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Meeting Abstracts  |   March 2008
A Fracture Pain Model in the Rat: Adaptation of a Closed Femur Fracture Model to Study Skeletal Pain
Author Affiliations & Notes
  • Katie T. Freeman, B.S.
    *
  • Nathan J. Koewler, B.A.
    *
  • Juan M. Jimenez-Andrade, Ph.D.
  • Ryan J. Buus, B.A.
  • Monica B. Herrera, M.D.
    §
  • Carl D. Martin, B.A.
    *
  • Joseph R. Ghilardi, B.S.
  • Michael A. Kuskowski, Ph.D.
    #
  • Patrick W. Mantyh, Ph.D.
    **
  • * Junior Scientist, Neurosystems Center and Department of Diagnostic and Biological Sciences, University of Minnesota. † Research Associate, Neurosystems Center and Department of Diagnostic and Biological Sciences, University of Minnesota. Current Position: Assistant Professor, Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona. § Postdoctoral Associate, Neurosystems Center and Department of Diagnostic and Biological Sciences, University of Minnesota. Current Position: Research Associate, Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona. ‡ Assistant Scientist, ∥ Senior Scientist, Research Service, VA Medical Center, Minneapolis, Minnesota. # Senior Statistician, Geriatric Research, Education and Clinical Center, VA Medical Center, Minneapolis, Minnesota. ** Professor, Neurosystems Center and Departments of Diagnostic and Biological Sciences, Psychiatry, Neuroscience, and Cancer Center, University of Minnesota. Current position: Professor, Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona.
Article Information
Meeting Abstracts   |   March 2008
A Fracture Pain Model in the Rat: Adaptation of a Closed Femur Fracture Model to Study Skeletal Pain
Anesthesiology 3 2008, Vol.108, 473-483. doi:10.1097/ALN.0b013e3181649351
Anesthesiology 3 2008, Vol.108, 473-483. doi:10.1097/ALN.0b013e3181649351
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FRACTURE pain is a common form of pain in the young and, even more so, in the old.1–3 In young individuals, (aged <30 yr), the majority of fractures are due to sports, motor vehicle–related accidents,4 and combat-related injuries.5 Although young males have historically had a higher incidence of fractures than young females,6 with the increasing number of women participating in sports7 and military roles,8 this 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.9–11 In humans, peak bone mass is reached at 25–30 yr of age, after which bone loss exceeds bone formation.12,13 As humans age (>30 yr), there is an increase in bone loss (osteopenia) that, if it becomes severe enough, is termed osteoporosis  .12,13 Osteopenia and osteoporosis are characterized by low bone mineral density and compromised bone strength, which predisposes individuals to an increased risk of fractures.12,14 Whereas women and men (aged >50 yr) are equally likely to have osteopenia, women are three times more likely to have osteoporosis.15 In the United States, approximately 8 million women have osteoporosis, 22 million have low bone mineral density of the hip,16 and 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)18,19 and are associated with a decreased quality of life and significantly contribute to morbidity and mortality in this population.9–11 This 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.21,22 Because bone healing is slow18,19 and 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.23 Furthermore, 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.26–28 However, NSAIDs have been shown to inhibit bone healing after fracture in rodents.29–32 In addition, in older patients where bone loss occurs12,13 and in many young patients from the military with explosion-induced fracture5 who 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.33,34 For 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 model35,36 that has been previously used to study bone regeneration after fracture.37 Although 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 pain38 and bone healing,29,39 advantages 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.40 Because 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
Animals
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.37,41 A-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.42 The 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)42 and subsequently adapted by Simon et al.  32 The 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.  43 and 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.
Pain-related Behaviors
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.35,36 Briefly, 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.45 Weight bearing was used as an endpoint in this study because it has been widely used in humans to evaluate bone healing after fracture.46,47 Briefly, 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:
Experimental Groups
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.48 Behavioral 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.50,51 Locomotor 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.53 Briefly, 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.41 Images 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.53 Briefly, 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.54 Briefly, 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.
Statistical Analysis
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.
Results
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.
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive  (A  and  B  ), pin  (C  and  D  ), and pin + fracture 2 days after fracture  (E  and  F  ).  Scale bar  = 3.0 mm. 
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive 
	(A  and  B  ), pin 
	(C  and  D  ), and pin + fracture 2 days after fracture 
	(E  and  F  ).  Scale bar  = 3.0 mm. 
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive  (A  and  B  ), pin  (C  and  D  ), and pin + fracture 2 days after fracture  (E  and  F  ).  Scale bar  = 3.0 mm. 
×
Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats  (closed squares  ) exhibited a greater time spent spontaneously guarding  (A  and  B  ), a greater number of spontaneous flinches  (C  and  D  ), and reduced weight bearing of the fractured limb  (E  and  F  ) as compared with pin rats  (open triangles  ) or age-matched naive rats  (closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats 
	(closed squares  ) exhibited a greater time spent spontaneously guarding 
	(A  and  B  ), a greater number of spontaneous flinches 
	(C  and  D  ), and reduced weight bearing of the fractured limb 
	(E  and  F  ) as compared with pin rats 
	(open triangles  ) or age-matched naive rats 
	(closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats  (closed squares  ) exhibited a greater time spent spontaneously guarding  (A  and  B  ), a greater number of spontaneous flinches  (C  and  D  ), and reduced weight bearing of the fractured limb  (E  and  F  ) as compared with pin rats  (open triangles  ) or age-matched naive rats  (closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
×
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).
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior  (A  and  B  ), reduced number of flinches  (C  and  D  ), and improved hind limb weight bearing  (E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects  (hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  vehicle-treated rats). 
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior 
	(A  and  B  ), reduced number of flinches 
	(C  and  D  ), and improved hind limb weight bearing 
	(E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects 
	(hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  vehicle-treated rats). 
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior  (A  and  B  ), reduced number of flinches  (C  and  D  ), and improved hind limb weight bearing  (E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects  (hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  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 ).
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).
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs  (A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis  (C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin  (E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs  (G  and  H  ) and three-dimensional micro–computed tomography images  (I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin)  (K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs 
	(A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis 
	(C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin 
	(E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs 
	(G  and  H  ) and three-dimensional micro–computed tomography images 
	(I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin) 
	(K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs  (A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis  (C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin  (E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs  (G  and  H  ) and three-dimensional micro–computed tomography images  (I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin)  (K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
×
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.56 To 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).
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female  (A  and  C  ) and male  (B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers  (A  D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  D  = 50 μm. 
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female 
	(A  and  C  ) and male 
	(B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers 
	(A  –D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  –D  = 50 μm. 
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female  (A  and  C  ) and male  (B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers  (A  D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  D  = 50 μm. 
×
Discussion
The Rat Model of Fracture-induced Pain
Jimenez-Andrade et al.  36 and Koewler et al.  35 have 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 pain38 and bone healing research.32,43 We 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.57–60 In comparing the current results in the rat model with our previous results in the mouse model,35,36 it 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.36 In 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.45 This 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.61–63 In 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 reconstruction64 and tooth extraction,65 no sex differences were noted after intracranial surgery.66 In 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,67 the hot plate assay,67 and the abdominal constriction test, which measures visceral pain.67 It 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.68 Thus, 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.69 Musculoskeletal pain is responsible for 40% of all chronic pain states and 54% of all long-term disability and work absenteeism.70 Despite 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.71,72 Our 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.74 For 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.44,74 Based 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.2,26 Therefore, stabilization of the fracture site by internal or external fixation in humans44,75–77 results 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.78–80 Therefore, blockade of prostaglandin production by NSAIDs26 or sequestration of nerve growth factor by anti–nerve growth factor antibodies35,36 results 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.81 Last, 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,27,28 have been shown to inhibit fracture healing in both mice31 and rats,32 although these results are less clear in humans.84–86 These data, together with recent reports that show selective prostaglandin agonists of the prostaglandin receptor E2 accelerate bone healing after fracture,87,88 indeed 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.2 However, 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.89 In 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.90,91 In elderly patients and patients with traumatic brain injury, opioid side effects tend to be more pronounced.33,34 After osteoporotic fractures in the elderly, minimum bed rest is desired to minimize inactivity-induced loss of bone and muscle mass.20 Yet 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.
References
Moholkar K, Ziran B: Local complications, Rockwood and Green’s Fractures in Adults, 6th edition. Edited by Rockwood CA, Green DP, Bucholz RW. Philadelphia, Lippincott Williams & Wilkins, 2006, pp 563–612Moholkar, K Ziran, B Rockwood CA, Green DP, Bucholz RW Philadelphia Lippincott Williams & Wilkins
Yates D, Smith M: Orthopaedic pain after trauma, Textbook of Pain, 3rd edition. Edited by Wall PD, Melzack R. Edinburgh, New York, Churchill Livingstone, 1994, pp 409–21Yates, D Smith, M Wall PD, Melzack R Edinburgh, New York Churchill Livingstone
Ross PD: Clinical consequences of vertebral fractures. Am J Med 1997; 103:30S–42SRoss, PD
Court-Brown CM, Koval KJ: The epidemiology of fractures, Rockwood and Green’s Fractures in Adults, 6th edition. Edited by Bucholz RW, Heckman JD, Court-Brown CM. Philadelphia, Lippincott Williams & Wilkins, 2006, pp 95–143Court-Brown, CM Koval, KJ Bucholz RW, Heckman JD, Court-Brown CM Philadelphia Lippincott Williams & Wilkins
Owens BD, Kragh JF Jr, Macaitis J, Svoboda SJ, Wenke JC: Characterization of extremity wounds in Operation Iraqi Freedom and Operation Enduring Freedom. J Orthop Trauma 2007; 21:254–7Owens, BD Kragh, JF Macaitis, J Svoboda, SJ Wenke, JC
Koval KJ, Cooley M: The epidemiology of fractures: Experience in the United States, Rockwood and Green’s Fractures in Adults, 6th edition. Edited by Bucholz RW, Heckman JD, Court-Brown C. Philadelphia, Lippincott Williams & Wilkins, 2006, pp 123–43Koval, KJ Cooley, M Bucholz RW, Heckman JD, Court-Brown C Philadelphia Lippincott Williams & Wilkins
Hame SL, LaFemina JM, McAllister DR, Schaadt GW, Dorey FJ: Fractures in the collegiate athlete. Am J Sports Med 2004; 32:446–51Hame, SL LaFemina, JM McAllister, DR Schaadt, GW Dorey, FJ
Feuerstein M, Berkowitz SM, Peck CA Jr: Musculoskeletal-related disability in US Army personnel: Prevalence, gender, and military occupational specialties. J Occup Environ Med 1997; 39:68–78Feuerstein, M Berkowitz, SM Peck, CA
Center JR, Nguyen TV, Schneider D, Sambrook PN, Eisman JA: Mortality after all major types of osteoporotic fracture in men and women: An observational study. Lancet 1999; 353:878–82Center, JR Nguyen, TV Schneider, D Sambrook, PN Eisman, JA
Johnell O, Kanis J: Epidemiology of osteoporotic fractures. Osteoporos Int 2005; 16 (suppl 2):S3–7Johnell, O Kanis, J
Johnell O, Kanis JA: An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int 2006; 17:1726–33Johnell, O Kanis, JA
Newton-John HF, Morgan DB: The loss of bone with age, osteoporosis, and fractures. Clin Orthop Relat Res 1970; 71:229–52Newton-John, HF Morgan, DB
Mazess RB: On aging bone loss. Clin Orthop Relat Res 1982:239–52Mazess, RB
Dennison E, Mohamed MA, Cooper C: Epidemiology of osteoporosis. Rheum Dis Clin North Am 2006; 32:617–29Dennison, E Mohamed, MA Cooper, C
Looker AC, Orwoll ES, Johnston CC Jr, Lindsay RL, Wahner HW, Dunn WL, Calvo MS, Harris TB, Heyse SP: Prevalence of low femoral bone density in older U.S. adults from NHANES III. J Bone Miner Res 1997; 12:1761–8Looker, AC Orwoll, ES Johnston, CC Lindsay, RL Wahner, HW Dunn, WL Calvo, MS Harris, TB Heyse, SP
Wehren LE, Siris ES: Beyond bone mineral density: Can existing clinical risk assessment instruments identify women at increased risk of osteoporosis? J Intern Med 2004; 256:375–80Wehren, LE Siris, ES
Orsini LS, Rousculp MD, Long SR, Wang S: Health care utilization and expenditures in the United States: A study of osteoporosis-related fractures. Osteoporos Int 2005; 16:359–71Orsini, LS Rousculp, MD Long, SR Wang, S
Nilsson BE, Edwards P: Age and fracture healing: A statistical analysis of 418 cases of tibial shaft fractures. Geriatrics 1969; 24:112–7Nilsson, BE Edwards, P
Gruber R, Koch H, Doll BA, Tegtmeier F, Einhorn TA, Hollinger JO: Fracture healing in the elderly patient. Exp Gerontol 2006; 41:1080–93Gruber, R Koch, H Doll, BA Tegtmeier, F Einhorn, TA Hollinger, JO
Zuckerman JD: Hip fracture. N Engl J Med 1996; 334:1519–25Zuckerman, JD
Visser M, Harris TB, Fox KM, Hawkes W, Hebel JR, Yahiro JY, Michael R, Zimmerman SI, Magaziner J: Change in muscle mass and muscle strength after a hip fracture: Relationship to mobility recovery. J Gerontol A Biol Sci Med Sci 2000; 55:M434–40Visser, M Harris, TB Fox, KM Hawkes, W Hebel, JR Yahiro, JY Michael, R Zimmerman, SI Magaziner, J
Hannan EL, Magaziner J, Wang JJ, Eastwood EA, Silberzweig SB, Gilbert M, Morrison RS, McLaughlin MA, Orosz GM, Siu AL: Mortality and locomotion 6 months after hospitalization for hip fracture: Risk factors and risk-adjusted hospital outcomes. JAMA 2001; 285:2736–42Hannan, EL Magaziner, J Wang, JJ Eastwood, EA Silberzweig, SB Gilbert, M Morrison, RS McLaughlin, MA Orosz, GM Siu, AL
Magaziner J, Hawkes W, Hebel JR, Zimmerman SI, Fox KM, Dolan M, Felsenthal G, Kenzora J: Recovery from hip fracture in eight areas of function. J Gerontol A Biol Sci Med Sci 2000; 55:M498–507Magaziner, J Hawkes, W Hebel, JR Zimmerman, SI Fox, KM Dolan, M Felsenthal, G Kenzora, J
Magaziner J, Simonsick EM, Kashner TM, Hebel JR, Kenzora JE: Predictors of functional recovery one year following hospital discharge for hip fracture: A prospective study. J Gerontol 1990; 45:M101–7Magaziner, J Simonsick, EM Kashner, TM Hebel, JR Kenzora, JE
Sernbo I, Johnell O: Consequences of a hip fracture: A prospective study over 1 year. Osteoporos Int 1993; 3:148–53Sernbo, I Johnell, O
Greenfield GQ: Orthopaedic pain, Weiner’s Pain Management: A Practical Guide for Clinicians, 7th edition. Edited by Boswell MV, Cole BE. Boca Raton, CRC, 2006, pp 465–76Greenfield, GQ Boswell MV, Cole BE Boca Raton CRC
Balano KB: Anti-inflammatory drugs and myorelaxants: Pharmacology and clinical use in musculoskeletal disease. Prim Care 1996; 23:329–34Balano, KB
Mason L, Moore RA, Edwards JE, Derry S, McQuay HJ: Topical NSAIDs for chronic musculoskeletal pain: Systematic review and meta-analysis. BMC Musculoskelet Disord 2004; 5:28Mason, L Moore, RA Edwards, JE Derry, S McQuay, HJ
Simon AM, Manigrasso MB, O’Connor JP: Cyclo-oxygenase 2 function is essential for bone fracture healing. J Bone Miner Res 2002; 17:963–76Simon, AM Manigrasso, MB O’Connor, JP
Gerstenfeld LC, Thiede M, Seibert K, Mielke C, Phippard D, Svagr B, Cullinane D, Einhorn TA: Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J Orthop Res 2003; 21:670–5Gerstenfeld, LC Thiede, M Seibert, K Mielke, C Phippard, D Svagr, B Cullinane, D Einhorn, TA
Murnaghan M, Li G, Marsh DR: Nonsteroidal anti-inflammatory drug-induced fracture nonunion: An inhibition of angiogenesis? J Bone Joint Surg Am 2006; 88 (suppl 3):140–7Murnaghan, M Li, G Marsh, DR
Simon AM, O’Connor JP: Dose and time-dependent effects of cyclooxygenase-2 inhibition on fracture-healing. J Bone Joint Surg Am 2007; 89:500–11Simon, AM O’Connor, JP
Feinberg SD: Prescribing analgesics: How to improve function and avoid toxicity when treating chronic pain. Geriatrics 2000; 55:44, 49–50Feinberg, SD
Ivanhoe CB, Hartman ET: Clinical caveats on medical assessment and treatment of pain after TBI. J Head Trauma Rehabil 2004; 19:29–39Ivanhoe, CB Hartman, ET
Koewler NJ, Freeman KT, Buus RJ, Herrera MB, Jimenez-Andrade JM, Ghilardi JR, Peters CM, Sullivan LJ, Kuskowski MA, Lewis JL, Mantyh PW: Effects of a monoclonal antibody raised against nerve growth factor on skeletal pain and bone healing following fracture of the C57BL/6J mouse femur. J Bone Miner Res 2007; 22:1732–42Koewler, NJ Freeman, KT Buus, RJ Herrera, MB Jimenez-Andrade, JM Ghilardi, JR Peters, CM Sullivan, LJ Kuskowski, MA Lewis, JL Mantyh, PW
Jimenez-Andrade JM, Martin CD, Koewler NJ, Freeman KT, Sullivan LJ, Halvorson KG, Barthold CM, Peters CM, Buus RJ, Ghilardi JR, Lewis JL, Kuskowski MA, Mantyh PW: Nerve growth factor sequestering therapy attenuates non-malignant skeletal pain following fracture. Pain 2007; 133:183–96Jimenez-Andrade, JM Martin, CD Koewler, NJ Freeman, KT Sullivan, LJ Halvorson, KG Barthold, CM Peters, CM Buus, RJ Ghilardi, JR Lewis, JL Kuskowski, MA Mantyh, PW
Manigrasso MB, O’Connor JP: Characterization of a closed femur fracture model in mice. J Orthop Trauma 2004; 18:687–95Manigrasso, MB O’Connor, JP
Walker K, Fox AJ, Urban LA: Animal models for pain research. Mol Med Today 1999; 5:319–21Walker, K Fox, AJ Urban, LA
Gerstenfeld LC, Alkhiary YM, Krall EA, Nicholls FH, Stapleton SN, Fitch JL, Bauer M, Kayal R, Graves DT, Jepsen KJ, Einhorn TA: Three-dimensional reconstruction of fracture callus morphogenesis. J Histochem Cytochem 2006; 54:1215–28Gerstenfeld, LC Alkhiary, YM Krall, EA Nicholls, FH Stapleton, SN Fitch, JL Bauer, M Kayal, R Graves, DT Jepsen, KJ Einhorn, TA
Malkmus SA, Yaksh TL: Intrathecal catheterization and drug delivery in the rat. Methods Mol Med 2004; 99:109–21Malkmus, SA Yaksh, TL
Sevcik MA, Ghilardi JR, Peters CM, Lindsay TH, Halvorson KG, Jonas BM, Kubota K, Kuskowski MA, Boustany L, Shelton DL, Mantyh PW: Anti-NGF therapy profoundly reduces bone cancer pain and the accompanying increase in markers of peripheral and central sensitization. Pain 2005; 115:128–41Sevcik, MA Ghilardi, JR Peters, CM Lindsay, TH Halvorson, KG Jonas, BM Kubota, K Kuskowski, MA Boustany, L Shelton, DL Mantyh, PW
Bonnarens F, Einhorn TA: Production of a standard closed fracture in laboratory animal bone. J Orthop Res 1984; 2:97–101Bonnarens, F Einhorn, TA
Gerstenfeld LC, Al-Ghawas M, Alkhiary YM, Cullinane DM, Krall EA, Fitch JL, Webb EG, Thiede MA, Einhorn TA: Selective and nonselective cyclooxygenase-2 inhibitors and experimental fracture-healing: Reversibility of effects after short-term treatment. J Bone Joint Surg Am 2007; 89:114–25Gerstenfeld, LC Al-Ghawas, M Alkhiary, YM Cullinane, DM Krall, EA Fitch, JL Webb, EG Thiede, MA Einhorn, TA
Santy J, Mackintosh C: A phenomenological study of pain following fractured shaft of femur. J Clin Nurs 2001; 10:521–7Santy, J Mackintosh, C
Medhurst SJ, Walker K, Bowes M, Kidd BL, Glatt M, Muller M, Hattenberger M, Vaxelaire J, O’Reilly T, Wotherspoon G, Winter J, Green J, Urban L: A rat model of bone cancer pain. Pain 2002; 96:129–40Medhurst, SJ Walker, K Bowes, M Kidd, BL Glatt, M Muller, M Hattenberger, M Vaxelaire, J O’Reilly, T Wotherspoon, G Winter, J Green, J Urban, L
Oni OA, Hui A, Gregg PJ: The healing of closed tibial shaft fractures. J Bone Joint Surg Br 1988; 70-B:787–90Oni, OA Hui, A Gregg, PJ
Skak SV, Jensen TT: Femoral shaft fracture in 265 children: Log-normal correlation with age of speed of healing. Acta Orthop Scand 1988; 59:704–7Skak, SV Jensen, TT
Mathes WF, Kanarek RB: Wheel running attenuates the antinociceptive properties of morphine and its metabolite, morphine-6-glucuronide, in rats. Physiol Behav 2001; 74:245–51Mathes, WF Kanarek, RB
Morgan MM, Fossum EN, Stalding BM, King MM: Morphine antinociceptive potency on chemical, mechanical, and thermal nociceptive tests in the rat. J Pain 2006; 7:358–66Morgan, MM Fossum, EN Stalding, BM King, MM
Hsieh YL: Effects of ultrasound and diclofenac phonophoresis on inflammatory pain relief: Suppression of inducible nitric oxide synthase in arthritic rats. Phys Ther 2006; 86:39–49Hsieh, YL
Raghavendra V, Agrewala JN, Kulkarni SK: Melatonin reversal of lipopolysaccharides-induced thermal and behavioral hyperalgesia in mice. Eur J Pharmacol 2000; 395:15–21Raghavendra, V Agrewala, JN Kulkarni, SK
Weijnen JA, Slangen JL: Effects of ACTH-analogues on extinction of conditioned behavior. Prog Brain Res 1970; 32:221–35Weijnen, JA Slangen, JL
Mach DB, Rogers SD, Sabino MC, Luger NM, Schwei MJ, Pomonis JD, Keyser CP, Clohisy DR, Adams DJ, O’Leary P, Mantyh PW: Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur. Neuroscience 2002; 113:155–66Mach, DB Rogers, SD Sabino, MC Luger, NM Schwei, MJ Pomonis, JD Keyser, CP Clohisy, DR Adams, DJ O’Leary, P Mantyh, PW
Hukkanen M, Konttinen YT, Rees RG, Gibson SJ, Santavirta S, Polak JM: Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related peptide containing sensory fibers. J Rheumatol 1992; 19:1252–9Hukkanen, M Konttinen, YT Rees, RG Gibson, SJ Santavirta, S Polak, JM
Jimenez-Andrade JM, Ortiz MI, Perez-Urizar J, Aguirre-Banuelos P, Granados-Soto V, Castaneda-Hernandez G: Synergistic effects between codeine and diclofenac after local, spinal and systemic administration. Pharmacol Biochem Behav 2003; 76:463–71Jimenez-Andrade, JM Ortiz, MI Perez-Urizar, J Aguirre-Banuelos, P Granados-Soto, V Castaneda-Hernandez, G
Allen MR, Hock JM, Burr DB: Periosteum: Biology, regulation, and response to osteoporosis therapies. Bone 2004; 35:1003–12Allen, MR Hock, JM Burr, DB
Carlton SM, Zhou S: Attenuation of formalin-induced nociceptive behaviors following local peripheral injection of gabapentin. Pain 1998; 76:201–7Carlton, SM Zhou, S
Bhave G, Karim F, Carlton SM, Gereau RW: Peripheral group I metabotropic glutamate receptors modulate nociception in mice. Nat Neurosci 2001; 4:417–23Bhave, G Karim, F Carlton, SM Gereau, RW
Banik RK, Woo YC, Park SS, Brennan TJ: Strain and sex influence on pain sensitivity after plantar incision in the mouse. Anesthesiology 2006; 105:1246–53Banik, RK Woo, YC Park, SS Brennan, TJ
Banik RK, Subieta AR, Wu C, Brennan TJ: Increased nerve growth factor after rat plantar incision contributes to guarding behavior and heat hyperalgesia. Pain 2005; 117:68–76Banik, RK Subieta, AR Wu, C Brennan, TJ
Wiesenfeld-Hallin Z: Sex differences in pain perception. Gend Med 2005; 2:137–45Wiesenfeld-Hallin, Z
Rollman GB, Lautenbacher S: Sex differences in musculoskeletal pain. Clin J Pain 2001; 17:20–4Rollman, GB Lautenbacher, S
Riley JL III, Robinson ME, Wise EA, Myers CD, Fillingim RB: Sex differences in the perception of noxious experimental stimuli: A meta-analysis. Pain 1998; 74:181–7Riley, JL Robinson, ME Wise, EA Myers, CD Fillingim, RB
Taenzer AH, Clark C, Curry CS: Gender affects report of pain and function after arthroscopic anterior cruciate ligament reconstruction. Anesthesiology 2000; 93:670–5Taenzer, AH Clark, C Curry, CS
Averbuch M, Katzper M: Gender and the placebo analgesic effect in acute pain. Clin Pharmacol Ther 2001; 70:287–91Averbuch, M Katzper, M
Thibault M, Girard F, Moumdjian R, Chouinard P, Boudreault D, Ruel M: Craniotomy site influences postoperative pain following neurosurgical procedures: A retrospective study. Can J Anaesth 2007; 54:544–8Thibault, M Girard, F Moumdjian, R Chouinard, P Boudreault, D Ruel, M
Cicero TJ, Nock B, Meyer ER: Gender-related differences in the antinociceptive properties of morphine. J Pharmacol Exp Ther 1996; 279:767–73Cicero, TJ Nock, B Meyer, ER
Kest B, Sarton E, Dahan A: Gender differences in opioid-mediated analgesia: Animal and human studies. Anesthesiology 2000; 93:539–47Kest, B Sarton, E Dahan, A
Weevers HJ, van der Beek AJ, Anema JR, van der Wal G, van Mechelen W: Work-related disease in general practice: A systematic review. Fam Pract 2005; 22:197–204Weevers, HJ van der Beek, AJ Anema, JR van der Wal, G van Mechelen, W
Woolf AD, Pfleger B: Burden of major musculoskeletal conditions. Bull World Health Organ 2003; 81:646–56Woolf, AD Pfleger, B
Seeman E: The periosteum: A surface for all seasons. Osteoporos Int 2007; 18:123–8Seeman, E
Seeman E, Delmas PD: Bone quality: The material and structural basis of bone strength and fragility. N Engl J Med 2006; 354:2250–61Seeman, E Delmas, PD
Kidd BL: Osteoarthritis and joint pain. Pain 2006; 123:6–9Kidd, BL
Crandall M, Miaskowski C, Kools S, Savedra M: The pain experience of adolescents after acute blunt traumatic injury. Pain Manag Nurs 2002; 3:104–14Crandall, M Miaskowski, C Kools, S Savedra, M
Camuso MR: Far-forward fracture stabilization: External fixation versus  splinting. J Am Acad Orthop Surg 2006; 14:S118–23Camuso, MR
Hedequist D, Starr AJ, Wilson P, Walker J: Early versus  delayed stabilization of pediatric femur fractures: Analysis of 387 patients. J Orthop Trauma 1999; 13:490–3Hedequist, D Starr, AJ Wilson, P Walker, J
Bone LB, Johnson KD, Weigelt J, Scheinberg R: Early versus  delayed stabilization of femoral fractures: A prospective randomized study. 1989. Clin Orthop Relat Res 2004; 422:11–16Bone, LB Johnson, KD Weigelt, J Scheinberg, R
Haegerstam GA: Pathophysiology of bone pain: A review. Acta Orthop Scand 2001; 72:308–17Haegerstam, GA
Hukkanen M, Konttinen YT, Santavirta S, Paavolainen P, Gu XH, Terenghi G, Polak JM: Rapid proliferation of calcitonin gene-related peptide-immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience 1993; 54:969–79Hukkanen, M Konttinen, YT Santavirta, S Paavolainen, P Gu, XH Terenghi, G Polak, JM
Levine JD, Fields HL, Basbaum AI: Peptides and the primary afferent nociceptor. J Neurosci 1993; 13:2273–86Levine, JD Fields, HL Basbaum, AI
Ekman EF, Koman LA: Acute pain following musculoskeletal injuries and orthopaedic surgery: Mechanisms and management. Instr Course Lect 2005; 54:21–33Ekman, EF Koman, LA
de Mos M, de Bruijn AG, Huygen FJ, Dieleman JP, Stricker BH, Sturkenboom MC: The incidence of complex regional pain syndrome: A population-based study. Pain 2006; 129:12–20de Mos, M de Bruijn, AG Huygen, FJ Dieleman, JP Stricker, BH Sturkenboom, MC
Sandroni P, Benrud-Larson LM, McClelland RL, Low PA: Complex regional pain syndrome type I: Incidence and prevalence in Olmsted county, a population-based study. Pain 2003; 103:199–207Sandroni, P Benrud-Larson, LM McClelland, RL Low, PA
Bhattacharyya T, Levin R, Vrahas MS, Solomon DH: Nonsteroidal antiinflammatory drugs and nonunion of humeral shaft fractures. Arthritis Rheum 2005; 53:364–7Bhattacharyya, T Levin, R Vrahas, MS Solomon, DH
Koester MC, Spindler KP: Pharmacologic agents in fracture healing. Clin Sports Med 2006; 25:63–73Koester, MC Spindler, KP
Wheeler P, Batt ME: Do non-steroidal anti-inflammatory drugs adversely affect stress fracture healing? A short review. Br J Sports Med 2005; 39:65–9Wheeler, P Batt, ME
Li M, Ke HZ, Qi H, Healy DR, Li Y, Crawford DT, Paralkar VM, Owen TA, Cameron KO, Lefker BA, Brown TA, Thompson DD: A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing. J Bone Miner Res 2003; 18:2033–42Li, M Ke, HZ Qi, H Healy, DR Li, Y Crawford, DT Paralkar, VM Owen, TA Cameron, KO Lefker, BA Brown, TA Thompson, DD
Paralkar VM, Borovecki F, Ke HZ, Cameron KO, Lefker B, Grasser WA, Owen TA, Li M, DaSilva-Jardine P, Zhou M, Dunn RL, Dumont F, Korsmeyer R, Krasney P, Brown TA, Plowchalk D, Vukicevic S, Thompson DD: An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A 2003; 100:6736–40Paralkar, VM Borovecki, F Ke, HZ Cameron, KO Lefker, B Grasser, WA Owen, TA Li, M DaSilva-Jardine, P Zhou, M Dunn, RL Dumont, F Korsmeyer, R Krasney, P Brown, TA Plowchalk, D Vukicevic, S Thompson, DD
Ensrud KE, Blackwell T, Mangione CM, Bowman PJ, Bauer DC, Schwartz A, Hanlon JT, Nevitt MC, Whooley MA: Central nervous system active medications and risk for fractures in older women. Arch Intern Med 2003; 163:949–57Ensrud, KE Blackwell, T Mangione, CM Bowman, PJ Bauer, DC Schwartz, A Hanlon, JT Nevitt, MC Whooley, MA
Mahowald ML, Singh JA, Majeski P: Opioid use by patients in an orthopedics spine clinic. Arthritis Rheum 2005; 52:312–21Mahowald, ML Singh, JA Majeski, P
Massey GM, Dodds HN, Roberts CS, Servoss TJ, Blondell RD: Toxicology screening in orthopedic trauma patients predicting duration of prescription opioid use. J Addict Dis 2005; 24:31–41Massey, GM Dodds, HN Roberts, CS Servoss, TJ Blondell, RD
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive  (A  and  B  ), pin  (C  and  D  ), and pin + fracture 2 days after fracture  (E  and  F  ).  Scale bar  = 3.0 mm. 
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive 
	(A  and  B  ), pin 
	(C  and  D  ), and pin + fracture 2 days after fracture 
	(E  and  F  ).  Scale bar  = 3.0 mm. 
Fig. 1. Representative radiographs showing a naive, pin, and pin + fracture femur in the female and male adult Sprague-Dawley rat. A stainless steel pin was implanted into the intramedullary space of the femur 21 days before fracture to provide mechanical stability to allow bone healing. Closed mid-diaphyseal fractures of the left femur were produced in female and male rats using a three-point impactor device. Radiographic images of femurs from naive  (A  and  B  ), pin  (C  and  D  ), and pin + fracture 2 days after fracture  (E  and  F  ).  Scale bar  = 3.0 mm. 
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Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats  (closed squares  ) exhibited a greater time spent spontaneously guarding  (A  and  B  ), a greater number of spontaneous flinches  (C  and  D  ), and reduced weight bearing of the fractured limb  (E  and  F  ) as compared with pin rats  (open triangles  ) or age-matched naive rats  (closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats 
	(closed squares  ) exhibited a greater time spent spontaneously guarding 
	(A  and  B  ), a greater number of spontaneous flinches 
	(C  and  D  ), and reduced weight bearing of the fractured limb 
	(E  and  F  ) as compared with pin rats 
	(open triangles  ) or age-matched naive rats 
	(closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
Fig. 2. Pain-related behaviors after a closed fracture of the femur in female and male rats. Female and male pin + fracture rats  (closed squares  ) exhibited a greater time spent spontaneously guarding  (A  and  B  ), a greater number of spontaneous flinches  (C  and  D  ), and reduced weight bearing of the fractured limb  (E  and  F  ) as compared with pin rats  (open triangles  ) or age-matched naive rats  (closed circles  ). There were no differences in pain-related behaviors between female and male rats at nearly all time points. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  pin). 
×
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior  (A  and  B  ), reduced number of flinches  (C  and  D  ), and improved hind limb weight bearing  (E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects  (hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  vehicle-treated rats). 
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior 
	(A  and  B  ), reduced number of flinches 
	(C  and  D  ), and improved hind limb weight bearing 
	(E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects 
	(hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  vehicle-treated rats). 
Fig. 3. Morphine reverses pain-related behaviors after fracture in female and male rats. Cumulative doses of morphine sulfate (0.3, 1.0, 3.0, and 10.0 mg/kg, subcutaneous) were administered 20 min before behavioral evaluation and 30 min between doses. Subcutaneous administration of morphine reduced fracture-induced guarding behavior  (A  and  B  ), reduced number of flinches  (C  and  D  ), and improved hind limb weight bearing  (E  and  F  ) day 7 after fracture in a dose-dependent manner. Cumulative dose of 10 mg/kg resulted in side effects  (hatched bars  ) as determined by reduction vertical stands and open field activity. Data are presented as mean ± SEM (*  P  > 0.05, Bonferroni adjusted,  vs.  vehicle-treated rats). 
×
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs  (A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis  (C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin  (E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs  (G  and  H  ) and three-dimensional micro–computed tomography images  (I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin)  (K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs 
	(A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis 
	(C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin 
	(E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs 
	(G  and  H  ) and three-dimensional micro–computed tomography images 
	(I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin) 
	(K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
Fig. 4. Soft callus formation, which results in stabilization of the fracture site, is evident at days 7 and 14 after fracture by histologic but not radiographic or micro–computed tomography analysis. At day 7 after fracture, although there is little mineralized callus around the fracture site in female and male rats as shown by radiographs  (A  and  B  ) and three-dimensional micro–computed tomography images of the mid-diaphysis  (C  and  D  ), there is extensive soft callus formation around the fracture site as shown by longitudinal cross-section (7 μm thick) of the femur stained with hematoxylin and eosin  (E  and  F  ). At day 14 after fracture, calcification of the callus around the fracture site has begun in female and male rats as shown in the radiographs  (G  and  H  ) and three-dimensional micro–computed tomography images  (I  and  J  ) of the mid-diaphysis. Additional soft callus formation has occurred at day 14 after fracture (hematoxylin and eosin)  (K  and  L  ). This soft callus provides mechanical stabilization of the fractured bone and may in part be responsible for attenuation of acute fracture pain.  Scale bar  = 3.0 mm. 
×
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female  (A  and  C  ) and male  (B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers  (A  D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  D  = 50 μm. 
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female 
	(A  and  C  ) and male 
	(B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers 
	(A  –D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  –D  = 50 μm. 
Fig. 5. The femoral periosteum of female and male rats receives a significant innervation by calcitonin gene-related peptide (CGRP)+and 200-kd neurofilament H (NF-200)+sensory fibers. Whole mount preparations of periosteum isolated from femurs of naive female  (A  and  C  ) and male  (B  and  D  ) rats were immunohistochemically labeled with antibodies against CGRP, a neuropeptide found in predominantly unmyelinated and thinly myelinated sensory fibers and NF200, which labels myelinated primary afferent sensory nerve fibers. The periosteum of naive female and male rats was densely innervated by CGRP+and NF200+sensory fibers  (A  D  ), which formed a net-like meshwork that may be involved in detecting mechanical distortion of underlying mineralized bone. Confocal images (30-μm z-series) were projected from 120 optical sections acquired at 0.25-μm intervals.  Scale bar  A  D  = 50 μm. 
×