Supplementary Materials for

Repetitive blast exposure in mice and combat veterans causes persistent

cerebellar dysfunction

James S. Meabon, Bertrand R. Huber, Donna J. Cross, Todd L. Richards,

Satoshi Minoshima, Kathleen F. Pagulayan, Ge Li, Kole D. Meeker, Brian C. Kraemer,

Eric C. Petrie, Murray A. Raskind, Elaine R. Peskind, David G. Cook*

*Corresponding author. E-mail: dgcook@u.washington.edu

Published 13 January 2016, Sci. Transl. Med. 8, 321ra6 (2016)

DOI: 10.1126/scitranslmed.aaa9585

The PDF file includes:

Methods

Tabulated data for Figs. 1B, 2E, 3 (C and D), 4 (A and B), 5 (B and C), 6D, 7 (B,

D, H, and I), and 8.

References (92–101)

www.sciencetranslationalmedicine.org/cgi/content/full/8/321/321ra6/DC1

Supplementary Materials

Supplementary Methods

FDG-PET imaging and analyses

[18F]FDG-PET images of metabolic activity were acquired on a GE Advance scanner (axial resolution 4.25

mm full-width-half-maximum [FWHM] at the center of the field of view) following administration of 7–10 mCi of

[18F]-FDG. Reconstructed PET images (2.25 mm isotropic voxels) were pixel-intensity-normalized to global

brain uptake, spatially normalized to Talairach atlas space(92) and smoothed with a 2.25 mm3 Gaussian kernel

(Neurostat/3D-SSP, University of Washington)(93-95).

Unbiased voxel-wise whole-brain analyses: Whole-brain voxel-wise correlation analyses of associations

between log10-transformed numbers of blast-mTBIs during military service and brain metabolic activity were

performed using NEUROSTAT, as described previously(90). The algorithm performs an r-to-z transform and

the statistical significance of the resultant z-score values was evaluated using random Gaussian fields and a

Euler characteristic algorithm(91) to control for multiple comparisons and maintain a Type I error rate of p<0.05

(corresponding to Z > 4.0).

Volume-of-interest (VOI) analyses: To confirm the findings from of the whole-brain voxel-wise correlations, an

independent VOI analysis was performed on 3D-SSP surface projected image sets where the mean values of

the metabolic activity for predefined right and left cerebellar hemisphere VOIs were calculated and

associations between mean cerebellar VOI values and log10 numbers of blast-mTBIs during military service

were evaluated using Spearman r correlation with a significance level of 0.05, two-tailed.

Diffusion tensor imaging and analyses

Diffusion tensor imaging (DTI) was acquired on a 3.0 T Philips Achieva whole body scanner (Philips

Medical Systems, Best, Netherlands) with a 32-channel radiofrequency head coil. The DTI image acquisition

protocol used a single-shot spin-echo echo-planar imaging sequence with TR=10.76 sec; TE=93.5 msec; flip

angle=80 degrees; matrix size=128•128; field-of-view (FOV)=256•256; slice thickness=2mm; 64 gradient

directions; and b-factors=0 and 3,000s/mm2.

Diffusion tensor image pre-processing: DTI head motion, eddy current, and B0-field inhomogeneity-induced

geometric distortion corrections were performed using the Oxford FMRI Software Library (FSL) DTI toolbox.

DTIPrep was then used to identify and remove image slices with large within-slice intensity differences,

wrapping abnormalities, or other artifacts(97).

DTI tractography analysis: DTI data was analyzed with tractography to test for correlations between DTI

parameters and subject blast-related mTBIs.

Tractography analysis: Diffusion images were converted from Philips style DICOM format to nearly raw raster

data (nrrd) data file/header (nhdr) format (http://teem.sourceforge.net/nrrd/format.html) using custom software

in g-Fortran. Software SLICER 4.3.1 (http://www.slicer.org/) was used to create the fiber tracts in vtk format

using the following steps:

1. Diffusion data were loaded into Slicer in nhdr format.

2. Slicer module: Diffusion/diffusion weighted images/DWI to DTI estimation were used to create the

tensors.

3. Slicer module: Diffusion/diffusion tensor images/diffusion tensor scalar measurements were used to

create a fractional anisotropy image.

4. Slicer module: Editor was used to create a label map and a region of interest located in the cerebellum

near the dentate nucleus based on Figure 8 which shows the location of the region of interest.

5. Slicer module: Diffusion/diffusion tensor images/tractography label map seeding were used to create

the fiber tracts which are connected to the seed region of step 4 using 1mm spacing.

Custom software using g-Fortran was used to read in the fiber tract vtk file generated by Slicer steps 1 – 5

and quantify mean diffusivity, fractional anisotropy, radial diffusivity, and axial diffusivity at 3 different regions

along the fiber tract defined by using the fractional anisotropy threshold of 0.2 in a cerebellar white matter

region near the dentate nucleus. The software generates a quantitative value for each individual for each VOI

for each DTI parameter. For each DTI parameter the average for each subject was calculated among VOIs 1-

3, which was then entered into a correlation statistical analysis with respect to the log10(number of blast-related

mTBIs). Each VOI had a volume of approximately 1cm3 and was positioned manually for each subject

(corresponding approximately to Montreal Neurologic Institute atlas x, y, z coordinates: [18.0, -37.5, -32.5],

[0.0, -15.0, -32.5], and [-20.0, -37.5, -32.5] for VOI1-3, respectively). As a validation of the mean diffusivity

results with tractography, mean diffusivity images were also calculated using FSL diffusion toolbox

(http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FDTP), which uses the tensor model to fit the tensor to calculate mean

diffusivity at each voxel within the brain. No skeletonization was performed.

Modeling blast overpressure

The shock tube design and its use are described in detail elsewhere(9). Briefly, blast exposure and sham

animals were anesthetized with 2% isoflurane delivered with a non-rebreathing anesthesia machine (1Lpm

oxygen). Animals were secured in place with their dorsal aspect against a wire mesh gurney and placed into

the shock tube with the ventral body surface oriented perpendicular to the oncoming blast wave in accordance

with well-established methods(39, 98). Each blast-exposed animal was yoked to a non-blasted sham control

animal that was mounted in the shock tube and held under anesthesia for the identical amount of time as its

paired BOP-exposed mouse. At the conclusion of experiments animals were humanely euthanized via

pentobarbital injection per IACUC approved methods.

In vivo dextran labeling, histopathology, and immunofluorescent microscopy

Dextran permeability studies were performed by injecting 100 µl of 400mg/ml 10kDa dextran labeled

with tetramethylrhodamine (Life Technology, Grand Island, NY) into the retro-orbital sinus after isoflurane

induction immediately before BOP or sham treatment. Dextran-labeled mice were allowed to recover up to 4

hours after blast/sham exposure before perfusion/fixation. Mice used for tissue imaging studies were

euthanized by sodium pentobarbital IP injection and transcardially perfused with phosphate-buffered saline

(PBS) prior to 4% paraformaldehyde perfusion. Brains were placed in 4% paraformaldehyde for 24-72 hr,

ethanol re-equilibrated, and then sectioned for immunofluorescent microscopy or paraffin-embedded for

immunohistochemistry. Immunofluorescence studies were conducted on fixed tissue as previously

described(9). Microglial marker Iba-1 (Wako, Richmond, VA), activated microglial/macrophage marker CD68

(AbD Serotec, Raleigh, NC), astrocyte marker anti-GFAP (Millipore, Billerica, ME), neuronal marker anti-

neurofilament-heavy chain (Aves, Tigard, OR), mouse monoclonal Tau396 (Life Technologies, Grand Island,

NY), rabbit anti-phospho Tau 396 (AnaSpec, Freemont, CA) and IP3R1 (Cell Signaling, Danvers, MA) were

used overnight at 4˚C. IF secondary antibodies (anti-chicken/AF633, anti-mouse/AF488, and anti-rabbit/AF555;

Molecular Probes, Grand Island, NY) were used as appropriate. Laser scanning confocal imaging was

conducted using a Leica TCS SP2 confocal/multiphoton hybrid microscope with tunable emission gating and

using sequential, between stack, single photon excitation at 488, 543, and 633. Z-plane images acquired the

full volume thickness of each slice imaged (typically 50 µm) using system optimized stepping. Within an

experiment, all directly compared sections/slides were identically and simultaneously prepared. GFAP

fluorescence in cerebellar white matter was evaluated by assessing the mean fluorescence intensity of both

the total area of interest (AOI) interior to the granule cell layer (minus the deep cerebellar nuclei) and by

examining the mean intensity of four ROI’s bracketing the deep cerebellar nuclei. Fluorescence intensities from

blast-exposed mice were normalized to sham-control values. For morphological analyses confocal images of

Iba-1 positive microglia, 285μm2 (x, y plane) X 50 m (z-plane) regions of white matter superior to the deep

cerebellar nuclei were imaged and subjected to quantitative analyses and data processing of microglial

morphometrics using Imaris 8.0.2 software (Bitplane, Zurich, Switzerland). Microglial volumes were generated

from filament reconstructions using the convex hull add-on developed in MatLab (MathWorks, Natick, MA).

Empty basket quantification

Cerebellar sections from blast/sham-exposed mice (1X and 3X) were immunostained for neurofilament-

heavy chain and the Purkinje cell specific marker, IP3R1. The distinctive Purkinje cell layer was then examined

in orthogonal viewing mode to identify neurofilament-positive baskets with an absence of IP3R1-positive

Purkinje cell body staining. The frequency of Purkinje cell loss was then calculated as the average number of

empty baskets per linear lobule length. Empty basket frequencies were then normalized to sham control

values.

Western blot analyses

Blast/sham brains were dissected in 4oC PBS. Protein lysates were prepared as previously described(99)

with the following minor modifications: phosphatase inhibitor cocktail sets 2 and 3 (Sigma, St. Louis, MO) were

added (10 µl/ml) to lysis buffer and tissues were homogenized twice by hand using an Eppendorf tube-fitting

pestle (Eppendorf, Hauppauge, NY) before centrifuge clarification. Criterion 4-20% TGX gels (Bio-Rad,

Hercules, CA) were loaded with 20 µg/lane and probed either with antibodies recognizing: APP 22C11 (EMD

Millipore, Billerica, MA); GFAP (Covance, Princeton, NJ); GABABR1 (NeuroMAb, Davis, CA); and Rabbit mAb

PSD-95 (Cell Signaling, Danvers, MA); CD68 (AbD Serotec, Raleigh, NC),and Pyruvate kinase (Rockland,

Gilbertsville, PA). Probes for pyruvate kinase were conducted after stripping. SYPRO Ruby protein blot stain

was done in accord with the manufacturer’s protocol (Life Technologies, Grand Island, NY). Densitometry was

performed with an ImageQuant TL (GE, Piscataway, NJ). Protein levels were standardized by pyruvate kinase

(PK) or Sypro Ruby (SR) loading control values.

Rotarod performance

Mice were tested with an accelerating rotarod (Accuscan; Columbus, OH) using established protocols(100,

101). During three consecutive trials the rotarod was set to accelerate from 0 to 8 rpm, 0 to 16 rpm and 0 to 24

rpm over 120 sec. Speed was held constant at the maximum rpm for 30 sec and decelerated over 50 sec to a

stop. Amount of time mice successfully negotiated the acceleration phase was recorded. Animals staying on

the rotarod longer than the acceleration phase were assigned a latency of 120 sec. Each rotating rod was

cleaned in between trials and any mouse still on the rod at the end of a trial was removed from the rod and

placed at the bottom of the chamber during cleaning. Mice received no training trials and were tested only once

at 24 hrs or 30 days after blast-sham treatments.

Tabular Data

Figure 1B

log10 blast # R Cerebellum L Cerebellum PHQ9 AUDIT-C CAPStotal

FDG uptake

(normalized to global) FDG uptake

(normalized to global)

0.7782 0.84000 0.83000 1 4 5

1.0000 0.83000 0.83000 0 8 30

1.0414 0.89000 0.89000 9 7 52

2.0086 0.86000 0.84000 25 6 88

1.1761 0.83000 0.83000 13 3 80

0.6990 0.85000 0.83000 9 4 61

1.7160 0.75000 0.76000 16 3 73

1.3010 0.85000 0.85000 2 7 63

1.3222 0.79000 0.78000 11 6 78

0.8451 0.85000 0.87000 7 9 66

1.3010 0.78000 0.77000 5 1 *

0.9542 0.78000 0.80000 25 8 100

1.4472 0.88000 0.88000 6 3 80

2.0086 0.81000 0.81000 5 7 62

1.7243 0.82000 0.82000 11 10 106

1.0414 0.85000 0.86000 2 3 13

0.7782 0.86000 0.84000 16 6 77

1.8195 0.83000 0.82000 3 5 12

0.8451 0.80000 0.81000 15 3 45

0.0000 0.86000 0.86000 11 6 48

1.2553 0.72000 0.70000 17 1 78

1.7160 0.82000 0.80000 2 4 23

0.0000 0.98000 0.99000 3 5 4

0.0000 0.88000 0.89000 7 0 58

0.6021 0.86000 0.85000 4 6 21

0.3010 0.81000 0.81000 5 5 33

1.0414 0.79000 0.81000 8 5 44

1.0792 0.81000 0.82000 16 6 104

0.6990 0.90000 0.89000 25 1 78

0.4771 0.86000 0.85000 1 2 8

1.0792 0.87000 0.88000 19 4 *

1.3424 0.87000 0.87000 2 5 *

0.4771 0.89000 0.87000 15 6 78

* denotes missing value

Figure 2E

Lobules

Subjects 1/2 3 4/5 6 7 8 9 10

Mean Dextran Fluorescence (normalized to shams)

blast 1.784314 1.207071 1.059896 0.942424 0.971014 0.928251 1.091691 0.902256

blast 0.664819 1.003453 0.88424 1.078248 1.025436 0.933884 0.771979

blast 1.526938 1.146527 1.218466 1.744668 2.305611 2.015624 1.815775 1.620113

blast 0.630334 0.702065 0.743924 0.814152 0.764508 0.785399 0.823588 0.768745

blast 1.214318 1.693867

blast 2.648391 3.094461 1.649901

blast 1.588571 2.486429 3.1365 2.902 2.003158 2.1266 2.08383 1.953077

sham 1 1 1 1 1 1 1 1

sham 1.102418 1.140046 1.152484 1.137596 0.994812 1.106719 1.099137 1.064079

sham 1.066856 0.976067 0.914949 0.89658 1.005509 1.041618 0.974887 1.032783

sham 0.974427 1.063717 0.990769 1.047762 0.857475 0.966859 1.020264

sham 0.830725 0.90946 0.86885 0.975055 0.951917 0.994187 0.959117 0.882873

sham 1 1 1

sham 1 1 1 1 1 1 1 1

Fixed floating sections for analysis were randomly selected for analysis with missing values

indicating missing or damaged lobes.

Figure 3C

Subjects Dorsal Ventral

Empty baskets

(norm to shams)

Sham 0 0

Sham 0 2.726094

Sham 0 0.836127

Sham 1.87308 3.650296

Sham 0.740447 0

Sham 5.142189 1.489495

Sham 0.162757 0

Sham 0 0

Sham 1.722139 0

Sham 0.359388 1.297988

blast 1X 2.855226 2.523866

blast 1X 0.726867 1.488013

blast 1X 1.213606 10.084789

blast 1X 0 0

blast 1X 0.506935 1.563321

blast 1X 0.392815 3.239688

blast 1X 0.48899 2.234242

blast 3X 4.108697 12.87212

blast 3X 7.435581 10.539592

blast 3X 3.711222 16.518534

blast 3X 0.526246 12.896594

blast 3X 28.804845 18.600983

blast 3X 4.680474 10.249802

blast 3X 13.782526 7.186242

blast 3X 1.943979 5.139382

blast 3X 11.717144 0

blast 3X 3.712494 13.375494

blast 3X 7.974118 20.317155

blast 3X 5.081841 2.489292

Figure 3D

Lobules

Subjects 1/2 3 4/5 6 7 8 9 10

Empty baskets (normalized to shams)

Sham 0 0 0 0 0 0 0 0

Sham 0 0 0 0 4.141844 2.901143 2.23337 0

Sham 0 0 0 0 0 1.886633 0 0

Sham 0 9 0 0 3.858156 5.212224 2.073367 0

Sham 0.400624 0 0 0

Sham 0 0 5.022378 8.858236 0 5.693263 0

Sham 0 0 0 0.352242 0 0 0 0

Sham 0 0 0 0 0 0 0 0

Sham 9 0 3.977622 0 0 0 0 0

Sham 0 0.388898 0 0 0 10

Sham 0 0

blast 1X 19.9332 0 0 1.907428 10.43131 0 0 0

blast 1X 5.58 0 0 0.377243 0 0 2.157075 8.203125

blast 1X 0 0 0 2.626517 10.9732 4.331284 15.09952 19.62617

blast 1X 0 0 0 0 0 0 0

blast 1X 0 0 2.426715 0 0 1.593374 4.368415 0

blast 1X 0 0 0 0.85014 4.235269 3.170322 0

blast 1X 0 0 0 1.058285 0 0 0 17.21312

blast 3X 5.15534 2.124013 10.23354 8.461693 26.64434

blast 3X 62.5778 5.952448 0 0 0 1.410648 37.54476 19.62617

blast 3X 16.55934 2.96648 1.138916 4.43536 5.802287 28.15857 56.61598

blast 3X 0 0 0 1.138916 22.64486 9.462191 0

blast 3X 20.03774 13.31472 31.71017 7.98994 9.814506 38.32155 36.97096

blast 3X 27.44262 0 0 4.248315 30.2438 3.812246 6.333538 0

blast 3X 139.1825 0 0 0 0 1.331261 12.07962 32.55814

blast 3X 0 0 0 4.207211 8.801418 0 5.556624 14.78873

blast 3X 24.54583 11.58446 2.725655 0 0

blast 3X 0 6.273629 5.878042 2.551444 20.26226 11.57338 0

blast 3X 0 0 7.110535 14.04311 39.39096 2.956935 8.764392 59.7561

blast 3X 16.59233 13.15529 0 1.516927 0 0 12.68636 0 Fixed floating sections were randomly selected for analysis with missing values indicating Missing, damaged, folded lobes.

Figure 4A

rpm

subjects 8 16 24

Latency to Fall (sec)

Sham 120 88.4 78.2

Sham 120 97.3 63.1

Sham 120 67.6 36.3

Sham 95.8 79 70.7

Sham 120 120 106.4

Sham 120 114.3 84.2

Sham 88 96.8 67.9

Sham 120 120 52.8

Sham 120 120 102.5

Sham 96.6 72.8 70.8

Sham 105.5 83.2 75.8

Sham 120 120 112.3

Sham 120 72 31.9

Sham 90.7 96.9 70

Sham 120 79.7 78.2

Sham 120 67.3 63.5

Sham 71.9 69 62.6

Sham 52.4 86 48.3

Sham 120 88.4 86.8

Sham 110.2 72.1 84.2

Blast 1X 120 96.1 80.8

Blast 1X 108.9 92.8 41.6

Blast 1X 120 88.2 69.9

Blast 1X 120 101 58.3

Blast 1X 120 118.7 82.2

Blast 1X 120 101.1 63.2

Blast 1X 72.6 80 53.4

Blast 1X 120 114.1 54.6

Blast 1X 120 120 79.9

Blast 1X 120 84.5 69.4

Blast 1X 120 117.2 57.7

Blast 1X 106.9 105.3 58.6

Blast 1X 118.5 71.6 59

Blast 1X 120 89.7 57.1

Blast 1X 120 120 43.9

Blast 1X 115.1 105.5 67.4

Blast 1X 42.3 34.2 30.6

Blast 1X 120 82.4 56.9

Blast 1X 25.4 31.3 65

Figure 4B

rpm

subjects 8 16 24

Latency to Fall (sec)

Sham 109 83.9 45.8

Sham 120 101.5 61.4

Sham 99.8 89.2 75.5

Sham 99 79 62.3

Blast 3X 67 65.4 6.9

Blast 3X 120 80.2 53.9

Blast 3X 47.8 58.4 36.5

Blast 3X 114.9 26.6 35

Blast 3X 46.3 43.7 Missing value: Animal fell prior to start Figure 5B

subjects PSD-95 GABABR1

Optical density

(normalized to shams)

Sham 0.989461 1.070110266

Sham 0.984716 1.038418777

Sham 0.855057 0.989957233

Sham 1.143354 0.697608914

Sham 1.027411 1.203904811

Blast 1X 30d 0.566415 1.551902748

Blast 1X 30d 0.670382 0.998979837

Blast 1X 30d 0.748859 0.606089655

Blast 1X 30d 0.671195 0.902287412

Blast 1X 30d 0.534053 0.943656548

Figure 5C

subjects delay(days) PSD-95

Optical density (normalized to

shams)

Sham 30 1.004544

Sham 30 0.984716

Sham 30 1.027411

Sham 30 1.002476

Sham 30 0.954178

Sham 30 0.953046

Sham 30 1.092776

Sham 1 0.729202

Sham 1 1.483452

Sham 1 0.696398

Sham 1 1.15887

Sham 1 0.932077

Sham 30 1.009166

Sham 30 1.070246

Sham 30 0.989461

Sham 30 1.143354

Sham 30 0.988359

Sham 30 0.92521

Sham 30 0.855057

Blast 1X 1 1.054044

Blast 1X 1 0.983278

Blast 1X 1 1.080724

Blast 1X 1 1.235472

Blast 1X 30 0.671195

Blast 1X 30 0.934973

Blast 1X 30 0.895597

Blast 1X 30 0.670382

Blast 1X 30 0.534053

Blast 1X 30 1.162934

Blast 1X 30 1.086741

Blast 1X 30 0.566415

Blast 1X 30 0.842939

Blast 1X 30 0.736549

Blast 1X 30 0.748859

Blast 3X 30 0.820317

Blast 3X 30 0.870856

Blast 3X 30 0.90662

Blast 3X 30 0.949261

Figure 6D

subjects delay(days) APP

Optical density (normalized to

shams)

Sham 30 1.2097

Sham 30 1.2213

Sham 30 1.0972

Sham 30 0.7848

Sham 30 0.687

Sham 1 1.0565

Sham 1 0.7675

Sham 1 0.8915

Sham 1 1.1911

Sham 1 1.0933

Sham 7 0.9557

Sham 7 0.8422

Sham 7 1.1412

Sham 7 1.0354

Sham 7 1.0255

Sham 30 0.9848

Sham 30 1.0765

Sham 30 0.8975

Sham 30 1.0126

Sham 30 1.0285

Blast 1X 1 1.1696

Blast 1X 1 1.2866

Blast 1X 1 1.2103

Blast 1X 1 1.1324

Blast 1X 30 0.8945

Blast 1X 30 0.9506

Blast 1X 30 1.0514

Blast 1X 30 1.1087

Blast 1X 30 1.087

Blast 1X 7 1.0775

Blast 1X 7 1.2657

Blast 1X 7 1.2479

Blast 1X 7 1.0078

Blast 3X 30 1.3334

Blast 3X 30 1.2663

Blast 3X 30 1.1587

Blast 3X 30 1.1227

Blast 3X 30 1.4224

Figure 7B

subjects GFAP white mater

GFAP Fluorescence (normalized to

shams)

sham 1.11675

sham 0.98462

sham 1.18033

sham 0.94345

sham 0.95379

sham 1.03268

sham 0.8568

sham 0.93159

Blast 1X 1.76382

Blast 1X 1.44567

Blast 1X 1.86189

Blast 1X 1.61983

Blast 1X 1.67415

Blast 1X 1.22193

Blast 1X 1.68083

Blast 1X 1.27991

Blast 1X 1.53189

Blast 1X 1.30535

Blast 3X 1.18162

Blast 3X 1.24694

Blast 3X 1.45968

Figure 7D

subjects GFAP DCN/DN

GFAP Fluorescence (normalized to

shams)

sham 1.12935

sham 0.86489

sham 1.243

sham 0.95826

sham 1.05102

sham 1.00356

sham 0.81757

sham 0.93235

Blast 1X 1.75096

Blast 1X 1.5157

Blast 1X 1.96678

Blast 1X 1.62331

Blast 1X 2.07259

Blast 1X 1.64598

Blast 1X 1.97754

Blast 1X 1.34609

Blast 1X 1.65176

Blast 1X 1.40042

Blast 3X 1.31537

Blast 3X 1.65986

Blast 3X 1.66893

Figure 7H

Subjects Blast # Convex hull

Volume (m3)

sham 0 42778.29

sham 0 32446.51

sham 0 39329.84

sham 0 26270.99

sham 0 37700.39

sham 0 39462.01

sham 0 50017.08

sham 0 39129.05

sham 0 31224.70

sham 0 33931.59

sham 0 41452.88

sham 0 40906.71

blast 1 27544.92

blast 1 29906.49

blast 1 48163.16

blast 1 29770.94

blast 1 47022.66

blast 1 27355.14

blast 1 30926.58

blast 1 26264.62

blast 3 20008.23

blast 3 37199.45

blast 3 34437.75

blast 3 33752.25

blast 3 24540.07

blast 3 27058.11

Figure 7I

Subjects Blast # CD68 Optical density (normalized to shams)

sham 0 .602917

sham 0 1.127397

sham 0 1.418697

sham 0 .850989

sham 0 .324485

sham 0 .997787

sham 0 .619358

sham 0 .930195

sham 0 2.128176

blast 1 1.238102

blast 1 1.674517

blast 1 .866983

blast 1 .692043

blast 1 1.021488

blast 3 3.588532

blast 3 2.349994

blast 3 5.903601

blast 3 1.316978

blast 3 1.559089

Figure 8

Log10blast# MD

FA Radial Axial Density PHQ9 AUDIT-C CAPStotal

mm2/sec mm2/sec mm2/sec fibers/cm3

0.48 0.00047 0.65957 0.000499 0.000413 11.14583 24.0 5.0 97.0

0.7 0.000493 0.704005 0.000548 0.000385 9.208333 5.0 3.0 72.0

1.18 0.000454 0.654049 0.000498 0.000366 7.770833 23.0 1.0 100.0

1.85 0.000447 0.663254 0.000491 0.00036 8.270833 19.0 2.0 111.0

1.04 0.000498 0.670689 0.000559 0.000375 12.77083 7.0 0 58.0

0.78 0.00049 0.668359 0.000551 0.000368 16.15625 11.0 4.0 80.0

0.7 0.000491 0.69927 0.000528 0.000416 14.875 14.0 2.0 71.0

1.48 0.000478 0.67739 0.000539 0.000356 10.79167 12.0 9.0 64.0

1.72 0.000484 0.635796 0.000535 0.000381 11.875 1.0 5.0 26.0

1.26 0.000481 0.699582 0.000544 0.000353 10.25 17.0 .0 57.0

0.85 0.000478 0.631569 0.00049 0.000454 14.16667 5.0 5.0 41.0

1.82 0.000481 0.653213 0.000507 0.000428 10.58333 2.0 4.0 13.0

1.04 0.000473 0.665953 0.000515 0.000389 14.35417 6.0 2.0 16.0

1.45 0.00048 0.673372 0.000542 0.000356 9.729167 25.0 2.0 104.0

0.85 0.000482 0.65876 0.000547 0.000353 11.04167 3.0 6.0 63.0

1.3 0.000488 0.623459 0.000491 0.000481 16.41667 0 5.0 33.0

1.72 0.000449 0.683008 0.000498 0.00035 14.97917 8.0 3.0 72.0

0.7 0.000487 0.653206 0.000514 0.000434 6.708333 7.0 5.0 58.0

2.01 0.000461 0.645851 0.000496 0.000391 10.79167 13.0 1.0 81.0