Ultrastructural changes in bones of the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mice, was developed as an animal model for senile osteoporosis. In the present study, we investigated the bone morphology, together with serum calcium and bone mineral density (BMD) 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 compositions and bone morphology. As compared with SAMR1, BMD, the femoral weight, femoral calcium and phosphorus levels were significantly reduced in SAMP6 at 2 and 5 months of age. The number of osteoblasts in trabecular bones was also significantly reduced. Swollen mitochondria and myelin-like structures were found in osteoblasts and osteocytes of SAMP6 mice at 2 and 5 months of age. There was a greater proportion of resting surface and less forming surface in the femoral endosteal surfaces of SAMP6 mice. The amount of trabecular bone in the lumbar vertebra and the distal metaphysis of the femur was reduced. The number of the mast cells in bone marrow of the tibia significantly increased in SAMP6 mice. These findings indicate that the lower bone mass in SAMP6 was due to the reduction in osteoblast formation and suggested that mast cells in bone marrows play a role in the pathogenesis of senile osteoporosis.     

Characterization of Senescence-Accelerated Mouse Prone 6 (SAMP6) as an Animal Model for Brain Research

The senescence-accelerated mouse (SAM) was developed by selective breeding of the AKR/J strain, based on a graded score for senescence, which led to the development of both senescence-accelerated prone (SAMP), and senescence-accelerated resistant (SAMR) strains. Among the SAMP strains, SAMP6 is well characterized as a model of senile osteoporosis, but its brain and neuronal functions have not been well studied. We therefore decided to characterize the central nervous system of SAMP6, in combination with different behavioral tests and analysis of its biochemical and pharmacological properties. Multiple behavioral tests revealed higher motor activity, reduced anxiety, anti-depressant activity, motor coordination deficits, and enhanced learning and memory in SAMP6 compared with SAMR1. Biochemical and pharmacological analyses revealed several alterations in the dopamine and serotonin systems, and in long-term potentiation (LTP)-related molecules. In this review, we discuss the possibility of using SAMP6 as a model of brain function.     

Catechins attenuate eccentric exercise-induced inflammation and loss of force production in muscle in senescence-accelerated mice

Catechins have a great variety of biological actions. We evaluated the potential benefits of catechin ingestion on muscle contractile properties, oxidative stress, and inflammation following downhill running, which is a typical eccentric exercise, in senescence-accelerated prone mice (SAMP). Downhill running (13 m/min for 60 min; 16° decline) induced a greater decrease in the contractile force of soleus muscle and in Ca(2+)-ATPase activity in SAMP1 compared with the senescence-resistant mice (SAMR1). Moreover, compared with SAMR1, SAMP1 showed greater downhill running-induced increases in plasma CPK and LDH activity, malondialdehyde, and carbonylated protein as markers of oxidative stress; and in protein and mRNA expression levels of the inflammatory mediators such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in muscle. SAMP1 exhibited aging-associated vulnerability to oxidative stress and inflammation in muscle induced by downhill running. Long-term (8 wk) catechin ingestion significantly attenuated the downhill running-induced decrease in muscle force and the increased inflammatory mediators in both plasma and gastrocnemius muscle. Furthermore, catechins significantly inhibited the increase in oxidative stress markers immediately after downhill running, accompanied by an increase in glutathione reductase activity. These findings suggest that long-term catechin ingestion attenuates the aging-associated loss of force production, oxidative stress, and inflammation in muscle after exercise.     

Tea catechin ingestion combined with habitual exercise suppresses the aging-associated decline in physical performance in senescence-accelerated mice

Catechins, which are abundant in green tea, possess a variety of biologic actions, and their clinical application has been extensively investigated. In this study, we examined the effects of tea catechins and regular exercise on the aging-associated decline in physical performance in senescence-accelerated prone mice (SAMP1) and age-matched senescence-accelerated resistant mice (SAMR1). The endurance capacity of SAMR1 mice, measured as the running time to exhaustion, tended to increase over the 8-wk experimental period, whereas that of SAMP1 mice decreased by 17%. On the other hand, the endurance capacity of SAMP1 mice fed 0.35% (wt/wt) catechins remained at the initial level and was significantly higher than that of SAMP1 mice not fed catechins. In SAMP1 mice fed catechins and given exercise, oxygen consumption was significantly increased, and there was an increase in skeletal muscle fatty acid beta-oxidation. The mRNA levels of mitochondria-related molecules, such as peroxisome proliferator-activated receptor-gamma coactivator-1, cytochrome c oxidase-II, III, and IV in skeletal muscle were also higher in SAMP1 mice given both catechins and exercise. Moreover, oxidative stress measured as thiobarbituric reactive substances was lower in SAMP1 groups fed catechins than in the SAMP1 control group. These results suggest that long-term intake of catechins, together with habitual exercise, is beneficial for suppressing the aging-related decline in physical performance and energy metabolism and that these effects are due, at least in part, to improved mitochondrial function in skeletal muscle.     

降钙素对骨质疏松大鼠骨密度形态计量学与骨代谢的影响

目的探讨降钙素(密盖息)对骨质疏松大鼠骨密度、骨形态计量学影响以及与血钙、磷、维生素D代谢和生长因子的关系。方法用摘除大鼠双侧卵巢的方式制备骨质疏松模型(OVX),实验动物分为4个组:模型对照组、密盖息治疗组,盐酸雷洛昔芬治疗组,假手术组。应用HOLOGIC第4代双能X线4500W骨密度仪测定大鼠腰椎、股骨上段骨密度值(BMD);以骨形态计量学测股骨骨小梁面积、矿化沉积率;用ELISA法测定血清IGF-1水平和血清25OHVitD浓度以及血淋巴细胞维生素D受体(VDR)含量。结果密盖息治疗组、盐酸雷洛昔芬治疗组均较OVX组腰椎、股骨上段骨密度增高,组间比较差异有显著性(P<0.01)。密盖息治疗组较盐酸雷洛昔芬治疗组股骨上段骨密度增高,两组之间差异有显著性(P<0.01)。密盖息治疗组骨小梁面积明显增加、矿化沉积率增高。密盖息治疗组、盐酸雷洛昔芬治疗组血清IGF-1浓度值、血清25-OHVitD浓度值升高,与OVX组比较差异有显著性(P<0.01)。各组血淋巴细胞VDR含量无明显变化,与OVX组比较差异无显著性(P>0.05)。结论密盖息能够预防腰椎、股骨上段骨密度丢失,使骨小梁面积明显增加、矿化沉积率增高并且血清IGF-1及血清25-OHVitD浓度值升高,但对VDR含量无明显作用。     

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.     

No effects of lifelong creatine supplementation on sarcopenia in senescence-accelerated mice (SAMP8)

Oral creatine supplementation can acutely ameliorate skeletal muscle function in older humans, but its value in the prevention of sarcopenia remains unknown. We evaluated the effects of lifelong creatine supplementation on muscle mass and morphology, contractility, and metabolic properties in a mouse model of muscle senescence. Male senescence-accelerated mice (SAMP8) were fed control or creatine-supplemented (2% of food intake) diet from the age of 10 to 60 wk. Soleus and extensor digitorum longus muscles were tested for in vitro contractile properties, creatine content, and morphology at weeks 25 and 60. Both muscle types showed reduced phosphocreatine content at week 60 that could not be prevented by creatine. Accordingly, age-associated decline in muscle mass and contractility was not influenced by treatment. Aged soleus muscles had fewer and smaller fast-twitch glycolytic fibers irrespective of treatment received. It is concluded that lifelong creatine supplementation is no effective strategy to prevent sarcopenia in senescence-accelerated mice.     

Differential effects of jump versus running exercise on trabecular architecture during remobilization after suspension-induced osteopenia in growing rats

High-impact exercise is considered to be very beneficial for bones. We investigated the ability of jump exercise to restore bone mass and structure after the deterioration induced by tail suspension in growing rats and made comparisons with treadmill running exercise. Five-week-old male Wistar rats (n = 28) were randomly assigned to four body weight-matched groups: a spontaneous recovery group after tail suspension (n = 7), a jump exercise group after tail suspension (n = 7), a treadmill running group after tail suspension (n = 7), and age-matched controls without tail suspension or exercise (n = 7). Treadmill running was performed at 25 m/min, 1 h/day, 5 days/wk. The jump exercise protocol consisted of 10 jumps/day, 5 days/wk, with a jump height of 40 cm. Bone mineral density (BMD) of the total right femur was measured by dual-energy X-ray absorptiometry. Three-dimensional trabecular bone architecture at the distal femoral metaphysis was evaluated using microcomputed tomography. After 5 wk of free remobilization, right femoral BMD, right hindlimb muscle weight, and body weight returned to age-matched control levels, but trabeculae remained thinner and less connected. Although both jump and running exercises during the remobilization period increased trabecular bone mass, jump exercise increased trabecular thickness, whereas running exercise increased trabecular number. These results indicate that restoration of trabecular bone architecture induced by jump exercise during remobilization is predominantly attributable to increased trabecular thickness, whereas running adds trabecular bone mass through increasing trabecular number, and suggest that jumping and running exercises have different mechanisms of action on structural characteristics of trabecular bone.     

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.     

Gender specific LRP5 influences on trabecular bone structure and strength

A mutation in LRP5 (low-density lipoprotein receptor-related protein 5) has been shown to increase bone mass and density in humans and animals. Transgenic mice expressing the LRP5 mutation (G171V) demonstrate an increase in bone mass as compared to non-transgenic (NTG) littermates. This study evaluated LRP5 gene and gender-related influences on the structural and biomechanical strength properties of trabecular and cortical bone in femurs and vertebrae (L5) of 17-week-old mice. Micro-computed tomography was used to evaluate the trabecular bone structure of distal femurs and vertebrae ex vivo. Mechanical testing of the trabecular bone in the distal femur was done to determine biomechanical strength. Differences due to genotype and gender were tested using two-way ANOVA at a significance level of p<0.05. Trabecular bone structural parameters (BV/TV, trabecular thickness, number, etc.) at the distal femur, femoral neck, and vertebral body sites were greater in the transgenic as compared to the NTG mice. In addition, vertebral cortical thickness and trabecular strength parameters (ultimate and yield loads, stiffness, ultimate and yield stresses) in the distal femur were greater in the transgenic mice as compared to NTG. The increasing trends of cortical thickness were also noted in the transgenic mice as compared to NTG. Within LRP5 (G171V) mutant mice, there were significant gender-related differences in some of the trabecular bone structural parameters at all the sites (distal femur, femoral neck, and vertebral body). However, unlike trabecular structural parameters, the gender-specific differences were not found in the trabecular strength of LRP5 transgenic mice. In summary, these findings suggest that the LRP5 (G171V) mutation results in greater trabecular bone structure and strength at both the distal femurs and vertebral bodies as compared to NTG. In addition, only the trabecular structure parameters were affected by gender within the LRP5 (G171V) mutation.     

Nanoindentation and whole-bone bending estimates of material properties in bones from the senescence accelerated mouse SAMP6

The senescence accelerated mouse, strain P6 (SAMP6) has been described as a model of senile osteoporosis. Recent results from whole-bone bending tests indicate that, despite having increased moments of inertia, SAMP6 long bones are weak and brittle compared to SAMR1 controls. In the current study we determined material properties of cortical bone from SAMP6 and SAMR1 femora and tibiae by two methods-nanoindentation and whole-bone bending tests combined with simple beam theory. We hypothesized that: (1) SAMP6 mice have reduced cortical bone material properties compared to SAMR1 controls; and (2) modulus estimated from whole-bone bending tests correlates well with modulus determined by nanoindentation. Results from nanoindentation indicated that modulus and hardness are approximately 10% higher in SAMP6 mice compared to SAMR1 controls (p<0.001), a finding consistent with slightly higher mineralization in SAMP6 bones. Despite their superior elastic and hardness properties, the bending failure properties of SAMP6 bones were markedly inferior--ultimate stress and toughness were reduced by 40% and 60%, respectively (p<0.001). Comparisons between the two testing methods for determining modulus showed poor agreement. Modulus estimated from whole-bone bending tests was not correlated with modulus determined by nanoindentation (p=0.054; r2=0.03) and the absolute values differed by a factor of five between the two methods (bending [wet], 6GPa; nanoindentation [dry], 31GPa). Moreover, relative differences between groups were inconsistent between the two methods. We conclude: (1) cortical bone from the SAMP6 mouse has increased modulus and hardness but poor material strength and toughness, which underscores the relevance of the SAMP6 mouse for studies of skeletal fragility, and (2) values of elastic modulus of bone tissue estimated using simple beam theory and bending tests of mouse femora and tibiae are inaccurate and should be interpreted with caution.     

Ultrastructure of the water-clear cell in the parathyroid gland of SAMP6 mice

Although the parathyroid water-clear cell is very rare, it has clinical significance because of its association with parathyroid hyperplasia or adenoma. SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid glands in SAMP6 and age-matched normal mouse SAMR1. The parathyroid water-clear cells, which contained numerous vacuoles and the crystalloid inclusions, were found in SAMP6 mice at 5, 8 and 12 months of age. It was noted that the number of water-clear cells increased with aging, which are fairly consistent with the change of the serum parathyroid hormone (PTH) level. We did not find any water-clear cells in the parathyroid glands of SAMR1 mice. The existence of water-clear cells may represent hyperfunction of the parathyroid glands in SAMP6.     

Differential expression of the liver proteome in senescence accelerated mice

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.     

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon.

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.     

Constructive effect of exogenous melatonin against osteoporosis after ovariectomy in rats

OBJECTIVE: To analyze histomorphometric, densitometric and biochemical effects of melatonin on osteoporosis in ovariectomized rats. STUDY DESIGN: Wistar rats were divided into 6 groups. Group C: control; Group I: bilateral ovariectomy (OVX); Group II: OVX + vehicle; Group III: OVX + 10 mg/kg/day melatonin (MLT); Group IV: OVX + 30 mg/kg/day MLT; Group V: sham + 10 mg/kg/day MLT. Cortex, trabecula, osteoblast and osteoclast numbers were evaluated on vertebra and femur histomorphometrically. Hydroxyproline analysis was used to determine collagen content of femur and vertebrae. Bone mineral density and bone mineral content were measured. RESULTS: Trabecular thickness and trabecular area of vertebra and femur and cortical thickness of femur showed remarkable decrease after OVX, but increased after MLT treatment in the OVX+MLT groups. Following OVX, no statistically significant difference was found in number of osteoblasts or osteoclasts, trabecular number or levels of hydroxyproline after treatment with MLT. OVX caused significant decrease in bone mineral density, but treatment with MLT was unable to reverse this effect. CONCLUSION: MLT may trigger microscopic changes in bone, and time of application is critical for clinical recovery. It can be effective in helping treat postmenopausal osteoporosis. However, it is contraindicated in women who have normal-functioning ovaries.     

Abnormal platelet amyloid-β precursor protein metabolism in SAMP8 mice: Evidence for peripheral marker in Alzheimer's disease

Senescence-accelerated mouse strains have proved to be an accelerated-aging model, which mimics numerous features with Alzheimer's disease (AD). Three, six, and nine-month senescence-accelerated resistant 1 and senescence-accelerated prone 8 (SAMP8) mice were used in the current study, to unravel potential mechanisms for dementia and explore new diagnostic approaches for AD. The amyloid-β (Aβ40) and Aβ42 levels were elevated in hippocampi and platelets from SAMP8, along with a reduced α-secretase expression and an enhanced β-secretase expression extent with age, compared to control mice. Furthermore, hippocampal Aβ40 and Aβ42 of SAMP8 were positively correlated with platelet of these mice with aging progression. In addition, β-γ-secretase-modulated proteolytic proceeding of amyloid precursor protein in platelet might work through the PI3K/Akt/GSK3β pathway. These results indicate that platelet could be a potential early marker in the periphery to study the age-correlative aggregation of the amyloid-β peptide in patients with AD, while still requiring the considerable study.     

Long-term effect of losartan administration on blood pressure, heart and structure of coronary artery of young spontaneously hypertensive rats

Alterations in geometry and structure of coronary arteries have marked consequences on blood flow to the respective area. We evaluated long-term effect of losartan on blood pressure (BP), heart weight/body weight (HW/BW), geometry and structure of septal branch of coronary artery (RS) of young SHR and Wistar rats. Four-week-old Wistar rats and SHR were used. Losartan was administered (20 mg/kg/day) in drinking water by gavage for 5 weeks. BP was measured by plethysmographic method. Cardiovascular system was perfused with a fixative (120 mm Hg). RS was processed for electron microscopy. Wall thickness of intima + media (WT), inner diameter (ID), cross-sectional area of intima + media (CSA), volume densities (VD) of endothelial cells (EC), extracellular matrix (ECM) of intima, smooth muscle cells (SMC) and ECM of media were evaluated. BP of 4-week-old SHR did not differ from that of Wistar rats. BP, HW/BW, WT, CSA, WT/ID, CSAs of SMC, ECM of media were increased in 9-week-old SHR, whereas their VD and CSA of EC were decreased. Losartan administration decreased BP and HW/BW in both groups. Geometry of RS was affected only in SHR (reduction of WT, CSA, WT/ID and increased of ID, circumferential tension, VD and CSA of EC). Losartan administration reduced BP and myocardial mass in both groups and beneficially affected geometry and structure of coronary artery in SHR.     

Effects of age on plasma levels of calcium-regulating hormones and bone status in male SAMP8 mice

This study was undertaken to examine whether the plasma levels of calcium-regulating hormones and bone status alter with age in male senescence accelerated mice (SAM), SAMP8. Age-matched senescence-resistant mice, SAMR1, were used as controls. The blood and femur samples were collected at 2.5 months of age (M) and then monthly from 3 to 12 M for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. With advancing age, the plasma calcitonin (CT) levels decreased progressively, and the plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) levels increased in both SAMR1 and SAMP8. The plasma calcium concentrations were maintained within a narrow range throughout the experimental period, while the plasma phosphorus (P) concentrations decreased with age in both strains. In contrast to SAMR1, the curves of age-related changes in the plasma CT levels and P concentrations were lower, and those in the plasma PTH levels were higher in SAMP8. The femoral bone densities and calcium contents increased gradually with age from the beginning of the experiment and peaked at 6 M in both strains, then declined. Those peaks were lower in SAMP8 than in SAMR1. These results indicate that the male SAMP8 develops osteoporotic signs earlier than SAMR1, and is proved to be a satisfactory animal model for longitudinal studies related to osteoporosis for men.