Bone 81 (2015) 400–406

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Original Full Length Article

Behavioral signs of pain and functional impairment in a mouse model ofosteogenesis imperfecta

Dareen M. Abdelaziz a,b, Sami Abdullah c, Claire Magnussen b,d,e, Alfredo Ribeiro-da-Silva b,d,e,f,Svetlana V. Komarova a,c, Frank Rauch c, Laura S. Stone a,b,d,e,g,⁎a Faculty of Dentistry, McGill University, Montreal, QC, Canadab Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, Canadac Shriners Hospitals for Children-Canada and McGill University, Montreal, QC, Canadad Department of Pharmacology & Therapeutics, Faculty of Medicine, McGill University, Montreal, QC, Canadae Integrated Program in Neuroscience, McGill University, Montreal, QC, Canadaf Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canadag Department of Anaesthesiology, Faculty of Medicine, McGill University, Montreal, QC, Canada

⁎ Corresponding author at: 740 Dr. Penfield Ave, Suit0G1, Canada.

E-mail address: laura.s.stone@mcgill.ca (L.S. Stone).

http://dx.doi.org/10.1016/j.bone.2015.08.0018756-3282/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 January 2015Revised 29 July 2015Accepted 3 August 2015Available online 13 August 2015

Keywords:Col1a1Jrt/+HypersensitivityOsteogenesis imperfectaPainSkeletal deformityCollagen mutation

Osteogenesis imperfecta (OI) is a congenital disorder causedmost often by dominantmutations in the COL1A1 orCOL1A2 genes that encode the alpha chains of type I collagen. Severe forms of OI are associated with skeletaldeformities and frequent fractures. Skeletal pain can occur acutely after fracture, but also arises chronicallywithout preceding fractures. In this study we assessed OI-associated pain in the Col1a1Jrt/+ mouse, a recentlydeveloped model of severe dominant OI. Similar to severe OI in humans, this mouse has significant skeletalabnormalities and develops spontaneous fractures, joint dislocations and vertebral deformities. In this model,we investigated behavioral measures of pain and functional impairment. Significant hypersensitivity tomechan-ical, heat and cold stimuli, assessed by von Frey filaments, radiant heat paw withdrawal and the acetone tests,respectively, were observed in OI compared to control wildtype littermates. OI mice also displayed reducedmotor activity in the running wheel and open field assays. Immunocytochemical analysis revealed no changesbetween OI and WT mice in innervation of the glabrous skin of the hindpaw or in expression of the pain-related neuropeptide calcitonin gene-related protein in sensory neurons. In contrast, increased sensitivity tomechanical and cold stimulation strongly correlated with the extent of skeletal deformities in OI mice. Thus,we demonstrated that the Col1a1Jrt/+mousemodel of severeOI has hypersensitivity tomechanical and thermalstimuli, consistent with a state of chronic pain.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Osteogenesis imperfecta (OI) or brittle bone disease is associatedwith bone fragility, bone deformities, short stature andmanyother skel-etal manifestations of widely varying severity. Extra-skeletal abnormal-ities associated with OI include joint hyperlaxity, muscle weakness,brittle teeth, bluish-gray sclera and hearing defects. The prevalence ofOI at birth is about 1 in 10,000 [1]. In the large majority of individualswith OI, the disorder is caused by mutations in genes encoding collagentype I, COL1A1 and COL1A2.

Skeletal pain is amajor issue in OI patients [2]. Evenmild forms of OIpain can be associated with decreased health-related quality of life [3].However, scientific data on the topic remain scarce. Themost extensivestudy on pain in OI obtained a 1-week pain diary in 35 children and

e 3200, Montreal, Quebec H3A

concluded that both acute and chronic pain were common and inter-fered with activities of daily life [2]. Thus, even though the importanceof pain in OI is universally acknowledged, little is known about theunderlying mechanisms and no mechanistic studies on pain in OIanimal models have been reported.

Here we used the recently developed Col1a1Jrt/+mousemodel of OIto study OI-related pain. Col1a1Jrt/+ (OI)mice exhibit small stature, lowbonemineral density and fragile bones similar to type IV OI patients [4].In mice, evoked responses to sensory noxious stimuli (nociception)of different modalities (mechanical, heat and cold) serve as proxybehavioral indices suggestive of increased pain sensitivity [5]. Otherbehavioral measures of functional impairment that may be secondaryto pain include decreased motor activity and limping.

OI and WT mice were tested for behavioral signs of sensory hyper-sensitivity and functional impairment. The OImice displayed significanthypersensitivity and reducedmotor ability compared toWTmice. Therewas a significant correlation between some evoked behavioral re-sponses and skeletal deformities in OI mice. In contrast, no differences

401D.M. Abdelaziz et al. / Bone 81 (2015) 400–406

were observed in measures of either cutaneous innervation density orsensory neuron plasticity. This study provides insights into the relation-ship between bone health and behavioral indices of pain and functionalimpairment in WT and OI mice.

2. Materials and methods

2.1. Animals

All experiments were approved by the Animal Care Committee atMcGill University and conformed to the ethical guidelines of theCanadian Council on Animal Care and the guidelines of the Committeefor Research and Ethical Issues of IASP [6]. The Col1a1Jrt/+ mice weredeveloped by screening of N-ethyl-N-nitrosourea-inducedmutagenesisresulting in T to C transition in the COL1A1 gene leading to an 18 aminoacid deletion in themain triple helical domain of Col1a1, as described in[4]. The Col1a1Jrt/+mice were bred on a FVB background. Mice were agift from Dr. Jane Aubin's laboratory, University of Toronto. The breed-ing colony was maintained at the Animal Care Facility of the ShrinersHospitals for Children®—Canada. Animals had unrestricted access tofood and water, and were on a 12-hour alternating light and darkcycle. A total of 29 (3–6 month old males); 15 WT and 14 Col1a1Jrt/+mice were used in the study.

2.2. Behavioral assessment

Animalswere transferred to the Alan Edwards Centre for Research onPain and habituated for one week prior to testing. Behavioral measure-ments were taken weekly for 7 consecutive weeks and at week 10.Animals were acclimatized for one hour prior to each testing session.

2.2.1. Behavioral measures of sensitivity to mechanical, heat and coldstimuli

2.2.1.1. Mechanical hypersensitivity. Mechanical hypersensitivity wasperformed using von Frey filaments (Stoelting Co., Wood Dale, IL).Animals were placed on an elevated wire mesh grid and coveredindividually with glass compartments. Filaments were applied to theplantar surface of the hind paw to the point of bending. A positiveresponse was defined as a withdrawal from the filament within twoseconds. The fifty percent withdrawal threshold in grams was calculat-ed according to the up-and-downmethod [7]. A decrease inwithdrawalthreshold corresponds to an increase in mechanical sensitivity.

2.2.1.2. Heat hypersensitivity. Heat hypersensitivity was measured usingthe IITC Life Science Inc. plantar analgesiameter as previously described[8]. A radiant heat beamwas directed to the planter surface of the hindpaw and latency to withdrawal was recorded in seconds. A cut off wasset to 22.7 s to avoid tissue damage. Decreases in withdrawal latencycorrespond to increased sensitivity to heat stimuli.

2.2.1.3. Cold hypersensitivity. Cold hypersensitivity was adapted fromChoi et al. [9]. Cold hypersensitivity test was performed by placing adrop of acetone (~25 μL) on the planter surface of the hind paw usinga syringe loaded needle. Total time spent engaging in acetone-evokedbehaviors such as lifting and shaking of the paw was recorded for oneminute. Increases in acetone-evoked behaviors are suggestive of in-creased sensitivity to cold.

2.2.2. Behavioral measures of functional impairment

2.2.2.1. Open field assay The open field assay. Was performed by placingmice individually in a transparent Plexiglas chamber with 40 cm highwalls and a square base of 27 × 27 cm. A video camera was placed onthe top of the chamber to track the animals' motor activity with ANY-

maze software (Stoelting, USA) for 5 min. Total distance traveled wasdetermined.

2.2.2.2. Rearing. Rearing was determined by an observer blind to geno-type as the time spent standing on the animals hind limbs in an uprightposition during the open field assay. Rearing time was recorded inseconds.

2.2.2.3. Limping score. A limping score modified from [10], was given toeach mouse by an observer blind to genotype after 5 min of non-forced ambulation in the open field setting. The assigned scale wasas follows: 0 = complete lack of limb use, 1 = partial non-use ofthe limb in locomotor activity, 2 = limping and guarding behavior,3 = substantial limping, 4 = normal use. A score was given to eachlimb according to the scale. An average score of all four limbs wasgiven to each animal.

2.2.2.4. Voluntary running wheel test. The voluntary running wheel testwas performed by placing an individual mouse in a home cage contain-ing a rotating wheel with sensor, wireless transmitter and receivingsoftware (Med Associates, Inc.). Voluntary running behavior was mea-sured as the number of wheel rotations detected during a one hourtest period.

2.3. Radiography

Live digital radiographs (Faxitron® MX-20) were taken at weeks 3,8 & 11. Mice were anesthetized with an intraperitoneal (i.p.) injectionof 0.01 mL/g of ketamine (100 mg/mL), xylazine (20 mg/mL) andacepromazine (10 mg/mL). Animals were scanned in a prone positionafter taping to the exact location for each scan. Radiographs were ana-lyzed by two independent observers blind to genotype. Skeletal abnor-malities including atlanto-occipital joint dislocation, scoliosis-like spinedeformity, reduced cervical intervertebral disc space, reduced length oflong bones, deformation and fracture of olecranon processes, deforma-tion of coxae, arthritic knees, hindpaw and forepaw bone deformities,loss of transverse processes of caudal vertebrae, hyperplastic callusformation, and osteophytes were recorded for each mouse and a skele-tal deformity score was calculated as a total number of all observedabnormalities.

2.4. Tissue processing for micro-computed tomography andimmunohistochemistry

Mice were deeply anesthetised and perfused transcardially withvascular rinse (5% 0.2 M phosphate buffer, 0.95% NaCl, 0.026% KCl,0.05% NaHCO3 in distilledwater, 0.1% NaNO2 added at day of perfusion)followed by 50mL of 4% paraformaldehyde in phosphate buffer (0.1 M,pH 7.4). Femurs were cleaned thoroughly, stored in 70% ethanol at 4°Cthen scanned by Micro Computed Tomography. Glabrous hindpawskin was cryoprotected in 30% sucrose in phosphate-buffered saline(PBS) overnight at 4°C, embedded in optimumcutting temperatureme-dium (OCT; Tissue Tek®) and sectioned (40 μm). Upper lumbar dorsalroot ganglia (DRG) were dissected, post-fixed in 4% paraformaldehydein phosphate buffer for one day at 4°C, cryoprotected in 30% sucrosein PBS for 4 days at 4°C, embedded in OCT, sectioned at a thickness of10 μm and thaw-mounted onto gelatin-coated slides.

2.5. Micro-computed tomography

Right femurs were scanned in PBS using cone beam computed to-mography (CT) (Skyscan 1172) at a voxel size of 6 μm. Scan parametersincluded a 0.45-degree increment angle, 3 frames averaged, an 84-kVpand 118-mA X-ray source with a 0.5-mmAl filter to reduce beam hard-ening artifacts. Trabecular bone was analyzed in a region starting at0.5 mm proximal to the distal femoral growth plate (to avoid primary

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spongiosa) and scanning a 1mmsection of bone in a proximal direction.Trabecular bone was manually selected along the inner cortical surface.Scans were quantified using the system's analysis software (SkyScanCT Analyser, Version 1.11.8.0). To analyze cortical bone, scanning wasperformed starting at a distance 44% of the total femur length fromthe distal end and scanned for 1 mm proximally. Average outer bonediameter and average diameter of the bone marrow cavity were deter-mined from cross-sectional areas assuming a circular bone cross-section. Cortical thickness was calculated as the difference of thesetwo diameters divided by 2.

2.6. Immunohistochemistry

2.6.1. SkinSkin sections to be labeled for protein-gene-product 9.5 (PGP 9.5), a

general neuronal marker, were collected in PBS containing 0.2% TritonX-100 (PBS-T), treated with 50% ethanol for 30 min. Sections werewashed for 30 min with PBS-T between incubations. To block non-specific binding of the secondary antibody, the sections were pre-treated with 10% normal goat serum (NGS), diluted in PBS-T for 1 h andthen incubated overnight at 4 °C with a rabbit polyclonal anti-PGP 9.5antibody (Ultraclone; 1:800) in 5% NGS. The following day, sectionswere incubated for 2 h with a highly cross-adsorbed goat anti-rabbitIgG conjugated to Alexa 594 (Molecular Probes; 1:800). All sectionswere washed in PBS for 30 min, mounted on gelatin-coated slidesand cover-slipped with Aqua-Poly/Mount (Polysciences). Changes inPGP 9.5 innervationwere determined by analyzing the density of fibersin the upper dermis of the paw skin. Digital images were captured witha high resolution camera attached to a Zeiss Axioplan 2e imagingfluorescence microscope using a 40× oil immersion objective. Foreach mouse, three sections of the skin were chosen at random, andin each section six images were taken of the upper dermis, totaling18 pictures per animal. Images exported as TIFF were analyzed forfiber density using an MCID Elite image analysis system (ImagingResearch). The area is roughly 0.035 mm2 from the dermal–epidermaljunction along the entire sectionwas outlined. PGP 9.5 immunoreactive(ir) fibers were automatically detected and converted to 1 pixel inthickness to measure the total fiber length (in micrometers) per scanarea. Representative images of PGP 9.5 fibers in the upper dermiswere taken on a Zeiss LSM 510 confocal microscope equipped with aHe–Ne laser. Several images were taken at different focal planes (z-stacks) using a 63× oil immersion objective, and merged into one hor-izontal projection.

2.6.2. Dorsal root gangliaSections were pre-treated for 60 min at room temperature in

blocking buffer (0.3% Triton X-100, 1% NDS, 1% BSA and 0.01% NaAzide in 0.01 M PBS) and placed overnight at 4 °C in primary antibody(sheep-derived anti-alpha-Calcitonin Gene Related Peptide (α-CGRP)antibody (1:500; Cat #CA1137, Enzo Life Sciences, Farmingdale, NY))in blocking buffer. Sections were then rinsed 3 × 10 min in PBS,incubated for 90 min at room temperature in secondary antibody(Cy3-AffiniPure conjugated goat anti-sheep IgG (1:500; JacksonImmuno Research, West Grove, PA)) diluted in blocking buffer,rinsed 3 × 10 min with PBS, immersed in DAPI, cover-slipped withAqua-Poly/Mount and examined using a fluorescence microscope(Olympus BX-5). Serial sections from 6 DRGs (3 per side) weredistributed over successive slides, so that each slide has a represen-tation of the upper, middle and lower parts of the DRGs. Imageswere obtained with a 20× objective. The number of immunoreactiveCGRP neurons were counted and divided by the total number ofneurons determined by DAPI staining in ImageJ software (WayneRasband, NIH). Quantification was done by an observer blind togenotype.

2.7. Data analyses

All data are expressed as means ± S.E.M., with n indicating numberof animals, p b 0.05 was considered statistically significant. Two-wayrepeated-measures ANOVA was used for comparing groups over time(Figs. 1,2,3). Non-parametric Spearman correlation analyses wereused to assess correlations between evoked pain behavior modalitiesand skeletal deformities scores in Col1a1Jrt/+ mice (Fig. 4). Studentt-test was used to compare between two groups at a single time point(Figs. 1,5). Statistical analyses were performed using GraphPad Prismsoftware version 6.00 for Windows, California, USA.

3. Results

3.1. Characterization of bone deformities in OI mice

OI mice weighed less than their WT littermates. The average weightof WT mice was 32.9 ± 1.8 g, while the average weight of OI mice was23.3 ± 1.5 g (Fig. 1A). WT animals gained 2.0 ± 1. 6 g over 11 weekswhile OI mice gained 1.1 ± 0.9 g. Radiographic assessment of OI micerevealed a smaller skeleton and widespread skeletal deformities. Thenumber of skeletal deformities per animal (i.e. skeletal deformityscore) was significantly and persistently increased in OI compared toWT mice (Fig. 1B). The skeletal deformities included atlanto-occipitaljoint dislocation (Fig. 1C), deformity and fracture of olecranon processesand osteophytes (Fig. 1D), hyperplastic callus formation (Fig. 1E),scoliosis-like spine deformity (Fig. 1 F), improper healing of the femurat the hip joint (Fig. 1G), reduced length of long bones (not shown),deformity of coxae (Fig. 1H), arthritic knees (Fig. 1I), hindpaw bonedeformities and tarsal–metatarsal fracture (Fig. 1 J). Micro-CT scans re-vealed a significant decrease in OI femur length (Fig. 1 K), bone volume/tissue volume (Fig. 1 L), cortical thickness (Fig. 1 M), and trabecularnumber (Fig. 1 N) compared to WT femurs. The bone deformities re-ported here are consistent with previous reports [4,11].

3.2. Increased sensitivity to mechanical, heat and cold stimuli in OIcompared to WT mice

We tested sensitivity to mechanical, heat and cold stimulation inOI and WT mice. OI mice have decreased paw withdrawal thresholdsto mechanical stimulation in comparison to WT (Fig. 2A). TestingOI mice for heat sensitivity revealed decreased withdrawal latencycompared to WT mice (Fig. 2B). OI mice showed significantly in-creased behavioral responses to acetone compared to WT (Fig. 2C).Overall, these results indicate that OI mice are hypersensitive to sen-sory stimuli.

3.3. Increased functional impairment in OI compared to WT

We tested the mice for signs of physical functional impairmentthat may be secondary to OI-induced pain and skeletal deformities.OI mice traveled less distance in the open field glass chamber com-pared toWTmice (Fig. 3A). In the same experimental setting, rearingtime (time spent standing on their hind limbs) (Fig. 3B) and limpingscore (4= normal walking, 0= no limb use) (Fig. 3C) were recordedduring a 5 minute observation. OI mice exhibited substantial limpingas well as decreased rearing attempts compared to WT mice. Volun-tarily running was measured with a home cage running wheel forone hour. While both OI and WT mice improved their performancefollowing their initial exposure to the running wheels, likely due tothe presence of a training component in the test, the overall countsof rotations were significantly reduced in OI compared to WT mice(Fig. 3D).

Fig. 1. Skeletal characteristics and bone histomorphometric parameters in OI andWTmice. (A)Weight in grams over the 11 week experiment period inWT and OI animals. (B) The totalnumber of skeletal deformities per animalwas quantified by giving a score of 1 to each deformed, fractured or arthritic bone/joint atweeks 3, 8 and 11. Data aremean±S.E.M., n=14–15/group. ****p b 0.0001 indicates significant group differences between OI and WT mice assessed by two-way repeated-measures ANOVA. (C–J) Radiographs demonstrating examples ofskeletal deformities observed in OI mice: (C) atlanto-occipital joint dislocation (arrow) and reduced cervical intervertebral disc space, (D) ulnar fracture (lower arrow) and osteophytein humerus (upper arrow), (E) hyperplastic callus formation of olecranon process, (F) scoliosis-like spine, (G) improper healing of femur at hip joint, (H) severe hip bone deformation,(I) arthritic knee and, (J) tarsal–metatarsal fracture. (K–N) Bone histomorphometric parameters quantified by micro-CT scans: (K) Femur length; (L) bone volume/tissue volume;(M) cortical thickness; (N) trabecular number. Data aremeans± S.E.M., n= 14–15/group. **p b 0.01 and ****p b 0.0001 indicate significant differences betweenOI andWTmice assessedby unpaired t-test. These results are consistent with previous studies characterizing the skeletal and histomorphometric phenotype of the Col1a1Jrt/+mice [4,11].

Fig. 2. Hypersensitivity to sensory stimuli in the hind paw of OI mice compared to WT littermates. (A) Changes in mechanical sensitivity assessed as 50% withdrawal threshold in grams(g) using von Frey filaments. (B) Changes in heat sensitivity assessed as withdrawal time (s) in response to radiant heat stimulation. (C) Changes in cold sensitivity assessed as time(s) spent responding to acetone application. Direction of arrow indicates increased hypersensitivity (i.e. more pain). Data are means ± S.E.M., n = 14–15/group. **p b 0.01 and ****p b

0.0001 indicate significant differences between OI and WT groups assessed by two-way repeated-measures ANOVA.

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Fig. 3. Behavioral measures of functional impairment in OI andWTmice. (A) Distance traveled and (B) time spent rearing (upright position on hind limbs) in an open field duringthe 5 minute test period. (C) A limb use score was given to each limb and averaged (4 = normal walking, 0 = limb non-use). (D) Running wheel rotations per hour of voluntaryrunning. Direction of arrow indicates increased functional impairment. Data are means ± S.E.M., n = 14–15/group. *P b 0.05, **P b 0.01, ****P b 0.0001 indicate significantdifferences between OI and WT mice assessed by two-way repeated-measures ANOVA.

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3.4. Hypersensitivity to mechanical and cold stimuli correlates with skeletaldeformities in OI mice

To assess if behavioral measures in OI mice are related to bonehealth, we performed correlation analyses between the number ofskeletal deformities, sensory thresholds and functional impairment.We found significant correlation between the total number of skeletaldeformities and sensitivity to mechanical stimulation (Fig. 4A) and toacetone-evoked behaviors (Fig. 4C) at week 3 (Fig. 5). No correlationswere observed with heat sensitivity (Fig. 4B) or with any functionalimpairment measures (data not shown).

3.5. Peripheral innervation in glabrous hindpaw skin and sensory neuronalplasticity in OI and WT mice

To assess if changes in peripheral innervation in OI mice might con-tribute to increased sensory sensitivity in the hindpaw, immunohisto-chemistry using the pan-neuronal marker PGP 9.5 was performed andnerve fiber density was quantified in the upper dermis of the hindpaw.

Fig. 4. Correlations between skeletal deformities and evoked pain-like behavior in OI mice. Skesitivities (C) at week 3 of behavioral testing. The solid line is plotted using linear regressionmodby Spearman non-parametric correlation analysis, ns = non-significant.

PGP 9.5-immunoreactive nerve fibers were not different between WTandOImice (Fig. 5A,B). In order to detect potential pain-related changesin sensory neuron cell bodies, we stained the upper lumbar dorsal rootganglia (DRG) for CGRP, a neuropeptide involved in pain transmission.The percent of CGRP-immunoreactive sensory neurons was not differ-ent between WT and OI mice (Fig. 5C,D).

4. Discussion

Our data demonstrate increased sensitivity to mechanical, heat andcold stimuli and significant functional impairment in the Col1a1Jrt/+mouse model of OI compared to their WT littermates. Consistent withprevious studies, Col1a1Jrt/+mice exhibited skeletal deformities includ-ing fracture-prone bones, kyphosis and small stature, similar to theclinical features of severe OI [4,11]. The changes in sensory thresholdsin Col1a1Jrt/+ mice were not associated with changes in nerve fiberdensity in the skin or with changes in CGRP-expression in dorsal rootganglia. Mechanical and cold hypersensitivity significantly correlatedwith bone deformities in Col1a1Jrt/ mice. These data suggest that the

letal deformities scores were correlated with mechanical (A), heat (B) and cold hypersen-el with 95% confidence interval (dotted lines). n=14, r= correlation coefficient assessed

Fig. 5. Nerve fiber density in the upper dermis of the glabrous skin of the hindpaw and quantification of CGRP-immunoreactivity in sensory neurons. (A) Photomicrograph showingrepresentative labeling of PGP 9.5-immunoreactive (−ir) fibers in the upper dermis of OImice. The dashed line represents the dermal–epidermal junction. (B) Total PGP 9.5-irfiber length(n) per area of upper dermis (mm2). (C) Representative photomicrograph of CGRP-immunoreactivity in theDRG. Arrowheads indicate examples of CGRP-ir neurons. (D) Average percent-age of immunoreactive neurons normalized to total number of neurons. Data are means ± S.E.M., n = 3/group, no significant difference.

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Col1a1Jrt/+mice represent a clinically-relevant animalmodel for under-standing the mechanisms and exploring therapeutic interventions inOI-related pain.

4.1. Col1a1Jrt/+ mice as a model for osteogenesis imperfecta

Col1a1Jrt/+ mice were recently established as a model of severedominant OI [4]. These mice present multiple skeletal abnormalitiesincluding fractures, hyperplastic callus formation at previously frac-tured sites, bone hypertrophy, severely deformed bones and arthriticjoints, kyphosis and scoliosis, which are consistent with skeletal find-ings in severely affected patients [12]. In addition, a 3-fold increase inthe marker of osteoclast activity CTX was reported in the Col1a1Jrt/+mice compared to WT [11]. These data are consistent with studies inhumans with OI [13], as well as with other OI mouse models, such asthe BrtlIV mice [14] or the oim/oim mouse [15]. Thus, the Col1a1Jrt/+micemimic the features of inheritance and skeletal phenotype observedin patients with severe OI.

4.2. Possible causes of pain in Col1a1Jrt/+ mice

We report significant and persistent increases in sensory sensitivityto mechanical, heat and cold stimuli in OI mice compared to control.Multiple mechanisms can contribute to the pain phenotype observedin this model. Pain in the Col1a1Jrt/+model may be related to multiplefractures [16]. Bone abnormalities and fracture disrupt the periosteum,rich in sensory innervation, and allow stimulation of the peripheralnerve endings supplying bone tissues [17]. Another source of paincould be condensation of vertebral bodies and kyphosis, which mightcompress the sensory nerve roots innervating the hind limbs, leadingto hindpaw hypersensitivity [18]. In addition, the knee arthritis ob-served in Col1a1Jrt/+ mice might contribute to both the hindpawsensory hypersensitivity and motor impairment [19]. It is also wellestablished that osteoclast activity contributes to pain in various animalmodels of bone-related pain [20–22], andmay be relevant to thismodel.In addition, muscle weakness might contribute to functional impair-ment in the OI mice [23]. It is unlikely, however, that functional/motor

impairment is responsible for the observed hypersensitivity in thesensory assays because faster and/or more vigorous motor responsesindicate hypersensitivity to sensory stimuli; motor impairment wouldbe expected to have the opposite impact. Finally, inflammation due torecurrent fractures or abnormal healing processes might contribute tothe pain phenotype [24].

4.3. Pain and collagen mutations

Diseases related to collagen disturbances in quality or quantitysuch as OI and Ehlers–Danlos syndrome are often associated with mus-culoskeletal pain. Collagen is one of the main components of tendons,ligaments and myofibrils and is important for tissue healing and scarformation. In our study, total nerve fiber density in the skin was unal-tered in Col1a1Jrt/+mice, suggesting that this collagen mutation doesnot alter dermal innervation. In addition, the mutation did not affectthe dorsal root ganglia expression of CGRP, a neuropeptide associatedwith nociceptive neuroplasticity. Overexpression of CGRP in DRGneurons is detected in several animal models of experimentalmusculoskeletal pain including degenerative disc disease [25], tibialfracture [26], osteoarthritis [27] and osteoporosis [18]. The absence ofdetectible changes in sensory neurons supports the hypothesis thatskeletal deformities and not changes in the nervous system are the pri-mary drivers of hypersensitivity and functional impairment in thismodel.

4.4. Limitations and future directions

The combination of OI-like skeletal, sensory and functional changesin the Col1a1Jrt/+ mice suggest that this is a suitable model forunderstanding the mechanisms behind OI-related pain. For example,future experiments could use this model to examine the efficacy ofbisphosphonates or other disease-modifying treatments to understandthe relationships between bone- and pain-related outcomes, to explorethe relative contributions of inflammatory vs. neuropathic pain mecha-nisms or to identify the most effective analgesic agents. The currentstudy has several limitations: First, only male mice were used; future

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studies will need to examine OI-related pain in both male and femalemice. Second, investigations into the role of neuronal plasticity werelimited to total innervation in the skin and to one pain-related neuro-peptide in the sensory neuron cell bodies. Studies in other tissues suchas muscles, synovial membranes and bones will be of great interest, aswould be studies examining signs of plasticity in the spinal cord or insupraspinal regions.

4.5. Conclusion

In spite of themultidisciplinary approaches toOI therapy and theuseof analgesics, pain is one of the main complaints that drive patients toseek treatment. The pain in OI is both acute and chronic in nature andis not necessarily associated with recurrent fractures [2]. The mobilitylimitations secondary to pain and physical and emotional distress fur-ther decrease quality of life [3,28]. Therefore, molecular mechanismsunderlying chronic non-fracture pain in OI patients need to be investi-gated. The Col1a1Jrt/+ mutation results in OI-like changes in skeletal,sensory and functional parameters. We propose the Col1a1Jrt/+ as atranslational tool to investigate targeted therapeutic options for OI-related pain and to lay the groundwork for the establishment of painmanagement protocols for OI patients.

Financial support

This study was supported by operating grants from the CanadianInstitutes for Health Research (SVK (MOP-77643), AR (MOP-136903,MOP-79411), LSS (MOP-126046)), The Shriners of North America (FR)and infrastructure and travel support from the Quebec Pain ResearchNetwork and the Quebec Network for Oral and Bone Health Research.DMA was supported by the Ministry of Higher Education, Egypt. CM issupported by a PhD studentship from the Louise and Alan EdwardsFoundation. FR received support from the Chercheur-Boursier Clinicienprogram of the Fonds de Recherche du Québec. SVK holds the CanadaResearch Chair in Osteoclast Biology. The sponsor(s) had no role instudy design; in the collection, analysis and interpretation of data; inthe writing of the report; or in the decision to submit the article forpublication.

Disclosure

None.

Acknowledgments

We thank Dr. Magali Millecamp, Lina Naso, Renée Bernatchez andRobert Samberg for technical assistance. Author's roles: Study design:FR, LSS, SVK and DMA. Study conduct, data collection and analysis:DMA, SA and CM. Data interpretation: DMA, LSS, SVK, FR and AR.Drafting manuscript: DMA. All the authors revised and approved thefinal version of the manuscript. DMA, SA and CM take responsibilityfor the integrity of the data analysis.

References

[1] F.H. Glorieux, Osteogenesis imperfecta, Best Pract. Res. Clin. Rheumatol. 22 (2008)85–100.

[2] P. Zack, L. Franck, C. Devile, C. Clark, Fracture and non-fracture pain in children withosteogenesis imperfecta, Acta Paediatr. 94 (2005) 1238–1242.

[3] V. Balkefors, E. Mattsson, Y. Pernow, M. Saaf, Functioning and quality of life in adultswith mild-to-moderate osteogenesis imperfecta, Physiother. Res. Int. 18 (2013)203–211.

[4] F. Chen, R. Guo, S. Itoh, L. Moreno, E. Rosenthal, T. Zappitelli, R.A. Zirngibl, A.Flenniken, W. Cole, M. Grynpas, L.R. Osborne, W. Vogel, L. Adamson, J. Rossant, J.E.

Aubin, First mouse model for combined osteogenesis imperfecta and Ehlers–Danlossyndrome, J. Bone Miner. Res. 29 (2014) 1412–1423.

[5] E.J. Cobos, E. Portillo-Salido, “Bedside-to-bench” behavioral outcomes in animalmodels of pain: beyond the evaluation of reflexes, Curr. Neuropharmacol. 11(2013) 560–591.

[6] M. Zimmermann, Ethical guidelines for investigations of experimental pain inconscious animals, Pain 16 (1983) 109–110.

[7] S.R. Chaplan, F.W. Bach, J.W. Pogrel, J.M. Chung, T.L. Yaksh, Quantitative assessmentof tactile allodynia in the rat paw, J. Neurosci. Methods 53 (1994) 55–63.

[8] K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and sensitive method formeasuring thermal nociception in cutaneous hyperalgesia, Pain 32 (1988) 77–88.

[9] Y. Choi, Y.W. Yoon, H.S. Na, S.H. Kim, J.M. Chung, Behavioral signs of ongoing painand cold allodynia in a rat model of neuropathic pain, Pain 59 (1994) 369–376.

[10] P. Honore, N.M. Luger, M.A.C. Sabino, M.J. Schwei, S.D. Rogers, D.B. Mach, P.F.O'Keefe, M.L. Ramnaraine, D.R. Clohisy, P.W. Mantyh, Osteoprotegerin blocks bonecancer-induced skeletal destruction, skeletal pain and pain-related neurochemicalreorganization of the spinal cord, Nat. Med. 6 (2000) 521–528.

[11] A. Roschger, P. Roschger, P. Keplingter, K. Klaushofer, S. Abdullah, M. Kneissel, F.Rauch, Effect of sclerostin antibody treatment in a mouse model of severe osteogen-esis imperfecta, Bone 66 (2014) 182–188.

[12] R.H. Engelbert, W.J. Gerver, L.J. Breslau-Siderius, Y. van der Graaf, H.E. Pruijs, J.M. vanDoorne, F.A. Beemer, P.J. Helders, Spinal complications in osteogenesis imperfecta:47 patients 1–16 years of age, Acta Orthop. Scand. 69 (1998) 283–286.

[13] F. Rauch, R. Travers, A.M. Parfitt, F.H. Glorieux, Static and dynamic bonehistomorphometry in children with osteogenesis imperfecta, Bone 26 (2000)581–589.

[14] T.E. Uveges, P. Collin-Osdoby, W.A. Cabral, F. Ledgard, L. Goldberg, C. Bergwitz, A.Forlino, P. Osdoby, G.A. Gronowicz, J.C. Marini, Cellular mechanism of decreasedbone in Brtl mouse model of OI: imbalance of decreased osteoblast function andincreased osteoclasts and their precursors, J. BoneMiner. Res. 23 (2008) 1983–1994.

[15] H. Zhang, S.B. Doty, C. Hughes, D. Dempster, N.P. Camacho, Increased resorptiveactivity and accompanying morphological alterations in osteoclasts derived fromthe oim/oim mouse model of osteogenesis imperfecta, J. Cell. Biochem. 102 (2007)1011–1020.

[16] C.D. Martin, J.M. Jimenez-Andrade, J.R. Ghilardi, P.W. Mantyh, Organization ofa unique net-like meshwork of CGRP+ sensory fibers in the mouse periosteum:implications for the generation and maintenance of bone fracture pain, Neurosci.Lett. 427 (2007) 148–152.

[17] J.M. Jimenez-Andrade, W.G. Mantyh, A.P. Bloom, H. Xu, A.S. Ferng, G. Dussor, T.W.Vanderah, P.W. Mantyh, A phenotypically restricted set of primary afferent nerve fi-bers innervate the bone versus skin: therapeutic opportunity for treating skeletalpain, Bone 46 (2010) 306–313.

[18] M. Suzuki, S. Orita, M. Miyagi, T. Ishikawa, H. Kamoda, Y. Eguchi, G. Arai, K.Yamauchi, Y. Sakuma, Y. Oikawa, G. Kubota, K. Inage, T. Sainoh, Y. Kawarai, K.Yoshino, T. Ozawa, Y. Aoki, T. Toyone, K. Takahashi, M. Kawakami, S. Ohtori, G.Inoue, Vertebral compression exacerbates osteoporotic pain in an ovariectomy-induced osteoporosis rat model, Spine 38 (2013) 2085–2091 (Phila Pa 1976).

[19] V.L. Harvey, A.H. Dickenson, Behavioural and electrophysiological characterisationof experimentally induced osteoarthritis and neuropathy in C57Bl/6 mice, Mol.Pain 5 (2009) 18.

[20] M. Nagae, T. Hiraga, H.Wakabayashi, L. Wang, K. Iwata, T. Yoneda, Osteoclasts play apart in pain due to the inflammation adjacent to bone, Bone 39 (2006) 1107–1115.

[21] M. Nagae, T. Hiraga, T. Yoneda, Acidic microenvironment created by osteoclastscauses bone pain associated with tumor colonization, J. Bone Miner. Metab. 25(2007) 99–104.

[22] B.W. Strassle, L. Mark, L. Leventhal, M.J. Piesla, X. Jian Li, J.D. Kennedy, S.S. Glasson,G.T. Whiteside, Inhibition of osteoclasts prevents cartilage loss and pain in a ratmodel of degenerative joint disease, Osteoarthr. Cartil. 18 (2010) 1319–1328.

[23] A. Pouliot-Laforte, L.N. Veilleux, F. Rauch, M. Lemay, Validity of an accelerometer as avertical ground reaction force measuring device in healthy children and adolescentsand in children and adolescents with osteogenesis imperfecta type I, J. Musculoskelet.Neuronal. Interact. 14 (2014) 155–161.

[24] A.D. Widgerow, S. Kalaria, Pain mediators and wound healing—establishing theconnection, Burns 38 (2012) 951–959.

[25] M. Miyagi, M. Millecamps, A.T. Danco, S. Ohtori, K. Takahashi, L.S. Stone, ISSLS Prizewinner: increased innervation and sensory nervous system plasticity in a mousemodel of low back pain due to intervertebral disc degeneration, Spine 39 (2014)1345–1354 (Phila Pa 1976).

[26] T. Wei, W.W. Li, T.Z. Guo, R. Zhao, L. Wang, D.J. Clark, A.L. Oaklander, M. Schmelz,W.S. Kingery, Post-junctional facilitation of Substance P signaling in a tibia fracturerat model of complex regional pain syndrome type I, Pain 144 (2009) 278–286.

[27] D. Yu, F. Liu, M. Liu, X. Zhao, X. Wang, Y. Li, Y. Mao, Z. Zhu, The inhibition ofsubchondral bone lesions significantly reversed the weight-bearing deficit and theoverexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal hornin the monosodium iodoacetate induced model of osteoarthritis pain, PLoS ONE 8(2013) e77824.

[28] C.L. Hill, W.O. Baird, S.J. Walters, Quality of life in children and adolescents withosteogenesis imperfecta: a qualitative interview based study, Health Qual. LifeOutcomes 12 (2014) 54.