Ezio Battaglione

A Deep Look Into Erionite Fibres: an Electron Microscopy Investigation of their Self-Assembly.

The exposure of humans to erionite fibres of appropriate morphology and dimension has been unambiguously linked to the occurrence of Malignant Mesothelioma. For this reason, a detailed morpho-structural investigation through Electron Microscopy techniques has been performed on erionite samples collected at two different localities, Durkee (ED) and Rome (ER), Oregon, USA. The sample from Rome has been also investigated after a prolonged leaching with Gamble’s solution (ER4G) in order to evaluate the possible occurrence of morpho-structural modifications induced by this Simulated-Lung-Fluid (SLF). Here we report how the micrometric erionite fibres evolve in irregular ribbon- or rod-like bundles as a function of different nano-structural features. The reasons for the observed morphological variability have been explained by considering the structural defects located at ED surface fibrils (bi-dimensional ribbons) and the presence of nontronite, an iron-bearing clay mineral embedding the ER fibrils (mono-dimensional rods). ER4G shows a decrease in width of the rod-like fibres due to their partial digestion by SLF leaching, which synchronously dissolves nontronite. The reported results represent a valuable background toward the full comprehension of the morphological mechanisms responsible for potentially damage of lung tissue through the potential relocation of fibers to extrapulmonary sites, increasing the carcinogenic risk to humans.

Age and diabetes related changes of the retinal capillaries: An ultrastructural and immunohistochemical study

Normal human aging and diabetes are associated with a gradual decrease of cerebral flow in the brain with changes in vascular architecture. Thickening of the capillary basement membrane and microvascular fibrosis are evident in the central nervous system of elderly and diabetic patients. Current findings assign a primary role to endothelial dysfunction as a cause of basement membrane (BM) thickening, while retinal alterations are considered to be a secondary cause of either ischemia or exudation. The aim of this study was to reveal any initial retinal alterations and variations in the BM of retinal capillaries during diabetes and aging as compared to healthy controls. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in diabetic retina. Transmission electron microscopy (TEM) was performed on 46 enucleated human eyes with particular attention to alterations of the retinal capillary wall and Müller glial cells. Inflammatory cytokines expression in the retina was investigated by immunohistochemistry. Our electron microscopy findings demonstrated that thickening of the BM begins primarily at the level of the glial side of the retina during aging and diabetes. The Müller cells showed numerous cytoplasmic endosomes and highly electron-dense lysosomes which surrounded the retinal capillaries. Our study is the first to present morphological evidence that Müller cells start to deposit excessive BM material in retinal capillaries during aging and diabetes. Our results confirm the induction of pro-inflammatory cytokines TNF-α and IL-1β within the retina as a result of diabetes. These observations strongly suggest that inflammatory cytokines and changes in the metabolism of Müller glial cells rather than changes in of endothelial cells may play a primary role in the alteration of retinal capillaries BM during aging and diabetes.

Bacterial Biofilm in Salivary Gland Stones: Cause or Consequence?

Objective The pathogenesis of salivary calculi is not yet clear; however, 2 theories have been formulated: (1) “the classic theory,” based on calcium microdeposits in serous and ductal acinous cells, successively discharged into the ducts; (2) “the retrograde theory,” based on a retrograde migration of food, bacteria, and so on from the oral cavity to the salivary duct. The aim of the present study is to highlight the role of bacteria and biofilm in stone formation. Study Design Case series without comparison. Setting Laboratory of the Department of Anatomical Pathology. Subjects and Methods Traditional optic microscopy and scanning electron microscopy were carried out on 15 salivary gland calculi that were collected from 12 patients. A qPCR (quantitative real-time polymerase chain reaction) assay was performed to highlight the presence of bacterial DNA on each stone. Results Optic microscopy showed formations that—due to their size, shape, and Gram and Giemsa staining—seemed to be Gram-positive bacterial cells. PAS- (periodic acid–Schiff) and alcian-PAS-positive staining matrix was present around them. The ultrastructural observation of the material processed for scanning electron microscopy showed the presence of structures resembling bacterial cells in the middle of the stones, surrounded by soft, amorphous material. Results of qPCR showed the presence of bacterial DNA in the internal part of the tissue sample. Conclusions The presence of bacteria and/or bacterial products resembling biofilm in salivary gland stones supports the “retrograde theory.” This evidence may support the hypothesis that biofilm could be the causative effect of lithiasic formations.

Primary vaginal leiomyosarcoma, a rare tumour: case report and review

Primary vaginal leiomyosarcomas (pvLMS) are rare, recurrent tumours accounting for ca. 2% of all vaginal cancers. The etiology is still unknown, the prognosis is poor and there is no consensus guideline on its management. Diagnosis is usually made during the 5th decade due to the presence of a vaginal mass or nodule [1-2]. Current medical literature reports about 200 cases (PubMed®); only 3 studies have considered the ultrastructure [2-4]. Herein a pvLMS is presented and discussed. A nodular, 25 x 23 x 28 mm-mass, infiltrating the urethra but not the rectovaginal septum, was widely excised from the superior vaginal wall of a 58-year-old previously hysterectomized woman. Macroscopic images and MRI were performed. Iliac lymph nodes and HMB-45 were negative. The sample was fixed and prepared for light microscopy, transmission (TEM) and scanning (SEM) electron microscopy. Semithin sections showed a storiform pattern of spindle shaped cells with blunt-ended nuclei. Cells arranged in interwoven fascicles within a dense and richly vascularised stroma (neoangiogenesis). Some atypic mitotic figures and focal necrosis were seen. SEM evidenced a dense collagenous stroma with numerous microvessels. TEM showed neoplastic and pleomorphic cells with complex cytoplasm projections containing paranuclear crowds of dilated mitochondria, free ribosomes and a well-developed rough endoplasmic reticulum. Nuclei were large, mostly hyperchromatic, usually indented, with prominent nucleoli and nucleolonema. The dense intercellular space contained dense bundles of collagen fibers. A high and reactive endothelium lined blood vessels. After 4 follow-ups, the patient is fine and without recurrence. Best outcomes occur when the tumour is small, localized, and can be removed surgically with wide, clear margins, as it was for this case. As there are different kinds of LMS, biopsy followed by immunohistochemistry and electron microscopy still represents a good diagnostic choice.

FE-SEM and VP-SEM imaging of human calcified aortic valves: conventional vs Ionic Liquid innovative techniques

Conventional FE-SEM protocol for calcified aortic valves (CAVs) consist of following steps: glutaraldehyde fixation, OsO4 post-fixation, dehydration in alcohol series, critical point drying and finally sputter coating. CAVs can be observed in their native state (fixed in glutaraldehyde with and without post-fixation in OsO4) by Variable Pressure-SEM (range 6- 650 Pa). Gas presence allows an inferior resolution (low signal to noise ratio), however there is the possibility to perform EDS elemental analysis without background noise due to sputter coating. Recently Ionic liquids (IL, salts in the liquid state at room temperature) were used as suppliers of electronic conductivity with insulating properties, so we have tested their ability to replace sputter coating on CAVs in high vacuum condition. Samples fixed in glutharaldehyde 2,5% in PBS with and without OsO4 post-fixation treated with ionic liquid (Hitachi HILEM® IL 1000) were compared with samples treated with conventional FE-SEM procedures. Several IL concentration (range from 5% to 20%) were tested, different operating voltages (range from 3 to 20Kv) were used. This novel technology requires a high degree of customization for each sample type. In our opinion fixation in glutaraldehyde with OsO4 post-fixation is recommended to preserve finest details, moreover residual liquid elimination is important to increase resolution and avoid beam interference as linear markings. Setting of a proper accelerating voltage allows to correctly visualize the surface topography. Processing CAVs with IL with respect to conventional FE-SEM is useful for several reasons. Mainly this method is time saving (and cost saving), secondary the same sample can be processed for transmission electron microscopy after SEM observations (allowing correlative microscopy), finally EDS can be performed without background noise due to sputter coating. Perhaps now this technique can not completely replaces the conventional SEM in terms of resolution but in our opinion rapid technical improvement can further reduce this gap.

Cholesteatoma affected incus bone surface shows unusual iron-rich crystals, microvesicles and altered bone turnover

Cholesteatoma is a noncancerous cystic lesion consisting in an abnormal growth of keratinizing squamous epithelium that invades the middle ear cavity. Due to its capacity of intracranial complications, cholesteatoma is cause of pediatric morbidity and death in countries with scarce hygiene and low possibility to access to advanced medical care (1). In order to understand cholesteatoma etiopathogenesis, we performed a SEM morphological analysis of 11 incus bones affected by cholesteatoma. Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h, then they were gently sonicated (to remove excess of keratinizing squamous epithelium, that would have prevented surface observation) and finally they were prepared with standard method for scanning electron microscopy observation. Five consecutive fields at 100X magnification aligned in 3 raws, the first one proximal and the last one distal to surgical removal point were analized. Images were obtained in secondary electron mode and in backscattering, bidimensional EDX analysis and mapping was also carried on. Incus bone surface analysis reveals the existence of an environment in which abnormal bone turnover takes place, in fact area of marked erosion were present together with areas of new bone formation. Resorbing bone surfaces with their characteristic lacunae were observed, resting surfaces (smooth and with collagen fibre bundles evident) were found and forming bone surface (collagen bundles in which calcium salts were just deposited) were also observed. Unusual flower-like apatite crystals rich in iron were uncovered in one sample. Iron presence may be due to cholesteatoma itself, being it made up of corneocytes that are iron-rich cells (2). Microvesicles of cellular origin, alone or clustered in groups or in about to fusing together, were found. Macrophages, lymphocytes osteoblast and osteoclast were observed in fully activated stage. The picture of these cell near to each other is the morphological representation of the complex cytochemical dialog existing among them. Taken all together our morphological results let us hypothesize that cholesteatoma creates an environment of chronic infection with peculiar biochemical characteristics that alters normal bone turnover on incus bone.This work was supported by grants from MIUR.

SEM study of incus surface erosion due to cholesteatoma action

Cholesteatoma is a noncancerous cystic lesion derived from an abnormal growth of keratinizing squamous epithelium in the temporal bone (1). It causes significant problems due to its erosive and expansile properties, resulting in the destruction of the ossicles. Over 5 million people worldwide are affected of cholesteatomas and gradually loss hearing. (2). In order to provide a prognostic tool useful during surgical procedures, we are performing a SEM morphological analysis of incus surface erosion due to cholesteatoma action; then we will investigate (on the same samples) the relationship among data from SEM analysis and genetical, proteomical, biochemical and histological data. Up to now we have observed 10 incus from patients with cholesteatoma. Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h, than they were prepared for scanning electron microscopy. Samples were gently sonicated before sputter coating, to remove excess of keratinizing squamous epithelium, that would have prevented erosion observation. The total surface area of the observed side was measured (3,54±0,21 mm2). The mean distance from the surgical removal point to the bone far end was measured in order to define a ROI (2,37±0,31 mm2). Five consecutive fields at 100X magnification aligned in 3 raws, the first one proximal and the last one distal to surgical removal point were analized. A total of 60 field for each raw were observed. Degree of erosion was classified as: No erosion=0, light =1, mild=2 high=3. Presence of biofilm was also recorded. Our early data suggest that although a gradient proximal to distal exists, looking to the distribution of eroded areas, grade 3 erosion is not limited only to the area proximal to cholesteatoma (first raw) but is also present in raw 2 and sometimes scattered since raw 3. Grade 3 erosion was observed around nutrient foramina of the bone (65%). Biofilm of bacteria was observed in 50% of analyzed fields, this is con- sistent with results reported in literature. Our data suggest that relapse of cholestea- toma is due to erosive activity of cells far from surgical removal point.

A multi-scale investigation of biological niches within human calcified aortic valves helps to understand the pathological biomineralization process

Calcific aortic valve stenosis (CAVS) is the most common form of heart valve disease in the industrialized countries, being an important public health problem [1]. Ectopic calcifications within aortic valve leaflets are strictly associated with CAVS, interfering with cusps opening, they lead to ventricular outflow obstruction [2]. Up to date no proven medical therapy stops CAVS course progression, so valve replacement is the only possible treatment of severe CAVS. Unfortunately, the degenerative valve calcification process, affects also bioprosthetic implants [3]. Being the molecular mechanisms leading to valve calcification still not understood, our aim was to carry on a multi-scale investigation using Scanning Electron Microscopy, Transmission Electron Microscopy and Energy Dispersive X-ray Spectrometry, to provide new insights into calcification process. Severely calcified aortic (tricuspid type, n = 29; bicuspid type, n = 3) and mitral valves (n = 4) were obtained from patients of both sexes (males=25) and different ages (mean age 72±10, range 41-90 years old) undergoing valve replacement due to severe aortic and mitral valve stenosis. We detected bioapatite crystals in two different mineralization sites: niches and extracellular matrix. This suggests the action of two different growth processes: the first occurs in biological niches and it is ascribed to a purely physico-chemical process; the second has the extracellular matrix acting ass the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear made up by the same subunits. We suggest that bioapatite nanocrystals in heart valve may activate a strong inflammatory process leading to irreversible pathological condition that, once activated,may aggravate the inflammatory response against bioapatite nanocrystals leading to a severe calcification process.

Introducing medical students to scientific research: an early electron-microscopy laboratory attendance experience

In the light of importance that “evidence-based medicine” has assumed in recent years (Snelgrove et al. 2009), we offer to the students of the first-year medical-degree the chance of an early exposure to the work in the ultrastructural research laboratory “Pietro M. Motta”. On an elective basis, students attended the laboratory in small groups. They were guided and supported by a qualified researcher, a post-graduate student, a graduate student and a technical-staff unit. During the week of attendance students performed several activities: at first they have visited the laboratory where the technicalities of the equipment were illustrated, than they have taken part to a lecture on the methods used to prepare the biological samples for Scanning and Transmission Electron Microscopy. In the following days preparation of samples for Scanning and Transmission Electron Microscopy was carried on and a guided discussion on scientific articles concerning the samples used in the experiments was conducted (Familiari et al. 2006). Later, samples were observed using a light microscope and both transmission and scanning electron microscope. At the end of the week students had taken hands-on in various stages of preparation, observation and analysis of the samples. The discussion with the researcher and the post-graduate doctors/ students provided the attendant students with key concepts regarding scientific work that are the basis of theory and practice of biomedical research projects, not only of ultrastructural type. The students’ early exposure to the work and methodology characteristic of ultrastructural research may prove useful, not only when promoting indepth understanding of microscopic anatomy, but also as a motivational base upon which to instil a correct approach to scientific research in future doctors.

Comparison of conventional cervical cytological sampling (Ayre¿s spatula, endocervical brush) and the PapCone® as seen by scanning electron microscopy

Introduction Efforts to improve Pap smear performance have recently focused on reducing the number of false negative smears, i.e. cases in which premalignant or malignant cells have been misdiagnosed as normal. Considering that the basis for a good quality cervical smear is a correct sampling method, a variety of technologies or clinical strategies have been proposed to improve Pap testing including various devices for collecting cytological cervical samples, such as the PapCone® (Otto Bock, Duderstadt, Germany). The PapCone® is a cone-shaped polyurethane (foam) sampling device designed by the University Hospital Gottingen (Germany) to obtain simultaneously cells from the ectoand the endocervix. It has the properties of a soft brush and at the same time it acts as a soft spatula by being pressed to the ectocervix. Aim We evaluated the ultrastructure of human cervical cells by means of scanning electron microscopy (SEM), comparing the samples obtained with the Papcone® with the traditional wooden Ayre’s spatula (S) and the endocervical cytobrush (C). Materials and methods Twenty-two adult fertile (under 40 y), primiparous women were submitted to traditional Pap test (by S and/or C) and to PapCone® sampling 3 months after, with the related informed consent. Ultrastructural features of the 3 devices were analysed by SEM before and after sampling. Specimens were fixed in 2.5% glutaraldehyde in 0.1M PBS, stored at 4°C, postfixed in 1% osmium tetroxide, and dehydrated in increasing ethanol concentrations. Specimens were critical-point dried with carbon dioxide, mounted on aluminum stubs, coated with platinum, and observed with a Hitachi S-4000 FE-SEM (20 kV). Results SEM allowed to recognize large and flattened ectocervical cells with microplicae from cylindrical and smaller endocervical cells. However, S and C appeared less useful in case of abundant presence of mucus. The PapCone® surface was porous, partly occluded by membranes, and divided by trabecles lodging sampled cells. S showed a finely regular surface, sometimes showing wooden chips, and cells appearing clustered in usually overlapping groups. C often entrapped cells at or among the bristles, wherein red cells were usually noted. PapCone® showed lower bleeding and less overlapping cell layers in comparison with S and C. Conclusion PapCone® is a good and low traumatic method for sampling cervical cells, that can be useful especially in case of flogosis or cervical bleeding.