Effect of integration time on the morphometric,densitometric and mechanical properties of the mouse tibia

Micro-Computed Tomography (microCT) images are used to measure morphometric and densitometric properties of bone, and to develop finite element (FE) models to estimate mechanical properties. However, there are concerns about the invasiveness of microCT imaging due to the X-rays ionising radiation induced by the repeated scans on the same animal. Therefore, the best compromise between radiation dose and image quality should be chosen for each preclinical application. In this study, we investigated the effect of integration time (time the bone is exposed to radiation at each rotation step during microCT imaging) on measurements performed on the mouse tibia. Four tibiae were scanned at 10.4 µm voxel size using four different procedures, characterized by decreasing integration time (from 200 ms to 50 ms) and therefore decreasing nominal radiation dose (from 513 mGy to 128 mGy). From each image, trabecular and cortical morphometric parameters, spatial distribution of bone mineral content (BMC) in the whole tibia and FE-based estimations of stiffness and strength were obtained. A high-resolution scan (4.3 µm voxel size) was used to quantify measurement errors. Integration time had the largest effect on trabecular morphometric parameters (7–28%). Lower effects were observed on cortical parameters (1–3%), BMC (1–10%) distribution, and FE-based estimations of mechanical properties (1–3%). In conclusion, the effect of integration time on image-based measurements has been quantified. This data should be considered when defining the in vivo microCT scanning protocols in order to find the best compromise between nominal radiation exposure and accuracy in the estimation of bone parameters.     

Influence of gait loads on implant integration in rat tibiae: Experimental and numerical analysis

Implanted rat bones play a key role in studies involving fracture healing, bone diseases or drugs delivery among other themes. In most of these studies the implants integration also depends on the animal daily activity and musculoskeletal loads, which affect the implants mechanical environment. However, the tissue adaption to the physiological loads is often filtered through control groups or not inspected. This work aims to investigate experimentally and numerically the effects of the daily activity on the integration of implants inserted in the rat tibia, and to establish a physiological loading condition to analyse the peri-implant bone stresses during gait. Two titanium implants, single and double cortex crossing, are inserted in the rat tibia. The animals are caged under standard conditions and divided in three groups undergoing progressive integration periods. The results highlight a time-dependent increase of bone samples with significant cortical bone loss. The phenomenon is analysed through specimen-specific Finite Element models involving purpose-built musculoskeletal loads. Different boundary conditions replicating the post-surgery bone–implant interaction are adopted. The effects of the gait loads on the implants integration are quantified and agree with the results of the experiments. The observed cortical bone loss can be considered as a transient state of integration due to bone disuse atrophy, initially triggered by a loss of bone–implant adhesion and subsequently by a cyclic opening of the interface.     

Bone ingrowth on the surface of endosseous implants. Part 1: Mathematical model

Osseointegration, understood as an intimate apposition and interdigitation of bone to a biomaterial, is usually regarded as a major condition for the long-term clinical success of bone implants. Clearly, the anchorage of an implant to bone tissue critically relies on the formation of new bone between the implant and the surface of the old peri-implant bone and depends on factors such as the surface microtopography, chemical composition and geometry of the implant, the properties of the surrounding bone and the mechanical loading process. The main contribution of this work is the proposal of a new mathematical framework based on a set of reaction-diffusion equations that try to model the main biological interactions occurring at the surface of implants and is able to reproduce most of the above mentioned biological features of the osseointegration phenomenon. This is a two-part paper. In this first part, a brief biological overview is initially given, followed by the presentation and discussion of the model. In addition, two-dimensional finite element simulations of the bone-ingrowth process around a dental implant with two different surface properties are included to assess the validity of the model. Numerical solutions show the ability of the model to reproduce features such as contact/distance osteogenesis depending upon the specific surface microtopography. In Part 2 [Moreo, P., García-Aznar, J.M., Doblaré, M., 2008. Bone ingrowth on the surface of endosseous implants. Part 2: influence of mechanical stimulation, type of bone and geometry. J. Theor. Biol., submitted for publication], two simplified versions of the whole model are proposed. An analytical study of the stability of fixed points as well as the existence of travelling wave-type solutions has been done with both simplified models, providing a significant insight into the behaviour of the model and giving clues to interpret the effectiveness of recently proposed clinical therapies. Furthermore, we also show that, although the mechanical state of the tissue is not directly taken into account in the model equations, it is possible to analyse in detail the effect that mechanical stimulation would have on the predictions of the model. Finally, numerical simulations are also included in the second part of the paper, with the aim of looking into the influence of implant geometry on the osseointegration process.     

Impact loading history modulates hip fracture load and location: A finite element simulation study of the proximal femur in female athletes

Sideways falls impose high stress on the thin superolateral cortical bone of the femoral neck, the region regarded as a fracture-prone region of the hip. Exercise training is a natural mode of mechanical loading to make bone more robust. Exercise-induced adaptation of cortical bone along the femoral neck has been previously demonstrated. However, it is unknown whether this adaption modulates hip fracture behavior. The purpose of this study was to investigate the influence of specific exercise loading history on fall-induced hip fracture behavior by estimating fracture load and location with proximal femur finite element (FE) models created from magnetic resonance images (MRI) of 111 women with distinct exercise histories: 91 athletes (aged 24.7 ± 6.1 years, >8 years competitive career) and 20 women as controls (aged 23.7 ± 3.8 years). The athletes were divided into five groups based on typical loading patterns of their sports: high-impact (H-I: 9 triple-jumpers and 10 high jumpers), odd-impact (O-I: 9 soccer and 10 squash players), high-magnitude (H-M: 17 power-lifters), repetitive-impact (R-I: 18 endurance runners), and repetitive non-impact (R-NI: 18 swimmers). Compared to the controls, the H-I, O-I, and R-I groups had significantly higher (11–26%, p < 0.05) fracture loads. Also, the fracture location in the H-I and O-I groups was significantly more proximal (7–10%) compared to the controls. These results suggest that an exercise loading history of high impacts, impacts from unusual directions, or repetitive impacts increases the fracture load and may lower the risk of fall-induced hip fracture.     

Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model

Cryopreservation of female reproductive cells allows preservation of fertility and provides materials for research. Although freezing protocols have been optimized, and there is a high survival rate after thawing, the in vitro fertilization (IVF) pregnancy rate is still lower in cycles with cryopreserved oocytes, thus highlighting the importance of identifying intrinsic limiting factors characterizing the cells at time of freezing. The aim of the present study is to investigate in the mouse model the impact of reproductive aging and postovulatory aging on oocyte biological competence after vitrification. Metaphase II oocytes were vitrified soon after retrieval from young and reproductively old mice. Part of the oocytes from young animals was vitrified after 6 h incubation (in vitro aged oocytes). All classes of oocytes showed similar survival rate after vitrification. Moreover, vitrification did not alter chromosomal organization in young cells, whereas in vitro aged and old oocytes presented an increase of slightly aberrant metaphase configurations. Compared to fresh young oocytes, in vitro aged and old oocytes showed increased ROS levels which remained unchanged after vitrification. By contrast, cryopreservation significantly increased ROS production in young oocytes. Both the aging processes negatively impacted oocyte ability to undergo pronucleus formation and first cleavage after vitrification by stimulating cellular fragmentation. These results could be helpful for establishing the correct time table for cryopreservation in the laboratory routine and improving its application in reproductively old females. Moreover, our observations highlight the importance of oxidative stress protection during vitrification procedures.     

Computational analysis of glenohumeral joint growth and morphology following a brachial plexus birth injury

Children affected with brachial plexus birth injury (BPBI) undergo muscle paralysis. About 33% of affected children experience permanent osseous deformities of the glenohumeral joint. Recent evidence suggests that some cases experience restricted muscle longitudinal growth in addition to paralysis and reduced range of motion at the shoulder and elbow. It is unknown whether altered loading due to paralysis, muscle growth restriction and contracture, or static loading due to disuse is the primary driver of joint deformity after BPBI. This study uses a computational framework integrating finite element analysis and musculoskeletal modeling to examine the mechanical factors contributing to changes in bone growth and morphometry following BPBI. Simulations of 8 weeks of glenohumeral growth in a rat model of BPBI predicted that static loading of the joint is primarily responsible for joint deformation consistent with experimental measures of bone morphology, whereas dynamic loads resulted in normal bone growth. Under dynamic loading, glenoid version angle (GVA), glenoid inclination angle (GIA), and glenoid radius of curvature (GRC) (−1.3°, 38.2°, 2.5 mm respectively) were similar to the baseline values (−1.8°, −38°, 2.1 mm respectively). In the static case with unrestricted muscle growth, these measures increased in magnitude (5.2°, −48°, 3.5 mm respectively). More severe joint deformations were observed in GIA and GRC when muscle growth was restricted (GVA: 3.6°, GIA: −55°, GRC: 4.0 mm). Predicted morphology was consistent with literature reports of in vivo glenoid morphology following postganglionic BPBI. This growth model provides a framework for understanding the most influential mechanical factors driving glenohumeral deformity following BPBI.     

Long-term study of bone remodelling after femoral stem: a comparison between dexa and finite element simulation

A hip replacement with a cemented or cementless femoral stem produces an effect on the bone called adaptive remodelling, attributable to mechanical and biological factors. The objective of all of cementless prostheses designs has been to achieve a perfect transfer of loads in order to avoid stress-shielding, which produces an osteopenia. In order to quantify this, the long term and mass-produced study with dual energy X-ray absorptiometry (DEXA) is necessary. Finite element (FE) simulation makes possible the explanation of the biomechanical changes which are produced in the femur after stem implantation. The good correlation obtained between the results of the FE simulation and the densitometric study allow, on one hand, to explain from the point of view of biomechanical performance the changes observed in bone density in the long-term, where it is clear that these are due to a different transfer of load in the implanted model compared to the healthy femur; on the other hand, it validates the simulation model, in a way that it can be used in different conditions and at different time periods, to carry out a sufficiently precise prediction of the evolution of the bone density from the biomechanical behaviour in the interaction between the prosthesis and femur.     

Cervical spine morphology and ligament property variations: A finite element study of their influence on sagittal bending characteristics

Cervical spine finite element models reported in biomechanical literature usually represent a static morphology. Not considering morphology as a model parameter limits the predictive capabilities for applications in personalized medicine, a growing trend in modern clinical practice. The objective of the study was to investigate the influence of variations in spinal morphology on the flexion-extension responses, utilizing mesh-morphing-based parametrization and metamodel-based sensitivity analysis. A C5-C6 segment was used as the baseline model. Variations of intervertebral disc height, facet joint slope, facet joint articular processes height, vertebral body anterior-posterior depth, and segment size were parametrized. In addition, material property variations of ligaments were considered for sensitivity analysis. The influence of these variations on vertebral rotation and forces in the ligaments were analyzed. The disc height, segmental size, and body depth were found to be the most influential (in the cited order) morphology variations; while among the ligament material property variations, capsular ligament and ligamentum flavum influenced vertebral rotation the most. Changes in disc height influenced forces in the posterior ligaments, indicating that changes in the anterior load-bearing column of the spine could have consequences on the posterior column. A method to identify influential morphology variations is presented in this work, which will help automation efforts in modeling to focus on variations that matter. This study underscores the importance of incorporating influential morphology parameters, easily obtained through computed tomography/magnetic resonance images, to better predict subject-specific biomechanical responses for applications in personalized medicine.     

A collagen-based interface construct for the assessment of cell-dependent mechanical integration of tissue surfaces

The interface between any newly engineered tissue and pre-existing tissue is of great importance to tissue engineering; however, this process has so far been largely ignored, with few reports regarding the mechanical strength of newly integrated connective tissues surfaces. A new model system has been developed to generate a well-defined interface between two collagen lattices: one pre-contracted by resident fibroblasts and the other a cell-free wrapping gel. This construct can be cultured for prolonged periods (>2 weeks) and can also be fitted onto a mechanical testing system to measure the interface adhesive strength at the end of the culture time. Interface adhesive strength shows a six-fold increase after 1 week in culture, compared with the time-zero baseline. Observations of cell migration across the interface suggest that cell translocation in the three-dimensional matrix might play an important role in the integration process. In this new controlled geometry, normal and shear stresses at the interface can be analysed by finite element modelling and the areas at which debonding starts can be defined. The current experimental design permits solid multiple (homogeneous or heterogeneous) interface formation in vitro with a well-defined geometry and the possibility of measuring mechanical linkage. This design should enable many other factors affecting cell-driven interface strengthening to be investigated. This study was supported by the Fifth Framework Programme of the European Commission, “Biomechanical Interactions in Tissue Engineering and Surgical Repair (BITES)”.     

Analysis of the independent power of age-related,anthropometric and mechanical factors as determinants of the structure of radius and tibia in normal adults. A pQCT study

To compare the independent influence of mechanical and non-mechanical factors on bone features, multiple regression analyses were performed between pQCT indicators of radius and tibia bone mass, mineralization, design and strength as determined variables, and age or time since menopause (TMP), body mass, bone length and regional muscles’ areas as selected determinant factors, in Caucasian, physically active, untrained healthy men and pre- and post-menopausal women. In men and pre-menopausal women, the strongest influences were exerted by muscle area on radial features and by both muscle area and bone length on the tibia. Only for women, was body mass a significant factor for tibia traits. In men and pre-menopausal women, mass/design/strength indicators depended more strongly on the selected determinants than the cortical vBMD did (p<0.01-0.001 vs n.s.), regardless of age. However, TMP was an additional factor for both bones (p<0.01-0.001). The selected mechanical factors (muscle size, bone lengths) were more relevant than age/TMP or body weight to the development of allometrically-related bone properties (mass/design/strength), yet not to bone tissue “quality” (cortical vBMD), suggesting a determinant, rather than determined role for cortical stiffness. While the mechanical impacts of muscles and bone levers on bone structure were comparable in men and pre-menopausal women, TMP exerted a stronger impact than allometric or mechanical factors on bone properties, including cortical vBMD.     

A Density Distribution Algorithm for Bone Incorporating Local Orthotropy,Modal Analysis and Theories of Cellular Solids

An algorithm for bone remodeling is presented which allows for both a redistribution of density and a continuous change of principal material directions for the orthotropic material properties of bone. It employs a modal analysis to add density for growth and a local effective strain based analysis to redistribute density. General re-distribution functions are presented. The model utilizes theories of cellular solids to relate density and strength. The code predicts the same general density distributions and local orthotropy as observed in reality.     

螺旋粉虱产卵行为及产卵分泌物的形态和化学成分

螺旋粉虱Aleurodicus dispersus Russell具独特的产卵过程--成虫将卵产在叶片上排列成螺旋状并覆盖丝状蜡泌物。本研究利用数码摄像机拍摄记录螺旋粉虱的产卵过程,并采用体视显微镜、扫描电镜及气相色谱/质谱联用研究产卵分泌物的结构和化学成分。结果表明：单头雌成虫产卵时可形成典型螺旋轨迹,当其他雌成虫加入产卵时,其产卵行为包括跨越螺旋轨迹后继续在外围扩大螺旋圈、仅在螺旋轨迹表面穿过及在螺旋轨迹外围直接产卵3种情况。 在体视镜及扫描电镜下产卵分泌物呈交错的白色丝状结构。 另用GC/MS联用检测仪检测发现产卵分泌物主要是一些饱和长链烃（66.21%）和芳香酯类（26%）,同时有少量酸和酚。 其中烃类化合物含有3种碘代物（10.64%）,其他为直链烃类,碳原子数介于C₈～C₃₀;芳香酯类化合物有3种,碳原子数介于C₁₆～C₂₄,其中含量最大的是邻苯二甲酸二丁酯（21.19%）。 最后讨论了产卵分泌物在吸引初孵爬虫、抵御天敌及阻碍其他植食性者进入螺旋圈的可能性。     

Proteomic profiling of adipose tissue from Zmpste24-/- mice, a model of lipodystrophy and premature aging, reveals major changes in mitochondrial function and vimentin processing

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.     

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.     

On the importance of 3D,geometrically accurate,and subject-specific finite element analysis for evaluation of in-vivo soft tissue loads

Pressure ulcers are a type of local soft tissue injury due to sustained mechanical loading and remain a common issue in patient care. People with spinal cord injury (SCI) are especially at risk of pressure ulcers due to impaired mobility and sensory perception. The development of load improving support structures relies on realistic tissue load evaluation e.g. using finite element analysis (FEA). FEA requires realistic subject-specific mechanical properties and geometries. This study focuses on the effect of geometry. MRI is used for the creation of geometrically accurate models of the human buttock for three able-bodied volunteers and three volunteers with SCI. The effect of geometry on observed internal tissue deformations for each subject is studied by comparing FEA findings for equivalent loading conditions. The large variations found between subjects confirms the importance of subject-specific FEA.     

Finite element simulation of the behavior of the periodontal ligament: A validated nonlinear contact model

Due to its significance in tooth movement, the stress/deformation field of periodontium and the alveolar bone remodeling process, periodontal ligament (PDL) cannot be excluded from the studies investigating dental biomechanics regarding its excessive deformability. Therefore, many analytical and numerical researches are carried out to simulate its response and to create a constitutive model via experiments intending to discover the material properties of PDL. The aim of this study is to formulate a user specified contact model that can be used in conjunction with finite element (FE) software and reflects PDL’s influence on neighboring structures based on the currently available information, without requiring an actual volumetric finite element mesh of ligament. The results show good agreement with available experimental tooth mobility data. Smooth stress fields are obtained on the tooth root and alveolar bone, which is a significant aspect in bone-remodeling studies. The advantage of simulating PDL as a contact model at the interface of tooth root and the alveolar process instead of a solid-meshed FE model with poor geometric morphology and/or very dense mesh is expected to save pre/post-processing workforce, to increase the accuracy and to contribute to the smoothness of interface stress distributions.     

Subject-specific planning of femoroplasty: A combined evolutionary optimization and particle diffusion model approach

A potential effective treatment for prevention of osteoporotic hip fractures is augmentation of the mechanical properties of the femur by injecting it with agents such as (PMMA) bone cement – femoroplasty. The operation, however, is only in research stage and can benefit substantially from computer planning and optimization. We report the results of computational planning and optimization of the procedure for biomechanical evaluation. An evolutionary optimization method was used to optimally place the cement in finite element (FE) models of seven osteoporotic bone specimens. The optimization, with some inter-specimen variations, suggested that areas close to the cortex in the superior and inferior of the neck and supero-lateral aspect of the greater trochanter will benefit from augmentation. We then used a particle-based model for bone cement diffusion simulation to match the optimized pattern, taking into account the limitations of the actual surgery, including limited volume of injection to prevent thermal necrosis. Simulations showed that the yield load can be significantly increased by more than 30%, using only 9 ml of bone cement. This increase is comparable to previous literature reports where gross filling of the bone was employed instead, using more than 40 ml of cement. These findings, along with the differences in the optimized plans between specimens, emphasize the need for subject-specific models for effective planning of femoral augmentation.     

Engineered bone culture in a perfusion bioreactor: a 2D computational study of stationary mass and momentum transport

Successful bone cell culture in large implants still is a challenge to biologists and requires a strict control of the physicochemical and mechanical environments. This study analyses from the transport phenomena viewpoint the limiting factors of a perfusion bioreactor for bone cell culture within fibrous and porous large implants (2.5 cm in length, a few cubic centimetres in volume, 250 μm in fibre diameter with approximately 60% porosity).

A two-dimensional mathematical model, based upon stationary mass and momentum transport in these implants is proposed and numerically solved. Cell oxygen consumption, in accordance theoretically with the Michaelis–Menten law, generates non linearity in the boundary conditions of the convection diffusion equation. Numerical solutions are obtained with a commercial code (Femlab® 3.1; Comsol AB, Stockholm, Sweden). Moreover, based on the simplification of transport equations, a simple formula is given for estimating the length of the oxygen penetration within the implant.

Results show that within a few hours of culture process and for a perfusion velocity of the order of 10^? 4 m s^? 1, the local oxygen concentration is everywhere sufficiently high to ensure a suitable cell metabolism. But shear stresses induced by the fluid flow with such a perfusion velocity are found to be locally too large (higher than 10^? 3 Pa). Suitable shear stresses are obtained by decreasing the velocity at the inlet to around 2 × 10^? 5 m s^? 1. But consequently hypoxic regions (low oxygen concentrations) appear at the downstream part of the implant.

Thus, it is suggested here that in the determination of the perfusion flow rate within a large implant, a compromise between oxygen supply and shear stress effects must be found in order to obtain a successful cell culture.     

Study of thermal behavior of a biological tissue: An equivalence of Pennes bioheat equation and Wulff continuum model

Equivalence of Pennes bioheat equation (PBHE) and Wulff continuum model (WCM) is established for a 1-D planar tissue. The derived condition of equivalence is specific to tissue without metabolic heat generation. Mathematical analysis is carried out to relate blood perfusion rate and local mean blood velocity that are needed in the analysis of WCM. It is found that the local mean blood velocity in a tissue is a direct function of square root of blood perfusion rate. This functional dependence is also established numerically by having same solution obtained from PBHE and WCM. Analysis is also done to check how closely the derived relation can be used for practical cases of breast tissue with and without a tumor. Blood velocity is a very important physiological quantity. Its measurement is a difficult process and requires a state-of-the-art technique. The proposed relation allows its computation merely from the knowledge of blood perfusion rate.     

Time-course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age-related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial-encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age-related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8-hydroxyguanosine (8-OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial-encoded genes in complexes I, III, IV, and V of the respiratory chain in 12- and 18-month-old C57BL6 mice compared to 2-month-old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up-regulation of mitochondrial genes in 12- and 18-month-old C57BL6 mice, mRNA expression in 24-month-old C57BL6 mice was decreased, suggesting that compensation maintained by the up-regulated genes cannot be sustained and that the down-regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over-expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8-OHG and cytochrome c revealed increased 8-OHG and cytochrome c in 12-month-old C57BL6 mice, suggesting that age-related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double-labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8-OHG suggest that specific neuronal populations undergo oxidative damage. Further, double-labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age-related diseases such as Alzheimer's disease and Parkinson's disease.