Morphometric modelling of ageing in the human pubic symphysis : sexual dimorphism in an Australian population

Despite the prominent use of the pubic symphysis for age estimation in forensic anthropology, little has been documented regarding the quantitative morphological and micro-architectural changes of this surface. Specifically, utilising post-mortem computed tomography data from a large, contemporary Australian adult population, this study aimed to evaluate sexual dimorphism in the morphology and bone composition of the symphyseal surface; and temporal characterisation of the pubic symphysis in individuals of advancing age. The sample consisted of multi-slice computed tomography (MSCT) scans of the pubic symphysis(slice thickness: 0.5 mm, overlap: 0.1 mm) of 200 individuals of Caucasian ancestry aged 15–70 years, obtained in 2011. Surface rendering reconstruction of the symphyseal surface was conducted in OsiriX1 (v.4.1) and quantitative analyses in Rapidform XOSTM and OsteomeasureTM. Morphometric variables including inter-pubic distance, surface area, circumference, maximum height and width of the symphyseal surface and micro-architectural assessment of cortical and trabecular bone compositions were quantified using novel automated engineering software capabilities. The major results of this study are correlated with the macroscopic ossification and degeneration pattern of the symphyseal surface, demonstrating significant age-related changes in the morphometric and bone tissue variables between 15 and 70 years. Regardless of sex, the overall dimensions of the symphyseal surface increased with age, coupled with a decrease in bone mass in the trabecular and cortical bone compartments. Significant differences between the ventral, dorsal and medial cortical surfaces were observed, which may be correlated to bone formation activity dependent on muscle activity and ligamentous attachments. Our study demonstrates significant sexual dimorphism at this site, with males exhibiting greater surface dimensions than females. These baseline results provide a detailed insight into the changes in the structure of the pubic symphysis with ageing and sexually dimorphic features associated with the cortical and trabecular bone profiles.

Near infrared spectroscopy for rapid determination of Mankin score components : a potential tool for quantitative characterization of articular cartilage at surgery

PURPOSE The purpose of this study was to demonstrate the potential of near infrared (NIR) spectroscopy for characterizing the health and degenerative state of articular cartilage based on the components of the Mankin score. METHODS Three models of osteoarthritic degeneration induced in laboratory rats by anterior cruciate ligament (ACL) transection, meniscectomy (MSX), and intra-articular injection of monoiodoacetate (1 mg) (MIA) were used in this study. Degeneration was induced in the right knee joint; each model group consisted of 12 rats (N = 36). After 8 weeks, the animals were euthanized and knee joints were collected. A custom-made diffuse reflectance NIR probe of 5-mm diameter was placed on the tibial and femoral surfaces, and spectral data were acquired from each specimen in the wave number range of 4,000 to 12,500 cm(-1). After spectral data acquisition, the specimens were fixed and safranin O staining (SOS) was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis, with spectral preprocessing and wavelength selection technique, the spectral data were then correlated to the structural integrity (SI), cellularity (CEL), and matrix staining (SOS) components of the Mankin score for all the samples tested. RESULTS ACL models showed mild cartilage degeneration, MSX models had moderate degeneration, and MIA models showed severe cartilage degenerative changes both morphologically and histologically. Our results reveal significant linear correlations between the NIR absorption spectra and SI (R(2) = 94.78%), CEL (R(2) = 88.03%), and SOS (R(2) = 96.39%) parameters of all samples in the models. In addition, clustering of the samples according to their level of degeneration, with respect to the Mankin components, was also observed. CONCLUSIONS NIR spectroscopic probing of articular cartilage can potentially provide critical information about the health of articular cartilage matrix in early and advanced stages of osteoarthritis (OA). CLINICAL RELEVANCE This rapid nondestructive method can facilitate clinical appraisal of articular cartilage integrity during arthroscopic surgery.

Experimental and numerical investigation of strain-rate dependent mechanical properties of single living cells

The objective of this project is to investigate the strain-rate dependent mechanical behaviour of single living cells using both experimental and numerical techniques. The results revealed that living cells behave as porohyperlastic materials and that both solid and fluid phases within the cells play important roles in their mechanical responses. The research reported in this thesis provides a better understanding of the mechanisms underlying the cellular responses to external mechanical loadings and of the process of mechanical signal transduction in living cells. It would help us to enhance knowledge of and insight into the role of mechanical forces in supporting tissue regeneration or degeneration.

Dynamic input to determine hip joint moments, power and work on the prosthetic limb of transfemoral amputees: ground reactions vs knee reactions

Study Design: Comparative analysis Background: Calculations of lower limbs kinetics are limited by floor-mounted force-plates. Objectives: Comparison of hip joint moments, power and mechanical work on the prosthetic limb of a transfemoral amputee calculated by inverse dynamics using either the ground reactions (force-plates) or knee reactions (transducer). Methods: Kinematics, ground reactions and knee reactions were collected using a motion analysis system, two force-plates and a multi-axial transducer mounted below the socket, respectively. Results: The inverse dynamics using ground reactions under-estimated the peaks of hip energy generation and absorption occurring at 63 % and 76 % of the gait cycle (GC) by 28 % and 54 %, respectively. This method over-estimated a phase of negative work at the hip (from 37 %GC to 56 %GC) by 24%. It under-estimated the phases of positive (from 57 %GC to 72 %GC) and negative (from 73 %GC to 98 %GC) work at the hip by 11 % and 58%, respectively. Conclusions: A transducer mounted within the prosthesis has the capacity to provide more realistic kinetics of the prosthetic limb because it enables assessment of multiple consecutive steps and a wide range of activities without issues of foot placement on force-plates. CLINICAL RELEVANCE The hip is the only joint that an amputee controls directly to set in motion the prosthesis. Hip joint kinetics are associated with joint degeneration, low back pain, risks of fall, etc. Therefore, realistic assessment of hip kinetics over multiple gait cycles and a wide range of activities is essential.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

Application of higher-order spectra for automated grading of diabetic maculopathy

Diabetic macular edema (DME) is one of the most common causes of visual loss among diabetes mellitus patients. Early detection and successive treatment may improve the visual acuity. DME is mainly graded into non-clinically significant macular edema (NCSME) and clinically significant macular edema according to the location of hard exudates in the macula region. DME can be identified by manual examination of fundus images. It is laborious and resource intensive. Hence, in this work, automated grading of DME is proposed using higher-order spectra (HOS) of Radon transform projections of the fundus images. We have used third-order cumulants and bispectrum magnitude, in this work, as features, and compared their performance. They can capture subtle changes in the fundus image. Spectral regression discriminant analysis (SRDA) reduces feature dimension, and minimum redundancy maximum relevance method is used to rank the significant SRDA components. Ranked features are fed to various supervised classifiers, viz. Naive Bayes, AdaBoost and support vector machine, to discriminate No DME, NCSME and clinically significant macular edema classes. The performance of our system is evaluated using the publicly available MESSIDOR dataset (300 images) and also verified with a local dataset (300 images). Our results show that HOS cumulants and bispectrum magnitude obtained an average accuracy of 95.56 and 94.39 % for MESSIDOR dataset and 95.93 and 93.33 % for local dataset, respectively.

Multi-site genetic analysis of diffusion images and voxelwise heritability analysis: A pilot project of the ENIGMA-DTI working group

The ENIGMA (Enhancing NeuroImaging Genetics through Meta-Analysis) Consortium was set up to analyze brain measures and genotypes from multiple sites across the world to improve the power to detect genetic variants that influence the brain. Diffusion tensor imaging (DTI) yields quantitative measures sensitive to brain development and degeneration, and some common genetic variants may be associated with white matter integrity or connectivity. DTI measures, such as the fractional anisotropy (FA) of water diffusion, may be useful for identifying genetic variants that influence brain microstructure. However, genome-wide association studies (GWAS) require large populations to obtain sufficient power to detect and replicate significant effects, motivating a multi-site consortium effort. As part of an ENIGMA-DTI working group, we analyzed high-resolution FA images from multiple imaging sites across North America, Australia, and Europe, to address the challenge of harmonizing imaging data collected at multiple sites. Four hundred images of healthy adults aged 18-85 from four sites were used to create a template and corresponding skeletonized FA image as a common reference space. Using twin and pedigree samples of different ethnicities, we used our common template to evaluate the heritability of tract-derived FA measures. We show that our template is reliable for integrating multiple datasets by combining results through meta-analysis and unifying the data through exploratory mega-analyses. Our results may help prioritize regions of the FA map that are consistently influenced by additive genetic factors for future genetic discovery studies. Protocols and templates are publicly available at (http://enigma.loni.ucla.edu/ongoing/dti-working-group/).

Discovery and replication of dopamine-related gene effects on caudate volume in young and elderly populations (N1198) using genome-wide search

The caudate is a subcortical brain structure implicated in many common neurological and psychiatric disorders. To identify specific genes associated with variations in caudate volume, structural magnetic resonance imaging and genome-wide genotypes were acquired from two large cohorts, the Alzheimer's Disease NeuroImaging Initiative (ADNI; N=734) and the Brisbane Adolescent/Young Adult Longitudinal Twin Study (BLTS; N=464). In a preliminary analysis of heritability, around 90% of the variation in caudate volume was due to genetic factors. We then conducted genome-wide association to find common variants that contribute to this relatively high heritability. Replicated genetic association was found for the right caudate volume at single-nucleotide polymorphism rs163030 in the ADNI discovery sample (P=2.36 × 10 -6) and in the BLTS replication sample (P=0.012). This genetic variation accounted for 2.79 and 1.61% of the trait variance, respectively. The peak of association was found in and around two genes, WDR41 and PDE8B, involved in dopamine signaling and development. In addition, a previously identified mutation in PDE8B causes a rare autosomal-dominant type of striatal degeneration. Searching across both samples offers a rigorous way to screen for genes consistently influencing brain structure at different stages of life. Variants identified here may be relevant to common disorders affecting the caudate.

Tracking Alzheimer's disease

Population-based brain mapping provides great insight into the trajectory of aging and dementia, as well as brain changes that normally occur over the human life span.We describe three novel brain mapping techniques, cortical thickness mapping, tensor-based morphometry (TBM), and hippocampal surface modeling, which offer enormous power for measuring disease progression in drug trials, and shed light on the neuroscience of brain degeneration in Alzheimer's disease (AD) and mild cognitive impairment (MCI).We report the first time-lapse maps of cortical atrophy spreading dynamically in the living brain, based on averaging data from populations of subjects with Alzheimer's disease and normal subjects imaged longitudinally with MRI. These dynamic sequences show a rapidly advancing wave of cortical atrophy sweeping from limbic and temporal cortices into higher-order association and ultimately primary sensorimotor areas, in a pattern that correlates with cognitive decline. A complementary technique, TBM, reveals the 3D profile of atrophic rates, at each point in the brain. A third technique, hippocampal surface modeling, plots the profile of shape alterations across the hippocampal surface. The three techniques provide moderate to highly automated analyses of images, have been validated on hundreds of scans, and are sensitive to clinically relevant changes in individual patients and groups undergoing different drug treatments. We compare time-lapse maps of AD, MCI, and other dementias, correlate these changes with cognition, and relate them to similar time-lapse maps of childhood development, schizophrenia, and HIV-associated brain degeneration. Strengths and weaknesses of these different imaging measures for basic neuroscience and drug trials are discussed.

On the biomechanical significance of inter-lamellar interfaces in the intervertebral disc

The intervertebral disc (IVD) is a unique soft tissue structure which provides structural support and flexibility in the axial skeleton of vertebrates. From a structural perspective, the disc behaves somewhat like a thick walled pressure vessel, where the walls are comprised of a series of composite annular rings (lamellae). However, a prior study (Marchand and Ahmed, 1990) found a high proportion of circumferentially discontinuous lamellae in human lumbar IVDs. The presence of these discontinuities raises important structural questions, because discontinuous lamellae cannot withstand high nucleus pressures via the generation of circumferential (hoop) stress. A possible alternative mechanism may be that inter-lamellar cohesion allows shear stress transfer between adjacent annular layers. The aim of the present study was therefore to investigate the importance of inter-lamellar shear resistance in the intervertebral disc. This work found that inter-lamellar shear resistance has a strong influence on the compressive stiffness of the intervertebral disc, with a change in interface condition from tied (no slip) to frictionless (no shear resistance) reducing disc compressive stiffness by 40%. However, it appears that substantial inter-lamellar shear resistance is present in the bovine tail disc. Decreases in inter-lamellar shear resistance due to degradation of bridging collagenous or elastic fibre structures could therefore be an important part of the process of disc degeneration.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction: Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods: PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results: Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions: This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

Inter-lamellar shear resistance confers compressive stiffness in the intervertebral disc: An image-based modelling study on the bovine caudal disc

The intervertebral disc withstands large compressive loads (up to nine times bodyweight in humans) while providing flexibility to the spinal column. At a microstructural level, the outer sheath of the disc (the annulus fibrosus) comprises 12–20 annular layers of alternately crisscrossed collagen fibres embedded in a soft ground matrix. The centre of the disc (the nucleus pulposus) consists of a hydrated gel rich in proteoglycans. The disc is the largest avascular structure in the body and is of much interest biomechanically due to the high societal burden of disc degeneration and back pain. Although the disc has been well characterized at the whole joint scale, it is not clear how the disc tissue microstructure confers its overall mechanical properties. In particular, there have been conflicting reports regarding the level of attachment between adjacent lamellae in the annulus, and the importance of these interfaces to the overall integrity of the disc is unknown. We used a polarized light micrograph of the bovine tail disc in transverse cross-section to develop an image-based finite element model incorporating sliding and separation between layers of the annulus, and subjected the model to axial compressive loading. Validation experiments were also performed on four bovine caudal discs. Interlamellar shear resistance had a strong effect on disc compressive stiffness, with a 40% drop in stiffness when the interface shear resistance was changed from fully bonded to freely sliding. By contrast, interlamellar cohesion had no appreciable effect on overall disc mechanics. We conclude that shear resistance between lamellae confers disc mechanical resistance to compression, and degradation of the interlamellar interface structure may be a precursor to macroscopic disc degeneration.

Clinical and pathological findings associated with congenital hypovitaminosis A in extensively grazed beef cattle

To determine the cause of exceptionally high mortality (41.4%) in perinatal calves on a beef cattle property 50 km south-west of Julia Creek in north-western Queensland. Investigations were based on clinical assessment of affected calves and laboratory analysis of pre- and postmortem specimens taken from 12 calves aged from 6 to 36 h of age. Associations between gross and histopathological findings and biochemical analyses conducted on serum and tissue samples were examined in relation to clinical observations. Clinical signs varied, but commonly included mild to severe ataxia, difficulty finding a teat and sucking, blindness (partial or complete, as judged by avoidance of obstacles) and depression with prominent drooping of the head. Gross and histopathological findings included herniation of the cerebellar vermis through the foramen magnum, squamous metaplasia of interlobular ducts in the parotid salivary glands and Wallerian degeneration of the optic nerves. Biochemical analysis of serum and liver samples available from four of the calves revealed low or undetectable levels of both vitamin A and vitamin E. Although vitamin E is known to have a sparing effect on vitamin A, the role (if any) played by deficiency of this vitamin was uncertain. The combination of clinical signs, postmortem findings, histopathological features and biochemical findings indicate that gestational vitamin A deficiency was highly likely to have been an important contributor to perinatal calf mortalities in this herd.

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.