Conventional Histological Techniques Research Articles

Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies.

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

Comparative analysis of the processes involved in the loss of elaiophores in two species of the christianelloid clade (Malpighiaceae).

Alicia anisopetala and Callaeum psilophyllum are two closely related species that belong to the christianelloid clade of the family Malpighiaceae. Both species are pollinated by oil-collecting bees and exhibit variations at specimen and population level in the number of elaiophores per flower. These floral glands that secrete non-volatile oils constitute an ancestral trait for the family. There is evidence that the observed variations in the number of elaiophores can be the result of processes of connation or reduction associated with differences in their vascularization. In order to identify which process occurs in each species, we conducted an anatomical study in natural populations of both species distributed along a wide range of their geographical distributions in Argentina. We collected flowers of different individuals, counted the number of elaiophores per flower, carried out exomorphological observations, and used conventional histological techniques to examine the vascularization of these glands. The floral anatomy of both species does not show any modifications in other whorls related to the fusion or reduction of elaiophores. Our results indicate that the process of loss of elaiophores in A. anisopetala is caused by incomplete connation and in C. psilophyllum by reduction, suggesting that the processes that lead to the loss of elaiophores in Malpighiaceae are homoplastic and would not reflect phylogenetic signals.

Simultaneous deep ultraviolet transmission and scattering microscopy for virtual histology.

In recent years, the emergence of a variety of novel optical microscopy techniques has enabled the generation of virtual optical stains of unlabeled tissue specimens, which have the potential to transform existing clinical histopathology workflows. In this work, we present a simultaneous deep ultraviolet transmission and scattering microscopy system that can produce virtual histology images that show concordance to conventional gold-standard histological processing techniques. The results of this work demonstrate the system's diagnostic potential for characterizing unlabeled thin tissue sections and streamlining histological workflows.

Step Serial Sectioning in Head and Neck Squamous Cell Carcinoma of Unknown Primary

Patients with suspected head and neck squamous cell carcinoma of unknown primary (HNSCCUP) may undergo tonsillectomy and tongue base mucosectomy (TBM) to help identify clinicoradiologically occult primary disease. It is hypothesized that when these diagnostic specimens are analyzed, conventional histopathological (CH) techniques risk missing small primary tumors that may be hidden in the tissue blocks. To establish the outcomes of a step serial sectioning (SSS) histopathological technique vs CH when analyzing diagnostic tissue specimens from TBM and tonsillectomy performed for HNSCCUP. The MOSES prospective multicenter noninterventional cohort study was conducted over a 25-month period from November 2019 at secondary and tertiary care ear, nose, and throat departments in the United Kingdom and included adults with clinicoradiologically occult HNSCCUP who were undergoing TBM. Conventional histopathological techniques performed on TBM and tonsillectomy specimens at participating centers, followed by SSS performed at the central laboratory. Identification of cancer on central histopathological review of TBM and tonsillectomy specimens. Tissue from 58 eligible patients was analyzed (median [range] age, 58 [47-82] years; 10 women [17%]), with 20 480 sections cut in the laboratory and 4096 sections directly examined by a pathologist (median [range], 64 [28-135] per patient). The overall identification rate for TBM following SSS according to study protocol was 50.0% (95% CI, 37.5%-62.5%) and by subgroups was 42.9% (95% CI, 21.4%-67.4%) when performed following a negative bilateral tonsillectomy, 46.7% (95% CI, 24.8%-69.9%) at the same time as bilateral tonsillectomy, and 57.1% (95% CI, 36.5%-75.5%) following historic tonsillectomy. Conventional histopathological techniques at central review identified 2 undiagnosed primary tumors and revised the diagnosis of 2 other cases (1 nonmalignant and another down staged). Step serial sectioning identified a single additional tumor: an ipsilateral synchronous tongue base tumor for which a contralateral tumor had been identified on CH. Multifocal disease was seen in 5 (8.6%); all were human papillomavirus-related and in the tongue base. In this multicenter cohort study of patients undergoing TBM for HNSCCUP, SSS was associated with added considerable histopathological workload with minimal additional diagnostic benefit. A second opinion for conventional histological techniques may be more beneficial. Synchronous primary disease should be considered when planning diagnostic oropharyngeal surgery for these patients.

Comparative morpho-anatomy of the sporophyte of the most austral American species of Didymoglossum (Polypodiopsida: Hymenophyllaceae)

Didymoglossum (Hymenophyllaceae) is a monophyletic genus that includes small, epiphytic or epilithic plants. Three morphologically very similar species are found in the southern sector of the Yungas and Parana forests: D. hymenoides, D. krausii and D. reptans. The aim of this research was to characterise the morphoanatomy to better delimitate the taxa. Also, to evidence the presence of fungal endophytes. Conventional histological techniques were applied to fresh and FAA-fixed material. The anatomical structure of the different organs, in the three species, is similar; with collateral protostelic rhizomes, covered with simple or branched rhizoids; stipes teretes with glandular trichomes; uniestratified laminae formed by epidermal cells, with collateral vascular bundles; false veins formed by macrosclereids and stegmata or only by macrosclereids. The eglandular and glandular trichomes on the lamina differ in the species studied. The basal and middle portion of the involucre are pluristratified, with a vascular bundle at each margin, while the apical portion is formed by a one layer of epidermal cells; the receptacle is pluristratified with glandular trichomes on its surface. The presence of stegmata, collenchymatous tissue, sclerenchyma, mucilage fibers in phloem, pores in lamina and cuticle on the epidermis is evidenced and reported. Septate fungal endophytes were observed in epidermal cells of the rhizome, stipe and lamina of D. krausii and D. reptans. Species are differentiated by the following characters: shape of the lamina, diameter of the rhizome, number of false veins and the types of trichomes and pores. A key for the studied species is presented.

Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos

Leucocytozoon parasites (Haemosporida, Leucocytozoidae) are haemosporidians whose diversity, exo-erythrocytic development and potential vectors are the least studied. The knowledge about their exo-erythrocytic development and pathogenicity is fragmentary, resulting in an incomplete comprehension of the impact of these parasites on avian hosts. For a long time, Leucocytozoon infections were considered benign to wild birds, even though they were virulent in poultry and responsible for some wild bird population declines. This study aimed to investigate the presence of Leucocytozoon species exo-erythrocytic stages in song thrushes Turdus philomelos using conventional histological techniques (sections stained by H&E) and chromogenic in situ hybridization (CISH). Tissues from ten birds (seven naturally infected and three opportunistic samplings) were examined using both methods. Parasite lineages were identified from blood samples using PCR-based techniques. Leucocytozoon species meronts were found in five individuals (in four birds using H&E staining protocol, and in three in CISH-treated histological sections). Meronts were found mainly in the kidneys, but some meronts were also present in the lungs. It was possible to observe different maturation stages of meronts in the same bird individual, indicating an asynchronous development. Cytomeres were readily visible in developing meronts. One megalomeront-like structure was present close to a blood vessel in the heart. It was covered with a prominent capsular-like wall. No inflammatory reaction or necrosis was seen in the tissues surrounding the meronts or the megalomeront-like structure. We could confirm the transmission of three Leucocytozoon lineages (lTUPHI14, lSTUR1 and lTUPHI13) in Europe, and add evidence of the transmission of two Plasmodium lineages, including Plasmodium circumflexum (pTURDUS1), and Haemoproteus asymmetricus (hTUPHI01). We call for further research to better understand Leucocytozoon parasite exo-erythrocytic development.

MIOEPITHELIAL CELLS: STRUCTURE, FUNCTION, AND THEIR ROLE IN NORMAL AND NEOPLASTIC TRANSFORMATION OF THE BREAST

Abstract. Myoepithelial cells (MECs) are characteristic of exocrine glands and are responsible for synthesizing the extracellular matrix, particularly the structural components of the basal membrane. MECs are of epithelial origin and exhibit contractile function, which aids in milk ejection from the ducts during lactation. Scientific literature suggests that MECs act as suppressors of tumors and play an important diagnostic role in mammary gland pathology, especially in distinguishing diff erent neoplastic processes.Research objective: T o elucidate the histological features and immunohistochemical verifi cation of myoepithelial cells in normal breast tissue and their role in neoplastic transformation of the mammary gland.Materials and Methods. The histological examination of myoepithelial cells of the mammary gland was conducted on archival slides from the Department of Histology, Cytology, and Embryology, Danylo Halytsky Lviv National Medical University. Histological sections, stained with hematoxylin and eosin, as well as immunohistochemical markers p63 (Clone 4A4, Masterdiagnostica) and cytokeratin 5/6Ab-2 (D5/16 B4, Thermoscientifi c) for confi rming the myoepithelial origin of the cells, were analyzed under a light microscope (UlabXSP-137TLED) with 10x and 40x objectives and a 10x eyepiece. Photographs were taken using an XCAM 1080P camera.Research Results. The structural units of the mammary gland (MG) are ductal-l obular formations, the terminal portions of which have an alveolar shape and are formed by secretory exocrine cells with underlying myoepithelial cells – basket-like cells. Identifi cation of myoepithelial cells using conventional histological staining techniques such as hematoxylin and eosin does not always provide a clear understanding of their topography. All myoepithelial cells are located on the basal membrane and contribute to the synthesis of its components (laminin, entactin, etc.). The myoepithelial cell layer, which is consistently present in the normal mammary gland, can appear diff erently: resembling smooth muscle or epithelioid cells, having a stellate or spindle- shaped form, or not being visualized at all. It has been shown that p63 is a sensitive and specifi c nuclear myoepithelial marker, expressed in myoepithelial cells of the normal mammary gland, in residual lobular acini, sclerosing adenosis, carcinoma in situ, and not detected in invasive carcinomas. It can be included in immunohistochemical panels aimed at identifying myoepithelial cells in diagnostically challenging breast pathologies.Conclusions. Myoepithelial cells in the mammary glands play a key role in their function, facilitating the process of milk secretion. However, in certain cases, they also play an important role in the development of neoplastic transformations. Intercellular connections between secretory and myoepithelial cells indicate that the last ones suppress both the progression of carcinoma in situ to invasive breast cancer and the carcinomainduced angiogenesis. The use of the immunohistochemical marker p63 is mandatory in the panel of antibodies to identify myoepithelial cells in the mammary gland and diff erentiate between benign and malignant lesions.

Histochemical and immunohistochemical analysis of testicular morphology and spermatogenesis in Gymnotus carapo (Teleostei: Gymnotidae).

This work aims to study the testicular morphology and spermatogenesis of Gymnotus carapo to provide information on their reproductive biology which is useful in managing this species as a fishing resource. The testicles were isolated and fixed in 10% formalin; subsequently, they were processed for scanning electron microscopy with conventional histological technique. To analyze the cell proliferation of germline cells and Sertoli cells, immunodetection of the proliferating cell nuclear antigen (PCNA) protein was performed. In G. carapo spermatogenesis, the spermatogenic line is organized into cysts. Spermatogonia A is characterized by more bigger and solitary cells. Spermatogonia B are smaller cells; their nucleus has a larger area concerning the cytoplasm and is grouped in tubules. Spermatocytes (I-II) are smaller than spermatogonia in the prophase of meiotic division. Spermatids are cells with dense, rounded nucleus. The sperm were found in the lumen of the tubule. By immunostaining PCNA, it was possible to observe the proliferative activity of germ line cells and Sertoli cells during the cyst reorganization phase. These results are the basis for future studies focusing on the analysis compared to females of the reproductive cycle of G. carapo.

Placentation in the Mexican scincid lizard Plestiodon brevirostris (Squamata: Scincidae).

Viviparity is the reproductive pattern in which females gestate eggs within their reproductive tract to complete their development and give birth to live offspring. Within extant sauropsids, only the Squamata (e.g., snakes, lizards, and amphisbaenians) evolved viviparity, representing 20% of the existing species. The genus Plestiodon is represented by 43 species and is one of the most widely distributed genera of the Scincidae in Mexico. The goal of this research has been to determine the placental morphology and ontogeny during gestation in the lizard Plestiodon brevirostris. Specimens were dissected to obtain the embryonic chambers and the embryos were categorized to carry out the correlation between the development stage and the placenta development. The embryonic chambers were processed using the conventional histological technique for light microscopy. The identified embryonic stages were 4, 29, 34, 36, and 39. A thin eggshell surrounds the egg in early developmental stages; however, this structure is already absent in the embryonic hemisphere during the developmental stage 29. The results revealed that P. brevirostris is a lecithotrophic species, but a close maternal-fetal relationship is established by tissue apposition. Ontogenically, the placental types that form in the embryonic hemisphere are the chorioplacenta, choriovitelline placenta, and chorioallantoic placenta; whereas the omphaloplacenta is formed in the abembryonic hemisphere. The structure of the chorioallantoic placenta in P. brevirostris suggests that it may play a role during gas exchange between the mother and the embryo, due to the characteristics of the epithelia that comprise it. The structure of embryonic and maternal epithelia of the omphaloplacenta suggests a role in the absorption of the eggshell during gestation and possibly in the transport or diffusion of some nutrients. In general, it is evident that ontogeny and placental characteristics of P. brevirostris match those of other species of viviparous lecithotrophic scincids.

A Neglected Normal Anatomical Structure in Mammal: A Study in Bats and Tree Shrews

The myodural bridge is a dense connective tissue connecting muscles and ligaments to the spinal dura mater in the atlanto-occipital interspace. Some researchers believe that the myodural bridge may play a vital physiological role. It is possible, for instance, that the prevention of spinal dura mater infoldings might be involved in regulated cerebrospinal fluid circulation. For instance, it is possible to prevent spinal dura mater infoldings, regulating cerebrospinal circulation. Bats are nocturnal and the only mammals that can perform a genuine and sustained flight, whereas tree shrews are arboreal mammals that often climb to a high altitude of about 10,000 feet. Both animals have lifestyles that are different from other previously studied mammals. The study of these two animals will shed further light on the existence of the myodural bridge in mammals. Gross anatomical dissection was used to observe the connections between the deep muscles of the neck and the dura mater at the level of the atlanto-occipital interspace. The existing structures were analyzed using conventional and special histological staining techniques. The suboccipital regions in bats and tree shrews contained the rectus capitis dorsal major (RCDma), rectus capitis dorsal minor (RCDmi), oblique capitis anterior (OCA), and oblique capitis posterior (OCP). Dense connective tissue connects the RCDmi to the posterior atlanto-occipital membrane (PAOM) and the latter to the spinal dura mater. The myodural bridge in these mammals shares a similar structure to the myodural bridge in humans. Histological analyses confirmed that the connective fibers of the myodural bridge were primarily type I collagen fibers. In this study, it is supplemented by the existence of the myodural bridge in mammals. This further demonstrates that myodural bridge widely exists in the normal anatomy of mammals. This provides morphological support for a comparative anatomical study of the physiological function of the myodural bridge.

Comparative stem anatomy of three species of Coryphantha (Cactoideae-Cactaceae)

Background: The genus Coryphantha has the second largest number of species of the tribe Cacteae-Cactaceae. Morphologically, it has been reported that the stem presents mainly globose, ovoid and cylindric variations. However, at the anatomical level, descriptions are scarce and focused on particular tissues (epidermis or xylem). The aim of this work is to contribute to the anatomical knowledge of the genus.
 Questions and/or Hypotheses: What are the anatomical characteristics that differentiate one species from another? Does the presence of crystals allow differentiation between species?
 Study species: Coryphantha clavata, Coryphantha cornifera and Coryphantha radians.
 Site and years of study: San Luis Potosí and Querétaro, Mexico; 2017, 2020.
 Methods: Three individuals per species were collected, and conventional histological techniques were applied. Anatomical descriptions were made for different tissues, and structural characteristics were measured for each species.
 Results: The species exhibited the greatest differences in the epidermis and hypodermis, such as the presence of subepidermal stomata (C. radians), three-layered hypodermis (C. clavata), and non-collenchymatous hypodermis (C. cornifera). Crystals were not observed in the epidermises but instead in the hypodermes, which exhibited crystal shapes of druses (C. cornifera and C. radians), prisms and round bodies (C. clavata). In the cortical tissue, C. clavata had abundant mucilage cells. The observed xylem and phloem tissues exhibited characteristics similar to those of other Coryphantha species.
 Conclusions: The anatomical characteristics of the epidermis and hypodermis are considered of high systematic value and could be used to distinguish between species of the genus.

Enhanced study of facial soft tissues using a novel large scale histology technique.

The safety and effectiveness of facial cosmetic surgery procedures are dependent on detailed 3D understanding of the complex surgical anatomy of the face. Traditional, small sample size anatomical dissection studies have limitations in providing definitive clarification of the fascial layers of the face, and especially in their relationship with the facial nerve, and their reaction to surgical manipulation. The objective study of large tissue areas is required to effectively demonstrate the broader architecture. Conventional histology techniques were modified to handle extraordinarily large tissue samples to fulfill this requirement. Full-thickness soft tissue samples (skin to bone) of maximum length 18 cm, width 4cm, and tissue thickness 1cm, were harvested from 20 hemifaces of 15 fresh human cadavers (mean age at death=81 years). After fixation, the samples were processed with an automated processor using paraffin wax for 156 h, sectioned at 30 μm, collected on gelatin-chromium-coated glass slides, stained with a Masson's Trichrome technique and photographed. Using this technique, excellent visualization was obtained of the fascial connective tissue and its relationship with the facial mimetic muscles, muscles of mastication and salivary glands in 73 large histological slides. The resulting slides improved the study of the platysma and superficial musculo-aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the facial nerve in relations to the deep fascia. Additionally, surgically induced changes in the soft-tissue organization were successfully visualized. This technique enables improved insight into the broad structural architecture and histomorphology of large-scale facial tissues.

EANO - EURACAN - SNO Guidelines on circumscribed astrocytic gliomas, glioneuronal, and neuronal tumors.

In the new WHO 2021 Classification of CNS Tumors the chapter "Circumscribed astrocytic gliomas, glioneuronal and neuronal tumors" encompasses several different rare tumor entities, which occur more frequently in children, adolescents, and young adults. The Task Force has reviewed the evidence of diagnostic and therapeutic interventions, which is low particularly for adult patients, and draw recommendations accordingly. Tumor diagnosis, based on WHO 2021, is primarily performed using conventional histological techniques; however, a molecular workup is important for differential diagnosis, in particular, DNA methylation profiling for the definitive classification of histologically unresolved cases. Molecular factors are increasing of prognostic and predictive importance. MRI finding are non-specific, but for some tumors are characteristic and suggestive. Gross total resection, when feasible, is the most important treatment in terms of prolonging survival and achieving long-term seizure control. Conformal radiotherapy should be considered in grade 3 and incompletely resected grade 2 tumors. In recurrent tumors reoperation and radiotherapy, including stereotactic radiotherapy, can be useful. Targeted therapies may be used in selected patients: BRAF and MEK inhibitors in pilocytic astrocytomas, pleomorphic xanthoastrocytomas, and gangliogliomas when BRAF altered, and mTOR inhibitor everolimus in subependymal giant cells astrocytomas. Sequencing to identify molecular targets is advocated for diagnostic clarification and to direct potential targeted therapies.

A Comparison of PCA-LDA and PLS-DA Techniques for Classification of Vibrational Spectra

Vibrational spectroscopies provide information about the biochemical and structural environment of molecular functional groups inside samples. Over the past few decades, Raman and infrared-absorption-based techniques have been extensively used to investigate biological materials under different pathological conditions. Interesting results have been obtained, so these techniques have been proposed for use in a clinical setting for diagnostic purposes, as complementary tools to conventional cytological and histological techniques. In most cases, the differences between vibrational spectra measured for healthy and diseased samples are small, even if these small differences could contain useful information to be used in the diagnostic field. Therefore, the interpretation of the results requires the use of analysis techniques able to highlight the minimal spectral variations that characterize a dataset of measurements acquired on healthy samples from a dataset of measurements relating to samples in which a pathology occurs. Multivariate analysis techniques, which can handle large datasets and explore spectral information simultaneously, are suitable for this purpose. In the present study, two multivariate statistical techniques, principal component analysis-linear discriminate analysis (PCA-LDA) and partial least square-discriminant analysis (PLS-DA) were used to analyse three different datasets of vibrational spectra, each one including spectra of two different classes: (i) a simulated dataset comprising control-like and exposed-like spectra, (ii) a dataset of Raman spectra measured for control and proton beam-exposed MCF10A breast cells and (iii) a dataset of FTIR spectra measured for malignant non-metastatic MCF7 and metastatic MDA-MB-231 breast cancer cells. Both PCA-LDA and PLS-DA techniques were first used to build a discrimination model by using calibration sets of spectra extracted from the three datasets. Then, the classification performance was established by using test sets of unknown spectra. The achieved results point out that the built classification models were able to distinguish the different spectra types with accuracy between 93% and 100%, sensitivity between 86% and 100% and specificity between 90% and 100%. The present study confirms that vibrational spectroscopy combined with multivariate analysis techniques has considerable potential for establishing reliable diagnostic models.

Cerebral Vascular Density and Its Possible Correlation with Alzheimer Disease Progression in Elderly Individuals with Rheumatoid Arthritis

BackgroundRheumatoid Arthritis (RA) is characterized by chronic systemic inflammation, primarily affecting the joints. In RA patients, high concentrations of inflammatory cytokines are found within the synovial fluid and serum (PMID: 1700672). These cytokines reduce the blood brain barrier (BBB) integrity by decreasing the expression of tight junction proteins (PMID: 29910805). In a RA animal model such decreased BBB integrity has been shown to affect the transport of amyloid beta peptide (Aβ) between the peripheral circulation and the brain, thereby promoting accumulation of Aβ within the cerebral vasculature followed by the endothelial cell degeneration (PMID: 22029666). In humans, a similar pathologic process is a major risk factor for Alzheimer’s Disease (AD) which accelerates neuronal loss and cognitive impairment associated with AD (PMID: 29377008). Assessment of cerebral vascular density is an emerging modality for detection and monitoring the progression of cerebrovascular disease (CVD) (PMID: 15564412). Advancements in computed tomography (CT) imaging technology and the development of novel contrast agents should provide more accurate assessment for CVD in humans.The main objective of this study was to test high‐resolution CT imaging using a recently developed BriteVu contrast agent in conjunction with conventional histological techniques for postmortem assessment of CVD and pathology in the cerebral cortex and hippocampus of two male cadavers. The first, 74‐year‐old (D1) was diagnosed with RA and AD, while the second, 90‐year‐old (D2) had RA.ResultsThe D1 and D2 cadavers were fixed and perfused with BriteVu contrast agent. Heads were removed and scanned with a VimagoGT30 CT scanner at 300 µm. The images were subjected to volumetric analysis using Radiant software. The results showed that scans of the cerebral cortex from D2, specifically the middle frontal, inferior parietal, and superior temporal gyri, had significantly lower mean Hounsfield unit densities (MHUD) than those from the same regions of D1. The MHUD from the hippocampal scans revealed no significant difference. The lower MHUD count in the D2 cortex in comparison to D1 most likely reflects a lower vascular density in the respective brain region. The AD related pathology in both donors was assessed by histochemical and immunohistochemical techniques. To that end, the brains were extracted and fixed in formalin solution for 12‐16 weeks. The brain hippocampal and cortical sections were paraffin processed followed by H&E and immunohistochemical staining for Aβ 1‐42, phospho‐tau, and phospho‐TDP‐43. Analysis of the data confirmed the AD diagnosis in D1 and demonstrated distinct AD pathology in D2. The AD staging could be characterized as mild in D1 and intermediate in D2.Conclusionsi) The vascular density within the cerebral cortex of elderly individuals with RA could correlate with the AD progression. ii) The adaptation of the reported CT imaging technique for its use in live individuals could provide more sensitive and accurate method for assessing AD in RA patients at the early AD stage(s).

Anatomía del esporofito de Anemia phyllitidis var. phyllitidis (Anemiaceae) de un bosque de ribera (Tucumán, Argentina)

Antecedentes y Objetivos: En Argentina, el género Anemia se encuentra representado por siete especies, una de ellas, A. phyllitidis, con dos variedades. Los antecedentes anatómicos para A. phyllitidis var. phyllitidis son escasos y están referidos a la ontogenia de estomas, estructura de epidermis y tipo de estela del pecíolo. Esta variedad, que habita en bosques riparios, es afectada por el incremento de presiones ecológicas estacionales. Debido a que la información que se registra acerca de la anatomía del esporofito es escasa, el objetivo de este trabajo fue caracterizarlo.Métodos: Cinco especímenes fueron colectados en la ribera del arroyo El Parque (Tucumán-Argentina). Una parte fue herborizada y la otra se fijó en FAA. Posteriormente fueron utilizadas técnicas histológicas convencionales y se determinaron la densidad estomática y el tamaño de estomas y tricomas. Se realizaron pruebas histoquímicas para la detección de almidón (lugol) y compuestos fenólicos (cloruro férrico al 10%).Resultados clave: Anemia phyllitidis var. phyllitidis muestra raíces diarcas y rizoma dictiostélico cubierto por tricomas glandulares. El pecíolo posee tricomas glandulares, epidermis y subepidermis formada por fibras lignificadas, haz vascular con xilema rodeado por floema, periciclo bistrato rodeado por endodermis y en el tejido cortical almidón y compuestos fenólicos. Las pinnas estériles poseen estomas pericíticos, anomocíticos y desmocíticos (tamaño promedio 49.8 µm × 38.7 µm); tricomas glandulares; lámina dorsiventral, hipostomática y haces vasculares con periciclo y endodermis. Las pinnas fértiles son anatómicamente semejantes al raquis. Algunas células epidérmicas de los distintos órganos evidenciaron la presencia de sílice.Conclusiones: El esporofito de A. phyllitidis var. phyllitidis mostró rasgos anatómicos, morfológicos y fisiológicos que revelan su adaptación al hábitat ribereño donde crece. Al mismo tiempo, se profundiza la descripción de esta variedad y se aportan datos inéditos del género, como la presencia de estegmata en las pinnas fértiles.

Myriad pillars formed by intussusceptive angiogenesis as the basis of intravascular papillary endothelial hyperplasia (IPEH). IPEH is intussusceptive angiogenesis made a lesion.

Intussusceptive angiogenesis (IA) is the process by which pre-existing blood vessels split, expand and remodel through intravascular pillar formation. In previous works, we studied the morphologic characteristics of intravascular papillary endothelial hyperplasia (IPEH) and suggested the participation of IA in the histogenesis of the lesion. Our current goal is to demonstrate that myriad papillae in IPEH are in fact myriad pillars, the hallmarks of IA. For this purpose, specimens of 14 cases of IPEH were used for conventional histologic techniques, immunohistochemistry and immunofluorescence in confocal microscopy. The studies showed the following pillar characteristics: a) structural composition by an endothelial cell (EC) cover and a connective core, b) characteristic pillar image and its appearance and disappearance in whole-mounted and series of individual views in confocal microscopy (requirements for pillar identification), c) arrangement in masses, alignments and meshes, and d) formation from vein intimal ECs, which extend and originate loops that encircle vein wall components (interstitial tissue structures: ITSs) and fibrin. The encircling ECs form the pillar cover and the encircled ITSs or fibrin form the initial core. Intraluminal endothelial bridges also originate from the vessel wall and from the pillars (nascent and thin pillars). In conclusion, the formation of myriad pillars, predominantly in veins, is the basis of IPEH. This lesion may therefore be considered an excessive expression of IA: IA becomes a lesion.

Molecular Spectroscopic Imaging Offers a Systematic Assessment of Pathological Aortic Valve and Prosthesis Tissue in Biomineralization

Pathological ECM remodelling and biomineralization in human aortic valve and bioprosthesis tissue were investigated by Fourier transformed infrared (FT-IR) spectroscopic imaging and multivariate data analysis. Results of histological von Kossa staining to monitor hydroxyapatite biomineralization correlated to the definition of mineralized tissue using FT-IR spectroscopic imaging. Spectra exhibit signals of carbonate and phosphate groups of hydroxyapatite. Proteins could be identified by the amide I and amide II bands. Proteins were detected in the calcified human aortic valve tissue, but no absorption signals of proteins were observed in the mineralized bioprosthesis sample region. A shift of the amide I band from 1654 cm−1 to 1636 cm−1 was assumed to result from β-sheet structures. This band shift was observed in regions where the mineralization process had been identified but also in non-mineralized bioprosthesis tissue independent of prior implantation. The increased occurrence of β-sheet conformation is hypothesized to be a promoter of the biomineralization process. FT-IR spectroscopic imaging offers a wealth of chemical information. For example, slight variations in band position and intensity allow investigation of heterogeneity across aortic valve tissue sections. The exact evaluation of these properties and correlation with conventional histological staining techniques give insights into aortic valve tissue remodelling and calcific pathogenesis.

Fast acetone tissue processing of human organs provides tissue characteristics equal to conventional processing

ABSTRACT Routine preparation of paraffin embedded tissue for histopathological diagnosis, here termed conventional histological technique (CT), whether performed manually or using an automated system, requires approximately 12 h. We developed earlier a rapid acetone dehydration technique (AT) for processing biopsies of nervous tissue that meets requirements for preserving tissue morphology and staining properties, and reduces processing time to 3.3 h. We compared the morphology and staining properties of human organ biopsies including adrenal gland, liver, ovary, pancreas, prostate, testis and thyroid prepared using both AT and CT. Following fixation with 10% formaldehyde and processing by either AT or CT, sections were stained using routine and special staining, and immunohistochemical methods. We evaluated nuclear and cytoplasmic staining, staining intensity, sharpness of images and presence of artifacts such as cracking and folding. AT preserved the morphology and staining properties of the tissues as well as CT. Consequently, the rapid AT procedure is a promising alternative technique for tissue processing.

Examination of ex-vivo viability of human adipose tissue slice culture.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 μm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco’s Modified Eagle’s Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.