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Year: 2011
Insula-specific responses induced by dental pain: a protonmagnetic resonance spectroscopy study
Gutzeit, A; Meier, D; Meier, M L; von Weymarn, C; Ettlin, D A; Graf, N; Froehlich, JM; Binkert, C A; Brügger, M
Gutzeit, A; Meier, D; Meier, M L; von Weymarn, C; Ettlin, D A; Graf, N; Froehlich, J M; Binkert, C A; Brügger,M (2011). Insula-specific responses induced by dental pain: a proton magnetic resonance spectroscopy study.European Radiology, 21(4):807-815.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Gutzeit, A; Meier, D; Meier, M L; von Weymarn, C; Ettlin, D A; Graf, N; Froehlich, J M; Binkert, C A; Brügger,M (2011). Insula-specific responses induced by dental pain: a proton magnetic resonance spectroscopy study.European Radiology, 21(4):807-815.
Gutzeit, A; Meier, D; Meier, M L; von Weymarn, C; Ettlin, D A; Graf, N; Froehlich, J M; Binkert, C A; Brügger,M (2011). Insula-specific responses induced by dental pain: a proton magnetic resonance spectroscopy study.European Radiology, 21(4):807-815.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:Gutzeit, A; Meier, D; Meier, M L; von Weymarn, C; Ettlin, D A; Graf, N; Froehlich, J M; Binkert, C A; Brügger,M (2011). Insula-specific responses induced by dental pain: a proton magnetic resonance spectroscopy study.European Radiology, 21(4):807-815.
Insula-specific responses induced by dental pain: a protonmagnetic resonance spectroscopy study
Abstract
OBJECTIVES: To evaluate whether induced dental pain leads to quantitative changes in brainmetabolites within the left insular cortex after stimulation of the right maxillary canine and to examinewhether these metabolic changes and the subjective pain intensity perception correlate. METHODS:Ten male volunteers were included in the pain group and compared with a control group of 10 otherhealthy volunteers. The pain group received a total of 87-92 electrically induced pain stimuli over15 min to the right maxillary canine tooth. Contemporaneously, they evaluated the subjective painintensity of every stimulus using an analogue scale. Neurotransmitter changes within the left insularcortex were evaluated by MR spectroscopy. RESULTS: Significant metabolic changes in glutamine(+55.1%), glutamine/glutamate (+16.4%) and myo-inositol (-9.7%) were documented during painstimulation. Furthermore, there was a significant negative correlation between the subjective painintensity perception and the metabolic levels of Glx, Gln, glutamate and N-acetyl aspartate. CONCLUSION: The insular cortex is a metabolically active region in the processing of acute dentalpain. Induced dental pain leads to quantitative changes in brain metabolites within the left insular cortexresulting in significant alterations in metabolites. Negative correlation between subjective pain intensityrating and specific metabolites could be observed.
1
Insula-Specific Responses Induced by Dental Pain.
A Proton Magnetic Resonance Spectroscopy Study A. Gutzeit MD1, D. Meier PhD2, M. Meier MSc3, C.v. Weymarn PhD1, D. Ettlin D MD4,
N. Graf PhD5, J.M. Froehlich PhD1, C. A. Binkert MD, MBA1, M. Brügger PhD3
1 Department of Radiology, Cantonal Hospital Winterthur, Brauerstrasse 15, 8401
Winterthur, Switzerland
2 Institute for Biomedical Engineering, University and ETH Zurich, Switzerland.
3 Institute of Psychology, Division Neuropsychology, University of Zurich, Zurich,
Switzerland.
4 Center for Dental and Oral Medicine and Cranio-maxillofacial Surgery, Clinic for
Removable Prosthodontics, Masticatory Disorders and Special Care Dentistry,
University of Zürich, Zürich, Switzerland.
5Clinical Trials Center, Center for Clinical Research, University Hospital of Zurich,
8091 Zurich, Switzerland
Correspondence address: Andreas Gutzeit, Cantonal Hospital Winterthur,
Department of Radiology, Brauerstrasse 15, 8401 Winterthur, Switzerland,
Phone number: +41 (0) 522664109, Fax number: +41(0) 522664603,
email: [email protected]
Manuscript type: Original research
Key words: Dental stimulation, trigeminal pain, proton MR spectroscopy, insular
cortex, pain-rating
2
Insula-Specific Responses Induced by Dental Pain. A Proton Magnetic Resonance Spectroscopy Study
3
Abstract:
Objectives: To evaluate whether induced dental pain leads to quantitative changes in
brain metabolites within the left insular cortex after stimulation of the right maxillary
canine, and further to examine whether these metabolic changes and the subjectively
rated pain intensity perception correlate.
Methods: This prospective clinical study was performed with approval of the
institutional review board and informed consent was given by all volunteers.Ten male
volunteers were included in the pain group and compared with a control group of 10
other healthy volunteers. The pain group received a total of 87-92 electrically induced
pain stimuli over 15 min to the right maxillary canine tooth. Contemporaneously, they
evaluated the subjective pain intensity of every stimulus using an analogue scale.
Neurotransmitter changes within the left insular cortex were evaluated by means of
MR spectroscopy before, during and after pain stimulation.
Results: Significant metabolic changes in glutamine (+55.1%), glutamine/glutamate
(+16.4%) and myo-inositol (-9.7%) were documented during pain stimulation in
comparison to baseline values. Furthermore, there was a significant negative
correlation between the subjective pain intensity perception and the metabolic levels
of Glx, Gln, glutamate and N-acetyl aspartate.
Conclusion: The insular cortex is a metabolically active region in the processing of
acute dental pain. Induced dental pain leads to quantitative changes in brain
metabolites within the left insular cortex resulting in significant alterations in
metabolites. Negative correlation between subjective pain intensity rating and specific
metabolites could be observed.
Key words: Dental stimulation, trigeminal pain, proton MR spectroscopy, insular
cortex, pain rating
4
5
Introduction:
Brain-specific mechanisms of experimentally induced acute pain have already been
described in several studies, inter alia with the assistance of functional magnetic
resonance imaging (fMRI), neuroelectrophysiological and neurosurgical procedures
[1-8]. Structures involved in processing the multidimensional aspect of pain
perception are often summarised with the term "pain matrix", and include several
regions consistently found within investigations [1, 8, 9], hence accommodating the
complex neurosignature pattern of pain [10]. This network's main components are
primary and secondary somatosensory cortices, insular cortex, anterior cingulate,
prefrontal cortices as well as the thalamus and cerebella cortices [1,8-9]. Among
these, the insular cortex is described as one of the most consistently activated
structures and seems to play a pivotal role within the cortical pain circuitry. Hence,
the significance of the insular cortex as a core structure in pain processing has been
established [1, 9, 11-16].
However, these results rest predominantly upon fMRI and PET studies, which are not
able to provide direct access to reaction mechanisms on the neurotransmitter level,
as becomes feasible with 1H Magnetic Resonance Spectroscopy (MRS). 1H-MRS is a
non-invasive procedure enabling the measurement and dedicated post-processing of
brain metabolites such as N-acetyl aspartate (NAA), glutamate (Glu), glutamine
(Gln), glutamine/glutamate (Glx), myo-inositol (mI), choline (Cho), gamma-
aminobutyric acid (GABA) and creatinine (Cr) [17].
Grachev et al. first described pain-related MR-spectroscopic changes in patients
suffering chronic low back pain by observing a significant drop in NAA within the
dorsolateral prefrontal regions [18]. These results could be confirmed indirectly by
Siddall et al. [19]. In 2009, Kupers et al. [20] were able to describe for the first time
that under acute thermal pain a significant increase in GABA occurred in the rostral
6
anterior cingulate cortex. Focusing on the anterior insular cortex, a very recently
published article describes for the first time pain-related alterations in MR spectra due
to thermal pain while stimulating the left dorsal forearm [21].
Based on preliminary work on the examination of toothache-related blood oxygen
level-dependent (BOLD) responses demonstrating strong perfusion alterations, the
current study was designed to observe more in detail whether this crucial pain
processing brain area might react also on the neurotransmitter level [22]. Therefore,
the specific aims were to answer the questions, are there basal metabolic changes in
the insular cortex under acute pain stimulation (in comparison to a non-stimulated
control group), and second,is it possible to find a link between the subjectively
experienced pain intensity and the corresponding individual quantifiable
spectroscopic metabolites.
7
Materials and Methods
This prospective clinical study was performed between January 2009 and July 2009
with the approval of the institutional review board. Written informed consent had been
obtained from all 24 volunteers. The inclusion criteria required the test tooth to be
caries-free, vital and without attachment loss. None of the volunteers had a known
psychiatric mental disease. All volunteers had no contraindications with respect to the
usual MRI examinations and received detailed information about the experimental
procedure. Only male volunteers were included in order to avoid different pain
perception evaluation known from females due to differing hormonal phases during
the menstrual cycle [23].
Pain-stimulated volunteer group
Fourteen healthy volunteers (14 men, mean age: 32 years, age range 24-51) were
included. Four of these volunteers had to be excluded secondarily mostly because of
claustrophobia or technical problems during dental stimulation inside the MRI (Fig. 1).
Finally, 10 volunteers were exploitable in the pain group.
Before the experiment, for each volunteer a dental splint of the maxillary dentition
with Blu-Mousse® (Thixotropic Vinyl Polysiloxane, Edgewood, MD, USA) was
constructed. Stainless steel electrodes were embedded in each splint opposite the
labial and palatal surface centre of the right maxillary canine. They served as anode
and cathode during the electrical stimulation. In order to minimise electrical
resistance, we punched out a round piece of hydrogel (AG602-6, AMGEL
Technologies, Lystrup, Denmark) 3 mm in diameter. This was placed between the
anode and cathode and was additionally covered with a thin layer of toothpaste
(Signal, Microgranuli; Unilever, Zug, Switzerland) (Fig. 2).
8
Sensory testing and training
One to two weeks before the MR experiment, sensory testing was performed with
each volunteer to define the respective thresholds for sensory perception (SPT), pain
perception (PPT) and pain tolerance (PTT), as well as to familiarise them with the
electrical stimulation set-up. Additionally, the accurate handling of the MR-compatible
analogue scale was explained and secured in a training session. Moreover, the
volunteers were asked whether the stimuli were perceived distinctly at the test tooth
only and not in the periodontal tissue. Electrical stimulation was performed with the
portable Compex Motion system [24] and the protocol was controlled by the
presentation software (www.neurobs.com/presentation) via a parallel port using a
self-made interface. Biphasic pulse forms of 1 ms duration were applied with
interstimulus intervals randomised between 5 and 8 seconds. Stimulus strength was
increased from 1 mA to a maximum of 100 mA in 1-mA steps. Subjects indicated the
relevant thresholds by hand signals previously specified.
Proton MR spectroscopy measurements
The experiments were performed using 3 T MRI (Achieva, Release 2.6.1; Philips
Healthcare, Best, Netherlands). Subjects were placed in the MRI in the supine
position using a T/R head coil. To prevent uncontrolled movements, the head was
fixed with a special plastic foam wedge between the head and the coil.
The standardised MRI protocol consisted of a scout, a reference acquisition and a
coronal and transverse T1-weighted sequence of the head. This T1 sequence had
the following parameters: TR 600 ms, TE 10 ms, voxel size 0.9 mm x 1.12 mm x 4
mm. The single voxel spectroscopy acquisitions (planned on the T1-weighted
9
images) covered the whole left insular cortex by using a PRESS technique with: TR
2000 ms, TE 30 ms, bandwidth 2000 Hz, voxel size 20 mm x 20 mm x 37.6 mm, NSA
(number of signal averages) 80. This spectroscopic sequence was measured once
before stimulus delivery, and three times during and after the stimulation phase,
respectively. The duration of every single MR- spectroscopic measurement lasted
3.16 min. Data were received after the postprocessing analysis with the LC Model,
and are described in more detail below (Fig. 3).
Pain-stimulated volunteer group
Before starting the MR measurement, SPT and PPT were re-tested once inside the
MRI to exclude sensory perception-related differences compared with the sensory
testing and training appointment. The paradigm was segmented into a pre-pain
phase lasting 3 minutes, a pain-stimulated phase lasting 9 minutes (consisting of 3
sequential MR spectroscopic measurements) and a post-pain phase lasting as well 9
minutes (3 MR spectroscopic measurements). During the pain-stimulated phase, an
electrical stimulus 150% above PPT was delivered. After every electrical stimulus,
each volunteer rated his subjective pain sensation using a special MR compatible
analogue scale (Medoc Ltd, Ramat Yishai, Israel) . In parallel, proton MR
spectroscopic data were acquired during the whole paradigm in the left insular cortex
(Fig. 4).
MR spectroscopic data analysis and post-processing
The raw 1H MRS data were transferred to a workstation and processed by using the
LC Model [17] with the relevant parameters set for the echo time (30 ms) and field
strength (3 Tesla). LC models were provided with the basis set containing the above-
mentioned single in vitro metabolite model spectra. These data were fitted within the
1
chemical shift range between 0.5 and 4.2 ppm leading to a list of metabolites, which
allows for further statistical analysis differentiating the changes among the pre-
stimulation, stimulation and post-stimulation phases.
Pain-free control group
Using the same rationale regarding ethical approval and volunteer
instruction/consent, ten healthy control subjects (10 men, mean age 34, age range
20-41) not belonging to the pain-stimulated group were measured additionally. To
exclude possible metabolic influences of the dental splint itself or MR-related
interferences, a similar dental impression (Fig. 2) was constructed. This control group
was measured using the same parameters and conditions, but without any pain
stimulation during the examination.
Statistical analysis
Metabolites were quantified using the fully automated and user-independent spectral
evaluation tool LC MODEL described above. All metabolites were measured relative
to CRE and analysed to test the differences with a repeated measures ANOVA.
Baseline, pain-stimulation phase and post-stimulation phase were calculated and
compared as “within-subject” factor. Additionally, a “between-subject factor”,
comprising the pain and the control group was calculated in order to ensure that only
the pain stimulation and not the dental splint itself induced metabolic changes within
the left insular cortex. Post-hoc t-tests were calculated to address specifically the
differences among the baseline, the mean of the pain stimulation measurements and
mean of the post-stimulation measurements when a significant main effect occurred.
Spearman’s rho was calculated to investigate the correlation between the mean of
1
the three pain measurements of single metabolites and the mean of the analogue
scale ratings. The significance level was set at p<0.05. In consideration of the
explorative nature of the study, p values were not corrected for multiple comparisons.
All statistical calculations were performed using SPSS software 17.0.1 release for
Windows (SPSS Inc, Chicago IL, USA).
Results:
The repeated measures ANOVA revealed a significant main effect "stimulation
phase" (within subjects factor) for Gln (p=<0.001), Glx (p=0.005) and mI (p<0.001) as
well as a significant interaction effect "group x stimulation phase" for the metabolites
Gln (p=0.008), Glx (p=0.025) and mI (p<0.001), suggesting that the course of these
metabolites was significantly different between the pain and the control group.
Unlike the non-stimulated control group, subjects receiving pain stimuli showed a
significant increase of Glx and Gln and a significant decrease of mI during the pain-
stimulation phase. In the post-stimulation phase, these metabolic changes returned
almost to baseline values (Fig. 5). Table 1 shows all the pairwise comparisons in the
pain group revealing significant differences between the baseline and the pain phase
as well as between the pain and the post-pain phase for the metabolites Gln, mI and
Glx. Thus, Gln after stimulation showed a significant average increase of 55.1%
compared with the metabolite measurement before stimulation (p=0.017). For Glx,
the increase during stimulation compared with the baseline was 16.4% (p=0.036). mI
presented a significant decrease during stimulation compared with the baseline of
9.7% (p=0.005). There was no significant difference between baseline and the post-
pain phase for the metabolites Glx (p=0.451) and mI (p=0.899), while there was a
significant difference for Gln (p=0.048).
1
The changes during pain stimulation compared with the baseline were not significant
for NAA (+2.5%, p=0.274) and Glu (+3.3%, p=0.352) (Fig. 6). Using standard MR
spectroscopic acquisition techniques, GABA quantification was limited because of
large standard deviations (>45%), excluding it from further evaluation.
Regarding pain perception, there was a strong and significant negative correlation
between the subjective pain rating measured on an analogue scale and the relative
metabolic levels of Glx (r=-0.818; p=0.004), Gln (r=0.806; p=0.005), Glu (r=-0.733;
p=0.016) and NAA (r=-0.685; p=0.029). Thus, higher values for Glx, Glu, Gln and
NAA went together with a lower subjective pain perception and vice versa (Fig. 7).
Discussion
Most knowledge on cortical pain processing has been gained through functional
studies such as fMRI or PET studies [1, 25, 26]. The advantage of these methods is
that “global changes” in brain activity under pain can be determined and as a result
conclusions can be drawn on which parts of the brain are more active in pain
processing. On the other hand, the disadvantage of these methods is that these
activation patterns cannot be characterised on neurotransmitter levels.
To our knowledge, only one publication so far has addressed metabolic changes
under acute experimentally evoked pain in the insular cortex [21] and one has
investigated chronic pain [27].
1
In the present study, we were able to show that healthy volunteers under acute
dental pain showed a significant average increase in Gln (55.1%) and Glx (16.4%)
compared with the baseline. Conversely, mI showed a significant decrease (9.7%)
during stimulation compared with the baseline. In the post-pain phase, all of these
metabolites recovered rapidly to the baseline level. However, the changes during
pain stimulation of NAA and glutamate compared with the baseline presented a
tendency towards increasing but without reaching significance. The other evaluated
metabolites such as Cho and Cr showed no significant alterations during the entire
period of the survey. Thus our study demonstrates the pivotal role of the insular
cortex within the cortical pain circuitry under acute pain stimulation.
Concerning other brain areas in patients suffering chronic low back pain, a NAA
decrease in the dorsolateral prefrontal cortex and thalamus has been described [18,
28]. NAA is only found in neuronal tissue and considered a marker of neuronal
activity [29]. A drop in NAA is related to chronic pain representing destruction of brain
cells. Consistently, in recent studies, it could be shown that the morphology of the
brain and in particular the ratio of grey to white matter can change after only a few
days of pain stimulation [30]. This confirms that acute and chronic pain might lead to
morphological and metabolic changes within short time frames [30-32].
The insula cortex, as already mentioned, is considered to be very important in the
processing of pain. According to the few spectroscopy-related studies conducted so
far, Glu and Gln appear to be important metabolic factors within the cortical pain
circuitry. Thus, Mullins et al. [33] were able to show that a painful cold-pressure
stimulus in the lower extremities led to a significant increase in both of these
metabolites of over 9% within frontal cingulate gyri. Consistent with Mullins’ results,
we also observed a significant increase in Glx (16.4%) despite using another
1
paradigm and measuring within a different brain region. Our results demonstrate the
feasibility of quantification according to pain-related changes in the insular cortex.
Our data confirm the previous results of Mullins et al. [33] with a comparable increase
in Glx levels while other metabolites differ. It should be emphasised that tooth pain is
probably a stronger pain stimulation paradigm compared with peripherally induced
pain [34]. Thus, this fact could be a possible explanation of why we found such strong
metabolite alterations in the insular cortex.
Glu and Gln are well known to be excitatory amino acids in the brain [35,36]. Glu is
produced in the synapses of the nerve cells, partially taken up by astroglial cells and
subsequently converted into Gln. Following this, both metabolites are transported
back into the nerve cells where they are available once again as the so-called
“glutamate pool”. It is supposed that the greater part of energy utilisation in the brain
is mainly used for the Glu/Gln cycle [35-37]. With the new generation of 3 T MRI the
proportions of Glu and Gln (located in the specific spectral range between 2.2-2.4
and 3.6-3.8 ppm) are more accurately differentiated and distinguished. Increases in
Glu and Gln levels have already been documented in certain disorders outside of
pain sensations such as schizophrenia [38,39]. Therefore, using MRS techniques
concentrations of Glu/Gln are reliably assessed representing different clinical and
functional cortically related situations such as pain. For the time-being we can not
explain the significant decrease of mI during pain stimulation also because similar
investigations are lacking. But a recently published study confirmed that even chronic
pain patients with fibromyalgia also present significantly decreased mI values in
comparison to a healthy control group. [40].
A further important point in this study was the fact that we were able to correlate the
subjectively perceived pain sensation with physiological metabolic changes. There
was a strong and significant negative correlation between the subjective pain
1
perception measured on an analogue scale and the metabolic levels of Glx, Glu, Gln
and NAA (Fig. 7). At first, we could not explain this relationship, especially because
our results partly contradict the study by Mullins et al. [33]. To be precise, in their
pain-related measurements, they were able to determine a strong correlation
between the subjective pain ratings and the quantitative increase in Glu and Gln. It
should, however, be emphasized that the pain rating in this cited study was
performed only at the end of the single experiment while in our model this rating
occurred concomitantly with the pain stimulation. In fact, such a late post hoc rating
can be problematic because of biasing influences. To explain again, in our pain
model, the volunteers were exposed to an average of 90 individual pain stimuli to a
single tooth and a subsequent immediate pain rating after each stimulus was
performed. Interesting as well are the results by Petrou et al. [27] reporting patients
with chronic pain due to fibromyalgia. They also described a negative correlation for
NAA/Cho levels in the insular cortex and the basal ganglia proportional to acute pain
stimulation thresholds. These results are partly in line with our findings. The question
arises whether the differences between healthy subjects and chronic pain patients
with regard to the cortical processing of acute pain might be comparable.
Overall, a strong correlation of the subjective perception of pain intensity and the
metabolic changes with inverse proportionality of the metabolic elevation is difficult to
explain. We hypothesise that volunteers with a high sensitivity to pain probably tend
to have a higher turnover of their Glx pool. Our results actually confirm that there is a
relationship between stronger pain perception and the subsequent lower relative
release of Glx. However, this theory should be the subject of further studies, and we
cannot provide definite evidence in favour of this hypothesis.
1
Our results contradict the studies by Kupers et al. [20], who recently investigated the
metabolic changes in the gyrus cinguli under acute pain. They reported no significant
changes in Glu and Gln but merely an increase in GABA occurred by explaining this
phenomenon with GABA having a partial overlap (of its chemical shift) with Glu and
Gln at 2.0 to 2.5 ppm.
Several studies indicate that sub-classification of the insular cortex in a posterior,
medial and anterior part revealed a pain context-dependent specification in terms of
more sensory or more cognitive-related activation patterns during nociceptive stimuli
[11,12]. Functional separation of the various insular cortex subregions from a pain
physiological point of view might therefore be addressed in future investigations.
Our study showed some limitations. First, the number of volunteers is relatively small.
However, the aim of this investigation was to identify quantitative changes in the
brain metabolism within the insular region, as a first feasibility study.
Second, a further limitation was the fact that the subjectively rated pain stimulus was
conducted using an MR-compatible analogue scale without visual feedback, which
was technically impossible. This was because we aimed with higher priority to
prevent arbitrary head movements by placing cushions between the cranium and the
coil impeding the use of a visual analogue scale. To circumvent this issue, volunteers
were intensively trained on the analogue scale during the training session before
imaging.
Conclusions:
It could be demonstrated that the insular cortex is a metabolically highly active
region in the processing of acute dental pain. During stimulation, Gln and Glx
presented a significant increase compared with the baseline, whereas mI
1
resulted in a significant decrease. There was a strong and significant negative
correlation between subjective pain intensity rating and increasing levels of
Glx, Gln, Glu and NAA. Acute dental pain results in significant metabolic
changes in the insular cortex which are addressable using MR spectroscopy,
opening up new insights into the cortical pain circuitry on a neurotransmitter
level.
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Table 1: Pairwise comparisons between the baseline, the pain phase and the post-
pain phase (n=10).
95% Confidence interval Metabolite Comparison Mean difference
Lower bound Upper bound
Baseline – Pain phase -0.028 -0.081 0.026 NAA
Pain phase – Post-pain phase 0.026 -0.010 0.062
Baseline – Post-pain phase -0.002 -0.042 0.039
Baseline – Pain phase -0.235* -0.416 -0.053 Gln
Pain phase – Post-pain phase 0.154* 0.021 0.286
Baseline – Post-pain phase -0.081* -0.161 -0.001
Baseline – Pain phase 0.083* 0.033 0.134 mI
Pain phase – Post-pain phase -0.086* -0.131 -0.041
Baseline – Post-pain phase -0.003 -0.050 0.045
Baseline – Pain phase -0.037 -0.124 0.049 Glu
Pain phase – Post-pain phase 0.076 -0.013 0.166
Baseline – Post-pain phase 0.039 -0.024 0.102
Glx Baseline – Pain phase -0.272* -0.521 -0.023
Pain phase – Post-pain phase 0.230* 0.025 0.435
Baseline – Post-pain phase -0.042 -0.163 0.079
*p<0.05
2
2
Figure 1: Flow chart representing an overview of the included and excluded
volunteers. Four volunteers were excluded secondarily due to claustrophobia (1
volunteer) and technical problems with dental stimulation (3 volunteers).
2
Figure 2: Blu-Mousse® dental splint obtained from every volunteer: The stainless
steel electrodes (arrow) were placed in each dental splint to serve as an anode and
cathode during the electrical stimulation at the right maxillary canine tooth.
2
Figure 3: Exemplary Proton MR Spectroscopic measurement within the insular cortex
from a single volunteer using the LC model. This is a commercially available
technique for post-processing analysis of MR spectroscopy. The program used fully
automated processes to perform functions such as water signal suppression,
correction of phase and residual eddy-current effects and low frequency filtering of
residual water signal. Upper spectrum with residual noise shows that the baseline is
quite stable, confirming high quality of measurement. The second spectrum
represents the fitted spectrum of all metabolites (in red) calculated by using the LC
Model. The lower two spectra represent the detailed analysis of Gln and Glu (in red)
based on their specific spectral distribution of resonance peaks in ppm. The three
images of the head showed the exact position of the voxel within the brain/left insular
cortex.
2
Figure 4: Graphical representation of the experimental setting and paradigm. It shows
the palatal and labial electrode with contact to the right maxillary canine. After short
intermittent electrical stimulation (90 repetitions) over approximately 15min (pain
stimulation phase), pain-stimulated nerve reactions are transmitted via the right
maxillary nerve into the brain. The spectroscopic measurements of relevant
metabolites (Gln, Glu, mI, NAA, Cr, Cho, GABA) were obtained in the left insular
cortex.
Figure 5: Statistically significant metabolite changes of Glx (a), Gln (b) and mI (c)
before (baseline), during (stimuli) and after pain stimulation (post stimuli phase). Bars
show the mean levels and whiskers the standard error. Black bars represent the pain
free control group, grey bars the pain-stimulated volunteer group. Unlike the control
group subjects undergoing pain stimuli showed a significant increase of Glx,and Gln
and a significant decrease of mI after dental stimulation.
2
Figure 6: Statistically non-significant metabolite changes of NAA (a) and Glu (b)
before, during and after pain stimulation. Bars show the mean levels and whiskers
the standard error. Black bars represent the pain-free control group, grey bars the
pain-stimulated group. There is an increase in NAA and Glu in the pain group during
stimulation and a subsequent decrease in the post-stimulation phase. The change of
NAA and Glu levels in the pain group, however, are not significantly different from the
change in NAA levels in the control group.
2
Figure 7: Mean subjective pain rating (using an analogue scale; x-axis) of each
volunteer rated continuously after each stimulus over 15 min of pain stimulation in
comparison to mean metabolic levels of the three pain-stimulation measurements (y-
axis): levels of NAA (filled circles), Gln (empty circles), Glu (filled triangles) and Glx
(empty triangles). There was a strong and significant negative correlation (p<0.05)
between the subjective pain perception and the metabolic levels of NAA, Glu, Gln
and Glx.
