ben ari et al. supplementary information · ben-ari et al. – supplementary information page 3...

BEN ARI ET AL. – SUPPLEMENTARY INFORMATION

S1. Supplementary Experimental Methods

S1.1. BCAS Microsurgery

On the day of the first surgery, the animals were brought into an SPF environment in

the Center for Cardiac Research, Hadassah Medical Center and entered into an animal

operation room. Each animal was first administered analgesia with Rimadyl 5 mg/kg

i.p. (0.25 ml for 25 g animal body weight) and then anesthetized using a mixture of

ketamine and xylazine (ketamine 70 mg per kg animal body weight, xylazine 10 mg per

kg animal body weight, mixture administered 0.25 ml per 25 g animal body weight).

After the animals were anesthetized, the upper chest and neck area were shaved and

disinfected with chlorhexidine solution and then a longitudinal section was made with

a surgical blade at the right side of the neck, and the underlying fascia was separated,

exposing the muscles below. The sternocleidomastoid muscle was deflected, and the

common carotid artery was exposed. Two Ethicon sutures 4-0 were inserted below

the exposed part of the artery and the specialized constricting coil was wrapped

around the part of the artery between the two sutures. The coil is made of gold plated

piano spring stainless steel, with 0.18 mm internal diameter and is 2.5 mm total length.

After the coil implantation, the two silk sutures were removed, and the cut was

sutured. Sham operated animals underwent the same procedure but no coil was

implanted. Following implantation, operated animals were given another i.p. injection

of Rimadyl, and were monitored until they regained consciousness. After that the

animals were returned to their home cages and were followed up for the next 3 days

after surgery.

Fifteen out of 17 sham operated animals survived the protocol (two were taken out of

the experiment due to aggressive behavior). In contrast, 6 out of 21 BCAS mice died

throughout the experiment (28.5% mortality rate). Three of the deaths were within

48 hours of surgery; the rest were later in the experiment.

Ben-Ari et al. – Supplementary Information

In the current study, both groups gained weight during the experimental protocol. This

result is consistent with our previous finding that young adult female mice did not lose

weight during the experiment and with other results in which young adult male mice

did not lose weight after BCAS (Shibata et al., 2007).

S1.2. Behavioral Tests

S1.2.1. Sucrose preference test (SPT): SPT was conducted twice: first as a baseline

experiment, prior to any manipulation, and second as part of the behavioral-cognitive

battery of tests used to assess the effects of BCAS microsurgery. The first conduction

required introduction of sucrose solution prior to the actual testing, hence was

consisted of 2 days habituation through which mice were given free access to water

and 2% sucrose solution, but the amounts drunk were not measured, followed by 2

days of test, through which the amounts of water and sucrose solution consumed per

cage were measured every 24 hours. In the second conduction mice were given free

access to water and 2% sucrose solution for 24 hours and the amounts of water and

sucrose solution consumed per cage were measured. Sucrose preference was

calculated as the ratio of sucrose consumed divided by the total liquid consumption

(water and sucrose).

S1.2.2. Open field test (OFT): Mice were placed in a 50 X 50 cm arena surrounded by

40 cm walls for a 6 min test. The center of the arena was defined as a 25 X 25 square

in the middle of the arena. Velocity of movement and presence in the center of the

arena were measured. Open field (OF) test was performed during the first week of the

behavioral battery, using Ethovision 11 system, providing fully computerized, blinded

and unbiased measurement.

S1.2.3. Elevated plus maze (EPM): The test apparatus consists of two open arms (30*5

cm) bordered by a 1 cm high rim across from each other and perpendicular to two

closed arms bordered by a rim of 16 cm. The center of the maze, in which the 4 arm

converge, is a 5*5 cm platform. The entire maze is lifted 75 cm from the floor. Mice


were placed in the central platform of the maze and were allowed to explore it for 6

minutes. Durations and number of visits in both the open and closed arms were

recorded.

S1.2.4. Rotarod: This is a test of motor abilities, which require mice to balance on a

rotating cylinder. The test was consisted on 3 4-minute trials. During each trial rod

rotation gradually increased up to 40 rotations/minute. The amount of time mice

balanced on the rod in each trail was measured. Trials were divided by at least 20-

minute breaks, to avoid mice exhausting. The experimenters were blind to the

experimental conditions of each mouse.

S1.2.5. Stress induced hyperthermia (SIH): This test is conducted using a single probe

thermometer connected to a specialized rectal probe. Upon insertion, basal rectal

temperature was measured. In order to test the effect of acute stress on rectal

temperature the probe was inserted again 10 minutes later, and rectal temperature

was measured.

S2. Supplementary Results 1

Fig. S.1. Effect of BCAS microsurgery on flood flow change. Top Panel: External carotid blood 2

flow was recorded at a sample rate of 40/s with a laser Doppler flowmeter. Flux was measure 3

before and after coil placement. Top. Rostral flux measurement before BCAS (left) and after 4

BCAS (right) shows decrease in blood flow. Middle. Caudal flux measurement before (left) and 5

after BCAS (right), no change in blood flow. Bottom. Ratio between rostral to caudal flux in 6

arbitrary units (A.U.). B. Flow change (A.U.) ratio of rostral/caudal measurement. Bottom 7

panel: External carotid blood flow was recorded during sham operation after artery exposure. 8


Fig. S.2. Effect of BCAS microsurgery on body weight in young adult, male mice. 9

Change in body weight from baseline to the end of the 8th week following BCAS is 10

shown (t (28) =1.505, p = 0.14). 11

12

13

Fig. S.3. Effect of BCAS microsurgery on sucrose preference of young adult mice. Left: 14

Sucrose preference at baseline and after 8 weeks, F(1,11) = 0.81, p = 0.38. Right: 15

Change in sucrose preference from baseline to the end of the 8th week following BCAS 16

microsurgery, t (12) =-1.308, p = 0.21. (n=number of cages, 2-3 mice in each cage).17

0

1

2

3

4

5

Weig

ht

gain

(g

)

Control (n=15) BCAS (n=15)

-0.45

-0.3

-0.15

0

0.15

0.3

Su

cro

se p

refe

ren

ce d

iffe

ren

ce (

ml)


18

19

Fig. S.4. Effect of BCAS microsurgery on activity in the open field (OF). 20

Left: Velocity of young adult, sham and BCAS mice: t (29) =1.026, p = 0.31. 21

Right: Effect of BCAS microsurgery on time spent in center: t (29) =-0.212, p = 0.83. 22

23

Fig. S.5. 24

Left: Effect of BCAS microsurgery on time spent in the open arms of the EPM: t (29) 25

=1.107, p = 0.31. 26

Right: Effect of BCAS microsurgery on number of entries to open arms: t (29) =-0.095, 27

p = 0.92. 28

29

0

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10

15

20

25

Tim

e in

cen

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(sec)


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/s)


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a b


30

31

Fig. S.6. Effect of BCAS microsurgery on balance, grip strength and motor coordination 32

in the rotarod test: t (29) =-0.22, p = 0.82. 33

34

35

36

37

Fig. S.7. Effect of BCAS microsurgery on stress induced hyperthermia (SIH): t (28) 0.21, 38

p = 0.83 for comparison of temperature increase. 39

40

41

42

0

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50

75

100

Late

ncy t

o f

all

(sec)


0

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)



43

44

Fig. S.8. Upper panel: Immunofluorescence staining of doublecortin (DCX) in the 45

granule cell layer of the dentate gyrus of hippocampi of control (A) and BCAS operated 46

(B) young adult male mice (X20 magnification). Lower panel: DCX cell quantification 47

in BCAS versus control mice: t[1, 26] =1.71, p=0.099. 48

49

50


Supplementary Table S1. Correlations between cognitive behavioral test (RAWM) and DTI 51

Variable Correlation 1 2 3 4 5 6 7

1 Number of errors Pearson Correlation 1 .878** -.868** .701* .356 -.641 .051

Sig. (2-tailed) .000 .002 .035 .347 .063 .896

N 30 30 9 9 9 9 9

2 Duration of trial

(seconds) Pearson Correlation .878** 1 -.756* .433 .485 -.698* -.095

Sig. (2-tailed) .000 .018 .244 .186 .036 .809

N 30 30 9 9 9 9 9

3 fractional Anisotropy (Corpus callosum)

Pearson Correlation -.868** -.756* 1 -.781** -.559 .621 .422

Sig. (2-tailed) .002 .018 .008 .093 .055 .225

N 9 9 10 10 10 10 10

4 Mean Diffusivity (internal

capsule right side-mm2/s)

Pearson Correlation .701* .433 -.781** 1 .523 -.642* -.181

Sig. (2-tailed) .035 .244 .008 .121 .045 .618

N 9 9 10 10 10 10 10

5 Mean Diffusivity (internal capsule left side-mm2/s)

Pearson Correlation .356 .485 -.559 .523 1 -.639* -.220

Sig. (2-tailed) .347 .186 .093 .121 .047 .542

N 9 9 10 10 10 10 10

6 Fractional Anisotropy

(anterior commissure left side)

Pearson Correlation -.641 -.698* .621 -.642* -.639* 1 -.183

Sig. (2-tailed) .063 .036 .055 .045 .047 .613

N 9 9 10 10 10 10 10

7 Fractional Anisotropy (anterior commissure

right side) Pearson Correlation .051 -.095 .422 -.181 -.220 -.183 1

Sig. (2-tailed) .896 .809 .225 .618 .542 .613

N 9 9 10 10 10 10 10


Supplementary Table S.2. Correlations between cognitive behavioral test (RAWM) and FA of corpus callosum (controlled for treatment). 52

Correlations

Control Variables Number of

errors

Fractional Anisotropy

(Corpus callosum)

Treatment group - code Number of errors Correlation 1.000 -.850

Significance (2-tailed)

.007

df 0 6

Bootstrapa Bias 0.000 -.006

Std. Error 0.000 .076

BCa 99.9% Confidence Interval

Lower -.994

Upper -.537

fractional Anisotropy (Corpus callosum)

Correlation -.850 1.000

Significance (2-tailed)

.007

df 6 0

Bootstrapa Bias -.006 0.000

Std. Error .076 0.000

BCa 99.9% Confidence Interval

Lower -.994

Upper -.537

53

54


Supplementary Table S.3. Total, direct, and indirect effects 55

Model = 4

Y = RAWM_Err

X = Treat_co

M = DTI_FA_c

Sample size

9

**************************************************************************

Outcome: DTI_FA_c

Model Summary

R R-sq MSE F df1 df2 p

.81743829 .66820536 .00090242 14.09738727 1.00000000 7.00000000 .00712463

Model

coeff se t p LLCI ULCI

constant .30490000 .01343446 22.69537232 .00000008 .25775365 .35204635

Treat_co -.07566250 .02015169 -3.75464876 .00712463 -.14638202 -.00494298

**************************************************************************

Outcome: RAWM_Err

Model Summary



.90152983 .81 275603 1.53 821772 13.021 87755 2.0 0000000 6.00 000000 .006 56483

Model


constant 20.21638997 4.790087 4.220464 0.005558 2.383678 38.0491

DTI_FA_c -61.71331573 15.60468 -3.9548 0.007496 -119.807 -3.61965

Treat_co -1.98588375 1.444377 -1.37491 0.218294 -7.36306 3.391295

************************** TOTAL EFFECT MODEL ****************************

Outcome: RAWM_Err

Model Summary


.56978989 .32466051 4.75538100 3.36515728 1.00000000 7.00000000 .10923254

Model


constant 1.40000000 .97523136 1.43555679 .19426626 -2.02243795 4.82243795

Treat_co 2.68350000 1.46284704 1.83443650 .10923254 -2.45015693 7.81715693

***************** TOTAL, DIRECT, AND INDIRECT EFFECTS ********************

Total effect of X on Y

Effect SE t p LLCI ULCI


2.68350000 1.46284704 1.83443650 .10923254 -2.45015693 7.81715693

Direct effect of X on Y


-1.98588375 1.44437672 -1.37490706 .21829438 -7.36306277 3.39129527

Indirect effect of X on Y

Effect Boot SE BootLLCI BootULCI

DTI_FA_c 4.66938375 2.00023219 1.55306275 13.18463702

Partially standardized indirect effect of X on Y


DTI_FA_c 1.88115228 .61864593 .73006127 3.75241648

Completely standardized indirect effect of X on Y


DTI_FA_c .99145431 .32226103 .42541143 2.12973660

Ratio of indirect to total effect of X on Y


DTI_FA_c 1.74003494 12204561946981 -35.79705821 17.92975946

Ratio of indirect to direct effect of X on Y


DTI_FA_c -2.35128756 132.29639527 -1114.82770184 7.85603324


56 57 58

R-squared mediation effect size (R-sq_med)


DTI_FA_c .26566713 .31278849 -.51421867 .89557600

Normal theory tests for indirect effect

Effect se Z p

4.66938375 1.74342205 2.67828650 .00739999


Supplementary Table S.4. 59

Correlations

Iba_mm3_CC fa_cc_NEW

Spearman's rho Iba_mm3_CC Correlation Coefficient 1.000 -.810*

Sig. (2-tailed) .015

N 8 8

Bootstrapc Bias 0.000 -.023

Std. Error 0.000 .110

BCa 99% Confidence Interval

Lower -1.000

Upper -.556

fa_cc_NEW Correlation Coefficient -.810* 1.000

Sig. (2-tailed) .015

N 8 8

Bootstrapc Bias -.023 0.000

Std. Error .110 0.000


Lower -1.000

Upper -.556

*. Correlation is significant at the 0.05 level (2-tailed).

**. Correlation is significant at the 0.01 level (2-tailed).

c. Unless otherwise noted, bootstrap results are based on 1000 stratified bootstrap samples

60 61



Correlations

Control Variables Iba_mm3_CC fa_cc_NEW

Treat Iba_mm3_CC Correlation 1.000 -.666

Significance (2-tailed) .103

df 0 5

Bootstrapa Bias 0.000 .070

Std. Error 0.000 .323


Lower -.907

Upper -.142

fa_cc_NEW Correlation -.666 1.000

Significance (2-tailed) .103

df 5 0

Bootstrapa Bias .070 0.000

Std. Error .323 0.000


Lower -.907

Upper -.142

a. Unless otherwise noted, bootstrap results are based on 1000 stratified bootstrap samples

63 64 65



Run MATRIX procedure:

************* PROCESS Procedure for SPSS Release 2.16.1 ******************

Written by Andrew F. Hayes, Ph.D. www.afhayes.com

Documentation available in Hayes (2013). www.guilford.com/p/hayes3

**************************************************************************

Model = 4

Y = fa_cc_NE

X = Treat

M = Iba_mm3_

Sample size

8

**************************************************************************

Outcome: Iba_mm3_

Model Summary


.89780832 .80605978 3044467.542672 24.93736839 1.00000000 6.00000000 .00246770

Model


constant -2253.79965233 1857.52430627 -1.21333521 .27059542 -5862.88161203 1355.28230736

Treat 6363.26996234 1274.25116157 4.99373291 .00246770 3887.46016333 8839.07976134

**************************************************************************


Outcome: fa_cc_NE

Model Summary


.91214693 .83201202 .00067049 12.38201696 2.00000000 5.00000000 .01156633

Model


constant .36389521 .03076251 11.82917967 .00007600 .30192005 .42587037

Iba_mm3_ -.00001208 .00000606 -1.99381888 .10274747 -.00002429 .00000013

Treat -.00935484 .04293982 -.21785935 .83615067 -.09586282 .07715314

************************** TOTAL EFFECT MODEL ****************************

Outcome: fa_cc_NE

Model Summary


.83573383 .69845103 .00100297 13.89726587 1.00000000 6.00000000 .00976080

Model


constant .39112000 .03371497 11.60078082 .00002470 .32561340 .45662660

Treat -.08622000 .02312828 -3.72790368 .00976080 -.13115716 -.04128284




-.08622000 .02312828 -3.72790368 .00976080 -.13115716 -.04128284


Direct effect of X on

Y


-.00935484 .04293982 -.21785935 .83615067 -.09586282 .07715314

-0.09068

Indirect effect of X on Y


Iba_mm3_ -.07686516 .10193588 -.14164040 -.01414878



Iba_mm3_ -1.43958687 1.80956709 -2.36521383 .38686201



Iba_mm3_ -.74505699 .91498992 -1.33783264 -.05718512



Iba_mm3_ .89150034 1.04464593 -.00147737 1.76083763

Ratio of indirect to direct effect of X on Y


Iba_mm3_ 8.21661865 71.36725494 3.91419582 2291.84511358

R-squared mediation effect size (R-sq_med)


Iba_mm3_ .69685640 .15718017 .32030704 .87182861

******************** ANALYSIS NOTES AND WARNINGS *************************


Number of bootstrap samples for bias corrected bootstrap confidence intervals:

5000

Level of confidence for all confidence intervals in output:

90.00

NOTE: Some cases were deleted due to missing data. The number of such cases was:

23

NOTE: Kappa-squared is disabled from output as of version

2.16.

NOTE: Some bootstrap samples had to be replaced. The number of such replacements was:

210

------ END MATRIX ----

- 67



Run MATRIX procedure:

************* PROCESS Procedure for SPSS Release 2.16.1 ******************

Written by Andrew F. Hayes, Ph.D. www.afhayes.com

Documentation available in Hayes (2013). www.guilford.com/p/hayes3

**************************************************************************

Model = 6

Y = rawm_err

X = Treat

M1 = Iba_mm3_

M2 = fa_cc_NE

Sample size

7

************************************************************************** Outcome:

Iba_mm3_

Model Summary


.86359360 .74579390 3295630.992717 14.66907944 1.00000000 5.00000000 .01224739

Model


coeff se t p LLCI

ULCI constant -1707.80662850 2069.85996882 -.82508317 .44688739 -5877.81489395

2462.20163694 Treat 5817.27693850 1518.86197362 3.83002343 .01224739 2757.32743493

8877.22644207

************************************************************************** Outcome:

fa_cc_NE

Model Summary


.87935157 .77325919 .00083809 6.82064406 2.00000000 4.00000000 .05141140

Model coeff se t p LLCI

ULCI constant .36383268 .03518325 10.34107781 .00049350 .28886352

.43880185 Iba_mm3_ -.00001206 .00000713 -1.69114297 .16607018 -.00002726

.00000314 Treat -.00936966 .04803988 -.19503915 .85486845 -.11173401

.09299469

************************************************************************** Outcome:

rawm_err

Model Summary


.93983255 .88328522 1.70954791 7.56789539 3.00000000 3.00000000 .06527006



ULCI constant 30.01888555 8.36836422 3.58718678 .03709694 10.36019922

49.67757187 Iba_mm3_ -.00064936 .00042181 -1.53946071 .22132259 -.00164026

.00034154 fa_cc_NE -84.96050720 22.58212045 -3.76229094 .03283849 -138.00968273 -

31.91133167 Treat -.04589903 2.17997744 -.02105482 .98452402 -5.16703043

5.07523236

************************** TOTAL EFFECT MODEL **************************** Outcome:

rawm_err

Model Summary


.52893065 .27976763 6.32964450 1.94220392 1.00000000 5.00000000 .22220387


ULCI constant -1.53350000 2.86854281 -.53459199 .61583950 -7.31256110

4.24556110 Treat 2.93350000 2.10493495 1.39362976 .22220387 -1.30717148

7.17417148





2.93350000 2.10493495 1.39362976 .22220387 -1.30717148 7.17417148

Direct effect of X on Y


-.04589903 2.17997744 -.02105482 .98452402 -5.16703043 5.07523236

Indirect effect(s) of X on Y


Total: 2.97939903 2.05628242 -1.01632852 5.29783886

Ind1 : -3.77751010 2.61803294 -7.69532767 .13805706

Ind2 : 5.96085822 3.01138606 .21285908 10.22952428

Ind3 : .79605091 3.12433645 -1.97676864 8.61348040

(C1) -9.73836833 5.07966164 -17.68054573 -2.32519602

(C2) -4.57356102 3.85326350 -12.44526051 1.12338447

(C3) 5.16480731 5.67213919 -8.84873678 11.76242443



Total: 1.10094584 .76734913 -.92706090 1.87651558

Ind1 : -1.39586339 1.07184500 -4.11787693 .06644090

Ind2 : 2.20265295 1.13448897 .06224532 3.59479367

Ind3 : .29415628 1.18977157 -1.21662203 3.04467206



Total: .53720656 .36537294 -.37384660 .92245448


Ind1 : -.68111159 .52806105 -1.70692356 .02511230

Ind2 : 1.07478459 .57295175 .03327152 1.83292402

Ind3 : .14353357 .58492676 -.53649385 1.56923992



Total: 1.01564651 17185483335307 -5.44061287 2.26584128

Ind1 : -1.28771437 4346450313815 -7.18159504 3.75746526

Ind2 : 2.03199530 35481658679690 -10.33064502 7.30426086

Ind3 : .27136558 14033131252172 -1.83930930 4.35120871

Ratio of indirect to direct effect of X on

Y


Total: -64.91202085 15.36182856 -9.64552384 9.64993701

Ind1 : 82.30042765 24.38533742 -21.82739342 14.89478053

Ind2 : -129.86892621 41.19733078 -19.90501864 32.10184765

Ind3 : -17.34352229 5.44680855 -9.13105305 3.82488436

Indirect effect key

Ind1 : Treat -> Iba_mm3_ -> rawm_err

Ind2 : Treat -> Iba_mm3_ -> fa_cc_NE -> rawm_err

Ind3 : Treat -> fa_cc_NE -> rawm_err

Specific indirect effect contrast definitions

(C1) Ind1 minus Ind2




******************** ANALYSIS NOTES AND WARNINGS *************************

Number of bootstrap samples for percentile bootstrap confidence intervals:

20000

Level of confidence for all confidence intervals in output:

90.00

NOTE: Some cases were deleted due to missing data. The number of such cases was:

24

NOTE: Some bootstrap samples had to be replaced. The number of such replacements was:

16788

------ END MATRIX -----

69

70


S3. Supplementary References 71

72

73

74

1. Shibata, M., Ohtani, R., Ihara, M., Tomimoto, H. (2004). White matter lesions 75

and glial activation in a novel mouse model of chronic cerebral hypoperfusion. 76

Stroke. doi: 10.1161/01.STR.0000143725.19053.60 77

78

2. Shibata, M., Yamasaki, N., Miyakawa, T., Kalaria, R.N., Fujita, Y., Ohtani, R., 79

Ihara, M., Takahashi, R., Tomimoto, H. (2007). Selective impairment of working 80

memory in a mouse model of chronic cerebral hypoperfusion. Stroke. doi: 81

10.1161/STROKEAHA.107.490151 82

83

Wolf, G., Lotan, A., Lifschytz, T., Ben-Ari, H., Kreisel Merzel, T., Tatarskyy, P., … 84

Lerer, B. (2017). Differentially severe cognitive effects of compromised 85

cerebral blood flow in aged mice: Association with myelin degradation and 86

microglia activation. Frontiers in Aging Neuroscience, 9, 191. 87

http://doi.org/10.3389/fnagi.2017.00191 88

https://doi.org/10.1161/01.STR.0000143725.19053.60

https://doi.org/10.1161/STROKEAHA.107.490151

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