Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature

Mammalian hibernators undergo a remarkable phenotypic switch that involves profound changes in physiology, morphology, and behavior in response to periods of unfavorable environmental conditions. The ability to hibernate is found throughout the class Mammalia and appears to involve differential expression of genes common to all mammals, rather than the induction of novel gene products unique to the hibernating state. The hibernation season is characterized by extended bouts of torpor, during which minimal body temperature (Tb) can fall as low as -2.9 degrees C and metabolism can be reduced to 1% of euthermic rates. Many global biochemical and physiological processes exploit low temperatures to lower reaction rates but retain the ability to resume full activity upon rewarming. Other critical functions must continue at physiologically relevant levels during torpor and be precisely regulated even at Tb values near 0 degrees C. Research using new tools of molecular and cellular biology is beginning to reveal how hibernators survive repeated cycles of torpor and arousal during the hibernation season. Comprehensive approaches that exploit advances in genomic and proteomic technologies are needed to further define the differentially expressed genes that distinguish the summer euthermic from winter hibernating states. Detailed understanding of hibernation from the molecular to organismal levels should enable the translation of this information to the development of a variety of hypothermic and hypometabolic strategies to improve outcomes for human and animal health.     

Role of the pineal gland in hibernators: a concept proposed to clarify why hibernators have to leave torpor and sleep

The contention that torpor during hibernation might not be a kind of sleep but a kind of sleep deprivation may be correct, as it can be conceptually explained. Sleep is part of life and cannot be part of the torpor state, which has no reactions. The state of torpor has a time limit and, if this is not observed, death follows by freezing. The evolutional differences particularly in pineal gland physiology may explain the differences between the blueprints governing vital reflexes in humans and hibernators.     

Similar damage initiation but different failure behavior in trabecular and cortical bone tissue

The mechanical properties of bone tissue are reflected in its micro- and nanostructure as well as in its composition. Numerous studies have compared the elastic mechanical properties of cortical and trabecular bone tissue and concluded that cortical bone tissue is stiffer than trabecular bone tissue. This study compared the progression of microdamage leading to fracture and the related local strains during this process in trabecular and cortical bone tissue. Unmachined single bovine trabeculae and similarly-sized cortical bovine bone samples were mechanically tested in three-point bending and concomitantly imaged to assess local strains using a digital image correlation technique. The bone whitening effect was used to detect microdamage formation and propagation. This study found that cortical bone tissue exhibits significantly lower maximum strains (trabecular 36.6%±14% vs. cortical 22.9%±7.4%) and less accumulated damage (trabecular 16100±8800 pix/mm2 vs. cortical 8000±3400 pix/mm2) at failure. However, no difference was detected for the maximum local strain at whitening onset (trabecular 5.8%±2.6% vs. cortical 7.2%±3.1%). The differences in elastic modulus and mineral distribution in the two tissues were investigated, using nanoindentation and micro-Raman imaging, to explain the different mechanical properties found. While cortical bone was found to be overall stiffer and more highly mineralized, no apparent differences were noted in the distribution of modulus values or mineral density along the specimen diameter. Therefore, differences in the mechanical behavior of trabecular and cortical bone tissue are likely to be in large part due to microstructural (i.e. orientation and distribution of cement lines) and collagen related compositional differences.     

Black Bears With Longer Disuse (Hibernation) Periods Have Lower Femoral Osteon Population Density and Greater Mineralization and Intracortical Porosity

Intracortical bone remodeling is persistent throughout life, leading to age related increases in osteon population density (OPD). Intracortical porosity also increases with age in many mammals including humans, contributing to bone fragility and fracture risk. Unbalanced bone resorption and formation during disuse (e.g., physical inactivity) also increases intracortical porosity. In contrast, hibernating bears are a naturally occurring model for the prevention of both age‐related and disuse osteoporoses. Intracortical bone remodeling is decreased during hibernation, but resorption and formation remain balanced. Black bears spend 0.25–7 months in hibernation annually depending on climate and food availability. We found longer hibernating bears demonstrate lower OPD and higher cortical bone mineralization than bears with shorter hibernation durations, but we surprisingly found longer hibernating bears had higher intracortical porosity. However, bears from three different latitudes showed age‐related decreases in intracortical porosity, indicating that regardless of hibernation duration, black bears do not show the disuse‐ or age‐related increases in intracortical porosity which is typical of other animals. This ability to prevent increases in intracortical porosity likely contributes to their ability to maintain bone strength during prolonged periods of physical inactivity and throughout life. Improving our understanding of the unique bone metabolism in hibernating bears will potentially increase our ability to develop treatments for age‐ and disuse‐related osteoporoses in humans. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.     

Bone histomorphometric changes in the femoral neck of aging and ovariectomized rats

Background: This study characterizes the changes in cortical and cancellous bone and cross sectional moment of inertia of the femoral neck from aging and ovariectomized (ovx'd) rats to determine their role in the previously reported ovx-induced reduction of mechanical strength in the femoral neck. Methods: Undecalcified double-fluorescent lableled cross sections of femoral neck of 3.5-, 5.5-, 6.5-, and 8.5-month-old female rats and rats ovx'd at 3.5 months for 2, 3, and 5 months of 45 rats were studied. The estimated endocortical and trabecular surfaces, cortical and cancellous bone histomorphometry, and cortical moment of inertia were determined. Results: The femoral neck was adding cortical bone between 3.5 and 5.5 months of age by increasing cortical thickness and decreasing marrow cavity area. No change of cortical bone mass was found between 5.5 and 8.5 months and the same amount of cancellous bone was observed between 3.5 and 8.5 months of age. Ovariectomy-induced cancellous, but not cortical bone loss. The loss was due to a transient ovx-induced negative bone balance that by 5 months post-ovx produced a 42% loss in trabecular bone while the histomorphometry profiles were the same as controls. The crosssectional moment of inertia increased with age but did not differ significantly between ovx'd and controls. Conclusions: Our findings suggest that the ovx-induced cancellous bone loss could be a contributing factor to the reduced mechanical strength in the femoral neck of ovx'd rats reported previously. © 1995 Wiley-Liss, Inc.     

Seasonal changes in the intestinal immune system of hibernating ground squirrels

Hibernation is associated with a prolonged fast (5-8 mo) which has the potential to affect intestinal immunity. We examined several aspects of the intestinal immune system in summer (non-hibernating) and hibernating ground squirrels. Peyer's patches were largely unaffected by hibernation, but numbers of intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) were greater in hibernators compared with summer. Hibernator IEL were less mature as demonstrated by low numbers of cells expressing activation-associated markers and co-receptors. Compared with summer, the percentage of B cells was higher and percentage of T cells was lower in the hibernator LPL. Hibernation was associated with greater mucosal levels of IFN-gamma, TNF-alpha, IL-10 and IL-4, but IL-6 and TGF-beta were unchanged. Mucosal IgA levels were greater in entrance and torpid hibernators compared with summer. The results suggest that modifications of the intestinal immune system during hibernation may help preserve gut integrity throughout the winter fast.     

股骨头坏死松质骨生物力学特征的空间分布规律

目的 探究股骨头坏死松质骨微观结构参数和力学性能的空间分布规律,为临床诊断中科学评估病灶对病情的影响提供理论依据。方法 结合影像学测量和数值模拟方法,定量分析不同区域松质骨的微观结构参数和力学性能,分别从冠状面、矢状面和水平面投影方向上探究松质骨生物力学特征的空间分布特点。结果 在股骨头冠状面和矢状面投影方向上,松质骨的微观结构特征与力学性能大致呈Y型分布,Y形区域内松质骨的力学性能高于其他区域,该分布特点与股骨头内的主压应力束的位置相吻合。结论 位于股骨头Y形区域内的坏死灶对股骨头内应力分布的影响程度会更大,更有可能导致病情恶化。临床诊断中应充分考虑坏死灶和Y形区域的位置关系。     

骨质疏松患者股骨头不同区域骨结构与生物力学分析

目的探讨老年股骨颈骨折病人的股骨头样本各个区域的结构和生物力学性能差异,研究不同区域显微结构和生物力学特征及其对内固定物的影响。方法收集20个老年股骨颈骨折病人关节置换术后股骨头标本,以股骨头表面解剖标志点为参照,将股骨头按平分方法分为外侧、内侧、中间三部分。确定环钻的位置和钻取方向,用环钻于不同区域钻取直径10 mm、高10 mm圆柱形松质骨柱。通过Micro-CT系统扫描分析,分析不同区域内松质骨柱数据,包括骨体积分数(BVF)、骨小梁间隙(Tb.Sp)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨表面积体积比(BS/BV)、结构模型指数(SMI)。应用微有限元计算不同区域骨组织的力学差异。结果老年股骨头颈内骨质含量下降,显微结构和生物力学性能区域变化明显,中间区域的骨结构和力学性能明显优于内侧和外侧。结论股骨头中间部位骨骼结构和力学强度明显优于内侧和外侧,在临床治疗骨质疏松股骨颈骨折的时候需要充分考虑内固定的部位。     

Hibernation induces immune changes in the lung of 13-lined ground squirrels (Ictidomys tridecemlineatus)

During hibernation, significant changes occur in the systemic and intestinal immune populations. We found that the lungs of hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus) also undergo shifts in immune phenotype. Within the population of mononuclear cells, the percentage of T cells increases and the percentage of CD11b/c⁺ cells decreases in hibernators. E-selectin, which promotes endothelial attachment, increases during arousal from torpor. Levels of the anti-inflammatory cytokine interleukin (IL)-10 in the lung are lower during hibernation while levels of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α remain constant. Expression of suppressor of cytokine signaling (SOCS) proteins is also decreased in torpid hibernators. Our data point to a unique immune phenotype in the lung of hibernating ground squirrels in which certain immunosuppressive proteins are downregulated while some potentially inflammatory proteins are maintained or amplified. This indicates that the lung houses an immune population that can potentially respond to antigenic challenge during hibernation.     

Black bear femoral geometry and cortical porosity are not adversely affected by ageing despite annual periods of disuse (hibernation)

Disuse (i.e. inactivity) causes bone loss, and a recovery period that is 2-3 times longer than the inactive period is usually required to recover lost bone. However, black bears experience annual disuse (hibernation) and remobilization periods that are approximately equal in length, yet bears maintain or increase cortical bone material properties and whole bone mechanical properties with age. In this study, we investigated the architectural properties of bear femurs to determine whether cortical structure is preserved with age in bears. We showed that cross-sectional geometric properties increase with age, but porosity and resorption cavity density do not change with age in skeletally immature male and female bears. These findings suggest that structural properties substantially contribute to increasing whole bone strength with age in bears, particularly during skeletal maturation. Porosity was not different between skeletally immature and mature bears, and showed minimal regional variations between anatomical quadrants and radial positions that were similar in pattern and magnitude between skeletally immature and mature bears. We also found gender dimorphisms in bear cortical bone properties: females have smaller, less porous bones than males. Our results provide further support for the idea that black bears possess a biological mechanism to prevent disuse osteoporosis.     

A non-destructive technique for 3-D microstructural phenotypic characterisation of bones in genetically altered mice: preliminary data in growth hormone transgenic animals and normal controls

A non-destructive, three-dimensional technique for microstructural phenotypic characterisation of skeletal elements in genetically altered mice is presented. Preliminary data in bovine growth-hormone transgenic animals and control littermates are shown. The technique is based on microcomputed tomography (microCT) and digital postprocessing and allows for a differential quantitative analysis of the cortical and trabecular bone compartments in the axial and peripheral skeleton. The distal femora and the first lumbar vertebral bodies of six animals were CT scanned in the axial plane with an isotropic resolution of 20 microm. The periostal surface and the marrow spaces were segmented fully automatically, and the trabecular and cortical compartments were separated interactively. After 3-D reconstruction, various regions of interest (diaphyseal, metaphyseal and epiphyseal) were selected for the analysis. The femora and vertebrae of the transgenic animals showed obvious differences in size, shape, and trabecular arrangement compared with the control animals. The total bone mass was increased by a factor of two to three, but the trabecular bone was increased much more (up to 12 times) than the cortical bone. The transgenic animals showed an increased ratio of trabecular vs cortical bone (0.90 to 1.27 vs 0.14 to 0.36 in the femoral diaphysis) and an elevated trabecular bone volume fraction (49% to 73% vs 18% to 43% in the femoral metaphysis). The mean 3-D cortical thickness was similar in the normal and transgenic animals (values between 93 microm and 232 microm in the dia- and metaphyses), but the minimal cortical thickness was lower in the transgenic animals (22 to 31 microm vs 54 microm to 110 microm in the diaphysis). The technique presented is suitable for phenotypic characterisation of bone structure in genetically altered mice.     

Effect of ovariectomy on BMD, micro-architecture and biomechanics of cortical and cancellous bones in a sheep model

Osteoporotic/osteopenia fractures occur most frequently in trabeculae-rich skeletal sites. The purpose of this study was to use a high-resolution micro-computed tomography (micro-CT) and dual energy X-ray absorptionmeter (DEXA) to investigate the changes in micro-architecture and bone mineral density (BMD) in a sheep model resulted from ovariectomy (OVX). Biomechanical tests were performed to evaluate the strength of the trabecular bone. Twenty adult sheeps were randomly divided into three groups: sham group (n=8), group 1 (n=4) and group 2 (n=8). In groups 1 and 2, all sheep were ovariectomized (OVX); in the sham group, the ovaries were located and the oviducts were ligated. In all animals, BMD for lumbar spine was obtained during the surgical procedure. BMD at the spine, femoral neck and femoral condyle was determined 6 months (group 1) and 12 months (group 2) post-OVX. Lumbar spines and femora were obtained and underwent BMD scan, micro-CT analysis. Compressive mechanical properties were determined from biopsies of vertebral bodies and femoral condyles. BMD, micro-architectural parameters and mechanical properties of cancellous bone did not decrease significantly at 6 months post-OVX. Twelve months after OVX, BMD, micro-architectural parameters and mechanical properties decreased significantly. The results of linear regression analyses showed that trabecular thickness (Tb.Th) (r=0.945, R2=0.886) and bone volume fraction (BV/TV) (r=0.783, R2=0.586) had strong (R2>0.5) correlation to compression stress. In OVX sheep, changes in the structural parameters of trabecular bone are comparable to the human situation during osteoporosis was induced. The sheep model presented seems to meet the criteria for an osteopenia model for fracture treatment with respect to morphometric and mechanical properties. But the duration of OVX must be longer than 12 months to ensure the animal model can be established successfully.     

Characterization of bone quality using computer-extracted radiographic features.

Both bone mineral density (BMD) and trabecular structure are important determinates of bone mechanical properties. However, neither BMD or trabecular structural features can completely explain the variations in bone mechanical properties. In this study, we combine BMD and bone structural features to characterize bone mechanical behavior. Radiographs were obtained from 34 femoral neck specimens excised during total hip arthroplasties. Each neck radiograph was digitized and a region of interest (ROI) was selected from the medial side of the femoral neck. Textural features, the global Minkoswski dimension and trabecular orientation, were extracted from each ROI image using Minkowski dimension analysis. The BMD of each specimen was measured using dual-energy x-ray absorptiometry (DXA) and subsequently normalized by bone size as measured from a standard pelvis radiograph. Mechanical testing was performed on the trabecular bone cubes machined from each femoral neck to yield bone mechanical properties. Multiple regression was performed to select the best features to predict bone mechanical properties. The results suggest that, using multiple predictors including normalized BMD structural features, and patient age, the coefficients of determination (R2) improved over the use of BMD alone. For bone strength, the R2 was improved from 0.24 using conventional BMD to 0.48 using a four-predictor model. Similar results were obtained in the prediction of Young's modulus, i.e., the R2 was improved from 0.25 to 0.55 in going from the model using conventional BMD to a four-predictor model. This study demonstrates the contributions of normalized BMD, structural features, and age to bone mechanical properties, and suggests a potential method for the noninvasive evaluation of bone mechanical properties.     

局部注射辛伐他汀对骨质疏松大鼠股骨髁骨小梁的改建效应

背景：局部注射具有成骨作用的辛伐他汀,可显著增加骨质疏松大鼠股骨颈及股骨髁部的骨密度及力学强度,分析局部注射辛伐他汀对股骨髁骨小梁的影响。 目的：进一步研究骨质疏松大鼠股骨内局部注射辛伐他汀对股骨髁骨小梁的影响。为将辛伐他汀应用于临床骨质疏松局部治疗提供实验基础。 方法：18只雌性SD大鼠双侧卵巢切除后3个月,制备大鼠骨质疏松模型。实验大鼠随机数字表法均分为3组,分别在实验大鼠的右侧股骨髓腔内单次注射辛伐他汀溶液5 mg、10 mg,对照组单纯注射空白载体。分别在注射后1个月处死大鼠并取材。Micro-CT扫描并定量分析骨组织形态变化。 结果与结论：给药后1个月,Micro-CT扫描结果显示,辛伐他汀治疗组的骨微结构参数如骨皮质厚度、骨小梁密度及连接率明显优于对照组。说明疏松骨骼单次注射小剂量辛伐他汀可显著促进股骨髁部骨小梁改建,改善骨骼微结构,可为强化局部、防治骨质疏松骨折的新选择进一步提供实验基础。     

Hibernation "trigger" alters renal function in the primate

Macaque monkeys acclimatized to a restraint chair were fitted with indwelling venous and urinary catheters. After basal rates of urine production and creatinine clearance were determined, a 50 mg dose of plasma dialysate albumin fraction obtained from the woodchuck was administered intravenously in a total volume of 2.5 ml. Plasma fractions were collected during the winter interval of hibernation (hibernation "trigger" or HT), or during the summer active (SAWA) period. Although the SAWA fraction exerted no effects on renal function, HT caused a significant reduction in creatinine clearance. In addition, a tendency toward reduced urine flow and creatinine production occurred following the HT infusion. These findings suggest that over and above the hypothermia, aphagia and opioid-like behavioral depression induced by HT, the albumin fraction (HT) present endogenously in the woodchuck during winter torpor, exerts a direct action on the kidney of the primate, possibly on the mechanisms underlying glomerular filtration and the tubular reabsorption process.     

The relationship between fasting-induced torpor,sleep, and wakefulness in laboratory mice

Study ObjectivesTorpor is a regulated and reversible state of metabolic suppression used by many mammalian species to conserve energy. Whereas the relationship between torpor and sleep has been well-studied in seasonal hibernators, less is known about the effects of fasting-induced torpor on states of vigilance and brain activity in laboratory mice.MethodsContinuous monitoring of electroencephalogram (EEG), electromyogram (EMG), and surface body temperature was undertaken in adult, male C57BL/6 mice over consecutive days of scheduled restricted feeding.ResultsAll animals showed bouts of hypothermia that became progressively deeper and longer as fasting progressed. EEG and EMG were markedly affected by hypothermia, although the typical electrophysiological signatures of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and wakefulness enabled us to perform vigilance-state classification in all cases. Consistent with previous studies, hypothermic bouts were initiated from a state indistinguishable from NREM sleep, with EEG power decreasing gradually in parallel with decreasing surface body temperature. During deep hypothermia, REM sleep was largely abolished, and we observed shivering-associated intense bursts of muscle activity.ConclusionsOur study highlights important similarities between EEG signatures of fasting-induced torpor in mice, daily torpor in Djungarian hamsters and hibernation in seasonally hibernating species. Future studies are necessary to clarify the effects on fasting-induced torpor on subsequent sleep.     

Femoral peak bone mass and osteoclast number in an animal model of age-related spontaneous osteopenia

Background: SAMP6 was developed as a murine model of age-related spontaneous osteopenia characterized by low peak bone mass. A morphometric study of the growing femur in SAMP6 and sex-matched SAMP2 at 10 days to 4 months of age was done to examine the pathogenic process related to osteopenia. Methods: Age-related changes in cortical bone thickness, femur score, trabecular bone volume, thickness of epiphyseal growth plate, number of osteoclasts, and osteoclast surface were measured with a computerized image analyzer. Osteoclasts were examined cytomorphometrically after TRAP (tartrate resistant acid phosphatase) staining of the femoral sections. Results: Cortical bone thickness and femur score increased significantly with age, while trabecular bone volume decreased significantly. Comparing mean values of cortical bone thickness, femur score and trabecular bone volume, we noted significantly lower mean values in SAMP6 than in SAMP2 mice. These significant inter-stain differences first became evident in 20–40-day-old mice, but there was no significant difference in thickness of the epiphyseal growth plate between the two strains. The mean values of the number of osteoclasts per unit none surface length and of the osteoclast surface in SAMP6 were significantly greater than in age- and sex-matched SAMP2. Histograms of distribution of size of osteoclasts of 40-day-old male mice revealed that larger ones were more frequently seen in SAMP6. Furthermore, the ratio of osteoclasts/TRAP positive cells free in the bone marrow cavity was significantly higher in SAMP6 than in SAMP2. Conclusion: Activated bone resorption may play a role in the osteopenia seen in SAMP6. © 1995 Wiley-Liss, Inc.     

Mechanical properties of OI type III bone tissue measured by nanoindentation

Nanoindentation was used to characterize the intrinsic mechanical properties of bone tissue from eight (8) children with type III Osteogenesis Imperfecta (OI). The bone samples were harvested from the cortex portion at the site of bowing (the mid 2/3 of the shaft of the tibia/femur). Unlike normal bone tissue, OI type III cortical bone exhibited more isotropic material properties. Young's modulus and hardness values measured in the longitudinal direction did not show significant differences from the transverse measurements. No differences were observed in modulus or hardness in an analysis of the cortical and trabecular samples. The deformation patterns of the OI type III bone during nanoindentation were found to be similar to those of normal adult bone in an analysis of the ratio of modulus to hardness. No correlation was found between nanoindentation measurement and age in an analysis of regression.