scholarly journals Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro

2017 ◽  
Vol 23 (6) ◽  
pp. 1197-1206 ◽  
Author(s):  
Elena Ruggeri ◽  
Keith F. DeLuca ◽  
Cesare Galli ◽  
Giovanna Lazzari ◽  
Jennifer G. DeLuca ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Imaging Data ◽  
Hoechst 33258 ◽  
Anticentromere Antibody ◽  
Zygote Development ◽  
Tubulin Antibodies ◽  
Secondary Antibodies ◽  
Developmental Phases

AbstractConfocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ2 and Fisher’s exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.

scholarly journals The use of confocal microscopy in experimental studies and clinical practice of an endocrinologist: modern opportunities

2019 ◽  
Vol 65 (3) ◽  
pp. 174-183
Author(s):  
Natalya G. Mokrysheva ◽  
Sergey L. Kiselev ◽  
Natalia V. Klementieva ◽  
Anna M. Gorbacheva ◽  
Ivan I. Dedov
Keyword(s):  
Confocal Microscopy ◽  
Experimental Studies ◽  
Three Dimensional ◽  
Imaging Method ◽  
New Methods ◽  
Corneal Confocal Microscopy ◽  
Endocrine Ophthalmopathy ◽  
Fundamental Research

Confocal microscopy is a modern imaging method that provides ample opportunities for in vitro and in vivo research. The clinical part of the review focuses on well-established techniques, such as corneal confocal microscopy for the diagnosis of diabetic neuropathy or endocrine ophthalmopathy; new methods are briefly described (intraoperative evaluation of tissues obtained by removing pituitary adenomas, thyroid and parathyroid glands). In the part devoted to fundamental research, the use of confocal microscopy to characterize the colocalization of proteins, as well as three-dimensional intracellular structures and signaling pathways in vivo, is considered. Indicators of intracellular calcium are analyzed.

scholarly journals Radiosynthesis and characterization of [18F]BS224: a next-generation TSPO PET ligand insensitive to the rs6971 polymorphism

2021 ◽  
Author(s):  
Sang Hee Lee ◽  
Nunzio Denora ◽  
Valentino Laquintana ◽  
Giuseppe Felice Mangiatordi ◽  
Angela Lopedota ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Competitive Inhibition ◽  
Translocator Protein ◽  
High Signal ◽  
Imaging Data ◽  
Visible Image ◽  
Rat Models ◽  
High Affinity

Abstract Purpose Translocator protein 18-kDa (TSPO) positron emission tomography (PET) is a valuable tool to detect neuroinflammed areas in a broad spectrum of neurodegenerative diseases. However, the clinical application of second-generation TSPO ligands as biomarkers is limited because of the presence of human rs6971 polymorphism that affects their binding. Here, we describe the ability of a new TSPO ligand, [18F]BS224, to identify abnormal TSPO expression in neuroinflammation independent of the rs6971 polymorphism. Methods An in vitro competitive inhibition assay of BS224 was conducted with [3H]PK 11195 using membrane proteins isolated from 293FT cells expressing TSPO-wild type (WT) or TSPO-mutant A147T (Mut), corresponding to a high-affinity binder (HAB) and low-affinity binder (LAB), respectively. Molecular docking was performed to investigate the interaction of BS224 with the binding sites of rat TSPO-WT and TSPO-Mut. We synthesized a new 18F-labeled imidazopyridine acetamide ([18F]BS224) using boronic acid pinacol ester 6 or iodotoluene tosylate precursor 7, respectively, via aromatic 18F-fluorination. Dynamic PET scanning was performed up to 90 min after the injection of [18F]BS224 to healthy mice, and PET imaging data were obtained to estimate its absorbed doses in organs. To evaluate in vivo TSPO-specific uptake of [18F]BS224, lipopolysaccharide (LPS)-induced inflammatory and ischemic stroke rat models were used. Results BS224 exhibited a high affinity (Ki = 0.51 nM) and selectivity for TSPO. The ratio of IC50 values of BS224 for LAB to that for HAB indicated that the TSPO binding affinity of BS224 has low binding sensitivity to the rs6971 polymorphism and it was comparable to that of PK 11195, which is not sensitive to the polymorphism. Docking simulations showed that the binding mode of BS224 is not affected by the A147T mutation and consequently supported the observed in vitro selectivity of [18F]BS224 regardless of polymorphisms. With optimal radiochemical yield (39 ± 6.8%, decay-corrected) and purity (> 99%), [18F]BS224 provided a clear visible image of the inflammatory lesion with a high signal-to-background ratio in both animal models (BPND = 1.43 ± 0.17 and 1.57 ± 0.37 in the LPS-induced inflammatory and ischemic stroke rat models, respectively) without skull uptake. Conclusion Our results suggest that [18F]BS224 may be a promising TSPO ligand to gauge neuroinflammatory disease-related areas in a broad range of patients irrespective of the common rs6971 polymorphism.

scholarly journals In vivo, chromatin is a fluctuating polymer chain at equilibrium constrained by internal friction

2017 ◽  
Author(s):  
M. Socol ◽  
R. Wang ◽  
D. Jost ◽  
P. Carrivain ◽  
V. Dahirel ◽  
...  
Keyword(s):  
Internal Friction ◽  
Polymer Chain ◽  
Motion Tracking ◽  
Life Time ◽  
Physical Parameters ◽  
Imaging Data ◽  
Chromosome Conformation ◽  
Time Motion

AbstractChromosome mechanical properties determine DNA folding and dynamics, and underlie all major nuclear functions. Here we combine modeling and real-time motion tracking experiments to infer the physical parameters describing chromatin fibers. In vitro, motion of nucleosome arrays can be accurately modeled by assuming a Kuhn length of 35-55 nm. In vivo, the amplitude of chromosome fluctuations is drastically reduced, and depends on transcription. Transcription activation increases chromatin dynamics only if it involves gene relocalization, while global transcriptional inhibition augments the fluctuations, yet without relocalization. Chromatin fiber motion is accounted for by a model of equilibrium fluctuations of a polymer chain, in which random contacts along the chromosome contour induce an excess of internal friction. Simulations that reproduce chromosome conformation capture and imaging data corroborate this hypothesis. This model unravels the transient nature of chromosome contacts, characterized by a life time of ∼2 seconds and a free energy of formation of ∼1 kBT.

scholarly journals Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

2021 ◽  
Author(s):  
Aziz UR RAHMAN
Keyword(s):  
Confocal Microscopy ◽  
Drug Uptake ◽  
Live Cells ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Oriented Growth ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method

Abstract Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

scholarly journals Staphylococcus aureus infects osteoclasts and replicates intracellularly

2019 ◽  
Author(s):  
Jennifer L Krauss ◽  
Philip M Roper ◽  
Anna Ballard ◽  
Chien-Cheng Shih ◽  
James AJ Fitzpatrick ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Confocal Microscopy ◽  
Bone Tissue ◽  
Post Infection ◽  
Bone Structure ◽  
High Rate ◽  
Therapeutic Interventions ◽  
Intracellular Infection

AbstractOsteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. The majority of OM is caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, persist intracellularly, and promote the release of pro-osteoclastogenic cytokines, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. In this study, we examined the interaction between S. aureus and OCs, demonstrating internalization of GFP-labeled bacteria by confocal microscopy, both in vitro and in vivo. Utilizing an intracellular survival assay and flow cytometry during OC differentiation from bone marrow macrophages (BMMs), we found that the intracellular burden of S. aureus increases after initial infection in cells with at least 2 days of exposure to the osteoclastogenic cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). Presence of dividing bacteria was confirmed via visualization by transmission electron microscopy. In contrast, undifferentiated BMMs, or those treated with interferon-γ or IL-4, had fewer internal bacteria, or no change, respectively, at 18 hours post infection, compared to 1.5 hours post infection. To further explore the signals downstream of RANKL, we manipulated NFATc1 and alternative NF-κB, which controls NFATc1 and other factors affecting OC function, finding that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus and phagolysosome colocalization. The ability of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs and bone remodeling are most abundant.Author SummaryThe inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus. To date, the bone building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system. Therapeutic interventions that target osteoclasts specifically might reduce the severity of OM or improve antibiotic responses.

Three-dimensional confocal microscopy in living cells: historical development, practical application and limitations

1992 ◽  
Vol 50 (2) ◽  
pp. 1120-1121
Author(s):  
Barry R. Masters
Keyword(s):  
New York ◽  
Confocal Microscopy ◽  
Optical Microscopy ◽  
Cell Biology ◽  
Living Cells ◽  
Theory And Practice ◽  
List Type ◽  
Academic Press

Confocal microscopy is a rapidly evolving technique which is solving problems in the biological and material sciences. This tutorial focuses on the confocal microscopy of living cells. Both in vivo and in vitro applications of confocal microscopy will be reviewed. Applications of confocal microscopy of the eye will illustrate the concepts. Fluorescence and back scattered confocal microscopy are critically reviewed. A bibliography on confocal microscopy is given to aid the users of this technique.Books (Optical Theory, Image Formation, Fluorescence Techniques) •Theory and Practice of Scanning Optical Microscopy, (eds. T. Wilson, C. Sheppard), Academic Press, London, 1984.•Confocal Microscopy, (ed. by T. Wilson), Academic Press, London, 1990.•Handbook of Biological Confocal Microscopy, (ed. J.B. Pawley), Plenum Press, New York, 1989.•New Techniques of Optical Microscopy and Microspectroscopy (ed. R.J. Cherry), CRC Press, Inc., Boca Raton, Florida, 1991.•Noninvasive Techniques in Cell Biology, (eds. J.K. Foskett, S. Grinstein), Wiley-Liss, New York, 1990.

scholarly journals POS0378 CCN1: AN ANGIOGENIC ACTOR IMPLICATED IN THE STRUCTURAL DAMAGES OF RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 419.2-419
Author(s):  
J. Avouac ◽  
A. Steelandt ◽  
O. Amiar ◽  
A. Leblond ◽  
A. Cauvet ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Confocal Microscopy ◽  
Synovial Tissue ◽  
Tube Formation ◽  
Statistical Test ◽  
Wilcoxon Test ◽  
Serum Concentrations ◽  
Mrna And Protein Expression

Background:We have previously shown that decreased expression of the deacetylase sirtuin-1 (SIRT1) contributes to the proliferative, activated and proangiogenic profile of endothelial cells (EC) in rheumatoid arthritis (RA) (1). The matricellular protein CCN1, characterized by proangiogenic and immunomodulatory properties, may be directly implicated in these processes, since its expression is negatively regulated by SIRT1 (1).Objectives:To study the implication of CCN1 in RA pathogenesis.Methods:CCN1 expression was assessed in ECs (25 RA and 10 controls) by quantitative RT-PCR, western blot and ELISA, in the synovial tissue (5 RA and 5 controls) by immunohistochemistry and immunofluorescence, and in the serum (205 RA and 20 controls) by ELISA. Invalidation of CCN1 in RA ECs was achieved through the use of shRNA and neutralizing monoclonal antibodies. The functional consequences of CCN1 invalidation in RA ECs were studied i) in vitro by the analysis of proliferation (cell impedance), tube formation in Matrigel and migration in Boyden chambers; and ii) in vivo in the murine model of tumor neoangiogenesis.Results:CCN1 mRNA and protein expression were increased by 1.72- (p = 0.012) and 7.2-fold (p=0.008) in RA ECs compared to controls, respectively. CCN1 concentrations were significantly increased in RA EC culture supernatants (930±153 vs. 359±199 pg/mL, p=0.007). CCN1 was overexpressed in the synovial tissue of RA patients (Figure 1A) and confocal microscopy analyses revealed a prominent CCN1 expression in the vascular endothelium (CD31 +) and T cells (CD3 +) (Figure 1B).In vitro, recombinant TNF-α and IL-17 induced the mRNA and protein expression of CCN1 in RA ECs. CCN1 invalidation was associated with reduced proliferative capacities, delayed capillary tube formation and decreased migration of RA ECs (Figure 1E). In vivo, subcutaneous transplantation of CT26 tumor cells combined with RA ECs transfected with CCN1 shRNA to CB17 SCID mice was associated with a 51% reduction in tumor volume (p=0.008) and a 27% reduction in tumoral vascular density (p=0.032) compared with mice transplanted with MOCK transfected RA-ECs (Figure 1F).Serum concentrations of CCN1 were significantly reduced in the serum of RA patients compared to controls (233±118 vs. 279±75 pg/mL, p=0.045) (Figure 1C). However, serum CCN1 concentrations were significantly higher in the presence of bone erosions (253±139 vs. 202±7 pg/mL, p=0.002) (Figure 1D) and correlated with radiographic Larsen score (r=0.3, p=0.001) and HAQ (r=0.25, p=0.012).Conclusion:CCN1 is overexpressed in ECs and the synovial tissue of patients with RA. CCN1 also regulate the functional properties of RA ECs and their angiogenic potential in vivo. CCN1 could represent a new therapeutic target, which is being evaluated in experimental models of erosive arthritis.CCN1 may be a reliable biomarker of structural damages given the association between its serum concentrations and the extent of radiographic lesions. The performance of CCN1 serum levels to predict structural progression is under investigation.References:[1]Leblond et a, Ann Rheum Dis 2020.Figure 1.Implication of CCN1 in the pathogenesis of rheumatoid arthritis (RA). A, Representative immunohistochemistry staining for CCN1. B, Representative confocal microscopy analyses. C-D, CCN1 serum concentrations; statistical test: Student t test, ** p<0.01. E, Representative images of RA endothelial cell (EC) migration; Y-axis shows the number of migrated cells, statistical test: Wilcoxon test, * p<0.05. F, Representative subcutaneous tumors, Y-axis shows the fluorescence area in %, statistical test: Wilcoxon test, * p<0.05.Disclosure of Interests:None declared

scholarly journals Suppressive Effect of Huzhentongfeng on Experimental Gouty Arthritis: An In Vivo and In Vitro Study

2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Zi-cong Wu ◽  
Qiang Xue ◽  
Zhen-ling Zhao ◽  
Peng-jun Zhou ◽  
Qun Zhou ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Antioxidant Capacity ◽  
Radical Scavenging ◽  
Neutrophil Infiltration ◽  
Control Group ◽  
Raw264.7 Macrophages ◽  
Microscopy Analysis ◽  
Confocal Microscopy Analysis

Background. Huzhentongfeng (HZTF) is an extract from four Chinese medical herbs for treating gout. This study aims to evaluate its antigout activity and preliminary explore its mechanism in vivo and in vitro. Methods. The rats were intragastrically administered with HZTF for 5 days and then injected 0.1 ml (10 mg) of MSU crystals to their joints for generating a gout model to analyze the paw volume and histopathology of joint synovial tissues of rats with different doses. We also investigated the antioxidant capacity of HZTF in vitro using indication including lipid peroxidation, DPPH·, and ABTS+ radical-scavenging capacity; besides, we used qRT-PCR to measure the effect of HZTF on interleukin (IL)-1β, caspase-1, NLRP3, and NQO1 expression in hydrogen peroxide-stimulated RAW264.7 macrophages and IL-1β, IL-6, and tumor necrosis factor (TNF)-α in MSU crystal-induced THP-1 monocytes. Confocal microscopy analysis was used to observe the dimerization of ASC adapter proteins. In addition, we also established quality standard of HZTF by using the high-performance liquid chromatography (HPLC) method. Results. HZTF could significantly suppress the paw swelling and neutrophil infiltration induced by MSU intra-articular injection in rats compared with the control group. HZTF also showed inhibition effects of inflammatory cytokines (IL-1β, IL-6, and TNF-α) secretion at 25.00 and 50.00 μg/ml in MSU-induced THP-1 cells but showed no effects of IL-1β, IL-6, and TNF-α mRNA expression in MSU-induced THP-1 cells. Furthermore, confocal microscopy analysis showed that HZTF could prevent the oligomerization of ASC. Moreover, HZTF also showed effects in cell-free and cell-base tests of antioxidant capacity. Conclusion. The results prove that HZTF possessed the potential preventive effect against gout arthritis, and the effect may be attributed to its preventing effect on neutrophil infiltration and proinflammatory cytokines secretion such as IL-1β, IL-6, and TNF-α which were caused by the activation of inflammasome.

scholarly journals Cathelicidin LL-37 in Severe Streptococcus pyogenes Soft Tissue Infections in Humans

2008 ◽  
Vol 76 (8) ◽  
pp. 3399-3404 ◽  
Author(s):  
Linda Johansson ◽  
Pontus Thulin ◽  
Parham Sendi ◽  
Erika Hertzén ◽  
Adam Linder ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Confocal Microscopy ◽  
Bacterial Resistance ◽  
Resistance Mechanism ◽  
Soft Tissue Infections ◽  
Bacterial Surface ◽  
Life Threatening ◽  
Severe Soft Tissue

ABSTRACT Severe soft tissue infections, such as necrotizing fasciitis and severe cellulitis, caused by group A streptococci (GAS) are rapidly progressing life-threatening infections characterized by massive bacterial loads in the tissue even late after the onset of infection. Antimicrobial peptides are important components of the innate host defense, and cathelicidins have been shown to protect against murine necrotic skin infections caused by GAS. However, it has been demonstrated that the streptococcal cysteine protease SpeB proteolytically inactivates the human cathelicidin LL-37 in vitro. Here we have investigated the expression of LL-37 and its interaction with GAS and SpeB during acute severe soft tissue infections by analyses of patient tissue biopsy specimens. The results showed large amounts of LL-37, both the proform (hCAP18) and the mature peptide, in the tissue. Confocal microscopy identified neutrophils as the main source of the peptide. A distinct colocalization between the bacteria and LL-37 could be noted, and bacterial loads showed positive correlation to the LL-37 levels. Areas with high LL-37 levels coincided with areas with large amounts of SpeB. Confocal microscopy confirmed strong colocalization of GAS, SpeB, and LL-37 at the bacterial surface. Taken together, the findings of this study provide in vivo support of the hypothesis that SpeB-mediated inactivation of LL-37 at the streptococcal surface represents a bacterial resistance mechanism at the infected tissue site in patients with severe GAS tissue infections.

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