Hard and soft X-ray imaging to resolve human ovarian cortical structures.

Laboratory and synchrotron X-ray tomography are powerful tools for non-invasive studies of biological samples at micrometric resolution. In particular, the development of phase contrast imaging is enabling the visualization of sample details with a small range of attenuation coefficients, thus allowing in-depth analyses of anatomical and histological structures. Reproductive medicine is starting to profit from these techniques, mainly applied to animal models. This study reports the first imaging of human ovarian tissue where the samples consisted of surgically obtained millimetre fragments, properly fixed, stained with osmium tetroxide and included in epoxydic resin. Samples were imaged by the use of propagation phase contrast synchrotron radiation micro-computed tomography (microCT), obtained at the SYRMEP beamline of Elettra light source (Trieste, Italy), and X-ray absorption microCT at the Theoretical Biology MicroCT Imaging Laboratory in Vienna, Austria. The reconstructed microCT images were compared with the soft X-ray absorption and phase contrast images acquired at the TwinMic beamline of Elettra in order to help with the identification of structures. The resulting images allow the regions of the cortex and medulla of the ovary to be distinguished, identifying early-stage follicles and visualizing the distribution of blood vessels. The study opens to further application of micro-resolved 3D imaging to improve the understanding of human ovary's structure and support diagnostics as well as advances in reproductive technologies.

Neuronal Dysfunction Associated with Cholesterol Deregulation.

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith⁻Lemli⁻Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time.

Light element distribution in fresh and frozen-thawed human ovarian tissues: a preliminary study.

RESEARCH QUESTION: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? DESIGN: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. RESULTS: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. CONCLUSIONS: The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.

The Combination of Trypan Blue and Brilliant Blue G-Assisted Vitrectomy for Macular Pucker: Histopathological Findings.

PURPOSE: To report on the combined use of trypan blue (TB) and brilliant blue G (BBG) for staining the epiretinal membrane (ERM) and internal limiting membrane (ILM) during vitrectomy and to describe the histopathological findings. METHODS: 10 surgical specimens were removed from 10 eyes with macular pucker during vitrectomy using a commercially available combination of TB and BBG for ERM and ILM staining and peeling. Specimens were evaluated using light and transmission electron microscopy. RESULTS: In all cases the combination of TB and BBG was useful for identifying and delineating ERM and ILM. No complications related to the use of the dye were observed during or after surgery. Glial cells were present in all specimens. Hyalocytes were observed in 6 cases and myofibroblasts in 3 of them. In 7 cases native vitreous collagen fibrils were found on the ILM, while in 5 specimens newly formed collagen was present. No clinical evidence of toxicity was observed during the 3-month follow-up. CONCLUSION: The combined use of TB and BBG appeared to be very useful intraoperatively to improve the visualization of ERM and ILM, thus facilitating their complete removal. Anatomical and histopathological findings demonstrated the safety and the efficacy of this vital dye.

Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy.

Carbon nanotubes (CNTs) are promising products in industry and medicine, but there are several human health concerns since their fibrous structure resembles asbestos. The presence of transition metals, mainly iron, in the fibres seems also implicated in the pathogenetic mechanisms. To unravel the role of iron at mesothelial level, we compared the chemical changes induced in MeT-5A cells by the exposure to asbestos (crocidolite) or CNTs at different content of iron impurities (raw-SWCNTs, purified- and highly purified-SWCNTs). We applied synchrotron-based X-Ray Fluorescence (XRF) microscopy and soft X-ray imaging (absorption and phase contrast images) to monitor chemical and morphological changes of the exposed cells. In parallel, we performed a ferritin assay. X-ray microscopy imaging and XRF well localize the crocidolite fibres interacting with cells, as well as the damage-related morphological changes. Differently, CNTs presence could be only partially evinced by low energy XRF through carbon distribution and sometimes iron co-localisation. Compared to controls, the cells treated with raw-SWCNTs and crocidolite fibres showed a severe alteration of iron distribution and content, with concomitant stimulation of ferritin production. Interestingly, highly purified nanotubes did not altered iron metabolism. The data provide new insights for possible CNTs effects at mesothelial/pleural level in humans.

Investigating the mechanical properties of zona pellucida of whole human oocytes by atomic force spectroscopy.

The role of mechanics in numerous biological processes is nowadays recognized, while in others, such as the fertilization process, it is still neglected. In the case of oocytes the description of their mechanical properties could improve the comprehension of the oocyte-spermatozoon interaction and be helpful for application in in vitro fertilization (IVF) clinics. Herein the mechanical properties of whole human oocytes (HOs) immediately after retrieval are investigated by indentation measurements with atomic force spectroscopy under physiological conditions. Measurements are performed on immature (metaphase I - MI) and mature (metaphase II - MII) HOs. According to their morphological characteristics MII-HOs are classified as "suitable" and "rejected"; these latter would be usually rejected for intracytoplasmic sperm injection (ICSI). For all maturation stages we observe that the elastic response of the zona pellucida (ZP) outer layer was different and distinguishable from the rest of the ZP-HO. The elasticity of this ZP outer layer varies with maturation and quality: stiffness decreases from immature MI to good quality MII, up to poor-quality rejected MII. An indirect analysis with IVF outcome indicates that the ZP outer layer of analysed HOs donated by women who achieved pregnancy is stiffer than that of HOs from women with negative outcome. Our findings suggest that mechanical properties can represent important oocyte quality indicators that may be exploited for the design of innovative ICSI dedicated cell sorters.

Geranylgeraniol and Neurological Impairment: Involvement of Apoptosis and Mitochondrial Morphology.

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm.

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fenton's reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

Histopathological data of iron and calcium in the mouse lung after asbestos exposure.

This data article contains data related to the research article entitled, "Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice" [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice.

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (µ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, µ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

Ultrastructural Morphology of Sperm from Human Globozoospermia.

Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.

Two-dimensional gel electrophoresis analysis of the leiomyoma interstitial fluid reveals altered protein expression with a possible involvement in pathogenesis.

Uterine leiomyoma is the most common smooth benign neoplasm. In the present study, we analyzed the global interstitial fluid (IF) profile of leiomyoma vs. normal myometrium to identify protein dysregulation involved in leiomyoma pathogenesis. Two-dimensional gel electrophoresis and mass spectrometry were used to generate and compare the global interstitial fluid profiles of the leiomyoma and of the normal tissue. Two proteins were validated by immunohistochemistry. By comparing the interstitial fluid profile of the leiomyoma with that of the normal myometrium, the levels of seven proteins were found to be significantly different: four structural organization proteins (desmin, prelamin-A/C, transgelin and α-actinin-1), an inflammatory response (α1-antitrypsin), a response to oxidative stress (peroxiredoxin-2), and a folding protein (heat shock 70 kDa protein 1A/1B). Desmin, α1-antitrypsin and peroxiredoxin-2 were upregulated in the leiomyoma, whereas heat shock 70 kDa protein 1A/1B, α-actinin-1, prelamin-A/C and transgelin were downregulated. Desmin and α1-antitrypsin were further validated by immunohistochemistry. By identifying proteins with altered expression levels compared to the myometrium from several pathways of the leiomyoma pathogenesis, we found the leiomyoma interstitial fluid to have a characteristic proteomic profile. A better appreciation of the pathophysiology of the disease can be useful in the development of conservative treatments that serve as viable alternatives to hysterectomy.

The application of scanning near field optical imaging to the study of human sperm morphology.

BACKGROUND: The morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures. RESULTS: In this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols. CONCLUSIONS: Such findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together.

Innovative Target Therapies Are Able to Block the Inflammation Associated with Dysfunction of the Cholesterol Biosynthesis Pathway.

The cholesterol pathway is an essential biochemical process aimed at the synthesis of bioactive molecules involved in multiple crucial cellular functions. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids and other molecules such as ubiquinone. Several diseases are caused by defects in this metabolic pathway: the most severe forms of which cause neurological involvement (psychomotor retardation and cerebellar ataxia) as a result of a variety of cellular impairments, including mitochondrial dysfunction. These pathologies are induced by convergent mechanisms in which the mitochondrial unit plays a pivotal role contributing to defective apoptosis, autophagy and mitophagy processes. Unraveling these mechanisms would contribute to the development of effective drug treatments for these disorders. In addition, the development of biochemical models could have a substantial impact on the understanding of the mechanism of action of drugs that act on this pathway in multifactor disorders. In this review we will focus in particular on inhibitors of cholesterol synthesis, mitochondria-targeted drugs and inhibitors of the inflammasome.

Potential role of circulating microRNAs as early markers of preeclampsia.

OBJECTIVE: To identify microRNAs (miRNAs) differentially expressed at early stages of gestation (12-14 weeks) in the serum of pregnant women, who later developed severe preeclampsia (sPE) in the third trimester of pregnancy (n = 24) compared to women with normal pregnancy (n = 24). MATERIALS AND METHODS: Sera from 12-14-week-gestation whole blood were subjected to microarray analysis with TaqMan Low Density Array chips (human microRNA panel V3.0), and to quantitative real-time polymerase chain reaction. RESULTS: By using the TaqMan Low Density Array chip technology, 19 mature miRNAs appeared differentially expressed in the group of women who later developed sPE as compared to normal women. The expression of four miRNAs (miR-1233, miR-520, miR-210, miR-144) was validated by quantitative real-time polymerase chain reaction analysis. MiR-1233 was the most overexpressed in the serum of women who later developed sPE. CONCLUSION: Circulating miRNAs deserve further investigation in order to explore their potential role in the pathogenesis of preeclampsia. In particular, miR-1233 might represent a potential marker of early sPE.

The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy.

In this study, we have performed a morphological analysis of crocidolite fibres interaction with mesothelial cells (MET5A) by combining conventional electron microscopy with atomic force (AFM) and scanning near-field optical microscopy (SNOM). After 6-h exposure at a crocidolite dose of 5 µg cm(-2), 90% of MET5A cells interact with fibres that under these conditions have a low cytotoxic effect. SEM images point out that fibres can be either engulfed by the cells that lose their typical morphology or they can accumulate over or partially inside the cells, which preserve their typical spread morphology. By using AFM we are able to directly visualize the entry-site of nanometric-sized fibres at the plasma membrane of the spread mesothelial cells. More importantly, the crocidolite fibres that are observed to penetrate the plasma membrane in SNOM topography can be simultaneously followed beneath the cell surface in the SNOM optical images. The analysis of SNOM data demonstrates the entrance of crocidolite fibres in proximity of nuclear compartment, as observed also in the TEM images. Our findings indicate that the combination of conventional electron microscopy with novel nanoscopic techniques can be considered a promising approach to achieve a comprehensive morphological description of the interaction between asbestos fibres and mesothelial cells that represents the early event in fibre pathogenesis.

Apoptosis-related factors (TRAIL, DR4, DR5, DcR1, DcR2, apoptotic cells) and proliferative activity in ameloblastomas.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a recently identified death factor that acts as a potent apoptosis inducer in ameloblastomas. MATERIALS AND METHODS: The expression of TRAIL and its receptors (TRAIL-R), and the location of apoptotic cells were evaluated in 15 cases of ameloblastoma using immunohistochemistry and an in situ DNA nick-end labeling method. The proliferative activity of ameloblastomas was analyzed by determining the Ki-67 labeling index. RESULTS: TRAIL and TRAIL-R were diffusely expressed in ameloblastomas, without clear correlation with the location of apoptotic cells. Apoptosis and proliferation were opposite in the peripheral and central components of the ameloblastomas. In some ameloblastoma variants, apoptosis and proliferation seemed to modify in the same direction. CONCLUSION: TRAIL and its receptors might be involved in neoplastic transformation of odontogenic epithelium and might suggest some intrinsic regulation of neoplastic cell proliferation and death in ameloblastomas, thus explaining their slow growth and inability to metastasize.

Increased OPG expression and impaired OPG/TRAIL ratio in the aorta of diabetic rats.

Despite accumulating evidence showing that TNF-related apoptosis inducing ligand (TRAIL) plays a role in vascular biology and that its decoy receptor osteoprotegerin (OPG) is expressed in the vessel wall, modulation of these TNF and TNF-R family members in the early phases of diabetes mellitus has not been investigated. The expression of TRAIL and of OPG was examined both at the mRNA and protein levels in control and streptozotocin (SZT)-induced diabetic rats at early time points after the induction of diabetes mellitus. No differences in the steady-state mRNA levels of TRAIL were noticed by quantitative RT-PCR among the two groups of animals. On the other hand, diabetic rats showed a rapid and significant increase of the steady-state mRNA levels of OPG in the aortic wall of diabetic animals with respect to vehicle-treated (control) animals. These findings were confirmed at the protein level by analysing the amount of TRAIL and OPG proteins in aortic lysates by either Western blot or immunohistochemistry. Thus, an abnormal elevation of the OPG/TRAIL ratio in the vessel wall characterizes the early onset of diabetes mellitus and might represent a molecular mechanism involved in the vascular dysfunction characterizing diabetes mellitus.

Receptor activator of nuclear factor kappa B ligand (RANKL) modulates the expression of genes involved in apoptosis and cell cycle in human osteoclasts.

It has been clearly established that receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine involved in the differentiation of osteoclastic precursors of the monocytic/macrophagic lineage. However, relatively little information is available on the ability of RANKL to modulate the expression of genes controlling cell survival/apoptosis and proliferation in human osteoclastic cells in comparison to macrophages. For this purpose, CD14+ human peripheral blood mononuclear cells, which express the cognate high affinity receptor activator of nuclear factor kappa B (RANK), were differentiated along the macrophagic or osteoclastic lineage by adding macrophage-colony stimulating factor (M-CSF) or M-CSF plus RANKL in culture for 12 days. RANKL up-regulated the expression of the chemokine MIP1alpha, which potentiates osteoclastic differentiation and simultaneously activated both anti-apoptotic (Bcl-2) and pro-apoptotic (CIDEB, PYCARD, and BAK-1) genes. Moreover, RANKL markedly up-regulated cylin D2, while it significantly decreased the levels of cyclin A, cyclin-dependent kinase 2, and other cyclin-dependent kinases, in keeping with the notion that end-stage osteoclasts are nondividing cells. Finally, a long-term exposure of RANKL up-regulated the adaptor protein TRAF3 but not TRAF6.