Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer.

Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions-two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-ß (TGF-ß) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

Precision Phenotyping of Nectar-Related Traits Using X-ray Micro Computed Tomography.

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant-pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Macrophages Characterization in an Injured Bone Tissue.

Biomaterial use is a promising approach to facilitate wound healing of the bone tissue. Biomaterials induce the formation of membrane capsules and the recruitment of different types of macrophages. Macrophages are immune cells that produce diverse combinations of cytokines playing an important role in bone healing and regeneration, but the exact mechanism remains to be studied. Our work aimed to identify in vivo macrophages in the Masquelet induced membrane in a rat model. Most of the macrophages in the damaged area were M2-like, with smaller numbers of M1-like macrophages. In addition, high expression of IL-1ß and IL-6 cytokines were detected in the membrane region by RT-qPCR. Using an innovative combination of two hybridization techniques (in situ hybridization and in situ hybridization chain reaction (in situ HCR)), M2b-like macrophages were identified for the first time in cryosections of non-decalcified bone. Our work has also demonstrated that microspectroscopical analysis is essential for macrophage characterization, as it allows the discrimination of fluorescence and autofluorescence. Finally, this work has revealed the limitations of immunolabelling and the potential of in situ HCR to provide valuable information for in vivo characterization of macrophages.

Diisopropylfluorophosphate-induced status epilepticus drives complex glial cell phenotypes in adult male mice.

Organophosphate pesticides and nerve agents (OPs), are characterized by cholinesterase inhibition. In addition to severe peripheral symptoms, high doses of OPs can lead to seizures and status epilepticus (SE). Long lasting seizure activity and subsequent neurodegeneration promote neuroinflammation leading to profound pathological alterations of the brain. The aim of this study was to characterize neuroinflammatory responses at key time points after SE induced by the OP, diisopropylfluorophosphate (DFP). Immunohistochemistry (IHC) analysis and RT-qPCR on cerebral tissue are often insufficient to identity and quantify precise neuroinflammatory alterations. To address these needs, we performed RT-qPCR quantification after whole brain magnetic-activated cell-sorting (MACS) of CD11B (microglia/infiltrated macrophages) and GLAST (astrocytes)-positive cells at 1, 4, 24 h and 3 days post-SE. In order to compare these results to those obtained by IHC, we performed, classical Iba1 (microglia/infiltrated macrophages) and GFAP (astrocytes) IHC analysis in parallel, focusing on the hippocampus, a brain region affected by seizure activity and neurodegeneration. Shortly after SE (1-4 h), an increase in pro-inflammatory (M1-like) markers and A2-specific markers, proposed as neurotrophic, were observed in CD11B and GLAST-positive isolated cells, respectively. Microglial cells successively expressed immuno-regulatory (M2b-like) and anti-inflammatory (M2a-like) at 4 h and 24 h post-SE induction. At 24 h and 3 days, A1-specific markers, proposed as neurotoxic, were increased in isolated astrocytes. Although IHC analysis presented no modification in terms of percentage of marked area and cell number at 1 and 4 h after SE, at 24 h and 3 days after SE, microglial and astrocytic activation was visible by IHC as an increase in Iba1 and GFAP-positive area and Iba1-positive cells in DFP animals when compared to the control. Our work identified sequential microglial and astrocytic phenotype activation. Although the role of each phenotype in SE cerebral outcomes requires further study, targeting specific markers at specific time point could be a beneficial strategy for DFP-induced SE treatment.

Changes in the Viscoelastic Properties of the Vastus Lateralis Muscle With Fatigue.

We investigated the in vivo effects of voluntary fatiguing isometric contractions of the knee extensor muscles on the viscoelastic properties of the vastus lateralis (VL). Twelve young males (29.0 ± 4.5 years) performed an intermittent voluntary fatigue protocol consisting of 6 sets × 10 repetitions of 5-s voluntary maximal isometric contractions with 5-s passive recovery periods between repetitions. Voluntary and evoked torque were assessed before, immediately after, and 20 min after exercise. The shear modulus (µ) of the VL muscle was estimated at rest and during a ramped isometric contraction using a conventional elastography technique. An index of active muscle stiffness was then calculated (slope from the relationship between shear modulus and absolute torque). Resting muscle viscosity (η) was quantified using a shear-wave spectroscopy sequence to measure the shear-wave dispersion. Voluntary and evoked torque decreased by ∼37% (P < 0.01) immediately after exercise. The resting VL µ was lower at the end of the fatigue protocol (-57.9 ± 5.4%, P < 0.001), whereas the resting VL η increased (179.0 ± 123%, P < 0.01). The active muscle stiffness index also decreased with fatigue (P < 0.05). By 20 min post-fatigue, there were no significant differences from the pre-exercise values for VL η and the active muscle stiffness index, contrary to the resting VL µ. We show that the VL µ is greatly reduced and η greatly enhanced by fatigue, reflecting a more compliant and viscous muscle. The quantification of both shear µ and η moduli in vivo may contribute to a better understanding of the mechanical behavior of muscles during fatigue in sports medicine, as well as in clinical situations.

Heterogeneity assessment of histological tissue sections in whole slide images.

Computerized image analysis (IA) can provide quantitative and repeatable object measurements by means of methods such as segmentation, indexation, classification, etc. Embedded in reliable automated systems, IA could help pathologists in their daily work and thus contribute to more accurate determination of prognostic histological factors on whole slide images. One of the key concept pathologists want to dispose of now is a numerical estimation of heterogeneity. In this study, the objective is to propose a general framework based on the diffusion maps technique for measuring tissue heterogeneity in whole slide images and to apply this methodology on breast cancer histopathology digital images.

Building of a composite virtual slide from contiguous tissue samples.

BACKGROUND: Currently available microscope slide scanners produce whole slide images at various resolutions from histological sections. Nevertheless, acquisition area and so visualization of large tissue samples are limited by the standardized size of glass slides, used daily in pathology departments. The proposed solution has been developed to build composite virtual slides from images of large tumor fragments. MATERIALS AND METHODS: Images of HES or immunostained histological sections of carefully labeled fragments from a representative slice of breast carcinoma were acquired with a digital slide scanner at a magnification of 20×. The tiling program involves three steps: the straightening of tissue fragment images using polynomial interpolation method, and the building and assembling of strips of contiguous tissue sample whole slide images in × and y directions. The final image is saved in a pyramidal BigTiff file format. The program has been tested on several tumor slices. A correlation quality control has been done on five images artificially cut. RESULTS: Sixty tumor slices from twenty surgical specimens, cut into two to twenty six pieces, were reconstructed. A median of 98.71% is obtained by computing the correlation coefficients between native and reconstructed images for quality control. CONCLUSIONS: The proposed method is efficient and able to adapt itself to daily work conditions of classical pathology laboratories.

A general framework for the segmentation of follicular lymphoma virtual slides.

Follicular lymphoma (FL) is one of the most common types of non-Hodgkin's lmphomas in the world. Diagnosis of FL is based on morphological and immunohistochemical characteristics found on tissue sections. Our project's aim is to develop computer-aided analysis tools on virtual slide images (VSI) of lymphoid tissues with the purpose of improving the FL grading performed in malignant follicles. In this paper, we focus on the first step of our work, an automated system for detecting follicles in VSI of lymphoid tissues. To mimic the human expert process, the system works on low-resolution CD20 images and maps the follicle boundaries on high-resolution H&E images.

Towards a computer aided diagnosis system dedicated to virtual microscopy based on stereology sampling and diffusion maps.

An original strategy is presented, combining stereological sampling methods based on test grids and data reduction methods based on diffusion maps, in order to build a knowledge image database with no bias introduced by a subjective choice of exploration areas. The practical application of the exposed methodology concerns virtual slides of breast tumors.

Classification of low resolution virtual slides from breast tumor sections: comparison between global and local analysis.

In this paper, a comparison is made between a global and a local approach for computer-aided classification of virtual slides from breast tumor sections. The first approach classifies the images according to both color and texture information from the whole tissue, whereas the second one classifies them only on shape and texture from epithelial compartment. The originality of this study is that only low resolution virtual slides are used. As the cytological details are not visible at low resolution, the classification is only based on architectural aspect of each lesion. Experimental results on images from breast tumor sections show that some information is already present in low resolution virtual slides, allowing the classification in benign lesions versus malignant carcinomas.

Automated region of interest retrieval and classification using spectral analysis.

Efficient use of whole slide imaging in pathology needs automated region of interest (ROI) retrieval and classification, through the use of image analysis and data sorting tools. One possible method for data sorting uses Spectral Analysis for Dimensionality Reduction. We present some interesting results in the field of histopathology and cytohematology. In histopathology, we developed a Computer-Aided Diagnosis system applied to low-resolution images representing the totality of histological breast tumour sections. The images can be digitized directly at low resolution or be obtained from sub-sampled high-resolution virtual slides. Spectral Analysis is used (1) for image segmentation (stroma, tumour epithelium), by determining a "distance" between all the images of the database, (2) for choosing representative images and characteristic patterns of each histological type in order to index them, and (3) for visualizing images or features similar to a sample provided by the pathologist. In cytohematology, we studied a blood smear virtual slide acquired through high resolution oil scanning and Spectral Analysis is used to sort selected nucleated blood cell classes so that the pathologist may easily focus on specific classes whose morphology could then be studied more carefully or which can be analyzed through complementary instruments, like Multispectral Imaging or Raman MicroSpectroscopy.