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Anti–nerve growth factor does not change physical activity in normal young or aging mice but does increase activity in mice with skeletal pain

Majuta, Lisa A.a; Mitchell, Stefanie A.T.a; Kuskowski, Michael A.b; Mantyh, Patrick W.a,c,*

doi: 10.1097/j.pain.0000000000001330
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Anti–nerve growth factor (anti-NGF) therapy has shown significant promise in attenuating several types of skeletal pain. However, whether anti-NGF therapy changes the level of physical activity in individuals with or without skeletal pain is largely unknown. Here, automated day/night activity boxes monitored the effects of anti-NGF treatment on physical activity in normal young (3 months old) and aging (18-23 months old) mice and mice with bone fracture pain. Although aging mice were clearly less active and showed loss of bone mass compared with young mice, anti-NGF treatment had no effect on any measure of day/night activity in either the young or aging mice. By contrast, in mice with femoral fracture pain, anti-NGF treatment produced a clear increase (10%-27%) in horizontal activity, vertical rearing, and velocity of travel compared with the Fracture + Vehicle group. These results suggest, just as in humans, mice titrate their level of physical activity to their level of skeletal pain. The level of skeletal pain may in part be determined by the level of free NGF that seems to rise after injury but not normal aging of the skeleton. In terms of bone healing, animals that received anti-NGF showed an increase in the size of calcified callus but no increase in the number of displaced fractures or time to cortical union. As physical activity is the best nondrug treatment for many patients with skeletal pain, anti-NGF may be useful in reducing pain and promoting activity in these patients.

Anti–nerve growth factor does not increase physical activity in young or aging naive mice but attenuates the decline in physical activity in mice with skeletal pain.

aDepartment of Pharmacology, University of Arizona, Tucson, AZ, United States

bDepartment of Psychiatry, University of Minnesota, Minneapolis, MN, United States

cCancer Center, University of Arizona, Tucson, AZ, United States

Corresponding author. Address: Department of Pharmacology, University of Arizona, 1501 N. Campbell Ave, PO Box 245050, Tucson, AZ 85724, United States. Tel.: (520) 626-0742; fax: (520) 626-8869. E-mail address: pmantyh@email.arizona.edu (P.W. Mantyh).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

Received February 28, 2018

Received in revised form May 09, 2018

Accepted May 25, 2018

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1. Introduction

1.1. The problem of musculoskeletal pain

Skeletal pain is a leading cause of morbidity and mortality in both developing and developed countries.61,63,99 Despite the increase in burden of musculoskeletal pain, treatment options remain limited. The 2 major classes of analgesics used to treat musculoskeletal pain are NSAIDs and opiates, although both are accompanied by a variety of unwanted side effects. Long-term NSAID use may inhibit bone formation in patients at high risk for impaired bone healing80 as well as produce significant gastrointestinal and renal side effects.2,23,75,76 In developed market economies, most prescribed opiates are for the management of chronic musculoskeletal pain. However, opiates have a variety of adverse side effects including sedation, respiratory compromise, genitourinary retention, and dependence all of which negatively impact the individual's physical activity, functional status, and ability to return to work.45,83,96 The lack of efficacious and better tolerated analgesics for treating musculoskeletal pain has undoubtedly played a major role in the current opioid epidemic plaguing several developed countries, including the United States.

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1.2. The promise and unknowns of anti–nerve growth factor and skeletal pain

Preclinical studies have shown that administration of anti–nerve growth factor (anti-NGF) can attenuate a variety of skeletal pains including that due to osteoarthritis (OA),10,35,36,38,51,87,100 hip dysplasia,53 bone fracture,43,48,77,82,98,101 orthopedic surgery,59 bone cancer,31,64,66,86 and head and neck cancer.102 In humans, anti-NGF has been shown to significantly reduce the magnitude of OA and low back pain.9,17,18,44,52,68 Anti–nerve growth factor treatment has also been shown to produce significant relief of human bone cancer pain, although this relief was only observed in patients who had high bone pain and low opiate use on initial enrollment in the study.89

One side effect that was observed when using anti-NGF to treat patients with OA was that a subset of patients developed an accelerated form of OA that has been previously described as rapidly progressive osteoarthritis (RPOA).33 Risk factors for the development of RPOA in these studies included a high dose of anti-NGF, concomitant use of NSAIDs, and underlying joint pathologies.33 A major question is whether this RPOA is due to a direct biological effect of anti-NGF on the bone and/or joint8,33,34,85 or whether, in some patients, the significant pain relief offered by anti-NGF may lead to increased physical activity resulting in further injury to an already compromised arthritic joint.63,70

Currently, relatively little is known as to whether anti-NGF therapy increases the level of physical activity in humans or animals with or without skeletal pain.20,29,53,59,70 In this study, this question is explored in naive mice and mice with skeletal pain because of fracture of the femur. Activity was quantified using automated activity boxes that continuously record horizontal activity, vertical activity, and velocity of movement over a 20-hour day/night period.

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2. Methods

2.1. Animals

Experiments were performed on 70 adult male C57Bl/6J mice (numbers represent 3 independent experiments) (Jackson Laboratories, Bar Harbor, ME), approximately 8 to 10 weeks old (young adult) or 18 to 23 months old (aged adult), weighing 22 to 30 g at the time of stabilizing pin implantation surgery (aged, naive mice were 28-40 g). Forty-two mice were used to establish the timeline and effect of fracture on activity, rearing, and velocity of movement after anti-NGF or vehicle (16 anti-NGF, 16 vehicle, and 10 naive) administration while an additional 28 naive mice (15 young and 13 aged) were tested before and after the administration of anti-NGF. Animals were individually housed (AAALAC approved SPF facility, Lab Products IVC 750 cages, 6.75″ × 12.25″ × 5″, with 1/4″ corn cob bedding and nestlet) for at least 2 weeks before baseline recordings and continued throughout the duration of the experiment. After 1 week of acclimation to the housing facility, all mice were numbered using ear tag. For the fracture experiment, the first 32 odd-numbered mice were assigned to a group and even-numbered mice to another group before any testing or surgical procedures. Fracture mice underwent stabilizing pin implantation surgery after naive baselines were obtained. Mice were housed in accordance with National Institutes of Health Guidelines and kept in a vivarium maintained at 22°C with a 12-hour alternating light–dark cycle (7 AM-7 PM) and provided food and water ad libitum. All procedures adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain and were approved by the Institutional Animal Care and Use Committee at the University of Arizona (Tucson, AZ, USA).

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2.2. Stabilizing pin implantation surgery

An arthrotomy was performed as described previously.59,60 In short, after induction of general anesthesia with ketamine/xylazine (100-mg/kg ketamine and 10-mg/kg xylazine, subcutaneous [s.c.]), a 1-cm incision was made in the skin above the right femur. The skin over the knee was degloved, and the joint was exposed by transposing the patella medially through blunt dissection while the knee is flexed. A 0.5 mm in diameter hole was drilled in the center of the trochlear groove of the femur using a pneumatic dental high-speed hand piece. A precut 0.015 mm in diameter stainless steel wire (Small Parts, Inc, Logansport, IN) was inserted into the intramedullary canal for fracture stabilization. The drill site was sealed with a dental amalgam plug (Dentsply, Milford, DE). The knee was extended, and the patella returned to its proper position. To minimize medial patella luxation, the rectus femoris and vastus medialis muscles near the knee were secured back in position using a horizontal mattress suture. Wound closure was achieved with two 7-mm auto wound clips (Becton Dickinson, Sparks, MD). Animals recovered from anesthesia on heating pads and received injections of antibiotic (Baytril, 85 mg/kg, s.c.) and sterile saline (1 mL, s.c.). After recovering from surgery, animals remained individually housed to avoid fighting that increases the likelihood of displacement of the patella.

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2.3. Fracture production

A closed mid-diaphyseal fracture of the femur was produced 28 days after pin implantation. After induction of general anesthesia with ketamine/xylazine (100-mg/kg ketamine and 10-mg/kg xylazine, s.c.), a fracture of the right femur was produced using a 3-point bending device (BBC Specialty Automotive Center, Linden, NJ) based on the fracture apparatus described by Bonnarens and Einhorn (1984). The anesthetized mice were placed in a supine position with the femur (medial side up) directly over the support anvil of the bending device. The blunt guillotine blade was gently lowered onto the hind limb equidistant between the knee and the hip joints. A 168-g weight was dropped onto the guillotine from a height of 18 cm creating the closed fracture. Immediately after fracture, mice were radiographed to ensure localization of fracture to the mid-diaphysis of the femur (±1.5 mm).

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2.4. Radiography

While under general anesthesia on the day of fracture, high-resolution x-ray images of the mediolateral plane of the ipsilateral femur were obtained using a Faxitron MX-20 digital cabinet x-ray system (Faxitron/Bioptics, Wheeling, IL). Animals also had x-ray images taken under light anesthesia (50-mg/kg ketamine and 5-mg/kg xylazine, s.c.) on day 7 after surgery, as x-ray images easily show if patellar displacement has occurred, and days 0, 7, 14, 21, 42, and 63 after fracture. Also using Photoshop, x-ray images, as TIFF files, were used to calculate the area of the calcified callus (mm2) around the healing femur as well as determining when cortical union occurs. The days when cortical union occurred were scored by blinded individuals other than the investigator performing the measurement of physical activity.

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2.5. Exclusion criteria

Animals were excluded from the experiment if any of these conditions occurred: surgical complications, a loss of more than 20% of their presurgery weight, if patella displacement had occurred as identified through radiography, fractures located too far from the mid-diaphyseal region of the femur, dislodged pin, nonvisible fracture after impact, or fragmentation of the bone. The final number of mice used for the fracture experiment was 30 (1 mouse was lost because of bone fragmentation, and 1 mouse was removed because of fracture located too distally; both mice were from the Fracture + Anti-NGF group).

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2.6. Assessment of locomotor activity

For the fracture experiments, mice were assessed at day -7 (baseline before surgery), day 21 after surgery (to insure the return to baseline levels), and on days 1, 3, 7, and 14 after fracture for locomotor activity (horizontal activity and velocity) and vertical rearing episodes. For the anti-NGF in naive mice experiment, mice were assessed at naive baseline and 4 days after anti-NGF administration for locomotor activity (horizontal activity and velocity) and vertical rearing episodes. Animals were placed individually in plexiglass boxes (16 × 16 × 11.75 inches) containing a thin layer of bedding and a 1-inch square of Napa Nectar (Systems Engineering, Napa, CA). Locomotor activity and rearing episodes were assessed through open-field monitoring by arrays of photo-beam sensors that use beam breaks to determine the location of each animal at all times (Omnitech Electronics, Columbus, OH). Locomotor activity and rearing episodes were monitored for 20 hours beginning at 12:00 hours (noon) in a light (12-hour light/dark, 7 AM-7 PM) and temperature controlled (22°-26°C) testing room that remained closed to any other activity. Fusion software (Omnitech Electronics) was used to analyze and store the above parameters.

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2.7. Drug treatment

Nerve growth factor sequestering antibody {mAb911 at a dose of 10 mg/kg (intraperitoneal [i.p.]), a kind gift from Dr David Shelton and Dr Kris Poulsen, Rinat/Pfizer, San Francisco, CA} or vehicle (phosphate-buffered saline, 10 mL/kg) was given 1 hour after bone fracture and once every 5 days thereafter until day 15. This dose was chosen because it significantly reduced evoked pain behaviors in preclinical models of skeletal pain (McCaffrey, 2014; Majuta, 2015; Guedon, 2016). Mice were assigned to a group using even- or odd-numbered ear tags. Anti–nerve growth factor and vehicle solutions were prepared in equal volumes and labeled A and B by an individual not associated with testing the animals. The individual giving the injections was blinded as to the identity of each solution.

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2.8. Statistical analysis

All values are expressed as mean ± SEM. Analysis of variance was performed on data from each day (0, 1, 3, 7, and 14) separately for the “exploratory hour” and for the “first 3 hours of dark” variables for horizontal distance, vertical episodes, and average velocity. All variables were log-transformed before area under the curve computation. Analyses of variance with post hoc t-tests (unadjusted for multiple comparisons) were used to compare the 3 mouse groups (Naive, Fracture + Vehicle, and Fracture + Anti-NGF). Two-group t-tests were used when comparing the Fracture + Vehicle and Fracture + Anti-NGF groups on callus area as well as for the naive before and after anti-NGF administration. Union results were calculated using Kruskal–Wallis 1-way nonparametric analysis of variance. Significance level was set at P ≤ 0.05.

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3. Results

3.1. The fracture model and bone healing

Fracture pain was generated in animals by implantation of a stabilizing pin followed 28 days later by the production of a closed mid-diaphyseal fracture of the femur using a three-point bending device. Figure 1 shows a naive femur, the stages of fracture production (pin placement and fracture) and fracture healing (mineralized callus and fracture union) using high-resolution x-ray images (Figs. 1A–E, respectively). The fracture (Fig. 1C, arrows) must occur within 1.5 mm of the mid-diaphysis. The mineralized callus is radiographically apparent by day 14 after fracture (Fig. 1D).

Figure 1

Figure 1

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3.2. The importance of monitoring spontaneous activity and rearing when animals are normally the most active

In mice, there are marked diurnal variations in horizontal activity and vertical activity (Figs. 2A and B, respectively) as well as velocity (Fig. 2C) for all groups (Naive, Frac + Anti-NGF, and Frac + Vehicle) on day 3 after fracture. When mice are first placed in the activity boxes during the light phase at noon (12:00 hours), mice actively explore the novel environment for approximately 1 hour. After this initial exploration, most mice are at rest or have a marked reduction in activity until the start of the dark phase (19:00 hours) when mice again show significant increases in locomotor activity during the hours of night (19:00-07:00 hours). This period of increased activity during the daytime (12:00-13:00 hours) is consistent with the behavior of mice that rapidly explore a novel environment for potential threats, whereas the increased activity of the mice immediately after the room becomes dark at 19:00 hours is consistent with mice grooming, eating, drinking, making nests, exploring their environment, and foraging for food.

Figure 2

Figure 2

Representative tracings of distance and the pattern of horizontal locomotor activity and episodes of rearing from a Naive, Fracture + Vehicle, and Fracture + Anti-NGF mouse obtained over a 15-minute period during the most active portion of the dark phase (21:00-21:15 hours) are shown in Figure 3. Visually significant reductions in horizontal activity and rearing episodes are apparent for the Fracture + Vehicle mouse (Fig. 3B and E) 3 days after fracture when compared with Naive (Fig. 3A and D). When compared with the Fracture + Vehicle mouse, horizontal activity and the number of rearing episodes are increased with the Fracture + Anti-NGF mouse (Fig. 3C and F).

Figure 3

Figure 3

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3.3. The fracture-induced reduction in horizontal distance traveled, number of vertical episodes, and average velocity are attenuated by administration of anti–nerve growth factor

Anti–nerve growth factor (10 mg/kg, i.p.) or vehicle was administered on the day of fracture and on days 5, 10, and 15 after fracture. Comparisons of the initial exploratory hour (12:00-13:00 hours) and the combined time over the first 3 hours of the dark phase (19:00-22:00 hours) are presented in Figure 4. Mice treated with anti-NGF (Frac + Anti-NGF) traveled a significantly greater horizontal distance on days 1, 3, and 7 after fracture during the initial exploratory hour when compared with vehicle-treated (Frac + Vehicle) animals (Fig. 4A; vehicle day 1 = 4227 ± 544 cm, anti-NGF day 1 = 5763 ± 374 cm, P = 0.012; vehicle day 3 = 4671 ± 615 cm, and anti-NGF day 3 = 9027 ± 798 cm, P = 0.00004; vehicle day 7 = 7750 ± 898 cm, and anti-NGF day 7 = 9766 ± 557 cm, P = 0.038; vehicle day 14 = 10,458 ± 929 cm, and anti-NGF day 14 = 12,097 ± 639 cm, P = 0.1). The number of vertical rearing episodes for the Fracture + Anti-NGF animals on days 1 and 3 after fracture during the initial exploratory hour was also significantly increased when compared with Fracture + Vehicle (Fig. 4C; vehicle day 1 = 101 ± 20.3, anti-NGF day 1 = 155 ± 12.6, P = 0.03; vehicle day 3 = 144 ± 27.4, anti-NGF day 3 = 292 ± 22.1, P = 0.0001; vehicle day 7 = 300 ± 45.6, and anti-NGF day 7 = 349 ± 21.0, P = 0.08; vehicle day 14 = 388 ± 44.8, and anti-NGF day 14 = 445 ± 22.0, P = 0.1). As with horizontal distance traveled, the average velocity of the Fracture + Anti-NGF animals on days 1, 3, and 7 after fracture during the initial exploratory hour was also significantly increased when compared with Fracture + Vehicle (Fig. 4E; vehicle day 1 = 7.9 ± 0.4 cm/second, anti-NGF day 1 = 9.2 ± 0.3 cm/second, P = 0.02; vehicle day 3 = 7.8 ± 0.5 cm/second, and anti-NGF day 3 = 11.2 ± 0.4 cm/second, P = 0.000008; vehicle day 7 = 10.4 ± 0.5 cm/second, and anti-NGF day 7 = 11.9 ± 0.3 cm/second, P = 0.02; vehicle day 14 = 12.1 ± 0.6 cm/second, and anti-NGF day 14 = 13.0 ± 0.4 cm/second, P = 0.2).

Figure 4

Figure 4

Measuring the activity of mice during the dark phase (19:00-07:00 hours) enables the capture of more spontaneous behaviors. Animals treated with anti-NGF after fracture also exhibited significantly higher horizontal distance traveled on days 1 and 3 after fracture during peak nocturnal activity (first 3 hours of dark, 19:00-22:00 hours) compared with Fracture + Vehicle animals (Fig. 4B; vehicle day 1 = 12,723 ± 2061 cm, anti-NGF day 1 = 22,237 ± 1180 cm, P = 0.0002; vehicle day 3 = 21,935 ± 2314 cm, and anti-NGF day 3 = 31,359 ± 2422 cm, P = 0.006; vehicle day 7 = 32,274 ± 2211 cm, and anti-NGF day 7 = 32,878 ± 2346 cm, P = 0.9; vehicle day 14 = 31,957 ± 2778 cm, and anti-NGF day 14 = 38,451 ± 3319 cm, P = 0.1). The number of vertical rearing episodes for the anti-NGF animals on days 1, 3, and 14 after fracture during peak nocturnal activity were also significantly increased when compared with Fracture + Vehicle (Fig. 4D; vehicle day 1 = 494 ± 68.8, anti-NGF day 1 = 1020 ± 64.1, P = 0.002; vehicle day 3 = 853 ± 77.4, anti-NGF day 3 = 1236 ± 88.9, P = 0.004; vehicle day 7 = 1262 ± 68.2, and anti-NGF day 7 = 1438 ± 92.5, P = 0.2; vehicle day 14 = 1333 ± 69.3, and anti-NGF day 14 = 1609 ± 69.6, P = 0.009). Again as with horizontal distance traveled, the average velocity of the Fracture + Anti-NGF animals on days 1 and 3 after fracture during peak nocturnal activity was also significantly increased when compared with Fracture + Vehicle (Fig. 4F; vehicle day 1 = 9.3 ± 0.5 cm/second, anti-NGF day 1 = 11.2 ± 0.2 cm/second, P = 0.002; vehicle day 3 = 11.5 ± 0.6 cm/second, and anti-NGF day 3 = 13.2 ± 0.4 cm/second, P = 0.014; vehicle day 7 = 13.7 ± 0.4 cm/second, and anti-NGF day 7 = 13.3 ± 0.4 cm/second, P = 0.52; vehicle day 14 = 14.2 ± 0.5 cm/second, and anti-NGF day 14 = 13.9 ± 0.5 cm/second, P = 0.7).

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3.4. The effect of anti–nerve growth factor on horizontal distance traveled, number of vertical episodes, and average velocity in young naive mice vs aged naive mice

Previous reports have shown that bone loss occurs with aging in C57Bl/6 mice (Fig. 5A).25 This loss includes a decrease in cortical thickness of the femur, as well as loss of trabecular bone in the proximal and distal femur. In this experiment, anti-NGF (10 mg/kg, i.p.) was administered to young and aged, naive mice after baseline activity recordings (Fig. 5B) were made and retested again 4 days later. Their naive horizontal distance traveled, number of rearing episodes, and average velocity during the initial exploratory hour (first hour of testing, 12:00-13:00 hours) and during peak nocturnal activity (first 3 hours of dark, 19:00-22:00 hours) were compared with those values after anti-NGF (Figs. 5C–H, respectively). For both the young and aged mice, no significant difference was found between Naive and Naive + Anti-NGF for any activity (Horizontal distance Explor. naive young = 12,444 ± 676 cm, anti-NGF = 13,026 ± 780 cm, P = 0.939; Horizontal distance Explor. naive aged = 8916 ± 750 cm, anti-NGF = 8225 ± 621 cm, P = 0.920; Horizontal distance Dark naive young = 36,193 ± 2126 cm, anti-NGF = 36,463 ± 2158 cm, P = 0.9998; Horizontal distance Dark naive aged = 29,086 ± 3145 cm, anti-NGF = 28,566 ± 2544 cm, P = 0.9991; Vertical episodes Explor. naive young = 411 ± 27, anti-NGF = 434 ± 26, P = 0.918; Vertical episodes Explor. naive aged = 287 ± 25, anti-NGF = 260 ± 19, P = 0.895; Vertical episodes Dark naive young = 1559 ± 79, anti-NGF = 1614 ± 82, P = 0.975; Vertical episodes Dark naive aged = 1027 ± 94, anti-NGF = 1126 ± 118, P = 0.895; Average velocity Explor. naive young = 13.1 ± 0.4 cm/second, anti-NGF = 13.4 ± 0.4 cm/second, P = 0.980; Average velocity Explor. naive aged = 10.0 ± 0.6 cm/second, anti-NGF = 9.3 ± 0.4 cm/second, P = 0.786; Average velocity Dark naive young = 14.7 ± 0.4 cm/second, anti-NGF = 14.5 ± 0.4 cm/second, P = 0.992; and Average velocity Dark naive aged = 12.4 ± 0.4 cm/second, anti-NGF = 12.6 ± 0.4 cm/second, P = 0.991). By contrast, the naive and anti-NGF data, when comparing young vs aged, clearly shows significant differences between the young and the aged mice for horizontal distance traveled (exploratory hour only), number of rearing episodes, and average velocity (Figs. 5C–H, respectively, Horizontal distance Explor: naive young vs naive aged P = 0.0078, young anti-NGF vs aged anti-NGF P = 0.0002; Horizontal distance Dark: naive young vs naive aged P = 0.222, young anti-NGF vs aged anti-NGF P = 0.147; Vertical episodes Explor: naive young vs naive aged P = 0.0072, young anti-NGF vs aged anti-NGF P = < 0.0001; Vertical episodes Dark: naive young vs naive aged P = 0.0016, young anti-NGF vs aged anti-NGF P = 0.0044; Average velocity Explor: naive young vs naive aged P = 0.0002, young anti-NGF vs aged anti-NGF P = < 0.0001; and Average velocity Dark: naive young vs naive aged P = 0.0030, young anti-NGF vs aged anti-NGF P = 0.0158).

Figure 5

Figure 5

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3.5. The effects of anti–nerve growth factor on bone healing after fracture

X-ray images of the healing fractures on days 0, 14, 21, 42, and 63 were used to determine the size of the callus as well as when union has occurred (Fig. 6). The callus areas for the Fracture + Anti-NGF mice were significantly higher on days 14, 21, and 42 after fracture when compared with Fracture + Vehicle but were not significantly different at day 63 (Fig. 6A; vehicle day 14 = 5.99 ± 0.4 mm2, anti-NGF day 14 = 9.97 ± 0.6 mm2, P = 0.000006; vehicle day 21 = 6.10 ± 0.5 mm2, anti-NGF day 21 = 8.89 ± 0.5 mm2, P = 0.0006; vehicle day 42 = 3.28 ± 0.4 mm2, anti-NGF day 42 = 5.62 ± 0.4 mm2, P = 0.0004; vehicle day 63 = 3.15 ± 0.4 mm2, anti-NGF day 63 = 4.11 ± 0.3 mm2, P = 0.0596). There was no significant difference between the Fracture + Vehicle and Fracture + Anti-NGF groups for the percentage of mice with cortical union on any day after fracture (Fig. 6B; vehicle day 21 = 16.67 ± 9.3%, anti-NGF day 21 = 30.77 ± 13.3%, P = 0.384; vehicle day 42 = 60.0 ± 13.0%, anti-NGF day 42 = 80.77 ± 10.7%, P = 0.239; vehicle day 63 = 100 ± 0%, anti-NGF day 63 = 100 ± 0%).

Figure 6

Figure 6

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4. Discussion

4.1. Changes in physical activity with anti–nerve growth factor

In this study, anti-NGF administration in mice with significant skeletal pain due to bone fracture produced a 10% to 27% increase in horizontal activity, rears, and velocity of movement as compared to mice that had Fracture + Vehicle. The most parsimonious explanation of the increased physical activity is that anti-NGF blocks NGF-induced sensitization of nociceptors that innervate the bone.37,43,48,71 Thus, similar to humans, mice seem to be titrating their level of physical activity to their level of pain, so that when anti-NGF administration reduces skeletal pain, there is a concomitant increase in physical activity.

In most animals, normal aging is accompanied by significant loss of mass/strength and a decrease in physical activity. One possibility is that as bone mass and strength deteriorates with aging, there would be a concomitant increase in the expression and release of NGF as is seen in acutely injured tissues.15,54,55 This gradual increase of NGF in the aging bone would in turn drive the decrease in physical activity that is observed with normal aging. However, acute treatment of normal young or aging animals with anti-NGF did not increase physical activity in either group. These results suggest that the age-related decline in physical activity that is generally seen in normal, healthy humans and animals is probably driven more by a central nervous system–driven increase in fatigue rather than peripherally driven NGF-induced bone pain.

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4.2. The expression and release of nerve growth factor in the normal and injured skeleton

Recent data suggest that, in the normal, young mouse femur, there is significant expression of NGF by a remarkably restricted set of cells in bone and cartilage.12 Thus, although less than 2% of all cells in the femur display any NGF expression, robust NGF immunoreactivity was observed in mostly CD-31–negative blood vessel–associated cells that have pericyte-like morphology, a small subset of CD-31+ endothelial cells, an unidentified group of cells located at the subchondral bone/articular cartilage interface, and a few isolated, single cells in the bone marrow.12 Interestingly, most of the NGF-expressing blood vessel–associated cells were surrounded by both TrkA+ and p75+ sensory and sympathetic nerve fibers. These data show that there is a close association between NGF+ blood vessel–associated cells and TrkA+ and p75+ nerve fibers in the naive bone.7,11,12,21,41,42 The present finding that anti-NGF treatment does not alter activity in naive mice suggests that there is little ongoing basal release of NGF and little free NGF in the normal, uninjured skeleton. However, after significant skeletal injury (here bone fracture), NGF is released resulting in a marked NGF-induced sensitization and activation of the TrkA and p75 sensory nerve fibers. This NGF-induced sensitization of sensory nerve fibers is reduced when anti-NGF is present.

The above data suggest that there is significant expression, but little release, of free NGF in the normal uninjured femur. However, a largely unanswered question is whether there is marked expansion of the types of cells expressing and/or releasing NGF after injury, disease, or aging of the skeleton. Previous studies have shown that there is a marked inflammatory and immune response after injury to the skeleton.43,58,65 However, which specific cells in vivo express and release NGF in the injured skeleton is largely unknown. It has been reported that mast cells, macrophages, T- and B-lymphocytes, neutrophils, fibroblasts, pericytes, endothelial cells, stem cells, osteoblasts, osteoclasts, and osteocytes express NGF.5,24,27,28,69,74,95 Many of the above studies showing NGF expression in bone were performed in vitro and do not thoroughly assess the specificity of the immunostaining, so the specific cells that express and release authentic NGF in the injured skeleton still remain largely undefined.15,54,55 Understanding whether the expression and release of NGF changes with injury, disease, or aging in the in vivo skeleton would significantly increase our understanding of the mechanisms that drive skeletal pain.

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4.3. Effects of anti–nerve growth factor on bone healing and physical activity

In terms of short-term bone healing, anti-NGF treatment clearly does increase the size of the calcified callus, although this is in the context of a 10% to 27% increase in physical activity. At day 63 after fracture, the size of the calcified callus in mice with Fracture + Anti-NGF is the same as in mice with Fracture + Vehicle. These data together with our findings showing that Fracture + Anti-NGF had no increase in number of displaced fractures and no change in cortical union are in agreement with previous data showing that anti-NGF treatment does not impair fracture healing of bone or the mechanical strength of newly formed bone at the site of fracture.48,77 In other studies, a similar increase in size of the calcified callus after fracture was also observed in animals that had received neonatal capsaicin, resulting in a 50% reduction in sensory nerve fibers innervating the skeleton and a 50% reduction in pain behaviors (guarding and flinching) after bone fracture.40 These and other studies32,72 suggest that increased callus size is in part due to increased loading and use of the affected limb,63 and further research into the potential effects of NGF on bone formation and healing are clearly needed.33,34,77,79,94,95

The present report shows that anti-NGF increases physical activity in mice with facture pain but not in normal young or aging mice. However, is this increase in activity desirable in terms of skeletal healing, skeletal health, and improving the functional status of patients with skeletal pain? Although it may be counterintuitive, previous studies have repeatedly shown that physical activity is the best nondrug treatment for improving pain and the functional status of patients with a variety of chronic skeletal pains including osteoarthritis, low-back pain, fibromyalgia, and bone fracture.6,26,29,47,73,84 Loading of bone has been shown to decrease the expression of sclerostin, a protein expressed by bone osteocytes that inhibit bone formation.56,67,91,92 Previous data have also shown that loading of the bone after fracture increases callus formation promotes fracture healing and decreases bone and muscle loss.13,22,39 Although overuse of the injured skeleton can result in further injury to the skeleton, moderate exercise and use of the skeleton is a key component to maintaining both bone and muscle mass.

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4.4. Translating preclinical rodent data into human clinical studies

Currently, the most common endpoint used to measure skeletal pain in rodents is mechanical hyperalgesia of the skin of the hind paw.1,19,62,88 Skin hypersensitivity clearly does occur in some animals3,46,78,93 and humans3,4,14,50 with skeletal pain conditions. However, it remains unclear what specific mechanisms generate skin hypersensitivity and whether relief of skin hypersensitivity accurately predicts the extent of the relief of the underlying skeletal pain. Previous studies have shown that a therapy can relieve skin hypersensitivity but not reduce the underlying skeletal pain.30 In rodent models of osteoporosis, there are both cutaneous pain and deep musculoskeletal pain, but these 2 types of pain are differentially sensitive to pharmacological interventions.90 In addition, few human studies use skin hypersensitivity as a primary endpoint to predict the relief of skeletal pain.

Previous data collected in humans with musculoskeletal pain have shown that monitoring day/night activity using accelerometers provide an unbiased objective and more complete assessment of skeletal pain than simply asking patients, physicians, or caregivers for their daily assessment of the pain.16,57,73,81,97 Thus, although the patient assessment of pain is still important, it is frequently performed at a single time point each day and is impacted by recency, mood, depression, and memory.49,73 By contrast, automated recording of day/night physical activity data tends to be much more specific (date, time, and extent) and reliable than observational data alone and may be used to compare and contrast automated day/night physical activity obtained in preclinical models with human clinical trials using accelerometers.

A therapy that reduces skeletal pain, allowing for increased activity, with minimal organ or tissue side effects would be highly desirable for a large clinical population. This study supports the use of activity monitoring as a means to assess skeletal pain and highlights the importance of evaluating both outcomes together since each is significantly affected by the other. With new technologies now available to obtain continuous monitoring of activity and more frequent assessment of pain, such an approach is feasible and will lead to a better understanding of pain mechanisms and their modulation.

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Conflict of interest statement

P.W. Mantyh has served as a consultant and/or received research grants from Abbott (Abbott Park, IL), Adolor (Exton, PA), Array Biopharma (Boulder, CO), Johnson and Johnson (New Brunswick, NJ), Merck (White Plains, New York), Pfizer (New York, NY), Plexxikon (Berkeley, CA), Rinat (South San Francisco, CA), and Roche (Palo Alto, CA). The remaining authors have no conflicts of interest to disclose.

This research was supported by the National Institutes of Health (grant number NS023970) to P.W. Mantyh.

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Acknowledgements

The authors thank Dr Viada Glatt (University of Texas Health Science Center, San Antonio, TX) for generously providing image 5A. The anti-NGF used in this study was a kind gift from Drs Kris Poulsen and David Shelton (Rinat/Pfizer, San Francisco, CA).

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Video content associated with this article can be found online at http://links.lww.com/PAIN/A619.

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Keywords:

Nociceptors; Neurotrophins; Peripheral sensitization; Bone; Joint; Anti-NGF

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