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1 Bone 44 (2009) Contents lists vilble t ScienceDirect Bone journl homepge: Bone structure nd remodelling in stroke ptients: Erly effects of zoledronte Kenneth E.S. Poole,, Shobn Vedi, Irene Debirm b, Collette Rose, Jon Power, Nigel Loveridge, Elizbeth A. Wrburton c, Jonthn Reeve, Juliet Compston Division of Bone Reserch, Deprtment of Medicine, University of Cmbridge, Box 157, Addenbrooke's Hospitl, Cmbridge, Englnd, CB2 2QQ, UK b NIHR Biomedicl Reserch Centre, Deprtment of Medicine, University of Cmbridge, Box 157, Addenbrooke's Hospitl, Cmbridge, Englnd, CB2 2QQ, UK c Deprtment of Clinicl Neurosciences, University of Cmbridge, Box 157, Addenbrooke's Hospitl, Cmbridge, Englnd, CB2 2QQ, UK rticle info bstrct Article history: Received 7 August 2008 Revised 27 October 2008 Accepted 10 November 2008 Avilble online 11 December 2008 Edited by: R. Recker Keywords: Histomorphometry Stroke Zoledronte Bone formtion Bone resorption Introduction: We hve reported tht fter n cute stroke, intrvenous zoledronte prevented bone loss in the hemiplegic hip. Prticipnts from the tril lso volunteered for trns-ilic bone biopsy, to ssess the erly effects of stroke nd zoledronte on ilic bone remodelling. Methods: Ptients with cute stroke were rndomly ssigned to single intrvenous dose of zoledronte 4 mg or plcebo within 5 weeks of stroke. Biopsies from 14 ptients (3 femle, 11 mle, men ge 71±11) were suitble for nlysis. These were tken t men 10 weeks (±2) post-stroke, nd included 5 ptients who hd received zoledronte. Histomorphometry ws performed on undeclcified sections using light nd fluorescence microscopy. Sttic nd dynmic indices of remodelling were compred to locl reference rnge from helthy controls. Osteoclsts nd their precursors were identified on frozen sections using trtrte resistnt cid phosphtse (TRAP) stining. Dul-energy x-ry bsorptiometry (DXA) of the proximl femor ws performed t bseline nd 6 months lter. Results: The eroded surfce in cncellous bone (ES/BS) ws significntly higher in stroke ptients thn controls (5.7% vs. ref 1.6%, pb0.0001). Although ES/BS did not differ between zoledronte nd plcebotreted groups, there were significntly fewer osteoclsts nd their precursors in zoledronte-treted individuls (p=0.023). Bone formtion indices (osteoid surfce, OS/BS nd minerlising surfce, MS/BS) were significntly lower in stroke ptients thn controls nd lthough OS/BS ws higher in the zoledronte group thn the plcebo group (p=0.033), MS/BS ws not different (p =0.924). There were no differences between hemiplegic nd unffected sides for ny histomorphometric prmeter despite symmetric reductions in hip bone minerl density (p =0.013). Conclusion: Stroke ptients hd higher resorption indices nd lower bone forming surfces thn controls, consistent with uncoupling of bone remodelling. These findings re preliminry nd lrger study is required to evlute the contributions of gender, ge nd hemiplegic sttus to the remodelling imblnce. Zoledronte therpy ws ssocited with reduction in osteoclstic cell numbers consistent with its known mode of ction in bone Elsevier Inc. Open ccess under CC BY-NC-ND license. Introduction We previously demonstrted tht single intrvenous infusion of zoledronte given within 5 weeks of cute stroke protected ginst the deleterious effects of hemiplegi on hip bone minerl density [1]. After stroke, there is reduction in bone minerl density in the hemiplegic hip s ssessed by DXA. Despite the mgnitude nd Funding sources: This work ws supported by project grnt from the Ntionl Osteoporosis Society (UK). KESP cknowledges funding from the rthritis reserch cmpign (Clinicin Scientist, Fellowship) nd Medicl Reserch Council (Trining Fellowship). ID nd CR cknowledge funding from the Cmbridge NIHR Biomedicl Reserch Centre. The funding bodies hd no role in study design; in the collection, nlysis, nd interprettion of dt; in the writing of the report; nor in the decision to submit the pper for publiction. Corresponding uthor. Fx: E-mil ddress: kp254@nhs.net (K.E.S. Poole). rpidity of these effects, the underlying mechnisms t the bone tissue level in stroke ptients re unexplined. Biochemicl bone mrker studies hve suggested n increse in bone resorption nd reduced bone formtion in the first week fter stroke compred to controls [2], which persisted throughout the first yer [3,4]. Although bone density reduction following stroke is site specific (with the gretest losses t sites such s the ffected hip [5]), more generlised bone loss s result of bed-rest nd ltertions in clciotropic hormone regultion is lso described [6]. Yer-long tretment with dily orl risedronte fter cute stroke ws ssocited with suppression of bone resorption mrkers, but lso n unexpected increse in bone formtion mrkers [4]. Bone formtion might be suppressed fter stroke due to combintion of reduced PTH nd reduced ctive vitmin D [7], the result of rised ionised clcium inhibiting the prthyroid glnd [8]. The ims of this study were three-fold: to ssess indices of cncellous, endocorticl nd corticl bone turnover in the ilic bone 10 weeks Elsevier Inc. Open ccess under CC BY-NC-ND license. doi: /j.bone

2 630 K.E.S. Poole et l. / Bone 44 (2009) following stroke; to evlute the erly effects of zoledronte on bone remodelling; nd to compre histomorphometric prmeters in ptients with stroke to UK helthy reference rnge. Secondry ims were to investigte the reltive contributions of the side of biopsy (hemiplegic or contr-lterl), bseline 25 hydroxyvitmin D (25OHD), stroke severity nd functionl sttus to chnges in bone remodelling in stroke ptients. Methods Approvl for the study ws obtined from the Cmbridge Locl Reserch Ethics Committee (Cmbs LREC C, 2001). All the study prticipnts were dmitted to n cute stroke unit with first ever stroke nd were tking prt in the rndomised plcebo controlled tril of 4 mg intrvenous zoledronte vs. plcebo to prevent bone loss in hemiplegi. As prt of tht study protocol, ll ptients who were rndomised to either zoledronte or plcebo were invited to volunteer for single trns-ilic bone biopsy. Ptients received clcium (1 g) nd vitmin D (800 IU) dily during the tril. Full inclusion nd exclusion criteri for this tril hve been published [1]. Additionl exclusion criteri for the biopsy study were disorders of blood cogultion, wrfrin therpy, respirtory disese nd obesity. Ptients hd been independent nd mbulnt before dmission with stroke nd ll hd hemiplegi ffecting the lower limb. They were unble to wlk 1 week following stroke. All ptients underwent mesurement of bone minerl density of both hips by DXA t study entry, repeted t 6 months. Two scles were used to evlute ptients' ctivity nd motor power t study entry nd immeditely prior to biopsy. These were the Brthel Index (/100) [9] nd the long term score (/48) of the Scndinvin Stroke Scle [10]. Fifteen of the 31 tril subjects volunteered for single trns-ilic bone biopsy tht ws timed to occur pproximtely 10 weeks fter the stroke (men 10.4+/ 1.9 SD). In one subject, the biopsy specimen ws incomplete leving 14 smples for nlysis. The resons for not hving biopsy were s follows: 9 ptients declined, 5 were tking orl wrfrin therpy, nd 2 hd discontinued the study. The trns-ilic bone biopsies were tken between Jnury 2002 nd October 2003 from 4 women (1 unsuitble for nlysis) nd 11 men, ged between 56 nd 89 (men 77.1+/ 11) yers. In order to explore the side-specific effects of hemiplegi on bone turnover, it ws decided t the strt of the study to smple lterntely from the ffected nd unffected side, so 7 ptients hd biopsy from the hemiplegic side nd 7 from the contr-lterl side. Upon unblinding, five ptients hd received zoledronte nd 9 hd received plcebo infusions. Biopsies were obtined using 7.5 mm internl dimeter modified Bordier trephine using locl nesthetic infiltrtion nd intrvenous sedtion. In 11 ptients it ws possible to double demeclocycline lbelling before the biopsy (300 mg demeclocycline twice dily for 2 dys, then ten dy gp, followed by 300 mg twice dily for 2 dys, followed by biopsy 4 dys fter the lst dose) [11]. Biopsies were cut in hlf longitudinlly, coded by the lbortory technicin nd ll histomorphometric mesurements on blinded biopsies were mde by the sme observer (KESP) with the exception of ES/BS (done by SV). One hlf of the biopsy ws embedded in methylmethcrylte for histomorphometry (British Drug House Chemicls Ltd, Poole, Dorset), the other ws briefly immersed in polyvinyllcohol (PVA) nd chilled in hexne for 10 min t 70 C before mounting in PVA on brss chuck for TRAP stining nd immunohistochemistry on cryosections [12]. Undeclcified cryosections (8 μm) were cut onto sectioning tpe using Bright cryostt microtome (Huntingdon, UK) nd rected for trtrte resistnt cid phosphtse ctivity [13]. After ir drying, cryotpe sections were plced in freshly prepred buffered solution of mg of nphthol AS B1 phosphte, mg of sodium trtrte nd 37 ml of 0.1 M tri sodium citrte (buffered to ph 4.5 with 2.1% citric cid). They were then wshed rpidly in solution of 0.25 g of sodium fluoride in 250 ml distilled wter before plcing in regent consisting of mg of fst grnet in 35 ml of buffer (250 ml distilled wter nd 3.4 g sodium cette buffered to ph 6.2 with cetic cid). Tpe sections were then wshed in distilled wter twice nd cover slipped with n queous mounting medi. After methylmethcrylte embedding, 8 μm undeclcified sections were cut with Jung Polycut microtome (Leic, Milton Keynes, UK) nd stined with Von Koss/Vn Gieson stin for osteoid mesurement, trbeculr surfce nd structurl prmeters nd toluidine blue stin for eroded surfce nd wll thickness (using polrised light) before mounting on slides with DPX (Fisher Chemicls). Additionl 15 μm sections were exmined for fluorescent lbels using ultrviolet light microscopy nd 365-nm filter. For ll cncellous indices t lest two sections from levels seprted by more thn 150 μm ( reduced thickness due to the limittions of using hlf core biopsy) were nlysed from ech biopsy to void replicte smpling of single surfce event. Our detiled histomorphometric methods hve been published previously [14,15] but dditions re described here. For the present study, cncellous bone ws defined s the re bounded by, prllel to nd seprted from the endocorticl surfce by 250 μm. The remining surfce including the entire endosteum ws nlysed seprtely. Osteoid thickness (O.Th) ws mesured t 200 mgnifiction, bone surfce (BS) t 80, wll thickness (W.Th) using polrised light t 125 nd eroded surfce (ES) t 200. For O.Th, minimum number of 20 osteoid sems ws nlysed from ech biopsy. All osteoid widths greter thn 3 μm were included in the nlysis [16]. For W.Th minimum of 25 bone remodelling units ws mesured for ech biopsy. Minerlising surfce (MS/BS%) ws defined s the surfce extent of lbelled (double-lbelled+hlf single-lbelled surfce, dl+1/ 2 sl) surfce to cncellous bone surfce. Minerl pposition rte (distnce between lbels) in plcebo ptients included vlues of 0.3 μm/d for 2/7 biopsies tht hd single corticl lbels only [17]. Bone formtion rte (BFR/BS um 3 /um 2 /d) ws defined s the mount of new bone minerlised per dy per unit of cncellous bone surfce. Bsed on the geometric probbility density function, mesures of men pprent widths were trnsformed into 3D men pprent thicknesses by multiplying them by π/4 [18]. All histomorphometric mesurements re described using nomenclture pproved by the Americn Society for Bone nd Minerl Reserch (ASBMR [19]), with the exception of corticl osteoid re, Ct. OAr/BAr. Endocorticl histomorphometry The endocorticl surfces were defined for the purposes of this study s both inner bone surfces nd cncellous surfces up to 250 μm into the medullry cvity mesuring prllel to the plne of the inner surfces. Osteoid surfce (Ec. OS/BS) ws mesured on these surfces in the sme mnner s the cncellous OS/BS. Corticl histomorphometry Men corticl thickness (Ct. Th) ws mesured utomticlly fter drwing periostel nd endocorticl surfces from three sections nd using correction for section obliquity. Corticl osteoid re (Ct. OAr/ BAr) nd corticl porosity (%) were mesured s follows. The entire Von Koss stined cortex ws cptured t 100 mgnifiction (2 cortices per biopsy) using utomted montging softwre nd digitl cmer. Cnls were defined on the cortex imge using digitl drwing pen nd osteoid ws filled in. The osteoid re ws mesured directly using ImgeJ softwre (ImgeJ 1.32e, NIH, USA vilble to downlod t nd osteoid re s percentge of corticl bone re ws clculted. Assumptions of isotropy used to convert 2D to 3D vlues in cncellous bone re not met in corticl bone [19,20], so men corticl res in 2D cnnot be extrpolted to men volumes in 3D. Therefore, Ct. OAr/BAr is reported, cknowledging tht the correct ASBMR nomenclture is to use either 2D or 3D referents only in single publiction.

3 K.E.S. Poole et l. / Bone 44 (2009) Comprison with helthy reference dt Men histomorphometric prmeters from the stroke ptients were compred with men vlues from the UK helthy dult reference rnge of Vedi et l [14]. Sttisticl nlysis Dt were nlysed using the JMP sttisticl pckge JMP (v 4.0, SAS institute, Cry, NC, USA). Significnce ws determined by Student's t-test nd either 2-smple t-test or Dunnett's test when compring stroke ptients with reference dt. For non-normlly distributed dt, significnce ws determined by use of the Wilcoxon/ Kruskls Wllis 2 smple test. To clculte the individul short term precision of the mesurements, five repeted mesurements were mde on 1 dy. Ech mesurement ws tken from the sme section. Intr-observtion (precision) ws s follows: BV/TV 1.9%, OS/BS 4.2%, O.Th 2.4%, nd W.Th 3.1%. Since the reference histomorphometry dt were generted loclly, coefficients of inter-observer vrition were clculted fter both observers (KESP nd SV) recorded three mesurements for ech prmeter fter exmining 2 3 sections from the sme test biopsy. The inter-observer coefficients of vrition were BV/TV 9.1%, OS/BS 11.3%, O.Th 8.8%, nd W.Th 8.2%. Results Ptient demogrphics re shown in Tble 1. Sttic histomorphometry could be performed in 5 zoledronte ptients nd 9 plcebo ptients. The groups were comprble in terms of ge nd severity of stroke (s ssessed by the Scndinvin Stroke Scle, SSS nd Brthel Index, BI) t the time of biopsy. Qulittive ssessment of the stroke biopsies showed no evidence of woven bone, mrrow fibrosis or signs of cellulr toxicity. The smple sizes for comprison were very smll (n=7 per group), but there were no differences between the ilic biopsies from the hemiplegic side nd unffected side for ny prmeter (0.90Np(ll) N0.13). However, in the plcebo group there ws mrkedly greter loss of totl hip BMD on the hemiplegic side compred to the unffected side in the 6 months following stroke (Tble 1). Leg power impirment t 3 months fter stroke (b5 on the SSS) ws not ssocited with sttisticlly significnt differences in ny prmeter (0.8Np(ll) N0.13). The ES/BS ws not significntly different between groups, but the zoledronte treted group hd fewer TRAP + cells per mm 2 thn plcebo. The OS/BS ws significntly higher in zoledronte-treted ptients thn plcebo-treted ptients (Tble 2). The zoledrontetreted stroke ptients lso hd significntly higher endocorticl Tble 1 Demogrphics in stroke ptients [men (95% CI)] Mesurement Zoledronte 4 mg Plcebo p Vlue Demogrphics Age 67.2 (52.8, 81.6) 72.7 (64.3, 81.0) n (femle, mle) (ffected, 5 (1, 4) (2, 3) 9 (2, 7) (5, 4) unffected) Intervl (weeks) from stroke to 9.3 (7.5, 11.1) 10.8 (9.4, 12.1) biopsy Intervl (weeks) from zol to biopsy 5.6 (4.2, 7.1) SSS/48 29 (21, 37) 28 (22, 39) SSS Leg Power/5 4.2 (2.6, 5.8) 3.6 (2.2, 5.0) Brthel Index/ (40, 76) 57 (34, 80) 25OHD (mmol/l) 41.4 (20.5, 62.7) 42.9 (29.5, 56.7) DXA results Bseline totl hip BMD (g/cm 2 ) 1.08 (0.93, 1.22) 0.98 (0.87, 1.09) ΔTotl hip BMD b hemiplegic side (%) +1.4 ( 0.2, +3.0) 3.3 ( 6.4, 0.2) Δ Totl hip BMD b unffected side (%) +1.9 ( 2.0, +5.7) 0.5 ( 2.7, +1.7) b Student's t-test. Chnge in BMD over 6 months. Tble 2 Bsic sttic, endocorticl nd dynmic histomorphometry in stroke ptients [medin (IQR)] Mesurement n Zoledronte 4 mg n Plcebo p Vlue Sttic prmeters BV/TV (%) (14.0, 25.6) (16.6, 21.9) OS/BS (%) (4.73, 1.53) (1.59, 4.91) OV/BV (%) (0.48, 1.46) (0.18, 0.45) O.Th (um) (6.01, 7.68) (5.00, 7.35) ES/BS (%) (3.67, 7.46) (4.89, 6.54) W.Th (um) (28.4, 35.4) (24.4, 29.1) Frozen sections TRAP + cells (/mm 2 ) (0.01, 0.17) (0.11, 0.52) Endocorticl prmeters Ec. OS/BS (%) (9.0, 18.8) (2.7, 7.5) Ct. OAr/BAr (%) (0.10, 0.58) (0.06, 020) Ct. Porosity (%) (3.60, 7.38) (2.93, 8.16) Ct. Th (um) (523, 875) (559, 1166) Dynmic prmeters MS/BS (%) (0.29, 2.62) b (0.00, 2.86) c MAR (um/d) (0.11, 0.91) b (0.30, 0.86) d BFR/BS (um 3 /um 2 /d) (0.001, 0.03) b (0.00, 0.03) c b c d Wilcoxon Rnks Sums test. Includes zero vlue from 1 zoledronte ptient with no lbels present. Includes zero vlues from 2 plcebo ptients with only single corticl lbels. Assumed MAR of 0.3 um/d in 2 plcebo ptients with only single corticl lbels (Foldes method). OS/BS (but not corticl OAr/BAr) thn plcebo-treted ptients, with no difference in corticl porosity or thickness. Of the 11 demeclocycline lbelled ptients, 4 were from the zoledronte group nd 7 from the plcebo group. Among the 7 plcebo group biopsies, 2 smples displyed single corticl lbelling only (with no double lbels in cncellous or corticl bone). In those 2 smples, the MAR ws ssumed to be 0.3 μm/d [17]. Double lbelling ws seen in three biopsies from zoledronte treted ptients, but in one biopsy there were no lbels in ny comprtment (Tble 2). There ws no significnt difference in MS/BS or BFR/BS between zoledrontetreted nd plcebo-treted stroke ptients. However, vlues for these indices in both groups were substntilly lower thn in the helthy controls (Tble 3). MAR ws lso similr in the two ptient groups nd similr to tht seen in helthy controls. Discussion In ilic bone biopsies obtined within 3 months fter cute stroke, bone formtion t the tissue level ws reduced when compred to helthy controls, irrespective of whether ptients were receiving zoledronte therpy or plcebo. However, the number of osteoclstic cells ws lower in ptients treted with zoledronte thn with Tble 3 Histomorphometric prmeters in stroke ptients vs. helthy controls [men (SD)] Mesurement n All Stroke Pts n Controls p Vlue Age rnge Sex 3f, 11m 16f, 12m BV/TV (%) (4.6) (4.3) O.Th (um) (1.1) (2.1) ES/BS (%) (1.7) (0.7) b MS/BS (%) (1.3) c (5.5) b MAR (um/d) (0.3) d (0.1) Zol Pl OS/BS (%) (3.9) 3.0 (1.7) (11.8) b0.05 b OV/BV (%) (0.6) 0.3 (0.2) (2.2) b0.05 b W.Th (um) (4.0) 28.3 (3.4) (3.0) b0.05 b Significntly different from control men by 2 sided t-test. b Significntly different from control men by Dunnett's test. c Includes zero vlues from 1 zoledronte ptient (no lbels present) nd from 2 plcebo ptients (single corticl lbels). d Includes ssumed MAR of 0.3 um/d in 2 plcebo ptients with only single corticl lbels (Foldes method).

4 632 K.E.S. Poole et l. / Bone 44 (2009) plcebo, consistent with the known nti-resorptive effects of zoledronte. The increse in eroded surfce in both zoledronte nd plcebo treted ptients when compred to controls my thus reflect reduced bone formtion rther thn incresed resorption, resulting in the filure of bone formtion to occur within previously formed resorption cvities. The reson for the higher osteoid surfce in zoledronte-treted ptients thn in those receiving plcebo is uncler since the minerlising surfce ws similr in the two groups. The pprent difference in osteoid surfce my reflect the lrge vrince in the smll number of smples mesured or multiple testing of significnce. The lck of demeclocyline lbel in one zoledrontetreted ptient ws noted; potentil explntions for this include filure to bsorb or tke demeclocycline (for ny reson), suppression of bone remodelling by zoledronte nd bsent lbelling s occsionlly observed in norml subjects. The finding of reduced tissue level bone formtion erly fter stroke is in greement with studies demonstrting tht biochemicl mrkers of bone formtion were reduced (when compred to controls) in the erly phse of stroke recovery [2] nd throughout the first yer [3] in ddition to mrked erly increse in resorption mrkers. We did not mesure turnover mrkers in the present study nd bone histomorphometric dt hve not been previously reported in the stroke popultion. Becuse the biopsies were obtined within 3 months of stroke, it is ssumed tht bone formtion mesures reported here predomintely reflect the ctivity within cncellous Bsic Multicellulr Units (BMUs) tht were initited prior to stroke, with fewer mesurements from BMUs initited fter stroke. Since prestroke biopsies were not vilble, indices of bone formtion prior to stroke re unknown. Nevertheless, the mrkedly lower minerlising surfces in the stroke ptients, irrespective of tretment, when compred to helthy controls support the contention tht tissue level formtion is significntly suppressed erly fter stroke, whether in new or previously initited BMUs. Our results suggest tht one explntion for bone loss in stroke my be n increse in the remodelling spce through filure of the coupled formtion tht would normlly ccompny bone resorption. There my be locl or systemic fctors tht re responsible for this ltered dynmic; for instnce, reversl my be prolonged or osteoblst recruitment nd/or ctivity my be impired. Histomorphometric ssessment of biopsies obtined in the pivotl Phse III zoledronte study (HORIZON) demonstrted the expected reduction in remodelling rte but lso suggested tht osteoblstic ctivity, s ssessed by minerl pposition rte, ws incresed by zoledronte reltive to plcebo [21]. HORIZON lso showed preservtion of cncellous bone microrchitecture in zoledronte-treted ptients, consistent with n nti-resorptive effect, lthough no increse in the degree of mtrix minerlistion ws demonstrted. In the present study no significnt difference in minerl pposition rte ws seen between zoledronte nd plcebo-treted ptients, perhps due to the smll smple vilble here [21]. In the present study, there were no differences in indices of bone remodelling between the hemiplegic nd unffected sides (lthough the smple size ws very smll for this comprison), in contrst to the mrked DXA BMD chnges t the hemiplegic nd unffected hips observed in the ptients (Tble 1). Using DXA, hemiplegi hs been shown to hve both generlised nd loclised effects on the skeleton [22 24]. Ilic bone remodelling my therefore be representtive of the generlised (or indirect) skeletl effects of stroke, rther thn those directly due to ltered mechnicl loding or muscle forces. It hs been suggested tht such generlised bone loss fter stroke occurs becuse of chnges in clciotropic hormone secretion such s rised ionised clcium inhibiting prthyroid hormone [25]. The min limittion of the present study is the modest smple size, prticulrly for comprison of demeclocycline-derived indices. The lrge mesurement nd smpling vrince ssocited with bone histomorphometry mens tht with the numbers included in our study, only lrge differences between zoledronte nd plcebotreted ptients could relibly be demonstrted. The lck of contemporneous control group for comprison is shortcoming common to most histomorphometry studies, but this is mitigted by the similrities between our findings nd those of Recker et l [21]. The control dt in this study were derived from helthy subjects nd were obtined by ourselves using similr methods. Finlly, since the biopsies were obtined reltively erly fter stroke, the long-term stedy stte effects of stroke nd of zoledronte could not be determined in this study. In conclusion, our results indicte reduced tissue level bone formtion in ilic bone erly fter stroke with reduction in osteoclstic cells only in zoledronte-treted ptients. Further studies re required to elucidte further the effects of cute stroke on bone remodelling nd the mechnisms by which bisphosphontes influence both bone resorption nd formtion in stroke ptients. Acknowledgments KESP, SV, ID, NL nd EAW certify tht they hve no ffilitions or involvement in ny orgniztion or entity with direct finncil interest in the subject mtter or mterils discussed in the mnuscript. JR nd JEC hve received funding for dvisory work from Novrtis. The uthors cknowledge the dvice of Dr. Stephen Kptoge, PhD (Sttisticin). References [1] Poole KE, Loveridge N, Rose CM, Wrburton EA, Reeve J. 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