Targeted Stereology: Sampling heterogeneously distributed structures and lesions in the lung.

Stereology, the gold standard of lung morphometry, critically depends on sampling of tissue for analysis. Random sampling approaches guarantee each part of the organ an equal chance of being included in the analysis, hence they guarantee a representative sample of the whole. However, when biological or pathological structures of interest are rare and/or heterogeneously distributed over the whole lung, the random sampling approach can be inefficient or even result in meaningless data. In such cases, a targeted sampling approach can be useful which helps to relate the analytical items to an appropriate reference space. Targeted stereology greatly benefits from the increasing availability of multi-resolution imaging techniques at macroscopic and microscopic level as well as digital tools of segmentation. As such, the present article outlines two basic sampling scenarios: 1. In the first scenario, computed tomography and microscopy are subsequently used to segment the airway/arterial tree and perform stereological measurements on specific branches of the tree. 2. The second scenario deals with heterogeneous distribution of pathological lesions. This type of analysis can be divided into two stages: assessment of lesions of interest (LOI) within the lung and assessment of subcompartments within LOI. Taken together, targeted stereology has a thorough foundation in stereological theory and is not only able to significantly increase the efficiency of the analysis but also to yield new types of information that would be lost with the classical random sampling approach.

Hypoxic perfusion of pulmonary arterial vasa vasorum increases pulmonary arterial pressure.

The pathophysiology of pulmonary hypertension (PH) is not fully understood. Here, we tested the hypothesis that hypoxic perfusion of the vasa vasorum of the pulmonary arterial (PA) wall causes PH. Young adult pig lungs were explanted and placed into a modified ex vivo lung perfusion unit (organ care system, OCS) allowing the separate adjustment of parameters for mechanical ventilation, as well as PA perfusion and bronchial arterial (BA) perfusion. The PA vasa vasorum are branches of the BA. The lungs were used either as the control group (n = 3) or the intervention group (n = 8). The protocol for the intervention group was as follows: normoxic ventilation and perfusion (steady state), hypoxic BA perfusion, steady state, and hypoxic BA perfusion. During hypoxic BA perfusion, ventilation and PA perfusion maintained normal. Control lungs were kept under steady-state conditions for 105 min. During the experiments, PA pressure (PAP) and blood gas analysis were frequently monitored. Hypoxic perfusion of the BA resulted in an increase in systolic and mean PAP, a reaction that was reversible upon normoxic BA perfusion. The PAP increase was reproducible during the second hypoxic BA perfusion. Under control conditions, the PAP stayed constant until about 80 min of the experiment. In conclusion, the results of the current study prove that hypoxic perfusion of the vasa vasorum of the PA directly increases PAP in an ex situ lung perfusion setup, suggesting that PA vasa vasorum function and wall ischemia may contribute to the development of PH.NEW & NOTEWORTHY Hypoxic perfusion of the vasa vasorum of the pulmonary artery directly increased pulmonary arterial pressure in an ex vivo lung perfusion setup. This suggests that the function of pulmonary arterial vasa vasorum and wall ischemia may contribute to the development of pulmonary hypertension.

Comparison of the stage-dependent mitochondrial changes in response to pressure overload between the diseased right and left ventricle in the rat.

The right ventricle (RV) differs developmentally, anatomically and functionally from the left ventricle (LV). Therefore, characteristics of LV adaptation to chronic pressure overload cannot easily be extrapolated to the RV. Mitochondrial abnormalities are considered a crucial contributor in heart failure (HF), but have never been compared directly between RV and LV tissues and cardiomyocytes. To identify ventricle-specific mitochondrial molecular and functional signatures, we established rat models with two slowly developing disease stages (compensated and decompensated) in response to pulmonary artery banding (PAB) or ascending aortic banding (AOB). Genome-wide transcriptomic and proteomic analyses were used to identify differentially expressed mitochondrial genes and proteins and were accompanied by a detailed characterization of mitochondrial function and morphology. Two clearly distinguishable disease stages, which culminated in a comparable systolic impairment of the respective ventricle, were observed. Mitochondrial respiration was similarly impaired at the decompensated stage, while respiratory chain activity or mitochondrial biogenesis were more severely deteriorated in the failing LV. Bioinformatics analyses of the RNA-seq. and proteomic data sets identified specifically deregulated mitochondrial components and pathways. Although the top regulated mitochondrial genes and proteins differed between the RV and LV, the overall changes in tissue and cardiomyocyte gene expression were highly similar. In conclusion, mitochondrial dysfuntion contributes to disease progression in right and left heart failure. Ventricle-specific differences in mitochondrial gene and protein expression are mostly related to the extent of observed changes, suggesting that despite developmental, anatomical and functional differences mitochondrial adaptations to chronic pressure overload are comparable in both ventricles.

Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs.

Flow cytometry and fluorescence-activated cell sorting are widely used to study endothelial cells, for which the generation of viable single-cell suspensions is an essential first step. Two enzymatic approaches, collagenase A and dispase, are widely employed for endothelial cell isolation. In this study, the utility of both enzymatic approaches, alone and in combination, for endothelial cell isolation from juvenile and adult mouse lungs was assessed, considering the number, viability, and subtype composition of recovered endothelial cell pools. Collagenase A yielded an 8-12-fold superior recovery of viable endothelial cells from lung tissue from developing mouse pups, compared to dispase, although dispase proved superior in efficiency for epithelial cell recovery. Single-cell RNA-Seq revealed that the collagenase A approach yielded a diverse endothelial cell subtype composition of recovered endothelial cell pools, with broad representation of arterial, capillary, venous, and lymphatic lung endothelial cells; while the dispase approach yielded a recovered endothelial cell pool highly enriched for one subset of general capillary endothelial cells, but poor representation of other endothelial cells subtypes. These data indicate that tissue dissociation markedly influences the recovery of endothelial cells, and the endothelial subtype composition of recovered endothelial cell pools, as assessed by single-cell RNA-Seq.

Chronic hypoxia-induced alterations of the right ventricular myocardium are not aggravated by alimentary obesity in the mouse.

BACKGROUND AND AIMS: Obesity is a risk factor of cardiopulmonary disorders including left and right ventricular dysfunction and pulmonary hypertension (PH), and PH is associated with right ventricular (RV) hypertrophy and failure. Here, we tested the hypothesis that alterations of the RV capillary network under PH induced by chronic hypoxia are aggravated by alimentary obesity, thereby representing a predisposition for subsequent RV dysfunction. METHODS AND RESULTS: Male, 6-week-old C57BL/6N mice were assigned to one of the following groups: control diet (CD), CD/hypoxia (CD-Hyp), high-fat diet (HFD), HFD/hypoxia (HFD-Hyp). Mice were fed CD or HFD for 30 weeks, CD-Hyp and HFD-Hyp mice were exposed to normobaric hypoxia (13 % O2) during the last 3 weeks of the experiments. Hearts were prepared for light and electron microscopy and right atria and RVs were analyzed by design-based stereology. HFD and hypoxia independently increased RV and cardiomyocyte volume. These changes were further enhanced in HFD-Hyp. The ratio between RV and body weights was similar in CD and HFD but enhanced in both hypoxia groups to a similar extent. The total length of capillaries was elevated in proportion with the RV hypertrophy, thus the area of myocardium supplied by an average capillary was similar in all groups. Similarly, the thickness of the capillary endothelium was not altered by HFD or hypoxia. CONCLUSION: In conclusion, in experimental PH capillaries of the RV myocardium showed similar adaptations in lean and obese mice. Thus, under chronic hypoxic conditions, obesity had no adverse effect on the capillarization of the right ventricle.

Short-chain fatty acids improve inflamm-aging and acute lung injury in old mice.

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.

Obesity impacts hypoxia adaptation of the lung.

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% O2) 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 CO2 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.

Spermidine supplementation influences mitochondrial number and morphology in the heart of aged mice.

Aging is associated with cardiac hypertrophy and progressive decline in heart function. One of the hallmarks of cellular aging is the dysfunction of mitochondria. These organelles occupy around 1/4 to 1/3 of the cardiomyocyte volume. During cardiac aging, the removal of defective or dysfunctional mitochondria by mitophagy as well as the dynamic equilibrium between mitochondrial fusion and fission is distorted. Here, we hypothesized that these changes affect the number of mitochondria and alter their three-dimensional (3D) characteristics in aged mouse hearts. The polyamine spermidine stimulates both mitophagy and mitochondrial biogenesis, and these are associated with improved cardiac function and prolonged lifespan. Therefore, we speculated that oral spermidine administration normalizes the number of mitochondria and their 3D morphology in aged myocardium. Young (4-months old) and old (24-months old) mice, treated or not treated with spermidine, were used in this study (n = 10 each). The number of mitochondria in the left ventricles was estimated by design-based stereology using the Euler-Poincaré characteristic based on a disector at the transmission electron microscopic level. The 3D morphology of mitochondria was investigated by 3D reconstruction (using manual contour drawing) from electron microscopic z-stacks obtained by focused ion beam scanning electron microscopy. The volume of the left ventricle and cardiomyocytes were significantly increased in aged mice with or without spermidine treatment. Although the number of mitochondria was similar in young and old control mice, it was significantly increased in aged mice treated with spermidine. The interfibrillar mitochondria from old mice exhibited a lower degree of organization and a greater variation in shape and size compared to young animals. The mitochondrial alignment along the myofibrils in the spermidine-treated mice appeared more regular than in control aged mice, however, old mitochondria from animals fed spermidine also showed a greater diversity of shape and size than young mitochondria. In conclusion, mitochondria of the aged mouse left ventricle exhibited changes in number and 3D ultrastructure that is likely the structural correlate of dysfunctional mitochondrial dynamics. Spermidine treatment reduced, at least in part, these morphological changes, indicating a beneficial effect on cardiac mitochondrial alterations associated with aging.

Differential temporal development of alveoli and the alveolar capillary network in the postnatal rat lung.

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.

Evaluation of classifications of the monopodial bronchopulmonary vasculature using clustering methods.

Mammalian pulmonary arteries divide multiple times before reaching the vast capillary network of the alveoli. Morphological analyses of the arterial branches can be challenging because more proximal branches are likely biologically distinct from more peripheral parts. Thus, it is useful to group the arterial branches into groups of coherent biology. While the generational approach of dichotomous branching is straightforward, the grouping of arterial branches in the asymmetrically branching monopodial lung is less clear. Several established classification methods return highly dissimilar groupings when employed on the same organ. Here, we established a workflow allowing the quantification of grouping results for the monopodial lung and tested various methods to group the branches of the arterial tree into coherent groups. A mouse lung was imaged by synchrotron x-ray microcomputed tomography, and the arteries were digitally segmented. The arterial tree was divided into its individual segments, morphological properties were assessed from corresponding light microscopic scans, and different grouping methods were employed, such as (fractal) generation or (Strahler) order. The results were ranked by the morphological similarity within and dissimilarity between the resulting groups. Additionally, a method from the mathematical field of cluster analysis was employed for creating a reference classification. In conclusion, there were significant differences in method performance. The Strahler order was significantly superior to the generation system commonly used to classify human lung structure. Furthermore, a clustering approach indicated more precise ways to classify the monopodial lung vasculature exist.

Dietary Carbohydrates and Fat Induce Distinct Surfactant Alterations in Mice.

Obesity and type 2 diabetes are nutrition-related conditions associated with lung function impairment and pulmonary diseases; however, the underlying pathomechanisms are incompletely understood. Pulmonary surfactant is essential for lung function, and surfactant synthesis by AT2 (alveolar epithelial type 2) cells relies on nutrient uptake. We hypothesized that dietary amounts of carbohydrates or fat affect surfactant homeostasis and composition. Feeding mice a starch-rich diet (StD), sucrose-rich diet (SuD), or fat-rich diet (FaD) for 30 weeks resulted in hypercholesterolemia and hyperinsulinemia compared with a fiber-rich control diet. In SuD and FaD groups, lung mechanic measurements revealed viscoelastic changes during inspiration, indicating surfactant alterations, and interfacial adsorption of isolated surfactant at the air-liquid interface was decreased under FaD. The composition of characteristic phospholipid species was modified, including a shift from dipalmitoyl-phosphatidylcholine (PC16:0/16:0) to palmitoyl-palmitoleoyl-phosphatidylcholine (PC16:0/16:1) in response to carbohydrates and decreased myristic acid-containing phosphatidylcholine species (PC14:0/14:0; PC16:0/14:0) on excess fat intake, as well as higher palmitoyl-oleoyl-phosphatidylglycerol (PG16:0/18:1) and palmitoyl-linoleoyl-phosphatidylglycerol (PG16:0/18:2) fractions in StD, SuD, and FaD groups than in the control diet. Moreover, mRNA expression levels of surfactant synthesis-related proteins within AT2 cells were altered. Under the StD regimen, AT2 cells showed prominent lipid accumulations and smaller lamellar bodies. Thus, in an established mouse model, distinct diet-related surfactant alterations were subtle, yet detectable, and may become challenging under conditions of reduced respiratory capacity. Dietary fat was the only macronutrient significantly affecting surfactant function. This warrants future studies examining alimentary effects on lung surfactant, with special regard to pulmonary complications in obesity and type 2 diabetes.

Capillary Changes Precede Disordered Alveolarization in a Mouse Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the most common sequela of preterm birth, is a severe disorder of the lung that is often associated with long-lasting morbidity. A hallmark of BPD is the disruption of alveolarization, whose pathogenesis is incompletely understood. Here, we tested the vascular hypothesis that disordered vascular development precedes the decreased alveolarization associated with BPD. Neonatal mouse pups were exposed to 7, 14, or 21 days of normoxia (21% O2) or hyperoxia (85% O2) with n = 8-11 for each group. The right lungs were fixed by vascular perfusion and investigated by design-based stereology or three-dimensional reconstruction of data sets obtained by serial block-face scanning EM. The alveolar capillary network of hyperoxia-exposed mice was characterized by rarefaction, partially altered geometry, and widening of capillary segments as shown by three-dimensional reconstruction. Stereology revealed that the development of alveolar epithelium and capillary endothelium was decreased in hyperoxia-exposed mice; however, the time course of these effects was different. That the surface area of the alveolar epithelium was smaller in hyperoxia-exposed mice first became evident at Day 14. In contrast, the surface area of the endothelium was reduced in hyperoxia-exposed mouse pups at Day 7. The thickness of the air-blood barrier decreased during postnatal development in normoxic mice, whereas it increased in hyperoxic mice. The endothelium and the septal connective tissue made appreciable contributions to the thickened septa. In conclusion, the present study provides clear support for the idea that the stunted alveolarization follows the disordered microvascular development, thus supporting the vascular hypothesis of BPD.

Stereology and three-dimensional reconstructions to analyze the pulmonary vasculature.

The pulmonary vasculature consists of a large arterial and venous tree with a vast alveolar capillary network (ACN) in between. Both conducting blood vessels and the gas-exchanging capillaries are part of important human lung diseases, including bronchopulmonary dysplasia, pulmonary hypertension and chronic obstructive pulmonary disease. Morphological tools to investigate the different parts of the pulmonary vasculature quantitatively and in three dimensions are crucial for a better understanding of the contribution of the blood vessels to the pathophysiology and effects of lung diseases. In recent years, new stereological methods and imaging techniques have expanded the analytical tool box and therefore the conclusive power of morphological analyses of the pulmonary vasculature. Three of these developments are presented and discussed in this review article, namely (1) stereological quantification of the number of capillary loops, (2) serial block-face scanning electron microscopy of the ACN and (3) labeling of branching generations in light microscopic sections based on arterial tree segmentations of micro-computed tomography data sets of whole lungs. The implementation of these approaches in research work requires expertise in lung preparation, multimodal imaging at different scales, an advanced IT infrastructure and expertise in image analysis. However, they are expected to provide important data that cannot be obtained by previously existing methodology.

The plate body: 3D ultrastructure of a facultative organelle of alveolar epithelial type II cells involved in SP-A trafficking.

Plate bodies are facultative organelles occasionally described in the adult lungs of various species, including sheep and goat. They consist of multiple layers of plate-like cisterns with an electron dense middle bar. The present study was performed to elucidate the three-dimensional (3D) characteristics of this organelle and its presumed function in surfactant protein A (SP-A) biology. Archived material of four adult goat lungs and PFA-fixed lung samples of two adult sheep lungs were used for the morphological and immunocytochemical parts of this study, respectively. 3D imaging was performed by electron tomography and focused ion beam scanning electron microscopy (FIB-SEM). Immuno gold labeling was used to analyze whether plate bodies are positive for SP-A. Transmission electron microscopy revealed the presence of plate bodies in three of four goat lungs and in both sheep lungs. Electron tomography and FIB-SEM characterized the plate bodies as layers of two up to over ten layers of membranous cisterns with the characteristic electron dense middle bar. The membranes of the plates were in connection with the rough endoplasmic reticulum and showed vesicular inclusions in the middle of the plates and a vesicular network at the sides of the organelle. Immuno gold labeling revealed the presence of SP-A in the vesicular network of plate bodies but not in the characteristic plates themselves. In conclusion, the present study clearly proves the connection of plate bodies with the rough endoplasmic reticulum and the presence of a vesicular network as part of the organelle involved in SP-A trafficking.

Voluntary activity reverses spermidine-induced myocardial fibrosis and lipid accumulation in the obese male mouse.

Obesity due to high calorie intake induces cardiac hypertrophy and dysfunction, thus contributing to cardiovascular morbidity and mortality. Recent studies in aging suggest that oral supplementation with the natural polyamine spermidine has a cardioprotective effect. Here, the hypothesis was tested that spermidine or voluntary activity alone or in combination protect the heart from adverse effects induced by obesity. Therefore, C57Bl/6 mice (n = 8-10 per group) were subjected to control or high fat diet (HFD) and were left untreated, or either received spermidine via drinking water or were voluntarily active or both. After 30 weeks, the mice were killed and the left ventricle of the hearts was processed for light and electron microscopy. Design-based stereology was used to estimate parameters of hypertrophy, fibrosis, and lipid accumulation. HFD induced cardiac hypertrophy as demonstrated by higher volumes of the left ventricle, cardiomyocytes, interstitium, myofibrils and cardiomyocyte mitochondria. These changes were not influenced by spermidine or voluntary activity. HFD also induced myocardial fibrosis and accumulation of lipid droplets within cardiomyocytes. These HFD effects were enhanced in spermidine treated animals but not in voluntarily active mice. This was even the case in voluntarily active mice that received spermidine. In conclusion, the data confirm the induction of left ventricular hypertrophy by high-fat diet and suggest that-under high fat diet-spermidine enhances cardiomyocyte lipid accumulation and interstitial fibrosis which is counteracted by voluntary activity.

Combination of µCT and light microscopy for generation-specific stereological analysis of pulmonary arterial branches: a proof-of-concept study.

Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.

Novel cell contact between podocyte microprojections and parietal epithelial cells analyzed by volume electron microscopy.

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.

Aging impairs alveolar epithelial type II cell function in acute lung injury.

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.

Spermidine supplementation and voluntary activity differentially affect obesity-related structural changes in the mouse lung.

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.

A transmural gradient of myocardial remodeling in early-stage heart failure with preserved ejection fraction in the pig.

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.