A substance P receptor (NK1) antagonist enhances the widespread osteoporotic effects of sciatic nerve section

Bone 33 (2003) 927–936

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A substance P receptor (NK1) antagonist enhances the widespread osteoporotic effects of sciatic nerve section Wade S. Kingery,a,b,* Sarah C. Offley,a,b Tian-Zhi Guo,a,b M. Frances Davies,c,d J. David Clark,c,d and Christopher R. Jacobse,f a

Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA b Department of Orthopedic Surgery, Stanford University School of Medicine, Stanford, CA, USA c Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA d Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA e Department of Mechanical Engineering, Stanford University School of Engineering, Stanford, CA, USA f Rehabilitation Research and Development Center, Veterans Affairs Palo Alto Health CareSystem, Palo Alto, CA, USA Received 17 March 2003; revised 12 June 2003; accepted 22 July 2003

Abstract The long-term effects of sciatic nerve section on bone mineral density (BMD) were studied using dual-energy X-ray absorptiometry (DEXA) in skeletally mature rats. Unilateral sciatic neurectomy caused the rapid loss of cancellous bone in the proximal and distal femur and tibia in the ipsilateral hindlimb and, to a lesser extent, in the contralateral intact hindlimb. The reduction in BMD rapidly progressed for 4 weeks after sciatic section and then gradually stabilized with no evidence of recovery at 12 weeks. The development of osteoporosis in the contralateral intact hindlimb was a novel finding. There was no evidence of disuse in the normal contralateral hindlimb after unilateral sciatic section; grid-crossing activity over a 24-h interval was unchanged and there was no reduction in weight bearing on the contralateral normal hindpaw during the stance phase of ambulation. Unilateral peripheral nerve lesions have well-documented effects on substance P content and function in the corresponding contralateral intact nerve. We hypothesized that after sciatic section a reduction in substance P signaling might contribute to bone loss in the contralateral hindlimb. Daily administration of the substance P receptor (NK1) antagonist LY303870 for 2 weeks caused significant loss of cancellous bone in the denervated and the contralateral hindlimb, evidence that substance P signaling sustained bone density after nerve section. After sciatic neurectomy there was a 33% reduction in sciatic nerve stimulation-evoked extravasation in the contralateral intact hindlimb, indicating transmedian inhibition of substance P signaling after nerve injury. Furthermore, there was a 50% reduction in the substance P content in both tibias after unilateral sciatic section. Collectively, these data support the hypothesis that a widespread reduction in substance P content in bone contributes to the osteoporotic effects of sciatic neurectomy and that residual substance P signaling maintains bone integrity after nerve section in both the denervated and contralateral intact hindlimb. © 2003 Elsevier Inc. All rights reserved. Keywords: Nerve injury; Osteoporosis; Substance P; Dual-energy X-ray absorptiometry; Bone; Bone mineral density

Introduction Sciatic neurectomy causes paralysis and atrophy in denervated hindlimb muscles and it is one of the methods used to model disuse osteoporosis in rats [1]. The sciatic nerve

* Corresponding author. Physical Medicine and Rehabilitation Service (117), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Fax: ⫹1-650-852-3470. E-mail address: [email protected] (W.S. Kingery). 8756-3282/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2003.07.003

not only innervates hindlimb muscles, but it is also the source of a network of skeletal nerve fibers innervating cancellous bone in the femur and tibia. These osseal neurons contain a variety of peripherally released neurotransmitters, and functional receptors for these transmitters have been identified in bone cells [2]. It has been proposed that this neuro-osteogenic network can modulate bone remodeling and that the loss of this regulatory element contributes to the negative bone balance observed in denervated bone [3,4]. Not only does unilateral sciatic section disrupt neuronal

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signaling in the ipsilateral hindlimb, but numerous studies have identified alterations in contralateral nerve anatomy, function, and neurotransmitter expression after nerve section [5]. If local neurotransmitter release can regulate bone remodeling we postulated that unilateral sciatic section might impair neuronal signaling in the contralateral sciatic nerve and the loss of this signal could induce bone loss in the contralateral hindlimb. The current study examined the temporal effects of unilateral sciatic neurectomy on bone mineral density (BMD) in the bilateral hindlimbs of 10-month-old Sprague–Dawley male rats. The aim of this investigation was to determine how neural systems and transmitters modulate bone remodeling, which is the principal bone cellular process that influences bone quantity and strength in the adult skeleton [6]. Osteoporosis is an adult disease in which perturbations in bone remodeling activity represent the final pathway through which a multitude of diverse processes affect bone balance. Mature animals in the remodeling phase of bone metabolism were used to identify changes in neuronal signaling in osteoporotic bone [7]. In accordance with our hypothesis, after unilateral sciatic section bone loss occurred in both the denervated and contralateral intact hindlimb. As there was no evidence of disuse in the contralateral intact hindlimb, we attempted to identify a neural mechanism for this transmedian effect of nerve injury. Reductions in substance P content and signaling in the contralateral counterparts of damaged neurons have been observed by several investigators [8 –10]. Substance P regulation of bone metabolism has been previously proposed based on anatomic and cell culture data [11,12]. We postulated that substance P signaling might contribute to the maintenance of bone density and tested this hypothesis by assessing the effects of a 2-week course of the substance P NK1 receptor antagonist LY303870 on bilateral hindlimb bone density in the unilateral sciatic section model. It was not feasible to attempt to reverse sciatic section-induced bone loss with chronically infused substance P due to its extremely brief (30 –90 s) plasma halflife [13,14]. The effect of sciatic transection on substance P content and signaling in the contralateral intact hindlimb was also investigated in this study. Substance P content of the proximal tibia was quantified by enzyme immunoassay (EIA) after unilateral sciatic section. Substance P signaling in the sciatic nerve was measured using electrically evoked extravasation responses. Substance P is synthesized in the small-diameter primary afferent neurons and is released from the peripheral nerve terminals during electrical stimulation of the nerve. When substance P binds to the NK1 receptors on the postcapillary venules it causes endothelial cell junctions to open, allowing the leakage of large molecules such as albumen. Evans blue dye forms a stable complex with albumen that can be used as a marker to quantify neurogenic extravasation after nerve stimulation in

the rat. This technique was used to examine changes in substance P signaling in the intact contralateral hindpaw after sciatic neurectomy.

Materials and methods Our institute’s Subcommittee on Animal Studies approved these experiments. Adult (10 month old) male Sprague–Dawley rats (B&K Universal, Fremont, CA, USA) 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, USA), which contains 1.0% calcium, 0.5% phosphorus, and 3.3 IU/g vitamin D3. Surgery Sciatic nerve transection was performed under isoflurane anesthesia. The right nerve was exposed in the proximal 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. Dual-energy X-ray absorptiometry Bone mineral density was measured in vivo by dualenergy X-ray absorptiometry (DEXA) using a Hologic (Waltham, MA) QDR-4000 instrument adapted to measurement in small animals. A high-resolution mode (line spacing 0.0254 cm and resolution 0.0127 cm) was used with a collimator 0.9 cm in diameter. The instrument calibration was assessed by scanning a spine phantom every day. Prior to scanning the rats were anesthetized with dexmedetomidine (300 ␮g/kg, ip) 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 and the hip, knee, and ankle articulations placed in 90° flexion. Pilot experiments indicated that this position allowed maximal reproducibility in BMD measurements as previously reported by other investigators [15]. 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 (each 7 mm in length) and the diaphysis. The average tibia length in the 10-month-old Sprague–Dawley male rat was 9.0 cm and the average femur length was 8.2 cm. Drugs The NK1 receptor antagonist LY303870 was a generous gift from Dr. L. Phebus (Eli Lily Co., Indianapolis, IN, USA). This compound has nanomolar affinity for the rat

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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 [16 –18]. Spontaneous locomotor activity Rats were individually placed in clear plastic monitoring chambers each measuring 72 ⫻ 32 ⫻ 32 cm. The chamber floors were covered with 0.5 cm of soft bedding and the rats were fed and watered ad libitum while in the chamber. Spontaneous locomotor activity was measured via seven sets of photoelectric sensors evenly spaced along the length of the monitoring chamber at a level 3 cm above the floor (San Diego Instruments, San Diego, CA, USA). Total gridcrossing activity was recorded as the number of times the rat interrupted the photoelectric sensors during a 24-h monitoring session. The testing room was lighted for 10 h (8 AM to 6 PM) and dark for 14 h during the monitoring session and the room temperature was maintained at 23°C. Hindpaw weight bearing Each rat was individually placed into the entrance of an open-top narrow wooden chute (9.5 cm wide, 28.5 cm high, 86 cm long) and the entrance was then closed. The rat then walked the length of the level wooden chute to the opposite end that opened into a dark box enclosure. In the middle of the chute was a plastic plate (4.75 cm wide and 6 cm long) flush with the floor. The plate was attached to a commercial strain gauge (Salter, Fairfield, NJ, USA), which was connected to a computer chart recorder (MacLab8e, ADInstruments, Castle Hill, Australia). Clear windows on both sides at the middle of the chute allowed observation of the plate. When the rat’s hindpaw was placed on the plate during the stance phase of gait the peak weight was recorded. The mean of three consecutive tests was taken as maximum hindpaw weight bearing. The chute could be rotated 180° and the entrance and dark box enclosure exchanged, thereby allowing both hindpaws to be tested. Neurogenic extravasation procedures The rats were deeply anesthetized with isoflurane for extravasation studies. The left sciatic nerve was exposed in the thigh and tightly ligated proximal to the midthigh stimulation site. Then the incision was filled with warm mineral oil and a Plexiglas–platinum stimulating electrode with a sliding jaw was gently secured around the nerve (Harvard Apparatus, Holliston, MA, USA). Evans blue dye (50 mg/kg, Sigma, St Louis, MO, USA) was administered intravenously in a 50 mg/ml solution of 0.9% saline. Ten minutes after dye injection the left sciatic nerve was stimulated for 5 min (5 Hz, 0.5-ms

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pulse duration, 10 mA). Ten minutes later the rats were transcardially perfused with 1000 ml of saline (0.9%), suspended 100 cm above the heart (50 mm Hg perfusion pressure). Then the plantar and dorsal skin on each hindpaw was excised from the base of the heel to the tip of the third digit and the tissue was placed in 4 ml of 99% formamide in a shaker bath at 55°C for 72 h. The dye concentration was determined spectrophotometrically at a wavelength of 620 nm. EIA assay Under isoflurane anesthesia the sciatic nerve (approximately 3 cm, 32– 46 mg) and the proximal tibia (approximately 7 mm, 282–380 mg) were collected, immediately frozen, and then weighed. Nerve samples were minced in 1 ml of 3:1 ethanol/0.7 M HCl and sonicated for 20 s. Bone samples were minced in 2 ml of the same solution and were homogenized for 20 s. The samples were then shaken for 2 h at 4°C and centrifuged at 3000g for 20 min at 4°C. The supernatant was frozen and lyophilized and the lyophilized product was stored at ⫺80°C. Substance P content of samples was assayed in duplicate using an EIA kit (No. 900018, Assay Designs, Inc. Ann Arbor, MI, USA). Study design Bone mineral density measurements Only skeletally mature 10-month-old male Sprague– Dawley rats were used [19,20]. To confirm that BMD was stable over the 12-week experimental period, the right tibia and femur were DEXA scanned in a cohort of control rats at 16, 40, 48, and 56 weeks of age. The BMD was determined for the three regions of interest in each bone: proximal and distal metaphyseal areas and diaphysis. To confirm the in vivo precision of our DEXA scanning technique the coefficient of variation (CV ⫽ SD/mean ⫻ 100) was calculated for three consecutive BMD measurements, each time with repositioning, in 10-month-old male Sprague–Dawley rats. The CV of each animal was determined from the three consecutive scans and the CVs of eight rats were averaged. The CV was determined in each of the three regions of interest in the tibia and femur. Determining the effects of sciatic section and LY303870 administration on BMD After baseline bilateral hindlimb DEXA scanning all rats had their right sciatic nerve sectioned. Repeat DEXA scanning was performed 2, 4, 6, and 12 weeks after sciatic section. After the 4-week scan the rats were assigned to 2 weeks of treatment with either a substance P receptor antagonist (LY303870, 20 mg/kg/day, ip) or a control group (daily saline for 2 weeks, ip). After the 2-week treatment period all rats were DEXA scanned again (6 weeks after

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Fig. 1. Effect of aging on bone mineral density (BMD) in Sprague–Dawley male rats. Dual-energy X-ray absorptiometry (DEXA) was used to determine BMD in the proximal and distal femur and tibia, as well as the diaphyses, for these bones. BMD increased in all test regions between the ages of 16 and 40 weeks and remained stable over the ensuing 16 weeks. ***P ⬍ 0.001.

section) and then scanned 6 weeks later (12 weeks after section). Grid crossing and weight bearing after sciatic section Total spontaneous locomotor activity over a 24-h period was determined at baseline and 2, 4, and 8 weeks after unilateral right sciatic transection. Maximum right and left hindpaw weight bearing during the stance phase of gait was also measured at these time points. Determining the effects of sciatic section on neurogenic extravasation The extravasation response was quantified by measuring Evans blue dye content in the hindpaw skin after intravenous dye injection and sciatic nerve stimulation. Control rats were intravenously injected with Evans blue and then the sciatic nerve stimulated on the right side. After transcardial perfusion the paw skin was removed from both the stimulated and nonstimulated hindlimbs. Another group of control rats were injected with the substance P receptor antagonist LY303870 (40 mg/kg, ip) 1 h before sciatic stimulation to determine to what extent the extravasation response was mediated by substance P signaling. Another group of rats underwent unilateral sciatic transection and at 2 weeks after surgery the contralateral intact sciatic nerve was stimulated.

Determining the effects of sciatic section on substance P content of nerve and bone Substance P content of the contralateral sciatic nerve and the bilateral proximal tibia was quantified using EIA. Samples were taken from controls and at 2 weeks after unilateral sciatic transection. Statistical analysis A repeated-measures analysis of variance (ANOVA) was used to determine differences between times for the time– response data and post hoc differences were analyzed using Fisher’s PLSD test. Differences between treatment groups were determined using the unpaired Student t test. The in vivo reproducibility of DEXA scanning was evaluated by measuring the coefficient of variation (CV ⫽ SD/mean BMD ⫻ 100). All data are presented as means ⫾ SEM, and differences are considered significant at a P value less than 0.05.

Results BMD measurements Figure 1 illustrates the effects of aging on the mean BMD of male Sprague–Dawley rats (n ⫽ 8). The BMD was

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Fig. 2. Changes in BMD in the ipsilateral hindlimb after unilateral sciatic section. Rapid bone loss occurred in the proximal and distal femur and proximal tibia over the first 4 weeks after sciatic section. Four weeks after the sciatic section the rats were started on a 2-week course (signified by bar) of either LY303870 (20 mg/kg/day) or saline. Twelve weeks after sciatic section the LY303870-treated cohort had significantly lower BMD compared with the saline-treated group. *P ⬍ 0.05, **P ⬍ 0.01.

determined for the proximal and distal femur and tibia and their diaphyses. There was a significant increase in the BMD for each test region between the ages of 16 and 40 weeks. Between 40 and 56 weeks no significant changes were observed in bone density in any of the test regions and this age interval was used for all further experiments. The precision of in vivo DEXA scanning for these regions of interest in the tibia and femur was determined by measuring the coefficient of variation in eight rats. The intertest CV was established for each rat based on three consecutive DEXA scans with the rat repositioned between each scan. The CV in the proximal femur was 2.9 ⫾ 0.7%, in the femoral diaphysis 2.7 ⫾ 0.9%, in the distal femur 4.2 ⫾ 1%, in the proximal tibia 3.6 ⫾ 0.9%, in the tibial diaphysis 2.6 ⫾ 0.4%, and in the distal tibia 2.1 ⫾ 0.4%.

Changes in BMD, weight, locomotor activity, and weight bearing after sciatic section After baseline DEXA scanning the right sciatic nerve was sectioned and changes in bone density were followed over a 12-week period after surgery (Figs. 2, 3). Table 1 illustrates the temporal loss of BMD in the proximal and distal aspects of the femur after unilateral sciatic section. BMD rapidly declined in the proximal femur ipsilateral to neurectomy, with a reduction of 7% at 4 weeks and 11% at 12 weeks postoperatively. The femoral diaphysis had a nonsignificant reduction in BMD of only 3% by 12 weeks. The distal femur BMD declined a maximum of 16% at 12 weeks after sciatic section. A similar pattern of bone loss was observed in the contralateral intact femur, but to a lesser

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Fig. 3. Changes in BMD in the contralateral intact hindlimb after unilateral sciatic section. Rapid bone loss occurred in the proximal and distal femur and proximal tibia over the first 4 weeks after sciatic section, but to a lesser extent than was observed in the ipsilateral hindlimb. Four weeks after the sciatic section the rats were started on a 2-week course (signified by bar) of either LY303870 (20 mg/kg/day) or saline. Twelve weeks after sciatic section the LY303870-treated cohort had significantly lower BMD compared with the saline-treated group. *P ⬍ 0.05, **P ⬍ 0.01.

extent, with the proximal femur losing 6% of its bone density at Week 4 and Week 12, the femoral diaphysis BMD declining only 1%, and the distal femur BMD dropping by 9% at 12 weeks after surgery. Table 2 illustrates a similar pattern in the bilateral tibia after sciatic neurectomy. There was a 16% reduction in the ipsilateral and a 5% reduction in the contralateral proximal tibia BMD at Week 4. BMD was reduced in the ipsilateral tibial diaphysis (3% at Week 4) but not the contralateral diaphysis (0% at Week 4). There was a 4% reduction in ipsilateral distal tibia BMD at Week 4 and contralateral distal tibia BMD declined only 2%. Sciatic section did not cause weight loss during the study. The baseline mean weight was 670 ⫾ 32 g for all rats (n ⫽ 16). Twelve weeks after sciatic section the mean weight of the saline treatment group was 678 ⫾ 21 g and that of the LY303870 treatment group was 662 ⫾ 35 g.

Baseline spontaneous locomotor activity was 5996 ⫾ 189 grid crossings over a 24-h period (n ⫽ 8). At 2, 4, and 8 weeks after sciatic section there was no significant change in spontaneous locomotor activity (Fig. 4A). Prior to unilateral sciatic section maximum weight bearing for the ipsilateral hindpaw was 310 ⫾ 9 g and for the contralateral hindpaw it was 304 ⫾ 11 g (n ⫽ 8). At 2, 4, and 8 weeks after sciatic section there was a 27% reduction of maximum hindpaw weight bearing ispilaterally, but no significant reduction contralaterally (Fig. 4B). In summary, there was a reduction in BMD after unilateral sciatic section that rapidly progressed for 4 weeks and then stabilized or slowly progressed over the next 8 weeks. Bone loss was most prominent in the cancellous bone of the proximal and distal femur and tibia. Bone loss occurred in a widespread distribution in the bilateral hindlimbs, both ipsilateral and, to a lesser extent, contralateral to the nerve

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Table 1 Bilateral femoral bone mineral density (BMD) and the percentage change (%) at 4 and 12 weeks after unilateral sciatic nerve transection

Ipsilateral femur Baseline 4 weeks 12 weeks Contralateral femur Baseline 4 weeks 12 weeks

n

Proximal

Diaphysis

Distal

16 16 8

533 ⫾ 12 494 ⫾ 9 (27%)*** 470 ⫾ 11 (211%)***

449 ⫾ 9 442 ⫾ 9 (22%) 431 ⫾ 9 (23%)

414 ⫾ 9 361 ⫾ 11 (213%)*** 338 ⫾ 10 (216%)***

16 16 8

538 ⫾ 12 503 ⫾ 13 (26%)** 493 ⫾ 12 (26%)*

443 ⫾ 8 446 ⫾ 7 (20%) 435 ⫾ 7 (21%)

421 ⫾ 6 388 ⫾ 8 (28%)*** 378 ⫾ 8 (29%)***

Note. Data are expressed as mean ⫾ SEM BMD (mg/cm2). (2%) at 4 weeks represents the percentage decrease in BMD from baseline in 16 rats, and at 12 weeks represents the percentage decrease in BMD in 8 rats (comparing with the same 8 rats at baseline). Multiple comparisons of data were performed by analysis of variance (ANOVA) with Fisher’s PLSD test.

section. There was no evidence of disuse in the contralateral intact hindlimb after sciatic section; body weight, spontaneous locomotor activity, and weight bearing on the intact hindlimb were unchanged. LY303870 enhanced the osteoporotic effects of sciatic section Four weeks after unilateral sciatic transection rats were treated with either LY303870 (20 mg/kg/day, ip, n ⫽ 8) or saline (ip, n ⫽ 8) on a daily basis for 2 weeks. Figure 2 illustrates that LY303870-treated rats had greater bone loss in the ipsilateral proximal and distal femur and proximal tibia at 12 weeks than was observed in the saline treatment group. Similarly, in the contralateral intact hindlimb at 12 weeks after neurectomy the LY303870 treatment group had a greater reduction in BMD in the proximal and distal femur and tibia than the saline treatment group (Fig. 3). These results suggest that substance P signaling contributes to residual bone integrity after neurectomy. Widespread inhibition of neurogenic extravasation after sciatic section At 2 weeks after unilateral sciatic section the rats were injected intravenously with Evans blue dye and the contralateral intact sciatic nerve was stimulated to evoke protein extravasation. Control rats (n ⫽ 10) had a hindpaw dye content of 191 ⫾ 20 ng/mg wet wt skin after sciatic stimulation and without stimulation the contralateral hindpaw dye content was only 11 ⫾ 1 ng/mg (Fig. 5A). LY303870 (40 mg/kg, ip) given 1 h before sciatic stimulation reduced hindpaw dye content to 9.4 ⫾ 1.9 ng/mg (n ⫽ 8). These data demonstrate that the sciatic evoked extravasation response was entirely mediated by substance P signaling. After sciatic section there was a 33% decrease in the sciatic evoked extravasation response in the contralateral intact hindpaw (131 ⫾ 16 ng/mg, n ⫽ 7, P ⬍ 0.05), indicating a widespread reduction in substance P signaling after neurectomy.

Widespread loss of substance P in the proximal tibia after sciatic section Unilateral sciatic section reduced substance P content in the bilateral proximal tibia by 50% (Fig. 5B). Substance P content in normal control bone was 1.5 ⫾ 0.28 pg/mg bone (n ⫽ 12), and at 2 weeks after sciatic section substance P content in the ipsilateral proximal tibia was reduced to 0.76 ⫾ 0.12 pg/mg (P ⬍ 0.05, n ⫽ 9) and in the contralateral proximal tibia it was reduced to 0.73 ⫾ 0.08 pg/mg (P ⬍ 0.05, n ⫽ 8). Substance P content in the normal control sciatic nerve was 16.91 ⫾ 2.68 pg/mg nerve (n ⫽ 13), and at 2 weeks after unilateral sciatic neurectomy substance P content in the contralateral nerve was reduced to 10.62 ⫾ 0.76 pg/mg (P ⬍ 0.05, n ⫽ 8). This 37% reduction in substance P content of the contralateral sciatic nerve is probably the basis for the 33% reduction in the contralateral extravasation response observed after neurectomy.

Discussion The first aim of this investigation was to use in vivo DEXA scanning to characterize the osteoporotic effects of sciatic nerve section in the skeletally mature rat. We observed a reduction in BMD after unilateral sciatic section that rapidly progressed for 4 weeks and then stabilized or slowly progressed over the next 8 weeks (Figs. 2, 3; Tables 1, 2). A similar time course has been reported for the reduction in proximal tibial cancellous bone volume after sciatic section in 10-week-old rats [21]. The current study used DEXA scanning to demonstrate that neurectomyevoked bone loss was most prominent in the metaphyseal regions of the proximal and distal femur and tibia. These results concur with those of other investigators using DEXA scanning, bone histomorphometric analysis, biomechanical testing, and bone metabolic markers in skeletally mature rats [22,23]. These studies demonstrated an increase in bone reabsorption and a decrease in bone formation at 4 weeks after sciatic section, with a loss of cancellous but not cor-

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Table 2 Bilateral tibial bone mineral density (BMD) and the percentage change (%) at 4 and 12 weeks after unilateral sciatic nerve transection

Ipsilateral tibia Baseline 4 weeks 12 weeks Contralateral tibia Baseline 4 weeks 12 weeks

n

Proximal

Diaphysis

Distal

16 16 8

375 ⫾ 9 314 ⫾ 8 (216%)*** 299 ⫾ 10 (219%)***

305 ⫾ 4 294 ⫾ 5 (23%)** 287 ⫾ 4 (25%)**

467 ⫾ 4 450 ⫾ 4 (24%)*** 439 ⫾ 7 (26%)*

16 16 8

376 ⫾ 6 357 ⫾ 9 (25%)** 356 ⫾ 10 (24%)

305 ⫾ 4 306 ⫾ 5 (20%) 296 ⫾ 5 (23%)

467 ⫾ 5 460 ⫾ 5 (22%) 468 ⫾ 5 (20%)

Note. Data are expressed as mean ⫾ SEM BMD (mg/cm2). (2%) at 4 weeks represents the percentage decrease in BMD from baseline in 16 rats, and at 12 weeks represents the percentage decrease in BMD in 8 rats (comparing with the same 8 rats at baseline). Multiple comparisons of data were performed by analysis of variance (ANOVA) with Fisher’s PLSD test.

tical bone, resulting in reductions in BMD in the distal femur and proximal tibia, but not in the diaphyses. As we had hypothesized, a widespread reduction in cancellous bone density was observed in the bilateral hindlimbs after neurotomy, both ipsilateral and, to a lesser extent, contralateral to the nerve section (Tables 1, 2). The contralateral bone loss observed after unilateral sciatic section is a novel finding. Other investigators have not seen any reductions in the contralateral whole femur ash weight 3 to

Fig. 4. Testing for disuse in the contralateral intact hindlimb after unilateral sciatic section. (A) There was no significant change in locomotor activity at 2, 4, and 8 weeks after neurotomy. (B) At 2, 4, and 8 weeks after sciatic section there was a 25–27% reduction in weight bearing on the ipsilateral hindpaw versus baseline and no change in weight bearing on the contralateral hindlimb. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

4 weeks after unilateral sciatic section [24,25]. The reduction in contralateral hindlimb BMD we observed using DEXA scanning was less robust than the ipsilateral BMD reduction and a change of this magnitude might not be detected using a total bone ash weight method that lacks the sensitivity of an in vivo within-animal comparison and cannot selectively measure only cancellous bone loss. Another group used peripheral quantitative computed tomography to measure cancellous bone loss in the contralateral distal femur after unilateral sciatic section [26]. These investigators observed a 17% reduction in the contralateral distal femur BMD, a change that did not reach significance in their study but paralleled the 8% reduction in BMD we observed using DEXA scanning in the contralateral distal femur (Fig. 3, Table 1). There was no evidence of disuse in the contralateral intact hindlimb after sciatic section; body weight, spontaneous locomotor activity, and weight bearing on the intact hindlimb were unchanged (Fig. 4). Furthermore, there is reportedly no reduction in the weight of the soleus or gastrocnemius muscles in the contralateral intact hindlimb after a unilateral sciatic section [21]. Collectively these data confirm that contralateral hindlimb disuse does not occur after unilateral sciatic section; therefore, contralateral hindlimb osteoporosis after nerve section must be attributed to some other mechanism. These results support the premise that contralateral inhibition of neuronal signaling after sciatic section could induce bone loss in the contralateral hindlimb. When the substance P receptor antagonist LY303870 was administered daily for 2 weeks in rats who had undergone unilateral sciatic section 4 weeks earlier there was enhanced reduction of cancellous bone density in the hindlimb both ipsilateral to the neurectomy (Fig. 2) and contralateral to the nerve injury (Fig. 3). These results suggest that substance P signaling maintained residual bone integrity after sciatic transection and that blocking the substance P receptor with LY303870 resulted in bone loss. Neurotransmitter regulation of bone metabolism has been previously proposed based on anatomic and cell culture data [2,11,12]. Substance P-immunoreactive axons have been

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Fig. 5. Effect of unilateral sciatic section on contralateral hindlimb substance P signaling and content. (A) Two weeks after right sciatic section there was a 33% reduction in the contralateral extravasation response vs control. The evoked hindpaw dye content in LY303870 (40 mg/kg, ip)-pretreated control rats was the same as the dye content in control rats that had no nerve stimulation, indicating that the extravasation response was mediated by substance P signaling. (B) After right sciatic section there was a 50% reduction in substance P content in the bilateral proximal tibias. *P ⬍ 0.05, ***P ⬍ 0.001.

observed in the periosteum, epiphysis, and bone marrow, whereas innervation of cortical bone is sparse [27–30]. The neurons containing substance P usually enter the meduallary space in association with a blood vessel but then dissociate from the vessel and terminate as a free ending in the marrow [12,31]. Immunocytochemical studies have observed NK1 receptors on the plasma membrane and in the cytoplasm of osteoclasts, osteoblasts, and osteocytes [28]. Furthermore, substance P has an osteogenic effect on bone marrow cells, stimulating the formation of bone colonies [32]. Substance P also increases [3H]proline incorporation and protein accumulation in bone marrow-derived osteogenic cell cultures and stimulates bone formation in primary cultured rat calvarial osteoblasts [11,33]. Collectively these data suggest that the release of substance P in bone could directly stimulate osteoblastic bone formation. Substance P may also enhance the osteogenic actions of other osseal neurotransmitters such as calcitonin gene-related peptide (CGRP) and glutamate [34,35]. CGRP stimulates osteoblast proliferation and bone colony formation in vitro and partly prevents bone loss in ovariectomized rats [2,36]. Glutamate is released by both osteoblasts and skeletal nerve fibers and it has been proposed that glutaminergic signaling may

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have an anabolic effect on bone [2,37,38]. Substance P, CGRP, and glutamate are co-localized in and co-released from unmyelinated sensory neurons and have synergistic effects in vascular tissue and on nociceptive signaling [39,40]. Substance P also enhances the release of glutamate from nerve terminals [40,41]. These studies indicate that substance P activity in bone could be due to facilitation of the osteogenic effects of CGRP and glutamate. Several investigators have reported that unilateral saphenous nerve transection can reduce neurogenic extravasation responses in the contralateral intact hindlimb by 30 – 40% [8 –10]. This reduction is paralleled by a decrease in the content of substance P in the contralateral saphenous nerve [10]. We observed a similar transneuronal effect after unilateral sciatic nerve transection with a subsequent 33% reduction in the contralateral sciatic evoked extravasation response (Fig. 5A), a 37% reduction in the substance P content of the contralateral sciatic nerve, and a 50% reduction in the substance P content in the bilateral proximal tibia (Fig. 5B). The contralateral reduction of extravasation responses and the bilateral reduction in proximal tibia substance P content within 2 weeks of unilateral nerve section parallel the rapid loss of trabecular bone density observed in both hindlimbs after neurectomy. Furthermore, the reduction in the contralateral extravasation response develops within 2 weeks and persists for at least 26 weeks after saphenous nerve transection [8], corresponding to the temporal development and maintenance of bone loss after sciatic section. Collectively, these data indicate that nerve section caused a widespread reduction of substance P signaling in the contralateral intact hindlimb. In conclusion, after unilateral sciatic transection there was widespread rapid bone loss in both hindlimbs of the rat, which was exacerbated by chronic administration of the substance P NK1 receptor antagonist LY303870. After neurotomy there was no evidence of disuse of the contralateral hindlimb. However, the contralateral neurogenic extravasation response was diminished and there was a contralateral reduction in bone substance P content, evidence of a transmedian loss of substance P signaling after nerve injury. Collectively these data support the hypothesis that a reduction in bone substance P levels contributes to the remote osteoporotic effects of sciatic transection and that residual substance P signaling limits bone loss after nerve section.

Acknowledgments This study was supported by a grant from the National Institutes of Health (R01 GM65345).

References [1] Jee WS, Ma Y. Animal models of immobilization osteopenia. Morphologie 1999;83:25–34.

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[2] Lerner UH, Lundberg P. Kinins and neuro-osteogenic factors. In: Bilezikian JP, Raisz LG, Rodar GA, editors. Principles of bone biology. San Diego: Academic Press, 2001. [3] Goto T. Introduction to the innervation of bone. Microsc Res Tech 2002;58:59 – 60. [4] Lundberg P, Lerner UH. Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells. Microsc Res Tech 2002;58:98 –103. [5] Koltzenburg M, Wall PD, McMahon SB. Does the right side know what the left is doing? Trends Neurosci 1999;22:122–7. [6] Parfitt AM. The physiologic and clinical significance of bone histomorphometric data, in: Recker RR, editor. Bone histomorphometry: techniques and interpretation. Boca Raton, FL: CRC Press, 1983. [7] Kimmel DB. Animal models in osteoporosis research, in: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press, 2001. p. 163. [8] Allnatt JP, Dickson KE, Lisney SJ. Saphenous nerve injury and regeneration on one side of a rat suppresses the ability of the contralateral nerve to evoke plasma extravasation. Neurosci Lett 1990; 118:219 –22. [9] Kolston J, Lisney SJ, Mulholland MN, Passant CD. Transneuronal effects triggered by saphenous nerve injury on one side of a rat are restricted to neurones of the contralateral, homologous nerve. Neurosci Lett 1991;130:187–9. [10] Scott C, Perry MJ, Raven PE, Massey EJ, Lisney SJ. Capsaicinsensitive afferents are involved in signalling transneuronal effects between cutaneous sensory nerves. Neuroscience 2000;95:535– 41. [11] Goto T, Tanaka T. Tachykinins and tachykinin receptors in bone. Microsc Res Tech 2002;58:91–7. [12] Imai S, Matsusue Y. Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone. Microsc Res Tech 2002;58:61–9. [13] Schaffalitzky De Muckadell OB, Aggestrup S, Stentoft P. Flushing and plasma substance P concentration during infusion of synthetic substance P in normal man. Scand J Gastroenterol 1986;21:498 –502. [14] Yeo CJ, Jaffe BM, Zinner MJ. The effects of intravenous substance P infusion on hemodynamics and regional blood flow in conscious dogs. Surgery 1984;95:175– 82. [15] Ammann P, Rizzoli R, Slosman D, Bonjour JP. Sequential and precise in vivo measurement of bone mineral density in rats using dual-energy x-ray absorptiometry. J Bone Miner Res 1992;7:311– 6. [16] Gitter BD, Bruns RF, Howbert JJ, Waters DC, Threlkeld PG, Cox LM, et al. Pharmacological characterization of LY303870: a novel, potent and selective nonpeptide substance P (neurokinin 1) receptor antagonist. J Pharmacol Exp Ther 1995;275:737–744. [17] Hipskind PA, Howbert JJ, Bruns RF, Cho SSY, Crowell TA, Foreman MM, et al. 3-Aryl-1,2,-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists. J Med Chem 1996;39:736 – 48. [18] Iyengar S, Hipskind PA, Gehlert DR, Schober D, Lobb KL, Nixon JA, et al. LY303870, a centrally active neurokinin-1 antagonist with a long duration of action. J Pharmacol Exp Ther 1997;280:774 – 85. [19] Kalu DN, Liu CC, Hardin RR, Hollis BW. The aged rat model of ovarian hormone deficiency bone loss. Endocrinology 1989;124:7–16. [20] Wang L, Banu J, McMahan CA, Kalu DN. Male rodent model of age-related bone loss in men. Bone 2001;29:141– 8. [21] Zeng QQ, Jee WS, Bigornia AE, King JG Jr., D’Souza SM, Li XJ, et al. Time responses of cancellous and cortical bones to sciatic neurectomy in growing female rats. Bone 1996;19:13–21. [22] Iwamoto J, Takeda T, Katsumata T, Tanaka T, Ichimura S, Toyama Y. Effect of etidronate on bone in orchidectomized and sciatic neurectomized adult rats. Bone 2002;30:360 –7.

[23] Shen V, Liang XG, Birchman R, Wu DD, Healy D, Lindsay R, Dempster DW. Short-term immobilization-induced cancellous bone loss is limited to regions undergoing high turnover and/or modeling in mature rats. Bone 1997;21:71– 8. [24] Tuukkanen J, Wallmark B, Jalovaara P, Takala T, Sjogren S, Vaananen K. Changes induced in growing rat bone by immobilization and remobilization. Bone 1991;12:113– 8. [25] Weinreb M, Patael H, Preisler O, Ben-Shemen S. Short-term healing kinetics of cortical and cancellous bone osteopenia induced by unloading during the reloading period in young rats. Virchow’s Arch 1997;431:449 –52. [26] Yonezu H, Ikata T, Takata S, Shibata A. Effects of sciatic neurectomy on the femur in growing rats: application of peripheral quantitative computed tomography and Fourier transform infrared spectroscopy. J Bone Miner Metab 1999;17:259 – 65. [27] Bjurholm A. Neuroendocrine peptides in bone. Int Orthop 1991;15: 325–9. [28] Goto T, Yamaza T, Kido MA, Tanaka T. Light- and electron-microscopic study of the distribution of axons containing substance P and the localization of neurokinin-1 receptor in bone. Cell Tissue Res 1998;293:87–93. [29] Hukkanen M, Konttinen YT, Rees RG, Gibson SJ, Santavirta S, Polak JM. Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related peptide containing sensory fibers. J Rheumatol 1992;19:1252–9. [30] Hukkanen M, Konttinen YT, Rees RG, Santavirta S, Terenghi G, Polak JM. Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone. Int J Tissue React 1992;14:1–10. [31] Imai S, Tokunaga Y, Maeda T, Kikkawa M, Hukuda S. Calcitonin gene-related peptide, substance P, and tyrosine hydroxylase-immunoreactive innervation of rat bone marrows: an immunohistochemical and ultrastructural investigation on possible efferent and afferent mechanisms. J Orthop Res 1997;15:133– 40. [32] Shih C, Bernard GW. Neurogenic substance P stimulates osteogenesis in vitro. Peptides 1997;18:323– 6. [33] Adamus MA, Dabrowski ZJ. Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro. J Cell Biochem 2001;81:499 –506. [34] Chenu C. Glutamatergic innervation in bone. Microsc Res Tech 2002;58:70 – 6. [35] Irie K, Hara-Irie F, Ozawa H, Yajima T. Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech 2002;58:85–90. [36] Valentijn K, Gutow AP, Troiano N, Gundberg C, Gilligan JP, Vignery A. Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats. Bone 1997;21:269 –74. [37] Genever PG, Skerry TM. Regulation of spontaneous glutamate release activity in osteoblastic cells and its role in differentiation and survival: evidence for intrinsic glutamatergic signaling in bone. FASEB J 2001;15:1586 – 8. [38] Skerry TM. Identification of novel signaling pathways during functional adaptation of the skeleton to mechanical loading: the role of glutamate as a paracrine signaling agent in the skeleton. J Bone Miner Metab 1999;17:66 –70. [39] Holzer P. Peptidergic sensory neurons in the control of vascular functions: mechanism and significance in the cutaneous and splanchnic vascular beds. Rev Physiol Biochem Pharmacol 1992;121:49–146. [40] Kangrga I, Randic M. Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice. J Neurosci 1990;10:2026 –38. [41] Stacey AE, Woodhall GL, Jones RS. Neurokinin-receptor-mediated depolarization of cortical neurons elicits an increase in glutamate release at excitatory synapses. Eur J Neurosci 2002;16:1896 –906.