Retrospective, controlled observational case study of patients with central retinal vein occlusion and initially low visual acuity treated with an intravitreal dexamethasone implant

Background Patients with initially low visual acuity were excluded from the therapy approval studies for retinal vein occlusion. But up to 28 % of patients presenting with central retinal vein occlusion have a baseline BCVA of less than 34 ETDRS letters (0.1). The purpose of our study was to assess visual acuity and central retinal thickness in patients suffering from central retinal vein occlusion and low visual acuity (<0.1) in comparison to patients with visual acuity (≥0.1) treated with Dexamethasone implant 0.7 mg for macular edema. Methods Retrospective, controlled observational case study of 30 eyes with macular edema secondary to central retinal vein occlusion, which were treated with a dexamethasone implantation. Visual acuity, central retinal thickness and intraocular pressure were measured monthly. Analyses were performed separately for eyes with visual acuity <0.1 and ≥0.1. Results Two months post intervention, visual acuity improved only marginally from 0.05 to 0.07 (1 month; p = 0,065) and to 0.08 (2 months; p = 0,2) in patients with low visual acuity as compared to patients with visual acuity ≥0.1 with an improvement from 0.33 to 0.47 (1 month; p = 0,005) and to 0.49 (2 months; p = 0,003). The central retinal thickness, however, was reduced in both groups, falling from 694 to 344 μm (1 month; p = 0.003,) to 361 μm (2 months; p = 0,002) and to 415 μm (3 months; p = 0,004) in the low visual acuity group and from 634 to 315 μm (1 month; p < 0,001) and to 343 μm (2 months; p = 0,001) in the visual acuity group ≥0.1. Absence of visual acuity improvement was related to macular ischemia. Conclusions In patients with central retinal vein occlusion and initially low visual acuity, a dexamethasone implantation can lead to an important reduction of central retinal thickness but may be of limited use to increase visual acuity

Toluidine blue staining as a rapid measure for initiation of oocyte growth and fertility in Varroa jacobsoni Oud

A whole mount method was used to determine the moment of activation of oocyte growth in Varroa jacobsoni females. Ovaries of the mites were dissected and stained with toluidine blue. The coloration of the terminal oocyte indicates the uptake of euplasmatic and/or yolk material and, therefore, the initiation of the reproductive phase. In phoretic mites from adult bees, no staining of the ovary could be detected. V. jacobsoni females artificially introduced into freshly capped brood cells and removed for dissection 6 h later already showed clear blue staining of the terminal oocyte. The ovaries of V. jacobsoni females introduced 14 h after capping of the brood cell, however, remained uncolored after incubation in toluidine blue. We conclude that, in phoretic mites, oogenesis is arrested at a previtellogenic phase. Immediately after invasion of the brood cell, reproduction is activated by an as yet unknown host factor. This factor is present in freshly capped brood cells but not in brood cells 14 h after capping. Our new method offers new possibilities for the exact determination of oocyte growth and, therefore, for the study of V. jacobsoni fertility in different host and parasite populations.Coloration au bleu de toluidine pour une détermination rapide du début de la croissance des ovocytes et de la fertilité chez Varroa jacobsoni Oud. La fertilité réduite des acariens Varroa jacobsoni dans les cellules de couvain d'ouvrières (Apis mellifera L.) est l'un des facteurs clés liés à la tolérance à la varroose, comme cela a été montré chez l'hôte d'origine Apis cerana Fabr. et chez les abeilles africanisées du Brésil. Les signaux qui déclenchent l'activation de la reproduction de l'acarien après l'invasion de la cellule de couvain restent à ce jour inconnus. Nous avons utilisé une nouvelle méthode de coloration in toto pour déterminer avec précision le début de la croissance des ovocytes. Des femelles phorétiques de V. jacobsoni ont été prélevées sur des abeilles adultes et introduites artificiellement dans des cellules de couvain fraîchement operculées et dans des cellules de couvain operculées depuis 14-16 h. Toutes les 6 h, les acariens ont été retirés des cellules et pesés sur une balance de précision. Immédiatement après la pesée, les ovaires ainsi que la spermathèque et l'organe en forme de lyre, ont été soigneusement disséqués, fixés dans la formaline à 4 % et colorés durant 30 min au bleu de toluidine à 0,005 % . Les ovocytes ont été lavés dans un tampon PBS et leur coloration a été analysée au microscope. La coloration de l'ovocyte dépend de l'incorporation de vitellus ou d'autres constituants euplasmatiques et marque donc l'activation de l'ovaire. Tous les acariens présents sur le couvain ont vu leur poids augmenter d'environ 325 $\mu$g à plus de 400 $\mu$g durant les 24 h qui ont suivi l'introduction (Fig. 1), quel que soit le moment de l'introduction. Ce comportement alimentaire indique une disposition générale des femelles phorétiques de V. jacobsoni à entrer dans la phase de reproduction. Mais chez tous les acariens phorétiques examinés (n = 18) aucune coloration au bleu de toluidine n'a été détectée (Tab. I ; Fig. 2a). Ceci confirme les observations antérieures selon lesquelles l'ovaire des acariens phorétiques est bloqué à un stade de prévitellogenèse. Pourtant, dans les ovaires des acariens présents sur le couvain, une coloration bleu clair de l'ovocyte terminal était déjà visible 6 h après l'introduction artificielle des acariens dans les cellules fraîchement operculées (n = 41 ; Tab. I ; Fig. 2b). Sur les 10 femelles de V. jacobsoni introduites 14 à 16 h après l'operculation, nous n'avons trouvé un ovocyte coloré que dans un seul ovaire (Tab. I), c'est-à-dire que, chez 90 % des acariens de ce groupe, la maturation des ovocytes n'avait pas commencé. En conclusion, la reproduction de V. jacobsoni doit être stimulée par un signal qui n'est présent que dans les cellules fraîchement operculées. Si les femelles de V. jacobsoni sont introduites dans des cellules operculées depuis 14 h, la croissance des ovocytes n'est pas du tout démarrée, ce qui indique l'absence de signal d'activation à ce stade de l'hôte. La nature du signal et les mécanismes de stimulation de la reproduction de V. jacobsoni restent inconnus. Notre nouvelle méthode offre la possibilité de déterminer avec précision la croissance des ovocytes dans de grands échantillons de femelles de V. jacobsoni. C'est donc un outil approprié pour des études en conditions semi-naturelles de la fertilité de V. jacobsoni dans des populations qui présentent divers degrés de tolérance à la varroose

Stretch exercises for stem cells expand the skin

Responses of epidermal stem cells to stretch in a living organism. Stretching the skin of mice reveals that mechanical strain is communicated by a subpopulation of stem cells that proliferate and promote mechanical resistance, and so generate extra skin

Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers

Funding: This work received financial support from the Leverhulme Trust (RPG-2017-231), the European Union’s Horizon 2020 Framework Programme (FP/2014-2020)/ERC grant agreement no. 640012 (ABLASE), EPSRC (EP/P030017/1), the Humboldt Foundation (Alexander von Humboldt professorship) and the RS Macdonald Charitable Trust (St Andrews Seedcorn Fund for Neurological Research). M.S. acknowledges funding from the European Commission (Marie Skłodowska-Curie Individual Fellowship, 659213) and the Royal Society (Dorothy Hodgkin Fellowship, DH160102; Research Grant, RGF\R1\180070; Enhancement Award, RGF\EA\180051).Integrating micro- and nanolasers into live cells, tissue cultures and small animals is an emerging and rapidly evolving technique that offers noninvasive interrogation and labeling with unprecedented information density. The bright and distinct spectra of such lasers make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. The implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular approach consisting of two separate procedures. The first procedure instructs users on how to efficiently integrate different types of lasers into living cells, and the second procedure presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125-kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental designs and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the analysis pipeline. We illustrate three applications including the rapid, large-volume mapping of absolute refractive index by using polystyrene microbead lasers, the intracellular sensing of cardiac contractility with polystyrene microbead lasers and long-term cell tracking by using semiconductor nanodisk lasers. Our sample preparation and imaging procedures require 2 days, and setting up the hyperspectral confocal microscope for microlaser characterization requiresPeer reviewe

Transition of responsive mechanosensitive elements from focal adhesions to adherens junctions on epithelial differentiation

The skin’s epidermis is a multilayered epithelial tissue and the first line of defense against mechanical stress. Its barrier function depends on an integrated assembly and reorganization of cell–matrix and cell–cell junctions in the basal layer and on different intercellular junctions in suprabasal layers. However, how mechanical stress is recognized and which adhesive and cytoskeletal components are involved are poorly understood. Here, we subjected keratinocytes to cyclic stress in the presence or absence of intercellular junctions. Both states not only recognized but also responded to strain by reorienting actin filaments perpendicular to the applied force. Using different keratinocyte mutant strains that altered the mechanical link of the actin cytoskeleton to either cell–matrix or cell–cell junctions, we show that not only focal adhesions but also adherens junctions function as mechanosensitive elements in response to cyclic strain. Loss of paxillin or talin impaired focal adhesion formation and only affected mechanosensitivity in the absence but not presence of intercellular junctions. Further analysis revealed the adherens junction protein α-catenin as a main mechanosensor, with greatest sensitivity conferred on binding to vinculin. Our data reveal a mechanosensitive transition from cell–matrix to cell–cell adhesions on formation of keratinocyte monolayers with vinculin and α-catenin as vital players

Polarity signaling balances epithelial contractility and mechanical resistance

Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKC lambda controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKC lambda(-/-) cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity.Peer reviewe