Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea

As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils

Identification of a primary stroma and novel endothelial cell projections in the developing human cornea

Purpose: To investigate the initial events in the development of the human cornea, focusing on cell migration, and extracellular matrix synthesis and organization. To determine whether elastic fibers are present in the extracellular matrix during early human corneal development. Methods: Human corneas were collected from week 7 to week 17 of development. An elastic fiber-enhancing stain, tannic acid–uranyl acetate, was applied to all tissue. Three-dimensional serial block-face scanning electron microscopy combined with conventional transmission electron microscopy was used to analyze the corneal stroma. Results: An acellular collagenous primary stroma with an orthogonal arrangement of fibrils was identified in the central cornea from week 7 of corneal development. At week 7.5, mesenchymal cells migrated toward the central cornea and associated with the acellular collagenous matrix. Novel cell extensions from the endothelium were identified. Elastic fibers were found concentrated in the posterior peripheral corneal stroma from week 12 of corneal development. Conclusions: This study provides novel evidence of an acellular primary stroma in the early development of the embryonic human cornea. Cell extensions exist as part of a communication system and are hypothesized to assist in the migration of the mesenchymal cells and the development of the mature cornea. Elastic fibers identified in early corneal development may play an important role in establishing corneal shape

A comparative study of the elastic fibre system within the mouse and human cornea

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera. We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane

Moving singing for lung health online in response to COVID-19: experience from a randomised controlled trial

Introduction Singing for Lung Health (SLH) is a popular arts-in-health activity for people with long-term respiratory conditions. Participants report biopsychosocial benefits, however research on impact is limited. The ‘SHIELD trial’, a randomised controlled, single (assessor) blind, trial of 12 weeks SLH vs usual care for people with Chronic Obstructive Pulmonary Disease (COPD) (n=120) was set-up to help to address this. The first group (n=18, 9 singing and 9 controls) started face-to-face (5 sessions) before changing to online delivery (7 sessions) due to COVID-19 related physical distancing measures. As such, the experience of this group is here reported as a pilot study to inform further research in this area. Methods We conducted semi-structured interviews and thematic analysis regarding barriers, facilitators and key considerations for transitioning from face-to-face to online delivery. Pilot quantitative outcomes include attendance, pre and post measures of quality of life and disease impact (SF-36, CAT score), breathlessness (MRC breathlessness scale, Dyspnoea-12), depression (PHQ9), anxiety (GAD-7), balance confidence (ABC scale) and physical activity (clinical visit PROactive physical activity in COPD tool, combining subjective rating and actigraphy). Results Attendance was 69% overall, (90% of the face-to-face sessions, 53% online sessions). Analysis of semi-structured interviews identified three themes regarding participation in SLH delivered face-to-face and online, these where 1) perceived benefits; 2) digital barriers (online); 3) digital facilitators (online). Findings were summarised into key considerations for optimising transitioning singing groups from face-to-face to online delivery. Pilot quantitative data suggested possible improvements in depression (treatment effect -4.78 PHQ9 points, p< 0.05, MCID 5) and balance confidence (treatment effect +17.21 ABC Scale points, p=0.04, MCID 14.2). Discussion This study identifies key considerations regarding the adaptation of SLH from face-to-face to online delivery. Pilot data suggest online group singing for people with COPD may deliver benefits related to reducing depression and improved balance confidence

Developmental abnormalities in the cornea of a mouse model for Marfan syndrome

Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive. Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations might be a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea

Probing the ultrafast energy dissipation mechanism of the sunscreen oxybenzone after UVA irradiation

Oxybenzone is a common constituent of many commercially available sunscreens providing photoprotection from ultraviolet light incident on the skin. Femtosecond transient electronic and vibrational absorption spectroscopies have been used to investigate the non-radiative relaxation pathways of oxybenzone in cyclohexane and methanol after excitation in the UVA region. The present data suggest that the photoprotective properties of oxybenzone can be understood in terms of an initial ultrafast excited state enol -> keto tautomerization, followed by efficient internal conversion and subsequent vibrational relaxation to the ground state (enol) tautomer

Structural control of corneal transparency, refractive power and dynamics

The cornea needs to be transparent to visible light and precisely curved to provide the correct refractive power. Both properties are governed by its structure. Corneal transparency arises from constructive interference of visible light due to the relatively ordered arrangement of collagen fibrils in the corneal stroma. The arrangement is controlled by the negatively charged proteoglycans surrounding the fibrils. Small changes in fibril organisation can be tolerated but larger changes cause light scattering. Corneal keratocytes do not scatter light because their refractive index matches that of the surrounding matrix. When activated, however, they become fibroblasts that have a lower refractive index. Modelling shows that this change in refractive index significantly increases light scatter. At the microscopic level, the corneal stroma has a lamellar structure, the parallel collagen fibrils within each lamella making a large angle with those of adjacent lamellae. X-ray scattering has shown that the lamellae have preferred orientations in the human cornea: inferior-superior and nasal-temporal in the central cornea and circumferential at the limbus. The directions at the centre of the cornea may help withstand the pull of the extraocular muscles whereas the pseudo-circular arrangement at the limbus supports the change in curvature between the cornea and sclera. Elastic fibres are also present; in the limbus they contain fibrillin microfibrils surrounding an elastin core, whereas at the centre of the cornea, they exist as thin bundles of fibrillin-rich microfibrils. We present a model based on the structure described above that may explain how the cornea withstands repeated pressure changes due to the ocular pulse

Establishment of long-term ostracod epidermal culture

Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing

Cell–cell and cell–matrix interactions at the presumptive stem cell niche of the chick corneal limbus

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell–cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG–IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG–IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites