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.

Ultrastructural analysis of lamellar bodies in type II alveolar epithelial cells in the human lung.

Pulmonary surfactant is produced by type II alveolar epithelial cells (AEC2) and stored in lamellar bodies (LBs) prior to secretion. Here, we characterize AEC2 and their LBs in the human lung ultrastructurally and quantitatively. Five human lungs were analyzed by transmission electron microscopy, serial section electron tomography and stereology. A human lung contained about 24 billion AEC2 with a mean size of about 650 µm³. The number of AEC2 as well as the total volume of LBs per lung, about 1.9 mL, strongly correlated with total lung volume. A single AEC2 contained an LB volume of about 74 µm³. This amount was packed in about 324 LBs with a mean size of 0.24 µm³. Three morphologically distinct subpopulations of LBs were identified: 1.) isolated LBs which make up the majority (average 300 per AEC2), 2.) LBs connected to each other via pores (average 23 per AEC2), and 3.) LBs connected to the plasma membrane via a fusion pore (average 1 per AEC2). Along this sequence of subpopulations, the mean size of LBs increased. LBs that are connected either with each other or to the plasma membrane contained about 14% of an AEC2´s LB volume. This is in line with the concept of an intermediate surfactant pool, stored in LBs either directly or indirectly connected to the plasma membrane. In summary, this study provides quantitative reference data on surfactant-storing LBs in AEC2 as well as morphological evidence for an intermediate surfactant pool in the human lung.

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.

Three-dimensional characteristics of the alveolar capillary network in infant and adult human lungs.

BACKGROUND: A comprehensive understanding of vascular development in the human lung is still missing. METHODS: Therefore, samples of infant (n = 5, 26 days to 18 months postnatally) and adult (n = 5, 20 to 40 years) human lungs were subjected to unbiased stereological estimation of the total number of capillary loops. Serial sections were segmented to visualize the alveolar capillary network (ACN) in 3D. RESULTS: The number of capillary loops increased in parallel to lung volume from 26 days to 18 months, while in adults, it was not correlated to lung volume. In infant lungs, two capillary layers were separated by a connective tissue sheet with a growing number of interconnections. In adults, the mature ACN was almost, but not completely, single-layered. Here, the connective tissue was thinner but still centrally positioned, suggesting the persistence of interconnected parts of both layers of the previously double-layered ACN. CONCLUSIONS: Small parts of the capillaries remain double-layered and seem to be grouped around the thin connective tissue sheet, suggesting a different mechanism of microvascular maturation than simple fusion of the two layers. These spots are a potential basis for further alveolarization after completion of bulk formation. IMPACT: The 3D data offer a new conceptual approach to microvascular maturation of the lung. Microvascular maturation rather results from reduction than simple fusion of capillary fragments. Adult lungs maintain small double-layered capillary spots. These could offer a potential source of regeneration. The data are important to better understand normal and pathological lung development.

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.

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.

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.

A short primer on lung stereology.

The intention of this short primer is to raise your appetite for proper quantitative assessment of lung micro-structure. The method of choice for obtaining such data is stereology. Rooted in stochastic geometry, stereology provides simple and efficient tools to obtain quantitative three-dimensional information based on measurements on nearly two-dimensional microscopic sections. In this primer, the basic concepts of stereology and its application to the lung are introduced step by step along the workflow of a stereological study. The integration of stereology in your laboratory work will help to improve its quality. In a broader context, stereology may also be seen as a contribution to good scientific practice.

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.

Spermidine and Voluntary Activity Exert Differential Effects on Sucrose- Compared with Fat-Induced Systemic Changes in Male Mice.

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.

Influence of Medication-Induced Preconditioning or Remote Ischemic Preconditioning on the Intrinsic Vascular Extracellular RNA/Ribonuclease System in Cardioprotection.

BACKGROUND: It has been demonstrated that remote ischemic preconditioning (RIPC) increases ribonuclease (RNase) levels and protects the heart by reducing extracellular ribonucleic acid (eRNA). As medication-induced preconditioning (MIPC) is also a powerful tool for cardioprotection, we examined the influence of both types of preconditioning on the eRNA/RNase system. METHODS: In 17 male rats, RIPC (3 × 5 minute hind-leg ischemia) or MIPC (isoflurane and buprenorphine anesthesia) was performed. Five rats served as control and did not undergo preconditioning (non-MIPC). After preconditioning, eRNA levels and RNase activity were determined in plasma, and the hearts were mounted on a blood-perfused Langendorff ischemia/reperfusion apparatus. Hemodynamic, metabolic, and electron microscopic parameters were determined. Furthermore, MIPC with one anesthetic drug only (isoflurane, buprenorphine, or etomidate) was induced in another five rats. After 30 minutes, eRNA levels and RNase activity were determined and compared with an RIPC group (n = 5). RESULTS: The plasma of RIPC-treated rats had higher RNase activity and lower eRNA levels than that of MIPC-treated rats. In addition, RIPC increased RNase activity more than MIPC with one drug alone. The RNase activity and eRNA levels in these MIPC groups differed considerably. Hemodynamic parameters of RIPC- and MIPC-treated hearts were better preserved after 90-minute ischemia than those of non-MIPC hearts. No obvious differences were noted between MIPC and RIPC regarding hemodynamics, metabolism, or structural parameters. CONCLUSIONS: Our results suggest that RIPC does not have any additional cardioprotective benefit in this experimental system. However, the influence of RIPC on the eRNA/RNase system was greater than that of MIPC.

Voluntary Activity Modulates Sugar-Induced Elastic Fiber Remodeling in the Alveolar Region of the Mouse Lung.

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.

Experimentally induced intrauterine growth restriction in rabbits leads to differential remodelling of left versus right ventricular myocardial microstructure.

Intrauterine growth restriction (IUGR) is associated with foetal cardiac remodelling and dysfunction together with increased risk of cardiovascular disease in adulthood. Experimental data concerning effects of IUGR on cardiomyocyte and microvascularization anatomy are inconsistent and it is unknown whether both ventricles are similarly susceptible to in utero undersupply. Foetal IUGR was induced in pregnant rabbits at 25 days of gestation by selective ligation of uteroplacental vessels. Foetal echocardiography showed systolic and diastolic dysfunction of both ventricles and body and heart weight were significantly reduced in response to IUGR. Design-based stereology revealed a decrease in cardiomyocyte number in both ventricles which was only in the left ventricle accompanied by a significantly higher cardiomyocyte mean volume. The proportion of mono- and bi-nucleated cardiomyocytes was unaltered between the groups indicating a similar maturation status. The number and length of cardiac capillaries in IUGR offspring was diminished in left but not in right ventricles. Foetal left and right ventricles are differently affected by placental insufficiency. While cardiomyocyte numbers are diminished in both ventricles, hypertrophic remodelling of cardiomyocytes and alterations in microvascularization is rather a left ventricular adaptation to IUGR. These unequal structural changes may be related to loading and developmental differences of the left and right ventricles.

Effect of irradiation/bone marrow transplantation on alveolar epithelial type II cells is aggravated in surfactant protein D deficient mice.

Irradiation followed by bone marrow transplantation (BM-Tx) is a frequent therapeutic intervention causing pathology to the lung. Although alveolar epithelial type II (AE2) cells are essential for lung function and are damaged by irradiation, the long-term consequences of irradiation and BM-Tx are not well characterized. In addition, it is unknown whether surfactant protein D (SP-D) influences the response of AE2 cells to the injurious events. Therefore, wildtype (WT) and SP-D-/- mice were subjected to a myeloablative whole body irradiation dose of 8 Gy and subsequent BM-Tx and compared with age- and sex-matched untreated controls. AE2 cell changes were investigated quantitatively by design-based stereology. Compared with WT, untreated SP-D-/- mice showed a higher number of larger sized AE2 cells and a greater amount of surfactant-storing lamellar bodies. Irradiation and BM-Tx induced hyperplasia and hypertrophy in WT and SP-D-/- mice as well as the formation of giant lamellar bodies. The experimentally induced alterations were more severe in the SP-D-/- than in the WT mice, particularly with respect to the surfactant-storing lamellar bodies which were sometimes extremely enlarged in SP-D-/- mice. In conclusion, irradiation and BM-Tx have profound long-term effects on AE2 cells and their lamellar bodies. These data may explain some of the clinical pulmonary consequences of this procedure. The data should also be taken into account when BM-Tx is used as an experimental procedure to investigate the impact of bone marrow-derived cells for the phenotype of a specific genotype in the mouse.