Osteoporosis-related pain can result from local fractures,40 but is also seen in the absence of observable bone trauma.30 We recently reported cutaneous hypersensitivity and deep musculoskeletal pain in a mouse model of ovariectomy (OVX)-induced osteoporosis.37 Studies investigating bone pain have demonstrated that increased osteoclast activity produces acidic microenvironments within the bone and activation of primary afferent terminals.24,25 This activity promotes the release of proinflammatory mediators including nerve growth factor (NGF), which increases expression of the neuropeptide calcitonin gene-related peptide (CGRP) in dorsal root ganglia (DRG), and contributes to sensory hypersensitivity.33,38,43
In clinical studies, anti-NGF therapy has efficacy against skeletal pain arising from osteoarthritis and low back pain.1,18,29,39 Anti-NGF therapy is untested in osteoporosis-related pain. The aim of this study was to examine the efficacy of anti-NGF antibody therapy against cutaneous hypersensitivity, deep musculoskeletal pain, and physical function in a mouse model of OVX-induced osteoporosis.
2. Materials and methods
Female C57BL6 mice (Charles River Laboratories, Montreal, QC, Canada) were housed in an environmentally controlled facility with free access to food and water. Mice were randomly assigned to the sham-operated (n = 10) or OVX (n = 20) groups. All experiments were approved by the Animal Care Committee at McGill University and conformed to the ethical guidelines of the Canadian Council on Animal Care and the International Association for the Study of Pain (IASP).45
2.2. Surgical procedures and bone mineral density
Surgical procedures were performed at 5 to 7 weeks of age as described.17,28,37 In the sham-operated group, ovaries were exposed using the identical procedure but were left intact. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry densitometry.
2.3. Behavioral procedures
Animals were habituated in the apparatus for 60 minutes before testing. Testing was between 9:00 AM and 3:00 PM.21,37 The experimenter was blind to surgical and treatment groups, all testing was performed in parallel, and all animals were included. Cutaneous sensitivity, musculoskeletal discomfort, and physical function were each evaluated on different test days.
2.3.1. Cutaneous sensitivity to mechanical, cold, and heat stimuli
Mechanical sensitivity was assessed using the von Frey up–down method.3 Cold sensitivity was evaluated by (1) nociceptive behaviors following a drop of acetone to the left hind paw4,21,22 and (2) the latency to the first brisk hind paw withdrawal from a 4°C plate (cutoff 30 seconds; Hot/Cold Plate, 35100, Ugo Basile, Varese, Italy).4 Heat sensitivity was detected as the latency to withdrawal from a radiant heat stimulus (cutoff 22.7 seconds; IITC Life Science Inc, Woodland Hills, CA).10
2.3.2. Deep musculoskeletal discomfort and physical function
The grip test assay measuring resistance to anteroposterior stretching as a behavioral index of deep musculoskeletal pain (Stoelting Co, Wood Dale, IL).15,41 Physical function was evaluated using the rotarod (Ugo Basil SRL, Varese, Italy) and a 5-minute open-field test.13,21
2.4. Anti–nerve growth factor treatment
Animals received either anti-NGF or vehicle (OVX/anti-NGF, OVX/Vehicle, and Sham/Vehicle; n = 10 per group). Two 10 mg/kg doses of anti-NGF mouse monoclonal antibody (Exalpha Biologicals Inc, Shirley, MA) were administered through intraperitoneal (i.p.) injection on days 0 and 13. Vehicle treatment (0.01 mL/g i.p. sterile saline) followed the same schedule.
Immunohistochemistry was performed as previously described.23 Ten micrometer cryostat sections of upper (L1-3) and lower (L4-6) DRG were incubated with anti-CGRP (1:500, Cat#BML-CA1137, Lot#01101327; Enzo Life Sciences, Farmingdale, NY) and Guinea pig-derived anti-NPY (1:500; Cat#AB10341, Lot#GR12360; Abcam, Tokyo, Japan), and visualized with Cy3-goat anti-sheep IgG and Cy2-goat anti-guinea pig IgG (1:500; Jackson Immuno Research, West Grove, PA) with an Olympus BX-5. Ten sections each from the upper and lower DRG per mouse were randomly selected for analysis. The proportion of CGRP-immunoreactive (ir) and NPY-ir neurons among total DRG neurons was determined.
2.6. Data analyses
Body weight and BMD were analyzed by a 1-tailed unpaired t test or 1-way analysis of variance (ANOVA) followed by the Tukey test. Behavioural measures between baseline and day 28 were analyzed by 2-way ANOVA with group and time as factors, followed by the Dunnet multiple comparisons test. Immunohistochemistry and the day 56 behaviour (only a subset was retested on day 56) were analyzed by 1-way ANOVA followed by the Tukey test. The statistical analyses were performed using GraphPad Prism 6 software.
3.1. Effects of ovariectomy and anti–nerve growth factor treatment on body weight and bone mineral density
Ovariectomy mice trended towards an increase in body weight compared with sham-operated mice (Fig. 1A, P = 0.06). Vertebral and femoral BMD were significantly lower in OVX compared with sham-operated mice (Fig. 1B, C). Anti-NGF treatment in OVX mice had no effect (Fig. 1D–F).
3.2. Efficacy of anti–nerve growth factor treatment on behavioral indices of osteoporosis-related cutaneous hypersensitivity, deep musculoskeletal pain, and physical function
Hypersensitivity to mechanical, cold, and heat (Fig. 2A–D, baseline) was fully developed 8 weeks after OVX. Anti-NGF treatment (injected days 0 and 13) reversed mechanical (Fig. 2A) and cold (Fig. 2B, C) throughout the 28-day study. Hypersensitivity to heat was reduced at 1 and 14 days but not at 28-day post-anti-NGF (Fig. 2D).
Grip strength was decreased in OVX 8 weeks after surgery (Fig. 3A, baseline). Anti-NGF treatment had significant effects at 1 day and 28 days in OVX-operated mice (Fig. 3A). No effects of OVX surgery or anti-NGF treatment were observed in the rotarod and open-field assays (Fig. 3B, C).
3.3. Effect of anti–nerve growth factor treatment on expression of calcitonin gene-related peptide-ir and NPY-ir neurons in dorsal root ganglia
The significant increase in CGRP-ir in DRG neurons in OVX mice was attenuated by anti-NGF treatment (Fig. 4A). NPY-ir in DRG neurons was not affected by OVX or anti-NGF (Fig. 4B).
Here, we demonstrate attenuation of OVX-induced cutaneous hind paw hypersensitivity, deep musculoskeletal pain, and increased CGRP-ir after anti-NGF treatment. This is the first report to demonstrate efficacy of anti-NGF therapy in an osteoporosis model.
4.1. Effects of anti–nerve growth factor therapy on behavioral indices of osteoporotic pain
In osteoporotic mice, 2 administrations of anti-NGF therapy (days 0 and 13) reduced mechanical and cold hypersensitivity for up to 28 days, suggesting that the effects are long lasting. Heat hypersensitivity was reversed on days 1 and 14 but not day 28, suggesting a more acute effect. Anti-NGF partially reversed impaired grip strength, an index of deep musculoskeletal discomfort. Neither OVX nor treatment affected the rotarod and open-field assays for physical function; consistent with the emergence of physical disability after fracture or physical deformity rather than osteoporosis per se. The efficacy of anti-NGF therapy in osteoporosis-related pain is consistent with efficacy in other skeletal pain-related animal models including malignant bone cancer,8 knee arthritis,5 and bone fracture pain.6,12,16,35 Together, these studies suggest a critical role for NGF in driving bone- and joint-related pain.
4.2. Potential mechanisms: effects of anti–nerve growth factor therapy on sensory neurons and bone homeostasis
We previously reported increased innervation of bone marrow and neuroplasticity in the spinal cord in rodent models of osteoporosis-induced pain, suggesting that osteoporosis-related pain is associated with nerve sprouting and sensitization.14,33,38,44 Here, the proportion of CGRP-ir DRG neurons was increased after OVX. The acidic microenvironment24,25,31–33,38 and secretion of local proinflammatory cytokines34 may contribute to increased CGRP expression, resulting in neurogenic inflammation and hypersensitivity.24,25,31–33,38
In addition to roles in development and maintenance of sensory and sympathetic neurons,19 NGF plays a role in bone differentiation27 and bone fracture repair.11 Most sensory nerve fibers in bone express the high-affinity NGF receptor TrkA.8,12,26,36 In osteoporosis, proinflammatory cytokines, such as TNFα and IL-1β, increase in bone marrow; NGF regulates these substances.20 The efficacy of anti-NGF therapy in musculoskeletal-related pain may be mediated, in part, by a reduction in NGF-maintained inflammation.
4.3. Potential limitations and future directions
First, we investigated the effects of 1 concentration of anti-NGF antibodies, administered through 1 route (i.p.) with 1 dosing schedule following a protocol efficacious in mice with fracture or neuropathic pain.16,42 Additional studies are needed to optimize treatment parameters, and conclusions regarding potency, efficacy, and duration of action are premature. Second, because the literature is unclear regarding specific characteristics of osteoporosis-related pain in humans, the behavioural end points used here should be considered as proxy measures. Third, the study was performed in females; extension to male subjects is an important next step. Fourth, anti-NGF was only tested in OVX mice. However, no effects of this treatment have been reported in other preclinical studies on control animals9 and in clinical studies on healthy volunteers7; phase III clinical studies are ongoing.2
Finally, here, we observed changes in neuropeptide expression in DRG; future studies should directly examine sensory innervation in osteoporotic bone and osteoporosis-related changes in skin to fully understand sensory-bone interactions.
Here, we demonstrate anti-NGF efficacy in the OVX mouse model of osteoporosis-related pain. These data implicate NGF as a driver of long-term osteoporotic pain and suggest that anti-NGF treatment may be a useful therapy for this population.
The authors have no conflict of interest to declare.
This work was supported by the Canadian Institutes of Health Research Grants MOP102586, MOP126046, and MOP142291 to M. Millecamps and L.S. Stone.
Author contributions: M. Suzuki, M. Millecamps, S. Ohtori, C. Mori, and L.S. Stone performed study design. M. Suzuki performed experimental conduct. M. Suzuki performed data collection. M. Suzuki and L.S. Stone performed data analysis. M. Suzuki and L.S. Stone performed data interpretation. M. Suzuki and L.S. Stone performed drafting manuscript. All authors revised and approved final version of manuscript.
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