Background Studying mechanical properties of canine trabecular bone is important for a better understanding of fracture mechanics or bone disorders and is also needed for numerical simulation of canine femora. No detailed data about elastic moduli and degrees of anisotropy of canine femoral trabecular bone has been published so far, hence the purpose of this study was to measure the elastic modulus of trabecular bone in canine femoral heads by ultrasound testing and to assess whether assuming isotropy of the cancellous bone in femoral heads in dogs is a valid simplification.
Background
Studying mechanical properties of canine trabecular bone is important for a better understanding of fracture mechanics or bone disorders and is also needed for numerical simulation of canine femora. No detailed data about elastic moduli and degrees of anisotropy of canine femoral trabecular bone has been published so far, hence the purpose of this study was to measure the elastic modulus of trabecular bone in canine femoral heads by ultrasound testing and to assess whether assuming isotropy of the cancellous bone in femoral heads in dogs is a valid simplification.Methods From 8 euthanized dogs, both femora were obtained and cubic specimens were cut from the centre of the femoral head which were oriented along the main pressure and tension trajectories. The specimens were tested using a 100 MHz ultrasound transducer in all three orthogonal directions. The directional elastic moduli of trabecular bone tissue and degrees of anisotropy were calculated.
Methods
From 8 euthanized dogs, both femora were obtained and cubic specimens were cut from the centre of the femoral head which were oriented along the main pressure and tension trajectories. The specimens were tested using a 100 MHz ultrasound transducer in all three orthogonal directions. The directional elastic moduli of trabecular bone tissue and degrees of anisotropy were calculated.Results The elastic modulus along principal bone trajectories was found to be 11.2 GPa ± 0.4, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. The mean density of the specimens was 1.40 ± 0.09 g/cm 3 . The degrees of anisotropy revealed a significant inverse relationship with specimen densities. No significant differences were found between the elastic moduli in x, y and z directions, suggesting an effective isotropy of trabecular bone tissue in canine femoral heads.
Results
The elastic modulus along principal bone trajectories was found to be 11.2 GPa ± 0.4, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. The mean density of the specimens was 1.40 ± 0.09 g/cm 3 . The degrees of anisotropy revealed a significant inverse relationship with specimen densities. No significant differences were found between the elastic moduli in x, y and z directions, suggesting an effective isotropy of trabecular bone tissue in canine femoral heads.Discussion This study presents detailed data about elastic moduli of trabecular bone tissue obtained from canine femoral heads. Limitations of the study are the relatively small number of animals investigated and the measurement of whole specimen densities instead of trabecular bone densities which might lead to an underestimation of Young's moduli. Publications on elastic moduli of trabecular bone tissue present results that are similar to our data.
Discussion
This study presents detailed data about elastic moduli of trabecular bone tissue obtained from canine femoral heads. Limitations of the study are the relatively small number of animals investigated and the measurement of whole specimen densities instead of trabecular bone densities which might lead to an underestimation of Young's moduli. Publications on elastic moduli of trabecular bone tissue present results that are similar to our data.Conclusion This study provides data about directional elastic moduli and degrees of anisotropy of canine femoral head trabecular bone and might be useful for biomechanical modeling of proximal canine femora.
Conclusion
This study provides data about directional elastic moduli and degrees of anisotropy of canine femoral head trabecular bone and might be useful for biomechanical modeling of proximal canine femora.

Background The mechanical properties of canine trabecular bone in the femoral head are important for a better understanding of normal biomechanics of the bone and are needed for assessing changes occurring under pathological conditions like osteoarthritis of the hip, osteonecrosis or Legg-Calvé-Perthes disease of the femoral head. Particularly the elastic moduli are important for finite element modeling of the proximal femur. Several methods have been used in the literature for identifying the elastic modulus of bone, such as mechanical testing [ 1 - 4 ], combinations of micro-computed tomography and finite element modeling [ 4 - 11 ], and ultrasonography[ 12 ] including acoustic microscopy [ 13 ]. Some studies have investigated canine bone [ 13 - 19 ], but to our knowledge no publication has presented any details about directional elastic moduli of canine femoral heads including degrees of anisotropy. Neither is it clear whether assuming isotropy on the tissue level is a justified simplification for trabecular bone in canine femoral heads as Kabel et al.[ 9 ] reported in a study of whale bone specimens. Therefore, in this study we determined Young's moduli of trabecular bone obtained from healthy canine femoral heads by ultrasonography. We then calculated degrees of anisotropy and used statistical testing in order to estimate whether assuming isotropy of the trabecular tissue might be a valid simplification.
Background
The mechanical properties of canine trabecular bone in the femoral head are important for a better understanding of normal biomechanics of the bone and are needed for assessing changes occurring under pathological conditions like osteoarthritis of the hip, osteonecrosis or Legg-Calvé-Perthes disease of the femoral head. Particularly the elastic moduli are important for finite element modeling of the proximal femur. Several methods have been used in the literature for identifying the elastic modulus of bone, such as mechanical testing [ 1 - 4 ], combinations of micro-computed tomography and finite element modeling [ 4 - 11 ], and ultrasonography[ 12 ] including acoustic microscopy [ 13 ]. Some studies have investigated canine bone [ 13 - 19 ], but to our knowledge no publication has presented any details about directional elastic moduli of canine femoral heads including degrees of anisotropy. Neither is it clear whether assuming isotropy on the tissue level is a justified simplification for trabecular bone in canine femoral heads as Kabel et al.[ 9 ] reported in a study of whale bone specimens. Therefore, in this study we determined Young's moduli of trabecular bone obtained from healthy canine femoral heads by ultrasonography. We then calculated degrees of anisotropy and used statistical testing in order to estimate whether assuming isotropy of the trabecular tissue might be a valid simplification.Methods Eight dogs (weight 30–63 kg) were selected that had been euthanized for several medical reasons. From each dog both femora were obtained and were examined by a veterinarian for signs of metastatic malignant disease, Legg-Calvé-Perthes disease, osteoarthritis of the hip or bone necrosis. An x-ray of the whole femur was obtained with the femoral head and the intertrochanteric region placed directly on the film, and the main pressure and tensile trajectories were marked on the image. The bones were kept moist, wrapped in plastic bags and stored at -21°C. Each bone was placed on the x-ray image and the direction of tension and pressure trajectories were marked on the bone according to the x-ray template. An orthogonal coordinate system was defined (Fig.  2 ). The positive x-axis was oriented along the main pressure trajectories and the y-axis was aligned with the main tension trajectories. One cubic specimen of 10 × 10 × 10 mm was cut (Fig.  1 ) from each frozen femoral head using a precision bone saw (Exakt Makro 310 CP, EXAKT Apparatebau, Norderstedt, Germany). The edges of the cubes were cut parallel to the x, y and z axes of the coordinate system. The cubic specimens were weighed using a laboratory scale (Acculab ALC-110.4, Acculab Europe, Göttingen, Germany). Figure 2 Schematic representation of femoral head and neck. The main tensile and compressive trajectories and the orientation of the cubic specimen and coordinate system are shown. Figure 1 Cubic specimen cut from one canine femoral head. For sonographic testing, a specially designed device with a custom-made ultrasound transducer (Institute of Materials Science, University of Hannover) was used. An ultrasound frequency of over 2 MHz was chosen for measuring the material properties of trabecular bone tissue[ 20 ]. The ultrasound frequency was adjusted so that a clear signal could be detected by the ultrasound receiver. A frequency of 100 MHz was chosen because ultrasound signals remained undetectable when using lower frequencies even with maximum power. The cubic specimens were placed in a container after thawing and immersed in standard Ringer's solution at room temperature. An ultrasound receiver was placed at the surface of the cube opposite the transducer which was also directly touching the specimen surface, and the runtime through the bone material of each bone cube (n = 16) was recorded ten times in all three orthogonal directions. The edge lengths of each cube were measured using the digital image analysis system IMAGE C ®  (IMTRONIC GmbH, Berlin, Germany), and the specimen volumes were calculated. Specimen densities were determined by equation (1): where ρ is specimen density, m is specimen mass and V is the specimen volume The ultrasound wave runtimes were processed by excluding the minimum and maximum results of the ten subsequent measurements, and the average of the remaining results was calculated. The transmission velocity was calculated by equation (2) and the elastic modulus was determined using equation (3). where c long  is the transmission velocity, s denotes the edge length of the specimen and equals the distance between ultrasound transmitter and receiver which are placed in direct contact with the opposing specimen surfaces, t 1  and t 0  the time at reception and sending of the ultrasound wave, respectively. where E x,y,z  is Young's modulus along x, y and z axes and ρ is the specimen density calculated from equation (1). C long  is the ultrasound velocity calculated from specimen edge length and transmission time (see equation (2)) Mean density and mean Young's moduli of the specimens and standard deviations were determined using the statistical software package SPSS 12.0 (SPSS, Chicago, USA). Strong correlations between density and elastic moduli could be expected from equation (3), nonetheless Pearson correlations were calculated for confirmation. The directional elastic moduli were checked for significant differences using one-way ANOVA (analysis of variance). The degrees of anisotropy [ 4 , 21 ](E x /E z;  E x /E y ; E z /E y ) were calculated and subsequently checked for a significant relationship with specimen density using Pearson correlations.
Methods
Eight dogs (weight 30–63 kg) were selected that had been euthanized for several medical reasons. From each dog both femora were obtained and were examined by a veterinarian for signs of metastatic malignant disease, Legg-Calvé-Perthes disease, osteoarthritis of the hip or bone necrosis. An x-ray of the whole femur was obtained with the femoral head and the intertrochanteric region placed directly on the film, and the main pressure and tensile trajectories were marked on the image. The bones were kept moist, wrapped in plastic bags and stored at -21°C. Each bone was placed on the x-ray image and the direction of tension and pressure trajectories were marked on the bone according to the x-ray template. An orthogonal coordinate system was defined (Fig.  2 ). The positive x-axis was oriented along the main pressure trajectories and the y-axis was aligned with the main tension trajectories. One cubic specimen of 10 × 10 × 10 mm was cut (Fig.  1 ) from each frozen femoral head using a precision bone saw (Exakt Makro 310 CP, EXAKT Apparatebau, Norderstedt, Germany). The edges of the cubes were cut parallel to the x, y and z axes of the coordinate system. The cubic specimens were weighed using a laboratory scale (Acculab ALC-110.4, Acculab Europe, Göttingen, Germany).For sonographic testing, a specially designed device with a custom-made ultrasound transducer (Institute of Materials Science, University of Hannover) was used. An ultrasound frequency of over 2 MHz was chosen for measuring the material properties of trabecular bone tissue[ 20 ]. The ultrasound frequency was adjusted so that a clear signal could be detected by the ultrasound receiver. A frequency of 100 MHz was chosen because ultrasound signals remained undetectable when using lower frequencies even with maximum power. The cubic specimens were placed in a container after thawing and immersed in standard Ringer's solution at room temperature. An ultrasound receiver was placed at the surface of the cube opposite the transducer which was also directly touching the specimen surface, and the runtime through the bone material of each bone cube (n = 16) was recorded ten times in all three orthogonal directions. The edge lengths of each cube were measured using the digital image analysis system IMAGE C ®  (IMTRONIC GmbH, Berlin, Germany), and the specimen volumes were calculated. Specimen densities were determined by equation (1):where ρ is specimen density, m is specimen mass and V is the specimen volumeThe ultrasound wave runtimes were processed by excluding the minimum and maximum results of the ten subsequent measurements, and the average of the remaining results was calculated. The transmission velocity was calculated by equation (2) and the elastic modulus was determined using equation (3).where c long  is the transmission velocity, s denotes the edge length of the specimen and equals the distance between ultrasound transmitter and receiver which are placed in direct contact with the opposing specimen surfaces, t 1  and t 0  the time at reception and sending of the ultrasound wave, respectively.where E x,y,z  is Young's modulus along x, y and z axes and ρ is the specimen density calculated from equation (1). C long  is the ultrasound velocity calculated from specimen edge length and transmission time (see equation (2))Mean density and mean Young's moduli of the specimens and standard deviations were determined using the statistical software package SPSS 12.0 (SPSS, Chicago, USA). Strong correlations between density and elastic moduli could be expected from equation (3), nonetheless Pearson correlations were calculated for confirmation. The directional elastic moduli were checked for significant differences using one-way ANOVA (analysis of variance). The degrees of anisotropy [ 4 , 21 ](E x /E z;  E x /E y ; E z /E y ) were calculated and subsequently checked for a significant relationship with specimen density using Pearson correlations.Results The edge lengths of the bone specimens varied by ± 1 % (± 0.1 mm). Elastic moduli in the bone specimens ranging from 6.3 to 14.3 GPa were found. The elastic moduli in X, Y and Z direction were 11.2 ± 0.4 GPa, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. Minimum, maximum, mean values and standard deviations of bone sample density and directional Young's moduli are listed in Table  1 . Pearson correlations between density and directional Young's moduli (E x , E y  and E z ) were significant as could be expected from equation (3) (p < 0.005). The degrees of anisotropy ranged from 0.82 to 1.59 and were significantly correlated with specimen density (Table  2 , Figure  3 ). Figure 3 Degrees of anisotropy in dependence of specimen density. Correlation coefficients are listed in the symbol legend. Table 1 Minimum, maximum and mean values for sample densities and directional Young's moduli Moduli are arranged according to testing direction (along x, y and z axes, see fig. 2). Minimum Maximum Mean Standard deviation Density [g/cm 3 ] 1.19 1.51 1.40 0.09 E X  [MPa] 10600 11760 11217 376 E Y  [MPa] 6283 14285 10459 2071 E Z  [MPa] 6832 13097 10506 1839 Table 2 Degrees of anisotropy The minimum and maximum degrees of anisotropy (range), the mean values, standard deviations (SD) and the correlation coefficients (r) indicating correlation of the respective degree of anisotropy with specimen density ρ. p designates the significance probability (p-value). Range Mean SD r p E x /E z 0.89–1.59 1.10 0.18 -0,93 0.000 E z /E y 0.92–1.11 1.01 0.05 -0,58 0.009 E x /E y 0.82–1.73 1.11 0.22 -0,96 0.000
Results
The edge lengths of the bone specimens varied by ± 1 % (± 0.1 mm). Elastic moduli in the bone specimens ranging from 6.3 to 14.3 GPa were found. The elastic moduli in X, Y and Z direction were 11.2 ± 0.4 GPa, 10.5 ± 2.1 GPa and 10.5 ± 1.8 GPa, respectively. Minimum, maximum, mean values and standard deviations of bone sample density and directional Young's moduli are listed in Table  1 . Pearson correlations between density and directional Young's moduli (E x , E y  and E z ) were significant as could be expected from equation (3) (p < 0.005). The degrees of anisotropy ranged from 0.82 to 1.59 and were significantly correlated with specimen density (Table  2 , Figure  3 ).Minimum, maximum and mean values for sample densities and directional Young's moduli Moduli are arranged according to testing direction (along x, y and z axes, see fig. 2).MinimumMaximumMeanStandard deviationDensity [g/cm 3 ]E X  [MPa]E Y  [MPa]E Z  [MPa]Degrees of anisotropy The minimum and maximum degrees of anisotropy (range), the mean values, standard deviations (SD) and the correlation coefficients (r) indicating correlation of the respective degree of anisotropy with specimen density ρ. p designates the significance probability (p-value).RangeMeanSDrpE x /E zE z /E yE x /E yDiscussion This study presents detailed data about mechanical properties of canine femoral trabecular bone tissue and degrees of anisotropy. Despite the strengths of our work, some limitations have to be noted. The correct calculation of the sample volume depends on exactly cubic specimens, but deviations might occur due to errors in the sawing technique. We found a maximum variation of edge length of ± 0.1 mm (1%) in our specimens so this error appears to be negligible. The apparent densities of the specimens were calculated by weighing whole samples and measuring sample volumes instead of calculating the densities of ashed samples[ 2 , 19 ] or cleaning the bone marrow out of the specimens using water jets prior to measurement. According to Rho [ 20 ] ultrasonic waves at frequencies of >2 MHz travel along the trabecular material and allow calculation of the elasticity of the trabecular bone material rather than the elasticity of whole specimens which is investigated by conventional compression testing. Considering this statement, in our study using a 100 MHz ultrasound transducer the application of apparent densities rather than the densities of the trabecular bone material might lead to an conspicuous underestimation of the elastic modulus (equation (3)) because it is expected that the density of the trabecular bone tissue is higher than the density of whole specimens. However, Kang et al. [ 22 ] measured densities of cylindrical trabecular bone specimens from canine femoral heads and reported a mean density of 1.17 ± 17 g/cm 3  for whole bone cylinders after cleaning and 0.65 ± 0.09 g/cm 3  for ashed samples which is much lower than our results. The lower densities might be caused by the specimen volume that was used by the authors: they geometrically measured the volume of the cylindrical bone specimens so that a considerable intertrabecular volume is included and bone tissue densities are underestimated according to equation (1). Hence the results of Kang's publication can not be used for comparison or correction of our density data. It is noticeable that in our study we found a small relative standard deviation (SD/mean) in apparent densities and Young's modulus in x direction (6.6 % and 3.3 %) although we used femora from a heterogeneous selection of different breeds. However, the relative SD in y and z directions was computed to be 19.5 % and 17.5 %, respectively. It is not clear whether the broader range of elastic moduli in these directions reflects real differences or whether it might be caused by a less accurate positioning of the saw when cutting the specimens from the femoral heads. The significant relationship between the specimen density ρ and directional elastic moduli found in our study was to be expected because Young's modulus was calculated from ρ. This significant relationship was also described in a study using compression testing of bone cubes from human donors (0.74 <= r <= 0.84; p < 0.001) [ 2 ]. No significant differences were found between directional elastic moduli (p = 0.34). This result could support the concept of an "effective" isotropic elastic tissue modulus as described by Kabel et al.[ 9 ]. Several works have been published concerning elastic moduli of canine or human bone. Studies that investigated the apparent elastic modulus of human bone specimens using mechanical testing and finite element models found much lower elastic moduli[ 4 , 21 ] than we did in our work. Kang et al. [ 22 ] reported elastic moduli of trabecular specimens from canine femoral heads of 428 ± 237 MPa which is also much lower than what we observed in our study; their results were obtained by conventional compression testing. Several other studies have been carried out investigating elastic moduli of canine trabecular bone specimens[ 14 , 16 , 19 ]. Those results are not comparable with our data because the authors measured elastic moduli of whole specimens rather than Young's moduli on the tissue level. Additionally, Odgaard et al. reported that conventional compression testing underestimates Young's modulus by about 20%[ 23 ]. Keaveny et al. [ 24 ] found a percentage difference in modulus when using platens compression testing of up to 86%. They recommend using the endcap technique for obtaining more accurate data, which however restricts testing to one direction. Jacobs et al. [ 25 ] investigated porous samples made from bone cement. Using finite element modeling and mechanical testing, they found that the mean error when using parallel platen compression testing was 8 or 15% depending on FE mesh size and was reduced to 2 or 0.5% with the endcap technique. Only one study investigating elastic moduli of canine trabecular bone tissue is available to our knowledge: Jorgensen and Kundu [ 13 ] used a 1 GHz acoustic microscope for examining a trabecular strut obtained from a canine distal femur. They computed a mean Young's modulus of 19.9 ± 2.5 GPa which is higher than our results; this could be caused by a different trabecular structure and higher bone volume fraction in the distal femur. The authors state that anisotropy was clearly detected at micrometer level, but no further quantification is given. More studies are available reporting mechanical properties of human trabecular bone. Rho [ 20 ] found an elastic modulus of 14.9 ± 1.7 GPa in trabecular bone specimens from human tibiae using a 2.25 MHz transducer. This modulus is higher than in our data but within the same order of magnitude. Ashman and Rho [ 26 ] measured elastic moduli from three human trabecular bone specimens using an ultrasonic technique and found a mean elastic modulus of 13.0 GPa which is about 30% higher than in our study of canine bone. This observation is supported by Kuhn's [ 19 ] assertion that elastic moduli are higher in human trabecular bone than in canine bone. Our results are further supported by Zysset et al. [ 27 ] who used a nanoindentation technique and found average elastic moduli in trabecular lamellae of human femoral necks of 11.4 ± 5.6 GPa.
Discussion
This study presents detailed data about mechanical properties of canine femoral trabecular bone tissue and degrees of anisotropy. Despite the strengths of our work, some limitations have to be noted.The correct calculation of the sample volume depends on exactly cubic specimens, but deviations might occur due to errors in the sawing technique. We found a maximum variation of edge length of ± 0.1 mm (1%) in our specimens so this error appears to be negligible.The apparent densities of the specimens were calculated by weighing whole samples and measuring sample volumes instead of calculating the densities of ashed samples[ 2 , 19 ] or cleaning the bone marrow out of the specimens using water jets prior to measurement. According to Rho [ 20 ] ultrasonic waves at frequencies of >2 MHz travel along the trabecular material and allow calculation of the elasticity of the trabecular bone material rather than the elasticity of whole specimens which is investigated by conventional compression testing. Considering this statement, in our study using a 100 MHz ultrasound transducer the application of apparent densities rather than the densities of the trabecular bone material might lead to an conspicuous underestimation of the elastic modulus (equation (3)) because it is expected that the density of the trabecular bone tissue is higher than the density of whole specimens. However, Kang et al. [ 22 ] measured densities of cylindrical trabecular bone specimens from canine femoral heads and reported a mean density of 1.17 ± 17 g/cm 3  for whole bone cylinders after cleaning and 0.65 ± 0.09 g/cm 3  for ashed samples which is much lower than our results. The lower densities might be caused by the specimen volume that was used by the authors: they geometrically measured the volume of the cylindrical bone specimens so that a considerable intertrabecular volume is included and bone tissue densities are underestimated according to equation (1). Hence the results of Kang's publication can not be used for comparison or correction of our density data.It is noticeable that in our study we found a small relative standard deviation (SD/mean) in apparent densities and Young's modulus in x direction (6.6 % and 3.3 %) although we used femora from a heterogeneous selection of different breeds. However, the relative SD in y and z directions was computed to be 19.5 % and 17.5 %, respectively. It is not clear whether the broader range of elastic moduli in these directions reflects real differences or whether it might be caused by a less accurate positioning of the saw when cutting the specimens from the femoral heads.The significant relationship between the specimen density ρ and directional elastic moduli found in our study was to be expected because Young's modulus was calculated from ρ. This significant relationship was also described in a study using compression testing of bone cubes from human donors (0.74 <= r <= 0.84; p < 0.001) [ 2 ].No significant differences were found between directional elastic moduli (p = 0.34). This result could support the concept of an "effective" isotropic elastic tissue modulus as described by Kabel et al.[ 9 ].Several works have been published concerning elastic moduli of canine or human bone. Studies that investigated the apparent elastic modulus of human bone specimens using mechanical testing and finite element models found much lower elastic moduli[ 4 , 21 ] than we did in our work. Kang et al. [ 22 ] reported elastic moduli of trabecular specimens from canine femoral heads of 428 ± 237 MPa which is also much lower than what we observed in our study; their results were obtained by conventional compression testing. Several other studies have been carried out investigating elastic moduli of canine trabecular bone specimens[ 14 , 16 , 19 ]. Those results are not comparable with our data because the authors measured elastic moduli of whole specimens rather than Young's moduli on the tissue level. Additionally, Odgaard et al. reported that conventional compression testing underestimates Young's modulus by about 20%[ 23 ]. Keaveny et al. [ 24 ] found a percentage difference in modulus when using platens compression testing of up to 86%. They recommend using the endcap technique for obtaining more accurate data, which however restricts testing to one direction. Jacobs et al. [ 25 ] investigated porous samples made from bone cement. Using finite element modeling and mechanical testing, they found that the mean error when using parallel platen compression testing was 8 or 15% depending on FE mesh size and was reduced to 2 or 0.5% with the endcap technique.Only one study investigating elastic moduli of canine trabecular bone tissue is available to our knowledge: Jorgensen and Kundu [ 13 ] used a 1 GHz acoustic microscope for examining a trabecular strut obtained from a canine distal femur. They computed a mean Young's modulus of 19.9 ± 2.5 GPa which is higher than our results; this could be caused by a different trabecular structure and higher bone volume fraction in the distal femur. The authors state that anisotropy was clearly detected at micrometer level, but no further quantification is given.More studies are available reporting mechanical properties of human trabecular bone. Rho [ 20 ] found an elastic modulus of 14.9 ± 1.7 GPa in trabecular bone specimens from human tibiae using a 2.25 MHz transducer. This modulus is higher than in our data but within the same order of magnitude. Ashman and Rho [ 26 ] measured elastic moduli from three human trabecular bone specimens using an ultrasonic technique and found a mean elastic modulus of 13.0 GPa which is about 30% higher than in our study of canine bone. This observation is supported by Kuhn's [ 19 ] assertion that elastic moduli are higher in human trabecular bone than in canine bone. Our results are further supported by Zysset et al. [ 27 ] who used a nanoindentation technique and found average elastic moduli in trabecular lamellae of human femoral necks of 11.4 ± 5.6 GPa.Conclusion Our study provides detailed data about elastic moduli and degrees of anisotropy of canine femoral bone tissue. No significant differences between directional Young's moduli were found indicating that the concept of an effective isotropy of trabecular bone in canine femoral heads might be a justified simplification. The degrees of anisotropy were highly correlated with specimen densities. The results of elastic moduli are comparable to similar studies of canine and human trabecular bone tissue [ 13 , 20 , 26 , 27 ].
Conclusion
Our study provides detailed data about elastic moduli and degrees of anisotropy of canine femoral bone tissue. No significant differences between directional Young's moduli were found indicating that the concept of an effective isotropy of trabecular bone in canine femoral heads might be a justified simplification. The degrees of anisotropy were highly correlated with specimen densities. The results of elastic moduli are comparable to similar studies of canine and human trabecular bone tissue [ 13 , 20 , 26 , 27 ].Authors' contributions TP carried out the statistical analysis of the results, took part in the detailed design of the study and prepared the manuscript. AB designed the study, prepared the bone specimens, conducted the ultrasound measurements and calculated the elastic moduli. UV and AML obtained the canine femora, examined the bone for exclusion criteria and prepared the bone specimens. BAB, IN and HW designed the study concept from a technical and medical perspective, respectively, and corrected the manuscript.
Authors' contributions
TP carried out the statistical analysis of the results, took part in the detailed design of the study and prepared the manuscript. AB designed the study, prepared the bone specimens, conducted the ultrasound measurements and calculated the elastic moduli. UV and AML obtained the canine femora, examined the bone for exclusion criteria and prepared the bone specimens. BAB, IN and HW designed the study concept from a technical and medical perspective, respectively, and corrected the manuscript.


Background Nonsteroidal anti-inflammatory drugs (NSAID) use may protect against Alzheimer's disease (AD) risk. We sought examine the association between NSAID use and risk of AD, and potential effect modification by APOE-ε4 carrier status and ethnicity.
Background
Nonsteroidal anti-inflammatory drugs (NSAID) use may protect against Alzheimer's disease (AD) risk. We sought examine the association between NSAID use and risk of AD, and potential effect modification by APOE-ε4 carrier status and ethnicity.Methods The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD. Subjects comprised 691 AD patients (probands) and 973 family members enrolled at 15 research centers between 1996 and 2002. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. Ethnicity was similarly examined as an effect modifier.
Methods
The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD. Subjects comprised 691 AD patients (probands) and 973 family members enrolled at 15 research centers between 1996 and 2002. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. Ethnicity was similarly examined as an effect modifier.Results NSAID use was less frequent in cases compared to controls in the overall sample (adjusted OR = 0.64; 95% CI = 0.38–1.05). The benefit of NSAID use appeared more pronounced among APOE-ε4 carriers (adjusted OR = 0.49; 95% CI = 0.24–0.98) compared to non-carriers, although this association was not statistically significant. The pattern of association was similar in Caucasian and African Americans.
Results
NSAID use was less frequent in cases compared to controls in the overall sample (adjusted OR = 0.64; 95% CI = 0.38–1.05). The benefit of NSAID use appeared more pronounced among APOE-ε4 carriers (adjusted OR = 0.49; 95% CI = 0.24–0.98) compared to non-carriers, although this association was not statistically significant. The pattern of association was similar in Caucasian and African Americans.Conclusions NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.
Conclusions
NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.

Background Several cross-sectional [ 1 , 2 ], case-control [ 3 - 6 ], and prospective studies [ 7 - 9 ] have reported an inverse association between nonsteroidal anti-inflammatory drug (NSAID) use and the risk of Alzheimer's disease (AD), whereas others [ 10 - 12 ] have not. In this report, we present results of analyses of data from the Multi-Institutional Research in Alzheimer's Genetic Epidemiology (MIRAGE) Study in which we examined potential effect modification by APOE-ε4 carrier status and ethnicity on this association.
Background
Several cross-sectional [ 1 , 2 ], case-control [ 3 - 6 ], and prospective studies [ 7 - 9 ] have reported an inverse association between nonsteroidal anti-inflammatory drug (NSAID) use and the risk of Alzheimer's disease (AD), whereas others [ 10 - 12 ] have not. In this report, we present results of analyses of data from the Multi-Institutional Research in Alzheimer's Genetic Epidemiology (MIRAGE) Study in which we examined potential effect modification by APOE-ε4 carrier status and ethnicity on this association.Methods Subjects and data collection The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD sponsored by the National Institute on Aging since 1991. The details of MIRAGE Study data collection procedures, protocols for obtaining family histories, and reports of validity studies of the MIRAGE questionnaires have been published elsewhere. [ 13 - 15 ] Briefly, families were recruited through probands meeting NINCDS-ADRDA criteria [ 16 ] for probable or definite AD who were ascertained through research registries and memory clinics. After obtaining informed consent from non-demented family members, and a combination of consent or assent – along with informed consent by proxy – on living demented subjects, questions eliciting demographic data and information about presumptive risk factors for AD were obtained using standardized MIRAGE questionnaires. Questions pertaining to NSAID use were added to the questionnaire in 1996, and the data presented in this report were collected from May, 1996 through May, 2002. Questions about the proband were answered by a surrogate source within the family, typically the spouse or adult offspring. The same information was sought on non-demented first-degree family members of these probands over 50 years of age, usually a sibling or spouse (less commonly parents or children). 1020 family members in this analysis claimed to be cognitively normal, or were reported by family informants to be dementia-free. Of these, 982 were evaluated using the modified Telephone Interview of Cognitive Status (mTICS) [ 17 , 18 ], and normal cognitive status was confirmed in 973 (99.1%). Information on both patients and first-degree family members was supplemented where available by multiple informants, and medical and nursing home records. To elicit information on prior NSAID use, the following question was asked: "Have you ever taken a  nonsteroidal  anti-inflammatory medication (e.g. Advil, Motrin, etc.)  on a daily basis for more than 6 months ?" No distinction was made between aspirin and other classes of NSAIDs. For proxy reporting about a relative with AD, the question substituted "your relative" for "you". For any affirmative answer, a follow-up question asked for the dates at which the medications were first used and the names of all NSAIDs that had been used. A discrete "index date" was established within each family corresponding to the earliest date that the family or medical records reported AD symptoms to have begun in the proband. Subjects from each family (whether AD cases or non-demented family members) were considered to have been exposed to NSAIDs only if the starting date for NSAID use preceded this index date by at least one year. Age represented the age of cases and of non-demented relatives at the index date, and was treated as a continuous variable. As shown in Figure  1 , there were 756 probands and 1020 relatives over the age of 50 with APOE genotype who were queried about prior NSAID use. After exclusions for those subjects who had missing or unsure responses for the name of their medication, did not include a medication start date, or had missing data for the variables age, sex, education or ethnicity, there remained for analysis 682 probands and 982 relatives. Of the 982 relatives, nine were reported to be demented with the onset of their dementia prior to the index date for that family, and their diagnoses were verified by review of medical records as having probable or definite AD by research criteria, so these were classified with the probands as having AD. Figure 1 MIRAGE subjects ≥ 50 years (adjusted for family index date) with APOE genotype who completed the personal history questionnaire. Statistical analysis Analyses were performed using SAS version 8.2. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. Crude odds ratios were computed in the first instance, followed by adjusted estimates using generalized estimating equation (GEE) models [ 19 ] to account for the possibility that variables of interest (e.g., medication use, APOE status) could be correlated among individuals within families. Adjustments were made for the following covariates: age, sex, ethnicity (categorized as White, African-American, or other), education (less than versus equal to or greater than high school level), and APOE-ε4 carrier status (one or two ε4 alleles vs. none). We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. In addition, we formally evaluated these associations by adding an interaction term (ε4 * NSAID use) to the GEE model. Ethnicity was similarly examined as an effect modifier.
Methods
Subjects and data collection The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD sponsored by the National Institute on Aging since 1991. The details of MIRAGE Study data collection procedures, protocols for obtaining family histories, and reports of validity studies of the MIRAGE questionnaires have been published elsewhere. [ 13 - 15 ] Briefly, families were recruited through probands meeting NINCDS-ADRDA criteria [ 16 ] for probable or definite AD who were ascertained through research registries and memory clinics. After obtaining informed consent from non-demented family members, and a combination of consent or assent – along with informed consent by proxy – on living demented subjects, questions eliciting demographic data and information about presumptive risk factors for AD were obtained using standardized MIRAGE questionnaires. Questions pertaining to NSAID use were added to the questionnaire in 1996, and the data presented in this report were collected from May, 1996 through May, 2002. Questions about the proband were answered by a surrogate source within the family, typically the spouse or adult offspring. The same information was sought on non-demented first-degree family members of these probands over 50 years of age, usually a sibling or spouse (less commonly parents or children). 1020 family members in this analysis claimed to be cognitively normal, or were reported by family informants to be dementia-free. Of these, 982 were evaluated using the modified Telephone Interview of Cognitive Status (mTICS) [ 17 , 18 ], and normal cognitive status was confirmed in 973 (99.1%). Information on both patients and first-degree family members was supplemented where available by multiple informants, and medical and nursing home records. To elicit information on prior NSAID use, the following question was asked: "Have you ever taken a  nonsteroidal  anti-inflammatory medication (e.g. Advil, Motrin, etc.)  on a daily basis for more than 6 months ?" No distinction was made between aspirin and other classes of NSAIDs. For proxy reporting about a relative with AD, the question substituted "your relative" for "you". For any affirmative answer, a follow-up question asked for the dates at which the medications were first used and the names of all NSAIDs that had been used. A discrete "index date" was established within each family corresponding to the earliest date that the family or medical records reported AD symptoms to have begun in the proband. Subjects from each family (whether AD cases or non-demented family members) were considered to have been exposed to NSAIDs only if the starting date for NSAID use preceded this index date by at least one year. Age represented the age of cases and of non-demented relatives at the index date, and was treated as a continuous variable. As shown in Figure  1 , there were 756 probands and 1020 relatives over the age of 50 with APOE genotype who were queried about prior NSAID use. After exclusions for those subjects who had missing or unsure responses for the name of their medication, did not include a medication start date, or had missing data for the variables age, sex, education or ethnicity, there remained for analysis 682 probands and 982 relatives. Of the 982 relatives, nine were reported to be demented with the onset of their dementia prior to the index date for that family, and their diagnoses were verified by review of medical records as having probable or definite AD by research criteria, so these were classified with the probands as having AD. Figure 1 MIRAGE subjects ≥ 50 years (adjusted for family index date) with APOE genotype who completed the personal history questionnaire.
Subjects and data collection
The MIRAGE Study is a multi-center family study of genetic and environmental risk factors for AD sponsored by the National Institute on Aging since 1991. The details of MIRAGE Study data collection procedures, protocols for obtaining family histories, and reports of validity studies of the MIRAGE questionnaires have been published elsewhere. [ 13 - 15 ] Briefly, families were recruited through probands meeting NINCDS-ADRDA criteria [ 16 ] for probable or definite AD who were ascertained through research registries and memory clinics. After obtaining informed consent from non-demented family members, and a combination of consent or assent – along with informed consent by proxy – on living demented subjects, questions eliciting demographic data and information about presumptive risk factors for AD were obtained using standardized MIRAGE questionnaires.Questions pertaining to NSAID use were added to the questionnaire in 1996, and the data presented in this report were collected from May, 1996 through May, 2002. Questions about the proband were answered by a surrogate source within the family, typically the spouse or adult offspring. The same information was sought on non-demented first-degree family members of these probands over 50 years of age, usually a sibling or spouse (less commonly parents or children).1020 family members in this analysis claimed to be cognitively normal, or were reported by family informants to be dementia-free. Of these, 982 were evaluated using the modified Telephone Interview of Cognitive Status (mTICS) [ 17 , 18 ], and normal cognitive status was confirmed in 973 (99.1%). Information on both patients and first-degree family members was supplemented where available by multiple informants, and medical and nursing home records.To elicit information on prior NSAID use, the following question was asked: "Have you ever taken a  nonsteroidal  anti-inflammatory medication (e.g. Advil, Motrin, etc.)  on a daily basis for more than 6 months ?" No distinction was made between aspirin and other classes of NSAIDs. For proxy reporting about a relative with AD, the question substituted "your relative" for "you". For any affirmative answer, a follow-up question asked for the dates at which the medications were first used and the names of all NSAIDs that had been used.on a daily basis for more than 6 monthsA discrete "index date" was established within each family corresponding to the earliest date that the family or medical records reported AD symptoms to have begun in the proband. Subjects from each family (whether AD cases or non-demented family members) were considered to have been exposed to NSAIDs only if the starting date for NSAID use preceded this index date by at least one year. Age represented the age of cases and of non-demented relatives at the index date, and was treated as a continuous variable.As shown in Figure  1 , there were 756 probands and 1020 relatives over the age of 50 with APOE genotype who were queried about prior NSAID use. After exclusions for those subjects who had missing or unsure responses for the name of their medication, did not include a medication start date, or had missing data for the variables age, sex, education or ethnicity, there remained for analysis 682 probands and 982 relatives. Of the 982 relatives, nine were reported to be demented with the onset of their dementia prior to the index date for that family, and their diagnoses were verified by review of medical records as having probable or definite AD by research criteria, so these were classified with the probands as having AD.Statistical analysis Analyses were performed using SAS version 8.2. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. Crude odds ratios were computed in the first instance, followed by adjusted estimates using generalized estimating equation (GEE) models [ 19 ] to account for the possibility that variables of interest (e.g., medication use, APOE status) could be correlated among individuals within families. Adjustments were made for the following covariates: age, sex, ethnicity (categorized as White, African-American, or other), education (less than versus equal to or greater than high school level), and APOE-ε4 carrier status (one or two ε4 alleles vs. none). We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. In addition, we formally evaluated these associations by adding an interaction term (ε4 * NSAID use) to the GEE model. Ethnicity was similarly examined as an effect modifier.
Statistical analysis
Analyses were performed using SAS version 8.2. The primary independent and dependent variables were prior NSAID use and AD case status, respectively. Crude odds ratios were computed in the first instance, followed by adjusted estimates using generalized estimating equation (GEE) models [ 19 ] to account for the possibility that variables of interest (e.g., medication use, APOE status) could be correlated among individuals within families. Adjustments were made for the following covariates: age, sex, ethnicity (categorized as White, African-American, or other), education (less than versus equal to or greater than high school level), and APOE-ε4 carrier status (one or two ε4 alleles vs. none).We stratified the dataset in order to evaluate whether the association between NSAID use and AD was similar in APOE-ε4 carriers and non-carriers. In addition, we formally evaluated these associations by adding an interaction term (ε4 * NSAID use) to the GEE model. Ethnicity was similarly examined as an effect modifier.Results Characteristics of the 1664 subjects are listed in Table  1 . AD patients were more likely to be older and to be APOE-ε4 carriers compared to controls. The distributions of sex and education were not different between cases and controls. Table 1 Characteristics of AD patients and non-demented family CHARACTERISTIC AD (N = 691) NON DEMENTED (N = 973) AGE ADJUSTED PERCENT AD AGE ADJUSTED PERCENT NON-DEMENTED P-VALUE* Mean Age (SD) 70.0 (8.2) 65.0 (8.8) <0.0001 Sex (%male) 242 (35.0) 381 (39.2) 36.5 39.9 0.39 Greater than HS Ed (%) 403 (58.3) 643 (66.1) 59.3 65.1 0.10 African American (%) 215 (31.1) 204 (21.0) 28.4 21.3 0.01 Use of NSAIDs† (%) 24 (3.5) 66 (6.8) 3.5 6.7 0.08 APOE-ε4 carrier (%) 448 (64.8) 370 (38.0) 65.3 38.0 <0.0001 * Reported p-values use General Estimating Equations to account for correlation among observations. †Exposure to non-steroidal anti-inflammatory drugs had to precede index date by one year or more. Sixty-six out of 973 non-demented relatives (6.8%) and 24 of 691 cases (3.5%) reported previous NSAID use (odds ratio = 0.49; 95% CI = 0.31–0.80). After adjustment for age, sex, educational level, and ethnicity, the odds ratio (OR) of NSAID use among AD cases compared to non-users was 0.57 (95% CI = 0.35–0.93); it was 0.64 (95% CI = 0.38–1.05) when APOE carrier status was added to the GEE model (see Table  2 ). Table 2 Risk of AD with and without prior use of NSAIDs, stratified by APOE-ε4 carrier status EXPOSURE AD (N = 691) NON-DEMENTED FAMILY MEMBERS (N = 973) CRUDE ODDS RATIO (95% CI) AGE-ADJUSTED ODDS RATIO (95% CI) ADJUSTED ODDS RATIO (95% CI) Overall No NSAIDs 667 907 1.0 1.0 1.0 Use of NSAIDs 24 66 0.49 (0.31, 0.80) 0.55 (0.34, 0.88) 0.64 (0.38, 1.05)* Having no ε4 alleles No NSAIDs 231 556 1.0 1.0 1.0 Use of NSAIDs 12 47 0.61 (0.32, 1.18) 0.75 (0.38, 1.46) 0.78 (0.39, 1.52)** Having at least one ε4 allele No NSAIDs 436 351 1.0 1.0 1.0 Use of NSAIDs 12 19 0.51 (0.24, 1.06) 0.47 (0.24, 0.96) 0.49 (0.24, 0.98)** *Adjusted for age, sex, ethnicity, education, and APOE-ε4 status. **Adjusted for age, sex, ethnicity, and education. ***NSAID*APOE-ε4 interaction p-value = 0.04. The magnitude of inverse association was greater among APOE ε4 carriers (OR = 0.49; 95% CI = 0.24–0.98) than non-carriers (OR = 0.78; 95% CI = 0.39–1.52). However, formal evaluation of the interaction between NSAID use and APOE-ε4 carrier status did not reveal a significant difference (p = 0.40). The association between NSAID use and AD risk was similar among Caucasian and African Americans (data not shown).
Results
Characteristics of the 1664 subjects are listed in Table  1 . AD patients were more likely to be older and to be APOE-ε4 carriers compared to controls. The distributions of sex and education were not different between cases and controls.Characteristics of AD patients and non-demented family* Reported p-values use General Estimating Equations to account for correlation among observations.†Exposure to non-steroidal anti-inflammatory drugs had to precede index date by one year or more.Sixty-six out of 973 non-demented relatives (6.8%) and 24 of 691 cases (3.5%) reported previous NSAID use (odds ratio = 0.49; 95% CI = 0.31–0.80). After adjustment for age, sex, educational level, and ethnicity, the odds ratio (OR) of NSAID use among AD cases compared to non-users was 0.57 (95% CI = 0.35–0.93); it was 0.64 (95% CI = 0.38–1.05) when APOE carrier status was added to the GEE model (see Table  2 ).Risk of AD with and without prior use of NSAIDs, stratified by APOE-ε4 carrier statusOverallHaving no ε4 allelesHaving at least one ε4 allele*Adjusted for age, sex, ethnicity, education, and APOE-ε4 status.**Adjusted for age, sex, ethnicity, and education.***NSAID*APOE-ε4 interaction p-value = 0.04.The magnitude of inverse association was greater among APOE ε4 carriers (OR = 0.49; 95% CI = 0.24–0.98) than non-carriers (OR = 0.78; 95% CI = 0.39–1.52). However, formal evaluation of the interaction between NSAID use and APOE-ε4 carrier status did not reveal a significant difference (p = 0.40). The association between NSAID use and AD risk was similar among Caucasian and African Americans (data not shown).Discussion This study supports the findings of previous reports [ 1 - 6 ] suggesting that use of NSAIDs for at least six months is associated with a reduced risk of AD. This association appears to be more robust among APOE-ε4 carriers than non-carriers, although the difference in associations between these two groups was not statistically significant. The MIRAGE Study includes the largest number of well-characterized AD cases and family controls to date, and this large sample size permits adjustment for important potential confounders, as well as the power to examine effect modification by APOE genotype and ethnicity. The subjects without dementia were first-degree family members of AD cases, providing some degree of informal matching on age, socioeconomic status, and health-seeking behavior. However, these results must be interpreted in light of some methodological limitations. Data on NSAID use was collected with a single retrospective question that did not distinguish between aspirin use and non-aspirin NSAID use. Moreover, while non-demented participants reported on themselves, a proxy historian reported on most of the demented individuals. Differential reporting is a potential source of bias in a study that uses self-report on most of the non-demented subjects, yet relies on surrogate respondents for all of the subjects with AD. Asymmetric data collection is difficult to avoid when cases are cognitively impaired, but may be more accurate than expected in AD patients where the surrogate historian has a long association with the subject. We addressed this potential bias by performing an independent validation study to determine the accuracy of surrogate information on a number of questions, including the same questions used in this report about NSAID use. [ 15 ] This study found substantial reliability on the NSAID item (kappa = 0.70). While a validation study comparing proxy historians for non-demented persons does not perfectly mirror the situation in which proxy historians report on demented individuals, our study revealed excellent concordance for surrogate responses from most categories of relatives. This result is consistent with those of prior studies which found a similar association despite differences in study design (cross-sectional [ 1 , 2 ] vs. case-control [ 3 - 6 , 10 , 11 ] vs. prospective [ 7 - 9 , 12 ]), sampling frame (family members [ 10 ] vs. registry-based [ 11 ] vs. general population [ 1 - 9 , 12 ]), ascertainment of exposure, type of medication considered (aspirin [ 2 - 4 , 6 , 8 - 10 , 12 ] vs. non-aspirin NSAIDs [ 1 - 11 ] vs. 'any' NSAID [ 4 , 8 ]), duration of exposure (current [ 1 - 3 ] vs. any history of use, duration ranging from a week to at least six months [ 3 , 5 - 12 ]), and degree of matching or adjustment (usually adjusted for age, sex, and education, less frequently APOE genotype [ 7 - 9 , 12 ]). While many studies have examined the association between NSAID use and risk of AD, few have examined the impact of APOE genotype on this association. The Cache County Study [ 9 , 20 ], the Canadian Study for Health and Aging (CSHA) [ 8 ], and the Rotterdam Study [ 7 ] adjusted for APOE and tested for effect modification and found none. But they had fewer AD cases, and, in the CSHA had a smaller proportion of genotyped subjects. The Rotterdam Study [ 7 ] reported separate odds ratios for APOE-ε4 carriers and non-carriers, but this sample did not have any subjects who were both APOE-ε4 carriers and who reported long-term use of NSAIDs. They found no difference in risk between those with at least one ε4 allele compared to ε4 non-carriers among subjects who used NSAIDs between one month and two years. Our data suggest an enhanced protective benefit of NSAID use among those with ε4. A smaller protective effect was also evident among those lacking ε4. In our sample there were relatively few AD cases who were not ε4 carriers, thus the appearance of different patterns of association between NSAID use and AD risk among APOE genotype subgroups may be spurious. This difference could also have arisen as a result of bias and confounding. The genotype-specific association could be explained by differential inclusion of subjects into the study on the basis of APOE-ε4 carrier status and NSAID use. This might occur if there were differential mortality, according to APOE genotype, among those with AD who had a history of NSAID use; or if for any reason among NSAID users APOE-ε4 carriers were less likely than non-carriers to be diagnosed with AD (or conversely, if among non-users of NSAIDs AD was more likely diagnosed in APOE-ε4 carriers compared to non-carriers). Differential recall could also give rise to this observation. However, these explanations are unlikely because subjects were not selected on the basis of APOE genotype. It is also possible that APOE-ε4 carrier status is a proxy for differentially distributed unmeasured confounders related to NSAID use such as inflammatory disease processes. Alternatively, our results imply that NSAID use affects AD risk differently between APOE-ε4 carriers and non-carriers. For example, because ε4 carriers are inherently more vulnerable to AD, there is a greater opportunity for attributable risk reduction. This explanation does not imply biological interaction between NSAIDs and ε4. On the other hand, the ε4 isoform may have greater pro-inflammatory properties [ 21 ] and ε4 individuals may be more responsive to the benefits of NSAID use than those lacking ε4. Examination of this finding in prospective studies and clinical trials of sufficient power (such as the ADAPT Study [ 22 ], a prospective trial of anti-inflammatory use in the prevention of AD) to detect effect modification by APOE-ε4 carrier status is needed. Such confirmation would provide critical insights into the mechanisms by which APOE isoforms modulate AD risk and into novel therapeutic strategies.
Discussion
This study supports the findings of previous reports [ 1 - 6 ] suggesting that use of NSAIDs for at least six months is associated with a reduced risk of AD. This association appears to be more robust among APOE-ε4 carriers than non-carriers, although the difference in associations between these two groups was not statistically significant.The MIRAGE Study includes the largest number of well-characterized AD cases and family controls to date, and this large sample size permits adjustment for important potential confounders, as well as the power to examine effect modification by APOE genotype and ethnicity. The subjects without dementia were first-degree family members of AD cases, providing some degree of informal matching on age, socioeconomic status, and health-seeking behavior.However, these results must be interpreted in light of some methodological limitations. Data on NSAID use was collected with a single retrospective question that did not distinguish between aspirin use and non-aspirin NSAID use. Moreover, while non-demented participants reported on themselves, a proxy historian reported on most of the demented individuals.Differential reporting is a potential source of bias in a study that uses self-report on most of the non-demented subjects, yet relies on surrogate respondents for all of the subjects with AD. Asymmetric data collection is difficult to avoid when cases are cognitively impaired, but may be more accurate than expected in AD patients where the surrogate historian has a long association with the subject. We addressed this potential bias by performing an independent validation study to determine the accuracy of surrogate information on a number of questions, including the same questions used in this report about NSAID use. [ 15 ] This study found substantial reliability on the NSAID item (kappa = 0.70). While a validation study comparing proxy historians for non-demented persons does not perfectly mirror the situation in which proxy historians report on demented individuals, our study revealed excellent concordance for surrogate responses from most categories of relatives.This result is consistent with those of prior studies which found a similar association despite differences in study design (cross-sectional [ 1 , 2 ] vs. case-control [ 3 - 6 , 10 , 11 ] vs. prospective [ 7 - 9 , 12 ]), sampling frame (family members [ 10 ] vs. registry-based [ 11 ] vs. general population [ 1 - 9 , 12 ]), ascertainment of exposure, type of medication considered (aspirin [ 2 - 4 , 6 , 8 - 10 , 12 ] vs. non-aspirin NSAIDs [ 1 - 11 ] vs. 'any' NSAID [ 4 , 8 ]), duration of exposure (current [ 1 - 3 ] vs. any history of use, duration ranging from a week to at least six months [ 3 , 5 - 12 ]), and degree of matching or adjustment (usually adjusted for age, sex, and education, less frequently APOE genotype [ 7 - 9 , 12 ]).While many studies have examined the association between NSAID use and risk of AD, few have examined the impact of APOE genotype on this association. The Cache County Study [ 9 , 20 ], the Canadian Study for Health and Aging (CSHA) [ 8 ], and the Rotterdam Study [ 7 ] adjusted for APOE and tested for effect modification and found none. But they had fewer AD cases, and, in the CSHA had a smaller proportion of genotyped subjects. The Rotterdam Study [ 7 ] reported separate odds ratios for APOE-ε4 carriers and non-carriers, but this sample did not have any subjects who were both APOE-ε4 carriers and who reported long-term use of NSAIDs. They found no difference in risk between those with at least one ε4 allele compared to ε4 non-carriers among subjects who used NSAIDs between one month and two years.Our data suggest an enhanced protective benefit of NSAID use among those with ε4. A smaller protective effect was also evident among those lacking ε4. In our sample there were relatively few AD cases who were not ε4 carriers, thus the appearance of different patterns of association between NSAID use and AD risk among APOE genotype subgroups may be spurious. This difference could also have arisen as a result of bias and confounding. The genotype-specific association could be explained by differential inclusion of subjects into the study on the basis of APOE-ε4 carrier status and NSAID use. This might occur if there were differential mortality, according to APOE genotype, among those with AD who had a history of NSAID use; or if for any reason among NSAID users APOE-ε4 carriers were less likely than non-carriers to be diagnosed with AD (or conversely, if among non-users of NSAIDs AD was more likely diagnosed in APOE-ε4 carriers compared to non-carriers). Differential recall could also give rise to this observation. However, these explanations are unlikely because subjects were not selected on the basis of APOE genotype. It is also possible that APOE-ε4 carrier status is a proxy for differentially distributed unmeasured confounders related to NSAID use such as inflammatory disease processes.Alternatively, our results imply that NSAID use affects AD risk differently between APOE-ε4 carriers and non-carriers. For example, because ε4 carriers are inherently more vulnerable to AD, there is a greater opportunity for attributable risk reduction. This explanation does not imply biological interaction between NSAIDs and ε4. On the other hand, the ε4 isoform may have greater pro-inflammatory properties [ 21 ] and ε4 individuals may be more responsive to the benefits of NSAID use than those lacking ε4.Examination of this finding in prospective studies and clinical trials of sufficient power (such as the ADAPT Study [ 22 ], a prospective trial of anti-inflammatory use in the prevention of AD) to detect effect modification by APOE-ε4 carrier status is needed. Such confirmation would provide critical insights into the mechanisms by which APOE isoforms modulate AD risk and into novel therapeutic strategies.Conclusions NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.
Conclusions
NSAID use is inversely associated with AD and may be modified by APOE genotype. Prospective studies and clinical trials of sufficient power to detect effect modification by APOE-ε4 carrier status are needed.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions Study concept and design (LAF, RCG, LAC); acquisition of data (LAF, RCG, MIRAGE investigators); analysis and interpretation of data (AGY, MH); drafting of the manuscript (AGY, RCG, LAF); critical revision of the manuscript for important intellectual content (AGY, LAF, RCG, LAC); statistical expertise (LAC); obtained funding (LAF, RCG, LAC, MIRAGE investigators). All authors read and approved the final manuscript.
Authors' contributions
Study concept and design (LAF, RCG, LAC); acquisition of data (LAF, RCG, MIRAGE investigators); analysis and interpretation of data (AGY, MH); drafting of the manuscript (AGY, RCG, LAF); critical revision of the manuscript for important intellectual content (AGY, LAF, RCG, LAC); statistical expertise (LAC); obtained funding (LAF, RCG, LAC, MIRAGE investigators). All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: