Coronary optical frequency domain imaging (OFDI) for in vivo evaluation of stent healing: comparison with light and electron microscopy

Coronary late stent thrombosis, a rare but devastating complication, remains an important concern in particular with the increasing use of drug-eluting stents. Notably, pathological studies have indicated that the proportion of uncovered coronary stent struts represents the best morphometric predictor of late stent thrombosis. Intracoronary optical frequency domain imaging (OFDI), a novel second-generation optical coherence tomography (OCT)-derived imaging method, may allow rapid imaging for the detection of coronary stent strut coverage with a markedly higher precision when compared with intravascular ultrasound, due to a microscopic resolution (axial approximately 10-20 microm), and at a substantially increased speed of image acquisition when compared with first-generation time-domain OCT. However, a histological validation of coronary OFDI for the evaluation of stent strut coverage in vivo is urgently needed. Hence, the present study was designed to evaluate the capacity of coronary OFDI by electron (SEM) and light microscopy (LM) analysis to detect and evaluate stent strut coverage in a porcine model.

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Implementation of a virtual correlative light and transmission electron microscope

In the long run, the widespread use of slide scanners by pathologists requires an adaptation of teaching methods in histology and cytology in order to target these new possibilities of image processing and presentation via the internet. Accordingly, we were looking for a tool with the possibility to teach microscopic anatomy, histology, and cytology of tissue samples which would be able to combine image data from light and electron microscopes independently of microscope suppliers. With the example of a section through the villus of jejunum, we describe here how to process image data from light and electron microscopes in order to get one image-stack which allows a correlation of structures from the microscopic anatomic to the cytological level. With commercially available image-presentation software that we adapted to our needs, we present here a platform which allows for the presentation of this new but also of older material independently of microscope suppliers.

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

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.

Excessive erythrocytosis in adult mice overexpressing erythropoietin leads to hepatic, renal, neuronal, and muscular degeneration

To investigate the consequences of inborn excessive erythrocytosis, we made use of our transgenic mouse line (tg6) that constitutively overexpresses erythropoietin (Epo) in a hypoxia-independent manner, thereby reaching hematocrit levels of up to 0.89. We detected expression of human Epo in the brain and, to a lesser extent, in the lung but not in the heart, kidney, or liver of tg6 mice. Although no acute cardiovascular complications are observed, tg6 animals have a reduced lifespan. Decreased swim performance was observed in 5-mo-old tg6 mice. At about 7 mo, several tg6 animals developed spastic contractions of the hindlimbs followed by paralysis. Morphological analysis by light and electron microscopy showed degenerative processes in liver and kidney characterized by increased vascular permeability, chronic progressive inflammation, hemosiderin deposition, and general vasodilatation. Moreover, most of the animals showed severe nerve fiber degeneration of the sciatic nerve, decreased number of neuromuscular junctions, and degeneration of skeletal muscle fibers. Most probably, the developing demyelinating neuropathy resulted in muscular degeneration demonstrated in the extensor digitorum longus muscle. Taken together, chronically increased Epo levels inducing excessive erythrocytosis leads to multiple organ degeneration and reduced life expectancy. This model allows investigation of the impact of excessive erythrocytosis in individuals suffering from polycythemia vera, chronic mountain sickness, or in subjects tempted to abuse Epo by means of gene doping.

Effect of voluntary exercise on number and volume of cardiomyocytes and their mitochondria in the mouse left ventricle

Voluntary exercise (VE) has a beneficial influence on the heart and mean lifespan. The present study evaluates structural adaptations of cardiomyocytes and their mitochondria due to VE by new, unbiased stereological methods. Female, 7-9-week-old mice were randomly assigned to a control (CG, n = 7) or VE group (EG, n = 7). EG animals were housed in cages with free access to a running wheel and had a mean running distance of 6.7 (1.8) km per day. After 4 weeks, the hearts of all mice were processed for light and electron microscopy. We estimated the number and volume of cardiomyocytes by the disector method and the number and volume of mitochondria by estimation of the Euler number. In comparison to CG, VE did not have an effect on the myocardial volume of the left ventricle (CG: 93 (10), EG: 103 (17) (mm(3))), the number of cardiomyocytes (CG: 2.81 (0.27), EG: 2.82 (0.43) (x10(6))) and their number-weighted mean volume. However, the composition of the cardiomyocytes changed due to VE. The total volume of mitochondria (CG: 21.8 (4.9), EG: 32.2 (4.3) (mm(3)), P < 0.01) and the total number (CG: 3.76 (0.44), EG: 7.02 (1.13) (x10(10)), P < 0.001) were significantly higher in EG than in CG. The mean number-weighted mitochondrial volume was smaller in EG than in CG (P < 0.05). In summary, VE does not alter ventricular volume nor cardiomyocyte volume or number but the oxidative capacity of cardiomyocytes by an increased mitochondrial number and total volume in the left ventricle. These structural changes may participate in the beneficial effects of VE.

Impact of preservation solution on the extent of blood-air barrier damage and edema formation in experimental lung transplantation

A major aim in lung transplantation is to prevent the loss of structural integrity due to ischemia and reperfusion (I/R) injury. Preservation solutions protect the lung against I/R injury to a variable extent. We compared the influence of two extracellular-type preservation solutions (Perfadex, or PX, and Celsior, or CE) on the morphological alterations induced by I/R. Pigs were randomly assigned to sham (n = 4), PX (n = 5), or CE (n = 2) group. After flush perfusion with PX or CE, donor lungs were excised and stored for 27 hr at 4 degrees C. The left donor lung was implanted into the recipient, reperfused for 6 hr, and, afterward, prepared for light and electron microscopy. Intra-alveolar, septal, and peribronchovascular edema as well as the integrity of the blood-air barrier were determined stereologically. Intra-alveolar edema was more pronounced in CE (219.80 +/- 207.55 ml) than in PX (31.46 +/- 15.75 ml). Peribronchovascular (sham: 13.20 +/- 4.99 ml; PX: 15.57 +/- 5.53 ml; CE: 31.56 +/- 5.78 ml) and septal edema (thickness of alveolar septal interstitium, sham: 98 +/- 33 nm; PX: 84 +/- 8 nm; CE: 249 +/- 85 nm) were only found in CE. The blood-air barrier was similarly well preserved in sham and PX but showed larger areas of swollen and fragmented epithelium or endothelium in CE. The present study shows that Perfadex effectively prevents intra-alveolar, septal, and peribronchovascular edema formation as well as injury of the blood-air barrier during I/R. Celsior was not effective in preserving the lung from morphological I/R injury.

Exogenous surfactant application in a rat lung ischemia reperfusion injury model: effects on edema formation and alveolar type II cells

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.

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes.

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-Swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4 M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.

Effects of anticancer drug docetaxel on the structure and function of the rabbit olfactory mucosa

Docetaxel (DCT) is an anticancer drug which acts by disrupting microtubule dynamics in the highly mitotic cancer cells. Thus, this drug has a potential to affect function and organization of tissues exhibiting high cellular turnover. We investigated, in the rabbit, the effects of a single human equivalent dose (6.26mg/kg, i.v.) of DCT on the olfactory mucosa (OM) through light and electron microscopy, morphometry, Ki-67 immunostaining, TUNEL assay and the buried food test for olfactory sensitivity. On post-exposure days (PED) 5 and 10, there was disarrangement of the normal cell layering in the olfactory epithelium (OE), apoptotic death of cells of the OE, Bowman's glands and axon bundles, and the presence (including on PED 3) of blood vessels in the bundle cores. A decrease in bundle diameters, olfactory cell densities and cilia numbers, which was most significant on PED 10 (49.3%, 63.4% and 50%, respectively), was also evident. Surprisingly by PED 15, the OM regained normal morphology. Furthermore, olfactory sensitivity decreased progressively until PED 10 when olfaction was markedly impaired, and with recovery from the impairment by PED 15. These observations show that DCT transiently alters the structure and function of the OM suggesting a high regenerative potential for this tissue.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

INTRODUCTION The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

Diethylhexylphthalate extracted by typical newborn lipid emulsions from polyvinylchloride infusion systems causes significant changes in histology of rabbit

Background: Looking for a candidate substance inducing hepatobiliary dysfunction under parenteral nutrition (PN) in newborns, we recently discovered that newborn infusions extract large amounts of the plasticizer diethylhexylphthalate (DEHP) from commonly used polyvinylchloride (PVC) infusion lines. This plasticizer is well known to be genotoxic and teratogenic in animals and to cause changes in various organs and enzyme systems even in humans. The aim of this study was to examine the effect of DEHP, extracted in the same way and in the same amount as in newborns, on livers of young rabbits. Methods: Prepubertal rabbits received lipid emulsion through central IV lines continuously for 3 weeks either via PVC or polyethylene (PE) infusion systems. Livers were examined after 1 and 3 weeks by light and electron microscopy. Results: By light microscopy, hydropic degeneration, single-cell necrosis, fibrosis, and bile duct proliferation were observed more in the PVC group. Electron microscopy revealed multiple nuclear changes, clusters and atypical forms of peroxisomes, proliferation of smooth endoplasmic reticulum, increased deposition of lipofuscin, and a mild perisinusoidal fibrosis only in the PVC group. These changes, which are generally regarded as reaction upon a toxic stimulus, could be exclusively attributed to DEHP. Conclusions: This investigation proved that DEHP produces toxin-like changes in livers of young rabbits in the same dose, duration, and method of administration as in newborn infants. For this reason, it is likely that DEHP is the substance that causes hepatobiliary dysfunction in newborns under PN. Possible modes of action of DEHP are proposed.

Re-evaluation of pulmonary titanium dioxide nanoparticle distribution using the "relative deposition index": Evidence for clearance through microvasculature

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.

C1-esterase inhibitor reduces reperfusion injury after lung transplantation

BACKGROUND: Activation of the complement system and polymorphonuclear neutrophilic leukocytes plays a major role in mediating reperfusion injury after lung transplantation. We hypothesized that early interference with complement activation would reduce lung reperfusion injury after transplantation. METHODS: Unilateral left lung autotransplantation was performed in 6 sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 hours in situ at 15 degrees C. After reperfusion the right main bronchus and pulmonary artery were occluded, leaving the animal dependent on the reperfused lung (reperfused group). C1-esterase inhibitor group animals (n = 6) received 200 U/kg body weight of C1-esterase inhibitor as a short infusion, half 10 minutes before, the other half 10 minutes after reperfusion. Controls (n = 6) underwent hilar preparation only. Pulmonary function was assessed by alveolar-arterial oxygen difference and pulmonary vascular resistance. The release of beta-N-acetylglucosaminidase served as indicator of polymorphonuclear neutrophilic leukocyte activation. Extravascular lung water was an indicator for pulmonary edema formation. Biopsy specimens were taken from all groups 3 hours after reperfusion for light and electron microscopy. RESULTS: In the reperfused group, alveolar-arterial oxygen difference and pulmonary vascular resistance were significantly elevated after reperfusion. All animals developed frank alveolar edema. The biochemical marker beta-N-acetylglucosaminidase showed significant leukocyte activation. In the C1-esterase inhibitor group, alveolar-arterial oxygen difference, pulmonary vascular resistance, and the level of polymorphonuclear neutrophilic leukocyte activation were significantly lower. CONCLUSIONS: Treatment with C1-esterase inhibitor reduces reperfusion injury and improves pulmonary function in this experimental model.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.