The authors gratefully acknowledge funding by the DFG Collaborative Research Centers SFB 1181 and TRR 241. The Optical Imaging Centre Erlangen (OICE) and in particular Ralf Palmisano, Philipp Tripal and Tina Fraa&#223; (Project Z2 of the DFG CRC 1181) are acknowledged for expert technical support for light-sheet fluorescence microscopic imaging.

M.F. Neurath has served as an advisor for Pentax, Giuliani, MSD, Abbvie, Janssen, Takeda and Boehringer. The remaining authors do not disclose any conflict.

The here described experimental setting provides the opportunity to monitor the lung homing capacity of primary human immune cells under in vivo inflammatory conditions and thereby relevantly complements classically performed in vitro adhesion and chemotaxis assays. To take into account specific anatomic organ characteristics of the lung, important aspects of immune cell homing (including chemotaxis and cell distribution within the target organ) as well as the clinical relevance and transfera...

Classic inflammatory disorders of the lung, such as allergic asthma and chronic obstructive pulmonary disease (COPD), are well known to be driven by an increased recruitment of activated lymphocytes into the pulmonary tissue1,2. Lymphocyte-released cytokines (e.g., IL-4, IL-5, IL-9, IL-13, IFN-&#947; and TNF-&#945;) further promote chemotaxis of innate and adaptive immune cells, induce fibrotic airway remodeling or directly damage the lung parenchyma2. So far, the underlying mechanisms responsible for the pathological accumulation of lymphocytes within lung tissue are not yet fully understood. In analogy to tissue-selective T cell imprinting described for gut and skin homing, pulmonary dendritic cells (DCs) are obviously able to prime peripheral T cells for preferential lung infiltration, at least partly via the induction of CCR4 expression on the surface of lymphocytes3. Besides CCR4, airway-infiltrating T cells are also characterized by a particularly increased expression of the chemokine receptors CCR5 and CXCR3 compared to T cells within the peripheral blood1,4,5. Overall, existing data are consistent with the concept that lung homing of T lymphocytes under physiological or inflammatory conditions involves a number of different chemokine receptors and their respective ligands and thus crucially depends on a closely controlled collaboration between innate and adaptive immune cells1. Especially, during the initial phase of pathogen or allergen exposure, cells of the innate immune system respond to TLR stimulation or to IgE-mediated cross-linking by the immediate release of different chemoattractants, like LTB4, CCL1, CCL17, CCL22, CCL20, CXCL10 and PGD21,6,7. As a prime example, the interaction between PGD2 and the chemoattractant receptor CRTh2 is known to be of particular importance for chemotaxis of Th2 cells and thus appeared as promising therapeutic target in the clinical management of asthma. Indeed, patients with moderate asthma showed an improvement of symptoms and a significant increase of the forced expiratory volume in one second (FEV1) after treatment with a selective CRTh2 antagonist compared to the placebo group8,9. In a more progressed state of the inflammatory response, already recruited T cells are able to further amplify pulmonary lymphocyte accumulation via the release of IL-4 and IL-13 as potent stimuli for pulmonary DCs. Subsequently, these myeloid-derived innate cells up-regulate the expression of CCL17 and CCL22 in a STAT6-dependent manner1,10,11.&#160; Although the complexity of the described scenario still hinders a complete understanding of T cell lung homing, it offers a plethora of molecular targets for a potentially optimized therapeutic control of inflammatory or allergic pulmonary diseases. Therefore, there is an urgent need of innovative experimental techniques, which are able to further deepen and complement our knowledge in the field of T cell chemotaxis and lung homing.
Due to the fact that lung homing of lymphocytes within the human body is influenced by multiple cellular, humoral and physical parameters1, most of the existing experimental methods are not able to model the whole complexity of this immunological process. Instead, many standard protocols for the analysis of lung homing selectively focus on a specific aspect involved in the cascade of lymphocyte attraction, adhesion, migration and retention. Besides a purely descriptive determination of the mRNA or protein expression pattern of integrins and chemokine receptors on peripheral or lung-infiltrating lymphocytes and the complementary measurement of respective chemokine levels in blood, bronchoalveolar lavage (BAL) or pulmonary tissue12,13,14,15, well-established in vitro cell culture assays allow a functional characterization of lymphocyte adhesion or chemotaxis upon defined experimental conditions16,17,18. In principle, static in vitro adhesion assays monitor the binding capacity of cultured lymphocytes to an endothelial monolayer or to glass slides coated with recombinant endothelial adhesion molecules (e.g., MAdCAM-1, VCAM-1), while standard in vitro chemotaxis assays are usually applied in order to quantify the ability of lymphocytes to migrate along a chemokine gradient in a transwell system19. Both in vitro settings enable a controlled adjustment and modulation of experimental conditions, but on the other hand lack important variables known to critically impact on in vivo chemotaxis and adhesion of lymphocytes. Predominantly, static cell culture assays disregard the influence of shear forces caused by the permanent blood flow19 and potentially neglect the involvement of the surrounding immunological milieu and interacting non-lymphocyte immune cells, both present in a living organism. In order to overcome these limitations, the interpretation of results acquired in static in vitro chemotaxis or adherence assays need further validation in dynamic adhesion experiments under flow conditions20,21 and in in vivo models of inflammatory organ pathology19. Indeed, important conclusions on the regulation of T cell lung homing under inflammatory or allergic conditions could be drawn from animal studies analyzing genetically modified mice in defined models of different pulmonary diseases3,22,23. The quantitative comparison of lung infiltrating lymphocytes between wildtype mice and mice with a deficiency for a specific gene of interest represents a well-established and broadly used tool for defining the impact of particular cellular pathways or receptors on the disease-driven pattern of T cell distribution. However, in contrast to before discussed in vitro cell culture assays, a study design based on classical animal models lacks the ability to analyze and monitor primary human T cells directly derived from the blood or BAL of patients suffering from an inflammatory lung disease. Thus, it still remains challenging to functionally validate whether a diagnostically specified lung disease is able to imprint human lymphocytes for preferential lung tropism and how far clinical parameters might impact on this scenario. Recently, a very elegant in vivo approach was introduced in the context of inflammatory bowel diseases (IBD), which was able to overcome most of these limitations and opened new avenues for advanced translational studies on intestinal lymphocyte homing24. Taking advantage of protocols for solvent-based tissue clearing followed by cross-sectional light-sheet fluorescence microscopy as a powerful imaging tool, it was possible to visualize the infiltration and distribution of adoptively transferred human T cells in the intestine of colitic immunodeficient mice24. In particular, this experimental setting implemented two main innovations: (1) Primary human immune cells can be analyzed under experimentally defined in vivo conditions; (2) a rather large area of the diseased organ (about 1.5&#160;cm&#160;x&#160;1.5&#160;cm) can be imaged in high resolution quality, followed by 3D-reconstruction. Moreover, several recent studies successfully established the use of solvent-based tissue clearing and light-sheet fluorescence microscopy as important tools for advanced lung imaging25,26. In order to benefit from this technological progress in the field of pulmonary immunology, we now adopted the system for analysis of lung homing.
The here presented protocol provides a step-by-step introduction how to purify and fluorescently label primary human T cells for transfer into mice with induced pulmonary inflammation and, moreover, describes in detail the subsequent process of light-sheet fluorescence microscopic imaging, including organ preparation and image processing. Overall, we hope to support future translational studies in the field of inflammatory or allergic lung diseases by introducing a sophisticated, but nevertheless feasible, experimental model for monitoring human lymphocyte lung homing upon in vivo conditions.

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			<td>PAN-Biotech GmbH, Aidenbach, Germany</td>
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			<td>VWR International Germany GmbH, Darmstadt, Germany</td>
			<td>732-2758</td>
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			<td>Bitplane AG, Zurich, Switzerland</td>
			<td>n.a.</td>
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			<td>Abberior Instruments GmbH, G&#246;ttingen, Germany</td>
			<td>n.a.</td>
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			<td height="20" style="height:20px;">LaVision UltraMicroscope II</td>
			<td>LaVision BioTec GmbH, Bielefeld, Germany</td>
			<td>n.a.</td>
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			<td height="20" style="height:20px;">MACS MultiStand</td>
			<td>Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany</td>
			<td>130-042-303</td>
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			<td height="20" style="height:20px;">Multifly cannula 20 G</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>851638035</td>
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			<td>B. Braun Melsungen AG, Melsungen, Hessen, Germany</td>
			<td>9161502</td>
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			<td>neoLab Migge GmbH, Heidelberg, Germany</td>
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			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 2 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 2 ml, brown</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 15 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 50 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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advanced imaging of lung homing human lymphocytes in an experimental in vivo model of allergic inflammation based on light-sheet microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The presented protocol describes an experimental mouse model, which allows monitoring and quantifying the accumulation of adoptively transferred human T lymphocytes in the lung via light-sheet fluorescence microscopy.&#160;Figure 1A provides a schematic overview of the in vivo steps of the experimental schedule. In order to guarantee reliable results, it is of substantial importance to ensure a good quality of the isolated and fluorescently labeled human CD4+ T ce...

Watch this Scientific Journal Video about Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy at JoVE.com

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

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7.5K Views.  University Hospital of Erlangen. The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Video: Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...