Remodeling of the Vertebral Body in Hereditary Lordoscoliotic ...

Bulletin of the Osaka Medical College 52137-44, 2006 37Original ArticleRemodeling of the Vertebral Body in Hereditary Lordoscoliotic RabbitsRevealed by In Situ HybridizationIchiro BABA, Akihiro KIN, and Muneaki ABEDepartment of Orthopedic Surgery, Osaka Medical CollegeTakatsuki-city, Osaka 569-8686, JapanKey WordsScoliosis, Hereditary Lordoscoliotic Rabbit, In situ hybridization,RemodelingABSTRACTThe etiology of human idiopathic scoliosis remains still unknown, although several etiologicfactors and many animal models of scoliosis have been reported. Kin previously reportedradiological and histological findings of spinal deformities in the Hereditary Lordoscoliotic Rabbit(LSR), which develops thoracic lordosis or lordoscoliosis during growth. We investigated biologicalactivities of bone cells of the LSR vertebrae by in situ hybridization. Digoxigenin labeled in situhybridization probes of mRNA of osteopontin, type I collagen and bone morphogenetic protein(BMP)-2 were prepared. Hybridization was carried out on the sagittal section of lordotic orlordoscoliotic thoracic vertebra in the LSR. In the apical vertebral body and spinous process of thelordotic thoracic spine of LSR during developmental age, stronger positive signals for type I collagenand BMP-2 mRNA were detected in osteoblasts in the endosteum of the ventral portion than thosein the dorsal portion of the vertebra, which indicates osteogenesis. In the apical vertebral body ofthe lordotic thoracic spine, positive signals for osteopontin mRNA were detected in osteoclasts ofthe Howship’s lacunae and osteocytes around these pits in dorsal endosteal portion, which indicatesbone resorption. No such signals were detected in the ventral endosteal portion of the samevertebral body. The vertebral bones of the LSR showed a specific mode of remodeling response tothe force producing lordosis.IntroductionThe etiology of human idiopathic scoliosis stillremains controversial. Several etiologic factors,such as genetic factor(1,2,3,4,5,6,7), hormonalfactors(8), neuromuscular disease(9,10,11),subtle spinal growth abnormalities(12,13,14) havebeen reported. Many animal models (15, 16, 17)of scoliosis have also been reported.Kin (18) previously reported radiological andhistological findings of the Hereditary LordoscolioticRabbit (LSR), which develops thoraciclordotic or lordoscoliotic spinal deformity like ahuman idiopathic scoliosis, and the deformityappear during growth. To find the unknownetiology of the spinal deformity in LSR, weinvestigated biological activities of bone cells ofthe LSR vertebral body by in situ hybridization.Address correspondence to:Ichiro Baba, M.D., Department of Orthopedic Surgery, Osaka Medical College,2-7 Daigaku-machi, Takatsuki-city, Osaka 569-8686, JapanPhone: +81-726-83-1221 Fax: +81-726-83-6265 E-mail: ort039@poh.osaka-med.ac.jp

38Ichiro BABA, Akihiro KIN, and Muneaki ABEMaterials and MethodsLSRLSR is a type of Japanese White Rabbit whichwas incidentally found by a breeder of RabbitonInstitute(Hyogo, Japan) in 1982(Fig.1). Sincethat time, crossbreeding has been continued. InLSR, spinal deformities are not recognized atbirth, but appear at 4-6 weeks of age and developuntil 16 weeks. Most of the rabbits demonstratedlordosis, which was seen at the T7-10 levels (44degrees measured by Cobb’s method(19) atmaximum), and was often accompanied by slightscoliosis(40 degrees at maximum)(Fig.2). Nospinal anomalies were observed but severedeformities sometimes impaired their gait andability to eat. The incidence of the deformitiesranged from 50 to 60%, and no gender differenceswere observed.Tissue PreparationLSR were sacrificed at 6-14 weeks of age underintra venous general anesthesia and the thoracicFig.1Fig.2aFig.2Typical example of LSR.Fig.2bRadiographs showing scoliosis (a) andlordosis(b) of the thoracic spine in LSR.spines were harvested with surrounding softtissue and fixed for 5 days at 4 in 4% freshlymade paraformaldehyde(PFA) in 0.1M phosphatebuffer. Then specimens were dehydrated inethanol, cleared in chloroform, and decalcifiedwith Morse’s solution(10% sodium citrate and22.5% formic acid) until the specimen becamesoft(it took 3 to 4 weeks). Decalcified specimenswere dehydrate and embedded in paraffin.Sagittal sections of 6m thick were cut andmounted on 3-aminopropyl- triethoxysilanecoatedslides(20).Probe PreparationBone samples of the skull, spine, ribs andfemurs dissected from the fetus of Japanese WhiteRabbits 1 week before birth under intra venousgeneral anesthesia were frozen in liquid nitrogen.Messenger RNA was extracted from the specimenusing the Micro-Fast Track mRNA isolationkit(Invitrogen Corp., Sandiego, CA), according tothe manufacture’s instructions. ComplementaryDNA(cDNA) was made so that mRNA was reversetranscribed in 20l reaction mixture containingreverse transcriptase and random primer.Thereafter, 1l of cDNA was amplified in 25lpolymerase chain reaction(PCR) mixturecontaining 0.125U Taq DNA polymerase and12.5pmol of each primer for type I collagen,osteopontin(Osp) and bone morphogeneticprotein-2(BMP-2) by PCR. Oligonucleotides forPCR were as follows: type I collagen, 5’-CAAGGGAGAACGTGGTTACC-3’ (5’sense;312-331) and 5’-TTCTTCCGGGA GCCTTCAGG-3’(3’antisense;934-963); and Osp, 5’-CACCGCAGAATGCTATGTCC-3’(5’ sense;251-270) and 5’-GGCTCGATGGCTAGCTTGTC-3’(3’antisense;922-941); and BMP-2, 5’-ATGCCAAGTCCTGCTAGGAG-3’(5’sense;395-414)and 5’-GATCGGCTAATCCTGACA TG-3’(3’antisense;1110-1129). PCR conditions were asfollows a total of 30 cycles was performed withthe Astec DNA thermal cycler(Astec, Fukuoka,Japan) at 94 for 0.5 minutes, 55 for 1 minute,72 for 1 minute, and then at 72 for 5 minutesat the end of the procedure. PCR products weresubcloned into multicloning sites of the plasmid,and analyzed with the ABI Model 373A DNASequencer using the PRISM Ready ReactionDyeDeoxy Terminator Cycle SequencingKit(Perkin-Elmer Corporation) according to themanufacture’s instructions. The sequence resultswere identical to those of the rabbit type Icollagen (GenBank accession no.D49399), BMP-2Bulletin of the Osaka Medical College 52137-44, 2006

Etiology of Spinal deformity in LSR 39(GenBank accession no.D30751, X56848) andOsp(GenBank accession no.D11411) previouslyreported(21). Digoxigenin(DIG)-labeled singlestrandantisense and sense complementary RNAprobes were prepared using the DIG RNA labelingkit(Boehringer Mannheim GmbH, Biochemica,Manheim, Germany) according to themanufacturer’s instructions(20).In situ hybridizationIn situ hybridization was carried out accordingto the manufacturer’s protocol(BoehringerMannheim Yamanouchi, Tokyo, Japan) with minormodifications. Sections were deparaffinized,fixed in 4%PFA in 0.1M PB (pH7.4), pretreatedwith HCl to inactivate endogenous alkaline phosphatase,and acetylated with acetic anhydrate.Hybridization was carried out at 50 for 16husing DIG labeled probes at a concentration ofapproximately 0.5g/ml in hybridization bufferand was washed with Rnase A (10g/ml) at 37for 30 minutes. Hybridized probes were detectedusing a nucleic acid detection kit(BoehringerMannheim GmbH, Biochemica, Mannheim,Germany) according to the manufacture’sinstructions.Controls hybridized with the sense probe didnot show positive signals.ResultsHistological findingsThe thoracic spine of 4-week-old LSR has nodeformity (Fig.3). In the apical vertebra andintervertebral disc in the lordotic thoracic spine ofa 8-week-old LSR, the main histological findingswere a deviation of the nucleus pulposus andextension in ventral and compression in the dorsalportion of the annulus fibrosus(Fig.4). No markedFig.3Sagittal section of the normal thoracicvertebra of LSR of 4 weeks of age. H.E.stain (x 3) vb:vertebral body sc:spinalcordFig.4a Fig.4b Fig.4cFig.4(a)Sagittal section of the normal thoracic vertebra of LSR of 10 weeks of age.(b)(c)Sagittal section of the apical thoracic lordotic vertebra of LSR of 10 weeks of age. There isdeviation of the nucleus pulposus and extension in ventral and compression in the dorsal portionof the annulus fibrosus. No marked abnormality is observed in the epiphysis or in the growthplate of the vertebral body. H.E. stain (x 3)vb:vertebral body sc:spinal cord np:nuculeus pulposusBulletin of the Osaka Medical College 52137-44, 2006

40Ichiro BABA, Akihiro KIN, and Muneaki ABEabnormality was observed in the epiphysis or inthe growth plate of the vertebral body.In the apical vertebra of a 12-week-old LSR,proliferation of columnar cartilage composed of avertebral growth plate was found on the ventralside. On the dorsal side, narrowing of thevertebral growth plate and proliferation ofmarginal epiphysial cartilage cells were found.In the apical vertebra of a 24-week-old LSR, awedging deformity in the epiphysis was found.The intervertebral disc was degenerated and thenucleus pulposus was destroyed.Fig.5aFig.5b Fig.5c Fig.5d Fig.5eFig.5Type I collagen in situ hybridization.Sagittal section of the apical thoracic lordotic vertebra of LSR of 10 weeks of age.(a)Arrows indicate markedly strong positive signals for type I collagen mRNA. (x 3)(b)Arrows indicate markedly strong positive signals for type I collagen mRNA in osteoblasts inthe endosteum at the ventral side of the vertebral body. (x 50)(c)Arrows indicate positive signals for type I collagen mRNA in osteoblasts in the extraosteum atthe dorsal side of vertebral body. (x 50)Sagittal section of the normal thoracic vertebra of LSR of 10 weeks of age.(d) Arrows indicates slightly positive signals for type I collagen mRNA in osteoblasts in theendosteum at the ventral side of the vertebral body. (x 50)(e) Positive signals for type I collagen mRNA are not seen at the dorsal portion of the vertebralbody. (x 50)v:ventral side d:dorsal side cr:cranial side ca:caudal side sc:spinal canalBulletin of the Osaka Medical College 52137-44, 2006

Etiology of Spinal deformity in LSR 41In situ hybridizationIn the apical vertebral body and spinousprocess in the lordotic thoracic spine of LSRduring growing age, stronger positive signals fortype I collagen mRNA were detected inosteoblasts in the endosteum of the ventral thanthose in the dorsal portion of the vertebra(Fig.5).In the vertebra without deformity, no differencesof positive signals between the ventral and dorsalportion were observed. Positive signals for BMP-2mRNA showed a similar pattern as that observedin type I collagen mRNA pattern(Fig.6).In the apical vertebral body of the lordoticthoracic spine, positive signals for Osp mRNAwere detected in osteoclasts in Howship’s lacunaeand osteocytes around these pits in dorsalendosteal portion. No such signals were detectedin the ventral endosteal portion of the samevertebral body(Fig.7). In the vertebral bodywithout deformity, there were no differences inthe Osp mRNA expression pattern betweenventral and dorsal portion.DiscussionTo investigate the etiology of idiopathicscoliosis, several scoliotic animal models havebeen developed, including paravertebral muscleexcision(22), division of the intercostalnerves(22), resection of the ribs(23), resection ofthe costotransvers joints(24), rib elongation(25),rib shortening(26). Recent report showed thatthe scoliosis which develops in chickens afterpinealectomy was similar to human idiopathicscoliosis, and thus may be a useful model ofidiopathic scoliosis(17). Moreover surgicaltethering of the spinous apophysis and transverseapophysis on the same side of the spine producedscoliosis with characteristics similar to those ofhuman idiopathic scoliosis(27). However thesescoliotic animal models can not show deformitywithout surgery, therefore these are models ofsecondary spinal deformity. On the other handthe spinal deformities observed in LSR weresimilar to those of human idiopathic scoliosis; nospinal anomaly, no deformity at birth, thedeformities of the spine appears at 4-6weeks ofage and progresses without surgery, anddeformities include lordosis or lordoscoliosis withFig.6aFig.6bFig.6BMP mRNA in situ hybridization.Sagittal section of the apical thoracic lordotic vertebra of LSR of 10 weeks of age.(a)Arrows indicate positive signals for BMP mRNA in osteoblasts in the endosteum in the ventralside of the vertebral body. (x 50)(b)Positive signals for BMP mRNA are not seen in the endosteum at the dorsal portion dorsalside of the vertebral body. (x 50)v:ventral side d:dorsal side cr:cranial side ca:caudal sideBulletin of the Osaka Medical College 52137-44, 2006

42Ichiro BABA, Akihiro KIN, and Muneaki ABEFig.7a Fig.7b Fig.7cFig.7Osp mRNA in situ hybridization.Sagittal section of the apical thoracic lordotic vertebra of LSR at 10 weeks of age.(a) Asterisk indicates positive signals for Osp mRNA in osteoclasts at Howship’s lacunae andosteocytes around these pits in extraosteum at the ventral side of the vertebral body. (x 50)(b) Asterisks indicate positive signals for Osp mRNA in osteoclasts in Howship’s lacunae andosteocytes around these pits in endosteum at the dorsal side of the vertebral body. (x 50)Sagittal section of the normal thoracic vertebra of LSR at 10 weeks of age.(c) Positive signals for Osp mRNA are not seen in the endosteum at the dorsal side of thevertebral body. (x 50)v:ventral side d:dorsal side cr:cranial side ca:caudal siderotation from middle to lower thoracic levels(18).In the LSR with severe lordosis, rabbits cannot eator reproduce, so they have to rotate their spine.This posture may enforce their lordosis intoscoliosis. Therefore LSR is an useful animalmodel for studying etiology of human idiopathicscoliosis.Concerning histological findings observed inpresent study, in the early stage of the deformity,main changes included a deviation in the nucleuspulposus of the intervertebral disc and extensionin the ventral portion and compression in thedorsal region of the annulus fibrosus. In the latestage, proliferation of columnar cartilagecomposed of the vertebral growth plate was foundon the ventral side. On the dorsal side, narrowingof the vertebral growth plate and proliferation ofmarginal epiphyseal cartilage cells were found.Finally, wedging deformity in the epiphysis wasfound, the intervertebral disc was degeneratedand the nucleus pulposus was destroyed. Thesefindings were considered to be secondary changesresulting from progressive local lordosis of thethoracic spine.Dubousset et al.(28) found that scoliosisroutinely developed in pinealectomized chickens,and scoliosis was caused from decreasingmelatonin production. Sobajima et al. (29)reported that serum melatonin levels in LSR weresignificant higher than those of Japanese whiterabbit. And they suggest that causes of spinaldeformities in the LSR may be the result of thecontribution of melatonin receptors as well as thatof altered serum melatonin levels in the LSR.Although further studies will be required toinvestigate the expression of melatonin receptorin other tissues of the LSR as well as to delineatethe role of melatonin in the pathogenesis ofidiopathic scoliosis.Type I collagen is a main component ofconnective tissues of the skin, tendon and bone.In present study, strong positive signals for type Icollagen mRNA were detected in osteoblasts ofthe endosteum of the ventral portion of theBulletin of the Osaka Medical College 52137-44, 2006

Etiology of Spinal deformity in LSR 43lordotic thoracic vertebral body and spinousprocess, indicating osteogenesis. BMP-2 is one ofthe most potent protein of osteogenesis and hasbeen shown to induce bone formation successfullyat heterotopic locations. Again in present studyBMP-2 mRNA showed a similar pattern to type Icollagen mRNA, indicating osteogenesis. OspmRNA-positive osteoclasts in Howship’s lacunaeand osteocytes around the pits were detected inthe dorsal endosteal portion of the same vertebralbody, indicating bone resorption(30). These bonecells activity indicates the remodeling fordistraction force to the anterior side andcompression force to the posterior side in thethoracic lordotic vertebral body. The vertebralbones showed a specific mode of remodelingresponse to the force producing lordosis.Asymmetrical loading of the spine is onepossible etiology of idiopathic scoliosis.Carpintero et al.(27) demonstrated thatlordoscoliosis could be induced in a rabbit bysurgical tethering of the spinous apopyhsis andtransverse apophysis. Our results suggest thatgrowth imbalance which may exist between theanterior component and the posterior componentwith muscle and ligament, enforced developinglordosis. This findings suggest that asymmetricalloading of the spine can be an etiology ofidiopathic scoliosis. Whether asymmetricalloading is the cause of idiopathic scoliosis or theresult of spinal deformity, is still unclear. Becausethe etiology of spinal deformity in LSR may bemultifactorial same as human idiopathic scoliosis,further studies is necessary to know the etiologyof spinal deformity of LSR.Acknowledgments The authors thank ShintaroNomura, PhD (Department of Pathology, MedicalSchool of Osaka University), Akira Tsutsumi,MD(Department of Surgical Pathology, OsakaMedical College) for technical assistance andadvice.References1. De George FV, Fisher RL: Idiopathic scoliosis:genetic and enviromental aspects.J MedGenet. 1967;4:251-257.2. Wynne-Davis R: Familial(idiopathic)scoliosis:A family survey. J Bone JointSurg(Br). 1968;50:24-30.3. Risenborough Ej, Wynne-Davies R: A genericsurvey of idiopathic scoliosis in Boston,Massachusetts. J Bone Joint Surg (Am).1973;55:974-982.4. Wynne-Davis R: Genetic aspects of idiopathicscoliosis. Dev. Med Child Neurol.1973;15:809-811.5. Robin GC, Cohen T: Familial scoliosis:A clinicalReport. J Bone Joint Surg (Br). 1975;57:146-148.6. Miller NH: Cause and natural history ofadolescent idiopathic scoliosis. Orthop CliniNorth Am. 1999;30:343-352.7. Hadley MN: Spine update:Genetics of familialidiopathic scoliosis. Spine.2000;25:2416-2418.8. Machida M, Dubousset J, imamura Y, MiyashitaY, Yamada T, Kimura J: Melatonin: A possiblerole in pathogenesis of adolescent idiopathicscoliosis. Spine.1996;21:1147-1152.9. Yamada K, Yamamoto H, Nakagawa Y,TezukaA, Tamura T, Kawata S:Etiology of idiopathicscoliosis. Clini Orthop.1980;152:232-236.10. Zadeh HG, Sakka SA, Powell MP, Mehta MH:Absent superficial abdominal reflexes inchildren with scoliosis: An early indicator ofsyringomyelia. J Bone Joint Surg (Br).1995;77:762-767.11. Samuelsson L, Lindell D: Scoliosis as the firstsign of a cystic spinal cord. Eur Spine J.1995;4:284-290.12. Perdriolle R, Becchetti S, Vidal J, Lopez P:Mechanical process and growth cartilages:Essentioal factors in the progression ofscoliosis.Spine.1993;18:343-349.13. Stokes Ia, Spence H, Aronsson Dd, Kilmer N:Mechanical modulation of vertbral bodygrowth:Implication for scoliosis progression.Spine.1996;21:1162-1167.14. Porter Rw:Idiopathic scoliosis:The relationbetween the vertebral canal and the bodies.Spine. 2000;25:1360-1366.15. Langenskiöd A, Michelsson E: Experimentalprogressive scoliosis in the rabbit.J Bone JointSurg (Br). 1961;43:116-12016. Machida M, Dubousset J, Imamura Y, ImamuraY, Iwaya T, Yamada T, Kimura J: Aexperimental study in chickens of thepathogenesis of idiopathic scoliosis.Spine.1993;18:1609-1615.17. Kanemura T, Kawakami N, Deguchi M,Mimatsu K, Iwata H: Natural course ofexterimental scoliosis in pinealectomizedchickens. Spine.1997;22:1563-1567.18. Kin A: Radiological and histological studies onthe spinal deformity in hereditary lordoscolioticrabbits J. Jpn. Orthop. Assoc. 1994;68:458-469.(in Japanese)Bulletin of the Osaka Medical College 52137-44, 2006

44Ichiro BABA, Akihiro KIN, and Muneaki ABE19. Cobb JR: American Academy of OrthopaedicSurgeons Instructional Course Lectures.1948;5:261.20. Nomura S, Hirakawa K, Nagoshi J: Method fordetecting the expression on bone matrixprotein by in situ hybridization usingdecalcified mineralized tissue. ActaHistochem.Cytochem. 1993; 26, (4): 303-309.21. GenomeNet. http://www.genome.jp/Japanese/22. Bisgard Jd: Experimental thoracogenicscoliosis. J Thoracic Surg .1935;4,435.23. Langenskiöd A, Michelsson JE: Thepathogenesis of experimental progressivescoliosis. Acta Orthop Scand. Suppl. 1962;59,3-26.24. Karaharju EO: Deformation of vertebrae inexperimental scoliosis. Acta Orthop Scand.Suppl. 1967;105:7-7925. Sevastik J, Agadir M, Sevastik Befits of ribelongation on the spine. I. Distortion of thevertebral alignment in the rabbit.Spine.1990;15:822-82526. Sevatikglou JA, Aaro S, Lindholm TS,Dahlborn MD: Experimental scoliosis ingrowing rabbits by operations on the rib cage.Clin Orthop.1978;136:282-28627. Carpintero P, Mesa M, Garcia J, Carpintero P:Scoliosis induced by asymmetric lordosis androtation. Spine.1997;22,(19):2202-2206.28. Dubousset J, Queneau P, Thillard MJ:Experimental scoliosis induced by pineal anddiencephalic lesions in young chickens. Itsrelation between with clinical findings inidiopathic scoliosis. Orthop. Trans. 1983;7:7.29. Sobajima S, Kin A, Baba I, Kanbara K, SemotoY, Abe M: Implication for melatonin and itsreceptor in the spinal deformity of hereditaryLordoscoliotic Rabbit. Spine. 2003;28(6):554-558.30. Ikeda T, Yamaguchi A, Yokose S, Nagai Y,Yamato H, Nakamura T, Tsurukami H,Tanizawa T, Yoshiki S: Changes in biologicalactivity of bone cells in ovariectomized ratsrevealed by in situ hybridization. J. Bone andMineral Research.1996;11,(6): 780-788.Received December 14, 2005Accepted January 5, 2006Bulletin of the Osaka Medical College 52137-44, 2006