scholarly journals A crosscorrelation methodology for in vivo pharmacokinetic study by the trans-scale fluorescent system

2022 ◽  
Author(s):  
Yueqing Gu ◽  
Siwen Li ◽  
Qiao Lin ◽  
Yi Ma ◽  
Lu Qian ◽  
...  
Keyword(s):  
Pharmacokinetic Study ◽  
Time Course ◽  
Drug Disposition ◽  
Ex Vivo ◽  
Temporal Correlation ◽  
Confocal Imaging ◽  
Innovative Methodology ◽  
Micro Level ◽  
Spatio Temporal

Abstract Conventional single-organ-isolation-based pharmacokinetics study is short of time-course information and exists considerable inaccuracy due to the inter-individual differences and characteristic imparities between in vivo and ex vivo tissues/cells. The in vivo time-course and multi-organs study of model drugs in living subjects could afford precise spatio-temporal correlation. Herein, a revolutionized trans-dimensional fluorescence system was home built, with the macro-level detection part for simultaneous pharmacokinetic study in different organs, and one confocal imaging needle for micro-level visualizing cellular uptake of drugs with super-high resolution (0.472 μm). Correlating these simultaneous acquired trans-scale data, an innovative physiologically-based pharmacokinetics (PBPK) model was firstly created for predicting drug disposition in other species. Its accuracy and reliability was firmly supported by the high consistent predicted-data with the real-measured data in mice and in human, respectively. This study provides an innovative methodology and revolutionized instrument for in vivo real-time advancing assessment of druggability.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Progestin increases the expression of gonadotropins in pituitaries of male zebrafish

2016 ◽  
Vol 230 (1) ◽  
pp. 143-156 ◽  
Author(s):  
Cuili Wang ◽  
Dongteng Liu ◽  
Weiting Chen ◽  
Wei Ge ◽  
Wanshu Hong ◽  
...  
Keyword(s):  
Time Course ◽  
Ex Vivo ◽  
Mrna Levels ◽  
Adult Zebrafish ◽  
Male Adult ◽  
Positive Effects ◽  
Time Course Experiment ◽  
Nuclear Progesterone Receptor

Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 weeks exposure to 10nM estradiol (E2). However, an additional 24h 100nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24h for fshb and 12h for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knockout (pgr–/–) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr–/– were significantly lower than those of pgr+/+. Furthermore, ex vivo treatment of pituitary fragments of pgr–/– with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level.

scholarly journals Multimodality Approach to Characterizing the Distinctive Hallmarks of Lung Fibrosis: A Mouse Model with Bleomycin and Indocyanine Green

2021 ◽  
Author(s):  
Andrea Grandi ◽  
Erica Ferrini ◽  
Roberta Ciccimarra ◽  
Martina Mambrini ◽  
Laura Mecozzi ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Time Course ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Lavage Fluid ◽  
Pharmacological Therapy ◽  
Micro Ct ◽  
Oropharyngeal Aspiration ◽  
Multimodality Approach

Abstract Background.Idiopathic Pulmonary Fibrosis is a progressive disease with short life expectancy and no disease-modifying pharmacological therapy. The continuous refinement of animal models and the integration of in-vivo imaging techniques is fundamental for the selection of new antifibrotic drugs.Indocyanine Green (ICG), a fluorescent dye, was administered by oropharyngeal aspiration (OA) to mice with Bleomycin (BLM) to map the lung exposure.Methods.Female mice C57bl/6 were treated via OA with BLM+ICG or ICG. Animals were imaged at 7, 14 and 21 days either with the fluorescent system or Micro-CT. At each time point subsets of mice were sampled for ex-vivo assessment. Histological assessment of fibrosis by Ashcroft score, airspace enlargements and mean linear intercept (MLI) were evaluated at 7, 14 and 21 days. Leukocytes and cytokines were measured in bronchoalveolar lavage fluid. Results.Fluorescence imaging revealed a persistent lung signal in both groups until 21 days. In BLM+ICG group, Micro-CT detected a marked increase in hypo- and non-aerated tissues throughout the study. At later time points hyper-inflated tissue was detected. Histology revealed high Ashcroft score throughout the time-course with a prominent increase in airspace size and MLI at day 21. ICG mice had healthy lungs.Conclusions.We showed that ICG can be used as a tracer to map the distribution of BLM in lungs. However, BLM+ICG produced unexpected severe lung changes different from pure BLM model, such as emphysema-like features which progressively worsened. The multimodalities approach warranted characterization of the distinctive features of this new pulmonary fibrosis model and provided fundamentals for in-vivo translation.

scholarly journals Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat

2019 ◽  
Vol 173 (2) ◽  
pp. 280-292 ◽  
Author(s):  
Iman Hassan ◽  
Hisham El-Masri ◽  
Jermaine Ford ◽  
Amanda Brennan ◽  
Sakshi Handa ◽  
...  
Keyword(s):  
Thyroid Gland ◽  
Time Course ◽  
Ex Vivo ◽  
Response Analysis ◽  
In Vitro Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Serum T4

Abstract Thyroperoxidase (TPO) is an enzyme essential for thyroid hormone (TH) synthesis and a target site for a number of xenobiotics that disrupt TH homeostasis. An in vitro high-throughput screening assay for TPO inhibition, the Amplex UltraRed-TPO (AUR-TPO), has been used to screen the ToxCast chemical libraries for this action. Output from this assay would be most useful if it could be readily translated into an in vivo response, namely a reduction of TH in serum. To this end, the relationship between TPO inhibition in vitro and serum TH decreases was examined in rats exposed to 2 classic TPO inhibitors, propylthiouracil (PTU) and methimazole (MMI). Serum and gland PTU, MMI, and TH levels were quantified using tandem liquid chromatography mass spectrometry. Thyroperoxidase activity was determined in thyroid gland microsomes treated with PTU or MMI in vitro and ex vivo from thyroid gland microsomes prepared from exposed animals. A quantitative model was constructed by contrasting in vitro and ex vivo AUR-TPO results and the in vivo time-course and dose-response analysis. In vitro:ex vivo correlations of AUR-TPO outputs indicated that less than 30% inhibition of TPO in vitro was sufficient to reduce serum T4 by 20%, a degree of regulatory significance. Although further testing of model estimates using other TPO inhibitors is essential for verification of these initial findings, the results of this study provide a means to translate in vitro screening assay results into predictions of in vivo serum T4 changes to inform risk assessment.

scholarly journals Modeling Immune Checkpoint Inhibitor Efficacy in Syngeneic Mouse Tumors in an Ex Vivo Immuno-Oncology Dynamic Environment

2020 ◽  
Vol 21 (18) ◽  
pp. 6478
Author(s):  
Daniel T. Doty ◽  
Julia Schueler ◽  
Vienna L. Mott ◽  
Cassie M. Bryan ◽  
Nathan F. Moore ◽  
...  
Keyword(s):  
Real Time ◽  
Mouse Models ◽  
Immune Checkpoint ◽  
Checkpoint Inhibitors ◽  
Ex Vivo ◽  
Confocal Imaging ◽  
Checkpoint Blockade ◽  
Syngeneic Mouse

The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.

scholarly journals Frictional internal work of damped limbs oscillation in human locomotion

2020 ◽  
Vol 287 (1931) ◽  
pp. 20201410 ◽  
Author(s):  
Alberto E. Minetti ◽  
Alex P. Moorhead ◽  
Gaspare Pavei
Keyword(s):  
Energy Balance ◽  
Time Course ◽  
Ex Vivo ◽  
Biological Tissues ◽  
The Body ◽  
Lower Limbs ◽  
Metabolic Energy ◽  
External Work ◽  
Internal Work

Joint friction has never previously been considered in the computation of mechanical and metabolic energy balance of human and animal (loco)motion, which heretofore included just muscle work to move the body centre of mass (external work) and body segments with respect to it. This happened mainly because, having been previously measured ex vivo , friction was considered to be almost negligible. Present evidences of in vivo damping of limb oscillations, motion captured and processed by a suited mathematical model, show that: (a) the time course is exponential, suggesting a viscous friction operated by the all biological tissues involved; (b) during the swing phase, upper limbs report a friction close to one-sixth of the lower limbs; (c) when lower limbs are loaded, in an upside-down body posture allowing to investigate the hip joint subjected to compressive forces as during the stance phase, friction is much higher and load dependent; and (d) the friction of the four limbs during locomotion leads to an additional internal work that is a remarkable fraction of the mechanical external work. These unprecedented results redefine the partitioning of the energy balance of locomotion, the internal work components, muscle and transmission efficiency, and potentially readjust the mechanical paradigm of the different gaits.

scholarly journals P397 Response to biologics in IBD patients assessed by Computerized image analysis of Probe Based Confocal Laser Endomicroscopy with molecular labeling

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S406-S407
Author(s):  
M Iacucci ◽  
E Grisan ◽  
N Labarile ◽  
O Nardone ◽  
S C Smith ◽  
...  
Keyword(s):  
Predictive Value ◽  
Ex Vivo ◽  
Binding Activity ◽  
Biological Agent ◽  
Confocal Imaging ◽  
Confocal Laser Endomicroscopy ◽  
Confocal Laser ◽  
Tertiary Referral Centre ◽  
Fluorescent Intensity

Abstract Background The increase in therapeutic choices in inflammatory bowel diseases (IBD) imposed the identification of personalized therapeutic strategy. Confocal laser endomicroscopy (CLE) is a new endoscopic tool developed to obtain virtual in vivo histology. This study aimed to identify CLE in vivo and ex vivo features predictive of response for patients starting biologics. Methods We performed a prospective observational study at a single tertiary referral centre. 29 IBD patients (14 ulcerative colitis-UC and 15 Crohn’s Disease-CD) underwent colonoscopy with CLE before and 12-14 weeks after starting anti -TNF or 16 weeks after anti α4β7 integrin. CLE parameters analyzed were: crypt distribution along the mucosal surface, crypt area (CA), eccentricity, diameter, inter-cryptic distance (ICD), vessel tortuosity (VT), fluorescein leakage through the colonic mucosa (FLCM). Ex-vivo binding activity of fluorescein labelled biologics on corresponding biopsies was also assessed. Mosaicism reconstruction of CLE images were analyzed using a dedicated software algorithm (CellvizioViewer, Mauna Kea Technologies, ParisFrance). A dedicated Graphical User Interface was designed to enable a semiautomated analysis. Results After treatment, VT resulted significantly changed in overall IBD population (p<0.05), FLCM were reduced in UC patients (p<0.05), whilst CA, eccentricity and ICD decreased in CD patients (p< 0.05). The univariate regression analysis identified FLCM as the most accurate parameter for predicting responsiveness (AUROC) 83%, accuracy 83%, positive predictive value-PPV 94% and negative predictive value-NPV 57%). FLCM and ICD were the best discriminants in responders Vs non responders in UC patients (AUROC 85%, accuracy 85%, PPV 100% and NPV 71%); whilst VT, CA and ICD in CD patients (AUROC 95%, 86% and 83%; accuracy 90%, 90% and 88%; PPV 100%, 100% and 86%; and NPV 75%, 75% and 100%, respectively). Ex vivo confocal imaging revealed that UC patients, especially those responders, had higher basal fluorescent intensity signals with a significant reduction after biological treatment (p< 0.05), whereas in CD patients no significant change was found. An increased mucosal binding to the fluorescent labelled biological agent was associated to a higher likelihood of response to the treatment; this effect was higher in UC patients (AUROC 81%, accuracy 77%, PPV 100%, NPV 63%) compared to CD patients (AUROC 64%, accuracy 79%, PPV 80%, NPV 50%). Conclusion FLCM and ICD were the best discriminants of response in UC, while VT, CA and ICD in CD. A higher mucosal binding to a biological agent before treatment was observed in responders UC patients but not in CD patients.

Confocal imaging of basal cell cancers in vivo and in thick skin excisions ex vivo

2002 ◽  
Author(s):  
Milind Rajadhyaksha ◽  
Peter J. Dwyer ◽  
Gregg M. Menaker ◽  
Thomas J. Flotte ◽  
James M. Zavislan ◽  
...  
Keyword(s):  
Basal Cell ◽  
Ex Vivo ◽  
Confocal Imaging ◽  
Thick Skin

Cytokine Production and Cellular Localization in a Murine Model of Brain Inflammation

2001 ◽  
Vol 7 (S2) ◽  
pp. 1008-1009
Author(s):  
J. S. Cavallo ◽  
Q. Wu ◽  
A.M. Szczepanik ◽  
X. Ying ◽  
G.E. Ringheim
Keyword(s):  
Cellular Localization ◽  
Time Course ◽  
Drug Effects ◽  
Confocal Imaging ◽  
Brain Inflammation ◽  
Amyloid Peptide ◽  
In Vivo Model ◽  
Chemokine Production ◽  
Β Amyloid Peptide

Brain inflammation is typically involved in the pathogenesis of acute conditions such as stroke and brain trauma and in chronic neurodegenerative diseases, for instance, Alzheimer’s disease. Accordingly, a well-characterized in vivo model of brain inflammation can be a useful tool to evaluate specific drug effects focused on various inflammation targets in the pharmaceutical drug discovery process. A model of brain inflammation was induced by the injection of β-amyloid peptide (αβ) or the bacterial endotoxin lipopolysaccharide (LPS) into the lateral ventricles of CD-I mice. The time course and dose response kinetics of cytokine and chemokine production were characterized. Levels of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and the chemokine MCP-1 were significantly increased with respect to both time and dose in the cortex and hippocampus as determined by ELISA. Immunohistochemical assays were developed to detect IL-1β to determine cellular localization of this cytokine to microglia and astrocytes. Astrocytes were labeled with antibodies to glial fibrillary acidic protein (GFAP) and microglia were labeled with F4/80 antibodies. IL-1β was localized to cell type in frozen sections using double immunofluorescence (IMF) tags and were visualized with traditional fluorescence and confocal imaging.

P6308Use of cell-embedded fully implantable biosensor system for real-time, in-vivo monitoring of NF-Kappa B inflammatory pathway

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Rosero ◽  
F Machet ◽  
M Kowarz ◽  
E Brown ◽  
J Lopes-Vicente ◽  
...  
Keyword(s):  
Real Time ◽  
Cellular Response ◽  
Time Course ◽  
Ex Vivo ◽  
Recovery Period ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Pathway Activation ◽  
Biosensor System

Abstract Background 21st century health care remains anchored to sporadic measurements of traditional physiologic variables such as heart rate, blood pressure, weight, physical exam findings and general biochemical values. Currently there is no sensor capable of monitoring in intra-cellular protein and gene level signaling in real-time and in-vivo. This gap between biological signaling and its translation into clinically relevant therapeutics targeting the individual has limited precision medicine approaches to heart and vascular diseases. Inflammatory processes have been implicated in numerous cardiovascular diseases providing an ideal target for using Biologically based-Implantable Electronic Devices (BIED) approaches. Objective We aimed to test an implantable electro-photonic biosensor system in which living cells are integrated within the BIED and 1) serve as the primary sensor element providing in-vivo, real-time monitoring of intra-cellular processes such as gene expression, protein signaling, and target pathway activation, and 2) provide intelligent biologically based-processing in which the the output reflects biological response to an event. The engineered sensor cells provide real-time monitoring and respond to prespecified biologic signals using green fluorescence protein (GFP). The fluorescence is then detected via the BIED's photonic system and the cellular response data transmitted providing remote monitoring capabilities to facilitate the development of innovative personalized therapeutics. Methods A biologically-based implantable biosensor (BIED) platform which provides fluorescence detection, data acquisition and transmission from living cells integrated within the device was tested. The sensor cells communicate with the surrounding implant environment via a biomembrane. NRK rat cells engineered to express GFP in response to NF-κB pathway activation were cultured and housed within the sensor. Prior implant studies confirmed NRK sensor cells remained viable for 21 days in-vivo as part of a fully functional implanted BIED hardware system. Ex-vivo experiments characterized the expected magnitude and time course of NRK response to TNF-α and Lipopolysaccharide (LPS) exposure used to elicit a proinflammatory inflammatory response. The biosensor was implanted in the subcutaneous space of male Sprague Dawley rats (n=2) for a total of 11 days consisting of a baseline post-surgical recovery period of 7 days, with subsequent challenge with intraperitoneal LPS on Day 8 and 96 hours of post LPS monitoring. Results Rats implanted with the Biological based sensor and challenged with intraperitoneal LPS showed real-time expression of GFP under NF-κB transcriptional control following time course similar to ex-vivo experiments (p<0.05) (Figure 1). Figure 1. Implantable Cell-Embedded Biosensor Conclusion We present the first in-vivo use of a new class of BIEDs to detect biological cell response which may herald real-time personalize health management at the molecular and cellular level. Acknowledgement/Funding Clinical and Translational Sciences Institute-University of Rochester, Efferent Labs

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