Murine Model Of Senile Osteoporosis Research Articles

The effects of dietary fatty acids on bone, hematopoietic marrow and marrow adipose tissue in a murine model of senile osteoporosis

Purpose: Marrow adipose tissue (MAT) expansion and associated lipotoxicity are important drivers of age-related bone loss and hematopoietic bone marrow (HBM) atrophy. Fish oil and borage oil (rich in ω3 fatty acids) can partially prevent aged-related bone loss in SAMP8 mice. However, whether preservation of bone mass in this progeria model is associated with MAT volumes remains unknown.Results: MAT volume fraction (MAT%) showed a negative association with hematopoietic bone marrow (HBM%;r=-0.836, p<0.001) and bone (bone%;r=-0.344, p=0.013) volume fractions.Adjusting for multiple comparisons, bone% was higher and MAT% was lower in Fish oil (FO)-supplemented groups vs. controls (p<0.001). HBM% did not differ significantly between the four groups. However, in the group supplemented with FO, HBM comprised higher fractions and MAT constituted lower fractions of total marrow vs. controls (p<0.001).Conclusion: Feeding FO-enriched diet prevented age-related bone and HBM loss, by reducing MAT expansion. Our results further emphasize on the role(s) of MAT expansion in bone and HBM atrophy.Methods: SAMP8 mice (n>9 /group) were allocated into 4 categories and fed a control ration, FO-, sunflower oil (SFO)- and borage oil-enriched diets for lifetime. Femurs were scanned using microcomputed tomography (μCT) and bone, MAT, and HBM volumes were determined using an image analysis software.

Chewing ameliorates chronic mild stress-induced bone loss in senescence-accelerated mouse (SAMP8), a murine model of senile osteoporosis

Chronic mild stress is a risk factor for osteoporosis and chewing inhibits the stress response. We examined the effect of chewing on chronic stress-induced bone loss and bone microstructural deterioration in mice. The senescence-accelerated mouse strain P8 (SAMP8) was randomly divided into control, stress, and stress with chewing groups of fifteen animals each. Mice in the stress and stress with chewing groups were placed in a ventilated restraint tube for 60minutes, twice a day for 4weeks. The restrained mice were simultaneously subjected daily to one of the following stressors: water immersion, physical shaking and flashing lights. Mice in the stress with chewing group were allowed to chew a wooden stick during the experimental period. After the experiment, the bone response was evaluated using quantitative micro computed tomography, bone histomorphometry, and biochemical markers. Exposure of SAMP8 mice to chronic stress resulted in significant increase of the blood corticosterone and noradrenaline levels, and adrenal weight. The bone resorption was activated and the bone formation was suppressed. Trabecular bone volume and trabecular number were decreased in both the vertebra and distal femur of the stress group. Chewing under chronic stress prevented the increase in the blood corticosterone and noradrenaline levels, attenuated the reduced bone formation and increased bone resorption, improved the trabecular bone loss and bone microstructural deterioration induced by chronic mild stress. These findings indicate that chewing can ameliorate chronic stress-induced bone loss in SAMP8 mice. Thus, chewing may represent a useful method preventing and/or treating chronic stress-related osteoporosis.

Segmental variations in trabecular bone density and microstructure of the spine in senescence-accelerated mouse (SAMP6): A murine model for senile osteoporosis

The senescence-accelerated mouse strain P6 (SAMP6) is a model of senile osteoporosis, which possesses many features of senile osteoporosis in humans. So far, little is known about the systemic bone microstructural changes that occur at the cervical, thoracic, and lumbar vertebrae. In this study, we therefore investigated segmental variations of vertebral trabecular bone mineral density (BMD) and three-dimensional microstructure in SAMP6 and the normal control mouse (SAMR1) at 12months of age using quantitative micro computed tomography (micro-CT) and image analysis software. The vertebral height and vertebral cross-sectional area (CSA) increased, while vertebral trabecular BMD and trabecular bone volume fraction (BV/TV) decreased from the cervical to lumbar spine both in SAMR1 and SAMP6. As compared with SAMR1, the thoracic vertebral CSA had a tendency to be low and the lumbar vertebral CSA was significantly declined in SAMP6. The vertebral trabecular BMD, BV/TV, trabecular thickness (Tb.Th), and trabecular number (Tb.N) significantly decreased in cervical, thoracic and lumbar spine of SAMP6. Trabecular bone pattern factor (TBPf) was higher at the lumbar spine and the structure model index (SMI) of the lower thoracic and lumbar spine was higher in SAMP6. These results indicate that vertebral trabecular bone microstructures are remarkably heterogeneous throughout the spine in both SAMR1 and SAMP6. The decrease of vertebral trabecular bone density in SAMP6 advanced faster caudally than cranially within the spine, similar phenomena were observed in humans. These findings highlight the relevance of SAMP6 for studies of vertebral fragility associated with senile osteoporosis.

Borage and fish oils lifelong supplementation decreases inflammation and improves bone health in a murine model of senile osteoporosis

Fats are prevalent in western diets; they have known deleterious effects on muscle insulin resistance and may contribute to bone loss. However, relationships between fatty acids and locomotor system dysfunctions in elderly population remain controversial. The aim of this study was to analyze the impact of fatty acid quality on the age related evolution of the locomotor system and to understand which aging mechanisms are involved. In order to analyze age related complications, the SAMP8 mouse strain was chosen as a progeria model as compared to the SAMR1 control strain. Then, two months old mice were divided in different groups and subjected to the following diets : (1) standard “growth” diet — (2) “sunflower” diet (high ω6/ω3 ratio) — (3) “borage” diet (high γ-linolenic acid) — (4) “fish” diet (high in long chain ω3). Mice were fed ad libitum through the whole protocol. At 12months old, the mice were sacrificed and tissues were harvested for bone studies, fat and muscle mass measures, inflammation parameters and bone cell marker expression. We demonstrated for the first time that borage and fish diets restored inflammation and bone parameters using an original model of senile osteoporosis that mimics clinical features of aging in humans. Therefore, our study strongly encourages nutritional approaches as relevant and promising strategies for preventing aged-related locomotor dysfunctions.This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism.

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis

Bone marrow osteogenesis in senile osteoporotic bone is impaired and, as such, may have significant implications on the successful outcome of fracture repair. Here we utilize a well-established murine model of senile osteoporosis, the P6 strain of senescence-accelerated mice (SAMP6), to investigate fracture healing in aged osteoporotic bone. A femoral osteotomy was created in SAMP6 and in non-osteoporotic age-matched control R1 senescence-resistant mice (SAMR1). The course of fracture healing was evaluated over a period of 42 days using quantitative microCT and histological analysis. The differentiation capabilities of bone mesenchymal progenitor cells derived from SAMP6 and SAMR1 mice was examined, and their osteogenic potential determined. Although preliminary in vitro analysis confirmed that bone marrow-derived stem cells (BMSC) isolated from SAMP6 mice had a reduced osteogenic capacity, no significant deficit in fracture repair as determined by quantitative microCT could be detected. This was supported by histology assessment, where complete bridging of the fracture gap was evident by day 28 and was fully healed day 42 in both SAMP6 and SAMR1 mice. Further in vitro studies revealed that periosteal-derived progenitor cells (PDPC) isolated from SAMP6 mice had an osteogenic potential comparable to that observed in SAMR1 mice. In conclusion, fracture healing in SAMP6 mice is not detrimentally affected by impairment of BMSC osteogenesis, suggesting that bone marrow-mediated repair processes are dispensable for normal bone healing in this senile osteoporotic fracture model. Furthermore, the influence of PDPC in the repair process may partly explain the absence of any detectable deficits in fracture repair in SAMP6 mice.

Effect of intermittent treatment with human parathyroid hormone 1–34 in SAMP6 senescence-accelerated mice

We examined trabecular and cortical bone in the senescence-accelerated mouse prone 6 (SAMP6) murine model of senile osteoporosis after treatment with human PTH 1-34. Sixteen-week-old female SAMP6 mice were assigned to control and PTH groups. PTH (20 microg/kg) was administered sc 3 times a week for 12 weeks. The control mouse strain, senescence-accelerated mouse resistant 1 (SAMR1), was used for comparison. The femoral metaphysis and diaphysis were used to measure bone mineral density (BMD), analyze the trabecular and the cortical structure by micro-computed tomography, and for conducting the bone strength test. PTH significantly attenuated the loss of BMD, improved the trabecular bone microstructure, and increased the bone strength in the femoral metaphysis. We did not find any differences in the bone strength of the femoral diaphysis after PTH treatment, although the cortical bone volume and cortical thickness were improved. Although the cortical thickness increased, the cortical bone density decreased, likely because of the increase of cortical porosity in the distal metaphysis after administration of PTH.

Site-specific bone loss in senescence-accelerated mouse (SAMP6): A murine model for senile osteoporosis

The senescence-accelerated mouse strain P6 (SAMP6) is a model of senile osteoporosis, which possesses many features of senile osteoporosis in humans. So far, little is known about the systemic bone microstructural changes that occur at multiple skeletal sites. In this study, we therefore, investigated site (vertebra, femur and tibia) dependence of bone microstructure and bone mineral density (BMD) in SAMP6 and the normal control mouse (SAMR1) at 5 and 12 months of age using quantitative micro computed tomography (micro-CT) and image analysis software. As compared with SAMR1, the most prominent change in SAMP6 was the reduction of vertebral trabecular bone volume fraction (BV/TV) and trabecular BMD. Moderate decrease of trabecular bone mass was observed in the proximal tibia and distal femur. Increased marrow area and periosteal perimeter were investigated, though the cortical area and cortical thickness had no marked changes in the mid-tibial and mid-femoral cortical bones. These results indicate that bone microstructural properties in SAMP6 are remarkably heterogeneous throughout the skeleton, which is analogous to changes that occur in human bones. These findings further validate the relevance of SAMP6 as a model of senile osteoporosis.

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): A murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle–tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle–tendon junction, tendon, tendon–bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.

Finite element analysis of the mouse tibia: Estimating endocortical strain during three‐point bending in SAMP6 osteoporotic mice

To support future studies of tibial bending in a murine model of senile osteoporosis (SAMP6), we sought to determine the relationship between applied external bending force and peak endocortical strain in the tibiae of SAMP6 and control SAMR1 mice. The lower hindlimbs of mice were loaded by three-point bending in the lateral-medial plane with a support length of 10 mm. Force-periosteal strain relations were first determined using standard strain gauge methods. Finite-element analysis (FEA) models of the tibia-fibula were generated based on microcomputed tomography images. After choosing appropriate boundary conditions, FEA predictions of periosteal strains were within 15% of measured values. FEA revealed a narrow (3-4 mm) region of the central tibia with well-developed bending strains (tension medially, compression laterally); outside this region, we observed high shear strains. Both the strain gauge data and the finite-element simulations indicated that the tibia of the SAMP6 mouse was 20-25% stiffer than the SAMR1 tibia, consistent with a larger moment of inertia and higher cortical bone modulus. Thus, higher levels of force are required to produce the same target values of strain in the SAMP6 tibia. The ratio of periosteal to endocortical strain in the region of interest was similar for the two mouse strains (1.5-1.6). Based on these ratios, we scaled the strain gauge data to estimate the force-endocortical strain relations for the two mouse strains. In conclusion, FEA, with supporting strain gauge measurements, has provided unique insight regarding the strain environment throughout the tibia during three-point bending in mice.

Effects of Traditional Chinese Medicine on Bone Loss in SAMP6: A Murine Model for Senile Osteoporosis

We evaluated the effects of the traditional Chinese medicines, Hachimi-jio-gan, Juzen-taiho-to and Unkei-to, on bone loss in murine model of senile osteoporosis (SAMP6). Two-month-old SAMP6 were divided into control and experimental groups. The control mice had the tap water available as the only drinking fluid. The experimental mice were given 0.05% aqueous solution of Hachimi-jio-gan, Juzen-taiho-to or Unkei-to for three months. The solution intake of a mouse averaged 5 ml per day. The bones were studied morphologically and histomorphometrically, together with bone mineral density (BMD), serum parathyroid hormone (PTH) and estradiol levels. In the control group, BMD and the amount of bone forming surface were low, the serum PTH level was high when compared with the normal mice SAMR1. Many osteocytes and osteoblasts showed degenerative changes and numerous mast cells were observed in the bone marrow. Compared with controls, the serum estradiol level was higher in the Unkei-to group. However, we did not find any significant changes of bones. In the Hachimi-jio-gan and Juzen-taiho-to groups, the bone mass and the amount of bone forming surface increased. Most of the osteocytes and osteoblasts appeared normal. As compared with controls, the number of mast cells in bone marrow decreased in the Hachimi-jio-gan group. The serum PTH level had declined in the Juzen-taiho-to group. The present study provides certain evidence that Hachimi-jio-gan and Juzen-taiho-to are effective in preventing bone loss in SAMP6, while Unkei-to can only improve the ovary function.

Morphological study of the parathyroid gland and thyroid C cell in senescence-accelerated mouse (SAMP6), a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid gland and thyroid C cell, together with the serum parathyroid hormone (PTH) and calcitonin (CT) in SAMP6 and age-matched normal mice SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 month of age with regard to the serum PTH level and the morphology of the parathyroid glands. As compared with SAMR1, the serum PTH level was significantly higher in SAMP6 at 2, 5 and 12 months of age. In the parathyroid chief cells of SAMP6 at 2, 5 and 12 months of age, the Golgi complexes and the cisternae of the granular endoplasmic reticulum were well developed. Numerous secretory granules were located near the plasma membranes and mitoses were sometimes observed. There was no marked difference between SAMR1 and SAMP6 regarding the morphology of the thyroid C cells and the serum CT level. These findings suggest that the secretory activity of the parathyroid gland is stimulated in SAMP6 at 2, 5 and 12 months of age. The parathyroid follicle was sometimes found in SAMP6, and the significance of this structure was also discussed.

Preventive effects of traditional Chinese medicine on senile osteoporosis using the murine model

In this study, the preventive effects of the traditional Chinese medicine, Hachimi‐jio‐gan, Juzen‐taiho‐to and Unkei‐to were investigated, on bone loss in the murine model of senile osteoporosis, senescence‐accelerated mouse P6 (SAMP6). The experimental animals were given 0.05% solution of the Chinese medicines for 3 months. Bone mineral density and bone morphology were studied together with serum parathyroid hormone and estradiol levels. After the treatment with Unkei‐to, the serum estradiol level was increased without any marked change in the bone. Bone mass was significantly increased after the treatment with Hachimi‐jio‐gan or Juzen‐taiho‐to. These findings suggest that Hachimi‐jio‐gan and Juzen‐taiho‐are effective in preventing bone loss in SAMP6.

Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

A congenic mouse and candidate gene at the Chromosome 13 locus regulating bone density

Previously, we identified two significant quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected for bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains, SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. We recently designated the Chr 13 locus as Pbd2 (Peak bone density 2) and constructed a congenic strain, P6.P2-Pbd2(b), which carried a single genomic interval from the Chr 13 of SAMP2 on a SAMP6-derived osteoporotic background. In this study, we have constructed a congenic strain, P2.P6-Pbd2(a), carrying a SAMP6-derived susceptible interval on a SAMP2-derived resistance background. This congenic strain had a lower bone density than the background strain, SAMP2, based on three measurement methods, each utilizing a different principle for evaluating bone density: MD, DXA, and pQCT. Next, a candidate gene approach was used to find polymorphisms of Bmp6 (bone morphogenetic protein 6). The CAG trinucleotide repeat numbers in exon 1 of this gene differ among SAM strains. We found an association of CAG repeat length with relative peak bone mass in mice.

Chromosome 13 locus, Pbd2, regulates bone density in mice.

Bone density is inherited as a complex polygenic trait. Previously, we identified two quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected by bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains: SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. In this study, we report the effects of the Chr 13 QTL on peak bone density (Pbd2). First, we constructed a congenic strain P6.P2-Pbd2b, which carried a single genomic interval from the Chr 13 of SAMP2 on an SAMP6-derived osteoporotic background, to dissect this polygenic trait into single gene factors. This congenic strain had a higher bone density than the background strain using three measurement methods with different principles for bone density. Next, we measured the peak relative bone mass of the AKR/J strain and the 13 senescence-accelerated mouse (SAM) strains, which are considered to be a series of recombinant-like inbred (RI) strains derived from the AKR/J strain and other unspecified strains. We then determined the microsatellite marker haplotypes of these strains around the Pbd2 locus, in which three strains with a high relative bone mass shared the same haplotype over the 26-centimorgan (cM) region. In the Pbd2 locus, a high relative bone mass was associated with alleles of the unknown strain, whereas a low relative bone mass was associated with the alleles from the AKR/J strain. These results confirmed the existence of a Pbd2 locus regulating bone density in the SAM strains.

Expression of the c- fos gene induced by parathyroid hormone in the bones of SAMP6 mice, a murine model for senile osteoporosis

SAMP6 mice are a murine model for senile osteoporosis, characterized by low peak bone mass seen at 4 or 5 months of age. Parathyroid hormone (PTH)-induced c- fos expression was examined in the bones, bone-marrow cells and kidney tissues of 2-month-old male SAMP6 mice. SAMP2 mice, which have a higher peak bone mass, were used as controls. The expression of c- fos in the bone peaked at 30 min after 60 μg/kg of human PTH(1–34) administration. After peaking, the expression fell quickly in SAMP2 mice. This decrease in expression was delayed in SAMP6 mice and the expression was higher at 1 h than in SAMP2 mice. The phenomenon observed in the bone appears to be tissue specific as it was not seen in the bone-marrow cells or kidney tissue. Immunohistochemical studies showed that c-Fos protein was localized to the nuclei of some of the osteocytes and a few of the osteoblasts in the cortical bone, and that osteocytes expressing c-Fos protein increased after PTH treatment. These results suggest that osteocytes might contribute to the maintenance of higher levels of c- fos expression in the bones of SAMP6 mice and may be related to cortical osteopenia in these mice by modulating bone remodeling and/or modeling.

Identification of peak bone mass QTL in a spontaneously osteoporotic mouse strain.

The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.

1407 Age-related changes in levels of NGF, BDNF and NT-3 in the mouse brain

Inter-strain differences in bone mass and density during growth were followed in three strains of mice: SAM-P/2, SAM-R/1 and SAM-P/6 (a murine model of senile osteoporosis, Matsushita et al., Am J Pathol 1986;125:276–283). Photometrically, the inter-strain disparities first appeared in mice at about age 28 days and increased until age 60 days. During this period, tetracycline labelling revealed significant strain differences regarding rate of the appositional formation at the endosteal surface but not at the periosteal surface. The order coincided with sesults of the photometrical assay, that is, highest in SAM-P/2, followed by SAM-R/1 and SAM-P/6, respectively. Therefore, strain differences, especially the osteopenic state of SAM-P/6, occur, at least in part, by disparities in endosteal formation rates during cortical bone modelling.

1506 The effect of chronic cerebral hypoperfusion on rat cortical muscarinic cholinergic receptor binding studied by pet

Inter-strain differences in bone mass and density during growth were followed in three strains of mice: SAM-P/2, SAM-R/1 and SAM-P/6 (a murine model of senile osteoporosis, Matsushita et al., Am J Pathol 1986;125:276–283). Photometrically, the inter-strain disparities first appeared in mice at about age 28 days and increased until age 60 days. During this period, tetracycline labelling revealed significant strain differences regarding rate of the appositional formation at the endosteal surface but not at the periosteal surface. The order coincided with sesults of the photometrical assay, that is, highest in SAM-P/2, followed by SAM-R/1 and SAM-P/6, respectively. Therefore, strain differences, especially the osteopenic state of SAM-P/6, occur, at least in part, by disparities in endosteal formation rates during cortical bone modelling.

Effective intervention of low peak bone mass and bone modeling in the spontaneous murine model of senile osteoporosis, SAM-P/6, by Ca supplement and hormone treatment

SAM-P/6 is a recently developed strain of osteoporotic mice. In this study we tried to determine whether calcium, vitamin D 3, parathyroid hormone (PTH), and estrogen modified the peak bone mass of young SAM-P/6 mice, and whether the effect of these medications persisted after treatment had been discontinued. Calcium supplement, PTH, and estrogen treatment increased the peak bone mass of SAM-P/6 mice. To clarify the process by which bone mass was increased in these treated mice, we evaluated their bone formation and resorption by histomorphometry and measured the levels of ions and serum enzymes relevant to bone metabolism. We found that bone formation was increased by calcium supplementation, and bone resorption was decreased by estrogen treatment. Furthermore, the effectiveness of calcium supplement on peak bone mass was retained after treatment had been discontinued, but the effect of estrogen treatment on peak bone mass was reduced after estrogen treatment had been discontinued. The results of this study indicate that calcium supplementation and estrogen and PTH treatment each increased peak bone mass at the midpoint of the femur of SAM-P/6, and that the effect of calcium supplementation, but not that of estrogen treatment, persisted after treatment was discontinued.