scholarly journals An event-based paradigm for analyzing fluorescent astrocyte activity uncovers novel single-cell and population-level physiology

2018 ◽  
Author(s):  
Yizhi Wang ◽  
Nicole V. DelRosso ◽  
Trisha Vaidyanathan ◽  
Michael Reitman ◽  
Michelle K. Cahill ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Region Of Interest ◽  
Population Level ◽  
Analytical Framework ◽  
Model Organisms ◽  
Imaging Data ◽  
Fluorescent Indicators ◽  
Event Based

AbstractRecent work examining astrocytic physiology centers on fluorescence imaging approaches, due to development of sensitive fluorescent indicators and observation of spatiotemporally complex calcium and glutamate activity. However, the field remains hindered in fully characterizing these dynamics, both within single cells and at the population-level, because of the insufficiency of current region-of-interest-based approaches to describe activity that is often spatially unfixed, size-varying, and propagative. Here, we present a paradigm-shifting analytical framework that releases astrocyte biologists from ROI-based tools. Astrocyte Quantitative Analysis (AQuA) software enables users to take an event-based approach to accurately capture and quantify the irregular activity observed in astrocyte imaging datasets. We apply AQuA to a range of ex vivo and in vivo imaging data, and uncover previously undescribed physiological phenomena in each. Since AQuA is data-driven and based on machine learning principles, it can be applied across model organisms, fluorescent indicators, experimental modes, and imaging resolutions and speeds, enabling researchers to elucidate fundamental astrocyte physiology.

scholarly journals Marker for the pre-clinical development of bone substitute materials

2017 ◽  
Vol 3 (2) ◽  
pp. 711-715
Author(s):  
Michael de Wild ◽  
Simon Zimmermann ◽  
Marcel Obrecht ◽  
Michel Dard
Keyword(s):  
Bone Graft ◽  
Mechanical Stability ◽  
Clinical Performance ◽  
Ex Vivo ◽  
Region Of Interest ◽  
Defect Site ◽  
Novel Approach ◽  
Bone Substitute Materials ◽  
Clinical Experiments

AbstractThin mechanically stable Ti-cages have been developed for the in-vivo application as X-ray and histology markers for the optimized evaluation of pre-clinical performance of bone graft materials. A metallic frame defines the region of interest during histological investigations and supports the identification of the defect site. This standardization of the procedure enhances the quality of pre-clinical experiments. Different models of thin metallic frameworks were designed and produced out of titanium by additive manufacturing (Selective Laser Melting). The productibility, the mechanical stability, the handling and suitability of several frame geometries were tested during surgery in artificial and in ex-vivo bone before a series of cages was preclinically investigated in the female Göttingen minipigs model. With our novel approach, a flexible process was established that can be adapted to the requirements of any specific animal model and bone graft testing.

scholarly journals Arginine-Vasopressin Expressing Neurons in the Murine Suprachiasmatic Nucleus Exhibit a Circadian Rhythm in Network Coherence in vivo

2021 ◽  
Author(s):  
Adam Stowie ◽  
Zhimei Qiao ◽  
Daniella Do Carmo Buonfiglio ◽  
J. Christopher Ehlen ◽  
Morris Benveniste ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Suprachiasmatic Nucleus ◽  
Arginine Vasopressin ◽  
Single Cells ◽  
Circadian Variation ◽  
Population Level ◽  
Correlational Analysis ◽  
Constant Darkness ◽  
Peak Time

AbstractThe Suprachiasmatic Nucleus (SCN) is composed of functionally distinct sub-populations of GABAergic neurons such as vasoactive intestinal polypeptide (VIP)-, arginine vasopressin (AVP)-, gastrin releasing peptide (GRP)-, and neuromedin S (NMS)-expressing neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons or neuronal sub-populations and which aspects result from neuronal interaction within a network. In this study, we address this question utilizing in vivo miniaturized microscopy to measure fluorescent GCaMP-mediated calcium dynamics in AVP neurons in the intact SCN of awake, behaving mice. This approach permits analysis of rhythms of single cells, populations, and correlational analysis among groups of AVP neurons in a field of view across the circadian and diurnal day and night. We report that AVP neurons in the murine SCN exhibit a periodic oscillatory increase in calcium of approximately 14 seconds across the day and night, in both constant darkness and under a 12:12 light-dark (LD) cycle. Using in vivo optogentically-targeted single unit activity recording, we demonstrated that these slow calcium waves are likely the result of burst-firing characteristic of AVP neurons previously reported for other brain regions. Rhythmicity analysis of several fluorescence measures suggests that individual AVP neurons exhibit unstable and stochastic rhythms, with approximately 30% of the neurons rhythmic during any given day across lighting conditions, and weak or absent rhythmicity at the population level. Network-level cross-correlational analysis revealed that coherence among neuron pairs also exhibited stochastic rhythms with about 25% of pairs rhythmic at any time. Notably, this analysis revealed a stronger rhythm at the population level than was observed in single cell analysis. The peak time of maximal coherence among AVP neuronal pairs occurs between CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity with the SCN as a whole. These results are the first to demonstrate robust circadian variation in the coordination between apparently weakly rhythmic or arrhythmic neurons suggesting that, for AVP neurons, interactions between neurons in the SCN are more influential than individual or single subpopulation activity in the regulation of mammalian circadian rhythms.

scholarly journals In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Chiara Garrovo ◽  
Natascha Bergamin ◽  
Dave Bates ◽  
Daniela Cesselli ◽  
Antonio Paolo Beltrami ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Optical Imaging ◽  
Adult Stem Cells ◽  
Near Infrared ◽  
Cross Validation ◽  
Ex Vivo ◽  
Imaging Data ◽  
Imaging Results

Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD) optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

scholarly journals Gonadotrope Plasticity at Cellular and Population Levels

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 4729-4739 ◽  
Author(s):  
Zahara Alim ◽  
Cheryl Hartshorn ◽  
Oliver Mai ◽  
Iain Stitt ◽  
Colin Clay ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Ex Vivo ◽  
Single Cells ◽  
Anterior Pituitary Gland ◽  
Female Mice ◽  
Model Animal ◽  
Pituitary Glands ◽  
Acute Changes

Abstract Hormone-secreting cells within the anterior pituitary gland may form organized and interdigitated networks that adapt to changing endocrine conditions in different physiological contexts. For gonadotropes, this might reflect a strategy to cope with acute changes throughout different female reproductive stages. The current study examined gonadotropes in female mice at characteristically different hormonal stages: prepubertal, postpubertal, and lactating. Gonadotrope plasticity was examined at the level of the whole population and single cells at different stages by imaging both fixed and live pituitary slices. The use of a model animal providing for the identification of selectively fluorescent gonadotropes allowed the particular advantage of defining cellular plasticity specifically for gonadotropes. In vivo analyses of gonadotropes relative to vasculature showed significantly different gonadotrope distributions across physiological states. Video microscopy studies using live slices ex vivo demonstrated pituitary cell plasticity in the form of movements and protrusions in response to GnRH. As positive feedback from rising estradiol levels is important for priming the anterior pituitary gland for the LH surge, experiments provide evidence of estradiol effects on GnRH signaling in gonadotropes. The experiments presented herein provide new insight into potential plasticity of gonadotropes within the anterior pituitary glands of female mice.

scholarly journals Two-Dimensional Modeling of Nanomechanical Strains in Healthy and Diseased Single-Cells During Microfluidic Stress Applications

2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Zachary D. Wilson ◽  
Sean S. Kohles
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Extensional Flow ◽  
Molecular Engineering ◽  
Creeping Flow ◽  
Recent Analysis ◽  
Two Dimensional ◽  
Monitoring And Control ◽  
The Impact

Investigations in cellular and molecular engineering have explored the impact of nanotechnology and the potential for monitoring and control of human diseases. In a recent analysis, the dynamic fluid-induced stresses were characterized during microfluidic applications of an instrument with nanometer and picoNewton resolution as developed for single-cell biomechanics (Kohles, S. S., Nève, N., Zimmerman, J. D., and Tretheway, D. C., 2009, “Stress Analysis of Microfluidic Environments Designed for Isolated Biological Cell Investigations,” ASME J. Biomech. Eng., 131(12), p. 121006). The results described the limited stress levels available in laminar, creeping-flow environments, as well as the qualitative cellular strain response to such stress applications. In this study, we present a two-dimensional computational model exploring the physical application of normal and shear stress profiles (with 0.1, 1.0, and 10.0 Pa peak amplitudes) potentially available within uniform and extensional flow states. The corresponding cellular strains and strain patterns were determined within cells modeled with healthy and diseased mechanical properties (5.0–0.1 kPa moduli, respectively). Strain energy density results integrated over the volume of the planar section indicated a strong mechanical sensitivity involving cells with disease-like properties. In addition, ex vivo microfluidic environments creating in vivo stress states would require freestream flow velocities of 2–7 mm/s. Knowledge of the nanomechanical stresses-strains necessary to illicit a biologic response in the cytoskeleton and cellular membrane will ultimately lead to refined mechanotransduction relationships.

scholarly journals In Vivo and Ex Vivo Microscopy: Moving Toward the Integration of Optical Imaging Technologies Into Pathology Practice

2018 ◽  
Vol 143 (3) ◽  
pp. 288-298 ◽  
Author(s):  
Wendy A. Wells ◽  
Michael Thrall ◽  
Anastasia Sorokina ◽  
Jeffrey Fine ◽  
Savitri Krishnamurthy ◽  
...  
Keyword(s):  
Optical Imaging ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Clinical Care ◽  
Imaging Data ◽  
Imaging Technologies ◽  
Optical Images ◽  
Tissue Removal ◽  
Excised Tissue

The traditional surgical pathology assessment requires tissue to be removed from the patient, then processed, sectioned, stained, and interpreted by a pathologist using a light microscope. Today, an array of alternate optical imaging technologies allow tissue to be viewed at high resolution, in real time, without the need for processing, fixation, freezing, or staining. Optical imaging can be done in living patients without tissue removal, termed in vivo microscopy, or also in freshly excised tissue, termed ex vivo microscopy. Both in vivo and ex vivo microscopy have tremendous potential for clinical impact in a wide variety of applications. However, in order for these technologies to enter mainstream clinical care, an expert will be required to assess and interpret the imaging data. The optical images generated from these imaging techniques are often similar to the light microscopic images that pathologists already have expertise in interpreting. Other clinical specialists do not have this same expertise in microscopy, therefore, pathologists are a logical choice to step into the developing role of microscopic imaging expert. Here, we review the emerging technologies of in vivo and ex vivo microscopy in terms of the technical aspects and potential clinical applications. We also discuss why pathologists are essential to the successful clinical adoption of such technologies and the educational resources available to help them step into this emerging role.

scholarly journals In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sebastian Schnorrenberg ◽  
Tim Grotjohann ◽  
Gerd Vorbrüggen ◽  
Alf Herzig ◽  
Stefan W Hell ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cell ◽  
Model Organisms ◽  
Alpha Tubulin ◽  
Light Levels

Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previously, we introduced the reversibly switchable fluorescent protein rsEGFP2, which facilitated fast RESOLFT nanoscopy (<xref ref-type="bibr" rid="bib10">Grotjohann et al., 2012</xref>). In that study, as in most other nanoscopy studies, only cultivated single cells were analyzed. Here, we report on the use of rsEGFP2 for live-cell RESOLFT nanoscopy of sub-cellular structures of intact Drosophila melanogaster larvae and of resected tissues. We generated flies expressing fusion proteins of alpha-tubulin and rsEGFP2 highlighting the microtubule cytoskeleton in all cells. By focusing through the intact larval cuticle, we achieved lateral resolution of <60 nm. RESOLFT nanoscopy enabled time-lapse recordings comprising 40 images and facilitated recordings 40 µm deep within fly tissues.

TMOD-17. A NOVEL GLIOBLASTOMA INVASION MODEL USING HUMAN BRAIN SLICE CULTURES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi219-vi219
Author(s):  
Vidyha Ravi ◽  
Kevin Joseph ◽  
Jürgen Beck ◽  
Oliver Schnell ◽  
Ulrich Hofmann ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tumour Microenvironment ◽  
Human Model ◽  
Model Organisms ◽  
Murine Models ◽  
Loss Of Function ◽  
High Concentration ◽  
Ample Evidence ◽  
Human Microglia

Abstract OBJECTIVE Glioblastoma (GBM) is among the most common of malignant brain tumours, with a median post-surgical survival of less than one year. Over the past several decades, therapies that appeared promising in mice models have failed during clinical trials due to the differences encountered during translation of research from model organisms to humans. To partially mitigate these difficulties in translation, we present a human cortical organotypic culture based GBM model, which allows us to manipulate individual components of the tumour environment in order to investigate the influence of different cell types in the immunosuppressive tumour microenvironment. METHODS Human neocortical tissue (at least 2 cm away from the tumour core) or entry cortex from epilepsy surgery guided by intraoperative neuro navigation, was cultured for up to 14 days post resection using an optimized medium. The cultured tissue was further injected with patient derived human GBM cells to create an ex vivo human model of glioblastoma model. The role of astrocytes in the tumour microenvironment was studied using microglia loss of function model. RESULTS Our established human neo-cortical slice model can recapitulate an in-vivo characteristics of glioblastoma from functional and imaging aspect. Our data corroborate differences between astrocytes in human and murine models in different reactive states, shows that the glioblastoma microenvironment can be difficult to be accurately modelled using murine models. Results from our human microglia depletion model, provided ample evidence that complex interaction of astrocytes and microglia cells, promotes an immunosuppressive environment in Glioblastoma by releasing high concentration of IL10 and TGFbeta (p&lt; 0.001). CONCLUSION Our model therefore has potential applications to the fields of neuroscience, neuro-oncology, and pharmacotherapy.

scholarly journals Live Imaging, Identifying, and Tracking Single Cells in Complex Populations In Vivo and Ex Vivo

2013 ◽  
pp. 109-123 ◽  
Author(s):  
Minjung Kang ◽  
Panagiotis Xenopoulos ◽  
Silvia Muñoz-Descalzo ◽  
Xinghua Lou ◽  
Anna-Katerina Hadjantonakis
Keyword(s):  
Ex Vivo ◽  
Single Cells ◽  
Live Imaging

scholarly journals In Vitro or in Vivo Models, the Next Frontier for Unraveling Interactions between Malassezia spp. and Hosts. How Much do We Know?

2020 ◽  
Author(s):  
Maritza Torres ◽  
Hans De Cock ◽  
Adriana Marcela Celis Ramírez
Keyword(s):  
Galleria Mellonella ◽  
Fungal Infections ◽  
Ex Vivo ◽  
Model Organisms ◽  
In Vivo Models ◽  
Fungal Host ◽  
Alternative Models ◽  
Malassezia Spp

Malassezia is a lipid-dependent genus of yeasts known for being an important part of the skin mycobiota. These yeasts have been associated in the development of skin disorders and cataloged as a causal agent of systemic infections under specific conditions, making them opportunistic pathogens. Little is known about the host-microbe interaction of Malassezia spp., and unraveling this implies the implementation of infection models. In this mini review we present different models that have been implemented in the fungal infections study with greater attention in Malassezia spp. infections. These models range from in vitro (cell cultures and ex vivo tissue), to in vivo (murine models, rabbits, guinea pigs, insects, nematodes, and amoebas). We additionally highlight the alternative models that reduce the use of mammals as model organisms, which have been gaining importance in the study of fungal host-microbe interactions. This is due to the fact that these systems have shown to have reliable results, which correlate with those obtained from mammalian models. Example of alternative models are Caenorhabditis elegans, Drosophila melanogaster, Tenebrio molitor, and Galleria mellonella. These are invertebrates that have been implemented in the study of Malassezia spp. infections in order to identify differences in virulence between Malassezia species.

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