Increased endothelin-1 and diminished nitric oxide levels in blister fluids of patients with intermediate cold type complex regional pain syndrome type 1.

Abstract

In complex regional pain syndrome type 1 (CRPS1) pro-inflammatory mediators and vascular changes play an important role in the sustained development and outcome of the disease. The aim of this study was to determine the involvement of vasoactive substances endothelin-1 (ET-1) and nitric oxide (NO) during early chronic CRPS1.
Included were 29 patients with CRPS 1 who were diagnosed during the acute stage of their disease and observed during follow-up visits. Disease activity and impairment were determined and artificial suction blisters were made on the CRPS1 and the contralateral extremities for measurements of IL-6, TNF-alpha, ET-1 and nitrate/nitrite (NOx).
The levels of IL-6, TNF-alpha and ET-1 in blister fluid in the CRPS1 extremity versus the contralateral extremity were significantly increased and correlated with each other, whereas NOx levels were decreased.
The NOx/ET-1 ratio appears to be disturbed in the intermediate stage of CRPS, resulting in vasoconstriction and consequently in a diminished tissue blood distribution.

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BioMed Central
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BMC Musculoskeletal Disorders
Open Access
Research article
Increased endothelin-1 and diminished nitric oxide levels in blister
fluids of patients with intermediate cold type complex regional pain
syndrome type 1
J George Groeneweg*, Frank JPM Huygen, Claudia Heijmans-Antonissen,
Sjoerd Niehof and Freek J Zijlstra
Address: Department of Anesthesiology, subdivision Pain Treatment Center, Erasmus MC Rotterdam, The Netherlands
Email: J George Groeneweg* - j.groeneweg@erasmusmc.nl; Frank JPM Huygen - f.huygen@erasmusmc.nl; Claudia Heijmans-
Antonissen - c.heymans-antonissen@erasmusmc.nl; Sjoerd Niehof - s.niehof@erasmusmc.nl; Freek J Zijlstra - f.zijlstra@erasmusmc.nl
* Corresponding author
Abstract
Background: In complex regional pain syndrome type 1 (CRPS1) pro-inflammatory
mediators and vascular changes play an important role in the sustained development and
outcome of the disease. The aim of this study was to determine the involvement of
vasoactive substances endothelin-1 (ET-1) and nitric oxide (NO) during early chronic CRPS1.
Methods: Included were 29 patients with CRPS 1 who were diagnosed during the acute
stage of their disease and observed during follow-up visits. Disease activity and impairment
were determined and artificial suction blisters were made on the CRPS1 and the
contralateral extremities for measurements of IL-6, TNF-α, ET-1 and nitrate/nitrite (NOx).
Results: The levels of IL-6, TNF-α and ET-1 in blister fluid in the CRPS1 extremity versus
the contralateral extremity were significantly increased and correlated with each other,
whereas NOx levels were decreased.
Conclusion: The NOx/ET-1 ratio appears to be disturbed in the intermediate stage of
CRPS, resulting in vasoconstriction and consequently in a diminished tissue blood
distribution.
1. Background
Complex regional pain syndrome (CRPS) is a painful dis-
order which mainly occurs as a complication after surgery
or trauma, and the main characteristics are continuous
pain, marked changes in tissue blood flow and skin sur-
face temperature, oedema and sweating, movement disor-
ders and trophic changes of the skin [1,2]. The severity of
the symptoms is disproportionate to the initial event. The
diagnosis of CRPS is entirely based upon clinical criteria
[3,4]. In the Netherlands, the incidence of CRPS1 is
approximately 2.6% for different fractures, which results
in approximately 5100 new patients yearly, of whom a
substantial part will not recover completely [5-7]. The
female to male ratio is approximately 3:1, with a median
age of 52.7 years at onset [7]. The pathophysiology of
CRPS1 is not unravelled yet, but growing evidence indi-
cates the involvement of an exaggerated inflammatory
processes. Both central and peripheral mechanisms have
Published: 30 November 2006
BMC Musculoskeletal Disorders 2006, 7:91doi:10.1186/1471-2474-7-91
Received: 14 October 2006
Accepted: 30 November 2006
This article is available from: http://www.biomedcentral.com/1471-2474/7/91
© 2006 Groeneweg et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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been proposed to play a prominent role. Central sensitiza-
tion leading to exacerbation of pain is thought to result
from neuroimmune activation of cells in the peripheral
nervous system [5]. During the neurogenic inflammation,
neuropeptides [8] and cytokines and other mediators are
released [9]. This leads to a so-called 'warm dystrophy'
with signs of inflammation such as redness, increased skin
temperature, loss of function and pain [4,5,10].
During the chronic, disabling stage of the disease, in gen-
eral the following changes occur: i) signs of extravasation,
and oedema changes into atrophy, ii) regional blood flow
declines, and iii) increased skin temperatures change into
diminished temperatures. Permanent damage of nerve
endings can lead to less endothelium-dependent vasodila-
tion and result in a diminished regional blood distribu-
tion, the so-called 'cold dystrophy' [11,12]. Both
continuous pain and signs of 'cold dystrophy' can lead to
assumed irreversible disuse. The contribution of endothe-
lial derived factors could be crucial; endothelin-1 (ET-1) is
a proven potent vasoconstrictive agent that is also
believed to affect hyperalgesia [13], muscle weakness and
movement disorders [14], and oedema [15].
In contrast to skeletal muscle resistance vessels, ET-1 con-
tributes to the maintenance of skin microvascular tone
through both ETA and ETB receptor-mediated vasocon-
striction [16]. The initially produced pro-inflammatory
cytokines tumour necrosis factor alpha (TNF-α) and inter-
leukin-6 (IL-6) also play an important role in the initial
and intermediate phases of the disease [9,17]. TNF-α is
known to decrease eNOS mRNA levels by increasing the
rate of mRNA degradation [18,19]. In the human forearm,
TNF-α promotes ET-1 production [20]. On the other
hand, ET-1 also promotes TNF-α and IL-6 production in
skin-derived mast cells [21], one of the cells which seem
to play a prominent role in the acute phase of CRPS1 [22].
The present study aimed to investigate the involvement of
the vasoactive substances ET-1 and nitric oxide (NO) in
intermediate/cold type CRPS1 in relation to inflamma-
tory mediators.
2. Methods
2.1. Subjects
For this study 29 patients (6 males, 23 females; mean age
48 ± 11.3 (SD) years) with CRPS1 in one hand according
to the criteria of Bruehl [3], were included. The patients
had a mean duration of their disease of 2.8 ± 1.4 (SD)
years, all being in the intermediate phase between the
acute inflammatory stage and the early atrophic stage.
The mean disease activity of the patients was 35 on a scale
of 0–100, calculated using the impairment sum score
according to Oerlemans [23], representing a low to
medium disease activity.
The Medical Ethics Committee of the Erasmus MC
approved the study protocol (MEC 2004-159). The
research was carried out in accordance with the Declara-
tion of Helsinki (2000) of the World Medical Association
and written informed consent was obtained from all par-
ticipants.
2.2. Blister fluid collection
Artificial blisters were induced by means of a suction
method [9,17,24-27]. A 3-hole (5 mm diameter per hole)
skin suction chamber was positioned on the skin of the
upper extremity, on the dorsal side of the involved hand
and on the flexor side of the contralateral forearm. The lat-
ter site was used in all our previous studies to reduce the
inconvenience for the patient and to enable a comparison
between our studies [5,9,17,22,28,29].
A vacuum of 300 mm Hg negative pressure was applied
with an Atmoforte 350A aspirator pump (ATMOS Mediz-
intechnik, Lenzkirch, Germany), which was reduced after
15 minutes to 250 mm Hg and again, 15 minutes later,
reduced to 200 mm Hg. This negative pressure was main-
tained until blisters containing sufficient fluid had been
developed, but not longer than 2.5 hours. The contents of
the blisters were punctured and pooled from each side
into a 1.5 ml Eppendorff conical polypropylene tube and
centrifuged for 5 minutes at 1600 × g. The mean recovery
of supernatants from control blisters was 144 ± 17 (±
S.E.M.) µl fluid, and 106 ± 14 µl blister fluid from the
CRPS1 side. All samples were stored in 1 ml conical poly-
propylene tubes at -80°C until analysis.
2.3. Measurements of cytokines
In order to determine the contribution of pro-inflamma-
tory cytokines to the disease activity, both IL-6 and TNF-α
were determined [9,17]. Blister fluid samples were diluted
1:4 in appropriate calibrator diluent assay buffer for the
direct measurement of cytokines. Cytokine assays were
performed following the manufacturer's protocol (Pelik-
ine™ human ELISA compact kits for IL-6 (cat. no. M1906)
and TNF-α (cat. no. M1920), Sanquin, Amsterdam, The
Netherlands). The standard curve ranges and mean calcu-
lated zero signal plus 3 SD for IL-6 were 0–450 pg/ml and
0.2 pg/ml, respectively; and for TNF-α 0–1000 pg/ml and
1 pg/ml, respectively. The requested solutions were pro-
vided with the ELISA compact kits and additional tool kits
(Pelikine-Tool™ set (cat. no. M1980), Sanquin, Amster-
dam, The Netherlands). Following the manufacturer's
step-by-step instructions, at the end of the procedure the
absorbance per well was measured at 450 nm with a
Medgenix ELISA reader. Sample concentrations were cal-

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culated using the appropriate standard calibration lines
and the Softmax® software of the reader.
2.4. Measurement of endothelin-1
ET-1 concentrations were determined in paired blister
fluid samples which were available for 22 patients. Blister
fluids were diluted 1:2 in appropriate diluent assay buffer.
An ELISA-based commercial assay kit was purchased from
R&D Systems (cat. no. QET00, Abingdon, UK). The Quan-
tiGlo ET-1 chemiluminescent immunoassay is a solid
phase ELISA designed to measure ET-1 levels in human
fluids without extraction procedures. It contains synthetic
human ET-1 and antibodies raised against the synthetic
factor, which has been shown to accurately quantitate
human ET-1. A microplate luminometer was used to
measure the intensity of light emitted in RLUs (relative
light units). The standard curve ranged from 0–1000 pg/
ml. Raw data were transferred to a personal computer in
which a log concentration-logit RLU equation was per-
formed after which sample concentrations were calcu-
lated. The minimal detectable concentration was 0.16 pg/
ml.
2.5. Measurement of nitric oxide
Sufficient remaining sample volume was available from
17 paired blister fluid samples to perform the assay proce-
dure for the quantitative determination of nitrate and
nitrite concentrations. Samples were diluted 1:1 in appro-
priate diluent assay buffer. A complete assay kit was
obtained from R&D Systems (cat. no. DE1500, Abingdon,
UK). This assay determines NO based on the enzymatic
conversion of nitrate to nitrite by nitrate reductase. The
reaction is followed by a colorimetric detection of nitrite
as an azo dye product of the Griess reaction. At the end of
procedure the chromophoric azo derivative which
absorbs light at 540 nm is detected in a Medgenix ELISA
reader, after which sample concentrations are calculated
using the appropriate standard calibration line by means
of the Softmax® software of the reader. The standard curve
ranges for nitrite and nitrate were respectively 0–200 and
0–100 nmol/ml and the minimal detectable concentra-
tions were respectively 0.22 and 0.54 nmol/ml. Sample
concentrations were expressed as total NOx in nmol/ml.
2.6. Statistical analysis
The Wilcoxon signed ranks test was used for comparisons
between measurements in blister fluid obtained from the
CRPS1 and the contralateral extremity. Correlation coeffi-
cients between different parameters were determined by
the Spearman's test for untransformed data. SPSS (version
14) for Windows was used and the significance level was
set at p < 0.05.
3. Results
Blister fluid
Pro-inflammatory cytokines IL-6 and TNF-α and vasoac-
tive ET-1 and NOx were determined in blister fluid
obtained from the involved CRPS1 limb and the contra-
lateral limb.
In Figures 1 and 2 the distributions of blister concentra-
tions have been plotted as a correlation between CRPS1
samples (Y-axis) and contralateral samples (X-axis). Suc-
cessively (Fig. 1a) IL-6, (Fig. 1b) TNF-α, (Fig. 2a) ET-1 and
(Fig. 2b) NOx are displayed. In each plot the straight line
indicates per definition a CRPS to contralateral ratio of
1.00 in case no difference was observed. Both in the IL-6,
TNF-α and ET-1 distribution plots, the main number of
data-points are located in the upper-left triangle, indicat-
ing a CRPS to contralateral ratio above 1.00. On the con-
trary, in the NOx distribution plot, the main number of
data points are located in the lower-right triangle, indicat-
ing a CRPS to contralateral ratio less than 1.00.
Box plots of these data, indicating the median, 25–75%
interval and the ranges of values, excluding outliers are
presented in Figures 1c and 2c.
The Wilcoxon signed ranks test was used to assign differ-
ences in paired samples (blister fluid obtained from the
CRPS1 and the contralateral extremity in the same
patient).
Increments in the paired sample tests of IL-6, TNF-α and
ET-1 were significant (p-values of 0.001, 0.003 and 0.002,
respectively), whereas NOx in paired samples was signifi-
cantly decreased (p-value 0.044).
Spearman's correlation coefficients between blister fluid
parameters were determined. Significant correlations
between IL-6, TNF-α and ET-1 were found (0.79, 0.44 and
0.67 for IL-6/TNF-α, IL-6/ET-1 and TNF-α/ET-1, respec-
tively and p-values of p < 0.001, p = 0.039 and p = 0.001,
respectively).
4. Discussion
This is the first study to investigate levels of ET-1 and NOx
in blister fluids obtained from CRPS1-involved extremi-
ties. Apart from a study comparing ET-1 with disease
severity in the skin of psoriatic patients [30], only Eisen-
berg et al. have investigated the possible contribution of
ET-1 in CRPS [31]. In that study, ET-1 concentrations were
determined in venous plasma from the contralateral limb
of CRPS1 patients and did not differ significantly from
values found in patients with other painful conditions
and in healthy controls. The ET-1 levels found in controls
and patients (mean 2.7 and 3.2 pg/ml) were, however,
much higher than those reported in the literature [32,33].

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Distribution of IL-6 and TNF-α
Figure 1
Distribution of IL-6 and TNF-α. Distribution of a) IL-6 data and b) TNF-α data from 29 CRPS patients, from both the
involved and the contralateral extremity. In each plot, the straight line indicates per definition a CRPS to contralateral ratio of
1.00 in case no difference was observed. Values are plotted on logarithmic scales. c) Box plots of these data, indicating the
median, 25–75% interval and the ranges of values, excluding outliers. The Wilcoxon signed ranks test was used to assign differ-
ences in paired samples (blister fluid obtained from the CRPS1 and the contralateral extremity in the same patient).

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Distribution of Endothelin 1 and Nitric Oxide
Figure 2
Distribution of Endothelin 1 and Nitric Oxide. Distribution of a) ET-1 data from 22 CRPS patients and b) NOx data from
17 CRPS patients. In each plot, the straight line indicates per definition a CRPS to contralateral ratio of 1.00 in case no differ-
ence was observed. Values are plotted on linear scales.c) Box plots of these data, indicating the median, 25–75% interval and
the ranges of values, excluding outliers. The Wilcoxon signed ranks test was used to assign differences in paired samples (blister
fluid obtained from the CRPS1 and the contralateral extremity in the same patient).