The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 379-388 ◽  
Author(s):  
M.J. Carette ◽  
M.W. Ferguson
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Palatal Fusion ◽  
Morphological Criteria ◽  
Medial Edge ◽  
Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy

1993 ◽  
Vol 104 (4) ◽  
pp. 1175-1185 ◽  
Author(s):  
P. Buchenau ◽  
H. Saumweber ◽  
D.J. Arndt-Jovin
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Topoisomerase Ii ◽  
Metaphase Plate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

scholarly journals Astrogliosis in an Experimental Model of Hypovitaminosis B12: A Cellular Basis of Neurological Disorders due to Cobalamin Deficiency

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2261
Author(s):  
Zuzanna Rzepka ◽  
Jakub Rok ◽  
Justyna Kowalska ◽  
Klaudia Banach ◽  
Justyna Magdalena Hermanowicz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cobalamin Deficiency ◽  
Confocal Laser ◽  
In Vivo Model ◽  
Cellular Expression ◽  
Wide Range ◽  
Vitro Model

Cobalamin deficiency affects human physiology with sequelae ranging from mild fatigue to severe neuropsychiatric abnormalities. The cellular and molecular aspects of the nervous system disorders associated with hypovitaminosis B12 remain largely unknown. Growing evidence indicates that astrogliosis is an underlying component of a wide range of neuropathologies. Previously, we developed an in vitro model of cobalamin deficiency in normal human astrocytes (NHA) by culturing the cells with c-lactam of hydroxycobalamin (c-lactam OH-Cbl). We revealed a non-apoptotic activation of caspases (3/7, 8, 9) in cobalamin-deficient NHA, which may suggest astrogliosis. The aim of the current study was to experimentally verify this hypothesis. We indicated an increase in the cellular expression of two astrogliosis markers: glial fibrillary acidic protein and vimentin in cobalamin-deficient NHA using Western blot analysis and immunocytochemistry with confocal laser scanning microscopy. In the next step of the study, we revealed c-lactam OH-Cbl as a potential non-toxic vitamin B12 antagonist in an in vivo model using zebrafish embryos. We believe that the presented results will contribute to a better understanding of the cellular mechanism underlying neurologic pathology due to cobalamin deficiency and will serve as a foundation for further studies.

scholarly journals Enhancement of Vancomycin Activity against Biofilms by Using Ultrasound-Targeted Microbubble Destruction

2011 ◽  
Vol 55 (11) ◽  
pp. 5331-5337 ◽  
Author(s):  
Nianan He ◽  
Jian Hu ◽  
Huayong Liu ◽  
Tao Zhu ◽  
Beijian Huang ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Laser Scanning ◽  
Rabbit Model ◽  
Antibiotic Activity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Content Type ◽  
Implanted Medical Devices

ABSTRACTTreating biofilm infections on implanted medical devices is formidable, even with extensive antibiotic therapy. The aim of this study was to investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) could enhance vancomycin activity againstStaphylococcus epidermidisRP62A biofilms. Twelve-hour biofilms were treated with vancomycin combined with UTMD. The vancomycin and MB (SonoVue) were used at concentrations of 100 μg/ml and 30% (vol/vol), respectively, in studiesin vitro. After US exposure (0.08 MHz, 1.0 W/cm2, 50% duty cycle, and 10-min duration), the biofilms were cultured at 37°C for another 12 h. The results showed that many micropores were found in biofilms treated with vancomycin combined with UTMD. Biofilm densities (A570values) and the viable counts ofS. epidermidisrecovered from the biofilm were significantly decreased compared with those of any other groups. Furthermore, the highest percentage of dead cells was found, using confocal laser scanning microscopy, in the biofilm treated with vancomycin combined with UTMD. The viable counts of bacteria in biofilms in anin vivorabbit model also confirmed the enhanced effect of vancomycin combined with UTMD. UTMD may have great potential for improving antibiotic activity against biofilm infections.

scholarly journals Characterization of a mucoid-like Pseudomonas aeruginosa biofilm

Fine Focus ◽  
2015 ◽  
Vol 1 (2) ◽  
pp. 121-137
Author(s):  
Brandon M. Bauer ◽  
Lewis Rogers ◽  
Monique Macias ◽  
Gabriella Iacovetti ◽  
Alexander M. Woodrow ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Growth Conditions ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chronic Infections ◽  
Planktonic Bacteria ◽  
The Difference

Pseudomonas aeruginosa biofilms are implicated in chronic infections. A key element of P. aeruginosapathogenicity is the formation of a biofilm, a community of bacteria encased in an exopolymeric substance (EPS) that shields the bacteria from the host immune response and antibiotic treatment. A crucial step in biofilm production is a switch in motility from freely swimming, planktonic bacteria to twitching movement and then to attached and sedentary bacteria that develop into a mature pillar-shaped biofilm. A mucoid biofilm produces an excess of alginate and is clinically the most pathogenic and the most resistant to antibiotics. Biofilms from patients exhibit a wide variety of structure, motility, and levels of attachment. In vitrobiofilms do not exhibit such a wide variety of structure and physiology. The difference between in vivo and in vitro biofilms has made the translation of in vitro studies into in vivo treatments difficult. Under different growth conditions in our lab, the P. aeruginosa strain PAO1 demonstrates two phenotypes: a non-mucoid and a mucoid-like phenotype. Confocal laser scanning microscopy (CLSM) indicates the mucoid-like phenotype is intermediate in height to the non-mucoid phenotype and biofilms formed in a once-flow-through chamber. Both mucoid-like and non-mucoid phenotypes exhibit a significant increase in twitching between 24 and 72 hours of development. The mucoid-like phenotype had greater attachment at 72 hours compared to non-mucoid phenotype. Therefore, the two phenotypes observed in our lab may represent the effect of environment to stimulate development of two types of biofilms by PAO1.

Cyclodextrin Nanosponge Based Babchi Oil Hydrogel Ameliorates Imiquimod-Induced Psoriasis in Swiss Mice: An impact on Safety and Efficacy

2021 ◽  
Vol 13 ◽  
Author(s):  
Sunil Kumar ◽  
Babu Lal Jangir ◽  
Rekha Rao
Keyword(s):  
Essential Oil ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Epidermal Keratinocytes ◽  
Topical Formulation ◽  
Safe Alternative ◽  
Safety And Efficacy

Background: Psoriasis, a chronic autoimmune disease, involves the integration of biological and molecular events by hyperproliferation of the epidermal keratinocytes and generation of inflammation markers. Due to severe complications of synthetic corticosteroids, there is a strong need for potential and safe alternative . Babchi oil (natural essential oil; BO) may prove as a promising natural agent for psoriasis. Objective: The aim of the present work was to investigate the safety and efficacy of cyclodextrin nanosponge-based babchi oil (BONS) hydrogel on skin annexes. Methods: Babchi oil nanosponge hydrogel (BONS-HG) was fabricated and evaluated. Cell viability studies have been carried out on THP1 cell lines to evaluate cytocompatibility. Irritation potential and in vivo visualization of cutaneous uptake of BONS-HG were carried out using Hen’s Egg Chorioallantoic Membrane Test (HET-CAM) and confocal laser scanning microscopy (CLSM), respectively. The nano hydrogel was tested in vivo using imiquimod-induced psoriasis mouse model. Results: The in vitro irritation potential of BONS-HG indicated no sign of erythema or irritation, suggesting the safety of prepared hydrogel as topical formulation. CLSM studies advocated targeting of BO to epidermis and dermis. Along with histopathological assessment, evaluation of oxidative stress markers revealed the significant antipsoriatic activity (p< 0.001) of the prepared BONS-HG. Conclusion: The present study amalgamated the advantages of natural essential oil with this approach for skin targeting and provided an effective and safe topical alternative for psoriasis.

scholarly journals Glycoconjugate Expression Defines the Origin and Differentiation Pathway of Intestinal M-cells

1997 ◽  
Vol 45 (10) ◽  
pp. 1341-1350 ◽  
Author(s):  
Andreas Gebert ◽  
Wolfgang Posselt
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Lymphoid Tissues ◽  
M Cells ◽  
Glycosylation Pattern ◽  
Clonal Origin ◽  
Computer Based

Intestinal M-cells are specialized epithelial cells located in the domes of the gut-associated lymphoid tissues, which transport antigens from the lumen to the underlying lymphoid tissue, thereby initiating immune reactions. It is assumed that M-cells arise from stem cells in the crypts, from which they migrate to the top of the domes. To study the differentiation pathway of M-cells, we used the rabbit cecal lymphoid patch in which the M-cells express high levels of α1–2-linked fucose and N-acetyl-galactosamine residues in their apical membrane. Dome areas were labeled with fluorescein- and rhodamine-conjugated lectins specific for α1–2-linked fucose and N-acetyl-galactosamine in vivo and in vitro, and were observed with confocal laser scanning microscopy. Ultrathin sections were double labeled with lectin–gold conjugates and the labeling density was quantified by computer-based image analysis. All cecal patch M-cells expressed α1–2-linked fucose and N-acetyl-galactosamine, but the amount of the two saccharides varied considerably depending on the position of the M-cells at the base, flank, or top of the dome. In eight of 18 rabbits studied, radial strips of M-cells with common glycosylation patterns were observed, each strip associated with an individual crypt. Confocal microscopy revealed that lectin-labeled M-cells were not restricted to the dome epithelium but were also detected in the upper third of crypts surrounding the domes. The results show that M-cells are heterogeneous concerning the glycosylation pattern of membrane glycoconjugates. This pattern is modified as the M-cells differentiate and migrate from the base to the top of the dome. Radial strips of M-cells with a common proclivity of glycoconjugate expression suggest that those M-cells that derive from the same crypt have a clonal origin. The presence of (pre-) M-cells in the crypts surrounding the domes indicates that M-cells derive directly from undifferentiated crypt cells and do not develop from differentiated enterocytes.

scholarly journals 286SPERM-OOCYTE INTERACTION DURING IN VITRO FERTILIZATION IN THE HORSE

2004 ◽  
Vol 16 (2) ◽  
pp. 263
Author(s):  
J.L. Tremoleda ◽  
T.A.E. Stout ◽  
B.M. Gadella ◽  
B. Colenbrander
Keyword(s):  
In Vitro Fertilization ◽  
Laser Scanning ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Molecular Probes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Vitro Fertilization

In vitro fertilization (IVF) has proven to be a surprisingly unsuccessful way of producing horse embryos. The aim of this study was to investigate the interaction between sperm and the cumulus oocyte complex (COC) during IVF. In experiment 1, three IVF conditions were tested: (A) COCs recovered from slaughtered mares were categorized with respect to cumulus morphology (C: compact, n=86, or E: expanded, n=55) and matured in TCM199 containing 0.01IU/mL porcine FSH and equine LH (IVM); after IVM, the oocytes were denuded and those with a visible polar body were incubated with sperm (IVF) in the presence or absence of 150ng/mL progesterone (P4) to induce the acrosome reaction (AR); (B) IVM oocytes from C-COCS were denuded (n=52) or not (n=67) before IVF in the presence of P4;; (C) in vivo-matured oocytes (n=15) recovered by transvaginal ultrasound-guided aspiration from preovulatory follicles 32h after the donor mare was treated with hCG, were fertilized in vitro in the presence of P4. In all cases, IVF was performed with frozen-thawed, Percoll-selected sperm from a single stallion, at a final concentration of 1×106spermatozoa/ml in fertil-TALP for 20h (Parrish et al., 1988 Biol. Reprod. 38, 1171–1180). In experiment 2, the possibility that semen cryopreservation or stallion critically influenced IVF was examined by incubating denuded IVM oocytes with fresh or frozen/thawed sperm from the same (fresh;; n=17 for both C- and E-COCs and frozen-thawed; n=12 and 21 for C and E-COCs, respectively) or one other stallion (Fresh;; n=12 and 19 and frozen-thawed; n=12 and 19 for C and E-COCs, respectively), in the presence of P4 for 20h. In both experiments, the resulting sperm-oocyte complexes were fixed, permeabilized and labelled with fluorescein-conjugated peanut agglutinin (EY Laboratores, San Mateo, CA, USA) and ethidium homodimer (Molecular Probes, Eugene, OR, USA) to stain the acrosomal membrane and DNA, respectively, so that membrane status and position of the sperm within the oocyte investments could be detected by confocal laser scanning microscopy. The total number of sperm bound per oocyte was compared between treatments using one-way ANOVA with pair-wise multiple comparison (Bonferroni t-test). Despite binding to the zona pellucida (ZP), neither fresh nor frozen/thawed sperm from either stallion acrosome-reacted or penetrated any oocytes, irrespective of cumulus morphology at the onset of IVM, denudation prior to IVF or the presence of P4. However, more sperm bound to the ZP of cumulus-denuded IVM oocytes (65±32 and 62±28 [mean±sd] for C and E-COCs, respectively), than cumulus-intact IVM (5±4) or in vivo-matured oocytes (23±17: P&lt;0.001). None of the other factors investigated affected bound sperm numbers. In all cases, ZP-bound sperm failed to AR in the classical fashion, and all oocytes remained arrested at the MII stage. In summary, fertilization failed because sperm did not acrosome-react after binding to the ZP. It is concluded that failure to adequately activate stallion sperm is an important obstacle to successful IVF in horses.

scholarly journals Mechanism of Action of Hydroxychloroquine in the Antiphospholipid Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5023-5023
Author(s):  
Nadine Müller-Calleja ◽  
Davit Manukyan ◽  
Wolfram Ruf ◽  
Karl Lackner
Keyword(s):  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Laser Scanning ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
In Vivo Model

Abstract Objective. The antiphospholipid syndrome (APS) is characterized by thromboembolic events and/or recurrent abortions in the presence of pathogenic antiphospholipid antibodies (aPL). Hydroxychloroquine (HCQ) is recommended for thromboprophylaxis in patients with systemic lupus erythematosus and aPL. We aimed at identifying mechanisms responsible for the beneficial effects of HCQ in the APS. Methods. Monocytic cells were stimulated with human aPL in vitro and expression of tissue factor (TF) and tumor necrosis factor (TNFα) was quantified by qRT-PCR. For intracellular ROS detection, cells were stained with OxyBurst and analyzed by flow cytometry. Live cell imaging was performed by confocal laser scanning microscopy to show translocation of NADPH oxidase 2 (NOX2) and TLR8 on stimulation. Finally, the effect of HCQ was tested in an in vivo model of venous thrombosis. Results. HCQ prevents the induction of TF and TNFα by aPL in MonoMac1 cells, by blocking the activation of endosomal NOX2. This latter effect is not mediated by direct interference with the previously described uptake of aPL into the endosome but by inhibiting the recruitment of the catalytic subunit of NOX2 (gp91phox) into the endosome. Furthermore, HCQ protects mice from aPL induced and NOX2 mediated thrombus formation in vivo. Conclusion. We propose that interference with the assembly of endosomal NOX2 is a major effect of HCQ responsible for its antithrombotic effect in the APS. Since most if not all APS patients harbor aPL that activate endosomal NOX2 this probably explains the well established efficacy of HCQ in APS patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization and Comparison of Biofilm Development by Pathogenic and Commensal Isolates of Histophilus somni

2007 ◽  
Vol 189 (22) ◽  
pp. 8179-8185 ◽  
Author(s):  
Indra Sandal ◽  
Wenzhou Hong ◽  
W. Edward Swords ◽  
Thomas J. Inzana
Keyword(s):  
Extracellular Matrix ◽  
Surface Area ◽  
Laser Scanning ◽  
Systemic Disease ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Histophilus Somni ◽  
Biofilm Development

ABSTRACT Histophilus somni (Haemophilus somnus) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.

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