Bisphosphonates adhere to calcium hydroxyapatite in bone and inhibit osteoclast differentiation, activation, and survival. They are primarily used to prevent bone loss and fractures associated with osteoporosis, bone metastases, multiple myeloma, osteogenesis deformans, and other conditions associated with increased bone fragility.1,2 Bisphosphonate treatment also can reduce metastatic bone pain in some patients, but the effect is not immediate and bisphosphonates are not utilized as initial or primary analgesic treatments for metastatic pain.3
Complex regional pain syndrome (CRPS) is a chronic pain syndrome that most frequently develops after limb injuries and presents with regional nociceptive sensitization, vascular changes, and periarticular bone loss that exceeds the expected clinical course of the inciting injury in both magnitude and duration. CRPS symptoms gradually resolve over the first year in the majority of patients, but persistent CRPS is a serious problem resulting in chronic pain, weakness, contractures, and bone loss.4 The debate regarding the underlying mechanisms of CRPS has been dynamic and controversial, and despite extensive investigation, the pathophysiology of this condition remains undefined and it is uncertain whether any treatment for CRPS is effective.5,6 Promising data from 5 small randomized controlled trials suggest that bisphosphonates may be an effective CRPS treatment, especially early in the course of the disease.7–11 These trials examined heterogeneous bisphosphonate preparations, used differing diagnostic criteria and outcome measures, and did not examine long-term efficacy, but the consistently positive trial results warranted further investigation and several multicenter trials are currently evaluating bisphosphonate therapy in CRPS (NIH ClinicalTrials.Gov identifier NCT02504008 and EU Clinical Trial Register numbers 2014 001156-28 and 2014 001915-37).
Population-based studies indicate that distal limb fracture is the most common cause of CRPS,12,13 and we have developed a fracture model in the rat and mouse closely resembling CRPS. Rats with distal tibia fractures treated with 4-weeks cast immobilization develop hindpaw allodynia, unweighting, increased spinal Fos-immunoreactivity, increased hindpaw skin temperature, edema, facilitated neuropeptide signaling, periarticular bone loss, mast cell and keratinocyte proliferation, and increased keratinocyte expression of tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, and nerve growth factor (NGF) inflammatory mediators in the affected skin.14–22 Experimental maneuvers blocking these inflammatory mediators partially inhibited the nociceptive and vascular changes that develop after fracture, but had no effect on trabecular bone loss in the injured or contralateral limbs. Adaptive immunity also contributed to postfracture nociceptive sensitization. After tibia fracture, elevated levels of IgM complexes were observed in the skin and sciatic nerve of the injured limb, and fracture mice lacking B cells and immunoglobulin had attenuated postfracture nociceptive sensitization.23 Bisphosphonates can inhibit monocyte/macrophage/dendritic cell migration, proliferation, and differentiation in vitro and reduce monocyte expression of TNF, IL-1, and IL-6 cytokines.24–29 Bisphosphonate treatment also reduces serum levels of TNF, IL-1, and IL-6 in osteoporosis patients.30–32 Because bisphosphonates can potentially reverse pain, osteoclast activation, bone loss, inflammation, and antigen presentation, they present an attractive therapeutic approach in CRPS. The aim of the current study was to determine whether bisphosphonate treatments could inhibit the postfracture development of nociceptive sensitization, reverse established pain behaviors, preserve trabecular bone integrity, and prevent the expression of cutaneous inflammatory mediators and immunocomplex deposition in skin and nerve after fracture.
Our institute’s animal care and use committee approved these experiments. All animals were treated in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and followed the guidelines of the International Association for the Study of Pain (IASP). Ten-month-old male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) were used in all experiments. When they arrived in our institution, they were housed individually in the isolator cages with solid floors covered with 3 cm of soft bedding in a room maintained at 26°C with 14-hour light and 10-hour dark cycles. During experimental period, the animals were fed ad libitum Lab Diet 5012 (PMI Nutrition, LandOLakes, St Paul, MN) that contained 1.0% calcium, 0.5% phosphorus, and 3.3 IU/g of vitamin D3 per gram. The animals were allowed free access to drinking water.
Tibia fracture was performed under 2% to 4% isoflurane to maintain surgical anesthesia as we have previously described.14,15 The right hind limb was wrapped in a stockinet (2.5-cm wide), and the distal tibia was fractured using pliers with an adjustable stop (Visegrip, Newell Rubbermaid, Atlanta, GA) that had been modified with a 3-point jaw. The hind limb was wrapped in casting tape (Delta-Lite, Johnson & Johnson, New Brunswick, NJ), so the hip, knee, and ankle were flexed. The cast extended from the metatarsals of the hind paw up to a spica formed around the abdomen. The cast over the paw was applied only to the plantar surface; a window was left open over the dorsum of the paw and ankle to prevent constriction when postfracture edema developed. To prevent the animals from chewing at their casts, the cast material was wrapped in galvanized wire mesh. The rats were given subcutaneous saline and buprenorphine immediately after procedure (0.03 mg/kg) and on the first day after fracture for postoperative hydration and analgesia. At 4 weeks, the rats were anesthetized with isoflurane and the cast removed with a vibrating cast saw. All rats used in this study had union at the fracture site after 4 weeks of cast immobilization.
Drug Treatment Protocols
To test the hypothesis that bisphosphonate treatment can inhibit immune responses and pain behavior after tibia fracture, the following treatments were evaluated: (1) no fracture controls, (2) fracture + vehicle (Fx + vehicle) s.c. for 4 weeks, (3) fracture + alendronate 3 μg/kg/d s.c. for 4 weeks (Fx + ALN 3), (4) fracture + alendronate 60 μg/kg/d s.c. for 4 weeks (Fx + ALN 60), and (5) fracture + zoledronate 3 mg/kg/d orally for 4 weeks (Fx + ZOL 3). Both alendronate and zoledronate are nitrogenous bisphosphonates that act on bone metabolism by binding and blocking the enzyme farnesyl diphosphate synthase in the 3-hydroxymethyl-3-methylglutaryl coenzyme A reductase pathway. These dosages were selected after a thorough review of the bisphosphonate CRPS literature and discussions with lead scientists at Axsome Therapeutics, taking into consideration the fact that bisphosphonate oral bioavailability is only 1% and that zoledronate is 20 times more potent than alendronate in the inhibition of farnesyl diphosphate synthase.33,34 Alendronate (Sigma, St. Louis, MO) was diluted in normal saline and chronically perfused by ALZET Osmotic pumps (Durect, Cupertino, CA) placed s.c. over the dorsum of the rat trunk immediately after fracture. Zoledronate (a generous gift from Dr Herriot Tabuteau, Axsome Therapeutics, New York, NY) was dissolved in 4 mL distilled water, and starting the day after fracture the rats were gavaged daily with either zoledronate (3 mg/kg/d) or distilled water. The rats were fasted 6 hours before gavage treatment and returned to normal ad libitum food and water conditions 2 hours later. Calcein (15 mg/kg body weight) (Sigma, St. Louis, MO) was injected intraperitoneally at 14 days and 4 days before the time of killing. After cast removal at 4 weeks postfracture, the rats underwent behavioral testing for hindpaw von Frey allodynia, unweighting, edema, and warmth, and then they were euthanized by CO2 inhalation and the bilateral hindpaw skin, sciatic nerves, gastrocnemius and soleus muscles, popliteal lymph nodes, femurs, and the L5 lumbar vertebra were collected.
An additional study was performed looking at the effect of zoledronate treatment on established CRPS-like symptoms in fracture rats. After cast removal at 4 weeks postfracture, the rats underwent behavioral testing for nociception, edema, and warmth, and then the fracture rats were started on daily gavage treatments with either zoledronate (a 21 mg/kg loading dose the first day and 3 mg/kg/d thereafter) or distilled water for 3 weeks (weeks 4–7 postfracture) and behavioral testing was repeated each week.
Hindpaw Nociception, Temperature, and Edema
To measure hindpaw plantar mechanical allodynia in the fracture rats, an up-down von Frey testing paradigm35 was used as we have previously described.14,15 Hind paw mechanical nociceptive thresholds were analyzed as the difference between the treatment side and the contralateral untreated side.
An incapacitance device (IITC Inc/Life Science, Woodland Hills, CA) was used to measure hindpaw unweighting. The rats were manually held in a vertical position over the apparatus with the hind paws resting on separate metal scale plates, and the entire weight of the rat was supported on the hindpaws. The duration of each measurement was 6 seconds, and 10 consecutive measurements were taken at 20-second intervals. Eight readings (excluding the highest and lowest) were averaged to calculate the bilateral hind paw weight-bearing values.15,18 Right hindpaw weight-bearing data were analyzed as a ratio between the right hindpaw weighting and the mean of right and left hindpaws values [(2R/(R + L)) × 100%].
The temperature of the hindpaw was measured using a fine wire thermocouple (Omega Engineering, Stamford, CT) applied as previously described.14,15 Temperature testing was performed over the hindpaw dorsal skin between the first and second metatarsals (medial), the second and third metatarsals (central), and the fourth and fifth metatarsals (lateral). The measurements for each hindpaw were averaged for the mean paw temperature. Hindpaw edema was determined by measuring the hindpaw dorsal–ventral thickness over the midpoint of the third metatarsal with a LIMAB laser measurement sensor (Goteborg, Sweden) while the rat was briefly anesthetized with isoflurane.14,15 Temperature and hindpaw thickness data were analyzed as the difference between the fracture side and the contralateral intact side.
Ex vivo scanning was performed for assessment of trabecular and cortical bone structure using microcomputed tomography (micro-CT) (Viva CT 40, Scanco Medical AG, Bassersdorf, Switzerland). Specifically, trabecular bone was evaluated in the distal femur, and the L5 vertebral bone and cortical bone was examined at the midshaft of the femur. CT images were reconstructed in 1024 × 1024-pixel matrices for vertebral, distal femur, and midfemur samples and scored in 3-dimensional arrays. The resulting gray scale imaging was segmented using a constrained Gaussian filter to remove noise, and a fixed threshold (25.5% of the maximal gray scale value for vertebra and distal femur and 35% for the midfemur cortical bone) was used to extract the structure of the mineralized tissue. The micro-CT parameters were set at threshold = 255, σ = 0.8, support = 1 for vertebral samples; threshold = 255, σ = 0.8, support = 1 for distal femur; and threshold = 350, σ = 1.2, support = 2 for midfemur evaluation analysis.
Each L5 vertebral body was scanned using 223 transversely oriented 21-μm-thick slices (21-μm isotropic voxel size) encompassing a length of 4.68 mm. The trabecular bone region was manually identified, and all slices containing trabecular bone between the growth plates were included for our analysis. In the distal femur, 150 transverse slices of 21-μm thickness (21-μm isotropic voxel size) encompassing a length of 3.15 mm were acquired, but only 100 slices encompassing 2.1 mm of the distal femur were evaluated, starting where the growth plate bridge across the middle of the metaphysic ends. The region of interest (ROI) was manually outlined on each CT slice, extending proximally from the growth plate. Relative trabecular bone volume (BV/TV; %), trabecular number (Tb.N; mm−1), trabecular thickness (Tb.Th; µm), and trabecular separation (Tb.Sp; µm) were calculated by measuring 3-dimensional distances directly in the trabecular network and taking the mean over all voxels. The connectivity density (Conn.D; mm−3) based on the Euler number was also determined. By displacing the surface of the structure by infinitesimal amounts, the structure model index (SMI; 0–3) was also calculated. The SMI quantifies the plate versus rod characteristics of trabecular bone, in which a SMI of 0 represents a purely plate-like bone and SMI of 3 indicates a purely rod-like structure.
At the femur midshaft, 10 transverse CT slices were obtained, each 21-μm thick totaling 0.21 mm in length (21-μm isotropic voxel size), and these were used to compute the total area (T.Ar; mm2), cortical bone area (B.Ar; mm2), cortical thickness (Ct.Th; µm), and periosteum perimeter (B.Pm; mm).
Rat distal femurs were embedded, without decalcification, in methylmethacrylate (Sigma, St. Louis, MO), and sectioned longitudinally with a Leica/Jung 2255 microtome (Leica Microsystems, Wetzlar, Germany) at 4- and 8-µm-thick sections. The 4-um sections were stained with toluidine blue for collection of bone mass and architecture data with the light microscope, whereas the 8-µm sections were left unstained for measurements of fluorochrome-based indices. Static and dynamic histomorphometry were performed using an automatic image analysis system (Bioquant, Nashville, TN) linked to a microscope equipped with a transmitted and fluorescence light.
Trabecular bone in the distal femur was quantified at a magnification of ×200. The area measured was defined by the cortical bone on both sides and by a line beginning 1 mm distal to the growth plate and extending further proximally to the middle femur shaft. The following parameters were measured: total bone area (T.Ar; mm2), total trabecular bone area (B.Ar; mm2), total trabecular bone perimeter (B.Pm; mm), single-labeled bone perimeter (sL.Pm; mm), double-labeled bone perimeter (dL.Pm; mm), and interlabeled width (Ir.L.Wi; µm). The following parameters were calculated: trabecular bone volume (BV/TV; %), trabecular thickness (Tb.Th; µm), trabecular number (Tb.N; mm−1), trabecular separation (Tb.Sp; µm), single-labeled surface (sLS/BS; %), double-labeled surface (dLS/BS; %), trabecular bone surface (BS), mineral apposition rate (MAR; µm/d), mineralizing surface (MS/BS;%), bone formation rate (BFR/BS; 10–2 µm3/µm2/day), osteoclast surface (Oc.S), and eroded surface (ES). All nomenclature and calculations of the histomorphometric indices are according to Parfitt et al.36
Cytokine and NGF ELISA
Rats were euthanized with CO2, and the hindpaw dorsal skin was collected and frozen immediately on dry ice. All tissues were cut into fine pieces in ice-cold phosphate-buffered saline (PBS), pH 7.4, containing protease inhibitors (aprotinin [2 μg/mL], leupeptin [5 μg/mL], pepstatin [0.7 μg/mL], and phenylmethylsulfonyl fluoride [100 μg/mL, Sigma, St. Louis, MO]) followed by homogenization using a rotor/stator homogenizer. Homogenates were centrifuged for 5 minutes at 14,000g at 4°C. Supernatants were transferred to fresh precooled Eppendorf tubes. Triton X-100 was added at a final concentration of 0.01 %. The samples were centrifuged again for 5 minutes at 14,000g at 4°C. The supernatants were aliquoted and stored at −80°C. The TNF, IL-1, and IL-6 levels were measured using enzyme immunoassay (EIA) kits (R&D Systems, Minneapolis, MN). The NGF concentrations were determined by using the NGF Emax ImmunoAssay System kit (Promega, Madison, WI) according to the manufacturer’s instructions. Total protein contents in all tissue extracts were measured by the Coomassie Blue Protein Assay Kit (Pierce, Life Technologies, Waltham, MA). Each protein concentration was expressed as picogram per milligram total protein. The results of all assays were confirmed by repeating the experiments twice.
Fos Spinal Cord Immunohistochemistry
Rats were euthanized with CO2 and perfused intracardially with 200 mL 0.1 M PBS followed by 200 mL neutral 10% buffered formaldehyde. Spinal cord segments (L3–L5) were removed, postfixed in the perfusion fixative overnight, and cryoprotected in 30% sucrose at 4°C for 24 hours. Serial frozen spinal cord sections, 40 μm thick, were cut on a coronal plane by using a cryostat, collected in PBS, and processed as free-floating sections. Fos immunostaining was performed as previously described.17,18 Because the sciatic nerve projects heavily to the L3-L5 segments of the spinal cord, we analyzed the numbers of Fos-immunoreactive (Fos-IR) neurons at those levels.
To evaluate and compare the distribution of Fos-positive neurons in the lumbar spinal cord, an image analysis system (Bioquant, Nashville, TN) attached to a Nikon Eclipse 80i microscope was used. Digital images were captured using 10× magnification. The Fos-IR neurons were identified by dense black staining of the nucleus. The Fos-IR neurons were plotted and counted with Bioquant Automated Imaging module through 3 arbitrary defined regions of the spinal gray matter of the L3-L5 segments, according to the cytoarchitectonic organization of the spinal cord: the superficial laminae (laminae I-II), the nucleus proprius (laminae III-IV), and the deep laminae (laminae V-VI; neck) of dorsal horn. For each section, the Fos-IR neurons were counted for each lamina, the counts were pooled, and the average number was calculated giving a count that was the mean of all stained neurons in those 3 sections per each cytoarchitectonic region. The investigator responsible for plotting and counting of the Fos-IR neurons was blinded to groups.
Skin and Sciatic Nerve Immunoglobulin ELISA
Rats were euthanized with CO2, and hindpaw dorsal skin and sciatic nerve were collected and frozen immediately on dry ice. All tissues were cut into fine pieces in ice-cold PBS, pH 7.4, containing a cocktail of protease inhibitors (Roche Applied Science, Penzberg, Germany), and followed by homogenization using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, CT). The homogenates were centrifuged at 12,000g for 15 minutes at 4°C. The supernatants were aliquoted and stored at −80°C until required for ELISA performance. Total protein contents in all tissue extracts were measured by using the DC Protein Assay kit (Bio-Rad, Hercules, CA). The albumin, IgM, and IgG levels were determined in duplicate by using ELISA kits (GenWay Biotech, San Diego, CA) according to the manufacturer’s instructions. The results of all assays were confirmed by repeating the experiments twice.
Popliteal Lymph Node Dissection and Size Measurement
The popliteal lymph node is embedded in the adipose tissue of the popliteal fossa and is spherical. Rats were euthanized with CO2, and the bilateral popliteal lymph nodes were dissected free under a microscope. The lymph node diameters were measured using a caliper with the average diameter for each lymph node defined as the (short-axis diameter + long-axis diameter)/2.
The primary end point of the study was comparing vehicle treatment to bisphosphonate treatment on postfracture hindpaw von Frey thresholds, while also confirming that fracture rats have reduced von Frey thresholds relative to nonfracture control rats. Secondary outcomes of the study were (1) comparing control (no fracture) to fracture for hindpaw unweighting; vascular changes (temperature and paw thickness); bone metabolism parameters (BV/TV, Conn.D, BFR, Oc.S/BS, ES/BS); hindpaw skin inflammatory mediator levels (TNF, IL-1, IL-6, NGF); hindpaw skin and sciatic nerve albumin, IgM, and IgG levels; and popliteal lymph node diameters and (2) comparing vehicle treatment to bisphosphonate treatment on postfracture hindpaw unweighting; vascular changes (temperature and paw thickness); bone metabolism parameters (BV/TV, Conn.D, BFR, Oc.S/BS, ES/BS); hindpaw skin inflammatory mediator levels (TNF, IL-1, IL-6, NGF); hindpaw skin and sciatic nerve albumin, IgM, and IgG levels; and popliteal lymph node diameters.
Statistical analysis was performed using Prism 4.02 (GraphPad software, La Jolla, CA). Sample sizes were based on a power analysis of preliminary and published data generated from using each of the proposed assays in fracture animals. On the basis of this analysis, we calculate that the proposed experiments would require 8 animals per cohort to provide 80% power to detect 25% differences between groups. The standard deviation of hindpaw von Frey thresholds used for sample size calculation was 4 g, and the clinically important difference was set as 1 g. Animals were randomly assigned to experimental groups using computer-generated random numbers, and all testing was performed in a blinded fashion when possible. No animals were excluded after enrollment into the experimental cohorts. The normal distribution of the data was confirmed using the D’Agostino-Pearson omnibus normality test. All data were evaluated using a 1-way analysis of variance (ANOVA), except the time-course comparisons in Figure 2, followed by Holm-Sidak multiple comparison testing (significance level 5%) to compare between control and fracture rats that were treated with either bisphosphonate or vehicle. The time-course comparisons in Figure 2 were evaluated using a repeated-measures 2-way ANOVA followed by Holm-Sidak multiple comparison testing to compare between baseline and postfracture time points and between vehicle and zoledronate treatment groups. All data are presented as the mean ± SEM.
Alendronate Effects on Fracture-induced Changes in Body and Muscle Weight
All groups had similar mean body weights at the start of the experiment (control, 437.6 ± 7.9 g; fracture + vehicle (Fx + Vehicle), 435.0 ± 7.8 g; fracture + alendronate 60 μg/kg/d (Fx + ALN 60), 437.6 ± 6.1 g). The fracture rats had significantly lower body weights (345.2 ± 6.8 g) than the age-matched, vehicle-treated control rats 4 weeks after surgery, as we had previously observed.14,15 After 4 weeks of alendronate treatment, when the experiment was terminated, the alendronate-treated fracture rats had slightly higher observed mean body weights (Fx+ ALN 60, 351.6 ± 6.2 g) than the age-matched, vehicle-treated fracture rats (Fx + Vehicle, 345.2 ± 6.8 g), but the increased weights were not statistically significant. The weights of ipsilateral gastrocnemius muscles from each group were also measured. Fracture caused a significant decrease in muscle weights in bilateral hindlimbs (Fx + Vehicle: ipsilateral side, 1.20 ± 0.06 g; contralateral side, 1.87 ± 0.09 g) when compared with the control rats (control: ipsilateral side, 3.56 ± 0.07 g; contralateral side, 3.60 ± 0.08 g). Interestingly, 4 weeks of alendronate treatment (Fx + ALN 60) significantly increased the muscle weight in the fractured hindlimb (1.39 ± 0.04 g, P < .05) and in the contralateral side (2.26 ± 0.06 g, P < .01), suggesting a possible bisphosphonate anticatabolic effect in muscle after fracture.
Bisphosphonates Inhibited the Development of Postfracture Nociceptive Changes
The effects of alendronate and zoledronate treatment on fracture-induced hindpaw mechanical sensitivity, weight bearing, warmth, and edema were evaluated (Figure 1). Fracture rats were administered vehicle (saline) or alendronate (3 µg/kg/d or 60 µg/kg/d) by subcutaneous osmotic pumps, or zoledronate (3 mg/kg/d) by oral gavage. The zoledronate controls were fracture rats gavaged daily with 4 mL distilled water. As there were no differences between the saline subcutaneous pump treated fracture rats and the distilled water gavaged fracture rats in any outcome measure, data from all the vehicle-treated fracture rats were pooled in Figure 1 and in the statistical analysis.
Figure 1A illustrates that von Frey nociceptive thresholds in the ipsilateral hindpaw are reduced 4 weeks after fracture, but treatment with alendronate or zoledronate blocked the development of this mechanical allodynia. There was no significant difference between the contralateral hindpaw von Frey withdrawal threshold in the fracture cohorts and the intact controls (data not shown), indicating that the vehicle-treated fracture rats did not develop mechanical allodynia in the contralateral hindpaw. In addition, there was no significant difference between the contralateral hindpaw von Frey withdrawal threshold in fracture rats treated with vehicle compared with any of the bisphosphonate treatments (data not shown), indicating that the bisphosphonate treatments had no effect on the normal mechanical nociceptive thresholds in the contralateral hindpaw. Figure 1B shows that vehicle-treated fracture rats unweighted the ipsilateral hindpaw by 34% (P < .0001) and that a 4-week course of alendronate treatment reduced fracture-induced unweighting to 26% (3 µg/kg s.c. daily, P < .05) and 18% (60 µg/kg s.c. daily, P < .0001), respectively. A 4-week course of zoledronate treatment (3 mg/kg orally daily) reduced hindpaw unweighting to 14% (P < .0001).
At 4 weeks, postfracture ipsilateral hindpaw temperature (Figure 1C) and thickness (Figure 1D) were increased. The alendronate and zoledronate treatments had no significant effect on postfracture temperature (Figure 1C). Neither alendronate or zoledronate treatment had any effect on paw edema (Figure 1D).
The possibility of persistent beneficial or curative effects after stopping zoledronate treatment was examined (Figure 2). After the completion of a 4-week course of zoledronate (3mg/kg orally daily), the cast was removed and the rats underwent behavioral testing. Postfracture hindpaw mechanical allodynia, unweighting, and edema were partially reversed by zoledronate treatment at 4 weeks postfracture, but by 5 weeks postfracture (1 week after stopping the zoledronate treatment) the inhibitory treatment effects resolved for mechanical allodynia and edema, but the inhibitory effect on hindpaw unweighting persisted (Figure 2, A–D). At 6 weeks postfracture (2 weeks after stopping zoledronate treatment), no differences were observed between the vehicle and treatment groups for any outcome measures, indicating that zoledronate treatment did not have a curative effect on postfracture CRPS-like sequelae.
Oral Zoledronate Reversed Established Pain Behaviors
To determine whether bisphosphonate treatment could reverse established CRPS-like symptoms, zoledronate treatment (3 mg/kg orally daily for 3 weeks, over weeks 4–7 postfracture) was started at 4 weeks postfracture, immediately after the baseline behavioral testing was complete (Figure 2, E–H). Zoledronate treatment reduced hindpaw allodynia after 2 weeks of zoledronate treatment (6 weeks postfracture), and reduced unweighting after 1 week of treatment (5 weeks postfracture). There was no treatment effect on hindpaw warmth or edema.
Alendronate Treatment Reversed Postfracture Bone Loss in the Bilateral Distal Femurs and L5 Vertebra
The effects of tibia fracture and alendronate treatment on trabecular bone mass were examined in the bilateral distal femurs and L5 vertebra. Four weeks after fracture, there was a 52% decrease in BV/TV (P < .0001), an 11% decrease in Tb.N (P < .05), a 14% decrease in Tb.Th (P < .001), a 67% decrease in Conn.D (P < .0001), a 12% increase in Tb.Sp (P < .01), and a 34% increase in SMI (P < .001) in the ipsilateral distal femur of the fracture rats (Figure 3, A and D; Table). Alendronate treatment significantly increased the trabecular bone mass in the ipsilateral distal femur of the fracture rats. After 4 weeks of treatment, both the 3 µg/kg/d and 60 µg/kg/d doses of alendronate increased trabecular BV/TV in the ipsilateral femur of the fracture rats by 77% (P < .001) and 158% (P < .0001), respectively, increased Tb.N by 44% (P < .001) and 58% (P < .001), respectively, increased Tb.Th by 8% (P < .05) and 15% (P < .001), respectively, increased Conn.D by 170% (P < .0001) and 414% (P < .0001), respectively, decreased Tb.Sp by 32% (P < .001) and 37% (P < .001), respectively, and decreased SMI by 13% (P < .01) and 27% (P < .001), respectively, when compared with the vehicle-treated fracture rats (Fx + Vehicle; Figure 3, A and D; Table).
Effects of fracture without and with alendronate on trabecular bone mass in the contralateral distal femur were also investigated with micro-CT (Figure 3, B and E; Table). Four weeks after fracture, there was a 20.2% decrease in BV/TV (P < .01), a 12.6% decrease in Tb.N (P < .05), a 7% decrease in Tb.Th (P < .001), a 17.9% decrease in Conn.D (P > .05), a 11.4% increase in Tb.Sp (P < .01), and a 17% increase in SMI (P < .001) in the contralateral distal femur of the fracture rats. After 4 weeks of treatment, both the 3 µg/kg/d and 60 µg/kg/d doses of alendronate increased trabecular BV/TV in the contralateral femur of the fracture rats by 22% (P < .05) and 61% (P < .0001), respectively, increased Tb.N by 26% (P < .001) and 30% (P < .001), respectively, increased Tb.Th by 2% (P > .05) and 6% (P > .05), respectively, increased Conn.D by 34% (P < .05) and 118% (P < .0001), respectively, decreased Tb.Sp by 22% (P < .001) and 24% (P < .001), respectively, and decreased SMI by 5% (P < .01) and 18% (P < .001), respectively, when compared with the vehicle-treated fracture rats (Fx + Vehicle; Figure 3, B and E; Table).
Effects of fracture without and with alendronate on cortical bone mass were examined at the bilateral middle femurs. In this study, fracture and alendronate treatments did not affect the cortical bone parameters, cortical B.Ar/T.Ar and cortical Ct.Th (data not shown).
At 4 weeks after fracture, there was a modest loss of trabecular bone in the L5 vertebral body (Figure 3, C and F; Table). When compared with the intact control rats, there was a 26% decrease in BV/TV (P < .05), a 2% decrease in Tb.N (P > .05), a 10% decrease in Tb.Th (P > .05), a 26% decrease in Conn.D (P < .01), a 3% increase in Tb.Sp (P > .05), and a 46% increase in SMI (P < .01) at L5 in the fracture rats. After 4 weeks of alendronate treatment, both the 3 µg/kg/d and 60 µg/kg/d doses of alendronate increased the L5 trabecular BV/TV of the fracture rats by 42% (P < .01) and 80% (P < .0001), respectively, increased Tb.N by 6% (P > .05) and 13% (P < .001), respectively, increased Tb.Th by 11% (P > .05) and 22% (P < .001), respectively, increased Conn.D by 53% (P < .01) and 59% (P < .0001), respectively, decreased Tb.Sp by 6% (P > .05) and 14% (P < .001), respectively, and decreased SMI by 53% (P < .05) and 61% (P < .001), respectively, when compared with the vehicle-treated fracture rats (Fx + Vehicle; Figure 3, C and F; Table).
Alendronate Treatment Inhibited Osteoclast Bone Resorption Activity
Effects of fracture and alendronate (60 µg/kg/d) on trabecular bone mass in the ipsilateral distal femur and the L5 vertebral body were also studied by both static and dynamic histomorphometry (Figure 4, A–F). Effects of alendronate on trabecular parameters as measured by static histomorphometry were similar to our previous micro-CT findings (data not shown). In addition, fracture caused a decrease in BFR in both sites and an increase in bone resorption activity as indicated by Oc.S/BS, and ES/BS in the ipsilateral distal femur. Four weeks of alendronate treatment did not change BFR in fracture rats, but successfully inhibited bone resorption activity by reducing the values of Oc.S/BS and ES/BS (Figure 4, A–F).
Alendronate Treatment Inhibited Postfracture Increases in Hindpaw Cytokine and NGF Levels
Cytokine and NGF protein levels in hindpaw skin were determined by EIA (Figure 5). At 4 weeks postfracture, there was a 338% increase in TNF (P < .0001), an 85% increase in IL-1 (P < .05), a 55% increase in IL-6 (P > .05), and a 229% increase in NGF (P < .01) protein levels in the fracture hindpaw. Four weeks of alendronate treatment (60 µg/kg s.c. daily) inhibited postfracture increases in cytokine and NGF protein levels in the hindpaw skin. Alendronate treatment reduced postfracture TNF levels by 48% (P < .001), IL-1 levels by 65% (P < .001), IL-6 levels by 63% (P < .001), and NGF levels by 51% (P < .01).
Bisphosphonate Treatment Did Not Inhibit Fos Expression in Lumbar Spinal Cord After Fracture
To test whether bisphosphonate treatment can inhibit postfracture increases in immediate early gene expression in the spinal cord, the effects of alendronate treatment on spinal Fos expression were evaluated. Immunostaining for Fos was performed in the L3-L5 dorsal horns in control rats and fracture rats treated for 4 weeks with either vehicle or alendronate 60 μg/kg/d (Fx + ALN 60). Confirming our previous findings in the rat fracture model,17,18 at 4 weeks after the fracture, Fos expression increased in the ipsilateral dorsal horn (data not shown) compared with control, but the increase was statistically significant only in laminae I-II and laminae III-IV. In the contralateral dorsal horn of control rats, there was a nonsignificant increase in Fos expression seen in lamina I through lamina VI. Alendronate treatment failed to reduce the Fos expression in bilateral spinal horns in the fracture rats compared with vehicle-treated controls (data not shown). Contrariwise, alendronate treatment evoked an increase of Fos expression in the contralateral dorsal horn compared with the Fos immunostaining observed in unfractured controls (data not shown). Previous studies have reported occasional dissociations between opiate-induced or N-methyl-d-aspartate antagonist–induced analgesia and spinal Fos expression, and this may be attributable to the fact that Fos expression is not specifically related to nociception, but is also induced by motor activity, nonnoxious sensory stimuli, stress, or even arousal.37
Zoledronate Treatment Did Not Inhibit Immune Complex Deposition in Fracture Limb
To determine whether bisphosphonate treatment could prevent the postfracture deposition of immune complexes indicative of autoimmunity, IgM and IgG protein levels were measured by EIA at 4 weeks postfracture in the skin and sciatic nerve tissues of rats treated with zoledronate (3 mg/kg orally daily for 4 weeks) or vehicle (distilled water orally daily for 4 weeks). IgM levels were increased 357% in the skin and 166% in the sciatic nerve, and zoledronate treatment failed to inhibit this postfracture increase (Figure 6B). Similarly, IgG levels were increased 107% in the skin and 67% in the sciatic nerve, and zoledronate treatment failed to prevent this postfracture increase in immune complex deposition (Figure 6C). Postfracture albumin levels only increased by 42% in the skin and by 22% in the sciatic nerve, a much lesser extent than the increases in IgM and IgG levels observed after fracture, suggesting that changes in vascular permeability did not account for the skin and nerve immunoglobulin deposition observed after fracture (Figure 6A).
Zoledronate Treatment Did Not Inhibit Postfracture Popliteal Lymphadenopathy
At 4 weeks postfracture, the popliteal lymph node diameter was increased 54% in the fracture limb (Fx-ipsi + vehicle), compared with the contralateral popliteal lymph node diameters (Fx-contra + vehicle) or with Control nonfracture rat popliteal lymph node diameters (Figure 7). Zoledronate treatment (3 mg/kg orally daily for 4 weeks) had no effect on postfracture lymphadenopathy, compared with vehicle treatment (distilled water orally daily for 4 weeks).
Four weeks of daily bisphosphonate treatment with alendronate (3 or 60 μg/kg s.c. daily) or zoledronate (3 mg/kg orally daily), started at the time of fracture, prevented the development of postfracture nociceptive sensitization, but it was not curative. When zoledronate treatment was discontinued at 4 weeks postfracture, there was a reoccurrence of hindpaw von Frey allodynia and unweighting, returning to the same levels as observed in the vehicle-treated fracture rats (Figures 1 and 2). Osteoclastic bone resorption is commonly observed in CRPS-affected limbs, and it has been postulated that the inhibitory effects of bisphosphonates on osteoclast formation and activation may mediate analgesia in CRPS patients. Bisphosphonates are not metabolized in the systemic circulation, but are instead rapidly incorporated into remodeling bone and cleared from the systemic circulation by renal elimination. Alendronate and zoledronate disappear from the circulation and soft tissues within hours or days after administration, but are very slowly released from calcific tissue over a period of months to years.38,39 The reoccurrence of hindpaw allodynia and unweighting within a week of discontinuing zoledronate treatment suggests that osteoclastic inhibitory effects do not contribute to the zoledronate analgesia observed in the mouse fracture CRPS model (Figure 2).
When zoledronate treatment (3 mg/kg orally daily for 3 weeks) was started at 4 weeks postfracture, it slowly reversed established postfracture pain behaviors (Figure 2). The antinociceptive effects of zoledronate developed slowly over 1 to 2 weeks, suggesting an indirect mechanism of pain relief (Figure 2). Zoledronate treatment had no effect on contralateral hindpaw von Frey thresholds, and when the same dose of zoledronate (3 mg/kg orally) was given to nonfracture control rats, it had no effect on tail-flick latencies between 1 and 24 hours after administration (data not shown). Zoledronic acid is considered to be the most potent bisphosphonate and is currently approved only as an intravenous formulation.40 This is the first report indicating that orally administered zoledronate can provide effective analgesia in a chronic pain model. Collectively, these results suggest that bisphosphonates can prevent or reverse postfracture pain behaviors, but are not curative. Furthermore, the antihyperalgesic effects of bisphosphonates developed slowly over days or weeks, and there was no evidence that they provided effective analgesia for acute pain.
The therapeutic efficacy of bisphosphonate treatment in the rat CRPS fracture model concurs with the clinical trial results in CRPS patients demonstrating bisphosphonate therapeutic efficacy early in the course of the disease.7–11 The clinical trial data are equivocal regarding the curative effects of bisphosphonate treatment for CRPS,10,11 but results from the current translational study (Figure 2, A and B) suggest that 4 weeks of bisphosphonate treatment would not be curative.
After distal tibia fracture, there was a loss of trabecular bone volume and connectivity in the ipsilateral distal femur (Figure 3, A and D). This bone loss was attributable to a reduction in bone formation and an increase in osteoclast activation and bone resorption (Figure 4, A–C). Interestingly, there was also a postfracture reduction in trabecular bone volume and connectivity in the contralateral distal femur and L5 vertebra (Figure 3, B, C, E, and F), with an associated reduction in bone formation and increased osteoclast activity and bone resorption (Figure 4, D–F). A similar pattern of trabecular bone loss is observed in both the ipsilateral and contralateral limb and in the lumbar vertebra of lower limb fracture patients.41–43 In CRPS patients, a regional patchy periarticular osteopenia is usually observed on radiographs with a loss of trabecular bone density in the involved limb and in the contralateral limb,7,44–46 and the severity of trabecular bone loss is greater in fracture patients with CRPS signs and symptoms than in fracture patients without CRPS.47,48 Studies using technetium 99m-labeled diphosphonates that are taken up in areas of active bone remodeling demonstrate accelerated bone resorption in the periarticular trabecular bone of the affected limb and frequently in the contralateral limb as well.9,45,46 Bone biopsies in CRPS-affected limbs demonstrate demineralization and osteoclastic resorption,49 similar to our histomorphometric findings in the distal femur and L5 vertebra of the fracture rats (Figure 4).
Four weeks of alendronate treatment, started at the time of fracture, dose-dependently prevented postfracture trabecular bone volume loss and loss of connectivity in the bilateral distal femurs and in the L5 vertebra (Figure 3; Table). Alendronate treatment had minimal effect on bone formation rates (Figure 4, A and D), but did inhibit postfracture increases in osteoclast surface (Figure 4, B and E) and bone resorption (Figure 4, C and F). Similarly, bisphosphonate treatment inhibits the development of regional trabecular bone loss in both fracture patients and CRPS patients.7,50,51 Bisphosphonates also reduce urinary levels of type I collagen N-telopeptide in CRPS patients, suggesting an inhibitory effect on osteoclastic bone resorption.8,9
Levels of inflammatory mediators (TNF, IL-1, IL-6, and NGF) were elevated in the hindpaw skin at 4 weeks after fracture (Figure 5), consistent with our prior findings in fracture rats and mice. Postfracture increases in inflammatory mediators were inhibited or completely blocked by 4 weeks of alendronate treatment (Figure 5), in agreement with prior reports of bisphosphonate inhibitory effects on monocyte, TNF, IL-1, and IL-6 expression in vitro24–29 and on TNF, IL-1, and IL-6 levels in osteoporotic patients.30–32 Previously, we demonstrated that epidermal keratinocytes are the primary cellular source for the expression of cutaneous inflammatory mediators in fracture rats and mice and in CRPS patient skin.20,52–54 Interestingly, TNF and IL-6 levels are also increased in the skin and experimental skin blister fluid of CRPS-affected limbs.54–57 Treating fracture rats with TNF, IL-1, IL-6, or NGF receptor antagonists or inhibitors partially inhibits postfracture allodynia and unweighting, but unlike alendronate, these treatments were ineffective at preventing trabecular bone loss in the CRPS fracture model.16–18,58
Recently, we observed that B cells contributed to the development and maintenance of CRPS-like changes in the mouse fracture model and postulated that IgM autoantibodies directed at antigens in the fracture limb skin and nerves contribute to nociceptive sensitization in CRPS.23 Clinical support for this hypothesis includes a recent study demonstrating that a third of CRPS patients exhibit strongly positive antinuclear antibody tests, a standard diagnostic test for autoimmune disease,59 and a small randomized trial of low-dose IVIG demonstrated that some chronic CRPS patients had prolonged and dramatic symptom improvement after a single IVIG treatment.60 Consistent with our prior results in fracture mice, at 4 weeks postfracture in rats there was a dramatic increase in IgM and IgG deposition in the skin and sciatic nerve of the injured limb, and 4 weeks of zoledronate treatment failed to inhibit this increase (Figure 6, B and C). These results do not support the hypothesis that bisphosphonate treatment can inhibit postfracture autoimmunity. In addition, zoledronate treatment had no effect on postfracture popliteal lymphadenopathy (Figure 7). Collectively, these data suggest that bisphosphonate treatment would be ineffective in chronic CRPS patients with predominantly autoimmune-mediated symptoms.
There will always be caveats in extrapolating from pharmacologic studies in the rat fracture model to human CRPS. The current study examined bisphosphonate effects over the first 7 weeks after fracture, corresponding to the earliest phase of CRPS. Previously, we demonstrated that early CRPS patients (less than 3 months disease duration) had cutaneous mast cell and keratinocyte proliferation and keratinocyte expression of TNF and IL-6 cytokines, but more chronic CRPS patients (greater than 3 months disease duration) did not exhibit these cutaneous inflammatory changes.54 Similarly, we have observed that peripheral inflammatory changes predominate at 4 weeks postfracture in the rat CRPS model, but that by 4 months postfracture, the peripheral inflammatory changes resolve and spinal cord inflammatory changes become crucial contributors to chronic pain behaviors.61 If the mechanisms supporting CRPS evolve over time, the results of the current study may not be directly applicable to more chronic CRPS patients. Another concern with the current study is that different bisphosphonates were used for different aspects of the experimental design. Alendronate was used to examine bisphosphonate effects on postfracture cutaneous inflammatory mediator expression, osteoclastic activity, bone loss, and resorption, whereas zoledronate was used to examine bisphosphonate effects on postfracture immunocomplex deposition and popliteal lymphadenopathy. Both of these drugs are nitrogenous bisphosphonates that act on bone metabolism by binding and blocking the enzyme farnesyl diphosphate synthase in the 3-hydroxymethyl-3-methylglutaryl coenzyme A reductase pathway and both drugs provided effective analgesia in the fracture rat model, but it is a limitation in the study design that all experiments were not replicated using both drugs.
In conclusion, bisphosphonate therapy prevented the development of pain behaviors, osteoclast activation, trabecular bone loss, and cutaneous inflammation in the rat CRPS fracture model. Furthermore, oral zoledronate reversed established pain behaviors in the fracture model. These results support the hypothesis that bisphosphonates can provide effective multimodal treatment in the early stages of postfracture CRPS.
Name: Liping Wang, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Name: Tian-Zhi Guo, MD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Name: Tzuping Wei, PhD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Name: Wen-Wu Li, PhD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Name: Xiaoyou Shi, MD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Name: J. David Clark, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Name: Wade S. Kingery, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
We wish to acknowledge Dr. Herriot Tabuteau’s (Axsome Therapeutics, New York, NY) invaluable assistance in calculating the dosage, route of administration, and treatment design for zoledronate treatment in the fracture rat model.
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