The OPFOS microscopy family: High-resolution optical-sectioning of biomedical specimens

We report on the recently emerging (Laser) Light Sheet based Fluorescence Microscopy field (LSFM). The techniques used in this field allow to study and visualize biomedical objects non-destructively in high-resolution through virtual optical sectioning with sheets of laser light. Fluorescence originating in the cross section of the sheet and sample is recorded orthogonally with a camera. In this paper, the first implementation of LSFM to image biomedical tissue in three dimensions - Orthogonal-Plane Fluorescence Optical Sectioning microscopy (OPFOS) - is discussed. Since then many similar and derived methods have surfaced (SPIM, Ultramicroscopy, HR-OPFOS, mSPIM, DSLM, TSLIM...) which we all briefly discuss. All these optical sectioning methods create images showing histological detail. We illustrate the applicability of LSFM on several specimen types with application in biomedical and life sciences.Comment: 19 pages, 10 figures, to be published in Anatomical Research International (Hindawi

Morphology and function of Bast’s valve: additional insight in its functioning using 3D-reconstruction

The utriculo-endolymphatic valve was discovered by Bast in 1928. The function of Bast’s valve is still unclear. By means of orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy 3D-reconstructions of the valve and its surrounding region are depicted. The shape of the duct at the utricular side is that of a flattened funnel. In the direction of the endolymphatic duct and sac this funnel runs into a very narrow duct. The valve itself has a rigid ‘arch-like’ configuration. The opposing thin, one cell-layer thick, utricular membrane is highly compliant. We propose that opening and closure of the valve occurs through movement of the flexible base/utricular membrane away from and toward the relatively rigid valve lip

Neuroinflammation, Mast Cells, and Glia: Dangerous Liaisons

The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions

An international review of laser Doppler vibrometry:Making light work of vibration measurement

© 2016 In 1964, just a few years after the invention of the laser, a fluid velocity measurement based on the frequency shift of scattered light was made and the laser Doppler technique was born. This comprehensive review paper charts advances in the development and applications of laser Doppler vibrometry (LDV) since those first pioneering experiments. Consideration is first given to the challenges that continue to be posed by laser speckle. Scanning LDV is introduced and its significant influence in the field of experimental modal analysis described. Applications in structural health monitoring and MEMS serve to demonstrate LDV's applicability on structures of all sizes. Rotor vibrations and hearing are explored as examples of the classic applications. Applications in acoustics recognise the versatility of LDV as demonstrated by visualisation of sound fields. The paper concludes with thoughts on future developments, using examples of new multi-component and multi-channel instruments

ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases

Impact of Osteoblastic Hypoxia Signaling in Bone and Beyond: Skeletal regulation of systemic glucose homeostasis

Although the skeleton is equipped with a well-organized and dens vascular network, gradients of oxygenation exist due to its specific architecture and high cellularity in the marrow. To face these challenging conditions, cells within the bone environment are well-adapted and respond to low oxygen by activating a specialized transcriptional program, mediated by hypoxia-inducible factors (HIFs), to reduce their need for oxygen on the one hand and stimulate the supply of oxygen on the other. In the bone field, the hypoxia signaling pathway has been extensively studied and found to play a central role in bone formation and skeletal homeostasis. In fact, HIF and its downstream target vascular endothelial growth factor (VEGF) represent the first recognized couplers of osteogenesis and angiogenesis and fulfil a bone-anabolic role. Additionally, recent work for the first time allocated a role for HIF-induced upregulation of glycolysis in the development of a high bone mass phenotype in genetically modified mice. In addition to its classically recognized functions, the skeleton has also become increasingly recognized as an important regulator of whole-body energy metabolism and glucose homeostasis, with osteocalcin and insulin representing the prime known endocrine mediators of this interplay. Genetic evidence has indicated that osteoblasts may additionally influence global energy metabolism through mechanisms independent from these known effectors, although their nature remains elusive. In this thesis, we studied the effects of the hypoxia signaling pathway on bone formation and homeostasis at the tissue level, but also on the cellular level by investigating osteoblast biology and cellular energy metabolism. We also addressed the implications of altered hypoxia signaling in bone on processes extending beyond skeletal tissues. For these studies, we generated a model of increased HIF signaling in osteolineage cells, by osteoprogenitor-specific deletion of the Von Hippel Lindau (Vhl) gene, a negative regulator of the hypoxia pathway, using the Osx-Cre:GFP driver strain. First, we investigated the bone phenotype of these Vhl conditional knockout (cKO) mice in-depth, as described in Chapter 3 of the thesis. We found that mice in which the hypoxia signaling pathway was constitutively activated in osteoblast lineage cells showed a marked increase in bone mass with hypervascularization and disruption of the bone marrow environment. However, despite the high bone mass, adult Vhl cKO mice showed greatly decreased bone formation and mineralization rates. This was associated with impaired terminal differentiation of osteoblasts and an expansion of the pool of relatively immature osteolineage cells, which were likely responsible for the deposition of the rather disorganized, woven bone matrix. Despite normal osteoclast numbers, the presence of cartilaginous remnants extending into the diaphysis and reduced serum levels of collagen degradation products relative to bone volume were suggestive of a reduced bone turnover status in Vhl cKO mice. In the next Chapter 4, we aimed to shed light on the cell-intrinsic effects of increased HIF signaling in osteolineage cells, with a special focus on cellular energy metabolism. Both in vitro and in vivo, Vhl-deficient osteoblasts showed increased glucose consumption and glycolysis, associated with upregulated glucose transporters and glycolytic enzymes, and a reduced oxygen consumption rate. This effects resembles the Warburg effect that typifies cancer cells. The pharmacologic agent dichloroacetate (DCA), a glycolysis inhibitor, corrected the bio-energetic switch towards glycolysis and increased glucose consumption of Vhl-deficient cells in vitro. In search of the mechanisms causing the high bone mass phenotype in the Vhl mutant mice, we next generated a postnatally (PN)-induced Vhl cKO model and treated these mice with DCA in vivo. Our results showed that the high bone density observed in PN-Vhl cKO mice was not reverted upon DCA administration, suggesting that the altered osteogenic metabolism was not the prime cause of the high bone mass phenotype in our model of skeletal Vhl-deletion. While handling Vhl cKO mice we noticed their lean appearance. We therefore wanted to gain understanding of how increased hypoxia signaling in the skeleton could affect body composition and energy homeostasis. As such, in Chapter 5 we focused on the characterization of the systemic metabolic phenotype of Vhl cKO mice. Vhl cKO mice showed consistently reduced blood glucose levels and increased glucose tolerance from the age of 6 weeks onward. Hypoglycemia in the mutant mice was associated with lower body weights and reduced energy stores, including decreased peripheral fat accumulation and glycogen content in the liver, despite normal food intake and even reduced physical activity. The hypoglycemia could not be explained through abnormal osteocalcin or insulin signaling. Instead, glycemia levels inversely correlated with high overall uptake of glucose by the skeleton. Considering the highly glycolytic nature of Vhl-deficient osteolineage cells, we hypothesized that the low glycemia levels in the mutant mice were the result of a continual drain of glucose towards the skeleton. Hence, we investigated the metabolic phenotype of vehicle- versus DCA-treated PN-Vhl cKO mice. PN-Vhl cKO mice recapitulated the key systemic features of constitutive Vhl-deletion, including increased glucose tolerance and reduced glycemia, but without the potentially confounding or complicating aspects of lipodystrophy and reduced BW and body size, thereby representing a hypomorph model of osteoblastic Vhl-deletion. Intriguingly, when treated with DCA, the development of the metabolic phenotype was prevented in the PN-Vhl cKO mice, suggesting that cellular glucose utilization in bone may be a major determinant of systemic glucose homeostasis. Altogether, in this thesis we revealed that Vhl-deletion in osteoprogenitors leads to high bone mass, despite reduced terminal osteoblast differentiation and reduced bone formation and turnover at adult age. Additionally, we provide genetic and pharmacologic evidence supporting the notion that local glucose utilization in the skeleton contributes to systemic glucose clearance and homeostasis, a novel concept that may possibly have wide-ranging clinical implications with regard to bone and metabolic disorders.status: publishe