100
100-125
125-150
150-175
Step
1
2
3
4
5
6
7
Step
1
2
3
4
5
6
7
frozen
-1.23E-01
-3.27E-01
-4.27E-01
-5.40E-01
-6.20E-01
-4.17E-01
-3.83E-01
what is happening to the 1day b/w 75 and 100? Is that normal?
Avg Diam Change
-0.9871079709
3.3259043639
3.0058900817
3.2841686456
3.9276408372
4.5645420807
4.7150823893
4.5879518541
5.2139761954
5.7491278177
5.1238315807
3.8695582585
3.2480270256
2.9797415195
2.5068934894
2.1328854794
7day
1.03E-01
1.61E-01
2.29E-01
2.23E-01
1.08E-01
2.79E-02
1.46E-02
14day
1.00E-01
1.50E-01
1.80E-01
1.80E-01
1.31E-01
5.76E-02
3.62E-02
28day
1.25E-01
1.39E-01
1.80E-01
1.67E-01
9.49E-02
5.59E-02
3.31E-02
1
2
3
4
5
6
7
1
2
3
4
5
6
7
frozen
8.88E-02
1.46E-01
1.86E-01
1.67E-01
9.83E-02
3.71E-02
2.00E-02
0-25
25-50
50-75
75-100
100-125
125-150
150-175
0-25
25-50
50-75
75-100
100-125
125-150
150-175
20090609
1
lcc1
1.165
1
lcc2
1.14
I think?? This is one that didn't stretch all the way…
1
lcc3
1.14
20090611
1
lcc1
1.08
1
lcc2
1.11
1
lcc3
1.175
1.75E+02
4.49E+01
just out of curiosity… but I don't think these are very useful
7 day
circ stress stdev
1day 20090609 lcc1
1day 20090609 lcc2
1day 20090609 lcc3
1day 20090611 lcc1
1day 20090611 lcc2
1day 20090611 lcc3
3day 20090612 lcc1
3day 20090612 lcc2
3day 20090612 lcc3
3day 20090625 lcc4
3day 20090625 lcc5
3day 20090625 lcc6
14day 20090625 lcc1
14day 20090625 lcc2
14day 20090625 lcc3
14day 20090626 lcc1
14day 20090626 lcc2
frozen 20090629 lcc3
frozen 20090630 lcc1
frozen 20090630 lcc3
7day 20090630 lcc4
7day 20090630 lcc5
7day 20090701 lcc1
7day 20090701 lcc2
7day 20090701 lcc3
28day 20090702 lcc1
28day 20090702 lcc2
31day 20090706 lcc1
31day 20090706 lcc2
31day 20090706 lcc3
20.460513179
20.460513179
10.5523151898
10.5523151898
12.5005977896
12.5005977896
4.0711722336
4.0711722336
12.0547088812
12.0547088812
19.8882068246
19.8882068246
0.4514697422
0.4514697422
0.2627341575
0.2627341575
2.0158383085
2.0158383085
0.6386924828
0.6386924828
1.8037335564
1.8037335564
1.7060819486
1.7060819486
1.0531533199
1.0531533199
1.4304530024
1.4304530024
2.555309052
2.555309052
1.5427074461
1.5427074461
2.0821675067
2.0821675067
1.4380029123
1.4380029123
3.3103642797
3.3103642797
2.2002837591
2.2002837591
6.3900224929
6.3900224929
2.6256965189
2.6256965189
4.2555526543
4.2555526543
4.9707348152
4.9707348152
10.6885465963
10.6885465963
5.9833717467
5.9833717467
13.3519029478
13.3519029478
5.0963201128
5.0963201128
5.9593558911
5.9593558911
12.535372904
12.535372904
20.5755279844
20.5755279844
9.8023481332
9.8023481332
26.8618702171
26.8618702171
10.3923084731
10.3923084731
6.4505036153
6.4505036153
21.6955531054
21.6955531054
25.3517541344
25.3517541344
16.8888159071
16.8888159071
38.4572669719
38.4572669719
20.2830908809
20.2830908809
12.0242139903
12.0242139903
31.2112153531
31.2112153531
26.6433392535
26.6433392535
24.624279316
24.624279316
44.3171821735
44.3171821735
24.6968722232
24.6968722232
14.0524077919
14.0524077919
38.4607075067
38.4607075067
29.9646111096
29.9646111096
30.3926971641
30.3926971641
48.5799070011
48.5799070011
31.0506796711
31.0506796711
16.7192829384
16.7192829384
46.8430263204
46.8430263204
0.0898045293
0.0898045293
0.0480937963
0.0480937963
0.0329180316
0.0329180316
0.1055771208
0.1055771208
0.1052689502
0.1052689502
0.0770789027
0.0770789027
0.1055120363
0.1055120363
0.0660470079
0.0660470079
0.0791094083
0.0791094083
0.086193563
0.086193563
0.1000025997
0.1000025997
0.0666770538
0.0666770538
0.1194539466
0.1194539466
0.0668076998
0.0668076998
0.0844709253
0.0844709253
0.0825410676
0.0825410676
0.0942964449
0.0942964449
0.0643623223
0.0643623223
0.1606773212
0.1606773212
0.0941150319
0.0941150319
0.1054538817
0.1054538817
0.0594044974
0.0594044974
0.0961685199
0.0961685199
0.0638336822
0.0638336822
0.2038253743
0.2038253743
0.0816913276
0.0816913276
0.1475653344
0.1475653344
0.0404788656
0.0404788656
0.1292756746
0.1292756746
0.0741874749
0.0741874749
0.1991059341
0.1991059341
0.0780930112
0.0780930112
0.1638499533
0.1638499533
0.0227074857
0.0227074857
0.1755880984
0.1755880984
0.0970794052
0.0970794052
0.1811874768
0.1811874768
0.0897206423
0.0897206423
0.1486404533
0.1486404533
0.0196171926
0.0196171926
0.1833952812
0.1833952812
0.1111438912
0.1111438912
0.177894925
0.177894925
0.0959790347
0.0959790347
0.1413824762
0.1413824762
0.015481387
0.015481387
0.1899333344
0.1899333344
0.1227307543
0.1227307543
0
0
0
0
0
26.4486676767
26.4486676767
11.465401692
11.465401692
7.4753812481
7.4753812481
8.4396778657
8.4396778657
7.6225876471
7.6225876471
0
7.0764491182
7.0764491182
0
0
0
0
0
26.9042262343
26.9042262343
11.2978073511
11.2978073511
8.2401015675
8.2401015675
8.6021464218
8.6021464218
6.3361339932
6.3361339932
0
6.3987104073
6.3987104073
0
0
0
0
0
27.9986530463
27.9986530463
11.0702025637
11.0702025637
9.4341662568
9.4341662568
8.8917917472
8.8917917472
4.9334057302
4.9334057302
0
7.2644248574
7.2644248574
0
0
0
0
0
30.0822210544
30.0822210544
10.7863130022
10.7863130022
10.8492560685
10.8492560685
8.3625395392
8.3625395392
5.4056270459
5.4056270459
0
9.8516480961
9.8516480961
0
0
0
0
0
34.3777787489
34.3777787489
11.5718106674
11.5718106674
13.7201385688
13.7201385688
8.60600555
8.60600555
4.1573415511
4.1573415511
0
12.6261424016
12.6261424016
0
0
0
0
0
38.7653359488
38.7653359488
13.5268826364
13.5268826364
16.4443605741
16.4443605741
9.1555594728
9.1555594728
6.5239021369
6.5239021369
0
14.8574407244
14.8574407244
0
0
0
0
0
41.9065590557
41.9065590557
16.3379169555
16.3379169555
17.6141434187
17.6141434187
10.6711935582
10.6711935582
6.8906705338
6.8906705338
0
18.4812326275
18.4812326275
0
0
0
0
0
45.3872923376
45.3872923376
18.7048037402
18.7048037402
18.7866386411
18.7866386411
12.4991034179
12.4991034179
8.0994957074
8.0994957074
0
22.4471258365
22.4471258365
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.1017497405
0.1017497405
0.11009066
0.11009066
0.1424883672
0.1424883672
0.0939413337
0.0939413337
0.1105360563
0.1105360563
0.0247225492
0.0247225492
1day
3day
7day
14day
28day
frozen
step
1day
3day
7day
14day
28day
frozen
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1day
3day
7day
14day
28day
frozen
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1day
3day
7day
14day
28dat
frozen
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
St dev
18.798218459
24.6105673516
17.2327674387
41.5036532749
33.6776392955
10.1676697422
17.9197938621
6/23/09
6/30/09
7
St dev
0.5
1
1.5
2
2.5
0
50
100
150
200