Rosemarie Heyn

Structural changes of the zona pellucida during fertilization and embryo development

The zona pellucida (ZP) is a unique extracellular coat surrounding the maturing oocyte, during ovulation, fertilization, and early embryo development. It is formed by three/four glycoproteins. Ultrastructural data obtained with transmission (TEM) and scanning electron microscopy (SEM) were compared with molecular data on the glycoproteins network from ovulation to blastocyst formation. Molecular models are quite different to the morphology obtained with TEM, which shows a microfibrillar architecture, or with SEM, which shows a spongy or smooth surface. The saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide technique allows to show the ZP real microfilamentous structure and the related functional changes. These results support an ultrastructural supramolecular model, more similar and comparable to molecular models related with the glycoprotein network. A detailed mapping of single mammalian ZP proteins and their relationship within the supramolecular architecture of the zona matrix would clearly supply insights into the molecular basis of sperm-egg recognition. Differences in ZP glycoproteins among mammals do not affect structural morphology; further studies are needed to clarify the relationships between ultrastructural and molecular organizations.

Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smalller than 10 µm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue-azur B - basic fuchsine - alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining. Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 µm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue-azur B-basic fuchsine-alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

Three-dimensional microanatomical dynamics of the ovary in postreproductive aged women

OBJECTIVE To evaluate the microanatomical dynamics of the ovary during postreproductive life. DESIGN Retrospective, observational research study. SETTING Institutional (university). PATIENT(S) Eleven (43- to 72-year-old) women in perimenopause, menopause, or postmenopause. INTERVENTION(S) Biopsies of ovaries obtained from patients undergoing gynecologic surgery or diagnostic procedures. MAIN OUTCOME MEASURE(S) Analysis of the 3-dimensional microanatomy of the ovary by transmission and high-resolution scanning electron microscopy. RESULT(S) The surface epithelium gets gradually flatter and is always present, even in advanced age. The surface appears smoother because of a smaller number of papillae and crypts as well as a decreased number and shortening of microvilli on surface epithelial cells. Signs of atrophy and fibrosis are evident. Primordial follicles are usually absent in postmenopause, whereas corpora atretica, hemorrhagica, and albicantia, scar tissue, and simple follicular cysts are common after menopause. Apoptotic and necrotic cells appear frequently within the surface epithelium. Major common features are a marked reduction in number and caliber of blood vessels with thickening of the vascular walls and changes in endothelial cells. CONCLUSION(S) Scanning electron microscopy studies are a useful complement to ordinary gynecologic diagnostic methods. Variations among patients of the same age range or functional status should be considered.

Skin erythrodiapedesis during chronic venous disorders

BACKGROUND Extravasation of erythrocytes (erythrodiapedesis [ED]) is currently included among causes of skin damage in legs with chronic venous disorders (CVD) and ascribed to venular hypertension. ED is followed by erythrocyte disruption, degradation of hemoglobin, and storing of ferric iron into hemosiderin. The aim of this study was to evaluate the occurrence of ED in the skin of legs with different clinical stages of CVD. METHODS One hundred eighteen skin biopsies from legs with CVD underwent histologic evaluation for ED and hemosiderin deposition (HD). RESULTS ED was found in only 21/118 specimens. In particular, it was found in ulcer samples, in tissues surrounding varicophlebitis and, finally, in acute eczematous skin. ED was found in only 15/30 samples showing HD. CONCLUSION Our findings confirm the occurrence of ED during CVD. However, it was found only in concomitance of severe dermal inflammation. Hemosiderin deposition in the absence of actual ED could be explained with previous healed episodes of skin inflammation. However, ED is not likely the only cause of skin iron overload, which could also occur by a molecular mechanism. Further studies are needed to define the mechanism of iron deposition in the skin of legs afflicted with CVD.

The Value of Neurosurgical and Intraoperative Magnetic Resonance Imaging and Diffusion Tensor Imaging Tractography in Clinically Integrated Neuroanatomy Modules: A Cross-Sectional Study.

Neuroanatomy is considered to be one of the most difficult anatomical subjects for students. To provide motivation and improve learning outcomes in this area, clinical cases and neurosurgical images from diffusion tensor imaging (DTI) tractographies produced using an intraoperative magnetic resonance imaging apparatus (MRI/DTI) were presented and discussed during integrated second‐year neuroanatomy, neuroradiology, and neurosurgery lectures over the 2008–2011 period. Anonymous questionnaires, evaluated according to the Likert scale, demonstrated that students appreciated this teaching procedure. Academic performance (examination grades for neuroanatomy) of the students who attended all integrated lectures of neuroanatomy, was slightly though significantly higher compared to that of students who attended these lectures only occasionally or not at all (P=0.04). Significantly better results were obtained during the national progress test (focusing on morphology) by students who attended the MRI/DTI‐assisted lectures, compared to those who did so only in part or not at all, compared to the average student participating in the national test. These results were obtained by students attending the second, third and, in particular, the fourth year (P≤0.0001) courses during the three academic years mentioned earlier. This integrated neuroanatomy model can positively direct students in the direction of their future professional careers without any extra expense to the university. In conclusion, interactive learning tools, such as lectures integrated with intraoperative MRI/DTI images, motivate students to study and enhance their neuroanatomy education. Anat Sci Educ 6: 294–306.

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.

Training the “clinical eye”. Rubens’ Three Graces: how many pathologies?

Art can serve as a powerful resource for medical students to both train the so called “clinical eye” and to better understand disease [1]. Herein a paleopathological analysis is performed on one of Ruben’s final artworks, “The Three Graces” (1630-1635; oil on oak panel; 220.5 x 182 cm; Museo del Prado, Madrid). Rubens depicts the three Graces beside a fountain, under a garland of flowers in a landscape. The circular rhythm and elegant undulation are based on classical sculpture. Painted shortly after his marriage, it bears witness to the happiness of the artist’s life. The figure on the left is directly inspired by his second wife, Helene Fourment (23 years old); the central and right Graces probably illustrate Rubens’ sisters-in-law. Besides overweight, scoliosis, and hyperlordosis observed in all three Graces, the left Grace evidences flat feet; hyperextension of the right metacarpal joints; signs of rheumatoid arthritis (even fibromyalgia has been proposed); lateral deviation of the nipple (Mondor’s disease?); varicose thighs, and right hallux vagus. The central Grace (Clara Fourment?), in turn, shows cellulite and, interestingly, positive Trendelenburg sign. Finally, the Grace on the right -Susanna Fourment- has been subject of a long debate on signs of a locally advanced breast cancer in the left external upper quadrant. In fact, several specialists agree in the observation of signs of an open ulcer; redness of the surrounding skin (an inflammatory sign); nipple retraction; reduction of the left breast volume, and enlarged axillary lymph nodes [2-3]. Rubens was one of main Baroque and realist painters, i.e. he painted whatever his eyes captured. If the Graces were sisters, then they are likely to share genetic traits. The latter, together with all the other signs described, favour the working diagnosis of familial benign hypermobility syndrome. Observation has a key role in clinical medicine; the paleopathological observation in art show us how artists could record abnormalities long before doctors did [2]. Therefore, artworks still represent useful teaching tools for refining visual skills in traditional and innovative medical education.

Looking is not seeing: visual art as a useful eLearning tool for teaching clinical anatomy

An old Chinese proverb reads “A picture paints a thousand words…”. Even though arts were used as a pedagogical tool as early as 1902, arts have not traditionally been part of medical education. Anatomy teaching is undergoing significant changes; long-distance education associated to web 2.0 tools have enlarged communication and interaction possibilities between users and virtual communities (1,2). We are developing a project consisting of a series of asynchronous (pre-registered) short video tutorials covering a wide range of clinical anatomy-related topics which can be posted on an internet/intranet site and then actively and autonomously be followed. Videos include short introductory remarks and legends which might be developed in different languages and which make them useful even to disable eLearners. The relationship between Art and: anatomy, thyroid gland, osteomuscular alterations, breast, ageing, artists’ diseases, as well as the presence of anatomists and physicians in Art, are some of the topics developed. Each tutorial considers numerous paintings and some sculptures covering a long time span, from Prehistory to the Classical period, Late Middle Ages, the rich Renaissance period up until modern times. Artworks were chosen in order to be narrative in nature and rich in detail, thus stimulating reflection and self-discussion. These video tutorials could be a valuable teaching/learning complement to theoretical knowledge within medical students’ education or even within a larger “art-loving” audience. eLearners are guided by the teacher’s voice and then get immersed in an artwork, discovering it. Clear and/or hidden clinical anatomical features can be enhanced and made easier to assimilate. This nontraditional format brings a new lens through which students can learn valuable visual skills on human clinical anatomy. It may favor critical thinking, opening the mind to alternative ways of seeing, thus enhancing medical students’ abilities to more deeply observe and to better understand real clinical situations.

Visual thinking strategy in the medical curriculum: training the “clinical eye” in classrooms and art museums

Medicine intertwines with arts as part of the wider field of humanities since its origin; they integrate creating a new synergy. Clinical diagnosis involves the observation, description and interpretation of visual information. These skills can be developed and trained by analysing visual art works. Visual thinking strategy (VTS) has been recognized as a legitimate cognitive tool (1,2). Thanks to a collaboration between our medical faculty (Sant’Andrea Hospital) and the Borghese Gallery and Museum we are piloting an innovative experience with small-group interactive sessions within the course of Methodology (3rd-year students). This activity is carried out under the tutelage of art education curators and faculty members. Sessions focused on the analyses of selected original paintings. Students were evaluated through pre- and posttest questionnaires and with short oral presentations. Discussion was warm and enthusiastic and students appreciated the interaction with faculty on a non-medical level. By engaging with art works, students learn a broader conception of human-ness, enhancing mindfulness, rendering learning enjoyable and meaningful. Museums as teaching labs are valuable resources for enhancing learning beyond the classroom walls. VTS in medicine encourages critical thinking and problem solving, thus improving clinical abilities by means of observation and clinical reasoning, promoting empathy and getting used with team work. These activities can be integrated with art works shown in the classroom as clinical triggers: several figurative paintings are shown within our course of human and clinical anatomy (first and second-year students), i.e. self-portraits of Piero della Francesca and Mantegna’s “Virgin and child” well illustrate goiter; Raphael’s La Fornarina and Rubens’ “The three graces” show breast cancer. Rombouts’ “Allegory of the five senses” is a nice introductory image for the sense organs as Bigot’s “A doctor examining urine” for the urinary tract. An art-based approach to teaching observational skills should be included continuously within required courses in the medical curriculum.

Paracrystalline and crystalline inclusions of the human testis in cases of subfertility

Testicular crystalline inclusions, namely Charcot-Bottcher and Spangaro crystals of Sertoli cells, Lubarsch crystals of spermatogonia and Reinke crystals of Leydig cells, have been considered normal ultrastructural features of the post-pubertal human testis (Reinke, 1896; Chemes et al., 1977; Kaya et al., 1985). Nevertheless, their significance is not known. We have noted that these structures are dynamic and may disappear and reappear in some cases of subfertility. In particular, patients showing spermatogenesis arrest or germ cell aplasia usually do not show any crystalline inclusions. We study herein using transmission electron microscopy testicular biopsies of young infertile men (idiopathic infertility or varicocele) showing intracytoplasmic crystalline and/or paracrystalline inclusions. Sertoli cells’ paracrystalline inclusions consist of closely packed electron-dense longitudinal fibrils, sometimes including a granular and light core. Coarse ca. 5-25 μm long and 2-3 μm thick bundles composed of 5 or 10 nm thick filaments mainly locate in the basal cytoplasm near the nucleus. Reinke’s crystals, in turn, appear as variable-sized (2-5 μm long) polyhedral crystals with a honeycomb lattice and sharp edges consisting of 5-10 nm filaments filling wide areas of the cytoplasm. Alternatively, they may appear as filamentous/tubular, electron dense (0,5 μm long) units. In both cases they associate to mitochondria and dilated smooth endoplasmic reticulum. Due to their structure, whether these inclusions arise or not within the nucleus and are somehow transported to the cytoplasm is not yet clear. Although their exact molecular composition remains to be discovered, they are not likely to be associated to steroidogenesis but rather they may represent a kind of protein deposit.