Aryl Hydrocarbon Receptor Activation in Pulmonary Alveolar Epithelial Cells Limits Inflammation and Preserves Lung Epithelial Cell Integrity.

The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-ß-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes.

EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

OBJECTIVE: Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). METHODS: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. RESULTS: Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-ß (TGF-ß) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-ß/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-ß-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. CONCLUSION: Our data implicate TGF-ß regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

Airway inflammation accelerates pulmonary exacerbations in cystic fibrosis.

Airway inflammation underlies cystic fibrosis (CF) pulmonary exacerbations. In a prospective multicenter study of randomly selected, clinically stable adolescents and adults, we assessed relationships between 24 inflammation-associated molecules and the future occurrence of CF pulmonary exacerbation using proportional hazards models. We explored relationships for potential confounding or mediation by clinical factors and assessed sensitivities to treatments including CF transmembrane regulator (CFTR) protein synthesis modulators. Results from 114 participants, including seven on ivacaftor or lumacaftor-ivacaftor, representative of the US CF population during the study period, identified 10 biomarkers associated with future exacerbations mediated by percent predicted forced expiratory volume in 1 s. The findings were not sensitive to anti-inflammatory, antibiotic, and CFTR modulator treatments. The analyses suggest that combination treatments addressing RAGE-axis inflammation, protease-mediated injury, and oxidative stress might prevent pulmonary exacerbations. Our work may apply to other airway inflammatory diseases such as bronchiectasis and the acute respiratory distress syndrome.

Alveolar macrophages from EVALI patients and e-cigarette users: a story of shifting phenotype.

Exposure to e-cigarette vapors alters important biologic processes including phagocytosis, lipid metabolism, and cytokine activity in the airways and alveolar spaces. Little is known about the biologic mechanisms underpinning the conversion to e-cigarette, or vaping, product use-associated lung injury (EVALI) from normal e-cigarette use in otherwise healthy individuals. We compared cell populations and inflammatory immune populations from bronchoalveolar lavage fluid in individuals with EVALI to e-cigarette users without respiratory disease and healthy controls and found that e-cigarette users with EVALI demonstrate a neutrophilic inflammation with alveolar macrophages skewed towards inflammatory (M1) phenotype and cytokine profile. Comparatively, e-cigarette users without EVALI demonstrate lower inflammatory cytokine production and express features associated with a reparative (M2) phenotype. These data indicate macrophage-specific changes are occurring in e-cigarette users who develop EVALI.

Immunopathological insights into villitis of unknown etiology on the basis of transplant immunology.

Villitis of unknown etiology (VUE) is an inflammatory disease characterized by the infiltration of maternal CD8 +T cells into the placental villi. Although the pathogenesis of VUE is still debated, dysregulation of the immune system appears to be an important factor in the development of the disease. Interaction of maternal T cells with the fetal antigens seems to be the trigger for the VUE onset. In this context, graft vs host disease (GVHD) and allographic rejection seem to share similarities in the VUE immunopathological mechanism, especially those related to immunoregulation. In this review, we compared the immunological characteristics of VUE with allograft rejection, and GVHD favoring a better knowledge of VUE pathogenesis that may contribute to VUE therapeutics strategies in the future.

Regulation of hyperoxia-induced neonatal lung injury via post-translational cysteine redox modifications.

Preterm infants and patients with lung disease often have excess fluid in the lungs and are frequently treated with oxygen, however long-term exposure to hyperoxia results in irreversible lung injury. Although the adverse effects of hyperoxia are mediated by reactive oxygen species, the full extent of the impact of hyperoxia on redox-dependent regulation in the lung is unclear. In this study, neonatal mice overexpressing the beta-subunit of the epithelial sodium channel (ß-ENaC) encoded by Scnn1b and their wild type (WT; C57Bl6) littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Results showed that O2-induced lung injury in transgenic Scnn1b mice is attenuated following chronic O2 exposure. To test the hypothesis that reversible cysteine-redox-modifications of proteins play an important role in O2-induced lung injury, we performed proteome-wide profiling of protein S-glutathionylation (SSG) in both WT and Scnn1b overexpressing mice maintained at 21% O2 (normoxia) or FiO2 85% (hyperoxia) from birth to 11-15 days postnatal. Over 7700 unique Cys sites with SSG modifications were identified and quantified, covering more than 3000 proteins in the lung. In both mouse models, hyperoxia resulted in a significant alteration of the SSG levels of Cys sites belonging to a diverse range of proteins. In addition, substantial SSG changes were observed in the Scnn1b overexpressing mice exposed to hyperoxia, suggesting that ENaC plays a critically important role in cellular regulation. Hyperoxia-induced SSG changes were further supported by the results observed for thiol total oxidation, the overall level of reversible oxidation on protein cysteine residues. Differential analyses reveal that Scnn1b overexpression may protect against hyperoxia-induced lung injury via modulation of specific processes such as cell adhesion, blood coagulation, and proteolysis. This study provides a landscape view of protein oxidation in the lung and highlights the importance of redox regulation in O2-induced lung injury.

Molecular insights into the multifaceted functions and therapeutic targeting of high mobility group box 1 in metabolic diseases.

HMGB1 is a ubiquitously expressed protein localized in nucleus, cytoplasm, as well as secreted into extracellular space. Nuclear HMGB1 binds to DNAs and RNAs, regulating genomic stability and transcription. Cytoplasmic HMGB1 regulates autophagy through binding to core autophagy regulators. Secreted extracellular HMGB1 functions as a ligand to various receptors (RAGE and TLRs, etc.), regulating multiple signalling pathways, such as MAPK, PI3K and NF-κB signallings. Trafficking and localization of HMGB1 across cellular compartments could be regulated by its posttranslational modifications, which fine-tune its functions in metabolic diseases, inflammation and cancers. The current review examines the up-to-date findings pertaining to the biological functions of HMGB1, with focus on its posttranslational modifications and roles in downstream signalling pathways involved in metabolic diseases. This review also discusses the feasibility of targeting HMGB1 as a potential pharmacological intervention for metabolic diseases.

Tumor microenvironment in salivary gland carcinomas: An orchestrated state of chaos.

Salivary gland carcinomas (SGC) are rare tumors of heterogeneous morphology and many histological subtypes. Like other tumors, the SGC mass consists of a varied population of malignant cells and a diverse array of immune cells, cancer-associated fibroblasts, cytokines, chemokines, extracellular matrix proteins, and metalloproteinases, collectively known as the tumor microenvironment (TME). This chaotic network serves as an important physical mediator of cancer cell growth. In this review, we provided current insights into the TME of some of the most common SGC. Here, we highlighted the histological heterogeneity of these tumors, delineated the nature/intensity of inflammatory infiltrates, and the mechanisms involved in immunological escaping related to each SGC subtype.

Salivary gland cancer in the setting of tumor microenvironment: Translational routes for therapy.

Salivary gland carcinomas (SGC) are aggressive cancers that arise in minor and major salivary glands. Given the complexity and the multiple subtypes of this class of tumors, diagnosis and, treatment may be challenging for clinicians. Recently the tumor microenvironment, composed mainly of immune and stromal cells are been a target for treatment. Accumulating evidence indicates that cancer immunotherapies have made a significant impact on oncologic patients, however immunotherapeutic attempts in SGC have been shown limited improvement. Advances in the models that best translate aggressive SGC are needed for the development of clinical protocols grouping immunotherapies and other classes of drugs that will promote better responses in patients with advanced SGC stages. In this review, we introduced different experimental models for SGC with a focus on tumor microenvironment highlighting potential therapy applications for each model.

Enhanced epithelial sodium channel activity in neonatal Scnn1b mouse lung attenuates high oxygen-induced lung injury.

Prolonged oxygen therapy leads to oxidative stress, epithelial dysfunction, and acute lung injury in preterm infants and adults. Heterozygous Scnn1b mice, which overexpress lung epithelial sodium channels (ENaC), and their wild-type (WT) C57Bl6 littermates were utilized to study the pathogenesis of high fraction inspired oxygen ([Formula: see text])-induced lung injury. Exposure to high [Formula: see text] from birth to postnatal (PN) day 11 was used to model oxidative stress. Chronic exposure of newborn pups to 85% O2 increased glutathione disulfide (GSSG) and elevated the GSH/GSSG redox potential (Eh) of bronchoalveolar lavage fluid (BALF). Longitudinal X-ray imaging and Evans blue-labeled-albumin assays showed that chronic 85% O2 and acute GSSG (400 µM) exposures decreased alveolar fluid clearance (AFC) in the WT lung. Morphometric analysis of WT pups insufflated with GSSG (400 µM) or amiloride (1 µM) showed a reduction in alveologenesis and increased lung injury compared with age-matched control pups. The Scnn1b mouse lung phenotype was not further aggravated by chronic 85% O2 exposure. These outcomes support the hypothesis that exposure to hyperoxia increases GSSG, resulting in reduced lung fluid reabsorption due to inhibition of amiloride-sensitive ENaC. Flavin adenine dinucleotide (FADH2; 10 µM) was effective in recycling GSSG in vivo and promoted alveologenesis, but did not impact AFC nor attenuate fibrosis following high [Formula: see text] exposure. In conclusion, the data indicate that FADH2 may be pivotal for normal lung development, and show that ENaC is a key factor in promoting alveologenesis, sustaining AFC, and attenuating fibrotic lung injury caused by prolonged oxygen therapy in WT mice.

Hypoxia induces expression of angiotensin-converting enzyme II in alveolar epithelial cells: Implications for the pathogenesis of acute lung injury in COVID-19.

SARS-CoV-2 uptake by lung epithelial cells is a critical step in the pathogenesis of COVID-19. Viral entry is dependent on the binding of the viral spike protein to the angiotensin converting enzyme II protein (ACE2) on the host cell surface, followed by proteolytic cleavage by a host serine protease such as TMPRSS2. Infection of alveolar epithelial cells (AEC) in the distal lung is a key feature in progression to the acute respiratory distress syndrome (ARDS). We hypothesized that AEC expression of ACE2 is induced by hypoxia. In a murine model of hypoxic stress (12% FiO2), the total lung Ace2 mRNA and protein expression was significantly increased after 24 hours in hypoxia compared to normoxia (21% FiO2). In experiments with primary murine type II AEC, we found that exposure to hypoxia either in vivo (prior to isolation) or in vitro resulted in greatly increased AEC expression of both Ace2 (mRNA and protein) and of Tmprss2. However, when isolated type II AEC were maintained in culture over 5 days, with loss of type II cell characteristics and induction of type I cell features, Ace2 expression was greatly reduced, suggesting that this expression was a feature of only this subset of AEC. Finally, in primary human small airway epithelial cells (SAEC), ACE2 mRNA and protein expression were also induced by hypoxia, as was binding to purified spike protein. Hypoxia-induced increase in ACE2 expression in type II AEC may provide an explanation of the extended temporal course of human patients who develop ARDS in COVID-19.

SARS-CoV-2 innate effector associations and viral load in early nasopharyngeal infection.

COVID-19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS-CoV-2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID-19 testing for symptoms at drive-through COVID-19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real-time polymerase chain reaction assays and quantitative proteomics to 20 virus-positive and 20 virus-negative samples. ACE-2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78-63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10-1.23). Transcripts for two interferons (IFN) were elevated, IFN-λ1 (OR =71, CI =7.07-713) and IFN-λ2 (OR =40.2, CI =3.86-419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23-1.49 and OR =1.33 CI =1.20-1.47, respectively). Only transcripts for IP-10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01-2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN-γ OR =0.90 CI =0.33-0.79, IFN-λ2,3 OR =0.60 CI =0.48-0.74) suggesting viral-induced shut-off of host antiviral protein responses. However, proteins for IP-10 (OR =3.74 CI =2.07-6.77) and several interferon-stimulated genes (ISG) increased with viral load (BST-1 OR =25.1, CI =3.33-188; IFIT1 OR =19.5, CI =4.25-89.2; IFIT3 OR =245, CI =15-4020; MX-1 OR =3.33, CI =1.44-7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS-CoV-2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral-induced host shut-off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

SARS-CoV-2 Innate Effector Associations and Viral Load in Early Nasopharyngeal Infection.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

Oxidized Glutathione Increases Delta-Subunit Expressing Epithelial Sodium Channel Activity in Xenopus laevis Oocytes.

Epithelial sodium channels (ENaC) are heterotrimeric structures, made up of α, ß, and γ subunits, and play an important role in maintaining fluid homeostasis. When δ-ENaC subunits are expressed in place of (or in addition to) the α-ENaC subunit alongside ß- and γ- subunits, fundamental changes in the biophysical properties of ENaC can be observed. Using human ENaC cRNA constructs and the Xenopas laevis oocyte expression system, we show that oxidized glutathione (GSSG) differently effects αßγ-ENaC and αßγ-ENaC current. GSSG (400 µM) significantly decreased normalized whole cell current in oocytes expressing αßγ-ENaC, and conversely increased whole cell current in δ1ßγ-ENaC and δ2ßγ-ENaC expressing oocytes. GSSG treatment increased current in oocytes expressing all four subunits. Western blot and PCR analysis show that human small airway epithelial cells (hSAEC) express canonical αßγ-subunits alongside δ-ENaC subunits. Differences in single channel responses to GSSG in hSAECs indicate that airway epithelia redox sensitivity may depend on whether δ- or α- subunits assemble in the membrane. In silico analysis predict that six Cys amino acids in the δ-ENaC extracellular loop, and a single Cys in the N-terminal domain, are susceptible to post-translational modification by GSSG. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of oxidized glutathione and δ-ENaC in lung disorders.

High-mobility group box-1 increases epithelial sodium channel activity and inflammation via the receptor for advanced glycation end products.

Cystic fibrosis (CF) lung disease persists and remains life-limiting for many patients. Elevated high-mobility group box-1 protein (HMGB-1) levels and epithelial sodium channel hyperactivity (ENaC) are hallmark features of the CF lung. The objective of this study was to better understand the pathogenic role of HMGB-1 signaling and ENaC in CF airway cells. We hypothesize that HMGB-1 links airway inflammation [via signaling to the receptor for advanced glycation end products (RAGE)] and airway surface liquid dehydration (via upregulation of ENaC) in the CF lung. We calculated equivalent short-current (Isc) and single-channel ENaC open probability (Po) in normal and CF human small airway epithelial cells (SAEC) in the presence and absence of human HMGB-1 peptide (0.5 µg/mL). In normal SAECs, HMGB-1 increased amiloride-sensitive Isc and elevated ENaC Po from 0.15 ± 0.03 to 0.28 ± 0.04 (P < 0.01). In CF SAECs, ENaC Po increased from 0.45 ± 0.06 to 0.73 ± 0.04 (P < 0.01). Pretreatment with 1 µM FPS-ZM1 (a RAGE inhibitor) attenuated all HMGB-1 effects on ENaC current in normal and CF SAECs. Confocal analysis of SAECs indicates that nuclear size and HMBG-1 localization can be impacted by ENaC dysfunction. Masson's trichrome labeling of mouse lung showed that intraperitoneally injected HMGB-1 significantly increased pulmonary fibrosis. Bronchoalveolar lavage fluid from HMGB-1-treated mice showed significant increases in IL-1ß, IL-10, IL-6, IL-27, IL-17A, IFN-ß, and granulocyte-macrophage colony-stimulating factor compared with vehicle-injected mice (P < 0.05). These studies put forth a new model in which HMGB-1 signaling to RAGE plays an important role in perpetuating ENaC dysfunction and inflammation in the CF lung.

RAGE-induced changes in the proteome of alveolar epithelial cells.

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1ß and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. SIGNIFICANCE STATEMENT: In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have implications for our understanding of RAGE signaling during lung injury. Our study was the first proteomic comparison showing the effects of RAGE activation from alveolar epithelial cells that constitutively express RAGE and these results can affect a wide field of lung biology, pulmonary therapeutics, and proteomics.

Angiotensin II regulates δ-ENaC in human umbilical vein endothelial cells.

The amiloride-sensitive epithelial sodium channel (ENaC) has been characterized in a variety of non-epithelial tissues. In the current study we sought to understand the effect of angiotensin II on δ ENaC function using human umbilical vein endothelial cells (HUVECs). The δ ENaC subunit is found in humans, but notably absent in rat and most mouse epithelial tissues. In this study we report the presence of δ ENaC in HUVECS with a half-life of ~80min and a change in δ ENaC abundance when HUVECs were treated with angiotensin II. We also observed that angiotensin II increased apical membrane expression of δ ENaC and decreased protein ubiquitination. Equivalent short circuit current measurements showed angiotensin II increased δ ENaC ion transport in HUVEC cells. Treatment with the antioxidant apocynin attenuated angiotensin II mediated effects indicating an important role for angiotensin-derived H2O2 in δ ENaC subunit regulation. Whole cell recordings from oocytes injected with δßγ ENaC shows H2O2-sensitive current. These results suggest that δ ENaC subunits can make up functional channel in HUVEC cells that are regulated by angiotensin II in a redox-sensitive manner. The novel findings have significant implications for our understanding of the role of ENaC in vascular conditions in which oxidative stress occurs.

Three-dimensional printing of X-ray computed tomography datasets with multiple materials using open-source data processing.

Advances in three-dimensional (3D) printing allow for digital files to be turned into a "printed" physical product. For example, complex anatomical models derived from clinical or pre-clinical X-ray computed tomography (CT) data of patients or research specimens can be constructed using various printable materials. Although 3D printing has the potential to advance learning, many academic programs have been slow to adopt its use in the classroom despite increased availability of the equipment and digital databases already established for educational use. Herein, a protocol is reported for the production of enlarged bone core and accurate representation of human sinus passages in a 3D printed format using entirely consumer-grade printers and a combination of free-software platforms. The comparative resolutions of three surface rendering programs were also determined using the sinuses, a human body, and a human wrist data files to compare the abilities of different software available for surface map generation of biomedical data. Data shows that 3D Slicer provided highest compatibility and surface resolution for anatomical 3D printing. Generated surface maps were then 3D printed via fused deposition modeling (FDM printing). In conclusion, a methodological approach that explains the production of anatomical models using entirely consumer-grade, fused deposition modeling machines, and a combination of free software platforms is presented in this report. The methods outlined will facilitate the incorporation of 3D printed anatomical models in the classroom. Anat Sci Educ 10: 383-391. © 2017 American Association of Anatomists.

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.