Supplementary Figure 1S: Subjects were presented with the blue cross and
were cued to anticipate “high pain” (i.e., brief thermal heat temperature that
produces high pain sensation) if the color of the cross changed to RED or “low
pain” (i.e., brief thermal heat that produces low pain sensation) if the color of the
cross changed to GREEN. In addition, an uninformed YELLOW cue (not shown)
was introduced which signaled a painful stimulus of the uninformed intensity (i.e.,
brief thermal heat of either high or low intensity at 50 % probability). Each
temperature was delivered for 6 sec.
Experimental Paradigm:
A total of 28 (14 high pain, 14 low pain) temperatures were delivered. High
temperatures were preceded by the high anticipatory cue (i.e., cross changed
from BLUE to RED) 7 times and by the uninformed cue (i.e., cross changed from
BLUE to YELLOW) 7 times. Likewise low temperatures were preceded by low
anticipatory cue (i.e., cross changed from BLUE to GREEN) 7 times and by the
uninformed cue 7 times.
Functional Connectivity Analyses:
Data were preprocessed as for the main effects analyses with minor
modifications aimed at removing non-specific physiological signals by band-pass
filtering(0.009<ƒ<0.08). Individual time courses in these processed raw signal
datasets were extracted for the seed ROIs within bilateral anterior insula(right AI,
left AI from the anticipation task effects, Figure 1a) and from bilateral insular
cortices(right IC, left IC from the pain task effects, Figure 2a). Data points were
censored if they differed by more than two standard deviations from the average
echoplanar signal for the given seed ROI. We then used AFNI function 3dTfitter
to remove hemodynamic delay from the timecourses in the seed ROIs. The
resultant signal in the seed ROIs was then multiplied by the respective
regressors, i.e., right and left AI were multiplied by the anticipation regressor,
while right and left IC were multiplied by the pain regressor. This created four
interaction timecourses, which were then convolved with gamma-variate
hemodynamic using the AFNI function waver. Four multiple linear regression
models were run thereafter to examine connectivity of right and left anterior
insula(AI) during pain anticipation and of right and left insular cortex(IC) during
pain experience. The interaction timecourse was used as a regressor of interest
in each model. Task regressors as well as regressors controlling for baseline
differences, linear drift, head movement(roll, pitch, and yaw), and fluctuations in
white matter signal were added to each regression model as nuissance
variables. REML fitting was performed to reduce false positives due to timedependent cross correlations. The resulting correlation coefficient for the
timecourse of interest was calculated for each voxel. This provided correlation
maps for the timecourse in the seed ROIs and the timecourse from all other brain
voxels as a function of pain anticipation or pain experience. The Fisher Z
transforms of these correlation maps were then warped to conform to the
Talairach atlas(45) to allow for group comparisons of the Fisher Z transforms in
seed ROIs during pain anticipation and pain experience using independent twosample t-tests. This assessed differences in functional connectivity in right and
left AI during pain anticipation and right and left IC during pain experience
between MTBI and HC groups.
Functional Connectivity Results
Pain Anticipation(Supplementary Figure 2S): Using the right anterior insula time
series during pain anticipation from the anticipation task
effects(X/Y/Z:38/23/8;volume=4352mm3; see Supplementary Figure 2S, left
panel) as a seed, four regions showed significant between group differences in
functional connectivity with right AI(Figure 2S, Table 1S). Right orbitofrontal
cortex(OFC) and right dorsolateral prefrontal cortex(dlPFC) showed increased
connectivity, while ventromedial prefrontal cortex(vmPFC) and ventral anterior
cingulate cortex(vACC) showed decreased connectivity during anticipation of
pain in the MTBI group, in contrast to the HC group. Furthermore, using the left
anterior insula time series during pain anticipation from the anticipation task
effects(X/Y/Z:-32/24/10;volume=2688mm3; see Figure 2S, right panel) as a
seed, three regions showed significant between group differences in functional
connectivity with left AI(Figure 2S, Table 1S). The vmPFC, vACC and midbrain
showed lower connectivity during anticipation of pain in the MTBI group, in
contrast to the HC group. We found that only increased connectivity between
right AI and OFC remained significantly different between the groups after
covarying out traumatic and depressive symptoms severity.
Pain Experience(Supplementary Figure 3S): Using the right insula time series
from the pain experience task effects as a
seed(X/Y/Z:38/6/10;volume=12160mm3; see Supplementary Figure 3S, left
panel), several regions showed significant between group differences in
functional connectivity with the right insula(Figure 3S, Table 2S). The vmPFC
and one cluster within middle temporal gyrus showed increased connectivity,
while right caudate, medial thalamus(with the peak on the left), and several
clusters within the occipital lobe showed decreased connectivity during pain
experience in the MTBI group, in contrasts with the HC group. Furthermore,
using the left insula time series from the pain experience task effects as a
seed(X/Y/Z:-41/4/7;volume=4608mm3; see Figure 3S, right panel), vmPFC, left
IFG, left dlPFC and several temporal clusters showed increased connectivity and
one occipital region showed decreased connectivity with left insula during pain
experience in the MTBI compared to the HC group(Figure 3S, Table 2S). None
of the observed group differences in connectivity during pain experience survived
significance after co-varying out trauma and depressive symptoms severity.
Figure 2S. Whole-Brain Between-Group Differences in Functional
Connectivity with Anterior Insulas During Pain Anticipation. Left. Whole
brain between group differences in functional connectivity using the right anterior
insula(AI) time series during pain anticipation from the anticipation task
effects(from Figure 1a) as a seed. Compared to HC, right orbitofrontal
cortex(OFC) and right dorsolateral prefrontal cortex(dlPFC) showed increased
connectivity, while ventromedial prefrontal cortex(vmPFC) and ventral anterior
cingulate cortex(vACC) showed decreased connectivity during anticipation of
pain in the MTBI group(see Table 1S for details); Increased connectivity between
right AI and right OFC remained significant after controlling for anxiety and
depression. Right. Whole brain between group differences in functional
connectivity using the left anterior insula(AI) time series during pain anticipation
from the anticipation task effects(from Figure 1a) as a seed. Compared to HC,
vmPFC, vACC and midbrain showed decreased connectivity during anticipation
of pain in the MTBI group(see Table 1S for details). Right=Left
Figure 3S. Whole-Brain Between-Group Differences in Functional
Connectivity with Mid-Posterior Insulas During Pain. Left. Whole brain
between group differences in functional connectivity using the right insular
cortex(IC) time series during pain experience from the pain experience task
effects(from Figure 2a) as a seed. Compared to HC, vmPFC and one region
within middle temporal gyrus showed increased connectivity, while right caudate,
medial thalamus(with the peak on the left), and several regions within the
occipital lobe showed decreased connectivity during pain experience in the MTBI
group(see Table 2S for details). Right. Whole brain between group differences in
functional connectivity using the left insular cortex(IC) time series during pain
experience from the pain experience task effects(from Figure 2a) as a seed.
Compared to HC, vmPFC, left IFG, left dlPFC and several temporal regions
showed increased connectivity and one occipital region showed decreased
connectivity with left insula during pain experience in the MTBI group(see Table
2S for details). Right=Left.
TABLE 1S: Functional Connectivity with Anterior Insula: Between
Group differences during Pain Anticipation (Whole Brain)
Brain Region
Right Anterior Insula
Right Orbitofrontal Cortex
Right dlPFC
Left Middle Occipital Gyrus
Right Fusiform Gyrus
Right Cerebellum
Left Cerebellum
Left Anterior Insula
No significant clusters
vol
x
MTBI > HC
2368
1472
1344
1536
2048
1344
y
36
35
-30
34
40
-29
z
stat
50
18
-80
-55
-60
-68
-1
39
-9
-16
-38
-36
2.7*
2.3
2.3
2.2
2.5
2.3
MTBI < HC
Right Anterior Insula
Left Anterior Cingulate(vACC)
Right ventromedial PFC
Left Precentral Gyrus
Right Superior Temporal
Gyrus
Left Anterior Insula
Left Anterior Cingulate (vACC)
Right ventromedial PFC
Right Midbrain
4160
1920
1280
1344
-3
1
-24
32
24
54
-27
16
-10
15
62
-30
2.6
2.6
2.4
2.4
2304
1280
1472
-6
2
1
29
54
-17
-13
14
-11
2.7
2.5
2.4
dlPFC – dorsolateral prefrontal; * - remained significant after controlling for
traumatic and depressive symptoms severity and multiple comparison
correction
TABLE 2S: Functional Connectivity with Insula: Between Group
differences during Pain Experience (Whole Brain)
Brain Region
Right Insula
Left Middle Temporal Gyrus
Right ventromedial PFC
Left Insula
Left ventromedial PFC
Left dorsolateral PFC
Left IFG/ventral AI
Right Middle Temporal Gyrus
Left Middle Temporal Gyrus
vol
x
MTBI > HC
y
z
stat
1984
1472
-60
-6
-23
55
-6
8
3
2.6
3264
1344
1344
2368
1344
5120
3904
-15
-28
-30
52
61
-49
-59
53
20
13
-2
-34
-58
-19
10
46
-18
-19
-8
24
-7
2.4
2.2
2.5
2.4
2.5
2.4
2.7
MTBI < HC
Right Insula
Right Fusiform Gyrus
Left Lingual Gyrus
Right Middle Occipital Gyrus
Left Thalamus
Right Caudate
Left Insula
Left Lingual Gyrus
2944
2560
2048
4928
1984
1920
1664
1536
37
47
17
-4
-10
24
-3
12
-68
-40
-65
-75
-92
-89
-15
22
-15
-17
-15
-9
0
7
-5
11
2.5
2.4
2.3
2.3
2.3
2.3
2.6
2.3
4032
-1
-74
-6
-2.3
PFC – prefrontal cortex, IFG/AI – inferior frontal gyrus/anterior insula