Because of the relative lack of understanding of the mechanisms that drive skeletal pain, the purpose of this study was to adapt a previously validated closed femur fracture model to quantitatively evaluate skeletal pain in female and male rats.
Three-month-old female and male Sprague-Dawley rats were anesthetized, and a stainless steel pin was inserted into the intramedullary space of the left femur. Three weeks later, the rats were reanesthetized, and left femoral diaphyses were fractured using a standardized impactor device. At 1-21 days after fracture, skeletal pain was measured by quantitatively assessing spontaneous guarding, spontaneous flinching, and weight bearing of the fractured hind limb.
Females and males showed highly robust pain behaviors that were maximal at day 1 after fracture and returned gradually to normal nonfractured levels at days 14-21 after fracture. The magnitude of fracture pain was not significantly different at most time points between female and male rats. In both females and males, the pain-related behaviors were attenuated by subcutaneous morphine in a dose-dependent manner.
This model may help in developing a mechanism-based understanding of the factors that generate and maintain fracture pain in both females and males and in translating these findings into new therapies for treating fracture pain.
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FRACTURE pain is a common form of pain in the young and, even more so, in the old.1–3In young individuals, (aged <30 yr), the majority of fractures are due to sports, motor vehicle–related accidents,4and combat-related injuries.5Although young males have historically had a higher incidence of fractures than young females,6with the increasing number of women participating in sports7and military roles,8this sex difference in fracture incidence in the young will likely decline.
As humans age, bone fractures not only become more frequent but have a significant impact on quality of life, morbidity, and mortality.9–11In humans, peak bone mass is reached at 25–30 yr of age, after which bone loss exceeds bone formation.12,13As humans age (>30 yr), there is an increase in bone loss (osteopenia) that, if it becomes severe enough, is termed osteoporosis .12,13Osteopenia and osteoporosis are characterized by low bone mineral density and compromised bone strength, which predisposes individuals to an increased risk of fractures.12,14Whereas women and men (aged >50 yr) are equally likely to have osteopenia, women are three times more likely to have osteoporosis.15In the United States, approximately 8 million women have osteoporosis, 22 million have low bone mineral density of the hip,16and more than 1.5 million osteoporotic-related fractures occur each year.17
Osteoporotic fractures can be highly disabling (because they heal slower and therefore remain painful for a longer period of time)18,19and are associated with a decreased quality of life and significantly contribute to morbidity and mortality in this population.9–11This is especially true of hip fractures (90% of “hip” fractures are actually a fracture of the proximal head of the femur20) because these almost invariably result in loss of function, loss of mobility, and hospitalization.21,22Because bone healing is slow18,19and it is painful to walk on the fractured bone (resulting in loss of bone and muscle mass), rehabilitation is often incomplete so that only 60% of patients with osteoporosis-related hip fractures will regain their prefracture mobility at 6 months after fracture.23Furthermore, many of these patients now find walking painful, which contributes to loss of mobility, independent living, and social interactions so that approximately 20% of patients die within a year after osteoporotic-related fracture of the hip.24,25
A major problem in treating chronic fracture pain is that the number of available analgesic therapies is limited. Nonsteroidal antiinflammatory drugs (NSAIDs) are effective in attenuating many musculoskeletal pain states.26–28However, NSAIDs have been shown to inhibit bone healing after fracture in rodents.29–32In addition, in older patients where bone loss occurs12,13and in many young patients from the military with explosion-induced fracture5who have also experienced traumatic brain injury, opiate-induced side effects such as sedation, cognitive impairment, clouding of mental status, and depression tend to be more severe.33,34For these reasons, there is a significant need to develop novel mechanism-based analgesics to treat chronic fracture pain without the unwanted side effects of currently available analgesics.
We recently characterized fracture-induced pain behaviors in a murine closed femur fracture model35,36that has been previously used to study bone regeneration after fracture.37Although the mouse model has many advantages, the rat model has advantages of its own. In addition to being a commonly used animal species for studying pain38and bone healing,29,39advantages of the rat model may include a more accessible central nervous system for the study of electrophysiologic properties of pain-transmitting neurons and the ability to implant indwelling catheters for the delivery of potentially therapeutic compounds.40Because this model can be used to simultaneously assess bone pain and bone healing, it may aid in developing new mechanism-based therapies for treating acute and chronic fracture pain that lack the side effects of currently available analgesics.
Materials and Methods
All procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (Minneapolis, Minnesota) and were in accordance with the National Institutes of Health guidelines for care and use of laboratory animals. Experiments were performed in 30 adult male (330–370 g) and 30 female (220–250 g) Sprague-Dawley rats (Harlan, Indianapolis, IN). The rats were housed in conventional facilities with a 12-h light–dark cycle and were given food and water ad libitum .
Surgical and Fracture Procedure
Before femoral pin placement, rats received an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine to provide anesthesia. An incision of approximately 6 mm was made in the skin, and the proximal patellar ligament of the left femur was severed, revealing the synovial space of the knee joint as previously described.37,41A-20 gauge needle was used to core between the condyles and into the medullary canal of the left femur. Rats were immediately radiographed to ensure proper coring; any rat with a needle protruding outside of the medullary canal was killed. A precut 0.8-mm-diameter (length: 25 mm for females and 27 mm for males) stainless steel wire (pin) (Small Parts Inc., Miami Lakes, FL) was inserted into the medullary space for fracture stabilization. Dental amalgam was used to secure the pin and close the hole. Wound clips (MikRon Precision Inc., Gardena, CA) were used to close the incision and were removed 7 days after pin placement.
A closed mid-diaphyseal fracture of the left femur was produced 21 days after pin placement in rats during anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine, intraperitoneal) as originally described by Bonnarens and Einhorn.42The three-point impactor device (BBC Specialty Automotive Center, Linden, NJ) used to fracture was based on the original design of Bonnarens and Einhorn (illustrated in article)42and subsequently adapted by Simon et al. 32The left femur of the anesthetized rat was secured between two lower supports and an upper impactor head. A guillotine-like effect was created by dropping a rod-guided 411-g weight from a height of 20 cm onto the spring-loaded upper impactor head, creating a femoral fracture. Immediately after fracture, rats were radiographed to ensure localization of a mid-diaphyseal fracture. Exclusion criteria were adapted from Gerstenfeld et al. 43and included fractures located too far from the mid-diaphyseal region of the femur, dislodged pins, and nonvisible fracture after impact. Only one rat met the exclusion criteria and was immediately killed and was not used for the current study. After recovery from anesthesia after fracture, rats were allowed unrestricted movement and hind limb weight bearing.
Female and male rats were behaviorally analyzed before fracture (day 0) and at days 1, 2, 4, 7, 10, 14, 18, and 21 after fracture to assess ongoing (spontaneous) fracture pain–related behaviors (guarding and flinching) as previously described.35,36Briefly, the number of hind limb flinches and time spent guarding over a 2-min observation period were recorded as measures of ongoing pain, because these endpoints are similar to observations in patients who protect their fractured limb.44
Fracture-induced pain was also assessed by differences in the distribution of weight in the left (fractured hind limb) versus the right hind limb (intact hind limb) using an incapacitance meter as previously described.45Weight bearing was used as an endpoint in this study because it has been widely used in humans to evaluate bone healing after fracture.46,47Briefly, the mean force applied during 3 s by each hind limb was measured in five trials. Weight bearing on the left hind limb (fractured or pinned femur) was calculated as percentage of total weight bearing on fractured hind limb by the following equation:
To determine possible sex-related differences in pain-related behaviors after fracture, our experimental protocol consisted of three different groups for female and male rats: naive (n = 4), pin (n = 4), and pin + fracture (n = 16 for female and n = 15 for male). Rats were behaviorally analyzed (guarding, flinching, weight bearing distribution) at the time points previously described. At days 7 and 14 after fracture, female (n = 5 for each time point) and male (n = 4 for day 7 and n = 5 for day 14) rats were killed and processed to evaluate the callus histology (see Radiographic, Histologic, and Micro–Computed Tomography Analyses in the Materials and Methods section) of the fractured bones. To monitor the general health of the rats, body weights were recorded throughout the experiment.
To evaluate possible sex-related differences in the response to morphine, female and male fracture rats (n = 10 for female and n = 9 for male) at day 7 after fracture received cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) 20 min before behavioral testing. Rats first received sterile saline (vehicle) followed by four cumulative doses of morphine having a 30-min interval between each dose of morphine.48Behavioral analysis was completed within 30 min after injection to ensure that the animals were tested within the known therapeutic window of drug action in rats after subcutaneous injection.49
Morphine-induced side effects were determined by measuring the number of total spontaneous vertical stands and locomotor activity in an open field. Vertical stands were defined as the number of times the animal stood on both hind limbs, supporting their entire body weight, in a 2-min period.50,51Locomotor activity was measured by counting the number of floor units the animal crossed during a 2-min period. The animal was placed in the center of a circular container (108 cm diameter × 38 cm height equally divided into 29 floor units) at the beginning of the 2-min period.52
Radiographic, Histologic, and Micro–Computed Tomography Analyses
Radiographic images (Specimen Radiography System Model MX-20, Faxitron X-ray Corporation, Wheeling, IL; Kodak film Min-R 2000, Rochester, NY) of fractured femurs were obtained immediately after fracture and at all behavior time points.
Female and male rats with fracture were killed at days 7 and 14 after fracture and processed for histologic analysis as previously described.53Briefly, rats were perfused intracardially with 200 ml phosphate-buffered saline (PBS), 0.1 m, followed by 200 ml 4% formaldehyde–12.5% picric acid solution in 0.1 m PBS. The femurs were removed, postfixed for 4 h in the perfusion fixative, and placed in a PBS solution. Micro–computed tomography (μCT) images of fractured femurs of female and male rats were obtained with an eXplore Locus SP μCT (GE Healthcare, London, Ontario, Canada). The cone beam μCT scanner used a 2,300 × 2,300 charge-coupled device detector with current and voltage set at 80 μA and 80 KVp, respectively. A 360° scan was performed with a 3,000-ms integration time with images reconstructed at 29 μm3resolution. Three-dimensional images were created using MicroView analysis software (version 2.2; GE Healthcare).
After μCT analysis was performed, femurs of fracture rats were decalcified in 10% EDTA at 4°C for no more than 3 weeks. After complete bone demineralization, determined radiographically, bones were embedded in paraffin and serially sectioned on the longitudinal axis using a Leica Microsystems RM2135 microtome (Wetzlar, Germany) at a thickness of 7 μm. Five sections at least 150 μm apart spanning at least 0.75 mm from the center of the fracture callus of each animal were stained with hematoxylin and eosin.41Images of sections were digitally captured at 10× using a SPOT II digital camera with SPOT image capture software (Diagnostic Instruments, Sterling Heights, MI) attached to an Olympus BX41 microscope (Olympus America Inc., Melville, NY).
Euthanasia and Processing of Tissue for Periosteum Immunohistochemistry
Naive female (n = 6) and male (n = 6) rats were killed and perfused as described above. Periosteum from the diaphyseal shaft was removed as a whole mount and processed for immunohistochemistry as previously described.53Briefly, whole mount preparations were washed in PBS 3 × 10 min and incubated for 60 min at room temperature in a blocking solution of 3% normal donkey serum in PBS with 0.3% Triton-X 100 and then incubated overnight at room temperature with primary antibodies. Unmyelinated primary afferent sensory nerve fibers were labeled with polyclonal rabbit anti-rat calcitonin gene–related protein (CGRP, 1:15,000 dilution; Sigma Chemical Co., St. Louis, MO). Myelinated primary afferent sensory nerve fibers were immunostained for 200-kd neurofilament H (NF200, polyclonal chicken anti-mouse NF200, 1:2,000, Chemicon, Temecula, CA). Preparations were then washed in PBS and incubated for 3 h at room temperature with secondary antibodies conjugated to fluorescent markers (Cy3 1:600; Jackson ImmunoResearch, West Grove, PA). Finally, tissue was washed in PBS and dehydrated through an alcohol gradient (70, 90, and 100%), cleared in xylene, mounted (attached muscle layer in contact with the slide) on gelatin-coated slides, and coverslipped with di-n-butylphthalate-polystyrene-xylene. To confirm the specificity of the primary antibodies, controls included preabsorption with the corresponding synthetic peptide and omission of the primary antibody. Images of periosteal whole mount preparations were captured using an Olympus Fluoview FV1000 laser scanning confocal imaging system (software version 5.0; Olympus America Inc.).
Quantification of CGRP+and NF200+fibers in periosteal whole mounts preparations from rats was performed as previously described.54Briefly, digital confocal images for each periosteal layer (400× magnification; two random sections per rat) were acquired as described above. Images were viewed on a high-resolution monitor, and the number of intersections between nerve fibers and the vertical grids (7.35 μm spacing, Adobe Photoshop software version 7.0; San Jose, CA) was quantified. Results were expressed as number of intersections per mm2.
The percent of antinociception was calculated according to the following equation55:
SPSS version 15 statistics package (SPSS, Chicago, IL) was used to perform statistical analyses. Frequency distributions of the behavioral dependent variables guarding, flinching, and incapacitance appeared markedly nonnormal, each failing the Lilliefors test for normality (P < 0.05). Therefore, response measures of guarding, flinching, and incapacitance for pin and fracture groups were compared separately for each sex on each outcome measure at each postintervention time point using Mann–Whitney nonparametric t tests, with significance levels Bonferroni adjusted for multiple comparisons. With eight postintervention time points for each outcome measure, the Bonferroni-adjusted significance level for a single-comparison P value was therefore set at P < 0.006 (0.05/8 = 0.006).
Percent analgesic effect under differing morphine dosages was compared between sexes using a two-way repeated- measures analysis of variance, with sex as a between-group factor and dose as the repeated factor. A significant sex × dose interaction effect was observed (P = 0.018, Greenhouse-Geisser corrected). While post hoc comparisons of analgesic response at each dose level revealed significant sex differences at 1.0, 3.0, and 10.0 dose levels for both guarding and flinching responses (all P < 0.05, unadjusted), only sex comparisons of guarding response at 3.0 and 10.0 remained statistically significant after the more stringent Bonferroni adjustment for multiple comparisons.
Effect of Pin Placement on Bone
Radiographic evaluation indicated no significant bone remodeling after intramedullary pin placement (fig. 1). Age-matched naive and pin rats were radiographically similar in appearance at all time points examined. In addition, body weight (data not shown; P > 0.05, Bonferroni adjusted), and pain related behaviors (fig. 2) were not significantly different between naive and pin rats during the length of the experiment.
Fracture Production in Sprague-Dawley Rats
The three-point fracture protocol resulted in reproducible transverse or slightly oblique mid-diaphyseal femoral fractures (figs. 1E and F). There were no sex-related differences in the success rate of usable fractures. After the surgical procedure, 0 rats, both female and male, were excluded because of protruding pins. In the current study, of the 16 female rats fractured, 0 were excluded, and of the 16 male rats fractured, 1 was excluded using previously described exclusion criteria.43
Femoral Fracture Produces Pain-related Behaviors in Female and Male Rats
Spontaneous guarding, spontaneous flinching, and weight bearing in the left hind limb were analyzed in naive, pin, and pin + fracture rats. Pin + fracture rats exhibited a greater time spent guarding, increased number of flinches, and marked reduction in weight bearing as compared with naive and pin rats (fig. 2) from day 1 through day 14 after fracture for both female and male rats. In both female and male rats, spontaneous pain-related behaviors peaked at day 1 after fracture, decreased gradually, and continued through day 18 after fracture (figs. 2A–D). In regard to weight bearing, there was a marked reduction after fracture. The greatest reduction was observed at day 1 after fracture and remained present until day 10 for female rats and day 14 for male rats (figs. 2E and F). At day 18 after fracture, all pain-related behaviors in fracture rats were not significantly different from those in pin rats. There were no significant differences in the magnitude of the pain-related behaviors between female and male rats at nearly all time points evaluated (data not shown, P > 0.05, Bonferroni adjusted). Rats with an intramedullary pin (pin, figs. 2A–D) showed minimal number of flinches, time spent guarding, and hind limb weight bearing, which was not significantly different from that observed in naive rats (baseline values).
Morphine Treatment Reduces Fracture-induced Pain
Acute subcutaneous administration of morphine administered at day 7 after fracture significantly reduced ongoing guarding and flinching behaviors in a dose-dependent manner (fig. 3). In female rats, administration of morphine at 3.0 and 10.0 mg/kg significantly reduced the fracture-induced guarding, and only 10.0 mg/kg significantly reduced flinching behaviors (figs. 3A and C; P < 0.05, Bonferroni adjusted, vs. vehicle-treated rats). In male rats, a significant reduction in these pain-related behaviors was observed after 3.0 and 10.0 mg/kg morphine (figs. 3B and D; P < 0.05, Bonferroni adjusted, vs. vehicle-treated rats).
For guarding behavior, the percentage of analgesia induced by morphine in female rats was smaller than that in male rats after 1.0 (8.8% for female and 31.1% for male), 3.0 (34.3% for female and 48.2% for male), and 10 mg/kg (57.3% for female and 67.1% for male). For flinching behavior, the percentage of analgesia induced by morphine in female rats was smaller than that in male rats after 1.0 (2.3% for female and 21.1% for male), 3.0 (28.2% for female and 42.0% for male), and 10 mg/kg (54.1% for female and 63.3% for male). While post hoc comparisons of analgesic response at each dose level revealed significant sex differences at 1.0, 3.0, and 10.0 mg/kg for both guarding and flinching responses (all P < 0.05, unadjusted), only sex comparisons of guarding response at 3.0 and 10.0 mg/kg remained statistically significant after the more stringent Bonferroni adjustment for multiple comparisons. Morphine at 10 mg/kg resulted in side effects including decreased locomotor activity (likely caused by lethargy) and decreased vertical stands on hind limbs (likely caused by sedation), which made it difficult to interpret the antinociceptive effect of morphine at this dose (additional information regarding this is available on the Anesthesiology Web site at http://www.anesthesiology.org).
In addition, morphine treatment reversed the reduction of hind limb weight bearing in a dose-dependent manner in female and male rats (figs. 3E and F). Administration of 3.0 and 10.0 mg/kg morphine for female rats and 3.0 and 10.0 mg/kg for male rats significantly reversed the reduction in hind limb weight bearing (P < 0.05, Bonferroni adjusted, vs. vehicle-treated rats). For hind limb weight-bearing analysis, the percentage of analgesia induced by morphine in female rats was not significantly different as compared with male rats after 1.0 (11.9% for female and 28.2% for male), 3.0 (32.1% for female and 47.1% for male), and 10 mg/kg (80.7% for female and 75.7% for male) (P > 0.05, Bonferroni adjusted).
Soft Callus Formation and Mineralization after Fracture
Soft callus formation around fracture site in rats can be visualized at early time points after fracture by histologic analysis but not by x-ray and μCT (fig. 4). Femoral fracture resulted in formation of mineralized callus (radiopaque area around the fractured cortical walls), which was minimal at day 7 after fracture (figs. 4A and B). Three-dimensional μCT images of the same bone also show the relative absence of calcified callus at day 7 after fracture (figs. 4C and D). However, histologic analysis (hematoxylin and eosin) revealed the presence of a soft, cartilaginous callus around the fracture line as well as endochondral calcification at this time point in both groups (figs. 4E and F). At day 14 after fracture, mineralized callus was more visible as determined by radiographic and three-dimensional μCT analysis (figs. 4G–J). Histologic analysis shows a greater cartilaginous callus around the fracture line (figs. 4K and L).
Density of Sensory Nerve Fibers in Diaphyseal Periosteum of Female and Male Naive Rats
The periosteum is a fibrous and cellular sheath that covers the outer surface of nearly all the bones of the body.56To elucidate what sensory fibers could be involved in the detection of fracture-induced pain, we determined the density of CGRP+and NF200+nerve fibers in the femoral periosteum of female and male rats.
Confocal micrographs of whole mount mid-diaphyseal periosteum preparations show that CGRP+and NF200+nerve fibers have a linear and bifurcating pattern of fibers. These sensory fibers form a mesh-like network that envelopes the naive, unfractured bone (figs. 5A–D). Sensory fibers in the periosteum can be found as single nerve fibers or nerve bundles. The density of CGRP+fibers in the periosteum of naive female rats was not significantly different when compared with naive male rats (2,045 ± 132 and 1,928 ± 209 CGRP+fiber intersections per mm2in female and male rats, respectively). Likewise, there were no significant differences in the density of NF200+fibers in the periosteum between naive female and male rats (2,224 ± 403 and 2,093 ± 201 NF200+fiber intersections per mm2in female and male rats, respectively).
The Rat Model of Fracture-induced Pain
Jimenez-Andrade et al. 36and Koewler et al. 35have previously described models of bone fracture pain in C3H/HeJ and C57BL/6J mice, respectively. In the current study, we modified this model for use in the rat because this species has been widely used in pain38and bone healing research.32,43We directly measured flinching, guarding, and weight bearing of the fractured hind limb. This latter behavioral endpoint may have significant utility in assessing the effects of novel analgesics have on use and rehabilitation of the fracture limb because the ability of the patient to voluntarily bear weight on the affected extremity is frequently used as one measure of successful bone healing during and after rehabilitation.46,47
Previous reports examining other rodent preclinical models of acute and chronic pain have reported significant differences in the time course of spontaneous pain-related behaviors (guarding, lifting/licking) in the mouse versus the rat.57–60In comparing the current results in the rat model with our previous results in the mouse model,35,36it is remarkable how similar the guarding and flinching pain behaviors are in terms of the pain scores over time and the reduction in the pain scores that occurs with callus induced stabilization of the fractured bone. Guarding and flinching behaviors are spontaneous, nonevoked pain behavior because animals withdraw their paw (flinching) and then guard their paw (guarding) to minimize the use of the fractured hind limb.36In contrast, weight bearing measured by incapacitance meter is a measure of the load the animal is willing to place on the fractured hind limb as compared with the nonfractured hind limb.45This latter measure seems to be analogous to the amount of weight a human would be willing to place on a fractured bone without pain.46,47
Pain after Femoral Fracture in Male and Female Rats
The influence of sex on pain sensitivity is of great interest to pain research.61–63In the current study, we found that there was no significant difference between females and males when comparing fracture-induced pain behaviors, which included flinching, guarding, and weight bearing. In humans, recent data regarding the effects of sex on musculoskeletal pain are mixed. Therefore, whereas it was shown that women have greater postoperative pain than men after arthroscopic anterior cruciate ligament reconstruction64and tooth extraction,65no sex differences were noted after intracranial surgery.66In contrast to the fracture-induced pain related behaviors, small but significant sex differences were noted in the analgesic response of guarding behavior at 3.0 and 10 mg/kg morphine. The current results are consistent with sex differences in the antinociceptive response to morphine in the warm-water tail withdrawal assay,67the hot plate assay,67and the abdominal constriction test, which measures visceral pain.67It should be noted that we did not examine whether this effect was complicated by the estrous cycle of the female because the literature on this is not consistent.68Thus, there does seem to be a small male-versus -female difference in the ability of morphine to relieve fracture pain over and above any changes due to the estrous cycle.
Potential Mechanisms That Drive Fracture Pain
Worldwide, musculoskeletal pain is the main complaint of 30% of all medical consultations.69Musculoskeletal pain is responsible for 40% of all chronic pain states and 54% of all long-term disability and work absenteeism.70Despite these facts, it is remarkable how little is known about the specific mechanisms that drive skeletal pain. While bone is frequently thought of as a rather static organ, in fact bone is remarkably malleable and one of the most dynamic organs of the body in that it is constantly being remodeled in response to general use, loading of the bone, injury, and aging.71,72Our lack of knowledge of what drives skeletal pain is in large part due to the dearth of animal models that closely mirror common painful conditions such as fracture or osteoarthritis that are usually accompanied by significant skeletal pain.2,26,73
In the current article, we have characterized a rat model of skeletal pain and, using this model, along with the human clinical literature on fracture pain, suggest that there are several distinct but overlapping mechanisms that drive this pain. The initial pain that follows acute fracture of the human femur is most frequently described as sharp, stabbing, aching, burning, and very intense.74For example, patients often refer to the pain that follows fracture of the femur as the worst pain that they have ever felt in their life.44,74Based on the nature of the pain after fracture, we believe that this initial pain is due to mechanical activation of mechanosensitive nociceptors (C and A-δ nerve fibers) that innervate the periosteum, mineralized bone, and marrow.2,26Therefore, stabilization of the fracture site by internal or external fixation in humans44,75–77results in a significant attenuation of fracture pain. Second, within minutes to hours of the initial fracture, there is a marked influx of hematologic and inflammatory cells into the fracture site, which results in activation of nociceptors that express receptors for cytokines, chemokines, and inflammatory factors such as bradykinin, nerve growth factor, or prostaglandins that are frequently released upon tissue injury.78–80Therefore, blockade of prostaglandin production by NSAIDs26or sequestration of nerve growth factor by anti–nerve growth factor antibodies35,36results in a significant attenuation in fracture pain. Third, these factors may directly excite as well as sensitize and induce sprouting of mechanosensitive nociceptors in the bone and induce a central sensitization characterized by neurochemical and cellular changes in the dorsal horn of the spinal cord and brain that facilitate the transmission and perception of pain in the central nervous system.81Last, in cases where significant fracture-induced nerve injury occurs after fracture, the peripheral and central sensitization may be maintained and accompanied by inappropriate sprouting. These changes may contribute to a component of the chronic pain observed in individuals with complex regional pain syndrome. In fact, in approximately 45% of complex regional pain syndrome patients, fracture seems to be the precipitating event.82,83
Current Analgesics Used to Treat Fracture Pain
A major reason fracture pain remains a significant health problem is the limited repertoire of analgesics available to treat this pain without negatively influencing fracture healing and/or the ability of the patient to participate in effective rehabilitation. For example, NSAIDs, which are effective in reducing a variety of musculoskeletal pains,27,28have been shown to inhibit fracture healing in both mice31and rats,32although these results are less clear in humans.84–86These data, together with recent reports that show selective prostaglandin agonists of the prostaglandin receptor E2 accelerate bone healing after fracture,87,88indeed suggest that the use of NSAIDs and cyclooxygenase-2 inhibitors may delay fracture-induced bone healing.
Opiates are currently the mainstay for treating moderate to severe chronic pain.2However, opioids do have a variety of nonskeletal side effects that could inhibit bone healing. Opioid side effects include sedation, cognitive impairment, clouding of mental status, and depression, which can reduce mobility, resulting in loss of bone and muscle mass.89In young individuals with severe fractures, long-term opiate use can result in dependence and a reduced ability to promptly and fully participate in effective musculoskeletal rehabilitation that is necessary for early and effective bone healing.90,91In elderly patients and patients with traumatic brain injury, opioid side effects tend to be more pronounced.33,34After osteoporotic fractures in the elderly, minimum bed rest is desired to minimize inactivity-induced loss of bone and muscle mass.20Yet administration of strong opiates will, in general, reduce the ability of these patients to effectively engage in the exercise and rehabilitation necessary for more rapid bone healing. Together, these data highlight the need for the development of novel, mechanism-based therapies to treat skeletal pain that have negligible or a positive effect on bone healing. The current model seems to offer an attractive platform for the preclinical screening of novel therapies to treat fracture pain.
The authors thank Therese Schachtele (Executive Assistant, Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, Minnesota) for excellent administrative assistance.