Search

Rationale: ABCA3 mutations are known to cause fatal surfactant deficiency.Objective: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis.Methods: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting.Results: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively.Conclusion: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis. [ABSTRACT FROM AUTHOR]

Abstract In order to understand how nanoparticles (NPs J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Abstract Background Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO2) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO2 effects on surfactant ultrastructure were visualized. Methods A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 μg/ml) of TiO2 NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope. Results TiO2 NSP, but not MSP, induced a surfactant dysfunction. For TiO2 NSP, adsorption surface tension (γads) increased in a dose-dependent manner from 28.2 ± 2.3 mN/m to 33.2 ± 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (γmin) slightly increased from 4.8 ± 0.5 mN/m up to 8.4 ± 1.3 mN/m (p < 0.01) at high TiO2 NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both γads (63.6 ± 0.4 mN/m) and γmin (21.1 ± 0.4 mN/m). Interestingly, TiO2 NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae. Conclusion TiO2 nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.

Abstract Background Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 μm and 2.5 μm) and ultrafine ( ≤ 0.1 μm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number. Methods Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (∅ fine – 1.062 μm, ultrafine – 0.041 μm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy. Results Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations. Conclusion This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM.

Abstract Background Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells. Methods Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4°C and 50 min of reperfusion at 37°C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells. Results Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 μm3(0.10)) and CE+S (481 μm3(0.10)) compared with controls (323 μm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05). Conclusion Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant.

Abstract Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions. This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells. This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Abstract Background Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. Methods SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. Main Results After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. Conclusion Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Abstract Background Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. Results Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 μm) and nano-sized (0.078 μm) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 μm) and titanium dioxide (0.02–0.03 μm) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-α in the supernatants. We measured a 2–3 fold increase of tumour necrosis factor-α in the supernatants after applying 1 μm polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. Conclusion Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-α.

Abstract Background Translocation of nanoparticles (NP) from the pulmonary airways into other pulmonary compartments or the systemic circulation is controversially discussed in the literature. In a previous study it was shown that titanium dioxide (TiO2) NP were "distributed in four lung compartments (air-filled spaces, epithelium/endothelium, connective tissue, capillary lumen) in correlation with compartment size". It was concluded that particles can move freely between these tissue compartments. To analyze whether the distribution of TiO2 NP in the lungs is really random or shows a preferential targeting we applied a newly developed method for comparing NP distributions. Methods Rat lungs exposed to an aerosol containing TiO2 NP were prepared for light and electron microscopy at 1 h and at 24 h after exposure. Numbers of TiO2 NP associated with each compartment were counted using energy filtering transmission electron microscopy. Compartment size was estimated by unbiased stereology from systematically sampled light micrographs. Numbers of particles were related to compartment size using a relative deposition index and chi-squared analysis. Results Nanoparticle distribution within the four compartments was not random at 1 h or at 24 h after exposure. At 1 h the connective tissue was the preferential target of the particles. At 24 h the NP were preferentially located in the capillary lumen. Conclusion We conclude that TiO2 NP do not move freely between pulmonary tissue compartments, although they can pass from one compartment to another with relative ease. The residence time of NP in each tissue compartment of the respiratory system depends on the compartment and the time after exposure. It is suggested that a small fraction of TiO2 NP are rapidly transported from the airway lumen to the connective tissue and subsequently released into the systemic circulation.

Background: Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells.Methods: Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4 degrees C and 50 min of reperfusion at 37 degrees C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells.Results: Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 microm3(0.10)) and CE+S (481 microm3(0.10)) compared with controls (323 microm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05).Conclusion: Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant. [ABSTRACT FROM AUTHOR]

Obesity is mostly associated with adverse health consequences, but may also elicit favorable effects under chronic conditions. This "obesity paradox" is under debate for pulmonary diseases. As confounding factors complicate conclusions from human studies, this study used a controlled animal model combining diet-induced obesity and chronic hypoxia as a model for pulmonary hypertension and chronic obstructive pulmonary disease. Male C57BL/6 mice were fed control or high-fat diet for 30 wk, and half of the animals were exposed to chronic hypoxia (13% O 2 ) for 3 wk. Hypoxia induced right ventricular hypertrophy, thickening of pulmonary arterial and capillary walls, higher lung volumes, and increased hemoglobin concentrations irrespective of the body weight. In contrast, lung proteomes differed substantially between lean- and obese-hypoxic mice. Many of the observed changes were linked to vascular and extracellular matrix (ECM) proteins. In lean-hypoxic animals, circulating platelets were reduced and abundances of various clotting-related proteins were altered, indicating a hypercoagulable phenotype. Moreover, the septal ECM composition was changed, and airspaces were significantly distended pointing to lung hyperinflation. These differences were mostly absent in the obese-hypoxic group. However, the obesity-hypoxia combination induced the lowest blood CO 2 concentrations, indicating hyperventilation for sufficient oxygen supply. Moreover, endothelial surface areas were increased in obese-hypoxic mice. Thus, obesity exerts differential effects on lung adaptation to hypoxia, which paradoxically include not only adverse but also rather protective changes. These differences have a molecular basis in the lung proteome and may influence the pathogenesis of lung diseases. This should be taken into account for future individualized prevention and therapy. NEW & NOTEWORTHY An "obesity paradox" is discussed for pulmonary diseases. By linking lung proteome analyses to pulmonary structure and function, we demonstrate that diet-induced obesity affects lung adaptation to chronic hypoxia in various ways. The observed changes include not only adverse but also protective effects and are associated with altered abundances of vascular and extracellular matrix proteins. These results highlight the existence of relevant differences in individuals with obesity that may influence the pathogenesis of lung diseases.

The mammalian pulmonary vasculature consists of functionally and morphologically heterogeneous compartments. When comparing sets of lungs, for example, in disease models or therapeutic interventions, local changes may be masked by the overall heterogeneity of the organ structure. Therefore, alterations taking place only in a sub-compartment may not be detectable by global analysis. In the monopodial lung, the characterization of distinct vessel groups is difficult, due to the asymmetrical branching pattern. In this pilot study, a previously established method to classify segments of the monopodial pulmonary arterial tree into homogeneous groups was employed. To test its suitability for experimental settings, the method was applied to a hyperoxia (HYX, ≥95% oxygen) rabbit model of bronchopulmonary dysplasia and a normoxic control group (NOX, 21% oxygen). The method allowed the identification of morphological differences between the HYX and the NOX groups. Globally visible differences in lumen diameter were pinpointed to specific lung regions. Furthermore, local changes of wall dimension and cell layers in single compartments, that would not have been identifiable in an unfocused analysis of the whole dataset, were found. In conclusion, the described method achieves a higher precision in morphological studies of lung disease models, compared to a common, global analysis approach., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O 2 ) or normoxia (21% O 2 ). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

A chronic proinflammatory milieu (inflamm-aging) is observed in the elderly and associated with poorer prognosis in acute lung injury (ALI). Gut microbiome-derived short-chain fatty acids (SCFAs) are known to have immunomodulatory capabilities, but their function in the gut-lung axis in aging is poorly understood. Here, we analyzed the gut microbiome and its impact on inflammatory signaling in the aging lung and tested the effects of SCFAs in young (3 mo) and old (18 mo) mice that received either drinking water with a mixture of each 50 mM acetate, butyrate, and propionate for 2 wk or water alone. ALI was induced by intranasal lipopolysaccharide (LPS; n = 12/group) administration. Controls ( n = 8/group) received saline. Fecal pellets were sampled for gut microbiome analysis before and after LPS/saline treatment. The left lung lobe was collected for stereology and right lung lobes for cytokine and gene expression analysis, inflammatory cell activation, and proteomics. Different gut microbial taxa, such as Bifidobacterium , Faecalibaculum , and Lactobacillus correlated positively with pulmonary inflammation in aging, suggesting an impact on inflamm-aging in the gut-lung axis. The supplementation of SCFAs reduced inflamm-aging, oxidative stress, metabolic alteration, and enhanced activation of myeloid cells in the lungs of old mice. The enhanced inflammatory signaling in ALI of old mice was also reduced by SCFA treatment. In summary, the study provides new evidence that SCFAs play a beneficial role in the gut-lung axis of the aging organism by reducing pulmonary inflamm-aging and ameliorating enhanced severity of ALI in old mice.

Quantitative data about the internal lung structure are needed to better understand normal and pathological lung development. Aberrant lung development causes deficits in alveolar and microvascular development; however, the normal temporal relationship between these processes is still not fully understood. We hypothesized that alveolar and capillary development show a differential time pattern. Lungs of rats aged 3, 7, 14, 21 days (d) or 3 mo ( n = 8-10 each) were fixed by vascular perfusion and processed for light microscopy. Using design-based stereology number, the surface area and volume of alveoli, septal capillaries, and alveolar septa were quantified. The total number and the total volume of alveoli increased progressively during postnatal development. Interestingly, the numerical density of capillary loops was significantly higher in 14- and 21-d-old rats than before or after this age, causing a duplication of the total number of capillary loops between 1 and 2 wk of age. The mean thickness of alveolar septa started to decline slightly at the age of 14d and more pronounced at later stages. Although the septal epithelial surface area increased in proportion to alveolar number during the first 3 wk, the capillary endothelial surface area grew only slightly compared with the number of capillaries. In conclusion, the number of elements composing the alveolar capillary network expands massively during the first two postnatal weeks and exceeds the formation of alveoli. The thinning of the alveolar septa during further development suggests a reduction of the capillary network during alveolarization.

The polyamine spermidine is discussed as a caloric restriction mimetic and therapeutic option for obesity and related comorbidities. This study tested oral spermidine supplementation with regard to the systemic, hepatic and pulmonary lipid metabolism under different diet conditions. Male C57BL/6 mice were fed a purified control (CD), high sucrose (HSD) or high fat (HFD) diet with (-S) or without spermidine for 30 weeks. In CD-fed mice, spermidine decreased body and adipose tissue weights and reduced hepatic lipid content. The HSD induced hepatic lipid synthesis and accumulation and hypercholesterolemia. This was not affected by spermidine supplementation, but body weight and blood glucose were lower in HSD-S compared to HSD. HFD-fed mice showed higher body and fat depot weights, prediabetes, hypercholesterolemia and severe liver steatosis, which were not altered by spermidine. Within the liver, spermidine diminished hepatic expression of lipogenic transcription factors SREBF1 and 2 under HSD and HFD and affected the expression of other lipid-related enzymes. In contrast, diet and spermidine exerted only minor effects on pulmonary parameters. Thus, oral spermidine supplementation affects lipid metabolism in a diet-dependent manner, with significant reductions in body fat and weight under physiological nutrition and positive effects on weight and blood glucose under high sucrose intake, but no impact on dietary fat-related parameters.

Background: Interpretation of results from diet-induced-obesity (DIO) studies critically depends on control conditions. Grain-based chows are optimized for rodent nutrition but do not match the defined composition of purified diets used for DIO, severely limiting the comparability. Purified control diets are recommended but often contain high starch and only minor fiber amounts. It is unknown whether this composition leads to metabolic alterations compared with chow and whether the addition of refined fibers at the expense of starch affects these changes., Methods: In this experiment, 6-week-old C57BL/6N mice were fed (i) a conventional purified control diet (high-starch, low-fiber; Puri-starch), (ii) an alternative, custom-made purified control diet containing pectin and inulin (medium-starch, higher-fiber; Puri-fiber), or (iii) grain-based chow for 30 weeks ( N = 8-10)., Results: Puri-starch feeding resulted in significantly elevated levels of plasma insulin ( p = 0.004), cholesterol ( p < 0.001), and transaminases (AST p = 0.002, ALT p = 0.001), hepatic de novo lipogenesis and liver steatosis, and an altered gut microbiota composition compared with chow-fed mice. In contrast, Puri-fiber exerted only minor effects on systemic parameters and liver lipid homeostasis, and promoted a distinct gut microbiota composition., Conclusion: Carbohydrate-rich purified diets trigger a metabolic status possibly masking pathological effects of nutrients under study, restricting its use as control condition. The addition of refined fibers is suited to create purified, yet physiological control diets for DIO research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schipke, Brandenberger, Vital and Mühlfeld.)

Design-based stereology is the gold standard for obtaining unbiased quantitative morphological data on volume, surface area, and length, as well as the number of tissues, cells or organelles. In cardiac research, the introduction of a stereological method to unbiasedly estimate the number of cardiomyocytes has considerably increased the use of stereology. Since its original description, various modifications to this method have been described. A particular field in which this method has been employed is the normal developmental life cycle of cardiomyocytes after birth, and particularly the question of when, during postnatal development, cardiomyocytes lose their capacity to divide and proliferate, and thus their inherent regenerative ability. This field is directly related to a second major application of stereology in recent years, addressing the question of what consequences intrauterine growth restriction has on the development of the heart, particularly of cardiomyocytes. Advances have also been made regarding the quantification of nerve fibers and collagen deposition as measures of heart innervation and fibrosis. In the present review article, we highlight the methodological progress made in the last 20 years and demonstrate how stereology has helped to gain insight into the process of normal cardiac development, and how it is affected by intrauterine growth restriction.

Aims: Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure., Methods and Results: RT-qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5-/-). Before manifesting cardiac dysfunction, Atg5-/- mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high-frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load, and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5-/- cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5-/- cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload., Conclusion: Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Primordial follicles are important for the reproduction cycle and, therefore, also for the survival of the whole population of a species. Mammals have a large pool of primordial follicles, and it is thought that this pool represents the total number of oocytes. The aim of the present study was to determine the total primordial follicle number of juvenile ringed seals ( Pusa hispida ) from the Gulf of Bothnia and Greenland. Overall, 52 ovaries from two ringed seal populations (West Greenland (N = 6), Gulf of Bothnia, region in the Baltic Sea (N = 46)) were examined. All ovaries were cut into 2 mm thick slices and every slice was embedded in paraffin. Out of each tissue block, a 5 µm thick section was cut and stained with haematoxylin-eosin. The mean volume of the follicles and the total volume of primordial follicles per ovary were estimated by stereology and used to calculate the total estimated number of primordial follicles. The median of the total estimated number of primordial follicles seemed to be higher in Baltic individuals than in Greenland individuals (Gulf of Bothnia = 565,657; Greenland Sea = 122,475). This widens the total range of primordial follicles in ringed seals overall and might bear some potential for discussions regarding the influence of endocrine disruptors and environmental influences depending on different regions/populations and their exposure to various factors. Thus, this study aims to provide basic reference data of the number and mean volume of ringed seal primordial follicles.

Objectives: Esmolol-based cardioplegic arrest offers better cardioprotection than crystalloid cardioplegia but has been compared experimentally with blood cardioplegia only once. We investigated the influence of esmolol crystalloid cardioplegia (ECCP), esmolol blood cardioplegia (EBCP) and Calafiore blood cardioplegia (Cala) on cardiac function, metabolism and infarct size in non-infarcted and infarcted isolated rat hearts., Methods: Two studies were performed: (i) the hearts were subjected to a 90-min cardioplegic arrest with ECCP, EBCP or Cala and (ii) a regional myocardial infarction was created 30 min before a 90-min cardioplegic arrest. Left ventricular peak developed pressure (LVpdP), velocity of contractility (dLVP/dtmax), velocity of relaxation over time (dLVP/dtmin), heart rate and coronary flow were recorded. In addition, the metabolic parameters were analysed. The infarct size was determined by planimetry, and the myocardial damage was determined by electron microscopy., Results: In non-infarcted hearts, cardiac function was better preserved with ECCP than with EBCP or Cala relative to baseline values (LVpdP: 100 ± 28% vs 86 ± 11% vs 57 ± 7%; P = 0.002). Infarcted hearts showed similar haemodynamic recovery for ECCP, EBCP and Cala (LVpdP: 85 ± 46% vs 89 ± 55% vs 56 ± 26%; P = 0.30). The lactate production with EBCP was lower than with ECCP (0.6 ± 0.7 vs 1.4 ± 0.5 μmol/min; P = 0.017). The myocardial infarct size and (ECCP vs EBCP vs Cala: 16 ± 7% vs 15 ± 9% vs 24 ± 13%; P = 0.21) the ultrastructural preservation was similar in all groups., Conclusions: In non-infarcted rat hearts, esmolol-based cardioplegia, particularly ECCP, offers better myocardial protection than Calafiore. After an acute myocardial infarction, cardioprotection with esmolol-based cardioplegia is similar to that with Calafiore., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is a complex condition frequently occurring in preterm newborns, and different animal models are currently used to mimic the pathophysiology of BPD. The comparability of animal models depends on the availability of quantitative data obtained by minimally biased methods. Therefore, the aim of this study was to provide the first design-based stereological analysis of the lungs in the hyperoxia-based model of BPD in the preterm rabbit. Rabbit pups were obtained on gestation day 28 (three days before term) by cesarean section and exposed to normoxic (21% O 2 , n = 8) or hyperoxic (95% O 2 , n = 8) conditions. After seven days of exposure, lung function testing was performed, and lungs were taken for stereological analysis. In addition, the ratio between pulmonary arterial acceleration and ejection time (PAAT/PAET) was measured. Inspiratory capacity and static compliance were reduced whereas tissue elastance and resistance were increased in hyperoxic animals compared with normoxic controls. Hyperoxic animals showed signs of pulmonary hypertension indicated by the decreased PAAT/PAET ratio. In hyperoxic animals, the number of alveoli and the alveolar surface area were reduced by one-third or by approximately 50% of control values, respectively. However, neither the mean linear intercept length nor the mean alveolar volume was significantly different between both groups. Hyperoxic pups had thickened alveolar septa and intra-alveolar accumulation of edema fluid and inflammatory cells. Nonparenchymal blood vessels had thickened walls, enlarged perivascular space, and smaller lumen in hyperoxic rabbits in comparison with normoxic ones. In conclusion, the findings are in line with the pathological features of human BPD. The stereological data may serve as a reference to compare this model with BPD models in other species or future therapeutic interventions., Competing Interests: Chiara Catozzi, Francesca Ricci, Costanza Casiraghi, and Fabrizio Salomone are Chiesi Farmaceutici employees. The rest of the authors declare that they do not have any conflict of interest., (Copyright © 2021 Christian Mühlfeld et al.)

The causal genetic underpinnings of congenital heart diseases, which are often complex and multigenic, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. In this study, we identified mutations in myomesin 2 ( MYOM2 ) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere, and a hub gene within interactions of sarcomere genes. We show that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, our comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 [herein referred to as Drosophila myomesin and myosin binding protein (dMnM)] may be an ortholog of MYOM2 , as well as other myosin binding proteins . Its partial loss of function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc ( MYH6/7 ) exhibited synergistic genetic interactions. In summary, our results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart.This article has an associated First Person interview with Emilie Auxerre-Plantié and Tanja Nielsen, joint first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 μg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Obesity is associated with lung function impairment and respiratory diseases; however, the underlying pathophysiological mechanisms are still elusive, and therapeutic options are limited. This study examined the effects of prolonged excess fat intake on lung mechanics and microstructure and tested spermidine supplementation and physical activity as intervention strategies. C57BL/6N mice fed control diet (10% fat) or high-fat diet (HFD; 60% fat) were left untreated or were supplemented with 3 mM spermidine, had access to running wheels for voluntary activity, or a combination of both. After 30 wk, lung mechanics was assessed, and left lungs were analyzed by design-based stereology. HFD exerted minor effects on lung mechanics and resulted in higher body weight and elevated lung, air, and septal volumes. The number of alveoli was higher in HFD-fed animals. This was accompanied by an increase in epithelial, but not endothelial, surface area. Moreover, air-blood barrier and endothelium were significantly thicker. Neither treatment affected HFD-related body weights. Spermidine lowered lung volumes as well as endothelial and air-blood barrier thicknesses toward control levels and substantially increased the endothelial surface area under HFD. Activity resulted in decreased volumes of lung, septa, and septal compartments but did not affect vascular changes in HFD-fed mice. The combination treatment showed no additive effect. In conclusion, excess fat consumption induced alveolar capillary remodeling indicative of impaired perfusion and gas diffusion. Spermidine alleviated obesity-related endothelial alterations, indicating a beneficial effect, whereas physical activity reduced lung volumes apparently by other, possibly systemic effects.

Podocytes are highly specialized cells with a clear cell polarity. It is known that in health and disease, microvilli protrude from the apical surface of the podocytes into the urinary space. As a basis to better understand the podocyte microprojections/microvilli, the present study analyzed their spatial localization, extension, and contact site with parietal epithelial cells (PECs). Using different electron microscopic (EM) techniques, we analyzed renal corpuscles of healthy young adult male C57BL/6 mice fixed by vascular perfusion. Serial block-face scanning EM was used to visualize entire corpuscles, focused ion beam scanning EM was performed to characterize microprojection/microvilli-rich regions at higher magnification, and transmission EM of serial sections was used to analyze the contact zone between podocyte microprojections and PECs. Numerous microprojections originating from the primary processes of podocytes were present in the urinary space in all regions of the corpuscle. They often reached the apical surface of the PEC but did not make junctional contacts. At high resolution, it was observed that the glycocalyx of both cells was in contact. Depending on the distance between podocytes and PECs, these microprojections had a stretched or coiled state. The present study shows that microprojections/microvilli of podocytes are a physiological feature of healthy mouse kidneys and are frequently in contact with the apical surface of PECs, thus spanning the urinary space. It is proposed that podocyte microprojections serve mechanosensory or communicative functions between podocytes and PECs.

Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction. This study aimed to analyze whether early HFpEF is already associated with ultrastructural alterations and whether they differ quantitatively among the layers of the left ventricular wall. HFpEF was induced in pigs by deoxy-corticosterone acetate (DOCA) treatment along with a high-salt/high lipid diet over 3 months and compared with weight-matched normal pigs (n = 5 each). Samples of the left ventricle were taken and processed for light and electron microscopy. Interstitial fibrosis, subcellular composition of cardiomyocytes and mean cardiomyocyte diameter were evaluated by stereology in subendocardial, midmyocardial and subepicardial regions. DOCA enhanced the mean cardiomyocyte diameter in all locations of the ventricle wall to the same degree. The subcellular composition did not differ between the locations and was not altered by DOCA. The volume fraction of interstitium was smaller in the subendocardium of DOCA group than of control group. Within the interstitium, the volume fraction of collagen fibrils (between cardiomyocytes) was increased in the subendocardial and midmyocardial wall layers of the DOCA group but not in the subepicardial layer. Although the capillary length density and average supply area were not altered in response to DOCA in any of the wall layers, the volume fraction of blood vessels related to the interstitial space was enhanced in the subendocardium of the DOCA group but not in the other wall layers. In conclusion, cardiomyocyte changes due to DOCA were similar in subepicardial, midmyocardial and subendocardial regions but DOCA-induced changes in the interstitium appeared to be more pronounced in the subendocardial ventricular wall layers. This suggests a pivotal role of the subendocardial interstitium in the pathogenesis of HFpEF., (© 2019 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Thin type 1 alveolar epithelial (AE1) and surfactant producing type 2 alveolar epithelial (AE2) cells line the alveoli in the lung and are essential for normal lung function. Function is intimately interrelated to structure, so that detailed knowledge of the epithelial ultrastructure can significantly enhance our understanding of its function. The basolateral surface of the cells or the epithelial contact sites are of special interest, because they play an important role in intercellular communication or stabilizing the epithelium. The latter is in particular important for the lung with its variable volume. The aim of the present study was to investigate the three-dimensional (3D) ultrastructure of the human alveolar epithelium focusing on contact sites and the basolateral cell membrane of AE2 cells using focused ion beam electron microscopy and subsequent 3D reconstructions. The study provides detailed surface reconstructions of two AE1 cell domains and two AE2 cells, showing AE1/AE1, AE1/AE2 and AE2/AE2 contact sites, basolateral microvilli pits at AE2 cells and small AE1 processes beneath AE2 cells. Furthermore, we show reconstructions of a surfactant secretion pore, enlargements of the apical AE1 cell surface and long folds bordering grooves on the basal AE1 cell surface. The functional implications of our findings are discussed. These findings may lay the structural basis for further molecular investigations.

Acute lung injury (ALI) is characterized by enhanced permeability of the air-blood barrier, pulmonary edema, and hypoxemia. MicroRNA-21 (miR-21) was shown to be involved in pulmonary remodeling and the pathology of ALI, and we hypothesized that miR-21 knock-out (KO) reduces injury and remodeling in ALI. ALI was induced in miR-21 KO and C57BL/6N (wildtype, WT) mice by an intranasal administration of 75 µg lipopolysaccharide (LPS) in saline ( n = 10 per group). The control mice received saline alone ( n = 7 per group). After 24 h, lung function was measured. The lungs were then excised for proteomics, cytokine, and stereological analysis to address inflammatory signaling and structural damage. LPS exposure induced ALI in both strains, however, only WT mice showed increased tissue resistance and septal thickening upon LPS treatment. Septal alterations due to LPS exposure in WT mice consisted of an increase in extracellular matrix (ECM), including collagen fibrils, elastic fibers, and amorphous ECM. Proteomics analysis revealed that the inflammatory response was dampened in miR-21 KO mice with reduced platelet and neutrophil activation compared with WT mice. The WT mice showed more functional and structural changes and inflammatory signaling in ALI than miR-21 KO mice, confirming the hypothesis that miR-21 KO reduces the development of pathological changes in ALI.

Lung function declines with advancing age. To improve our understanding of the structure-function relationships leading to this decline, we investigated structural alterations in the lung and their impact on micromechanics and lung function in the aging mouse. Lung function analysis was performed in 3, 6, 12, 18, and 24 months old C57BL/6 mice ( n = 7-8/age), followed by lung fixation and stereological sample preparation. Lung parenchymal volume, total, ductal and alveolar airspace volume, alveolar volume and number, septal volume, septal surface area and thickness were quantified by stereology as well as surfactant producing alveolar epithelial type II (ATII) cell volume and number. Parenchymal volume, total and ductal airspace volume increased in old (18 and 24 months) compared with middle-aged (6 and 12 months) and young (3 months) mice. While the alveolar number decreased from young (7.5 × 10 6 ) to middle-aged (6 × 10 6 ) and increased again in old (9 × 10 6 ) mice, the mean alveolar volume and mean septal surface area per alveolus conversely first increased in middle-aged and then declined in old mice. The ATII cell number increased from middle-aged (8.8 × 10 6 ) to old (11.8 × 10 6 ) mice, along with the alveolar number, resulting in a constant ratio of ATII cells per alveolus in all age groups (1.4 ATII cells per alveolus). Lung compliance and inspiratory capacity increased, whereas tissue elastance and tissue resistance decreased with age, showing greatest changes between young and middle-aged mice. In conclusion, alveolar size declined significantly in old mice concomitant with a widening of alveolar ducts and late alveolarization. These changes may partly explain the functional alterations during aging. Interestingly, despite age-related lung remodeling, the number of ATII cells per alveolus showed a tightly controlled relation in all age groups., (Copyright © 2019 Schulte, Mühlfeld and Brandenberger.)

Generation of three-dimensional (3D) data sets from serial sections of tissues imaged by light microscopy (LM) allows identification of rare structures by morphology or fluorescent labeling. Here, we demonstrate a workflow for correlative LM and electron microscopy (EM) from 3D LM to 3D EM, using the same sectioned material for both methods consecutively. The new approach is easy to reproduce in routine EM laboratories and applicable to a wide range of organs and research questions.

The alveolar capillary network (ACN) has a large surface area that provides the basis for an optimized gas exchange in the lung. It needs to adapt to morphological changes during early lung development and alveolarization. Structural alterations of the pulmonary vasculature can lead to pathological functional conditions such as in bronchopulmonary dysplasia and various other lung diseases. To understand the development of the ACN and its impact on the pathogenesis of lung diseases, methods are needed that enable comparative analyses of the complex three-dimensional structure of the ACN at different developmental stages and under pathological conditions. In this study a newborn mouse lung was imaged with serial block-face scanning electron microscopy (SBF-SEM) to investigate the ACN and its surrounding structures before the alveolarization process begins. Most parts but not all of the examined ACN contain two layers of capillaries, which were repeatedly connected with each other. A path from an arteriole to a venule was extracted and straightened to allow cross-sectional visualization of the data along the path within a plane. This allows a qualitative characterization of the structures that erythrocytes pass on their way through the ACN. One way to define regions of the ACN supplied by specific arterioles is presented and used for analyses. Pillars, possibly intussusceptive, were found in the vasculature but no specific pattern was observed in regard to parts of the saccular septa. This study provides 3D information with a resolution of about 150 nm on the microscopic structure of a newborn mouse lung and outlines some of the potentials and challenges of SBF-SEM for 3D analyses of the ACN., (Copyright © 2019 Buchacker, Mühlfeld, Wrede, Wagner, Beare, McCormick and Grothausmann.)

Diabetes and respiratory diseases are frequently comorbid conditions. However, the mechanistic links between hyperglycemia and lung dysfunction are not entirely understood. This study examined the effects of high sucrose intake on lung mechanics and alveolar septal composition and tested voluntary activity as an intervention strategy. C57BL/6N mice were fed a control diet (CD, 7% sucrose) or a high sucrose diet (HSD, 35% sucrose). Some animals had access to running wheels (voluntary active; CD-A, HSD-A). After 30 weeks, lung mechanics were assessed, left lungs were used for stereological analysis and right lungs for protein expression measurement. HSD resulted in hyperglycemia and higher static compliance compared to CD. Lung and septal volumes were increased and the septal ratio of elastic-to-collagen fibers was decreased despite normal alveolar epithelial volumes. Elastic fibers appeared more loosely arranged accompanied by an increase in elastin protein expression. Voluntary activity prevented hyperglycemia in HSD-fed mice. The parenchymal airspace volume, but not the septal volume, was increased. The septal extracellular matrix (ECM) composition together with the protein expression of ECM components was similar to control levels in the HSD-A-group. In conclusion, HSD was associated with elastic fiber remodeling and reduced pulmonary elasticity. Voluntary activity alleviated HSD-induced ECM alterations, possibly by preventing hyperglycemia.

Background: Excess dietary fat and sugar are linked to obesity and metabolic syndrome. Polyamines such as spermidine are implicated in fat accumulation and may support activity-induced weight loss., Objective: This study tested interventional spermidine supplementation and voluntary activity against fat- and sucrose-induced systemic and gut microbiota changes., Methods: A 3-factorial study design (3 × 2 × 2) was used to test the factors diet, activity, and spermidine. Male 6-wk-old C57BL/6N mice were fed a control diet (CD; carbohydrate:protein:fat, 70%:20%:10% of energy; 7% sucrose), a high-fat diet (HFD; carbohydrate:protein:fat, 20%:20%:60% of energy; 7% sucrose), or a high-sucrose diet (HSD; carbohydrate:protein:fat, 70%:20%:10% of energy; 35% sucrose). Diet groups were left untreated (+0) or had unlimited access to running wheels (+A) or were supplemented with 3 mM spermidine via drinking water (+S) or a combination of both (+A+S) for 30 wk (n = 7-10)., Results: In comparison to the CD, the HFD enhanced body weights (by 36%, P < 0.001), plasma lipids (cholesterol by 24%, P < 0.001; triglycerides by 27%, P = 0.004), and glucose concentrations (by 18%, P < 0.001), whereas the HSD increased weight by 13% (P < 0.001) and fasting glucose by 17% (P < 0.001) but did not increase plasma lipids. Microbiota taxonomic composition changed upon the HFD and HSD (both P < 0.001); however, only the HSD increased microbial diversity (P < 0.001) compared with the CD. Activity influenced microbiota composition (P < 0.01) and reduced glucose concentrations in HSD-fed (P = 0.021) and HFD-fed (P < 0.001) mice compared with nonactive mice. The combination of activity and spermidine affected energy intake (P-interaction = 0.037) and reduced body weights of HSD+A+S mice compared with HSD+0 mice (P = 0.024)., Conclusions: In male C57BL/6N mice, dietary sucrose and fat caused diverse metabolic and microbiota changes that were differentially susceptible to physical exercise. Spermidine has the potential to augment activity-induced beneficial effects, particularly for sucrose-induced obesity., (© 2019 American Society for Nutrition.)

Background: Ex vivo lung perfusion (EVLP) is used by an increasing number of transplant centres. It is still controversial whether an acellular or cellular (erythrocyte enriched) perfusate is preferable. The aim of this paper was to evaluate whether acellular (aEVLP) or cellular EVLP (cEVLP) preserves functional lung ultrastructure better and to generate a hypothesis regarding possible underlying mechanisms., Methods: Lungs of 20 pigs were assigned to 4 groups: control, ischaemia (24 h), aEVLP and cEVLP (both EVLP groups: 24 h ischaemia + 12 h EVLP). After experimental procedures, whole lungs were perfusion fixed, samples for light and electron microscopic stereology were taken, and ventilation, diffusion and perfusion related parameters were estimated., Results: Lung structure was well preserved in all groups. Lungs had less atelectasis and higher air content after EVLP. No significant group differences were found in alveolar septum composition or blood-air barrier thickness. Small amounts of intraalveolar oedema were detected in both EVLP groups but significantly more in aEVLP than in cEVLP., Conclusions: Both EVLP protocols supported lungs well for up to 12 h and could largely prevent ischaemia ex vivo reperfusion associated lung injury. In both EVLP groups, oedema volume remained below the level of functional relevance. The group difference in oedema formation was possibly due to inferior septal perfusion in aEVLP.

Alterations of the pulmonary vasculature are an important feature of human lung diseases such as chronic obstructive pulmonary disease, pulmonary hypertension, and bronchopulmonary dysplasia. Experimental studies to investigate the pathogenesis or a therapeutic intervention in animal models of these diseases often require robust, meaningful, and efficient morphometric data that allow for appropriate statistical testing. The gold standard for obtaining such data is design-based stereology. However, certain morphological characteristics of the pulmonary vasculature make the implementation of stereological methods challenging. For example, the alveolar capillary network functions according to the sheet flow principle, thus making unbiased length estimations impossible and requiring other strategies to obtain mechanistic morphometric data. Another example is the location of pathological changes along the branches of the vascular tree. For developmental defects like in bronchopulmonary dysplasia or for pulmonary hypertension, it is important to know whether certain segments of the vascular tree are preferentially altered. This cannot be overcome by traditional stereological methods but requires the combination of a three-dimensional data set and stereology. The present review aims at highlighting the great potential while discussing the major challenges (such as time consumption and data volume) of this combined approach. We hope to raise interest in the potential of this approach and thus stimulate solutions to overcome the existing challenges.

Objectives: Blood cardioplegia (BCP) can be used in different ways to protect the heart from ischaemia-reperfusion injury during cardiac surgery. Because there could be differences between warm and cold intermittent cardioplegia with or without warm reperfusion, we investigated the influence of 2 blood cardioplegia solutions on cardiac function, metabolism and infarct size in stable and infarcted rat hearts., Methods: The hearts of 32 male Wistar rats were excised and inserted into a blood-perfused isolated heart apparatus. In 16 hearts, an acute myocardial infarction was induced by ligation of the left anterior descending coronary artery at least 30 min before aortic clamping. After aortic clamping, either Calafiore or Buckberg BCP was administered. During reperfusion, coronary blood flow, left ventricular developed pressure and dp/dt max were recorded, and oxygen consumption and lactate production were determined. The infarct size after 90 min of reperfusion was measured by triphenyl tetrazolium chloride staining. The hearts of rats without infarction were investigated using transmission electron microscopy., Results: In hearts without infarction, haemodynamic recovery was similar for Calafiore and Buckberg solutions: left ventricular developed pressure [Cala 62% of baseline (BL), Buck 58% BL] and dp/dt max (Cala 83% BL, Buck 89% BL). Coronary flow, which was slightly less in infarcted hearts, also recovered similarly after the administration of the 2 BCP solutions (Cala 65% BL, Buck 68% BL). During reperfusion, lactate production was similar (Cala 0.85 ml/min, Buck 1.0 ml/min), and the cellular oedema index and mitochondrial swelling were comparable between the 2 groups. In hearts with infarction, left ventricular developed pressure (Cala 58% BL, Buck 56% BL) and dp/dt max (Cala 79% BL, Buck 72% BL) showed similar recovery for reperfusion with Calafiore or Buckberg BCP. In addition, coronary flow recovered similarly (Cala 54% BL, Buck 57% BL). During reperfusion, myocardial oxygen consumption was lower in the Cala (67% BL) than in the Buck (82% BL) group, but lactate production was similar between the Cala (1.1 ml/min) and the Buck (1.1 ml/min) groups. Myocardial infarct size was also similar in the Cala group (24%) and in the Buck group (26%)., Conclusions: In stable perfused rat hearts and in an in vitro model of acute myocardial infarction, the 2 BCP solutions offer equally good myocardial protection.

Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (β-MyHC, MYH7 ) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype β-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either β-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7 -mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7 -pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca ++ -sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant β-MyHC while others had highly reduced Ca ++ -sensitivity suggesting substantial fractions of mutant β-MyHC. Single-cell MYH7 -mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7 -mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7 . Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7 -mRNA, a similar variation in β-MyHC-protein, and highly heterogeneous Ca ++ -sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.

The testis as a site for atherosclerotic changes has so far attracted little attention. We used the apolipoprotein E (ApoE)/low density lipoprotein (LDL) receptor deficient mouse model (KO) for atherosclerosis (20, 40, 60 and 87-week-old) to investigate whether Leydig cells or the capillary network are responsible for reduced serum testosterone levels previously observed in extreme ages of this model. In KO mice, overall testosterone levels were reduced whereas the adrenal gland-specific corticosterone was increased excluding a general defect of steroid hormone production. In addition to micro-CT investigations for bigger vessels, stereology revealed a reduction of capillary length, volume and surface area suggesting capillary rarefaction as a factor for diminished testosterone. Stereological analyses of interstitial cells demonstrated significantly reduced Leydig cell numbers and size. These structural changes in the testis occurred on an inflammatory background revealed by qPCR. Reduced litter size of the KO mice suggests hypo- or infertility as a consequence of the testicular defects. Our data suggest reduced testosterone levels in this atherosclerosis model might be explained by both, rarefication of the capillary network and reduced Leydig cell number and size. Thus, this study calls for specific treatment of male infertility induced by microvascular damage through hypercholesterolemia and atherosclerosis.

More frequent utilization of non-heart-beating donor (NHBD) organs for lung transplantation has the potential to relieve the shortage of donor organs. In particular with respect to uncontrolled NHBD, concerns exist regarding the risk of ischaemia/reperfusion (IR) injury-related graft damage or dysfunction. Due to their immunomodulating and tissue-remodelling properties, bone-marrow-derived mesenchymal stem cells (MSCs) have been suspected of playing a beneficial role regarding short- and long-term survival and function of the allograft. Thus, MSC administration might represent a promising pretreatment strategy for NHBD organs. To study the initial effects of warm ischaemia and MSC application, a large animal lung transplantation model was generated, and the structural organ composition of the transplanted lungs was analysed stereologically with particular respect to the blood-gas barrier and the surfactant system. In this study, porcine lungs (n = 5/group) were analysed. Group 1 was the sham-operated control group. In pigs of groups 2-4, cardiac arrest was induced, followed by a period of 3 h of ventilated ischaemia at room temperature. In groups 3 and 4, 50 × 10 6 MSCs were administered intravascularly via the pulmonary artery and endobronchially, respectively, during the last 10 min of ischaemia. The left lungs were transplanted, followed by a reperfusion period of 4 h. Then, lungs were perfusion-fixed and processed for light and electron microscopy. Samples were analysed stereologically for IR injury-related structural parameters, including volume densities and absolute volumes of parenchyma components, alveolar septum components, intra-alveolar oedema, and the intracellular and intra-alveolar surfactant pool. Additionally, the volume-weighted mean volume of lamellar bodies (lbs) and their profile size distribution were determined. Three hours of ventilated warm ischaemia was tolerated without eliciting histological or ultrastructural signs of IR injury, as revealed by qualitative and quantitative assessment. However, warm ischaemia influenced the surfactant system. The volume-weighted mean volume of lbs was reduced significantly (P = 0.024) in groups subjected to ischaemia (group medians of groups 2-4: 0.180-0.373 μm³) compared with the sham control group (median 0.814 μm³). This was due to a lower number of large lb profiles (size classes 5-15). In contrast, the intra-alveolar surfactant system was not altered significantly. No significant differences were encountered comparing ischaemia alone (group 2) or ischaemia plus application of MSCs (groups 3 and 4) in this short-term model., (© 2017 Anatomical Society.)