Research Paper

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Pathophysiological mechanisms of neuropathic

pain: comparison of sensory phenotypes in patients
and human surrogate pain models
Jan Vollerta,b,*, Walter Magerlb, Ralf Baronc, Andreas Binderc, Elena K. Enax-Krumovaa,d, Gerd Geisslingere,f,
Janne Gierthmühlenc, Florian Henrichb, Philipp Hüllemannc, Thomas Kleinb, Jörn Lötsche, Christoph Maiera,
Bruno Oertelf, Sigrid Schuh-Hoferb, Thomas R. Tölleg, Rolf-Detlef Treedeb
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Abstract
As an indirect approach to relate previously identified sensory phenotypes of patients suffering from peripheral neuropathic pain to
underlying mechanisms, we used a published sorting algorithm to estimate the prevalence of denervation, peripheral and central
sensitization in 657 healthy subjects undergoing experimental models of nerve block (NB) (compression block and topical lidocaine),
primary hyperalgesia (PH) (sunburn and topical capsaicin), or secondary hyperalgesia (intradermal capsaicin and electrical high-
frequency stimulation), and in 902 patients suffering from neuropathic pain. Some of the data have been previously published.
Randomized split-half analysis verified a good concordance with a priori mechanistic sensory profile assignment in the training (79%,
Cohen k 5 0.54, n 5 265) and the test set (81%, Cohen k 5 0.56, n 5 279). Nerve blocks were characterized by pronounced
thermal and mechanical sensory loss, but also mild pinprick hyperalgesia and paradoxical heat sensations. Primary hyperalgesia
was characterized by pronounced gain for heat, pressure and pinprick pain, and mild thermal sensory loss. Secondary hyperalgesia
was characterized by pronounced pinprick hyperalgesia and mild thermal sensory loss. Topical lidocaine plus topical capsaicin
induced a combined phenotype of NB plus PH. Topical menthol was the only model with significant cold hyperalgesia. Sorting of the
902 patients into these mechanistic phenotypes led to a similar distribution as the original heuristic clustering (65% identity, Cohen
k 5 0.44), but the denervation phenotype was more frequent than in heuristic clustering. These data suggest that sorting according
to human surrogate models may be useful for mechanism-based stratification of neuropathic pain patients for future clinical trials, as
encouraged by the European Medicines Agency.
Keywords: Quantitative sensory testing, German Research Network on Neuropathic Pain, Menthol, A-fiber block, UVB,
High-frequency electrical stimulation, Capsaicin, Lidocaine, Human surrogate pain models

1. Introduction
Quantitative sensory testing (QST) in accordance with the DFNS
(German Research Network on Neuropathic Pain) protocol
assesses the sensory function of afferent myelinated A-beta- and
unmyelinated and thinly myelinated C- and A-delta-fibers3 for both
Sponsorships or competing interests that may be relevant to content are disclosed
at the end of this article.
a
Department of Pain Medicine, BG University Hospital Bergmannsheil GmbH,
Ruhr-University Bochum, Bochum, Germany, b Center of Biomedicine and Medical
Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University,
Mannheim, Germany, c Division of Neurological Pain Research and Therapy,
Department of Neurology, Universitätsklinikum Schleswig-Holstein, Campus Kiel,
Kiel, Germany, d Department of Neurology, BG University Hospital Bergmannsheil
GmbH, Ruhr-University Bochum, Bochum, Germany, e Institute of Clinical
Pharmacology, Pharmazentrum Frankfurt/ZAFES, University Hospital of Goethe-
University, Frankfurt am Main, Germany, f Fraunhofer Institute for Molecular Biology
and Applied Ecology (IME), Project Group Translational Medicine and Pharmacol-
ogy (TMP), Frankfurt am Main, Germany, g Department of Neurology, Klinikum
Rechts der Isar, Technische Universität München, Munich, Germany
*Corresponding author. Address: Neurophysiologie, Zentrum für Biomedizin und
Medizintechnik Mannheim, Medizinische Fakultät Mannheim, der Universität
Heidelberg, Ludolf-Krehl-Str.13-17, 68167 Mannheim, Germany. Tel.: 149 621/
383-9926; fax: 149 621/383-9921. E-mail address: Jan.Vollert@rub.de (J. Vollert).
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http://dx.doi.org/10.1097/j.pain.0000000000001190
loss (ie, hypoesthesia) and gain of function (ie, hyperalgesia and
allodynia).45 Quantitative sensory testing plays an important role for
diagnosing patients with neuropathic pain19,56 and can offer indirect
insights into underlying mechanisms of pathophysiology.7,15,18,27
Peripheral neuropathic pain is induced by partial nerve
damage, leaving 2 classes of nociceptors as candidates to cause
the pain: damaged nociceptors and surviving undamaged
nociceptors.11 Damaged nociceptors are responsible for sensory
loss due to denervation, but possibly also for ongoing pain due to
ectopic activity either arising in the periphery or in denervated
second-order neurons. Surviving nociceptors can be peripherally
sensitized by inflammatory processes related to denervation and
reinnervation which may cause hyperalgesia. Input from both
damaged and undamaged nociceptors can induce central
sensitization which may also cause hyperalgesia. We thus have
4 candidate mechanisms to model in human surrogate models of
peripheral neuropathic pain: denervation, ectopic activity, pe-
ripheral sensitization, and central sensitization. Models of ectopic
activity relate to ongoing pain, whereas the other 3 mechanisms
relate to altered perception of evoked pain.
There are no established human surrogate models of ectopic
activity and ongoing pain,33 but human surrogate models for
denervation, peripheral sensitization, and central sensitization
have been used for pharmacological studies.8,33,42 The aim of
this study was to estimate the prevalence of 3 distinct types of

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predefined mechanisms (denervation, peripheral sensitization,
and central sensitization) in a large number of healthy subjects
submitted to various surrogate models of experimental pain (both
previously published8,16,20,25,34,37,40,41,53,62 and unpublished
data) using a sorting algorithm developed in patients with
peripheral neuropathic pain. Each of the predefined mechanisms
was represented by a pair of established surrogate models. This
served the purpose of emphasizing those QST characteristic
prototypical for the underlying mechanism and minimizing
idiosyncrasies of individual surrogate models:
(1) Transient functional denervation by nerve compression or
topical lidocaine, targeting either myelinated A-fibers or
unmyelinated C-fibers, or both.
(2) Peripheral sensitization by inducing primary hyperalgesia (PH) by
topical application of capsaicin or by ultraviolet B (UVB) radiation;
these models commonly induce heat and pressure hyperalgesia.
(3) Central sensitization by inducing secondary hyperalgesia (SH)
by cutaneous electrical high-frequency stimulation (HFS) or
intradermal injection of capsaicin; they induce mostly mechan-
ical hyperalgesia to pinprick.
We then used an algorithm that had been developed for
heuristic sorting of QST profiles of neuropathic pain patients to
sort sensory profiles of human surrogate models into mechanis-
tically defined groups,60 including 5 additional human surrogate
models with less clearly defined, controversial, or mixed un-
derlying mechanisms. The final aim was to assign neuropathic
pain patients to presumed underlying mechanisms according to
their similarity to the mechanism-related profiles derived from the
human surrogate models.
2. Methods
2.1. German Research Network on Neuropathic Pain
The German Research Network on Neuropathic Pain (DFNS,
http://www.neuropathischer-schmerz.de) was established in
2002 to investigate mechanisms and treatments of neuropathic
pain. Quantitative sensory testing of human surrogate models of
neuropathic pain using the complete DFNS QST protocol has
since been conducted at the following sites: Ruhr-University
Bochum; Goethe-University Frankfurt; Medical Faculty Man-
nheim, Heidelberg University; University of Schleswig-Holstein
Campus Kiel; and Technical University of Munich. Appropriate
ethics committee approval has been obtained at all sites.
Subjects were included according to our developed guidelines,24
and quality of the included QST data was assured by
standardized training courses for each investigator of each
center and verified by regular audits.61 Each center and
investigator used identical equipment and standardized instruc-
tions. A recent analysis of heterogeneity between centers has
shown that data of various centers can be analyzed as
a homogenous data set, if these quality criteria are fulfilled.59
2.2. Surrogate models
The following surrogate models were included in the analysis
(Table 1):
(1) A-fiber-conduction blockade by sustained pressure on the
nerve trunk (unpublished data, methods as in Ref. 65)
(2) Topical lidocaine 5% patch34
(3) Topical capsaicin application; using cream, watery solution, or
patch16,40,41
(4) Ultraviolet B light irradiation at 3 times minimal erythema dose
(published and unpublished data, methods as in Refs. 25 and 43)
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(5) Intraepidermal capsaicin injection (unpublished data, methods
as in Refs. 36 and 43)
(6) Cutaneous electrical high-frequency stimulation28,32,37
(7) Muscle electrical high-frequency stimulation53
(8) Topical application of capsaicin solution and lidocaine16
(9) Topical 40% menthol application9,62
2.3. Quantitative sensory testing
The standardized protocol of the DFNS was used for QST, which
assesses the function of small and large afferent fibers. The
protocol has been described in detail previously;52 in brief, the
following parameters were tested: thermal detection thresholds
for the perception of cold (cold detection threshold [CDT]) and
warmth (warm detection threshold), paradoxical heat sensations
(PHSs) during assessment of thermal sensory limen of alternating
warm and cold stimuli, thermal pain thresholds for cold and hot
stimuli, tactile mechanical detection threshold (MDT) and
vibration detection threshold, pain summation to repetitive
pinprick stimuli (wind-up ratio), mechanical pain sensitivity
(MPS) including thresholds for pinprick (mechanical pain thresh-
old) and blunt pressure (pressure pain threshold), and a stimulus–
response–function for pinprick sensitivity (MPS) during which
dynamic mechanical allodynia (DMA) was assessed by stroking
light touch stimuli. For all parameters, negative (loss of function)
as well as positive (gain of function) phenomena were assessed.
2.4. z-Transformation
An initial analysis of 180 healthy subjects revealed that all QST
parameters are either normally or log-normally distributed (with
the exception of DMA and PHS).51 To compare individual QST
data between subjects regardless of sex-, age-, or body-site
differences, the DFNS proposed presenting QST values as z-
values.44,48,51,52 These values are normalized to the mean and
SD of reference data obtained in healthy subjects so that a z-value
of zero represents the mean value of a healthy control cohort
matched in age and sex, and the respective body re-
gion.44,48,51,52 Z-scores above zero indicate gain of function
when the subject was more sensitive to the test stimuli compared
with the population mean (hyperesthesia or hyperalgesia),
whereas z-scores below zero indicate loss of function referring
to a lower sensitivity of the subject (hypoesthesia or hypoalgesia).
Although individual z-scores are considered as abnormal if
beyond 61.96,51 for groups of patients, z-scores of 61.0 have
been shown to be of clinical significance, as they include
a relevant number of patients with abnormal values.45
Dynamic mechanical allodynia and PHS, which do not normally
occur in healthy subjects and do not fit any known distribution,
can thus not be z-transformed. They are usually presented as
percentages of presence, but for use in statistical analysis, they
can be transformed to pseudo-z-values: DMA can be coded into
a 0/2/3-variable representing no DMA (coded as 0), DMA with
average pain ratings below 1 on a 0 to 100 numerical rating scale
(coded as 12), and DMA with average pain ratings between 1 and
100 (coded as 13). Paradoxical heat sensation can be trans-
formed to a binary 0/2-variable showing absence (coded as 0) or
presence (coded as 12) of pathological values.
As many surrogate models were applied at the volar lower arm
or upper thigh, which are not standardized reference areas, we
also performed intraindividual comparisons either relative to the
same area before treatment, or, if these data were unavailable, to
the contralateral untreated side using effect sizes (Cohen d13).
This measure normalizes changes in the mean value before and
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Table 1
Human surrogate models and participating laboratories.
Model n Sex: n (%) female Age: mean (range) Participating centers Test area
NBs
A-fiber block 24 12 (50) 25 (21-39) Kiel, Mannheim Hand dorsum
Topical lidocaine 41 20 (49) 34 (19-69) Bochum Volar forearm
PH
Topical capsaicin 273 147 (53) 25 (15-75) Bochum, Frankfurt, Mannheim Volar forearm, dorsal hand, foot
UVB irradiation 158 51 (32) 24 (19-42) Frankfurt, Mannheim Volar forearm, upper thigh
SH
Capsaicin injection 36 19 (53) 32 (23-68) Kiel, Mannheim, Munich Volar forearm
Cutaneous HFS 12 3 (25) 36 (24-57) Mannheim Volar forearm
Mixed
Topical capsaicin, SH area 37 15 (41) 24 (19-39) Mannheim Volar forearm
UVB, SH area 22 0 (0) 24 (24-24) Mannheim Upper thigh
Muscular HFS 15 7 (47%) 24 (19-27) Mannheim Lower back
Topical menthol 11 0 (0) 25 (23-28) Kiel Hand dorsum
Topical lidocaine 1 topical capsaicin 28 17 (61) 30 (20-75) Bochum Volar forearm
HFS, high-frequency stimulation; NB, nerve block; PH, primary hyperalgesia; SH, secondary hyperalgesia; UVB, ultraviolet B.

after treatment to SD. There are no general interpretations of
“good” effect sizes, but it is often considered that effect sizes
below 0.3 can be considered small, above 0.5 medium, and
above 0.8 are commonly considered as large treatment effects.12
2.5. Pattern-based sorting algorithm
As sorting method, we applied a method developed for assigning
patients to sensory phenotypes developed in our previous work,
where we also showed general validity of the method.5,60 The same
method was applied here for 2 reasons: first, continuity to our work in
patients suffering from neuropathic pain would allow for higher
comparability between the hypothesis-driven approach in human
surrogate models in the present article, and our earlier, hypothesis-
free work in patients suffering from neuropathic pain. Second, and
more importantly, the probabilistic version of our previously
published sorting algorithm allows for any given patient to be
assigned to more than one sensory phenotype (or to present with
more than one underlying mechanism); our previous article
established a robust cut-off criterion for this type of sorting.60 In
this study, the probabilistic sorting was used to assign individual
patients to appropriate mechanisms of pathophysiology.
Based on 6 surrogate models with a clearly described
mechanism (A-fiber block by nerve compression or by topical
lidocaine 5% for small fiber block, topical capsaicin or UVB
radiation for PH, and intraepidermal capsaicin injection or
cutaneous HFS for SH), we developed an algorithm determining
whether an individual QST profile is most similar to denervation,
PH, or SH. We used a method we recently established in patients
suffering from neuropathic pain,5,60 applying a standard normal
distribution, stripped from the normalization factor, so that the
resulting value ranges on a scale from 0% to 100%.60
For each participant n, over the QST profile of the z-values of
i 5 13 parameters, a probability can be calculated, showing
a percentage of similarity to the prototypic QST profile of fiber
block, PH, or SH:
0 11 0
 2
B B xin 2 mim CC
13
+ pi
Fð1Þ: i ¼ 1 n;m
13
with pin;m 5exp@ 2 @ q ffiffiffiffiffiffiffiffiffi AA
2s2im
With i 5 one of 13 QST parameters, m 5 one of 3 prototypic
sensory profiles and sim being the SD of the ith QST parameter for
the mth mechanism in the defining data set, mim being the mean z-
value of the same QST parameter and mechanism in the defining
data set, n 5 the nth participant in a set of participants, and finally
xin being the z-value found in the nth participant for the ith QST
parameter. By averaging the probability over the 13 QST
parameters, we quantify the similarity of the individual subject’s
QST profile to the mean profile of each of the 3 mechanisms.60
The resulting probability value is ranging from 0% to 100% and
can be calculated for m 5 3 mechanisms.
As a simple way of categorizing patients into mechanisms, we
suggest sorting each patient to the phenotype with the highest
probability value:
(1) Calculate pi according to F(1) for each of the 13 QST
parameters. Use m and s from Table 2 for nerve block (NB).
(2) Average the 13 probabilities. The resulting value is the
probability for this patient to show the NB phenotype.
(3) Repeat steps 1 and 2, using m and s from Table 2 for PH and
SH.
(4) Allocate the patient to the phenotype with the highest
probability value.
To show reproducibility of the QST profile of the subgroups
found, a random number generator was used to split the data
set of the 6 defining surrogate models in half, using about 50%
of the data to define the prototypic sensory profiles of each of
the 3 mechanisms and the other half to verify correct
allocation.
2.6. Healthy sensory profile and deterministic and
probabilistic algorithm
A fourth prototypical sensory profile is derived from the QST
reference data sets in healthy subjects without any experimental
models. According to the definition of the z-transformation, this
profile will have a mean z-value of zero and an SD of one for each
parameter. In our previous analysis,60 we showed that the
algorithm can separate patients suffering from neuropathic pain
from healthy subjects if a probability cut-off of 64% is applied. To
this point, we use a deterministic approach, that is, each
participant is sorted to exactly one mechanism. It is, however,
our belief that different mechanisms can coexist; thus, with the
cut-off determined for healthy subjects in our previous work

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 4 J. Vollert et al. 0 (2018) 1–13
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Mean QST z-values (m) and SDs (s, in brackets) for each of the 13 QST parameters separately for each of the mechanisms. Values for healthy subjects follow the definition of z-values: mean 5 0 and SD 5 1. PHS is coded as pseudonormally distributed with 0 5 absence and 2 5 presence, DMA is coded

CDT, cold detection threshold; CPT, cold pain threshold; DMA, dynamic mechanical allodynia; HPT, heat pain threshold; MDT, mechanical detection threshold; MPS, mechanical pain sensitivity; MPT, mechanical pain threshold; PH, primary hyperalgesia; PHS, paradoxical heat sensation; PNP, polyneuropathy,
transferred onto the surrogate models, we suggest 2 alternative

0.48 (0.86)
0.13 (0.49)
0.16 (0.54)
0.00 (1.00)
versions, namely a deterministic or a probabilistic algorithm. The
deterministic algorithm follows the steps below:

PHS
Calculate F(1) for each of the 13 QST parameters. Use m and s
from Table 2 for healthy.

0.06 (0.34)
0.81 (1.07)
1.12 (1.11)
0.00 (1.00)
(1) Average the 13 probabilities. The resulting value is the
DMA probability for this patient to show a healthy profile.
(2) Repeat steps 1 and 2, using m and s from Table 2 for NB, PH,
and SH.
0.22 (1.06)
20.23 (1.93)
21.08 (1.83)
0.00 (1.00)
(3) Allocate the participant to the phenotype with the highest
probability value.
By contrast, in the probabilistic algorithm, steps 1 to 3 remain
VDT

identical and step 4 is exchanged as follows:

23.24 (2.89)
20.32 (1.13)
20.80 (1.38)
0.00 (1.00)

(4) Sort the participant to all phenotypes with a probability

above 64%. If the only probability above this cut-off is for
being healthy or no phenotype reaches a probability above
MDT

this cut-off, the sensory profile of the subjects cannot be

distinguished from normal.
0.25 (1.29)
0.18 (1.01)
0.31 (1.15)
0.00 (1.00)

These 2 versions were used for all analyses below and are
PPT, pressure pain threshold; QST, quantitative sensory testing; SH, secondary hyperalgesia; TSL, temperature sensory limen; VDT, vibration detection threshold; WDT, warm detection threshold; WUR, wind-up ratio.

presented alongside, all QST profiles of all surrogate models

WUR

underwent both versions of the algorithm.

20.15 (1.38)
0.71 (1.21)
1.95 (1.19)
0.00 (1.00)

2.7. Comparison with sensory phenotypes in patients

Random split-half analysis of human surrogate models of nerve blocks, PH, and SH: training data set (n 5 265).
MPS

In our recent work,5,60 we have described 3 distinct sensory

phenotypes that appear in patients suffering from peripheral
0.86 (1.87)
1.54 (1.08)
3.12 (0.97)
0.00 (1.00)

neuropathic pain of various etiology or clinical origin. To analyze

how this previous hypothesis-free pattern searching method in
MPT

patients suffering from neuropathic pain may relate to our new

mechanistic sensory subgroup classification described above,
20.63 (0.97)
1.35 (1.66)
0.63 (1.45)
0.00 (1.00)

the n 5 902 patients from the initial cluster analysis study5

underwent the deterministic version of the new sorting algorithm
and the result was compared with each patient’s sensory
PPT

phenotype in the deterministic version of the previous sorting

algorithm developed for patients.60 Agreement between algo-
0.52 (1.23)
2.37 (1.14)
20.14 (1.24)
0.00 (1.00)

rithms was assessed using Cohen kappa.21 Cohen kappa is

a value that measures similarity between groups or concordance
HPT

of ratings on a scale ranging from 0 (no similarity) to 1 (perfect

identity). Although no universal guideline for interpreting Cohen
0.06 (1.13)
20.12 (1.31)
0.33 (1.16)
0.00 (1.00)

kappa exists, as a rule of thumb, it was suggested that values

pseudonormally distributed with 0 5 absence, 2 5 0 to 1 (on a 0-100 numerical rating scale), and 3 5 1 to 100.

below 0.4 would indicate poor similarity, between 0.4 and 0.75
CPT

good similarity, and above 0.75 excellent.21 We then applied the

new probabilistic sorting algorithm to estimate the prevalence of
22.55 (1.52)
21.03 (1.12)
22.28 (0.84)
0.00 (1.00)

the 3 predefined mechanisms in this cohort of patients; this allows

each patient to be assigned to more than one mechanism.
TSL

3. Results
21.36 (1.27)
20.23 (1.04)
21.87 (0.89)
0.00 (1.00)

The analysis comprised a total of n 5 657 healthy subjects who

WDT

participated in published and unpublished studies on human

surrogate models: 9 distinct models (2 of them including areas of
both PH and SH) at 5 centers (Table 1). Approximately 44% of
22.94 (2.36)
21.05 (1.59)
22.71 (1.07)
0.00 (1.00)

subjects were female (291/657); sex distribution was not

homogenous across models, as for the UVB, cutaneous HFS,
CDT

and menthol models, predominantly men were recruited. Age

ranges were mostly lower than in neuropathic pain populations.
Denervation (m [s]), n 5 33

These factors were accounted for by normalizing all QST data to

Healthy subjects (m [s])

sex-specific and age-specific reference data.

PH (m [s]), n 5 207
SH (m [s]), n 5 25

Figure 1 shows z-profiles of the 6 models that were chosen for

defining phenotype means. As human surrogate models of NBs,
both nerve compression and topical lidocaine led to substantial
Table 2

loss in thermal detection threshold and MDT (Fig. 1A). For CDT
and MDT in the A-fiber compression block, this loss was almost
complete, reaching a mean z-value beyond 25, that is, beyond 5

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Figure 1. Deep sensory profiling of human surrogate models of a priori
mechanisms. (A) NBs, (B) PH, and (C) SH. Positive z-scores indicate positive
sensory signs (hyperalgesia) and negative z-values indicate negative sensory
signs (hypoesthesia and hypoalgesia). Dashed lines: 95% CI for healthy
participants (21.96 , z , 11.96). Inserts show numeric pain ratings for DMA
on a logarithmic scale (0-100) and frequency of PHS (0-3). CDT, cold detection
threshold; CI, confidence interval; CPT, cold pain threshold; DMA, dynamic
mechanical allodynia; HPT, heat pain threshold; MDT, mechanical detection
threshold; MPS, mechanical pain sensitivity; NB, nerve block; PH, primary
hyperalgesia; PHS, paradoxical heat sensation; PNP, polyneuropathy; PPT,
pressure pain threshold; QST, quantitative sensory testing; SH, secondary
hyperalgesia; TSL, temperature sensory limen; VDT, vibration detection
threshold; WDT, warm detection threshold; WUR, wind-up ratio.
SDs of normal detection, whereas for topical lidocaine, z-values
were around 22 indicating highly significant but partial sensory
loss. Unlike topical lidocaine, selective A-fiber block was also
associated with signs of sensory gain, namely the frequent
occurrence of pinprick hyperalgesia and PHSs.
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www.painjournalonline.com 5
As human surrogate models of PH, topical capsaicin and UVB
sunburn both induced substantial heat and mechanical hyper-
algesia (Fig. 1B). Heat hyperalgesia was more pronounced for
capsaicin, whereas mechanical hyperalgesia was more pro-
nounced for UVB. Capsaicin also induced loss of cold detection
and cold pain.
As human surrogate models of SH, cutaneous HFS and
intradermal capsaicin injection led to mechanical hyperalgesia
and thermal sensory deficits (Fig. 1C). Mechanical hyperalgesia
and DMA were more pronounced in the capsaicin injection
model.
Table 3 compares mean z-scores normalized to published
reference data of healthy subjects with effect sizes (Cohen d) in
intraindividual comparison with untreated control areas. Intra-
individual comparisons mostly confirmed the patterns of negative
or positive sensory signs of z-values, but the loss of thermal
detection for intradermal capsaicin and cutaneous HFS (Fig. 1C)
seems to be overestimated in z-values due to the nonstandard
test area.
The QST profiles of the remaining models are presented in
Figure 2. Figure 2A shows the QST profiles of surrounding skin
from 2 models where SH is either controversial (UVB) or known to
be mild (topical capsaicin). Although the area of SH of topical
capsaicin displays sensory loss in the z-profile, these effects are
much smaller in the intraindividual comparisons with untreated
skin, again suggesting an overestimation in z-values due to the
nonstandard test area (Table 3). Topical menthol has been
introduced to induce cold hyperalgesia, and muscle HFS to
induce hyperalgesia of deep tissue (Fig. 2B); sensory changes in
both models were mild. The combined application of topical
capsaicin and lidocaine (Fig. 2C) shows the combined effects of
lidocaine and capsaicin-induced PH, that is, the capsaicin-
induced mechanical hyperalgesia is abolished, whereas the
thermal and tactile loss is exaggerated.
3.1. Sorting algorithm
By random number assignment, 49% of subjects from the 6
surrogate models with clearly defined mechanism (topical
lidocaine 5%, A-fiber block, topical capsaicin, UVB radiation,
capsaicin injection, and cutaneous HFS) were assigned to the
training data set (n 5 265), which defined mean values and SDs
for the sorting algorithm (Table 2). Individual allocation by the
deterministic sorting (DET) algorithm replicated the a priori
assignment of surrogate models in 79% of the cases for training
set and in 81% of the cases for the test set (remaining 279
subjects from the same surrogate models). Cohen kappa
coefficient of agreement (scale: 0 5 random classification, 1 5
perfect agreement between methods) was 0.54 (95% confidence
interval [CI]: 0.43-0.65) for the training set and 0.56 (95% CI: 0.46-
0.67) for the test set; both values may be categorized as “good,”
although no universal guideline for interpreting Cohen kappa
exists.21 Most common shifts were PH or SH to NB (18% and
27%, respectively), and least common shifts were NB to SH and
secondary to PH (both ,1%). Shifts between original
and algorithmic assignment are shown in Table 4.
3.2. Deterministic and probabilistic sorting
The z-profiles of each sensory subgroup are shown in Figure 3.
Forty-one subjects showed a sensory profile that was most
similar to untreated healthy skin, although part of a surrogate
model (Fig. 3A). The subjects sorted to the NB profile were mostly
characterized by loss of cold and mechanical detection (CDT and
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Table 3
z-scores and effect sizes (Cohen d) of human surrogate models.

Blue indicates loss of function and red indicates gain of function, for z-scores: boundaries 1.0 and 1.96, for Cohen d values: boundaries above 0.5 and 0.8.
CDT, cold detection threshold; CPT, cold pain threshold; HFS, high-frequency stimulation; HPT, heat pain threshold; MDT, mechanical detection threshold; MPS, mechanical pain sensitivity; MPT, mechanical pain threshold;
PPT, pressure pain threshold; TSL, temperature sensory limen; UVB, ultraviolet B; VDT, vibration detection threshold; WDT, warm detection threshold; WUR; wind-up ratio.

MDT, representing A-delta and A-beta fiber function, respec-
tively, Fig. 3B). Loss of warm detection was less pronounced
compared with cold detection because the topical lidocaine
block was less effective in comparison with the A-fiber block.
Loss of vibration detection was not detectable, which is due to
a limitation of the A-fiber block, which only affects a small area,
whereas vibration is poorly localized and can be sensed by rapidly
adapting mechanoreceptors (Pacinian corpuscles) situated
beyond this area. Primary hyperalgesia (Fig. 3B) was character-
ized by pronounced heat hyperalgesia and moderate mechanical
hyperalgesia, whereas SH presented most prominently by
mechanical hyperalgesia and some loss of thermal detection.
Figure 4 (6 defining models) and Figure 5 (5 additional models)
show results of deterministic and probabilistic sorting into the 3
prototypic mechanistic profiles. In the probabilistic sorting
algorithm, the percentage of sensory profiles compatible with
being from normal skin increased to 187 (28%) vs 41 (6%) in the
deterministic version of the algorithm. The highest frequency of
healthy profiles was found in muscular HFS and topical menthol,
which also had the mildest sensory changes in their averaged
QST profiles (Fig. 2B).
Quantitative sensory testing profiles compatible with the NB
profile increased to 352 cases (54%) in the probabilistic algorithm
vs 178 (27%) in the deterministic version. Apart from the defining
models of A-fiber block and topical lidocaine, the highest
frequency of the NB profile was found in cutaneous HFS and in
the menthol model.
Quantitative sensory testing profiles compatible with PH
increased from 380 (58%) in the deterministic to 470 (72%) in
the probabilistic algorithm. Beyond the defining models, this
profile was frequent in the skin area surrounding the UVB
radiation model (where profiles compatible with SH were rare),
topical menthol, and muscular HFS.
Quantitative sensory testing profiles consistent with SH were
more than twice as frequent in the probabilistic sorting (n 5 134;
20%) than in the DET (n 5 58; 9%). Beyond the defining capsaicin
injection and cutaneous HFS, this profile was only found in the
skin area surrounding topical capsaicin in a relevant frequency
(deterministic: 27% and probabilistic: 65%) confirming that SH
occurs also in this model.
3.3. Comparison with sensory phenotypes in patients
Figure 5F shows DET and probabilistic sorting results for 902
neuropathic pain patients that were the basis of the original
cluster analysis (Baron et al. 2017), and Table 5 compares
mechanistic sorting results with original heuristic cluster alloca-
tion. In 65% of the cases, patients from the sensory loss
phenotype were sorted to NB, patients from the thermal
hyperalgesia phenotype to the PH pattern, and patients from

Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
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Figure 2. Deep sensory profiling of additional human surrogate models. (A) SH
surrounding topical capsaicin or UVB-treated areas, (B) topical menthol (cold
hyperalgesia model) and muscle HFS (deep pain model), and (C) sequentially
combined topical application of lidocaine and capsaicin. Positive z-scores
indicate positive sensory signs (hyperalgesia) and negative z-values indicate
negative sensory signs (hypoesthesia and hypoalgesia). Dashed lines: 95% CI for
healthy participants (21.96 , z , 11.96). Inserts show numeric pain ratings for
DMA on a logarithmic scale (0-100) and frequency of PHS (0-3). CDT, cold
detection threshold; CI, confidence interval; CPT, cold pain threshold; DMA,
dynamic mechanical allodynia; HFS, high-frequency stimulation; HPT, heat pain
threshold; MDT, mechanical detection threshold; MPS, mechanical pain
sensitivity; PHS, paradoxical heat sensation; PNP, polyneuropathy; PPT,
pressure pain threshold; QST, quantitative sensory testing; SH, secondary
hyperalgesia; TSL, temperature sensory limen; UVB, ultraviolet B; VDT, vibration
detection threshold; WDT, warm detection threshold; WUR, wind-up ratio.
the mechanical hyperalgesia phenotype to the SH QST pattern
(Table 5). This corresponded to a Cohen kappa coefficient of
agreement (scale: 0 5 random classification, 1 5 perfect
Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
www.painjournalonline.com 7
agreement between methods) of 0.44 (95% CI: 0.28-0.60),
showing a substantial degree of agreement.21 The most
prominent shifts were from the 2 hyperalgesia phenotypes in
the heuristic patient clustering into the mechanistic phenotype
consistent with NB.
Interestingly, in the probabilistic sorting (Table 5), fewer
patients were sorted to the NB profile (512 vs 600 in DET),
whereas relatively more patients were sorted to PH (271 vs 186 in
DET) or SH (198 vs 116 in DET). But, the most prominent shift was
still towards the profiles consistent with NBs. Still, only one-third
of the patients could be uniquely assigned to 1 of the 3
mechanisms. Another third of the patient QST profiles was
consistent with multiple mechanisms (n 5 282), most frequently
a combination including the NB phenotype. The last third was not
sufficiently distinct from a normal skin QST phenotype to be
assigned to any mechanism (n 5 279).
Figure 6 illustrates the similarity in QST profiles grouped either
according to the original cluster analysis (Fig. 6A) or the
mechanistic sorting (Fig. 6B). Although the profiles of patients
sorted according to both algorithms are highly similar, the profiles
of patients and surrogate models in the mechanistic sorting
display some interesting differences (compare Fig. 3 with Fig.
6B): patients sorted to the NB exhibit hypoalgesia, whereas in
surrogate models, pain thresholds are normal or show mild gain.
Patients sorted to NB also have loss of vibration detection
threshold, which could not be modeled in the A-fiber block. For
PH, the models display almost no cold, but an isolated heat
hyperalgesia, whereas patients show both cold and heat hyper-
algesia. For SH, participants under surrogate models have more
gain in mechanical pain threshold than in MPS, which is reversed
in patients.
4. Discussion
These multicenter data confirmed principal properties of NB
(sensory loss), PH (heat hyperalgesia), and SH (pinprick hyper-
algesia) that have been already reported in human and animal
experiments.8,35,42,47,55 They also revealed additional sensory
alterations in these human surrogate models that had not been
previously described, that is, sensory gain in NBs and sensory
loss in hyperalgesia models. This probably reflects a selection or
reporting bias in previous studies that had not assessed these
sensory functions.
In a randomized split-half analysis, a sorting algorithm pre-
viously validated for sensory profiles of neuropathic pain
patients60 led to reproducible sorting of surrogate model sensory
profiles into patterns defined a priori according to known
mechanisms. Sorting of 902 neuropathic pain patients into these
mechanistic phenotypes led to a similar distribution as the original
heuristic clustering,5,60 laying the basis for a mechanism-based
treatment approach.64
4.1. Nerve blocks as human surrogate model of transient
functional denervation
The sensory profile of human surrogate models for transient
functional denervation (ie, A-fiber compression block and topical
lidocaine) was characterized by pronounced loss in thermal and
mechanical detection combined with PHS. These patterns have
been reported in previous single-center studies.34,65 Consistent
with its selective effect on myelinated nerve fibers, compression
NB had larger effects on MDT and CDT than on warm detection
threshold.28,65 Pinprick stimuli are often perceived as less painful
in complete A-fiber block,28,65 whereas mild pinprick
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Table 4
Cross-tabulation of dominant phenotype identified using the algorithm (columns) vs the original assignment (rows) for training
and test data set (before and after slash).
Algorithm
Original Nerve blocks
Nerve blocks 29/30
Primary hyperalgesia 35/42
Secondary hyperalgesia 8/5
Overall, concordance of the algorithm to original assignment was strong (79% of the cases for the training and 81% for the test data set, note that the even higher concordance for the test data set compared with training data
indicates a very robust sorting).
hyperalgesia has been associated with preserved A-delta fiber
function2,29 Topical lidocaine had mild effects compared with
complete conduction block by regional anesthesia.22,33 Interest-
ingly, in contrast to the present data, intradermal lidocaine 0.01%
and 0.1% was reported to induce also a heat hyperalgesia
Figure 3. Average profiles of 657 healthy subjects who had undergone human
surrogate models stratified on prototypic mechanism-based sorting. (A)
Healthy profiles and (B) NBs vs PH vs SH profiles. Positive z-scores indicate
positive sensory signs (hyperalgesia) and negative z-values indicate negative
sensory signs (hypoesthesia and hypoalgesia). Dashed lines: 95% CI for
healthy participants (21.96 , z , 11.96). Inserts show numeric pain ratings
for DMA on a logarithmic scale (0-100) and frequency of PHS (0-3). CDT, cold
detection threshold; CI, confidence interval; CPT, cold pain threshold; DMA,
dynamic mechanical allodynia; HPT, heat pain threshold; MDT, mechanical
detection threshold; MPS, mechanical pain sensitivity; NB, nerve block; PH,
primary hyperalgesia; PHS, paradoxical heat sensation; PNP, polyneuropathy,
PPT, pressure pain threshold; QST, quantitative sensory testing; SH,
secondary hyperalgesia; TSL, temperature sensory limen; VDT, vibration
detection threshold; WDT, warm detection threshold; WUR, wind-up ratio.
Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
PAIN®
Primary hyperalgesia Secondary hyperalgesia
4/2 0/0
162/178 10/4
0/1 17/17
probably due to sensitization of TRPV1 and TRPA1
receptors.38,39,49,63
4.2. Primary hyperalgesia as human surrogate model of
peripheral sensitization
The profile of surrogate models of peripheral sensitization (topical
capsaicin or UVB radiation) was characterized by pronounced
hyperalgesia to heat, pressure, and pinprick pain. Primary
nociceptive afferents are easily sensitized to heat stimuli, but much
less so to von-Frey or pinprick stimuli.55 Peripheral sensitization to
heat may be explained by phosphorylation of the heat-gated cation
channel TRPV1,58 but has also been shown to be induced by
TRPA1 agonist allyl-isothiocyanate.1 Although peripheral sensiti-
zation to blunt pressure has been reported before,31 pinprick
hyperalgesia in these models may indicate additional central
sensitization induced by enhanced peripheral nociceptive input to
the spinal cord. Sensory loss occurred mostly in the topical
capsaicin model and was restricted to thermal detection thresh-
olds; this has been discussed previously10,16 and may reflect
desensitization by the TRPV1 agonist capsaicin, which is the
intended mode of action clinically.26
4.3. Secondary hyperalgesia as human surrogate model of
central sensitization
Human surrogate models of central sensitization (intradermal
capsaicin and cutaneous electrical HFS) were characterized by
pronounced pinprick hyperalgesia, but also pronounced thermal
sensory loss. Combined studies in monkeys and humans using
intradermal capsaicin had shown pronounced increases in both
wide dynamic-range and high-threshold spinal neuron output
despite unchanged A- and C-nociceptor input.6,54 Tactile
sensory loss has been reported in human surrogate models of
SH and also in patients with pinprick hyperalgesia.23 An inverse
spinal gate with small fiber input inhibiting processing of large fiber
input46,66 has been suggested as a mechanism. Thermal sensory
loss is a new finding, suggesting that sensory loss in chronic pain
patients does not necessarily have to be due to structural
changes, but may also be a functional sign, and hence potentially
sensitive to analgesic treatment regimes.
4.4. Other human surrogate models
The presence of a zone of SH surrounding topical capsaicin and
UVB is controversial in the published literature.8,33,40,41 Our data
confirmed sensory profiles compatible with SH surrounding
topical capsaicin but not UVB, which mostly had characteristics
of PH or NB instead. These findings suggest that topical
capsaicin is useful to study both PH and SH, whereas the UVB
model seems to be limited to PH.
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Figure 4. Deterministic and probabilistic sorting of the defining human
surrogate models. Deterministic sorting (DET): each profile is sorted to its best
fit (this column always adds up to 100%); probabilistic sorting (the following 4
columns, each profile can fall in multiple categories, so that these columns are
not additive): each profile is sorted to each mechanism for which it reaches
a probability above 64%, as determined as optimal cut-off in an ROC analysis
of patients suffering from neuropathic pain and healthy subjects in Vollert et al.,
2017. H (grey), normal healthy skin, NB (blue), nerve block; PH (red), primary
hyperalgesia; SH (yellow), secondary hyperalgesia. ROC, receiver operating
curve.
Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
www.painjournalonline.com 9
Figure 5. Deterministic and probabilistic sorting of additional human
surrogate models and patients suffering from peripheral neuropathic pain.
Deterministic sorting (DET): each profile is sorted to its best fit (this column
always adds up to 100%); probabilistic sorting (the following 4 columns,
each profile can fall in multiple categories, so that these columns are not
additive): each profile is sorted to each mechanism for which it reaches
a probability above 64%, as determined as optimal cut-off in an ROC
analysis of patients suffering from neuropathic pain and healthy subjects in
Vollert et al., 2017. Patient data (F) are from Baron et al. 2017. H (grey),
normal healthy skin; NB (blue), nerve block; PH (red), primary hyperalgesia;
SH (yellow), secondary hyperalgesia.
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Table 5 The mechanical hyperalgesia patient phenotype shows a strong

Cross-tabulation of dominant phenotype identified using the overlap with surrogate models of SH, which supports an
deterministic algorithm developed in patients (columns) vs the interpretation of this phenotype to be a phenotype of reorganization
algorithm developed based on surrogate models (rows) for or central sensitization. Substantial thermal sensory loss suggests
n 5 902 patients. that also damaged nociceptors are involved, generating ongoing
pain and inducing central sensitization.4
Sensory Thermal Mechanical
loss hyperalgesia hyperalgesia The sensory loss patient phenotype shows a strong overlap
(n 5 (n 5 267) (n 5 279) with experimental NBs. These blocks were frequently used as
356) tools to identify normal sensory function of fiber classes (A vs C),
Deterministic but not yet widely recognized as mimicking aspects of
NB (n 5 600) 337 97 166 neuropathic pain.7,33 Both the clinical phenotype and the
PH (n 5 186) 6 156 24 surrogate models are dominated by loss of small and large fiber
SH (n 5 116) 13 14 89 functions. This supports an interpretation as denervation or
Probabilistic
deafferentation, where central neurons may develop denervation
NB (n 5 512) 202 130 180 super-sensitivity to other inputs.14
PH (n 5 271) 12 163 96 Roughly one-third of the patients were assigned a NB phenotype
SH (n 5 198) 38 26 134 according to the surrogate models vs one of the hyperalgesia
phenotypes in the heuristic patient cluster analysis. This systematic
Of the above: patients sorted to
a single mechanism (n 5 341) shift is consistent with the presence of partial nerve damage in most
NB, n 5 247 163 30 54 of the neuropathic pain conditions that may lead to coexistence of
PH, n 5 62 1 58 3 denervation and sensitization of the remaining pathways.11 These
SH, n 5 32 3 3 26 data cover, however, only peripheral neuropathic pain, and to this
Of the above: patients sorted to
point, we cannot make any extrapolations onto, for example,
multiple mechanisms central pain, nociceptive pain, or deep pains.
(n 5 282)
NB 1 PH (n 5 116) 4 84 28
4.6. Limitations and technical considerations
NB 1 SH (n 5 73) 28 2 43
PH 1 SH (n 5 17) 0 7 10 The validity of the prototypic mechanistic profiles is partly
NB 1 PH 1 SH (n 5 76) 7 14 55 demonstrated by the accuracy of allocation of the training set
Patients sorted to no 150 69 60 data to the appropriate a priori profile of roughly 80%. This shows
mechanism (n 5 279) general robustness, but also that the deterministic algorithm is
In the clear majority of the cases (65%), both algorithms concurred in the deterministic variant. not perfect and should be used with some caution on individual
NB, nerve block; PH, primary hyperalgesia; SH, secondary hyperalgesia. basis. For a better separation of mechanism-specific sensory
profiles from idiosyncrasies of individual experimental models,
Muscle HFS was introduced as a model for plasticity of deep more human surrogate models of PH and SH (eg, burn injury and
pain, and the overlying skin did not exhibit profiles compatible surgical incision) and of temporary functional denervation (eg,
with SH. Our data were from the lower back; more pronounced limb ischemia and capsaicin-induced defunctionalization) should
effects might occur when other muscles, for example, in the lower be studied using the methods of this article.
limb or face are stimulated. The models in this analysis do not explicitly cover the
Topical menthol had only mild effects with some character- endogenous pain-modulating systems30 nor ectopic activity.
istics of NBs and PH. Its main value is the pronounced cold Descending modulation may contribute to the SH phenotype, but
hyperalgesia, but the current data do not make a strong might also exhibit yet another sensory profile. Ectopic activity may
suggestion as to whether this is due to peripheral or central have been present in any of our models, but we have neither
sensitization or both. positive nor negative evidence about it.
The sensory profile of topical lidocaine plus topical capsaicin The percentage of sensory profiles compatible with normal
displayed an almost perfect combination profile of lidocaine block variability was higher in the surrogate models than in the patients
and capsaicin-induced PH, but it lacks the distinct mechanical (29% vs 20%). We think that this is due to the fact that for ethical
hyperalgesia characterizing SH. This finding suggests that reasons, all manipulations in humans were relatively minor (as
sensory profiling may be able to identify combined mechanisms compared to patients and animal models50).
and to distinguish between contributions of peripheral vs central
sensitization also in patients with neuropathic pain.
4.7. Summary and conclusions
With these prototypical sensory profiles for 3 predefined
4.5. Mechanistic significance of heuristically found mechanisms (denervation, peripheral, and central sensitization),
patient phenotypes
we provide a well-studied mechanistic background for our
The sorting according to surrogate model profiles was applied to previously described heuristic sensory phenotype clusters. Using
our previously published patient data, which leads to a similar the probabilistic sorting according to human surrogate model
distribution as the original heuristic clustering. This supports profiles, patients suffering from neuropathic pain can be
previous mechanistic interpretations of the clinically found tentatively stratified in future studies to presumed underlying
phenotypes: the thermal hyperalgesia patient phenotype shows mechanisms. The European Medicines Agency encourages such
strong overlap with surrogate models of PH. This supports an approach in its new guideline17 as a step towards increasing
previous interpretations as irritable nociceptor18 and peripheral response rates in clinical trials by a mechanism-based treatment
sensitization.57 Both evoked and ongoing pain is likely to be due approach towards neuropathic pain.64 It should be noted,
to surviving nociceptors in these patients. however, that the 3 classes of human surrogate models studied

Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
 Month 2018
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Figure 6. Average profiles of 902 patients with peripheral neuropathic pain
sorted either according to the deterministic algorithm derived from heuristic
patient clusters (A) or according to the deterministic version of the algorithm
derived from human surrogate models (B). Positive z-scores indicate positive
sensory signs (hyperalgesia) and negative z-values indicate negative sensory
signs (hypoesthesia and hypoalgesia). Dashed lines: 95% CI for healthy
participants (21.96 , z , 11.96). Inserts show numeric pain ratings for DMA
on a logarithmic scale (0-100) and frequency of PHS (0-3). CDT, cold detection
threshold; CI, confidence interval; CPT, cold pain threshold; DMA, dynamic
mechanical allodynia; HPT, heat pain threshold; MDT, mechanical detection
threshold; MPS, mechanical pain sensitivity; PHS, paradoxical heat sensation;
PNP, polyneuropathy, PPT, pressure pain threshold; QST, quantitative sensory
testing; TSL, temperature sensory limen; VDT, vibration detection threshold;
WDT, warm detection threshold; WUR, wind-up ratio. Patient data from Baron
et al., 2017, heuristic sorting algorithm according to Vollert et al., 2017.
here likely represent combined rather than single mechanisms.
This, however, is likely true also for studies in awake behaving
animals. Therefore, a reverse translation approach may be useful
for developing novel analgesic medications: they should initially
be tested in animal models of NB, PH, or SH. Medications
effective on these phenotypes can easily be validated in human
surrogate models and then transferred to subgroups of neuro-
pathic pain patients.
Conflict of interest statement
The authors have no financial or other relationships that might
lead to a conflict of interest.
This study was supported by the German Research Network
on Neuropathic Pain (DFNS).
The EUROPAIN project is a public-private partnership and has
received support from the Innovative Medicines Initiative Joint
Undertaking under grant agreement no 115007, resources for
Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
www.painjournalonline.com 11
which are composed of financial contribution from the European
Union’s seventh framework programme (FP7/2007–2013) and
European Federation of Pharmaceutical Industries and Associ-
ations (EFPIA) companies’ in kind contribution. The NEUROPAIN
project is an investigator-initiated European multicentre study
with R. Baron as principal investigator and 10 coinvestigator sites,
supported by an independent research grant from Pfizer Ltd.
Acknowledgements
The authors thank all consortia for building up the basis of this
study by patient and subject recruitment and assessment.
Article history:
Received 9 October 2017
Received in revised form 26 January 2018
Accepted 14 February 2018
Available online 24 February 2018
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