Pain 104 (2003) 75–84
`
`www.elsevier.com/locate/pain
`
`A substance P receptor (NK1) antagonist can reverse vascular and
`nociceptive abnormalities in a rat model of complex regional pain
`syndrome type II
`
`Wade S. Kingerya,b,*, M. Frances Daviesc,d, J. David Clarkc,d
`
`aDepartment of Orthopedic Surgery, Stanford University School of Medicine, Stanford, CA 94304, USA
`bPhysical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304, USA
`cDepartment of Anesthesia, Stanford University School of Medicine, Stanford, CA 94304, USA
`dAnesthesiology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA
`
`Received 23 September 2002; accepted 2 December 2002
`
`Abstract
`
`Sciatic nerve section in rats evokes chronic hindlimb edema, pain behavior, and hyperalgesia, a syndrome resembling complex regional pain
`syndrome (CRPS II) in man. Furthermore, there is an increase in spontaneous protein extravasation in the hindpaw skin of rats after sciatic
`transection, similar to the increased protein extravasation observed in the edematous limbs of CRPS patients. Now we demonstrate that sciatic
`nerve section also generates chronic hindlimb warmth, distal articular tenderness, allodynia, and periarticular osteoporosis, sequelae of nerve
`injury resembling those observed in CRPS. We postulated that facilitated substance P signaling may contribute to these vascular and
`nociceptive abnormalities and attempted to reverse these changes with the long acting substance P receptor (NK1) antagonist LY303870.
`Hindpaw spontaneous extravasation was inhibited by LY303870. Systemic administration of LY303870 also reversed hindpaw edema and
`cutaneous warmth. Intrathecal, but not systemic administration of LY303870 reversed soft tissue and articular mechanical hyperalgesia in the
`hindpaw. Collectively, these data further support the hypothesis that the sciatic nerve transection model closely resembles CRPS and that
`substance P contributes to the spontaneous extravasation, edema, warmth, and mechanical hyperalgesia observed in this model.
`q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.
`
`Keywords: Edema; Neurogenic extravasation; Nerve injury; Complex regional pain syndrome; Osteopenia; Osteoporosis; Substance P
`
`1. Introduction
`
`Nerve injuries can lead to the development of a complex
`regional pain syndrome type II (CRPS II or causalgia)
`(Bonica, 1979; Suntherland, 1978). This syndrome presents
`a baffling array of clinical findings, including an initial
`increase in skin temperature (Baron et al., 1996; Birklein
`et al., 1998; Chelimsky et al., 1995; Wasner et al., 2001),
`increased cutaneous protein extravasation (Oyen et al.,
`1993), distal limb edema (Veldman et al., 1993), hyper-
`algesia (increased sensitivity to painful stimuli) (Veldman
`et al., 1993), distal articular tenderness (Atkins et al., 1989;
`Bryan et al., 1991; Sarangi et al., 1991), allodynia (a painful
`response to a normally non-painful stimuli) (Blumberg and
`
`* Corresponding author. Tel.: þ 1-650-493-5000, ext. 64768; fax: þ 1-
`650-852-3470.
`E-mail address: wkingery@stanford.edu (W.S. Kingery).
`
`Janig, 1994; Verdugo et al., 1994), and periarticular
`osteoporosis (Bickerstaff et al., 1993; Laroche et al., 1997;
`Sarangi et al., 1993). In earlier studies we presented
`evidence that sciatic nerve transection in rats induced the
`gradual development of chronic paw edema, facilitated
`spontaneous cutaneous protein extravasation, and a saphe-
`nous nerve mediated hyperalgesia; a syndrome resembling
`CRPS in man (Kingery et al., 1999, 2001b). Treating sciatic
`transected rats with methylprednisolone, a glucocorticoid
`that can reverse edema and hyperalgesia in CRPS patients,
`also reversed paw edema, spontaneous extravasation, and
`hyperalgesia in the sciatic section model (Kingery et al.,
`2001a,b). Since methylprednisolone treatment also blocked
`electrically evoked cutaneous neurogenic extravasation in
`the rat, we postulated that the actions of glucocorticoids in
`the rat sciatic section model and in CRPS patients might be
`mediated by inhibitory effects on neurogenic inflammation.
`Neurogenic inflammation is mediated by activation of
`
`0304-3959/03/$30.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.
`doi:10.1016/S0304-3959(02)00467-0
`
`Grün. Ex. 1032
`Grünenthal v. Antecip
`PGR2018-00062
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`76
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`W.S. Kingery et al. / Pain 104 (2003) 75–84
`
`small diameter primary afferent neurons causing the release
`of substance P, which binds to the substance P receptors
`(NK1) on the vascular endothelium, resulting in vasodilata-
`tion and increased vascular permeability (Bowden et al.,
`1994; Holzer et al., 1994; McDonald, 1988; Newby et al.,
`1997; Quartara and Maggi, 1998). Increased neurogenic
`inflammation in the cutaneous tissues would cause vasodi-
`lation and increased protein extravasation with resultant
`warmth and edema in the affected extremity. Enhanced
`substance P signaling in the spinal cord could also
`contribute to spontaneous pain, allodynia, and hyperalgesia
`in CRPS. These hypotheses are supported by our exper-
`iments demonstrating that neurotoxic lesioning of capsaicin
`sensitive sensory afferents inhibited neurogenic extravasa-
`tion and prevented the development of hindpaw edema and
`hyperalgesia in the sciatic section rat model (Kingery et al.,
`2002). Collectively these data suggest
`that facilitated
`substance P signaling could contribute to the vascular and
`nociceptive abnormalities observed in CRPS.
`Given these findings, the aim of this study was to further
`define the nociceptive, vascular, and bone changes devel-
`oping after sciatic nerve injury in the rat CRPS model and to
`determine whether substance P signaling contributes these
`sequelae of nerve injury.
`
`2. Materials and methods
`
`These experiments were approved by our institute’s
`Subcommittee on Animal Studies and followed the
`guidelines of the IASP (Zimmermann, 1983). Adult (10
`month old) male Sprague – Dawley rats (B&K Universal,
`Fremont, CA) were used in all experiments. The animals
`were housed individually in gnotobiotic isolator cages with
`solid floors covered with 3 cm of soft bedding and were fed
`and watered ad libitum. During the experimental period the
`animals were fed Lab Diet 5012 (PMI Nutrition Institute,
`Richmond, IN), which contains 1.0% calcium, 0.5%
`phosphorus, and 3.3 IU/g of vitamin D3.
`
`2.1. Surgery
`
`Sciatic nerve transection was performed under isoflurane
`anesthesia. The right nerve was exposed at the middle of the
`thigh and a 1 cm segment of the nerve was excised to
`prevent nerve regeneration. The incision was then closed
`with wound clips, which were removed 10 days later. Sham
`sciatic section surgery followed the same procedure, but the
`nerve was not transected.
`
`2.2. Hindpaw volume
`
`The maximum dorsal – ventral thickness of the hindpaw
`was measured as we have previously described, using a
`manual caliper very lightly applied to the skin so there was
`contact but no tissue displacement (Kingery et al., 2001b).
`
`2.3. Hindpaw temperature
`
`The room temperature was maintained at 238C and
`humidity ranged between 25 – 45%. The temperature of the
`hindpaw was measured using a fine wire thermocouple
`(Omega, Stanford, CT) applied to the paw skin. The
`investigator held the thermister wire using an insulating
`Styrofoam block. Three sites were tested over the dorsum of
`the hindpaw;
`the space between the first and second
`metatarsals (medial),
`the second and third metatarsals
`(central), and the fourth and fifth metatarsals (lateral).
`After a site was tested in one hindpaw the same site was
`immediately tested in the contralateral hindpaw. The testing
`protocol was medial dorsum right then left, central dorsum
`right then left, lateral dorsum right then left, medial dorsum
`left then right, central dorsum left then right, and lateral
`dorsum left then right. The six measurements for each
`hindpaw were averaged for the mean temperature.
`
`2.4. Hindpaw mechanical nociception
`
`Three different hindpaw mechanical nociceptive assays
`were used; withdrawal
`thresholds to noxious pressure
`applied momentarily over the soft tissue between the first
`and second metatarsals using large von Frey fibers, with-
`drawal thresholds to gradually increasing noxious pressure
`applied over the articular surface of the interphalangeal joint
`of the first toe using hemostats, and withdrawal thresholds to
`very low pressures applied continuously for 6 – 8 s over the
`medial calcaneous at the border between the glabrous and
`hairy skin. After acute sciatic section the rats continued to
`demonstrate withdrawal response with each of the nocicep-
`tive assays, data indicating that the three nociceptive test
`sites were all innervated by the saphenous nerve.
`Soft
`tissue nociceptive withdrawal responses were
`measured with calibrated von Frey fibers (North Coast
`Medical, San Jose, CA), applied over the medial dorsum of
`the hindpaw, between the first and second metatarsals as we
`have previously have described (Kingery et al., 2001a, 1999).
`Briefly, each fiber was applied three consecutive times,
`pushing down on the hindpaw until the rat withdrew it’s paw
`or the fiber bowed. Four different fibers were used in
`graduating sequence (10, 23, 57, and 85 g, respectively), for a
`total of 12 consecutive fiber applications. The withdrawal
`threshold was the smallest fiber size which evoked at least
`two hindpaw withdrawal responses during three consecutive
`applications with the same fiber. Each fiber was applied for
`approximately 1 s and the interstimlus interval was approxi-
`mately 5 s.
`responses were
`Articular nociceptive withdrawal
`measured using a hemostatic forceps to apply gradual
`pressure over the interphalangeal joint of the first toe. The
`hemostat jaws were rubber coated and 2 mm wide. The
`hemostat was fixed to a commercial strain-gauge (Salter,
`Fairfield, NJ) which was connected to a computer chart
`recorder (MacLab8e, ADInstruments, Castle Hill, Austra-
`
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`W.S. Kingery et al. / Pain 104 (2003) 75–84
`
`77
`
`lia), allowing the examiner to manually apply gradually
`increasing pressure (approximately 50 g/s up to a maximum
`force of 500 g). A force of 500 g applied to the
`experimenter’s skin was mildly painful. The stimulus was
`applied once and the withdrawal threshold was the pressure
`that evoked any movement of the paw or a vocalization
`response.
`To measure mechanical allodynia an up – down von Frey
`testing paradigm was used (Chaplan et al., 1994). Rats were
`placed in a clear plastic cylinder (20 cm in diameter) with a
`wire mesh bottom and allowed to acclimate for 15 min. The
`paw was tested with one of a series of eight von Frey hairs
`ranging in stiffness from 0.41 to 15.14 g. The von Frey hair
`was applied against
`the skin over
`lying the medial
`calcaneous at the border between the glabrous and hairy
`skin. The fiber was pushed until it slightly bowed and then it
`was held in that position for 6 – 8 s. Stimuli were presented
`at an interval of several seconds. Hindpaw withdrawal from
`the fiber was considered a positive response. The initial fiber
`presentation was 2.1 g and the fibers were presented
`according to the up – down method of Dixon to generate
`six responses in the immediate vicinity of the 50% threshold
`(Dixon, 1980).
`
`2.5. Dual-energy X-ray absorptiometry (DXA)
`
`Bone mineral density (BMD) was measured by dual-
`energy X-ray absorptiometry (DXA) using a Hologic
`(Waltham, MA) QDR-4000 instrument adapted to measure-
`ment in small animals. A high-resolution mode (line spacing
`0.0254 cm and resolution 0.0127 cm) was used with a
`collimator of 0.9 cm diameter. The instrument calibration
`was assessed by scanning a spine phantom every day.
`During the experiments the animals were anesthetized
`with dexmedetomidine and were taped into position in clear
`plastic boxes that were filled with 3 cm of warm water. The
`animals were positioned on their backs with the hindlimb
`externally rotated with the hip, knee and ankle articulations
`at 908 flexion. Pilot experiments indicated that this position
`allowed maximal reproducibility as previously reported by
`other investigators (Ammann et al., 1992). The femur and
`tibia were each scanned and the fibula was excluded from
`the region of interest. Both the femur and tibia were divided
`into three regions of interest,
`the proximal and distal
`metaphyseal regions and the diaphysis. The bones were
`scanned longitudinally. The regions of interest were
`determined by measurements made directly on DXA scans
`appearing on the computer screen. The proximal and distal
`regions of interest were 2.0 cm high on the screen, which
`corresponded to 0.7 cm in the actual bone. The average tibia
`length in the 10 months old Sprague – Dawley male rats was
`9.0 cm, and the average femur length was 8.2 cm.
`
`2.6. Drugs
`
`The NK1 receptor antagonist LY303870 was a generous
`
`gift from Dr L. Phebus (Eli Lily Company, Indianapolis,
`IN). This compound has nanomolar affinity for the rat NK1
`receptor, has no affinity for 65 other receptors and ion
`channels, has no sedative, cardiovascular or core body
`temperature effects in rats at systemic doses up to 30 mg/kg,
`and is physiologically active for 24 h after a single systemic
`dose of 10 mg/kg (Gitter et al., 1995; Hipskind et al., 1996;
`Iyengar et al., 1997).
`
`2.7. Neurogenic extravasation procedures
`
`Evans blue dye (50 mg/kg, Sigma, St Louis, MO) was
`administered intravenously in a 50 mg/ml solution of 0.9%
`saline. Exactly 24 h later the rats were deeply anesthetized
`with isoflurane and transcardially perfused with 200 ml of
`saline (0.9%) and then the distal aspect of the right hindpaw
`was removed (severed in a line bisecting the most distal tori
`pad on the plantar surface) and placed in 4 ml of 99%
`formamide at room temperature for 14 days. The dye
`concentration in the formamide was then determined
`spectrophotometrically at a wavelength of 620 nm. The
`total amount of Evans blue dye extracted from the hindpaw
`was then calculated from a standard curve of Evans blue
`concentrations. Since Evans blue dye binds to serum
`albumin in vitro and in vivo, the dye content of the hindpaw
`skin provides an accurate measure of albumin extravasation
`into the interstitial space (Saria and Lundberg, 1983).
`
`2.8. Study design
`
`2.8.1. Vascular and nociceptive testing after sciatic section
`Baseline determinations were made of bilateral paw
`thickness, temperature, and mechanical nociceptive with-
`drawal thresholds. After two baseline tests all rats ðn ¼ 16Þ
`had their right sciatic nerve sectioned and sham surgery on
`the left side. After surgery repeat bilateral testing was
`performed weekly for 6 weeks and also at 8, 12, and 20
`weeks after sciatic section. Any rats demonstrating self-
`mutilation of the digits (autotomy) were dropped from the
`study. All data was analyzed as the difference between the
`sciatic transected side and the sham operated side.
`
`2.8.2. Bone mineral density testing after sciatic section
`Baseline DXA scanning was performed in the right tibia
`and femur and the BMD was determined for the three
`regions of interest in each bone; the proximal and distal
`metaphyseal areas and the diaphysis. After baseline testing
`all rats ðn ¼ 8Þ had their right sciatic nerve sectioned. After
`surgery repeat testing was performed at 2, 4, 6, 12, and 20
`weeks after sciatic section.
`Only skeletally mature 10 month-old male Sprague –
`Dawley rats were used in these experiments. Pilot data in
`normal male Sprague – Dawley rats demonstrated that BMD
`increased over time in both the tibia and femur up until the
`rats reached 40 weeks in age, after which no significant
`change in BMD was observed at 48 and 56 weeks . These
`
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`

`78
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`W.S. Kingery et al. / Pain 104 (2003) 75–84
`
`data are in agreement with other studies demonstrating that
`Spague – Dawley rats reach skeletal maturity by 9 months of
`age and BMD remains relatively stable for 4 – 5 months after
`that (Kalu et al., 1989; Wang et al., 2001).
`
`2.8.3. Measuring LY303870 inhibition of vascular sequelae
`of sciatic section
`The inhibitory effect of LY303870 on spontaneous
`protein extravasation was determined 2 weeks after sciatic
`section or sham surgery (n ¼ 8 per cohort). LY303870
`(20 mg/kg, i.p.) or saline was injected 1 h before Evans blue
`dye (50 mg/kg, i.v.) was administered. At 24 h after dye
`injection the animals were anesthetized,
`transcardially
`perfused with 200 ml of saline, the hindpaw was removed,
`placed in formamide, and 14 days later the dye content was
`spectrophotometrically determined. Pilot data indicated this
`dose of LY303870 reduced electrially evoked neurogenic
`extravasation for 24 h in the rat. The investigator perform-
`ing the extravasation procedure was blinded to treatment
`groups.
`Another experiment determined the inhibitory effects of
`LY303870 on sciatic section evoked hindpaw warmth.
`Three weeks after sciatic section the bilateral hindpaw
`temperatures were measured,
`then either LY303870
`(20 mg/kg) or saline was injected (i.p.) and 1 h later the
`bilateral hindpaw temperatures were retested by a blinded
`observer. This was designed as a cross-over controlled
`study, so the following week the rats that initially were
`injected with saline received LY303870, and the rats
`injected with the antagonist received saline. This same
`blinded cross-over protocol was used to test the effects of i.t.
`administered LY303870 (20 mg in 20 ml of saline) on
`hindpaw warmth after sciatic section. The bilateral paw
`temperatures were measured 10 min after i.t. injection.
`The anti-edematous effect of a 14 days course of
`LY303870 was tested 4 weeks after sciatic section. Two
`separate cohorts of rats were used, a LY303870 treatment
`group (20 mg/kg/day, i.p.) and a saline treatment group
`(daily saline, i.p.). The bilateral hindpaw thickness was
`measured, then either LY303870 or saline was injected and
`1 h later the bilateral hindpaw thickness was retested by a
`blinded observer. The rats were tested 1 h after the first
`injection, and then after 7 and 14 days of daily injections.
`
`2.8.4. Measuring LY303870 anti-nociceptive effect
`The anti-nociceptive effects of LY303870 were deter-
`mined at 3 – 4 weeks after sciatic section using three
`nociceptive assays; soft tissue and articular mechanical
`hyperalgesia and mechanical allodynia. The bilateral
`hindpaw nociceptive thresholds were measured, then either
`LY303870 (20 mg/kg) or saline was injected (i.p.) and 1 h
`later the bilateral hindpaw nociceptive thresholds were
`retested by a blinded observer. This was designed as a cross-
`over controlled study, so the following week the rats that
`initially were injected with saline received LY303870, and
`the rats injected with the antagonist received saline. This
`
`same blinded cross-over protocol was used to test the effects
`of i.t. administered LY303870 (20 mg in 20 ml of saline) on
`hindpaw nociceptive thresholds after sciatic section. The
`bilateral paw nociceptive thresholds were measured 10 min
`after i.t. injection. Rats that failed to develop mechanical
`allodynia after sciatic section were not used in these
`experiments.
`
`2.9. Statistical analysis
`
`A one-way repeated measures analysis of variance
`(ANOVA) was used to measure time dependent changes
`for continuous data (temperature, paw thickness, articular
`mechanical thresholds, and BMD) and post hoc testing was
`performed with a paired two-tailed Students’ t
`test
`to
`determine change from baseline. The Friedman ANOVA
`was used to test for time dependent changes for non-
`parametric data (von Frey fiber assays for hyperalgesia and
`allodynia) and post hoc testing was performed with
`Wilcoxon test. Differences between treatment groups were
`tested for using the paired two-tailed Student’s t test for
`continuous data and the Wilcoxon test for non-parametric
`data. All data are presented as the mean ^ standard error of
`the mean, and differences are considered significant at a P
`value less than 0.05.
`
`3. Results
`
`3.1. Hindpaw warmth and edema after sciatic section
`
`The chronic effects of sciatic section on hindpaw
`temperature and edema were measured in this experiment.
`After baseline testing of the bilateral paws, all the rats
`underwent right sciatic nerve sectioning ðn ¼ 16Þ and
`contralateral sham surgery, then temperature and caliper
`measurements were determined over a 20 week period.
`Seven rats were dropped from the study due to self-
`mulitation of the deafferented digits (autotomy). Fig. 1A
`illustrates that after sciatic section the right paw temperature
`was elevated (P , 0:001, n ¼ 9, F-value 3.16 compared
`with Fð10; 80Þ). One week after sciatic section, the hindpaw
`temperature was increased 4.1 ^ 0.48C degrees compared
`to the contralateral side ðP , 0:001Þ, then the right hindpaw
`temperature gradually dropped over the ensuing 12 weeks to
`a non-significant
`level. No temperature changes were
`observed in the contralateral paw compared to baseline.
`Fig. 1B illustrates that after sciatic section hindpaw
`thickness increased (P , 0:001, n ¼ 9, F-value 6.64
`compared with Fð10; 80Þ). The hindpaw thickness gradually
`increased until by 6 weeks the sectioned side was
`1.3 ^ 0.3 mm thicker
`than the contralateral hindpaw
`ðP , 0:01Þ. This edema persisted for the duration of the
`study. The thickness of the contralateral sham-operated paw
`did not change over time relative to baseline measurements.
`
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`

`W.S. Kingery et al. / Pain 104 (2003) 75–84
`
`79
`
`weeks the maximal reduction observed in the articular
`nociceptive threshold was 153 ^ 19 g compared to the
`contralateral side (n ¼ 9, P , 0:01). The articular mechan-
`ical hyperalgesia also gradually resolved over 20 weeks.
`After sciatic section mechanical allodynia developed
`over the glaborous-hairy border of the medial calcaneous
`(Fig. 2C, P , 0:05, n ¼ 9). The maximal reduction in the
`withdrawal
`threshold occurred 2 weeks after surgery
`(6.2 ^ 1.8 g less than on the contralateral side, n ¼ 9,
`P , 0:01) and this mechanical allodynia gradually resolved
`over 12 weeks.
`
`3.3. Periarticular osteopenia after sciatic section
`
`Fig. 3 illustrates the temporal development of BMD loss
`most prominent in the proximal and distal aspects of the
`femur and tibia after sciatic section ðn ¼ 8Þ. Bone mineral
`density declined in the proximal femur after surgery
`(P , 0:001, 7.16 F-value compared to Fð5; 35Þ), with a
`13% maximal reduction at 20 weeks. The femoral diaphysis
`had a smaller reduction in BMD of only 4% (P , 0:05, 2.49
`F-value compared to Fð5; 35Þ). The distal femur BMD
`declined a maximum of 15% at 12 weeks after sciatic
`section (P , 0:001, 9.13 F-value compared to Fð5; 35Þ). A
`similar pattern of bone loss was observed in the tibia, with
`the proximal tibia losing 18% of its bone density by week 6
`(P , 0:001, 16.34 F-value compared to Fð5; 35Þ), the tibial
`diaphysis BMD only declining 4% (P , 0:01, 3.98 F-value
`compared to Fð5; 35Þ), and the distal tibia BMD dropping by
`7% at 12 weeks after surgery (P , 0:01, 4.44 F-value
`compared to Fð5; 35Þ). These results indicate that bone loss
`after sciatic section progresses for 6 – 12 weeks after injury,
`occurs primarily in the periarticular regions of the femur and
`tibia, and persists for at least 20 weeks without evidence of
`recovery.
`
`3.4. LY303870 Inhibition of facilitated extravasation,
`warmth, and edema
`
`Spontaneous protein extravasation was determined by
`measuring hindpaw Evans blue dye content 24 h after
`intravascular dye injection. Two weeks after sciatic section
`there was an increase in spontaneous extravasation in the
`hindpaw (Fig. 4A, P , 0:05). The substance P receptor
`antagonist LY303870 (20 mg/kg,
`i.p.) administered 1 h
`before the dye injection reduced spontaneous extravasation
`in the neuropathic hindpaw by 53% (P , 0:001 vs. saline
`treated rats).
`i.p)
`Systemically administered LY303870 (20 mg/kg,
`given 1 h before testing caused a 66% reduction in the
`hindpaw warmth observed 3 – 4 weeks after sciatic section
`(Fig. 4B, P , 0:01 vs. saline treatment). Intrathecally
`administered LY303870 (20 mg,
`i.t.) had no effect on
`hindpaw temperature.
`Fig. 4C shows that one injection of LY303870 had no
`effect on hindpaw edema, but the chronic administration of
`
`Fig. 1. After baseline testing the right sciatic nerve was sectioned and
`bilateral hindpaw skin temperature (A) and hindpaw thickness (B) were
`determined over the ensuing 20 weeks ðn ¼ 9Þ. Measurements represent the
`difference between the sciatic section side and the contralateral paw, thus a
`positive value represents an increase in temperature or thickness on the
`section side. (A) Hindpaw skin temperature increased by 4.1 ^ 0.48C at 1
`week after sciatic section and then gradually returned to normal by 12
`weeks after nerve injury. (B) Hindpaw thickness gradually increased for 6
`weeks after sciatic section and remained elevated for the duration of the
`study.*P , 0:05, **P , 0:01, ***P , 0:001.
`
`3.2. Hindpaw nociceptive changes after sciatic section
`
`This experiment measured the effect of sciatic section on
`soft tissue (Fig. 2A) and articular (Fig. 2B) mechanical
`nociceptive thresholds. The development mechanical allo-
`dynia after sciatic section was also examined (Fig. 2C).
`After baseline testing sciatic nerve was sectioned in the right
`leg and sham surgery was performed on the left.
`Nociceptive testing was performed over a 20 week
`period after surgery.
`Fig. 2A illustrates that soft tissue mechanical nociceptive
`thresholds over the medial dorsum of the hindpaw dropped
`after sciatic section (P , 0:001, n ¼ 9). After 3 weeks the
`thresholds were 54 ^ 11 g lower on the neuropathic side
`than on the contralateral side ðP , 0:001Þ. The soft tissue
`mechanical hyperalgesia gradually resolved over 20 weeks.
`Mechanical hyperalgesia also developed over the first
`interphalangeal
`joint after sciatic section (Fig. 2B,
`P , 0:05, F-value 2.47 compared with Fð10; 80Þ). After 8
`
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`W.S. Kingery et al. / Pain 104 (2003) 75–84
`
`LY303870 (20 mg/kg daily i.p. injections) for 2 weeks
`gradually reduced edema by 75% compared to saline treated
`rats.
`
`3.5. LY303870 inhibition of mechanical hyperalgesia, but
`not allodynia
`
`At 3 – 4 weeks after sciatic section the anti-nociceptive
`effects of intraperitoneal (i.p.) (20 mg/kg) and i.t. (20 mg)
`administered LY303870 were determined.
`Intrathecal
`LY303870 increased soft tissue (Fig. 5A) and articular
`(Fig. 5B) mechanical nociceptive thresholds in the hyper-
`algesic paw (n ¼ 10, P , 0:05). The i.p.
`injection of
`LY303870 had no effect on soft
`tissue and articular
`mechanical nociceptive thresholds (Fig 5A, n ¼ 8).
`i.t.
`In contrast
`to the anti-hyperalgesic effects of
`LY303870, neither i.p. or i.t. LY303870 had any significant
`effect on mechanical allodynia (Fig. 5C, n ¼ 6).
`
`4. Discussion
`
`The first aim of this investigation was to further
`characterize the rat sciatic nerve section model of CRPS.
`Previous studies had shown that sciatic nerve transection led
`to the development of hindpaw hyperalgesia, edema, and
`increased spontaneous cutaneous extravasation persisting
`for at least 7 weeks after injury (Kingery et al., 1999,
`2001b). The current study demonstrates that sciatic section
`also caused hindpaw warmth, soft
`tissue and articular
`mechanical hyperalgesia, and mechanical allodynia that
`persisted for 8 – 12 weeks after injury (Figs. 1, 2). In
`addition, sciatic section dramatically reduced BMD in the
`periarticular regions of the femur and tibia but had only
`modest osteoporotic effect on the diaphyses (Fig. 3). This
`regional loss of bone density progressed for 6 – 12 weeks
`after nerve injury and persisted for at least 20 weeks. Sciatic
`section also caused an increase in thickness of the hindpaw
`which persisted for at least 20 weeks (Fig. 1B). Although
`increased hindpaw thickness could represent soft tissue
`proliferation, sciatic section also enhanced spontaneous
`cutaneous extravasation (Fig. 4A), evidence supporting the
`argument that plasma transudation into the tissue space was
`the mechanism of hindpaw thickening. The reversal of
`hindpaw thickening after LY303870 administration sup-
`ports this interpretation.
`The vascular abnormalities observed in the rat sciatic
`section model resemble those observed in CRPS patients.
`Skin blood flow is usually abnormal at some point during
`the disease and patients note differences in skin temperature
`and color between the affected and unaffected limbs in 98%
`of the cases (Veldman et al., 1993). Side-to-side skin
`temperature differences are usually greater than 0.58C, with
`the affected extremity usually warmer the first several
`months and then gradually becoming cooler over time
`(Baron et al., 1996; Birklein et al., 1998; Chelimsky et al.,
`
`Fig. 2. After baseline testing the right sciatic nerve was sectioned and
`bilateral hindpaw soft tissue (A) and articular (B) mechanical nociceptive
`withdrawal thresholds (in grams) were determined over the ensuing 20
`weeks ðn ¼ 9Þ. Mechanical allodynia was also measured (C), using the up –
`down von Frey paradigm (Chaplan et al., 1994). Measurements represent
`the difference between the sciatic section side and the contralateral paw,
`thus a negative value represents a decrease in the withdrawal threshold on
`the section side. (A) Soft tissue mechanical withdrawal thresholds to large
`von Frey fibers applied momentarily over the medial dorsum of the
`hindpaw. Thresholds significantly decreased by 2 weeks after sciatic
`section and then gradually returned to normal by 20 weeks after sciatic
`section. (B) Articular mechanical withdrawal thresholds to pinch over the
`interphalangeal joint of the first toe. Thresholds significantly decreased by 3
`weeks after sciatic section and then gradually returned to normal by 20
`weeks after nerve injury. (C) Soft tissue mechanical withdrawal thresholds
`to small von Frey fibers applied continuously for 6 – 8 s over the medial
`calcaneous at
`the border of the glabrous and hairy skin. Thresholds
`significantly decreased by 1 week after sciatic section and then gradually
`returned to normal by 12 weeks after sciatic section. *P , 0:05,
`**P , 0:01, ***P , 0:001.
`
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`W.S. Kingery et al. / Pain 104 (2003) 75–84
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`
`Fig. 3. Dual-energy X-ray absorptiometry (DXA) was used to measure bone mineral density (BMD). After baseline testing the right sciatic nerve was sectioned
`and the right hindlimb BMD was determined over the ensuing 20 weeks ðn ¼ 8Þ. Over a 4 – 6 week period after sciatic section there was a rapid decline in the
`BMD of the periarticular bone regions tested, including the proximal femur, distal femur, proximal tibia, and distal tibia. There was only mininmal loss of
`BMD in the femoral and tibial diaphyses, evidence that sciatic section induced osteoporosis is most prominent in periarticular bone. *P , 0:05, **P , 0:01,
`***P , 0:001.
`
`1995; Wasner et al., 2001). When skin blood flow in the
`affected extremity is assessed by laser Doppler flowmetry
`there is evidence of vasodilatation for the first several
`months after onset, and vasoconstriction later in the
`disease.(Kurvers et al., 1996; Wasner et al., 2001) Distal
`limb edema develops in 86% of CRPS patients and most
`patients note persistent edema years after onset (Veldman
`et al., 1993).
`The mechanical hyperalgesia and allodynia that devel-
`oped after sciatic section in the rat resemble nociceptive
`changes in CRPS. Using a pressure measurement device
`(dolorimeter) to apply painful pressure to finger and toe
`joints, 92 – 100% of CRPS patients exhibit articular
`mechanical hyperalgesia (Atkins et al., 1989; Bryan et al.,
`1991; Sarangi et al., 1991). Dolorimetry has been used
`clinically to quantify the natural progression of CRPS
`hyperalgesia (Bryan et al., 1991; Sarangi et al., 1993) and its
`response to treatment (Bickerstaff and Kanis, 1991; Field
`and Atkins, 1993; Kozin et al., 1981). Mechanical allodynia
`(pain due to a light touch) is observed in 8 – 41% of CRPS
`
`patients (Blumberg and Janig, 1994; Verdugo et al., 1994).
`The gradual spontaneous resolution of the hyperalgesia and
`allodynia by 20 weeks after sciatic section resembles the
`natural history of CRPS type II patients, with a dramatic
`reduction in pain reported in 57 – 68% of patients at 6
`months and in 90 – 91% of patients by 12 – 17 months after
`nerve injury (Echlin et al., 1948; Sunderland and Kelly,
`1948).
`The development of periarticular osteoporosis after
`sciatic section models the bone loss observed in CRPS.
`Radiographs of CRPS limbs demonstrate periarticular bone
`loss within 3 weeks after injury and late in the disease there
`may be subperiosteal bone reabsorption with a ground-glass
`appearance and tunneling of the cortices and endosteal
`surfaces(Bickerstaff et al., 1993). Technetium 99 m bone
`scanning frequently demonstrates increased periarticular
`uptake in the CRPS extremity on the late scan phase,
`indicative of periarticular bone remodeling (Blockx and
`Driessens,