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TRANSCRIPT
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: [email protected]
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