Prominent elevation of extracellular matrix molecules in intracerebral hemorrhage.

Background: Intracerebral hemorrhage (ICH) is the predominant type of hemorrhagic stroke with high mortality and disability. In other neurological conditions, the deposition of extracellular matrix (ECM) molecules is a prominent obstacle for regenerative processes and an enhancer of neuroinflammation. Whether ECM molecules alter in composition after ICH, and which ECM members may inhibit repair, remain largely unknown in hemorrhagic stroke. Methods: The collagenase-induced ICH mouse model and an autopsied human ICH specimen were investigated for expression of ECM members by immunofluorescence microscopy. Confocal image z-stacks were analyzed with Imaris 3D to assess the association of immune cells and ECM molecules. Sections from a mouse model of multiple sclerosis were used as disease and staining controls. Tissue culture was employed to examine the roles of ECM members on oligodendrocyte precursor cells (OPCs). Results: Among the lectican chondroitin sulfate proteoglycan (CSPG) members, neurocan but not aggrecan, versican-V1 and versican-V2 was prominently expressed in perihematomal tissue and lesion core compared to the contralateral area in murine ICH. Fibrinogen, fibronectin and heparan sulfate proteoglycan (HSPG) were also elevated after murine ICH while thrombospondin and tenascin-C was not. Confocal microscopy with Imaris 3D rendering co-localized neurocan, fibrinogen, fibronectin and HSPG molecules to Iba1+ microglia/macrophages or GFAP+ astrocytes. Marked differentiation from the multiple sclerosis model was observed, the latter with high versican-V1 and negligible neurocan. In culture, purified neurocan inhibited adhesion and process outgrowth of OPCs, which are early steps in myelination in vivo. The prominent expression of neurocan in murine ICH was corroborated in human ICH sections. Conclusion: ICH caused distinct alterations in ECM molecules. Among CSPG members, neurocan was selectively upregulated in both murine and human ICH. In tissue culture, neurocan impeded the properties of oligodendrocyte lineage cells. Alterations to the ECM in ICH may adversely affect reparative outcomes after stroke.

Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism.

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

Quantitative analysis of spinal cord neuropathology in experimental autoimmune encephalomyelitis.

Multiple sclerosis is an inflammatory and neurodegenerative condition that is frequently modeled using experimental autoimmune encephalomyelitis (EAE). Current methods of EAE histology include imprecise qualitative assessments and time-consuming analyses of selected regions. With increasing interest in neuroprotective or reparative therapies, it is important that potential therapeutics are evaluated in EAE through quantitative neuropathology. We describe a quantitative whole slide imaging immunofluorescence method that allows longitudinal sections of the entire EAE thoracic spinal cord to be investigated for the extent of neuroinflammation, axonal loss, and myelin density. This method should impact MS research by making histological comparisons of EAE increasingly robust.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Shear deformation and fracture of human cortical bone.

Bone can be viewed as a nano-fibrous composite with complex hierarchical structures. Its deformation and fracture behaviors depend on both the local structure and the type of stress applied. In contrast to the extensive studies on bone fracture under compression and tension, there is a lack of knowledge on the fracture process under shear, a stress state often exists in hip fracture. This study investigated the mechanical behavior of human cortical bone under shear, with the focus on the relation between the fracture pattern and the microstructure. Iosipescu shear tests were performed on notched rectangular bar specimens made from human cortical bone. They were prepared at different angles (i.e. 0°, 30°, 60° and 90°) with respect to the long axis of the femoral shaft. The results showed that human cortical bone behaved as an anisotropic material under shear with the highest shear strength (~50MPa) obtained when shearing perpendicular to the Haversian systems or secondary osteons. Digital image correlation (DIC) analysis found that shear strain concentration bands had a close association with long bone axis with an average deviation of 11.8° to 18.5°. The fracture pattern was also greatly affected by the structure with the crack path generally following the direction of the long axes of osteons. More importantly, we observed unique peripheral arc-shaped microcracks within osteons, using laser scanning confocal microscopy (LSCM). They were generally long cracks that developed within a lamella without crossing the boundaries. This microcracking pattern clearly differed from that created under either compressive or tensile stress: these arc-shaped microcracks tended to be located away from the Haversian canals in early-stage damaged osteons, with ~70% developing in the outer third osteonal wall. Further study by second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) microscopy revealed a strong influence of the organization of collagen fibrils on shear microcracking. This study concluded that shear-induced microcracking of human cortical bone follows a unique pattern that is governed by the lamellar structure of the osteons.

Inelastic deformation and microcracking process in human dentin.

Dentin is a mineralized collagen tissue with robust mechanical performance. Understanding the mechanical behavior of dentin and its relations to the dentinal structure can provides insight into the design strategies to achieve tooth functions. This study focuses on the inelastic deformation of human dentin and its underlying mechanisms. By combining four-point bending tests with fluorescent staining and laser scanning confocal microscopy, it was found that human dentin, especially root dentin, exhibited significant inelastic deformation and developed extensive microdamage in the form of microcracks prior to fracture. Dense and wavy microcracks spread uniformly across the tensile surface of root dentin, while compressive microcracks formed cross-hatched patterns. The presence of peritubular dentin in coronal dentin dramatically decreased the extent of microcracking, reducing inelasticity. Dentinal tubules were found to be initiation sites of both tensile and compressive microcracks. A unique crack propagation process was observed in root dentin under tension: numerous ring-shaped cracks formed at each dentinal tubule ahead of a growing crack tip. The advance of the tensile microcracks occurred by the merging of those ring-shaped cracks. The current findings on the microcracking process associated with inelastic deformation helps to understand the nature of strength and toughness in dentin, as well as the mechanical significance for structural variations across the whole tooth.

Sub-lamellar microcracking and roles of canaliculi in human cortical bone.

Bone is a tough biological material. It is generally accepted that bone's toughness arises from its unique hierarchical structure, which in turn facilitates distributed microcracking prior to fracture. Yet, there has been limited progress on the detailed roles of the structural elements in the microcracking process. The present study examines the structure-microcracking relations at the lamellar and sub-lamellar levels of human cortical bone subjected to compressive loading. Laser scanning confocal microscopy revealed a clear influence of the local structure and porosity of the Haversian systems' lamellae on microcrack development. In particular, crack initiation and growth under transverse compression were associated with stress concentration at canaliculi. Later stages of microcracking showed extensive sub-lamellar cracks forming cross-hatched patterns and regularly spaced 0.5-1.7 µm apart. The density, size and regularity of the crack patterns suggest enhanced inelastic deformation capacity through cracking control at the level of mineralized collagen fibril bundles. The present study thus improves the current understanding of the nature of inelastic deformation and microcracking in bone and further suggests that bone's resistance to fracture is achieved through microcrack control at multiple length scales.

During sideways falls proximal femur fractures initiate in the superolateral cortex: evidence from high-speed video of simulated fractures.

Results of recent imaging studies and theoretical models suggest that the superior femoral neck is a location of local weakness due to an age-related thinning of the cortex, and thus the site of hip fracture initiation. The purpose of this study was to experimentally determine the spatial and temporal characteristics of the macroscopic failure process during a simulated hip fracture that would occur as a result of a sideways fall. Twelve fresh frozen human cadaveric femora were used in this study. The femora were fractured in an apparatus designed to simulate a fall on the greater trochanter. Image sequences of the surface events related to the fractures were captured using two high-speed video cameras at 9111 Hz. The videos were analyzed with respect to time and load to determine the location and sequence of these events occurring in the proximal femur. The mean failure load was 4032 N (SD 370 N). The first surface events were identified in the superior femoral neck in eleven of the twelve specimens. Nine of these specimens fractured in a clear two-step process that initiated with a failure in the superior femoral neck, followed by a failure in the inferior femoral neck. This cadaveric model of hip fracture empirically confirms hypotheses that suggested that hip fractures initiate with a failure in the superior femoral neck where stresses are primarily compressive during a sideways fall impact, followed by a failure in the inferior neck where stresses are primarily tensile. Our results confirm the superolateral neck of the femur as an important region of interest for future hip fracture screening, prevention and treatment research.

The effect of adhesion promoter on the adhesion of PDMS to different substrate materials.

The effect of the adhesion promoter GE SS4120 on the adhesion strength of PDMS to different substrates was tested. The adhesion to silicon, glass and aluminium was significantly increased, while adhesion of PDMS to Teflon remained poor, and the adhesion strength of PDMS to PDMS decreased.

Strain redistribution and cracking behavior of human bone during bending.

Long bones often fail due to bending loads. Understanding the fracture process during bending is of great importance to the prevention and treatment of bone fractures. In this study, we investigated the origin of long bone's bending strength through the study of the dynamic strain redistribution happening during the post-yield stage of deformation and its relation to microdamage at the microstructural level. This was accomplished by comparing the behaviors of human long bones with standard cortical bone specimens in terms of strain redistribution, Poisson's ratios, microdamage morphologies, and macro-scale fracture patterns. It was found that human tibia failure in bending was very similar to that of standard beam cortical bone specimens with respect to the four previous aspects. Also, the examination of bone's Poisson's ratio indicated very different inelastic deformation mechanisms under tension and compression: bone volume expanded in tension but was nearly conserved in compression. Finally, as a result of strain redistribution, bone's bending strength mainly depended on its compressive strength, which was significantly influenced by the osteonal "porous" microstructure of human bone as compared to its tensile behavior. Thus, we concluded that bone microstructure at the Haversian system level plays an important role in bone deformation and fracture.