The Journal of Pain, Vol 10, No 11 (November), 2009: pp 1161-1169
`Available online at www.sciencedirect.com
`
`Role of NFkB in an Animal Model of Complex Regional Pain
`Syndrome–type I (CRPS-I)
`Marissa de Mos,* Andre´ Laferrie` re,y Magali Millecamps,y,z Mercedes Pilkington,x
`Miriam C. J. M. Sturkenboom,* Frank J. P. M. Huygen,{ and Terence J. Coderrey,z,x,#,,
`* Pharmacoepidemiology Unit, Department of Medical Informatics & Epidemiology and Biostatistics.
`y Department of Anesthesia, McGill University, Montre´ al, Quebec, Canada.
`z Alan Edwards Center for Research on Pain, McGill University, Montre´ al, Quebec, Canada.
`x Psychology, McGill University, Montre´ al, Quebec, Canada.
`{ Department of Anesthesiology, Erasmus Medical Center, Rotterdam, the Netherlands.
`# Neurology and Neurosurgery, McGill University, Montre´ al, Quebec, Canada.
`, McGill University Health Care Research Institute, Montre´ al, Quebec, Canada.
`
`Abstract: NFkB is involved in several pathogenic mechanisms that are believed to underlie the
`complex regional pain syndrome (CRPS), including ischemia, inflammation and sensitization. Chronic
`postischemia pain (CPIP) has been developed as an animal model that mimics the symptoms of CRPS-I.
`The possible involvement of NFkB in CRPS-I was studied using CPIP rats. Under sodium pentobarbital
`anesthesia, a tourniquet was placed around the rat left ankle joint, producing 3 hours of ischemia,
`followed by rapid reperfusion (IR injury). NFkB was measured in nuclear extracts of muscle and spinal
`cord tissue using ELISA. Moreover, the anti-allodynic (mechanical and cold) effect was tested for sys-
`temic, intrathecal, or intraplantar treatment with the NFkB inhibitor pyrrolidine dithiocarbamate
`(PDTC). At 2 and 48 hours after IR injury, NFkB was elevated in muscle and spinal cord of CPIP rats
`compared to shams. At 7 days, NFkB levels were normalized in muscle, but still elevated in spinal
`cord tissue. Systemic PDTC treatment relieved mechanical and cold allodynia in a dose-dependent
`manner, lasting for at least 3 hours. Intrathecal—but not intraplantar—administration also relieved
`mechanical allodynia. The results suggest that muscle and spinal NFkB plays a role in the pathogen-
`esis of CPIP and potentially of human CRPS.
`Perspective: Using the CPIP model, we demonstrate that NFkB is involved in the development of
`allodynia after a physical injury (ischemia and reperfusion) without direct nerve trauma. Since CPIP
`animals exhibit many features of human CRPS-I, this observation indicates a potential role for
`NFkB in human CRPS.
`ª 2009 by the American Pain Society
`Key words: Chronic postischemia pain, CPIP, pyrrolidine dithiocarbamate, PDTC, ischemia, reperfusion,
`inflammation, neuropathic pain.
`
`Complex regional pain syndrome (CRPS) is a painful
`
`and disabling complication of an injury, for exam-
`ple, a fracture or sprain, which affects the distal
`end of the injured extremity. CRPS patients can be classi-
`fied into 2 subtypes, based on the presence (type II) or ab-
`sence (type I) of direct nerve injury. The majority of CRPS
`
`Received October 8, 2008; Revised March 10, 2009; Accepted April 19,
`2009.
`Supported by the Dutch CRPS patient association (Stichting Esperance),
`the Canadian Institutes of Health Research, and the Louise and Allan
`Edwards Foundation.
`Address reprint requests to Dr. Marissa de Mos, Department of Medical
`Informatics, room 2157, Erasmus Medical Center, Dr. Molewaterplein
`50, 3015 GE Rotterdam, The Netherlands. E-mail: m.vrolijk-demos@
`erasmusmc.nl
`1526-5900/$36.00
`ª 2009 by the American Pain Society
`doi:10.1016/j.jpain.2009.04.012
`
`patients are considered to suffer from type I. CRPS is as-
`sumed to evolve from several pathological mechanisms,
`including oxidative stress,5,29 classic4,7,23,24 and neuro-
`genic4,7 inflammation, and autonomic17 and sensory
`nerve system alterations.54 A previously described auto-
`mated analysis of literature has revealed that the tran-
`scription factor nuclear
`factor kappa B (NFkB)
`is
`involved in all these disease mechanisms.21 For example,
`affected limbs of human CRPS patients show signs of
`chronic ischemia,29,53 which can induce NFkB activation,
`mediated by the formation of reactive oxygen species
`(ROS) and peroxinitrite.16,20 Inflammatory mediators, in-
`cluding tumor necrosis factor alpha (TNFa), interleukin-1
`(IL-1), and IL-6 have been demonstrated in blister23 and
`spinal cord fluid1 of CRPS patients, and can activate or
`are activated themselves by NFkB.9,26,48 Moreover, NFkB
`
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`interacts with neuropeptides such as calcitonin gene re-
`lated protein (CGRP)35 and substance P (SP) 36 that have
`been found abnormally expressed during CRPS.7,34 Fi-
`nally, animal studies have revealed that NFkB is involved
`in spinal plasticity39 and the development of neuro-
`pathic pain.50,51
`NFkB resides in the cytosol of many different cell types
`and can be activated by many triggers, including ultravi-
`olet radiation, free radicals, cytokines, and products of
`bacterial and viral infections.12 Upon activation, inhibi-
`tory kappa B (IkB) protein is cleaved from the NFkB com-
`plex, which subsequently forms dimers that are capable
`of passing through the nuclear membrane.
`In the
`nucleus, NFkB promotes the transcription of a wide vari-
`ety of genes. NFkB has been attracting considerable sci-
`entific attention over the past years as a key factor in
`inflammation, apoptosis, and neuronal-glial
`interac-
`tions.3 Excessive NFkB activity has been attributed to
`the pathogenesis of several chronic inflammatory disor-
`ders and oncological diseases.52 Since 2005, the NFkB
`pathway inhibitor bortezomib has been applied success-
`fully in the therapy of multiple myeloma and other malig-
`nancies.45 An NFkB inhibitor that is frequently applied in
`research settings is pyrrolidine dithiocarbamate (PDTC).
`PDTC is a chemical with metal chelating and antioxidant
`properties, and inhibits NFkB activity by blocking the
`phosphorylation of IkB.37,42 Systemically administrated
`PDTC and other dithiocarbamates have been shown to
`be protective and therapeutic in animal models for ische-
`mia and reperfusion (IR) injury22, acute inflammation,15
`and neuropathic pain.31
`The chronic posti-schemia pain (CPIP) model is an ani-
`mal model for the study of molecular mechanisms that
`underlie the sensory disturbances occurring in rats after
`IR injury of the hind paw. CPIP rats display several fea-
`tures that resemble human CRPS, including edema, hy-
`peremia, and the development of mechanical and cold
`allodynia without direct nerve injury.14 The CPIP model
`has therefore been proposed as animal model for CRPS
`type-I. The aim of the present study was investigate the
`involvement of NFkB in CPIP, and potentially the patho-
`genesis of CRPS, by measuring NFkB levels and assessing
`the anti-allodynic effect of NFkB inhibition by PDTC in
`rats after IR injury.
`
`Methods
`
`Study Design
`NFkB levels were measured in muscle and spinal cord
`tissue of CPIP animals and compared to sham animals
`at 2 hours (CPIP, N = 15; sham, N = 9), 48 hours (CPIP,
`N = 15; sham, N = 9–10) and 7 days (CPIP, N = 6; Sham,
`N = 7) after IR injury.
`The effect of NFkB inhibition by systemic PDTC admin-
`istration on allodynia was studied in CPIP rats using 4
`treatment groups (saline and 10, 30, and 100 mg/kg of
`PDTC; 10 rats per group) and in sham rats using 2 treat-
`ment groups (saline and 100 mg/kg of PDTC; 10 rats per
`group). PDTC/saline was administered intraperitoneally
`(i.p.) 48 hours after IR injury. Animals were tested for me-
`
`The Role of NFkB in CPIP
`
`chanical and cold allodynia in the ipsilateral hind paw
`just before treatment, and at 30, 60, 90, 120, and 180
`minutes after treatment.
`Additionally, to investigate the site of PDTC effects, in-
`trathecal or intraplantar administrations (250 mg per rat)
`were performed in 2 additional groups of animals (N = 10
`per group) and compared to saline treatment using both
`administration routes (N = 10 per group). Intrathecal in-
`jections (20 mL volume) were performed by L6 lumbar
`puncture under brief anesthesia with isofluorane,
`whereby intrathecal delivery was confirmed by observ-
`ing an injection induced tail-flick.40 Intraplantar injec-
`tions (50 mL volume) were performed in the ipsilateral
`foot of awake animals. Mechanical allodynia was mea-
`sured in both the ipsi- and the contralateral hindpaw
`at 30 and 60 minutes after PDTC administration.
`All treatment and testing procedures were performed
`by a single experimenter per test, who was blinded for
`the CPIP/sham status of rat as well as for the treatment
`status (PDTC or saline). PDTC was obtained from Sigma-
`Aldrich (St. Louis, MO) and was freshly dissolved daily
`in saline.
`
`Animals
`Male Long Evans rats (275–300 g, Charles River, Que-
`bec) arrived at least 5 days before the start of experi-
`ments. They were kept under a 12 hour/12 hour light-
`dark cycle (lights on at 7:00 h) with free access to food
`and water. All experiments were performed during the
`light cycle. Methods were approved by the Animal Care
`Committee at the McGill University, and conformed to
`the ethical guidelines of the Canadian Council on Animal
`Care.
`
`CPIP
`CPIP was induced by ischemia and reperfusion (IR) in-
`jury of the left hind paw as described by Coderre
`et al.14 Briefly, animals were anesthetized over a 3-hour
`period with a bolus (55 mg/kg, i.p.) and chronic i.p. infu-
`sion of sodium pentobarbital for 2 hours (27.5 mg/kg/h).
`After induction of anesthesia, a Nitrile 70 Durometer
`O-ring (O-rings West, Seattle, WA) with a 5.5 mm internal
`diameter was placed around the rat’s left ankle joint.
`After 3 hours the O-ring was cut, allowing reperfusion
`of the hind limb. Sham animals underwent anesthesia
`similar to the CPIP animals, but an O-ring was not placed
`around the ankle.
`
`Tissue Sampling and Preparation
`Animals were euthanized by decapitation under anes-
`thesia with isofluorane. Immediately, muscle samples of
`the superficial plantar layer (one each from the Flexor
`Hallucis Brevis, Flexor Digiti Minimi Brevis and Flexor
`Digitorium Brevis, each weighting between 29 and 50
`mg) and spinal cord samples at L5-L6 (each weighting
`12 to 20 mg) were obtained and quickly frozen in isopen-
`tane, kept on dry ice, and stored at –80C until process-
`ing. Spinal cord samples were sectioned to isolate the
`dorsal half, which contained predominantly the dorsal
`horns, and sectioned again at the midline to isolate
`
`

`

`de Mos et al
`
`ipsilateral and contralateral tissue. Samples were thawed
`at 4C and homogenized either mechanically (muscle) or
`by sonification (spinal cord) in 12 mL/mg tissue of RIPA
`buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1mM
`EDTA, 1% Igepal (Sigma-Aldrich), 1% Sodium deoxycho-
`late and .1% SDS (Ph 7.4), to which was added a 1%
`protease inhibitor cocktail (Sigma-Aldrich). Tissue ho-
`mogenates were centrifuged at 3,000 g for 10 minutes,
`and the supernatant was collected and processed for nu-
`clear fraction extraction following the recommended
`procedure of a commercially produced extraction kit
`(Chemicon Nuclear Extraction Kit; Millipore Corp Biller-
`ica, MA). Briefly, after spinning at 250 g for 5 minutes,
`samples were diluted 1/5 (vol/vol)
`in cytoplasmic
`lysis buffer and incubated at 4C for 20 minutes. The
`homogenates were mechanically sheared by repeatedly
`drawing and ejecting each sample through a series of
`25 ga, 26 ga and finally 27 ga needles and centrifuged
`at 8,000 g for 20 minutes at 4C. Subsequently, the pellets
`were resuspended in nuclear extraction buffer, mechan-
`ically disrupted with a 27 ga needle,
`incubated for
`60 minutes, and then centrifuged at 16,000 g for 6 min-
`utes at 4C, in order to obtain the supernatant that con-
`tained the nuclear fraction. Nuclear fractions were
`concentrated by centrifugal filtration at 14,000 g for 20
`minutes using cellulose filters with a 30 kDa cut-off (Mi-
`crocon YM-30; Millipore Corp). Nuclear fractions remain-
`ing after filtration were collected and diluted in buffer
`to a final volume of 100 mL for muscle and 50 mL for spinal
`samples. Total sample protein content was determined
`by the Bradford method.10 Nuclear extracts were stored
`at –80C until further analysis.
`
`Measurement of NFkB by ELISA
`NFkB measurements were performed using a commer-
`cially supplied NFkB transcription factor binding assay
`(Cayman Chemical, Ann Arbor, MI) according to the man-
`ufacturer’s suggested protocol. Briefly, duplicate 10 mL
`samples of nuclear extract were first incubated overnight
`at 4C in wells precoated with a dsDNA sequence corre-
`sponding to the NFkB consensus motif. The N-terminus
`of NFkB is highly conserved (92% sequence homology be-
`tween rat and human p50 subunit); thus, the NFkB con-
`sensus motif of the assay should bind both human and
`rat p50. After 5 washes, the samples were incubated
`overnight at 4C with a rabbit polyclonal antibody to
`the p50 subunit of NFkB at a final dilution of 1:100
`(sc-7178; Santa Cruz Biotechnology, Santa Cruz, CA).
`The detection antibody is an epitope corresponding to
`amino acids 120-239 mapping at the N-terminus of
`NFkB of human origin recommended for the detection
`of NFkB p50 and p105 and has high cross-reactivity be-
`tween mouse, rat, and human. Subsequently, samples
`were incubated for 60 minutes with an HRP-conjugated
`goat antirabbit secondary antibody (Cayman Chemical),
`followed by colorimetric detection (measured as absor-
`bance at 450 nm, Versamax; Molecular Devices, Sunny-
`vale, CA). After background subtraction, absorbance
`measures were referred to a standard curve obtained
`from a series of duplicate wells containing measured
`
`1163
`
`amounts of human recombinant p50 (Cayman Chemical)
`and then converted to an estimate of the quantity of
`p50/well, which was normalized by dividing the p50 esti-
`mate by the total amount of protein measured in the
`sample.
`
`Mechanical and Cold Allodynia
`Mechanical allodynia was assessed by measuring the
`50% withdrawal response to stimulation with von Frey
`filaments, according to a modified method as described
`by Chaplan et al.11 Briefly, rats were placed in Plexiglas
`cages with a wire grid bottom. Filaments (Stoelting,
`Wood Dale, IL) were applied to the plantar surface of
`the hind paw for approximately 5 seconds in either as-
`cending or descending strength, to determine the fila-
`ment closest to the threshold of response. CPIP rats
`that had not developed mechanical allodynia at 48 hours
`post-IR injury (nonresponders, 50% threshold > 10 g)
`were excluded from further measurements of mechani-
`cal allodynia after PDTC treatment.
`Cold allodynia was assessed using a modification of
`the acetone drop method as described by Choi et al.13
`A drop of acetone was placed on the plantar surface of
`the foot and the response was measured as the amount
`of seconds of nociceptive behavior observed during the
`first minute after acetone application. Again, nonre-
`sponders for cold allodynia at 48 hours post-IR injury
`(pain behavior for 1 second or less) were excluded from
`further measurements of cold allodynia after PDTC
`treatment.
`Mechanical and cold allodynia were tested in the same
`animals, with mechanical allodynia always tested first.
`When both sides were tested, the contralateral side
`was tested before the ipsilateral side.
`
`Statistics
`All statistical analyses were performed using the statis-
`tical package for social sciences (SPSS v.12.0; SPSS Inc, Chi-
`cago, IL) Significance was established at P < .05. Data
`were plotted as the mean 6 standard error of the
`mean (SEM).
`NFkB in tissue from the ipsi- and contralateral side of
`CPIP rats was compared to sham rats using a Mann-Whit-
`ney U test. Ipsi- vs contralateral differences within rats
`were compared using a Wilcoxon signed rank test.
`Baseline mechanical and cold allodynia test results
`were compared with one-way ANOVA. Post-treatment
`differences between groups were analyzed using
`repeated measures ANOVA with a Greenhous-Geisser
`correction for sphericity. In post hoc analyses, each treat-
`ment group was compared to the saline control group
`applying a Bonferoni correction.
`In CPIP rats only,
`decreases in cold allodynia relative (percentage) to
`pretreatment values were calculated and compared to
`the saline control group. For mechanical allodynia,
`a delta area under
`the curve (DAUC)
`relative to
`pretreatment values was calculated over the period of
`observation and compared with the saline group using
`one-way ANOVA followed by a post hoc LSD test.
`
`

`

`The Role of NFkB in CPIP
`
`NFkB seemed to be increased compared to shams, al-
`though this difference was only statistically significant
`for the spinal cord at 48 hours and 7 days after IR injury.
`Moreover, within CPIP rats there was no significant dif-
`ference in NFkB levels between ipsi- and contralateral
`sides in both muscle and spinal cord.
`
`Systemic PDTC Treatment
`Paw-withdrawal thresholds of the ipsilateral hind paw
`at baseline did not differ between CPIP (N = 40) and sham
`(N = 20) rats (13.2 1/- 3.6 g and 13.4 1/- 3.0 g respectively,
`P = .823). At 48 hours after IR injury, CPIP rats developed
`a decrease in paw-withdrawal threshold (mean 50% von
`Frey threshold of 6.83 6 3.64 g) compared to shams
`(mean 50% von Frey threshold of 11.88 6 3.42 g)
`(P < .0001). Within the CPIP group, 32 rats (80%) dis-
`played a 50% von Frey threshold < 10 and were regarded
`as responders for mechanical allodynia.
`Acetone responses at baseline were similar between
`CPIP and sham rats (1.075 6 1.8 seconds and 1.10 6 1.6
`seconds, respectively, P = .957). Compared to shams
`(2.05 6 3.1 seconds), acetone responses were increased
`in CPIP rats at 48 hrs after IR injury (3.78 6 4.3 seconds),
`although the difference was not significant (P = .113).
`In the CPIP group, 26 rats (65%) displayed pain behavior
`for more than 1 second, and were considered as
`responders for cold allodynia.
`The effect of systemic PDTC treatment at 48 hours
`after IR injury on mechanical allodynia is displayed in
`Fig 2A. A significant main effect of time was observed
`(F(4,121) = 14.3, P < .001), as well as a significant main ef-
`fect of treatment (F(3,28) = 8.1, P < .001), but there was
`no significant time  treatment interaction (F(13,121) =
`1.29, P = 0.229). In post hoc analyses, CPIP rats that had
`received the highest dosage of PDTC (100 mg/kg)
`showed a decrease in mechanical hypersensitivity com-
`pared to the saline group (P = .02) and the group that re-
`ceived the lowest dose of PDTC (10 mg/kg, P = .02). The
`DAUC showed an effect of treatment for the highest
`(100 mg/kg, P = .002) and middle (30 mg/kg, P = .021)
`doses of PDTC, compared to saline controls.
`In the
`sham rats, no significant effect of time was observed,
`but there was a significant main effect of treatment
`(F(1,17) = 7.3, P = .015) and a significant time  treat-
`ment interaction (F(6,102) = 3.1, P = .008). Also within
`sham rats, the DAUC differed between the PDTC and
`the saline-treated group (P = .028).
`Regarding the absolute values for cold allodynia, a sig-
`nificant main effect of time was observed (F(5,104) = 5.7,
`P < .001), but the main effect of treatment (F(3,22) = 2.6,
`P = .079) and the time  treatment
`interaction
`(F(14,104) = 1.7, P = .057) just failed to reach significance.
`In sham rats, there was no significant main effect of time
`(F(3,50) = 1.8, P = .164) or treatment (F(1,17) = 0.8, P =
`.377), nor was there a significant time  treatment inter-
`action (F(3,50) = 1.2, P = 0.318). For CPIP rats only, the rel-
`ative (percentage) decrease in cold allodynia after
`systemic PDTC treatment is depicted in Fig 2B. A signifi-
`cant main effect of treatment was observed (F(3,22) =
`4, P = .020), and post hoc analyses demonstrated
`
`1164
`
`Pre- and posttreatment values within 1 treatment group
`were compared using a Wilcoxon signed rank test.
`
`Results
`
`NFkB in Muscle and Spinal Cord
`The results of NFkB measurements in muscle and spinal
`cord are depicted in Fig 1. Because in sham rats NFkB
`levels at both sides were similar (P = .162 for muscle
`and P = .694 for spinal cord), the right and left side mea-
`sures of sham rats were combined in the comparison to
`CPIP rats. At both 2 and 48 hours after IR injury, NFkB
`was
`increased compared to sham rats
`in muscle
`(P = .004 at 2 hours and P = .020 at 48 hours) as well as spi-
`nal cord (P = .027 at 2 hours and P = .001 at 48 hours) from
`the ipsilateral side of CPIP rats. At 7 days after IR injury,
`NFkB levels in muscle did not differ between CPIP and
`sham rats. However, in spinal cord, the ipsilateral NFkB
`levels from CPIP rats were still elevated (P = .001). Re-
`markably, also on the contralateral side of CPIP rats,
`
`**
`
`*
`
`Sham
`CPIP ipsi
`CPIP contra
`
`2 hrs
`
`48 hrs
`
`7 days
`
`**
`
`*
`
`*
`
`Sham
`CPIP ipsi
`CPIP contra
`
`**
`
`*
`
`2 hrs
`
`48 hrs
`
`7 days
`
`0.14
`
`0.12
`
`0.10
`
`0.08
`
`0.06
`
`0.04
`
`0.02
`
`0
`
`0.50
`0.45
`0.40
`0.35
`0.30
`0.25
`0.20
`0.15
`0.10
`0.05
`0
`
`A
`
`NFκB in ng/μg protein
`
`NFκB in ng/μg proteinB
`
`Figure 1. (A) NFkB levels in muscle from the ipsilateral (ipsi)
`and contralateral (contra) hind paw of chronic post-ischemia
`pain (CPIP) rats compared to shams at 2 hours, 48 hours and 7
`days postreperfusion, measured by ELISA. CPIP ipsilateral at 2
`and 48 hours, N = 15; at 7 days, N = 6. CPIP contralateral at 2
`and 48 hours, N = 15; at 7 days, N = 6. Sham at 2 and 48 hours,
`N = 18; at 7 days, N = 14. *P < .05, **P < .005, Mann-Whitney U
`test compared to shams (B). NFkB levels in spinal cord from the
`ipsilateral (ipsi) and contralateral (contra) side of CPIP rats com-
`pared to shams at 2 hours, 48 hours and 7 days post reperfusion,
`measured by ELISA. CPIP ipsilateral at 2 and 48 hours, N = 15; at 7
`days, N = 6. CPIP contralateral at 2 and 48 hours, N = 15; at 7 days,
`N = 6. Sham at 2 and 48 hours, N = 18; at 7 days, N = 14. *P < .05,
`**P < .005, Mann-Whitney U test compared to shams.
`
`

`

`de Mos et al
`
`1165
`
`ΔAUC
`
`##
`
`#
`
`#
`
`Sham
`saline
`
`Sham
`PDTC
`100
`
`*
`
`*
`
`CPIP
`PDTC
`100
`*
`
`*
`
`1200
`
`600
`
`0
`
`grams*minutes
`
`-600
`
`CPIP
`saline
`
`CPIP
`PDTC
`10
`
`CPIP
`PDTC
`30
`
`*
`*
`
`*
`*
`
`*
`*
`
`16
`
`14
`
`12
`
`10
`
`8 6 4 2 0
`
`A
`
`50% Von Frey treshold
`
`B
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`Relative change (%)
`
`Sham saline
`CPIP saline
`
`Sham PDTC100
`PDTC10
`PDTC30
`
`PDTC100
`
`baseline
`
`before
`treatment
`
`30
`
`60
`
`90
`
`120
`
`180
`
`Time (min)
`
`Saline
`PDTC 10
`PDTC 30
`PDTC 100
`
`*
`
`30
`
`*
`
`60
`
`*
`
`*
`
`*
`
`90
`
`120
`
`180
`
`before
`treatment
`
`Time (min)
`
`Figure 2. (A) Mechanical paw-withdrawal threshold in chronic post-ischemia pain (CPIP) and sham rats after systemic pyrrolidine di-
`thiocarbamate (PDTC) treatment at 48 hours postreperfusion, compared to saline treatment. CPIP saline, N = 7; CPIP PDTC 10 mg/kg,
`N = 9; CPIP PDTC 30 mg/kg, N = 9; CPIP PDTC 100 mg/kg, N = 7; sham saline, N = 10; sham CPIP, N = 10. *P < .05, repeated measurements
`ANOVA followed by a Bonferoni test compared to the saline control group. #P < .05, ##P < .005, one-way ANOVA followed by an LSD
`test compared to the saline control group (B). Relative changes in acetone responses in the ipsilateral hind paw of CPIP rats after sys-
`temic saline or PDTC treatment at 48 hours postreperfusion, compared to acetone responses before treatment. CPIP saline, N = 9; CPIP
`PDTC 10 mg/kg, N = 6; CPIP PDTC 30 mg/kg, N = 4; CPIP PDTC 100 mg/kg, N = 7. *P < .05, repeated measurements ANOVA followed by
`a Bonferoni test compared to the saline control group.
`
`a decrease in cold allodynia in rats that had received the
`highest dose of PDTC compared to saline treatment (P =
`.035) and the lowest PDTC dose (P = .045).
`
`Intrathecal and Intraplantar PDTC
`Treatment
`At baseline, there was no difference in mechanical sen-
`sitivity between the right and left hind paw (13.5 6 2.15
`g and 13.3 6 2.20 g, P = .689), while after IR injury, 26 out
`of 40 rats (65%) had developed mechanical allodynia.
`The effects of intrathecal and intraplantar PDTC treat-
`ment at 48 hours after IR injury on mechanical allodynia
`are depicted in Fig 3. For intrathecal treatment (Fig 3A),
`there was a significant main effect of time (F(2,27) = 21.6,
`
`P < .001), but there was no significant main effect of
`treatment (F(1.14) = 2.6, P = .132) or time  treatment
`interaction (F(2,27) = 2.6, P = .094). However, in the
`PDTC treatment group, the mean 50% VF threshold
`was increased at both 30 minutes (P = .017) and 60 min-
`utes (P = .012) posttreatment, compared to the pretreat-
`ment
`threshold. No pre- vs postdifferences were
`observed in the saline-treatment group (P = .687). The
`DAUC was significantly larger for the PDTC group com-
`pared to the saline group (P = .034).
`For intraplantar PDTC treatment (Fig 3B), a significant
`main effect of time was observed (F(2,14) = 45.9,
`P < .001), but there was no significant main effect of
`treatment (F(1,8) = 0, P = .980) or time  treatment inter-
`action (F(2,14) = 1.9, P = .195). The mean 50% von Frey
`
`

`

`The Role of NFkB in CPIP
`
`ferent tissue types have demonstrated increased NFkB
`activity early after hypoxia, for example, in myocardial
`tissue, brain, hepatic tissue, and skeletal muscle.37,38,42,49
`In all of these studies, the extent of the damage caused
`by IR injury could be attenuated by administration of
`an NFkB inhibitor. Second, CPIP rats display signs of in-
`flammation.14 IR injury is known to provoke a well-docu-
`mented cascade of inflammatory events,8 and NFkB is an
`important mediator in such inflammatory responses.
`Third, CPIP rats develop neuropathic painlike symptoms,
`including mechanical and cold allodynia. Previously, in-
`creased NFkB activity has been demonstrated in animal
`neuropathic pain models, while these symptoms can be
`relieved by an NFkB inhibitor.32,47,51
`The hypothetical involvement of NFkB in CPIP is con-
`firmed by our present observations. NFkB elevation in
`muscle tissue is consistent with a previous report of mus-
`cular NFkB activation upon ischemia by arterial clamp-
`ing.37 On the contrary, increases in spinal NFkB levels
`following peripheral
`IR injury is to our knowledge
`a new finding, although spinal NFkB activation has
`been reported following peripheral nerve section44 and
`nerve inflammation.25,33 These peripheral triggers can in-
`duce an intraspinal cytokine release, a process that may
`be mediated by NFkB.47,51 However, neuropathic painlike
`symptoms and spinal NFkB activation in CPIP rats are sub-
`sequent to IR injury instead of traumatic nerve injury or
`direct immunological stimulation.14 Presumably, IR injury
`can induce pathological responses in the central nervous
`system (CNS) similar to those induced by mechanical or in-
`flammatory nerve damage, through direct activation of
`nociceptors by either reactive oxygen species (ROS) or
`ROS-induced inflammatory reactions. The observation
`in CPIP rats of prolonged spinal NFkB activity (until at
`least 7 days after IR injury), when peripheral levels were
`normalized, suggests that eventually the CNS pathology
`becomes independent of its initial peripheral trigger. This
`is consistent with previous observations showing that
`CPIP rats display ongoing mechanical allodynia at 7
`days after IR injury, while plasma extravasation in the af-
`fected hind paw had been normalized within 24 hours.14
`NFkB activity is also increased in the contralateral mus-
`cle tissue of CPIP rats, although not as profoundly as on
`the ipsilateral side, and within CPIP rats, a significant dif-
`ference between the ipsi- and contralateral sides was not
`observed. A possible explanation may be the spread of
`free radicals and subsequently activated inflammatory
`mediators from the side of IR injury to the opposite
`side by blood circulation. In support of this possibility,
`contralateral allodynia has been found in the CPIP rats
`in the past,14 although not consistently in all studies.41
`It may be that contralateral allodynia depends on spinal
`sensitization which may be mediated by NFkB that is
`increased in the contralateral spinal cord dorsal horn.
`The anti-allodynic effect of systemic PDTC administra-
`tion was clear and dose-dependent. Results from intra-
`thecal administration were less pronounced, and no
`effect was obtained by intraplantar treatment. Presum-
`ably, at 48 hours after IR injury, mechanical allodynia is
`mainly caused by the enhanced central, and not the pe-
`ripheral, NFkB activity. PDTC passes the blood brain
`
`ȈAUC
`
`#
`
`Saline
`ipsi
`
`PDTC
`ipsi
`
`Saline
`contra
`
`PDTC
`contra
`
`300
`
`150
`
`0
`
`-150
`
`grams*minutes
`
`*
`
`*
`
`Saline contra
`Saline ipsi
`
`PDTC contra
`PDTC ipsi
`
`baseline
`
`before
`treatment
`
`Time (min)
`
`30
`
`60
`
`ȈAUC
`
`Saline
`ipsi
`
`PDTC
`ipsi
`
`Saline
`contra
`
`PDTC
`contra
`
`300
`150
`
`0
`
`-150
`
`grams*minutes
`
`Saline contra
`Saline ipsi
`
`PDTC contra
`PDTC ipsi
`
`baseline
`
`before
`treatment
`
`Time (min)
`
`30
`
`60
`
`1166
`
`A
`
`16
`14
`12
`10
`
`02468
`
`50% Von Frey treshold
`
`B
`
`16
`14
`12
`10
`
`02468
`
`50% Von Frey treshold
`
`Figure 3. (A) Mechanical paw-withdrawal
`in
`thresholds
`chronic post-ischemia pain (CPIP) rats after intrathecal treat-
`ment with pyrrolidine dithiocarbamate (PDTC) 48 hours postre-
`perfusion. Saline ipsilateral (ipsi), N = 8; PDTC ipsilateral, N = 8;
`saline contralateral (contra), N = 8; PDTC contralateral, N=8.
`*P < .05, Wilcoxon signed rank test comparing each posttreat-
`ment value with its pretreatment value. #P < .05, one-way
`ANOVA compared to the saline control group (B). Mechanical
`paw-withdrawal thresholds in CPIP rats after intraplantar treat-
`ment with PDTC 48 hours postreperfusion. Saline ipsilateral
`(ipsi), N = 5; PDTC ipsilateral, N = 5; saline contralateral (contra),
`N = 5; PDTC contralateral, N = 5.
`
`thresholds did not differ significantly between before
`and after treatment. Additionally, the DAUC was not sig-
`nificantly different.
`
`Discussion
`We investigated the involvement of NFkB in CPIP. NFkB
`was increased in muscle and spinal cord from CPIP rats
`compared to shams at both 2 and 48 hours after IR injury.
`At 7 days after IR injury, NFkB was equalized to shams in
`muscle, but was still elevated in spinal cord. Systemic
`PDTC administration at 48 hours after IR injury relieved
`mechanical and cold allodynia in a dose-dependent man-
`ner. Mechanical allodynia was also relieved upon intra-
`thecal treatment, but not upon intraplantar treatment.
`Considering previous studies, a role of NFkB in CPIP
`was a plausible expectation. First, several studies in dif-
`
`

`

`de Mos et al
`
`barrier and systemic doses will likely produce CNS effects,
`whereas
`low concentration intraplantar
`injections
`should not. However, it is also possible that we were un-
`able to determine the effective dose for local treatment.
`In sham animals, we observed a slightly decreasing 50%
`von Frey threshold during the course of the experiment
`(Fig 1A), which we believe can be attributed to sensitiza-
`tion by repeated testing. This sensitization did not occur
`in sham rats that were systemically treated with PDTC.
`PDTC may have prevented mechanical sensitization
`upon repeated mechanical stimulation by reducing
`NFkB activity, which was also measured at lower levels
`in the peripheral and central tissues of sham rats.
`In CPIP rats, we observed a central and peripheral in-
`crease of NFkB activity together with allodynia that
`was relieved by administration of the NFkB inhibitor
`PDTC. However, we did not show directly the relation-
`ship between PDTC administration and decreasing
`NFkB activity. Although this might have completed the
`study, we considered such experiments of limited addi-
`tional value, since both a central and peripheral decrease
`in NFkB activity upon systemic PDTC treatment have al-
`ready been demonstrated convincingly by others. For ex-
`ample, Lille et al have demonstrated that systemic PDTC
`administration results in diminished NFkB binding activ-
`ity in muscle tissue,37 and Nurmi et al have demonstrated
`blocking of the otherwise increased NFkB activity upon
`middle cerebral artery occlusion when rats were pre-
`treated with systemically administered PDTC.42
`Few animal models have been used to study the molec-
`ular mechanisms that potentially underlie CRPS. Some of
`these rely on direct nerve injury and are therefore more
`representative for CRPS type II and not for CRPS type
`I.28,30 Animal models that claim to mimic CRPS-I involve
`tibia fracture and casting,19 local infusion of a free radi-
`cal donor,53 interarterial infusion of SP,18 and IR injury
`(CPIP model).14 The IR injury or CPIP model that was
`used in the present study resembles human CRPS-I in sev-
`eral aspects. CPIP rats display features that represent
`both inflammatory and neuropathic painlike symptoms
`of human CRPS. Moreover, CPIP rats express sympatheti-
`cally maintained pain,55 a phenomenon that in the past
`has been considered almost pathognomic for CRPS, al-
`though currently it is acknowledged to be present in
`only a subset of CRPS patients. Similar to CRPS-I patients,
`CPIP rats receive poor pain relief from classical anti-
`inflammatory and anti-neuropathic pain treatments,41
`but respond well to treatment with free radical scaven-
`gers.14 Th