CB1 receptor-dependent and - independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2 We investigated the effect of a synthetic cannabinoid , WIN 55,212-2 on excitatory postsynaptic currents -LRB- EPSCs -RRB- evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells . Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 μM . In rats and mice , this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and μM concentrations . When the function of CB1 cannabinoid receptors -LRB- CB1R -RRB- was impaired , either by the application of a CB1R antagonist AM251 , or by using CB1R knockout mice , WIN 55,212-2 in μM concentrations could still significantly reduced the amplitude of EPSCs . WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites , since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased . The inactive enantiomer , WIN 55,212-3 , mimicked the effect of WIN 55,212-2 applied in high doses . In further experiments we found that the CB1R-independent effect of 10 μM WIN 55,212-2 at glutamatergic synapses was fully abolished , when slices were pre-treated with ω-conotoxin GVIA , but not with ω-agatoxin IVA . 1 Introduction The type 1 cannabinoid receptors -LRB- CB1Rs -RRB- have been shown to control the release of different neurotransmitters , but the mechanisms underlying the regulation of synaptic communication could substantially vary between brain regions -LRB- Freund et al. , 2003 -RRB- . Pharmacological results , suggesting a presynaptic locus of action of cannabinoid receptor ligands , have been fully supported by immunohistochemical data . Several studies demonstrated at the electron microscopic level that CB1Rs decorated both inhibitory and excitatory axon terminals -LRB- Katona et al. , 1999 , 2006 ; Kawamura et al. , 2006 -RRB- . In addition , recent high-resolution quantitative studies established that CB1Rs were found all around the axon membrane , but were enriched in the perisynaptic annulus and on preterminal segments , whereas immunolabelling was weaker in the synaptic active zone -LRB- Nyiri et al. , 2005 ; Kawamura et al. , 2006 -RRB- . This subcellular distribution of CB1Rs might imply an action on several regulatory mechanisms of transmitter release , including the control of Ca2 + entry via voltage-dependent Ca2 + channels -LRB- primarily by receptors located in the perisynaptic annulus -RRB- , the reduction of axonal conduction -LRB- by receptors present on the preterminal segments -RRB- , or a direct action on exocytosis -LRB- Wilson et al. , 2001 ; Diana and Marty , 2003 -RRB- . In spite of the direct anatomical evidence , several pharmacological observations suggest that some synthetic cannabinoid agonists -LRB- mainly WIN 55,212-2 -RRB- could also have a CB1R-independent action on synaptic glutamate release . This possibility has been fuelled primarily by experiments using CB1R knockout mice . Our laboratory was the first to show that , in the absence of CB1Rs , WIN 55,212-2 was still able to reduce excitatory , but not inhibitory postsynaptic currents in CA1 pyramidal neurons -LRB- Hájos et al. , 2001 -RRB- . Moreover , WIN 55,212-2 was more potent in suppressing GABAergic than glutamatergic transmission -LRB- Hoffman and Lupica , 2000 ; Ohno-Shosaku et al. , 2002 ; Hájos and Freund , 2002 -RRB- , providing further support for the possible presence of CB1R-independent binding site at excitatory synapses . Importantly , AM251 , a CB1R antagonist prevented the reduction of synaptic inhibition after application of WIN 55,212-2 , whereas glutamatergic transmission could still be suppressed by about 50 % in the presence of AM251 -LRB- Hájos and Freund , 2002 -RRB- . In contrast to the above findings showing that hippocampal glutamatergic synapses were effectively regulated independent of CB1Rs , electrophysiological data from other groups suggested that CB1Rs were solely responsible for the cannabinoid modulation of excitatory synaptic transmission in the hippocampus -LRB- Ohno-Shosaku et al. , 2002 ; Domenici et al. , 2006 ; Takahashi and Castillo , 2006 -RRB- . To shed light on the reasons behind the contradictory findings regarding the involvement of CB1R-dependent vs. - independent mechanisms in the regulation of hippocampal excitatory synapses , we re-examined the effect of WIN 55,212-2 on monosynaptically evoked excitatory postsynaptic currents -LRB- EPSCs -RRB- in CA1 pyramidal cells . All these experiments were performed in a modified submerged recording conditions -LRB- Hájos et al. , 2005 -RRB- . 2 Methods Experiments were carried out according to the guidelines of the institutional ethical code and the Hungarian Act of Animal Care and Experimentation -LRB- 1998 . XXVIII . section 243/1998 . -RRB- . Male Wistar rats -LRB- 14 -- 18 days old -RRB- , as well as wild type and CB1R knockout mice -LRB- 15 -- 25 days old , CD1 strain -RRB- were used . The animals were deeply anaesthetized with isoflurane followed by decapitation . After opening the skull , the brain was quickly removed and immersed into ice-cold cutting solution containing -LRB- in mM : sucrose 252 ; KCl 2.5 ; NaHCO3 26 ; CaCl2 0.5 ; MgCl2 5 ; NaH2PO4 1.25 ; glucose 10 -RRB- . The solution had been bubbled with 95 % O2/5 % CO2 -LRB- carbogen gas -RRB- for at least 30 min before use . Thick horizontal slices -LRB- 350 μm from mice and 400 μm from rats -RRB- were prepared using a Leica VT1000S Vibratome . The CA3 region was removed to prevent epileptic burst firings . The slices were stored in an interface type chamber containing ACSF -LRB- in mM : 126 NaCl , 2.5 KCl , 26 NaHCO3 , 2 CaCl2 , 2 MgCl2 , 1.25 NaH2PO4 , and 10 glucose -RRB- at room temperature for at least 1 h before recording . After the initial incubation period , slices were transferred individually into a submerged type recording chamber . Whole-cell patch-clamp recordings were obtained at 30 -- 32 °C from CA1 pyramidal cells visualized by infrared DIC videomicroscopy -LRB- Zeiss Axioscope , Germany -RRB- . Patch electrodes were pulled from borosilicate glass capillaries with an inner filament -LRB- 1.5 mm O.D. ; 1.12 mm I.D. , Hilgenberg , Germany -RRB- using a Sutter P-87 puller . Electrodes -LRB- ∼ 3 -- 6 MΩ -RRB- were filled with a solution containing -LRB- in mM -RRB- 80 CsCl , 60 Cs-gluconate , 3 NaCl , 1 MgCl2 , 10 HEPES , 2 Mg-ATP , and 5 QX-314 -LRB- pH 7.2 -- 7.3 adjusted with CsOH ; osmolarity 275 -- 290 mOsm -RRB- . Excitatory postsynaptic currents -LRB- EPSCs -RRB- were recorded at a holding potential of − 65 mV . Slices were perfused with ACSF containing 70 -- 100 μM picrotoxin to block inhibitory neurotransmission . The solution was bubbled with carbogen gas at room temperature and perfused at a flow rate of 3 -- 4.5 ml/min in a slice chamber optimized for laminar flow to ensure the stability of the amplitude of evoked currents and a better oxygenation of submerged slices -LRB- Hájos et al. , 2005 -RRB- . To evoke EPSCs , the stimulating electrode was placed in the stratum radiatum of CA1 . Pairs of electrical stimuli separated by 50 ms were delivered via a theta glass pipette -LRB- Sutter Instrument Company , Novato , CA -RRB- filled with ACSF at 0.1 Hz using a Supertech timer and isolator -LRB- Supertech LTD , Pécs , Hungary , http://www.superte.ch -RRB- . Access resistances -LRB- between 4 and 18 MΩ , compensated 65 -- 70 % -RRB- were frequently monitored and remained constant -LRB- ± 20 % -RRB- during the period of analysis . Signals were recorded with a Multiclamp 700A -LRB- Molecular Devices , Sunnyvale , CA -RRB- , filtered at 2 kHz , digitized at 6 kHz -LRB- National Instruments PCI-6024E A/D board , Austin , TX -RRB- , and analyzed off-line with the EVAN program -LRB- courtesy of Prof. I. Mody , UCLA , CA -RRB- . The drug was perfused in a given concentration until the maximal effect was reached . The time needed for maximal inhibition was usually 6 -- 8 min . To avoid the possible effect of a changing pH , we added the same amount of HCl to the control solution . The concentration response relationship for WIN 55,212-2 was obtained as follows : control EPSC amplitudes in a 2 -- 3 min time window were compared to those measured after 10 min drug application for the same period of time . Only those experiments were included that had stable amplitudes at least for 10 min before drug application . After each experiment , the tubing made of Teflon was washed with ethanol for 10 min and with ACSF for 15 min . Each data point represents the mean ± SEM of the maximal inhibition of the evoked EPSCs -LRB- n = 3 -- 7 -RRB- . EC50 values were estimated by fitting a curve to the points of the dose response plots obtained in rats or wild type mice using the equation of f -LRB- x -RRB- = a / -LRB- 1 + exp -LRB- − -LRB- x − c -RRB- / b -RRB- -RRB- + -LRB- 100 − a -RRB- / -LRB- 1 + exp -LRB- − -LRB- x − e -RRB- / d -RRB- -RRB- , where ` c ' and ` e ' give the values for high and low affinity binding sites , respectively . The data points obtained in the presence of AM251 or in CB1 knockout mice were fitted by the equation of f -LRB- x -RRB- = a / -LRB- 1 + exp -LRB- − -LRB- x − c -RRB- / b -RRB- -RRB- , where ` c ' gives the value of EC50 . The curve fitting was done using Origin 7.5 -LRB- OriginLab Corporation , MA -RRB- . The paired pulse ratio was calculated from the mean amplitude of the second EPSCs divided by the mean amplitude of the first EPSCs . The paired pulse ratio after drug treatment was compared with the control using Wilcoxon matched pairs test in Statistica 6.1 -LRB- Statsoft , Inc. , Tulsa , OK -RRB- . Data are presented as mean ± SEM . Picrotoxin , WIN 55,212-2 and WIN 55,212-3 were purchased from Sigma-Aldrich , AM251 was obtained from Tocris , while ω-conotoxin GVIA and ω-agatonix IVA from Alomone Labs . For all experiments , WIN-55,212-2 was dissolved in 0.1 N HCl giving a 20 mM stock solution stored at 4 °C . AM251 was dissolved in DMSO -LRB- 100 mM -RRB- and stored at − 20 °C . WIN 55,212-3 dissolved in DMSO -LRB- 100 mM -RRB- was stored at 4 °C . From these stock solutions , the final dilution of drugs was done in ACSF containing picrotoxin under constant stirring and the prepared solution was bath applied . In control solutions , the vehicle was diluted in the same concentration as in the solutions containing drugs . Bovine serum albumin -LRB- BSA -RRB- was added in a concentration of 0.1 mg/ml to the solutions used for experiments with WIN 55,212-3 . 3 Results The effects of the cannabinoid agonist WIN 55,212-2 on EPSCs evoked by focal stimulation of Schaffer collaterals were measured in hippocampal CA1 pyramidal cells . First we performed concentration response analyses for the inhibitory effects of WIN 55,212-2 on evoked EPSC in rat slices -LRB- Fig. 1a -RRB- . WIN 55,212-2 bath applied between the concentrations of 0.1 nM and 30 μM suppressed the amplitude of EPSCs in two steps . The apparent EC50 values from the fitted curve were 2.91 nM and 3.77 μM -LRB- Fig. 1c -RRB- . Then we investigated the WIN 55,212-2-sensitivity of EPSCs , when AM251 , a CB1R specific antagonist was added to the bath solution in the concentration of 2 μM . In spite of the presence of AM251 , the cannabinoid agonist could still reduce the amplitude of evoked currents , but only in the μM range -LRB- Fig. 1a -RRB- . The estimated EC50 value for this effect was 1.69 μM -LRB- Fig. 1c -RRB- . In the next set of experiments , we examined the concentration response relationship for the WIN 55,212-2-induced reduction of evoked EPSCs in mouse slices -LRB- Fig. 1b -RRB- . The sensitivity of synaptic currents for WIN 55,212-2 was tested between the concentrations of 0.01 nM and 30 μM . Similar to that observed in rat slices , the cannabinoid agonist also decreased the amplitude of EPSCs in two steps . The EC50 values estimated by fitting a curve to the points of the dose response plot were 1.91 nM and 12.1 μM -LRB- Fig. 1c -RRB- . To reveal whether WIN 55,212-2 could still suppress excitatory transmission in CB1R-independent manner in mice , we examined the effect of the cannabinoid agonist in CB1R knockout animals . As shown in Fig. 1b , WIN 55,212-2 effectively reduced the amplitude of EPSCs , but only in the μM range . The apparent EC50 value estimated from the curve fitting was 8.32 μM -LRB- Fig. 1c -RRB- . These results obtained both in rats and mice suggest that WIN 55,212-2 in nM concentrations inhibits excitatory synaptic transmission exclusively via CB1Rs , whereas in μM concentrations it has a mixed CB1R-dependent and - independent effect on glutamatergic transmission at Schaffer collateral synapses . By a comparison of the paired-pulse ratios of evoked EPSCs , we next investigated whether the CB1R-independent action of WIN 55,212-2 is presynaptic , i.e. whether it is inhibiting glutamate release similar to that seen earlier for CB1Rs . We first examined the effect of 10 nM WIN 55,212-2 on the paired-pulse ratio in rats and wild type mice . After drug application , the ratio significantly increased to 132.5 ± 9.4 % of control in rats and to 129.5 ± 14.2 % of control and mice -LRB- Fig. 2 ; n = 5 each , p < 0.05 , Wilcoxon test -RRB- . These data are in line with both electrophysiological and anatomical results , suggesting a presynaptic locus of CB1R-dependent action . To check that the changes in the paired-pulse ratio were due to the activation of CB1Rs , we contrasted these values before and after the application of 10 nM WIN 55,212-2 in the presence of AM251 . As expected , the paired-pulse ratio was not altered -LRB- 98.4 ± 6.1 % , n = 6 ; p > 0.1 , Wilcoxon test ; Figs. 2a , c -RRB- . Next we compared the paired-pulse ratio before and after the application of 30 μM WIN 55,212-2 . The ratio of evoked currents was significantly increased to 145.1 ± 5.3 % of control in rat slices -LRB- n = 5 ; p < 0.05 , Wilcoxon test -RRB- and to 141.3 ± 11.8 % in slices from wild type mice -LRB- n = 7 ; p < 0.05 , Wilcoxon test -RRB- . To reveal that the CB1R-independent action of WIN 55,212-2 also modifies transmitter release , we investigated the paired-pulse ratio after application of 30 μM WIN 55,212-2 , while 2 μM AM251 was included in the bath . The ratio of the amplitude of evoked EPSCs still significantly increased , to 136.4 ± 8.4 % of control -LRB- Figs. 2a , c ; n = 7 , p < 0.05 , Wilcoxon test -RRB- . Similar to these results , 30 μM WIN 55,212-2 also raised the paired pulse ratio to 121.1 ± 2.9 % of control in CB1R knockout mice -LRB- Figs. 2b , c ; n = 6 , p < 0.05 , Wilcoxon test -RRB- . Thus , the CB1R-independent effect of WIN 55,212-2 also appears to be presynaptic , reducing glutamate release from Schaffer collateral terminals . As reported earlier -LRB- Shen and Thayer , 1998 -RRB- , WIN 55,212-2 in μM concentrations could directly alter Ca2 + currents independent of CB1Rs , an effect that could be mimicked by its inactive enantiomer WIN 55,212-3 . To test whether at glutamatergic axon terminals a similar mechanism would be responsible for the reduction of EPSC amplitude , WIN 55,212-3 was bath applied in two different concentrations to rat slices . This inactive enantiomer significantly suppressed the amplitude of evoked EPSCs by 42.8 ± 10.7 % -LRB- n = 5 -RRB- and 54.7 ± 12.1 % -LRB- n = 3 -RRB- in 10 μM and 30 μM concentrations , respectively -LRB- Fig. 1c -RRB- . These effects were indistinguishable from those values , which were obtained in the presence of AM251 after application of 10 μM -LRB- 46.9 ± 7.8 % ; n = 5 -RRB- or 30 μM -LRB- 59.6 ± 3.2 % ; n = 4 -RRB- WIN 55,212-2 -LRB- p > 0.1 , Mann -- Whitney U-test -RRB- . These results suggest that the CB1R-independent action of WIN 55,212-2 on glutamatergic transmission might be due to the direct inhibition of Ca2 + entry into the presynaptic boutons . To get deeper insight into the mechanisms underlying the CB1R-independent effects of WIN 55,212-2 , we specifically examined the involvement of voltage-gated Ca2 + channels in this process . Rat slices were pre-incubated either in 250 nM ω-agatoxin IVA -LRB- a specific blocker of P/Q-type Ca2 + channels -RRB- or in 250 nM ω-conotoxin GVIA -LRB- a specific inhibitor of N-type Ca2 + channels -RRB- at least for an hour . After placing the pre-treated slices in the recording chamber , we bath applied 10 μM WIN 55,212-2 in the presence of 2 μM AM251 . In slices pre-treated with ω-agatoxin IVA , the amplitude of EPSCs was reduced by 39.7 ± 8.3 % -LRB- n = 4 , p < 0.05 , Wilcoxon test ; Fig. 3a -RRB- , whereas there was no change in the synaptic currents after application of WIN 55,212-2 in slices pre-incubated with ω-conotoxin GVIA -LRB- 95.8 ± 3.2 % of control , n = 6 , p > 0.1 , Wilcoxon test ; Fig. 3b -RRB- . The results of these experiments suggest that the CB1R-independent action of WIN 55,212-2 at glutamatergic synapses is mediated via inhibition of N-type Ca2 + channels . 4 Discussion Our data presented here demonstrate that excitatory synapses of Schaffer collaterals in CA1 pyramidal cells are inhibited by WIN 55,212-2 both via CB1R-dependent and independent mechanisms . In low nM concentrations , this cannabinoid ligand solely acts as a CB1R agonist reducing glutamate release . In contrast , WIN 55,212-2 in the μM range suppresses glutamatergic synaptic transmission via activation of CB1Rs as well as inhibiting N-type Ca2 + channels independent of CB1Rs . Shen et al. , -LRB- 1996 -RRB- were the first to show that excitatory transmission in the hippocampus could be reduced by WIN 55,212-2 , a finding that has been strengthened later by several other laboratories -LRB- Misner and Sullivan , 1999 ; Al-Hayani and Davies , 2000 ; Hájos and Freund , 2002 ; Ohno-Shosaku et al. , 2002 ; Hoffman et al. , 2003 -RRB- . Similarly to that observed in the hippocampus , WIN 55,212-2 was also shown to suppress excitatory synapses in other brain regions , including the cerebellum -LRB- Levenes et al. , 1998 ; Takahashi and Linden , 2000 -RRB- , neocortex -LRB- Domenici et al. , 2006 -RRB- , basolateral amygdala -LRB- Azad et al. , 2003 ; Domenici et al. , 2006 -RRB- , or striatum -LRB- Gerdeman and Lovinger , 2001 ; Huang et al. , 2001 -RRB- . In earlier studies , the lack of immunostaining for CB1Rs at excitatory terminals -LRB- Katona et al. , 1999 ; Hájos et al. , 2000 -RRB- , taken together with experiments showing that WIN 55,212-2 could significantly reduce glutamate release in CB1R knockouts -LRB- Hájos et al. , 2001 ; Kofalvi et al. , 2003 -RRB- , fuelled the concept that distinct cannabinoid receptors control synaptic excitation and inhibition . This interpretation was supported by the unequivocal demonstration of high densities of CB1Rs on GABAergic axons , while adjacent glutamatergic terminals remained negative -LRB- Katona et al. , 1999 ; Hájos et al. , 2000 ; Nyiri et al. , 2005 -RRB- , as well as by the complete disappearance of cannabinoid sensitivity of IPSCs in CB1R knockouts -LRB- Hájos et al. , 2000 , 2001 ; Varma et al. , 2001 ; Wilson et al. , 2001 -RRB- . Moreover , Hájos and Freund -LRB- 2002 -RRB- showed that AM251 could fully antagonise the effect of WIN 55,212-2 at GABAergic , but not at glutamatergic synapses , further strengthening the existence of a novel cannabinoid-sensitive binding site at hippocampal excitatory synapses . Recently this concept was substantially challenged both by anatomical and electrophysiological experiments , including studies from our own laboratory . First , using a different type of antibody , CB1Rs were convincingly shown to be present on glutamatergic terminals , although in much smaller quantities than on GABAergic axons -LRB- Katona et al. , 2006 ; Kawamura et al. , 2006 -RRB- . Specificity of the staining has been confirmed in CB1R knockout tissue . Second , excitatory transmission was found to be insensitive to the application of WIN 55,212-2 in distinct strains of transgenic mice lacking CB1Rs -LRB- Ohno-Shosaku et al. , 2002 ; Domenici et al. , 2006 ; Takahashi and Castillo , 2006 -RRB- . The discrepancy between earlier and recent data might be resolved by the present findings suggesting that at low nM concentrations WIN 55,212-2 specifically activates CB1Rs , whereas in the μM range the agonist could further reduce glutamate release via direct inhibition of presynaptic Ca2 + entry independent of CB1Rs -LRB- present study ; Shen and Thayer , 1998 ; Kofalvi et al. , 2007 -RRB- . This difference in the specificity of WIN 55,212-2 as a function of its concentration can be noticed already in studies reported by the Kano laboratory . WIN 55,212-2 in 100 nM caused a large reduction in the amplitude of EPSCs in wild type mice , but in CB1R knockouts less then 5 % suppression was found -LRB- Ohno-Shosaku et al. , 2002 -RRB- . However , in 2 μM concentration , WIN 55,212-2 inhibited excitatory transmission by about 20 % in CB1R knockout mice , an effect that was unaltered in the presence of CB1R antagonists -LRB- Kawamura et al. , 2006 -RRB- . Further support to the hypothesis that WIN 55,212-2 in μM concentrations can reduce excitatory transmission via CB1R-independent mechanisms come from the work of Hoffman et al. -LRB- 2005 -RRB- , where the authors showed that in the presence of AM251 , 3 μM WIN 55,212-2 significantly -LRB- appr . by 40 % -RRB- reduced excitation . This view , however , is not supported by two recent studies using CB1R knockout animals , in which 5 μM WIN 55,212-2 was found to be completely ineffective at excitatory synapses -LRB- Domenici et al. , 2006 ; Takahashi and Castillo , 2006 -RRB- . The explanation for these negative findings remains to be investigated . Another finding of the present study that deserves discussion is that the effective concentration of WIN 55,212-2 that significantly inhibited the amplitude of synaptic events in slice preparations was 100 fold lower than it was earlier reported by several laboratories using similar recording circumstances -LRB- Takahashi and Linden , 2000 ; Robbe et al. , 2001 ; Hájos and Freund , 2002 ; Hoffman et al. , 2005 -RRB- . Compared to earlier studies , we changed some conditions that could account for the distinct efficacy of WIN 55,212-2 , which allowed clearly separating CB1R-dependent and independent effects . The preparation and storage of slices , as well as the flow rate of the solution during recordings was modified : slices were cut in a sucrose containing solution and stored in an interface-type chamber before recordings , and a higher flow rate was used in the recording chamber , ensuring a better oxygenation of the tissue -LRB- Hájos et al. , 2005 -RRB- . Under these circumstances , the amplitude of evoked synaptic currents became more stable , and , more importantly , the efficacy of WIN 55,212-2 to suppress excitatory synapses was comparable with those measured in cell cultures -LRB- Ohno-Shosaku et al. , 2002 -RRB- or binding assays -LRB- Felder et al. , 1995 -RRB- . Several studies in different brain regions suggested that the CB1R-independent effect of WIN 55,212-2 might significantly alter synaptic communication among neurons -LRB- Hájos et al. , 2001 ; Pistis et al. , 2004 ; Kofalvi et al. , 2005 ; Matyas et al. , 2006 -RRB- , presumably through a direct blockade of Ca2 + entry at the presynaptic terminals -LRB- Shen and Thayer , 1998 ; Kofalvi et al. , 2007 -RRB- . In the present study , we provided evidence that the CB1R-independent effect of WIN 55,212-2 at glutamatergic synapses was mediated by inhibiting N-type Ca2 + channels . The question arises whether WIN 55,212-2 in high doses could also alter GABAergic transmission independent of CB1Rs , since GABA release from CB1R-expressing axon terminals is known to depend on N-type Ca2 + channel activation -LRB- Wilson et al. , 2001 -RRB- . Indeed , we found that in CB1R knockout mice 10 μM WIN 55,212-2 substantially reduced the amplitude of IPSCs to 62.8 ± 20.5 % of control -LRB- n = 4 -RRB- . In contrast , when CB1R function was intact , both low and high doses of WIN 55,212-2 led to a comparable reduction of IPSC amplitudes -LRB- in 3 nM : 47.9 ± 15.4 % of control , n = 4 ; in 10 μM : 45.2 ± 19.7 % of control , n = 4 -RRB- , similar to results obtained earlier -LRB- Hájos and Freund , 2002 -RRB- . The reason why WIN 55,212-2 in 10 μM concentration did not result in an additional suppression of inhibitory events in the presence of functional CB1Rs may be explained by the fact that CB1R-dependent inhibition of GABAergic currents is entirely due to blocking N-type Ca2 + channels , which occludes the CB1R - independent action of WIN 55,212-2 directly on the same Ca2 + channels . Testing this hypothesis , and other novel aspects of cannabinoid modulation of GABAergic transmission is the subject of another line of investigations in our laboratory . The question arises whether at glutamatergic synapses , under some experimental conditions , endocannabinoids can reduce the efficacy of neurotransmission via CB1R-dependent and - independent mechanisms , similar to high concentrations of WIN 55,212-2 , which can modify Ca2 + entry directly . Some results indeed imply that endocannabinoids could directly inhibit different types of voltage-gated Ca2 + channels independent of CB1Rs -LRB- Chemin et al. , 2001 ; Fisyunov et al. , 2006 -RRB- , yet other data suggest that endocannabinoids released upon depolarization of a postsynaptic neuron -LRB- or exogenously applied -RRB- are unable to suppress excitatory transmission in CB1R knockout mice -LRB- Ohno-Shosaku et al. , 2002 ; Straiker and Mackie , 2005 , but see Rouach and Nicoll , 2003 -RRB- . Nevertheless , the importance of identifying a CB1R-independent binding site for WIN 55,212-2 as N-type Ca2 + channels at excitatory terminals lies in resolving some contradictions in pharmacological and behavioural studies that emerged partly due to the use of WIN 55,212-2 in widely varying concentrations -LRB- see a thorough discussion of this issue in Haller et al. , 2007 -RRB- .

O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

glutamate
transmitter release
hippocampus

brain slices
pyramidal cell
cannabinoids


The

prevalence

of

vertebral

fracture

amongst

patients

presenting

with

non-vertebral

fractures

Introduction

Despite

vertebral

fracture

being

a

significant

risk

factor

for

further

fracture

vertebral

fractures

are

often

unrecognised

A

study

was

therefore

conducted

to

determine

the

proportion

of

patients

presenting

with

a

non-vertebral

fracture

who

also

have

an

unrecognised

vertebral

fracture

Introduction

Osteoporosis

may

be

defined

as

a

skeletal

disorder

characterized

by

compromised

bone

strength

predisposing

a

person

to

an

increased

risk

of

fracture

-LSB-

-RSB-

Overall

osteoporotic

or

low

trauma

fractures

are

common

it

has

been

estimated

that

one

in

two

women

and

one

in

five

men

aged

over

years

will

sustain

at

least

one

low

trauma

fracture

in

their

lifetime

-LSB-

-RSB-

The

fracture

sites

which

most

commonly

come

to

clinical

attention

include

the

distal

radius

-LRB-

Colles

-RRB-

proximal

femur

-LRB-

hip

-RRB-

humerus

and

ankle

In

the

Fracture

Liaison

Service

covering

Glasgow

of

presenting

fractures

were

at

these

sites

-LSB-

-RSB-

The

spine

is

another

key

fracture

site

however

it

has

been

estimated

that

only

of

vertebral

fractures

receive

clinical

attention

which

means

that

the

majority

of

patients

with

vertebral

fractures

remain

undetected

-LSB-

-RSB-

It

appears

that

only

those

patients

with

the

most

severe

vertebral

fractures

come

to

clinical

attention

it

is

likely

that

this

is

due

to

higher

levels

of

back

pain

and

disability

-LSB-

-RSB-

The

European

Prospective

Osteoporosis

Study

-LRB-

EPOS

-RRB-

assessed

vertebral

fracture

incidence

in

men

-LRB-

mean

age

years

-RRB-

and

women

-LRB-

mean

age

years

-RRB-

over

a

mean

of

years

The

age-standardised

incidence

of

morphometric

fracture

was

person

years

in

women

and

person

years

in

men

-LSB-

-RSB-

Vertebral

fracture

incidence

increased

markedly

with

age

in

both

men

and

women

Indeed

the

incidence

in

women

aged

was

person

years

rising

to

person

years

in

women

aged

-LSB-

-RSB-

Lateral

dorsal

and

lumbar

X-rays

were

used

in

the

European

Vertebral

Osteoporosis

Study

-LRB-

EVOS

-RRB-

population

to

identify

vertebral

fracture

over

an

period

The

investigators

found

an

incidence

rate

of

person

years

compared

with

person

years

for

hip

fracture

person

years

for

Colles

fracture

and

person

years

for

other

fractures

-LSB-

-RSB-

Low

trauma

fractures

have

substantial

consequences

Although

hip

fracture

has

the

greatest

mortality

and

morbidity

vertebral

fracture

is

also

associated

with

significant

mortality

and

morbidity

In

one

study

patients

with

vertebral

deformity

had

a

substantially

higher

risk

of

death

compared

to

patients

without

vertebral

deformity

regardless

of

age

sex

and

BMD

-LRB-

mortality

rate

versus

over

a

follow-up

-RRB-

The

risk

of

death

was

highest

in

those

patients

with

a

vertebral

deformity

who

had

a

subsequent

symptomatic

fracture

-LSB-

HR

-LRB-

-RRB-

-RSB-

-LSB-

-RSB-

In

terms

of

morbidity

around

of

patients

with

symptomatic

vertebral

fracture

complain

of

difficulty

in

standing

and

of

difficulty

in

bending

and

complain

of

constant

pain

-LSB-

-RSB-

It

is

well-established

that

the

presence

of

one

low

trauma

fracture

significantly

increases

the

risk

of

a

further

fracture

-LSB-

-RSB-

In

a

systematic

review

carried

out

by

Klotzbuecher

et

al.

-LSB-

-RSB-

women

with

a

single

pre-existing

vertebral

fracture

identified

in

eight

studies

at

baseline

by

vertebral

morphometry

and

in

another

seven

as

clinical

-LRB-

symptomatic

-RRB-

fractures

were

more

likely

to

have

another

vertebral

fracture

than

women

without

vertebral

fracture

Prior

vertebral

fracture

also

predicted

non-vertebral

fracture

in

women

with

vertebral

fracture

the

risk

of

hip

fracture

was

increased

by

of

Colles

by

and

of

any

non-vertebral

fracture

by

Fracture

risk

increased

even

further

with

each

additional

vertebral

fracture

for

example

the

odds

of

a

new

vertebral

fracture

in

women

with

five

or

more

prior

vertebral

fractures

were

greater

than

for

women

without

prior

vertebral

fracture

Given

that

vertebral

fracture

is

a

significant

risk

factor

for

further

fracture

and

that

vertebral

fractures

often

go

undiagnosed

this

study

set

out

to

determine

the

proportion

of

patients

attending

a

Fracture

Liaison

Service

following

a

non-vertebral

fracture

who

also

have

an

unrecognised

vertebral

fracture

The

standard

method

to

assess

vertebral

fracture

is

radiography

of

the

thoraco-lumbar

spine

-LSB-

-RSB-

However

there

is

no

gold

standard

for

the

definition

of

osteoporotic

vertebral

fracture

A

number

of

methods

have

been

developed

for

interpretation

of

spinal

X-rays

including

the

Genant

semi-quantitative

method

-LSB-

-RSB-

which

has

been

used

as

a

surrogate

gold

standard

in

a

number

of

key

osteoporosis

studies

This

approach

is

more

objective

and

reproducible

than

other

qualitative

methods

-LSB-

-RSB-

Vertebral

morphometry

using

dual-energy

X-ray

absorptiometry

-LRB-

DXA

-RRB-

also

known

as

morphometric

X-ray

absorptiometry

-LRB-

MXA

-RRB-

is

a

fast

low-radiation

technique

which

produces

images

that

are

of

sufficient

quality

to

be

used

to

diagnose

the

presence

of

vertebral

deformity

consistent

with

fracture

-LSB-

-RSB-

Methods

The

Fracture

Liaison

Service

in

South

Glasgow

has

been

previously

described

-LSB-

-RSB-

This

service

assumes

responsibility

for

all

patients

who

present

with

a

clinical

low

trauma

fracture

and

ensures

that

appropriate

assessment

diagnosis

and

treatment

recommendations

are

made

to

prevent

further

fracture

A

low

trauma

fracture

is

defined

as

one

sustained

from

a

standing

height

or

less

and

not

occurring

as

a

result

of

a

road

traffic

accident

Patients

with

skull

or

facial

fractures

are

not

routinely

offered

assessment

for

osteoporosis

-LSB-

-RSB-

All

patients

aged

years

or

over

identified

by

an

Osteoporosis

Specialist

Nurse

have

a

DXA

scan

carried

out

if

they

are

likely

to

be

candidates

for

bisphosphonate

therapy

It

should

be

noted

that

patients

aged

over

years

presenting

to

this

service

after

hip

fracture

do

not

have

a

DXA

scan

carried

out

since

most

of

these

patients

have

osteoporosis

and

require

therapy

All

patients

who

presented

with

a

low

trauma

fracture

and

who

underwent

DXA

evaluation

with

vertebral

morphometry

from

to

were

included

in

this

prospective

study

All

scans

were

carried

out

on

a

Lunar

Prodigy

densitometer

using

the

Dual

Energy

Vertebral

Assessment

-LRB-

DVA

-RRB-

morphometry

imaging

software

-LRB-

version

-RRB-

Vertebral

fractures

were

identified

by

direct

visualisation

using

the

Genant

semi-quantitative

grading

scale

-LSB-

-RSB-

All

scans

were

reviewed

by

and

vertebral

deformities

reported

by

the

same

physician

-LRB-

SJG

-RRB-

Although

Genant

's

semi-quantitative

grading

is

based

on

analysis

of

X-rays

and

is

not

validated

for

MXA

it

was

used

in

this

study

to

assess

vertebral

status

on

MXA

since

it

provides

a

practical

and

pragmatic

option

Statistical

analysis

Baseline

differences

that

potentially

explained

observed

differences

in

morphometric

vertebral

fracture

rates

-LRB-

sex

age

body

mass

index

T-score

and

prior

fracture

history

-RRB-

were

clustered

into

pre-defined

categories

Sex

male/femaleAge

Body

mass

index

also

compared

by

quartilesT-score

to

Prior

fracture

history

yes/no

Contingency

tables

for

each

set

of

categories

were

prepared

and

chi-squared

values

calculated

to

test

for

overall

interaction

Yates

continuity

correction

was

used

where

tables

resulted

Where

the

overall

result

was

significant

individual

pairwise

comparisons

and

t-testing

were

then

carried

out

Results

Data

was

collected

during

the

routine

work

of

the

FLS

between

September

and

March

The

data

was

analysed

between

July

and

July

Demographics

Data

were

available

for

patients

presenting

with

low

trauma

fracture

-LRB-

n

-RRB-

were

female

Of

all

patients

were

aged

years

were

aged

years

and

were

aged

years

or

over

Mean

body

mass

index

-LRB-

BMI

-RRB-

was

-LRB-

SD

-RRB-

-LRB-

n

-RRB-

had

a

BMI

-LRB-

n

-RRB-

between

-LRB-

n

-RRB-

between

and

-LRB-

n

-RRB-

According

to

WHO

definitions

of

patients

had

normal

lumbar

spine

BMD

-LRB-

T-score

or

above

-RRB-

were

osteopenic

-LRB-

T-score

to

-RRB-

and

were

osteoporotic

-LRB-

T-score

or

lower

-RRB-

Humerus

-LRB-

n

-RRB-

radius

ulna

-LRB-

n

-RRB-

and

hand/foot

-LRB-

n

-RRB-

were

the

most

common

presenting

fractures

-LRB-

see

Fig.

-RRB-

Other

fracture

sites

included

hip

-LRB-

n

-RRB-

clavicle

-LRB-

n

-RRB-

rib

-LRB-

n

-RRB-

tibia-fibula

-LRB-

n

-RRB-

pelvis

pubic

rami

-LRB-

n

-RRB-

and

olecranon

patella

scapula

sternum

and

shaft

of

femur

-LRB-

n

-RRB-

Fig.

fracture

and

prevalence

of

vertebral

deformity

for

each

type

of

fracture

Other

includes

olecranon

patella

scapula

sternum

and

shaft

of

femur

The

presenting

fracture

was

the

first

non-vertebral

low

trauma

fracture

to

come

to

clinical

attention

in

of

patients

-LRB-

n

-RRB-

the

second

in

-LRB-

n

-RRB-

the

third

in

-LRB-

n

-RRB-

and

the

fourth

in

-LRB-

n

-RRB-

Information

regarding

risk

factors

for

fracture

including

prior

history

of

fracture

early

menopause

BMI

history

of

smoking

or

excess

alcohol

thyrotoxicosis

history

of

maternal

hip

fracture

family

history

of

osteoporosis

steroid

use

rheumatoid

disease

history

of

recurrent

falls

and

partial

gastrectomy

was

also

collected

Of

all

patients

had

one

fracture

risk

factor

had

two

risk

factors

had

three

risk

factors

and

had

more

than

three

risk

factors

Risk

fractures

for

fracture

were

assessed

after

patients

had

presented

to

the

FLS

with

a

non-vertebral

fracture

therefore

every

patient

had

a

prior

history

of

fracture

Vertebral

deformity

consistent

with

fracture

The

overall

prevalence

of

vertebral

deformity

established

by

MXA

was

The

prevalence

of

vertebral

deformity

by

type

of

presenting

fracture

is

shown

in

Fig.

Patients

with

a

hip

fracture

were

significantly

more

likely

to

have

a

vertebral

deformity

identified

on

MXA

than

were

patients

who

presented

with

a

non-hip

fracture

-LRB-

p

-RRB-

Overall

of

patients

with

vertebral

deformities

had

deformities

in

the

thoracic

spine

and

had

lumbar

spine

deformities

whilst

had

deformities

in

both

regions

Of

the

patients

with

vertebral

deformities

identified

by

MXA

had

deformities

of

more

than

one

vertebra

Multiple

vertebral

deformity

prevalence

varied

considerably

according

to

the

site

of

the

presenting

fracture

-LRB-

see

Fig.

-RRB-

Fig.

fracture

and

prevalence

of

multiple

-LRB-

two

or

more

-RRB-

vertebral

deformities

Of

the

patients

with

vertebral

deformities

-LRB-

-RRB-

had

unreadable

scans

for

grading

Of

the

remaining

patients

-LRB-

-RRB-

had

Grade

or

Grade

deformities

Thirty-two

-LRB-

-RRB-

of

the

patients

with

vertebral

deformity

of

Grade

and

Grade

had

multiple

vertebral

fractures

Of

the

sub-group

of

patients

with

vertebral

deformity

of

Grade

and

had

deformities

in

the

thoracic

spine

in

the

lumbar

spine

and

in

both

regions

There

was

no

significant

difference

between

the

vertebral

deformity

population

and

the

sub-population

of

patients

with

Grade

or

deformities

in

terms

of

the

proportion

of

patients

with

multiple

vertebral

deformities

or

in

the

site

of

deformity

Table

shows

the

prevalence

of

vertebral

deformity

and

multiple

vertebral

deformities

by

lumbar

spine

T-scores

Patients

whose

lumbar

spine

T-scores

were

consistent

within

the

osteoporosis

range

-LRB-

T-score

-RRB-

were

significantly

-LRB-

p

-RRB-

more

likely

to

have

vertebral

deformities

-LRB-

-RRB-

than

patients

whose

T-scores

were

in

the

osteopenic

or

normal

ranges

-LRB-

and

respectively

-RRB-

They

were

also

significantly

more

likely

to

have

multiple

vertebral

deformities

than

those

with

a

normal

T-score

-LRB-

versus

p

-RRB-

Table

of

lumbar

spine

T-score

on

prevalence

of

vertebral

deformityLumbar

spine

T-scoreTotalVertebral

deformityMultiple

vertebral

deformitiesFracture

Grade

or

n

an

bn

aOsteoporotic

-LRB-

T-score

-RRB-

Osteopenic

-LRB-

T-score

-RRB-

deformity

Chi-squared

p

Multiple

vertebral

deformities

Chi-squared

p

a

within

each

T-score

group

with

vertebral

deformityb

of

vertebral

deformity

group

with

multiple

deformities

within

each

T-score

group

Patients

with

lower

BMI

-LRB-

-RRB-

were

significantly

more

likely

to

have

vertebral

deformity

than

patients

with

the

highest

BMI

-LRB-

-RRB-

-LRB-

versus

p

-RRB-

-LRB-

Table

-RRB-

A

very

similar

picture

was

seen

when

BMI

was

divided

into

quartiles

of

patients

in

the

lowest

quartile

had

a

vertebral

deformity

compared

with

of

those

in

the

highest

quartile

-LRB-

p

-RRB-

The

mean

BMI

for

patients

with

and

without

vertebral

deformity

did

not

differ

significantly

-LRB-

vs

-RRB-

nor

did

the

mean

BMI

of

patients

with

multiple

vertebral

deformities

-LRB-

-RRB-

Table

of

BMI

on

prevalence

of

vertebral

deformity

n

deformityMultiple

vertebral

deformitiesFracture

Grade

or

n

n

n

Underweight

Normal

2411133.03947.035.12054.151.32441.421.625

2911835.12226.518.6616.227.31729.314.430

deformity

Chi-squared

p

Multiple

vertebral

deformities

Chi-squared

p

Patients

with

a

history

of

one

or

more

non-vertebral

fractures

prior

to

the

fracture

they

presented

with

were

significantly

more

likely

to

also

have

vertebral

deformity

-LRB-

ies

-RRB-

-LRB-

Table

-RRB-

-LRB-

versus

p

-RRB-

Table

of

prior

fracture

history

on

prevalence

of

vertebral

deformity

TotalVertebral

deformityMultiple

vertebral

deformitiesFracture

Grade

or

n

n

n

or

deformity

Chi-squared

p

Multiple

vertebral

deformities

Chi-squared

p

-LRB-

ns

-RRB-

Table

also

includes

analysis

of

the

sub-group

of

patients

with

vertebral

deformities

of

Grade

and

Grade

Patients

with

an

osteoporotic

T-score

were

significantly

more

likely

to

have

vertebral

deformities

than

those

with

a

normal

or

osteopenic

T-score

-LRB-

vs

-LSB-

osteopenic

-RSB-

and

-LSB-

normal

-RSB-

p

-RRB-

Patients

with

BMI

were

significantly

more

likely

to

have

vertebral

deformities

than

those

with

BMI

of

over

-LRB-

vs

p

-RRB-

Patients

with

a

history

of

one

or

more

prior

non-vertebral

fractures

were

more

likely

to

have

vertebral

deformity

although

this

did

not

reach

significance

-LRB-

vs

p

-RRB-

Older

patients

were

slightly

-LRB-

although

not

statistically

significantly

-RRB-

more

likely

to

have

a

vertebral

deformity

of

patients

aged

over

had

a

vertebral

deformity

compared

with

and

of

patients

aged

years

and

years

respectively

Gender

did

not

appear

to

affect

the

likelihood

of

identifying

a

vertebral

deformity

-LRB-

of

women

and

of

men

-RRB-

although

women

were

somewhat

more

likely

to

have

multiple

vertebral

deformities

-LRB-

vs

p

ns

-RRB-

Discussion

One-quarter

of

patients

presenting

to

our

Fracture

Liaison

Service

with

a

non-vertebral

fracture

had

a

previously

undiagnosed

vertebral

deformity

of

which

almost

one-half

-LRB-

-RRB-

were

multiple

vertebral

deformities

This

is

similar

to

the

prevalence

seen

in

previous

studies

using

radiographic

or

MXA

detection

in

women

with

and

without

non-vertebral

fracture

Sub-analysis

of

patients

by

grade

of

deformity

revealed

that

almost

three-quarters

of

the

patients

had

vertebral

deformities

of

Grade

and

Grade

These

are

more

likely

to

be

vertebral

fractures

rather

than

vertebral

deformities

of

non-osteoporosis

aetiology

which

might

be

more

likely

where

Grade

deformities

are

present

In

the

Study

of

Osteoporotic

Fractures

-LRB-

SOF

-RRB-

a

prospective

study

of

almost

women

the

prevalence

of

vertebral

fracture

was

-LRB-

of

which

were

multiple

-RRB-

in

women

aged

over

years

who

did

not

have

a

non-vertebral

fracture

-LSB-

-RSB-

In

another

study

of

women

aged

over

years

without

a

non-vertebral

fracture

the

prevalence

of

vertebral

fracture

was

-LSB-

-RSB-

In

a

smaller

study

of

women

with

Colles

fracture

vertebral

fractures

were

identified

by

MXA

in

of

patients

-LSB-

-RSB-

In

this

present

study

patients

presenting

with

hip

fracture

spine

T-score

low

BMI

-LRB-

either

in

the

lowest

quartile

or

-RRB-

or

with

more

than

one

prior

non-vertebral

fracture

were

all

significantly

more

likely

to

have

evidence

of

a

prevalent

vertebral

deformity

However

we

found

that

of

patients

with

normal

BMD

at

the

lumbar

spine

had

a

prevalent

vertebral

deformity

-LRB-

of

which

were

multiple

-RRB-

This

is

comparable

to

that

seen

in

an

earlier

study

conducted

in

women

aged

over

years

without

a

non-vertebral

fracture

and

with

normal

BMD

at

the

spine

which

revealed

a

prevalence

of

vertebral

fracture

of

-LSB-

-RSB-

In

the

UK

current

treatment

decisions

for

patients

with

low

trauma

fracture

are

based

on

fracture

status

and

BMD

as

determined

by

DXA

-LSB-

-RSB-

In

England

patients

aged

under

receive

treatment

if

they

have

a

fracture

and

osteoporosis

is

confirmed

by

DXA

scanning

-LSB-

-RSB-

In

Scotland

patients

with

vertebral

fracture

and

BMD

outside

the

osteoporotic

range

as

defined

by

the

WHO

classification

do

not

routinely

receive

treatment

unless

they

have

two

or

more

vertebral

fractures

irrespective

of

BMD

or

one

vertebral

fracture

and

an

osteopenic

BMD

score

-LSB-

-RSB-

Identification

of

those

patients

who

already

have

an

-LRB-

often

unrecognised

-RRB-

prior

vertebral

fracture

is

particularly

important

as

they

are

at

high

risk

of

further

fracture

by

virtue

of

their

prior

vertebral

fracture

Although

these

patients

might

not

fulfil

the

criteria

for

therapy

on

the

basis

of

their

-LRB-

non-vertebral

-RRB-

fracture

history

and

BMD

they

might

be

candidates

for

therapy

if

their

underlying

positive

vertebral

fracture

status

was

known

In

this

study

five

of

the

patients

with

normal

BMD

-LRB-

-RRB-

had

two

or

more

previously

undiagnosed

vertebral

deformities

plus

a

new

non-vertebral

fracture

and

of

the

patients

with

osteopenic

BMD

-LRB-

-RRB-

had

one

or

more

previously

undiagnosed

vertebral

deformities

plus

a

non-vertebral

fracture

These

patients

would

not

have

been

candidates

for

anti-resorptive

treatment

if

MXA

had

not

been

performed

and

their

vertebral

deformities

identified

Therefore

one

in

patients

with

a

normal

T-score

and

almost

one

in

five

patients

with

an

osteopenic

T-score

would

experience

a

change

in

management

by

virtue

of

their

underlying

vertebral

deformity

or

deformities

Overall

-LRB-

-RRB-

of

the

patients

in

this

study

would

be

eligible

for

treatment

by

virtue

of

their

newly

identified

vertebral

deformity.

which

means

that

patients

who

present

with

a

non-vertebral

fracture

would

need

to

undergo

vertebral

morphometry

in

order

to

identify

one

patient

who

ought

to

be

managed

differently

This

Number

Needed

to

Screen

gives

an

indication

of

the

number

of

patients

for

whom

treatment

recommendations

would

change

after

the

identification

of

vertebral

fracture

These

data

also

provide

the

first

attempt

to

quantify

the

overlap

between

patient

populations

with

non-vertebral

and

vertebral

fracture

Estimates

suggest

that

million

post-menopausal

women

in

the

UK

have

experienced

a

prior

non-vertebral

fracture

-LSB-

-RSB-

and

million

have

at

least

one

prior

vertebral

fracture

-LSB-

-RSB-

In

this

study

of

patients

presented

with

their

first

non-vertebral

fracture

and

vertebral

morphometry

subsequently

identified

vertebral

deformities

in

one

in

five

-LRB-

-RRB-

of

these

patients

Nearly

one-half

of

vertebral

deformities

were

multiple

-LRB-

-RRB-

It

is

highly

likely

that

these

vertebral

fractures

had

occurred

prior

to

the

first

non-vertebral

fracture

suggesting

that

a

substantial

number

of

patients

will

have

experienced

a

vertebral

fracture

prior

to

any

non-vertebral

fracture

event

Our

results

support

the

recommendation

to

perform

vertebral

morphometry

in

any

patient

who

is

referred

for

DXA

testing

after

experiencing

a

non-vertebral

fracture

Treatment

decisions

will

then

better

reflect

any

given

patient

's

future

absolute

fracture

risk

The

Number

Needed

to

Screen

if

MXA

is

used

in

this

way

would

be

seven

to

identify

one

patient

with

vertebral

deformity

and

to

identify

one

patient

with

two

or

more

vertebral

deformities

Routinely

performing

lateral

vertebral

morphometry

does

raise

a

number

of

issues

Firstly

there

is

an

increase

in

radiation

exposure

for

the

patient

A

standard

AP

spine

and

hip

DXA

scan

is

associated

with

a

radiation

exposure

-LSB-

entrance

surface

dose

-LRB-

ESD

-RRB-

-RSB-

of

around

mGy

and

results

in

an

effective

dose

of

less

than

μSv

-LRB-

microsieverts

-RRB-

The

ESD

associated

with

lateral

vertebral

morphometry

is

mGy

-LSB-

-RSB-

corresponding

to

an

estimated

effective

dose

of

about

μSv

These

are

relatively

small

doses

compared

to

the

UK

natural

background

radiation

level

of

approximately

mSv

The

small

carcinogenic

potential

associated

with

these

scans

has

to

be

balanced

against

both

the

prognostic

importance

-LRB-

in

terms

of

future

fracture

risk

-RRB-

that

is

associated

with

the

identification

of

unrecognised

vertebral

deformities

and

also

the

much

higher

radiation

exposure

risk

associated

with

plain

spine

radiology

-LRB-

effective

dose

mSv

-RRB-

Lateral

vertebral

morphometry

does

have

limitations

for

example

it

is

less

reliable

than

conventional

lateral

spine

X-rays

at

the

upper

thoracic

spine

and

in

Grade

deformities

A

recent

study

-LSB-

-RSB-

comparing

MXA

with

lateral

spine

X-rays

found

that

vertebral

morphometry

using

MXA

allowed

diagnosis

of

vertebral

fracture

in

the

lumbar

and

mid

thoracic

spine

where

vertebral

fractures

are

most

common

MXA

was

less

reliable

at

the

upper

thoracic

spine

which

is

also

an

established

limitation

of

X-rays

An

earlier

study

-LSB-

-RSB-

compared

identification

of

vertebral

fracture

on

MXA

using

the

Genant

semi-quantitative

method

using

MXA

with

conventional

X-rays

in

post-menopausal

women

Of

the

potentially

evaluable

vertebrae

were

adequately

visualised

on

MXA

to

allow

grading

Prevalent

vertebral

fracture

was

identified

in

vertebral

bodies

using

X-ray

of

these

-LRB-

-RRB-

were

correctly

identified

on

MXA

-LRB-

-RRB-

Grade

or

and

-LRB-

-RRB-

of

Grade

fractures

Of

the

non-fractured

vertebrae

were

correctly

classified

as

normal

by

MXA

Although

the

Genant

semi-quantative

method

is

not

validated

for

MXA

it

has

been

used

in

other

studies

-LSB-

-RSB-

and

was

used

in

this

study

as

a

pragmatic

option

To

carry

out

quantitative

analysis

on

each

scan

would

have

been

time-consuming

and

impractical

Given

that

the

sample

population

had

already

had

at

least

one

non-vertebral

fracture

-LRB-

had

more

than

one

prior

fracture

-RRB-

were

drawn

from

the

FLS

population

and

that

almost

three-quarters

had

Grade

or

vertebral

deformity

it

seems

reasonable

to

expect

the

vertebral

deformity

to

be

due

to

osteoporosis

rather

than

other

diagnoses

such

as

degenerative

change

or

Scheurmann

's

disease

The

sensitivity

of

detection

of

a

vertebral

fracture

is

lower

both

where

there

is

significant

spine

scoliosis

and

also

where

there

is

marked

disc-space

osteoarthritic

change

In

these

settings

proceeding

to

plain

spine

radiology

would

be

recommended

-LSB-

-RSB-

Plain

spine

X-rays

were

not

carried

out

in

this

study

Furthermore

in

keeping

with

our

local

protocol

bone

densitometry

was

not

carried

out

in

older

patients

-LRB-

over

the

age

of

-RRB-

after

hip

fracture

Both

of

these

features

may

mean

that

the

overall

prevalence

of

vertebral

deformity

in

this

population

has

been

underestimated

In

conclusion

undiagnosed

vertebral

deformity

is

highly

prevalent

in

patients

aged

over

years

who

present

with

a

new

non-vertebral

fracture

Given

that

prior

vertebral

fracture

significantly

increases

the

risk

of

further

fracture

and

because

newer

diagnostic

techniques

make

identification

of

vertebral

deformity

consistent

with

fracture

relatively

straightforward

with

minimal

radiation

exposure

we

recommend

that

all

patients

undergoing

DXA

should

also

undergo

vertebral

morphometry

to

identify

prior

vertebral

deformities

consistent

with

fractures

O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O

low trauma fracture
number needed to screen
osteoporosis
vertebral fracture
vertebral morphometry