<title>1. INTRODUCTION</title> <p>Complex regional pain syndrome 1 (CRPS) is defined as
“a syndrome in which the central nervous system representations of the
somatosensory, somatomotor, and sympathetic systems are altered concomitantly
with important peripheral changes such as edema, signs of inflammation,
sympathetic-afferent coupling, and trophic changes” [##REF##16426757##1##]. The manner in which the peripheral and central
changes interact is only partly understood [##REF##16426757##1##]. During the chronic stage of CRPS, increased
vasoconstriction [##REF##7596630##2##],
tissue hypoxia [##REF##12855324##3##], and metabolic tissue acidosis [##REF##10770524##4##, ##REF##11071503##5##]
indicate that microcirculation is impaired, which affects the nutritive blood
flow in superficial and deep tissues [##UREF##0##6##, ##REF##15494189##7##].</p> <p>The endothelium modulates vascular
tone by releasing endothelium-derived vasodilators including nitric oxide (NO),
prostacyclin, bradykinin, and endothelium-derived hyperpolarizing factor; and
vasoconstrictors, such as endothelin-1 (ET-1) and angiotensin II, in response
to a number of biochemical and physical stimuli [##REF##12652159##8##]. Growing evidence [##REF##16924021##9##] suggests that endothelial
dysfunction is the main cause of diminished blood flow in chronic cold CRPS.</p> <p>Although numerous papers about CRPS have been
published since its first description in 1545 [##UREF##1##10##], literature regarding the associated pathological
alterations in blood vessels is scarce. The few papers on this topic describe
clearly visible abnormalities of the entire microvascular system, including an
increase in the number of capillaries [##REF##56891##11##, ##REF##6194403##12##],
endothelial swelling, and changes in the vessel laminal wall [##REF##6864679##13##]. The impressive capillary changes range from severely
thickened basal membrane with intimal vacuolization, perivascular edema, and debris from pericytes between the
basal membrane layers, to necrosis [##REF##77594##14##, ##REF##9674773##15##].
Greatly thickened multilaminated walls were found, considerably reducing the
inner diameter of the vessel [##REF##6194403##12##, ##REF##16427199##16##].
Endothelial cells with a shrunken appearance and capillaries with only
endothelial cell debris in the lumina were observed, while other capillaries
could be traced by the thickened basal membrane only, lacking the presence of
other cellular remnants [##REF##9674773##15##].</p> <p>Histochemical procedures [##REF##8866163##17##] and immunofluorescence [##REF##16427199##16##]
have been used to investigate the distribution of cutaneous nerve fibers, but
there are no reports of using immunohistochemical staining to evaluate
endothelial dysfunction in CRPS. Key elements in the function of the endothelium
are NO and ET-1. The loss of endothelium-dependent NO mediated vasodilation
occurs early during endothelial dysfunction [##UREF##2##18##]. Since NO has a half-life time of only 3–5 seconds [##REF##1852778##19##], we measured endothelial nitric oxide synthase
(eNOS), which is a constitutional endothelial cell enzyme that produces NO from
L-arginine. Increased production and/or activity of ET-1 may participate in
several pathologic states related to a dysfunctional endothelium [##REF##1886637##20##].</p> <p>The aim of this study was to examine the distribution
of eNOS and ET-1 in relation to vascular density represented by the endothelial
marker CD31-immunoreactivity in skin tissue from amputated limbs of patients
with CRPS.</p>
<title>2. METHODS</title> <title>2.1. Patients</title> <p>Human tissue was obtained from the surgically
amputated extremities of two patients diagnosed with CRPS type 1, according to
the Bruehl diagnostic criteria [##REF##10353502##21##], at the Pain Treatment Centre of the Erasmus MC. Both
patients gave written informed consent prior to the amputation. A lower limb
was amputated in patient A and an upper limb in patient B. Skin samples,
harvested immediately following the amputations, were taken from the dorsal
side of the foot of patient A and from the dorsal side of the hand of patient
B; both of these samples were categorized as distal tissue samples. Proximal
tissue samples were harvested from near the cutting face, taking tissue that
appeared to be the least diseased.</p> <p>Patient A was a 46-year-old female who developed CRPS
in her right leg after an electromyography recording in 1996. One year later,
symptoms appeared in the left leg, and in 1998, in her right hand. The
patients’ complaints persisted and despite intense rehabilitation, both legs
and the right hand became nonfunctional. There was edema in both legs, and
there were severe contractures and pain in the legs and hand. Walking was
impossible and she relied on a wheelchair for transport. During a visit to our
clinic in 2003, muscle force, reflexes, and coordination could not be tested
and there was hyperesthesia in both legs and the hand. She was treated with
paracetamol, amitriptyline, durogesic, mannitol, and baclofen, and received an epidural
block with marcaine and fentanyl. Due to continuous infections (erisipelas) in
both legs in June 2003, antibiotics were prescribed but the infections were
resistant to therapy. Therefore, the left leg had to be amputated in October
2003, followed by the right leg one week later. Postoperative healing was
complicated by pressure ulcers and she was released from the hospital using
paracetamol and tramadol for pain control. A video thermographic recording was
not made, but the examining physician described both legs as very cold.</p> <p>Patient B was a 38-year-old female who developed CRPS
in the right hand in December 2002 as a consequence of a metacarpal II
fracture. After 10 days in a plaster cast, she showed severe symptoms of CRPS
with pain, edema, and contractures. Despite extensive pharmacotherapy to reduce
pain and inflammation, improve peripheral blood flow, and relieve the
spasticity, the patient developed severe dystonia in the right hand. The
dystonia and pain made conventional nail care impossible; therefore, nail
clipping was performed under general anesthesia every few months. The patient
had contractures in her wrist and fingers, spasms of the musculature, burning
pain, allodynia and hyperalgesia, edema, increased transpiration, excessive
hair growth, and a darker skin color on the affected limb. A video
thermographic recording [##REF##14735366##22##, ##REF##18165590##23##]
showed that the CRPS extremity was 6.5°C colder than the contralateral
unaffected control. Shoulder functioning was normal. Spinal cord trial
stimulation in February 2004 was not successful. At the request of the patient,
who could no longer cope with the burden of frequent general anesthesia, upper
arm amputation was performed in May 2006. There were no postoperative complications
and the patient experienced a dramatic reduction in pain.</p> <title>2.2. Immunohistochemical staining for CD31 and eNOS</title> <p>In order to demonstrate the vascular state of the CRPS
skin tissues, the endothelial marker CD31 was visualized by immunoreactive (IR)
staining.</p> <p>Frozen skin tissue samples were cut in serial 6 <italic>μ</italic>m
sections, transferred to poly-L-lysine-coated microscope slides (Menzel-Glaser,
Omnilabo, Breda, the Netherlands), air dried, and stored at −80°C. For immunohistochemical staining,
sections were thawed, fixed in acetone for 10 minutes, and rinsed with
phosphate buffer saline (PBS, pH 7.8). The staining procedure was conducted in
a half-automatic stainer (Sequenza, Shandon Scientific, Zeist,
the Netherlands). The slides were incubated with 10% normal goat serum (NGS; Sanquin, Amsterdam, the Netherlands)
for 10 minutes and then with primary mouse antihuman antibodies against CD31
(JC/70A Dako, Glostrup, Denmark) or eNOS (SA-258 BIOMOL International
L.P., Exeter, UK) for 60 minutes. Both antibodies
had been diluted in 1% blocking buffer (Blocking Reagent, Roche Diagnostics
GmbH, and Mannheim, Germany). After each incubation
step, the slides were rinsed with PBS for 5 minutes. After incubation with the
primary antibodies, sections were rinsed and incubated with biotinylated goat
antimouse antibodies (BioGenex, Klinipath, Duiven, the Netherlands) and 10%
normal human serum (NHS; Sanquin) for 30 minutes. This step was followed by
incubation with alkaline phosphatase-conjugated streptavidin (Biogenex,
Klinipath) and 10% NHS for another 30 minutes. Slides were rinsed with both PBS
and TRIS buffers (TRIS HCl 0.1 mol/L, pH 8.5), and then incubated with new
fuchsine substrate (Chroma, Kongen, Germany) diluted in TRIS buffer. Finally, the sections were washed with PBS, counterstained
with Gill’s hematoxylin (Merck, Darmstadt, Germany) for 30 seconds, rinsed with
tap water, dried, and embedded in VectaMount (Vector, Burlingame, Calif, USA).</p> <title>2.3. Immunohistochemical double staining for ET-1 and CD31</title> <p>After fixing in acetone and washing with PBS,
endogenous peroxidase was blocked with 0.1% sodium azide and 0.03% hydrogen
peroxide in PBS for 30 minutes. Sections were rinsed and incubated with 10% NGS
for 10 minutes, followed by incubation with rat antihuman ET-1 (3G10, R&amp;D systems,
Abingdon, UK) for 60 minutes. The sections were rinsed and incubated with goat
antirat antibody conjugated with alkaline phosphatase (Sigma, St. Louis, Mo, USA) and 10% NHS for 30 minutes.
Thereafter, the slides were rinsed and incubated with mouse antihuman CD31 for
60 minutes. Then sections were rinsed and incubated with biotinylated goat
antimouse antibodies and 10% normal human serum (NHS) and 10% normal rat serum
(Sanquin) for 30 minutes. After washing, the slides were incubated with
streptavidin-conjugated peroxidase (Biogenex, Klinipath) and 10% NHS for
another 30 minutes. After rinsing with PBS and substrate TRIS buffer, slides
were incubated for 30 minutes in fast blue substrate (Sigma). Finally, sections
were rinsed and incubated with peroxidase nova red substrate (Vector) for 5
minutes, rinsed with PBS, and embedded in VectaMount.</p> <title>2.4. Quantification of the immunoreaction</title> <p>ENOS and CD31 staining was performed
on adjacent slides of serial sections. Slides were examined using a Leica
microscope fixed with a Leica DC500 camera for digitizing images. Semiquantitative
evaluation of the different markers was performed by counting the number of
positive blood vessels in the dermis of two sections each from both the distal
and proximal specimens. After measuring the total area of the dermis using the
Leica imaging analysis system, the number of positive blood vessels per square
millimeter was calculated. In the case of ET-1 and CD31 double staining, we
determined the number of ET-1 positive cells in the dermis and the percentage
of positive ET-1 blood vessels on the total number of blood vessels.</p>
<title>3. RESULTS</title> <title>3.1. Vascular status in CRPS</title> <p>The qualitative differences between the distal and
proximal specimens are shown in ##FIG##0##Figure 1(a)##. These differences were confirmed
by qualitative image analysis of the sections. In both patients, endothelial
immunoreactivity was more prominent in distal than in proximal specimens. In
patient A, the mean number of CD31-IR capillaries in distal tissue was 43 blood
vessels/mm<sup>2</sup> versus 14 blood vessels/mm<sup>2</sup> in proximal
tissue; in patient B, the means were 39 and 19 blood vessels/mm<sup>2</sup>,
respectively.</p> <title>3.2. eNOS immunoreactivity</title> <p>eNOS immunoreactivity of capillaries and other
small-diameter blood vessels was prominent in the distal specimens (see ##FIG##0##Figure 1(b)##).
The measure of eNOS-IR endothelium in patient A was 23 blood vessels/mm<sup>2</sup> in distal tissue versus 7 blood vessels/mm<sup>2</sup> in proximal tissue; in
patient B, the means were 20 and 13 blood vessels/mm<sup>2</sup>, respectively.</p> <title>3.3. Ratios</title> <p>The ratios of eNOS/CD31-IR vessels were similar. In
patient A, the mean ratios were 53% and 50% in distal and proximal tissues,
respectively, whereas in patient B, the mean ratios were 51% and 68%,
respectively.</p> <title>3.4. Endothelin-1 positive cells</title> <p>The mean number of ET-1-positive cells was determined
in both the dermis and the blood vessels (##FIG##1##Figure 2##). In patient A, there were
81 ET-1-positive dermis cells/mm<sup>2</sup> in distal tissue versus 22
ET-1-positive dermis cells/mm<sup>2</sup> in proximal tissue; in patient B,
there were 42 and 12 ET-1-positive dermis cells/mm<sup>2</sup>, respectively.
The mean percentages ET-1-positive blood vessels were similar; in patient A,
there were 70% ET-1-positive blood vessels in the distal tissue and 69% in the
proximal tissue; in patient B, the mean values were 78% and 63%, respectively.</p>
<title>4. DISCUSSION</title> <p>Because of the increased risk of an overreaction to a
skin biopsy [##REF##9674773##15##],
we could not take skin tissue samples from CRPS-affected limbs in the usual
manner. Therefore, we used specimens taken from amputated limbs, which are
seldom available. Proximal and distal specimens from the amputated leg or arm
from two different patients were dissected out immediately after the
amputation, and deep frozen in liquid nitrogen. As specimens from the
contra-lateral side or healthy tissue were not available for comparison, we
considered the distal specimens as the most-affected and the proximal specimens
as non- or least-affected tissue. This is the first study that investigates the
distribution of eNOS and ET-1 in tissue of CRPS patients, therefore we decided
to limit the study to skin tissue, following previous observations in skin
blister fluid obtained from CRPS patients. Expanding the study to muscle and/or
nerve tissue might have provided additional insights in mechanisms underlying
CRPS.</p> <p>Tissue blood distribution is altered in patients with CRPS. It is generally accepted that
during the course of this disease, and partly due to disuse, tissue ischemia
will occur, leading to chronic pain [##REF##16393336##24##]. However, until now, there was no evidence suggesting that these patients have endothelial
dysfunction or impaired angiogenesis in response to ischemia. We observed
regional differences in the expression of endothelial markers in the
CRPS-affected limbs. Information about the normal distribution of these markers
between proximal and distal upper or lower extremities is not available. Expression
of CD31, eNOS, and ET-1 was highest in the distal specimens, representing the
most affected samples of the diseased limbs. However, the eNOS/CD31 ratios were
similar, ranging from 51% to 68%. Palatka et al. [##REF##16036527##25##]
reported an eNOS/CD31 ratio of 92% in healthy mucosal biopsies, whereas this
ratio was 8% in tissue samples from patients with Crohn’s disease and 82% in
samples from patients with ulcerative colitis, which suggests that eNOS
activity is diminished in disease-affected endothelial cells. Although values
from mucosa may not be comparable to the skin tissue values in our study, the
suggestion is made that eNOS activity was diminished in our patients, but not
dramatically. Moreover, we observed no distinct regional differences,
coinciding with the observations that during the course of the disease, pain
spreads through the entire limb and that CRPS may also occur in the contra
lateral limb and/or the other extremities [##REF##16772794##26##]. It has been shown that during hypoxia, expression of
both endothelial cell markers CD31 and eNOS increases [##REF##17541946##27##]. In the case of ischemia, eNOS is essential for
promoting collateral growth in order to restore blood distribution to the
tissues [##REF##11641268##28##, ##REF##17692499##29##]. Therefore, in patients with CRPS, supplementing NO precursors will not provide
benefit because eNOS activity is compromised. Treating patients with chronic
CRPS with an endothelium-independent NO donor might be a more effective
strategy [##REF##18180642##30##].</p> <p>Our data confirmed the previously reported increase in capillary density in CRPS-affected tissue [##REF##56891##11##, ##REF##6194403##12##], and showed a higher number of ET-1-positive blood vessels/mm<sup>2</sup> in
distal versus proximal specimens. There were marked differences (3 to 4 fold)
in the number of ET-1-positive cells between the distal and proximal dermis.
ET-1 promotes proliferation and migration of endothelial cells via endothelin-B
receptors [##REF##11863336##31##]. These migrated cells are associated with swelling
and laminal wall disruptions [##REF##6864679##13##]. The pronounced increase in ET-1 expression in the
distal limbs confirms earlier work by our group in which we found elevated
levels of ET-1 in the superficial blister fluids harvested from the distal
region of CRPS limbs [##REF##16393336##24##]. However, alterations in the number of ET-1-positive
cells and skin blister fluid concentrations do not necessarily reflect an
increase in the number of endothelin-B receptor-binding CD31-positive
endothelial cells [##REF##11863336##31##]. Nevertheless, ET-1 blockers could provide relief.</p> <p>This study was limited to only two CRPS type 1 patients, and lacks appropriate control in the form of
contralateral tissue, or a normal distribution of these markers. Both patients
were chronic and have been treated with many medical and interventional
procedures. Conclusions can therefore only apply to potential mechanisms that
maintain chronic CRPS, like severe vasoconstriction, blood supply redistribution
due to abnormal blood flow shunting with hypoperfusion in nutritive vessels,
hypoxia, lactate increase, and acidosis [##REF##16426757##1##].</p> <p>In conclusion, we found an indication that endothelial
dysfunction plays a role in chronic CRPS. In comparison to proximal specimens, we
found an increased number of migrated endothelial cells as well as an increase
of eNOS activity in distal dermis specimens.</p>
<p>Recommended by Philipp Lepper</p> <p>
<italic>Background and Objectives</italic>. Impaired microcirculation during the chronic stage of complex regional pain syndrome (CRPS) is related to increased vasoconstriction, tissue hypoxia, and metabolic tissue acidosis in the affected limb. Endothelial dysfunction is suggested to be the main cause of diminished blood flow. The aim of this study was to examine the distribution of endothelial nitric oxide synthase (eNOS) and endothelin-1(ET-1) relative to vascular density represented by the endothelial marker CD31-immunoreactivity in the skin tissue of patients with chronic CRPS. <italic>Methods</italic>. We performed immunohistochemical staining on sections of skin specimens obtained from the amputated limbs (one arm and one leg) of two patients with CRPS. <italic>Results</italic>. In comparison to proximal specimens we found an increased number of migrated endothelial cells as well as an increase of eNOS activity in distal dermis specimens. <italic>Conclusions</italic>. We found indications that endothelial dysfunction plays a role in chronic CRPS.</p>
<title>ACKNOWLEDGMENTS</title> <p>We appreciate the skillful assistance of Dr. Joan
Holstege (Department of Pathology), Feikje Wesseldijk, and Emmy van Bodegraven.
The study was performed as a part of the TREND (Trauma-Related Neuronal
Dysfunction) knowledge consortium, which integrates research on CRPS1.
Personnel costs of George Groeneweg were funded by a Dutch government grant
(BSIK03016).</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>(a) CD31-immunoreactive vessels in skin tissue from
the amputated arm of CRPS patient A. CD31-positive blood vessels are stained
red. (b) eNOS staining of a serial section. eNOS-positive blood vessels are
stained red.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Double staining for CD31 and ET-1 in skin tissue from
the amputated leg of CRPS patient B. CD31-positive blood vessels are stained
red and ET-1-positive cells are stained blue.</p></caption></fig>
<graphic xlink:href="MI2008-680981.001"/> <graphic xlink:href="MI2008-680981.002"/>
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Mediators Inflamm. 2008 Jul 20; 2008:680981