Crystal ribcage: a platform for probing real-time lung function at cellular resolution.

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies.

R-Spondin2, a Positive Canonical WNT Signaling Regulator, Controls the Expansion and Differentiation of Distal Lung Epithelial Stem/Progenitor Cells in Mice.

The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/ß-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Mesenchymal cells in the Lung: Evolving concepts and their role in fibrosis.

Mesenchymal cells in the lung are crucial during development, but also contribute to the pathogenesis of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF), the most common and deadly form of fibrotic interstitial lung diseases. Originally thought to behave as supporting cells for the lung epithelium and endothelium with a singular function of producing basement membrane, mesenchymal cells encompass a variety of cell types, including resident fibroblasts, lipofibroblasts, myofibroblasts, smooth muscle cells, and pericytes, which all occupy different anatomic locations and exhibit diverse homeostatic functions in the lung. During injury, each of these subtypes demonstrate remarkable plasticity and undergo varying capacity to proliferate and differentiate into activated myofibroblasts. Therefore, these cells secrete high levels of extracellular matrix (ECM) proteins and inflammatory cytokines, which contribute to tissue repair, or in pathologic situations, scarring and fibrosis. Whereas epithelial damage is considered the initial trigger that leads to lung injury, lung mesenchymal cells are recognized as the ultimate effector of fibrosis and attempts to better understand the different functions and actions of each mesenchymal cell subtype will lead to a better understanding of why fibrosis develops and how to better target it for future therapy. This review summarizes current findings related to various lung mesenchymal cells as well as signaling pathways, and their contribution to the pathogenesis of pulmonary fibrosis.

Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration.

Lung regeneration deteriorates with aging leading to increased susceptibility to pathologic conditions, including fibrosis. Here, we investigated bleomycin-induced lung injury responses in young and aged mice at single-cell resolution to gain insights into the cellular and molecular contributions of aging to fibrosis. Analysis of 52,542 cells in young (8 weeks) and aged (72 weeks) mice identified 15 cellular clusters, many of which exhibited distinct injury responses that associated with age. We identified Pdgfra + alveolar fibroblasts as a major source of collagen expression following bleomycin challenge, with those from aged lungs exhibiting a more persistent activation compared to young ones. We also observed age-associated transcriptional abnormalities affecting lung progenitor cells, including ATII pneumocytes and general capillary (gCap) endothelial cells (ECs). Transcriptional analysis combined with lineage tracing identified a sub-population of gCap ECs marked by the expression of Tropomyosin Receptor Kinase B (TrkB) that appeared in bleomycin-injured lungs and accumulated with aging. This newly emerged TrkB + EC population expressed common gCap EC markers but also exhibited a distinct gene expression signature associated with aberrant YAP/TAZ signaling, mitochondrial dysfunction, and hypoxia. Finally, we defined ACKR1 + venous ECs that exclusively emerged in injured lungs of aged animals and were closely associated with areas of collagen deposition and inflammation. Immunostaining and FACS analysis of human IPF lungs demonstrated that ACKR1 + venous ECs were dominant cells within the fibrotic regions and accumulated in areas of myofibroblast aggregation. Together, these data provide high-resolution insights into the impact of aging on lung cell adaptability to injury responses.

Histopathological assessment of laterality defects in zebrafish development.

Laterality defects during embryonic development underlie the aetiology of various clinical symptoms of neuropathological and cardiovascular disorders; however, experimental approaches to understand the underlying mechanisms are limited due to the complex organ systems of vertebrate models. Zebrafish have the ability to survive even when the heart stops beating for a while during early embryonic development and those adults with cardiac abnormalities. Therefore, we induced laterality defects and investigated the occurrence of situs solitus, situs inversus, and situs ambiguus in zebrafish development. Histopathological analysis revealed heterotaxy in both embryos and juvenile fish. Additionally, randomization of left-right asymmetry of the brain and heart in individual zebrafish embryos under artificial experimental pressure further demonstrated the advantage of transparent zebrafish embryos as an experimental tool to select or reduce the embryos with laterality defects during early embryonic development for long-term studies, including behavioural and cognitive neuroscience investigations.

WNT Signaling in Lung Repair and Regeneration.

The lung has a vital function in gas exchange between the blood and the external atmosphere. It also has a critical role in the immune defense against external pathogens and environmental factors. While the lung is classified as a relatively quiescent organ with little homeostatic turnover, it shows robust regenerative capacity in response to injury, mediated by the resident stem/progenitor cells. During regeneration, regionally distinct epithelial cell populations with specific functions are generated from several different types of stem/progenitor cells localized within four histologically distinguished regions: trachea, bronchi, bronchioles, and alveoli. WNT signaling is one of the key signaling pathways involved in regulating many types of stem/progenitor cells in various organs. In addition to its developmental role in the embryonic and fetal lung, WNT signaling is critical for lung homeostasis and regeneration. In this minireview, we summarize and discuss recent advances in the understanding of the role of WNT signaling in lung regeneration with an emphasis on stem/progenitor cells.

Diffuse Traumatic Subarachnoid Hemorrhage Mimicking Aneurysmal Bleeding Secondary to Ophthalmic Artery Avulsion: Case Report and Review of the Literature.

BACKGROUND: Traumatic subarachnoid hemorrhage (SAH) is a common finding following traumatic brain injury. In some cases, it can be associated with hydrocephalus. This type of hemorrhage is mostly caused by the rupture of small vessels in the brain and is usually managed conservatively. CASE DESCRIPTION: We present a case of a 60-year-old woman who presented with traumatic luxation of the eye following a fall. This resulted in diffuse SAH (Fisher grade IV) with associated hydrocephalus. We also report on 3 previous similar cases found in the literature. Avulsion of the ophthalmic artery was found to be the cause of the traumatic SAH. Apart from cerebrospinal fluid diversion using an external ventricular drain, the case was managed conservatively. There was no evidence of delayed clinical or radiologic vasospasm. CONCLUSIONS: Traumatic avulsion of the ophthalmic artery may result in diffuse SAH, mimicking that of aneurysmal rupture. This case shows that management of early complications, such as hydrocephalus and seizures, should be the main aim. Surgical or endovascular treatment of the injured artery, however, would be unnecessary.

R-spondins: Multi-mode WNT signaling regulators in adult stem cells.

R-spondins (RSPOs) are secreted cysteine-rich glycoproteins that belong to a superfamily of thrombospondin type 1 repeat-containing proteins. RSPOs together with WNT proteins potentiate canonical WNT/ß-catenin signaling activity. Over the last several years, the understanding of the regulatory mechanisms and functional roles of RSPOs in many biological contexts has increased. Particularly, because a leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), a stem cell marker originally identified as a marker for intestinal stem cells, and two closely related proteins, LGR4 and LGR6, were identified as cognate receptors for RSPOs, significant research progress has been made in understanding the functional roles of RSPO/LGR signaling in stem cell biology. Given the crucial roles of canonical WNT signaling in self-renewal and differentiation of various types of stem cells, examination of RSPO function and underlying mechanism in these stem cells has provided new insight into the regulatory roles of WNT signaling in stem cell behavior. In this review, we summarize and discuss recent advances in the understanding of the signaling mechanism and roles of RSPOs in different stem cell contexts.

3D light-sheet assay assessing novel valproate-associated cardiotoxicity and folic acid relief in zebrafish embryogenesis.

Precise in vivo toxicological assays to determine the cardiotoxicity of pharmaceuticals and their waste products are essential in order to evaluate their risks to humans and the environment following industrial release. In the present study, we aimed to develop the sensitive imaging-based cardiotoxicity assay and combined 3D light-sheet microscopy with a zebrafish model to identify hidden cardiovascular anomalies induced by valproic acid (VPA) exposure. The zebrafish model is advantageous for this assessment because its embryos remain transparent. The 3D spatial localization of fluorescence-labeled cardiac cells in and around the heart using light-sheet technology revealed dislocalization of the heart from the outflow tract in two-day-old zebrafish embryos treated with 50 µM and 100 µM VPA (P < 0.01) and those embryos exposed to 20 µM VPA presented hypoplastic distal ventricles (P < 0.01). These two observed phenotypes are second heart field-derived cardiac defects. Quantitative analysis of the light-sheet imaging demonstrated that folic acid (FA) supplementation significantly increased the numbers of endocardial and myocardial cells (P < 0.05) and the accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate increased in response to the cellular changes occurring in embryonic heart development (P < 0.05). The present study disclosed the cellular mechanism underlying the role of FA in spontaneous cellular changes in cardiogenesis and in VPA-associated cardiotoxicity. The 3D light-sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicities in both humans and animals.