Beyond the Brain: Perinatal Exposure of Rats to Serotonin Enhancers Induces Long-Term Changes in the Jejunum and Liver.

Serotonin (5-hydroxytryptamine, 5HT) homeostasis is essential for many physiological processes in the central nervous system and peripheral tissues. Hyperserotonemia, a measurable sign of 5HT homeostasis disruption, can be caused by 5HT-directed treatment of psychiatric and gastrointestinal diseases. Its impact on the long-term balance and function of 5HT in the peripheral compartment remains unresolved and requires further research due to possible effects on human health. We explored the effects of perinatal 5HT imbalance on the peripheral organs responsible for serotonin metabolism-the jejunum, a synthesis site, and the liver, a catabolism site-in adult rats. Hyperserotonemia was induced by subchronic treatment with serotonin precursor 5-hydroxytryptophan (5HTP) or serotonin degradation inhibitor tranylcypromine (TCP). The jejunum and liver were collected on postnatal day 70 and analyzed histomorphometrically. Relative mRNA levels of 5HT-regulating proteins were determined using qRT-PCR. Compared to controls, 5HTP- and TCP-treated rats had a reduced number of 5HT-producing cells and expression of the 5HT-synthesising enzyme in the jejunum, and an increased expression of 5HT-transporter accompanied by karyomegaly in hepatocytes, with these differences being more pronounced in the TCP-treated animals. Here, we report that perinatal 5HT disbalance induced long-term cellular and molecular changes in organs regulating 5HT-metabolism, which may have a negative impact on 5HT availability and function in the periphery. Our rat model demonstrates a link between the developmental abnormalities of serotonin homeostasis and 5HT-related changes in adult life and may be suitable for exploring the neurobiological substrates of vulnerability to behavioral and metabolic disorders, as well as for modeling the adverse effects of the prenatal exposure to 5HT enhancers in the human population.

A Stereological Approach to Quantify Immunohistochemical Staining in Zebrafish Larvae.

Immunohistochemistry (IHC) is a powerful research tool to localize specific antigens in whole-mount or tissue sections of embryos with labeled antibodies based on antigen-antibody interactions. Stereological methods are nowadays an essential tool to quantify cells or other types of structures in an unbiased and reproducible manner. In this chapter, a general protocol for a stereological estimation of the relative volume density of each structural component (Vv), which can be applied to any organ/structural component, will be described.

Initial assessment of “Gissell's stain”: A novel histopathological method for the identification of Helicobacter pylori / Valoración inicial de la tinción de Gissell, un nuevo método histopatológico en la identificación del Helicobacter pylori

Introduction and Objectives: The histopathological identification of Helicobacter pylori using the routine method (haematoxylin–eosin) is not only very difficult but also has low sensitivity. Giemsa staining is often used in addition, but different protocols do not produce homogeneous results. Furthermore, the Gold Standard recommended by the European Helicobacter Pylori Study Group has been applied in very few studies, thus resulting in uncertain outcomes. Therefore, a new staining method is required to overcome these limitations. The aim of this study was to evaluate the diagnostic capacity and inter-observer agreement of “Gissell's stain”. Material and Methods: A cross-sectional study evaluated 99 gastric paraffin blocks from a private laboratory. Three sections were prepared from each block, and haematoxylin–eosin (HE), Giemsa and “Gissell's stain” methods were applied. The kappa statistics, sensitivity, specificity, and predictive values were calculated. Results: “Gissell's stain” obtained the highest inter-observer agreement (kappa=0.87) compared to the other two methods (HE, kappa=0.51; Giemsa, kappa=0.83). It also obtained the best sensitivity and negative predictive value (97.1% and 98.3%, respectively) compared with the other two methods (HE: 68.6% and 85.1%, respectively; Giemsa: 88.6% and 93.9%, respectively). Conclusions: Given its unique characteristics (fast, cheap, accessible, and easy to use), in addition to its statistical reliability, “Gissell's stain” has great potential for routine use in the identification of H. pylori.(AU)

Introducción y objetivos: La identificación histopatológica del Helicobacter pylori, utilizando hematoxilina-eosina (HE) como método de rutina es frecuentemente dificultosa y con una baja sensibilidad. El método de Giemsa es usado a veces de modo adicional, pero diferentes protocolos no producen resultados homogéneos. Además, el método recomendado por el grupo europeo de estudio de Helicobacter pylori ha sido empleado en pocos estudios, resultando en datos discrepantes. Por tanto, un método alternativo eficaz podría superar estas limitaciones. El presente estudio valora la agudeza diagnostica y el acuerdo interobservador en la aplicación del método de Gissell. Material y métodos: Un estudio interobservador de 99 biopsias gástricas provenientes de la practica privada. De cada caso se prepararon tres muestras teñidas correspondientemente con: HE, Giemsa y Gissell. Se procedió mediante estadística kappa a valorar la sensibilidad, especificidad y valores predictivos. Resultados: La técnica de Gissell obtuvo el mayor acuerdo interobservador con un kappa con 0,87, comparado con la HE (kappa = 0,51) y Giemsa (kappa = 0,83). Además, obtuvo la mayor sensibilidad y valor predictivo negativo comparado con los otros dos métodos. Conclusiones: Dadas sus características prácticas, tales como rapidez, economía, accesibilidad y facilidad de uso, además de su fiabilidad estadística, el método de Gissell posee un gran potencial para la identificación rutinaria del Helicobacter pylori.(AU)

Initial assessment of "Gissell's stain": A novel histopathological method for the identification of Helicobacter pylori.

INTRODUCTION AND OBJECTIVES: The histopathological identification of Helicobacter pylori using the routine method (haematoxylin-eosin) is not only very difficult but also has low sensitivity. Giemsa staining is often used in addition, but different protocols do not produce homogeneous results. Furthermore, the Gold Standard recommended by the European Helicobacter Pylori Study Group has been applied in very few studies, thus resulting in uncertain outcomes. Therefore, a new staining method is required to overcome these limitations. The aim of this study was to evaluate the diagnostic capacity and inter-observer agreement of "Gissell's stain". MATERIAL AND METHODS: A cross-sectional study evaluated 99 gastric paraffin blocks from a private laboratory. Three sections were prepared from each block, and haematoxylin-eosin (HE), Giemsa and "Gissell's stain" methods were applied. The kappa statistics, sensitivity, specificity, and predictive values were calculated. RESULTS: "Gissell's stain" obtained the highest inter-observer agreement (kappa=0.87) compared to the other two methods (HE, kappa=0.51; Giemsa, kappa=0.83). It also obtained the best sensitivity and negative predictive value (97.1% and 98.3%, respectively) compared with the other two methods (HE: 68.6% and 85.1%, respectively; Giemsa: 88.6% and 93.9%, respectively). CONCLUSIONS: Given its unique characteristics (fast, cheap, accessible, and easy to use), in addition to its statistical reliability, "Gissell's stain" has great potential for routine use in the identification of H. pylori.

Histological Techniques for the Study of the Dentogingival Junction: A Scoping Review Using the Anatomical Quality Assurance Checklist (AQUA) / Técnicas Histológicas para el Estudio de la Unión Dentogingival: Una Revisión de Alcance Utilizando el Anatomical Quality Assurance Checklist (AQUA)

SUMMARY: The dentogingival junction (DGJ) is an adaptation of the oral mucosa composed of epithelial and connective tissues intimately related with the mineralised tissues of the tooth. The histological evidence available is mainly based on studies in animals, separate evaluations of hard and soft tissues, and studies using conventional histological techniques that eliminate the enamel from preparations. The aim of this study was to carry out a review of the existing evidence on histological techniques available for study of the tooth and periodontium in conjunction in humans. A scoping review was carried out of the available literature referring to study of the tooth and the periodontium in conjunction in humans, in the Web of Science (WoS), EMBASE, Scopus and SciELO databases, using the terms "Histological Techniques"[Mesh]) and "Epithelial Attachment"[Mesh]. One hundred and fifty-nine articles were found, of which 54 were selected for full- text reading. Ten were finally included in the qualitative synthesis, and we applied the Anatomical Quality Assurance (AQUA) checklist for analysis the methodological quality of the selected articles. The results showed that the only articles with a low risk of bias in all five domains according to the AQUA criteria corresponded to Silva et al. (2011) and Agustín-Panadero et al. (2020). Finally, we conclude that the quality of the histological sections to observe tissues that simultaneously contain the tooth and the periodontium, is conditioned by the selected technique and by the care required in certain specific tasks during the histological processing of the samples.

La unión dentogingival (DGJ) es una adaptación de la mucosa oral compuesta por tejidos epitelial y conectivo íntimamente relacionados con los tejidos mineralizados del diente. La evidencia histológica disponible se basa principalmente en estudios en animales, evaluaciones separadas de tejidos duros y blandos y estudios utilizando técnicas histológicas convencionales que eliminan el esmalte de las preparaciones. El objetivo de este estudio fue realizar una revisión de la evidencia existente sobre las técnicas histológicas disponibles para el estudio del diente y el periodonto en conjunto en humanos. Se realizó un scoping review de la literatura disponible referente al estudio del diente y el periodonto en conjunto en humanos, en las bases de datos Web of Science (WoS), EMBASE, Scopus y SciELO, utilizando los términos "Histological Techniques"[Mesh]) y "Epithelial Attachment"[Mesh]. Se encontraron 159 artículos, de los cuales 54 fueron seleccionados para lectura de texto completo. Diez fueron finalmente incluidos en la síntesis cualitativa, y se aplicó la lista de verificación Anatómica Quality Assurance (AQUA) para el análisis de la calidad metodológica de los artículos seleccionados. Los resultados mostraron que los únicos artículos con bajo riesgo de sesgo en los cinco dominios según los criterios AQUA correspondían a Silva et al. (2011) y Agustín-Panadero et al. (2020). Finalmente, concluimos que la calidad de los cortes histológicos para observar los tejidos que contienen simultáneamente el diente y el periodonto, está condicionada por la técnica seleccionada y por el cuidado requerido en ciertas tareas específicas durante el procesamiento histológico de las muestras.

Temporal changes of periodontal tissue pathology in a periodontitis animal model.

PURPOSE: This study aimed to characterize the early stages of periodontal disease and determine the optimal period for its evaluation in a mouse model. The association between the duration of ligation and its effect on the dentogingival area in mice was evaluated using micro-computed tomography (CT) and histological analysis. METHODS: Ninety mice were allocated to an untreated control group or a ligation group in which periodontitis was induced by a 6-0 silk ligation around the left second maxillary molar. Mice were sacrificed at 1, 2, 3, 4, 5, 8, 11, and 14 days after ligature placement. Alveolar bone destruction was evaluated using micro-CT. Histological analysis was performed to assess the immune-inflammatory processes in the periodontal tissue. RESULTS: No significant difference in alveolar bone loss was found compared to the control group until day 3 after ligature placement, and a gradual increase in alveolar bone loss was observed from 4 to 8 days following ligature placement. No significant between-group differences were observed after 8 days. The histological analysis demonstrated that the inflammatory response was evident from day 4. CONCLUSIONS: Our findings in a mouse model provide experimental evidence that ligature-induced periodontitis models offer a consistent progression of disease with marginal attachment down-growth, inflammatory infiltration, and alveolar bone loss.

Utilidad de la coloración de Giemsa para diagnosticar Helicobacter pylori en pacientes con lesiones preneoplásicas / The usefulness of Giemsa staining to diagnose Helicobacter pylori in patients with preneoplastic lesions

Resumen Introducción: Helicobacter pylori es una bacteria asociada con enfermedades gastroduodenales inflamatorias y neoplásicas. La histopatología es uno de los métodos diagnósticos empleados para su detección, la cual tiene sensibilidad del 90% al 95% cuando hay alta densidad de H. pylori; sin embargo, la bacteria puede pasar desapercibida en infecciones de baja densidad porque la coloración de rutina de hematoxilina y eosina (H&E) no es una tinción específica para su detección y presenta variabilidad interobservador. El objetivo de este estudio fue determinar la utilidad de la tinción complementaria de Giemsa para el diagnóstico de H. pylori en lesiones preneoplásicas en las que la bacteria se encuentra en baja densidad. Materiales y métodos: se realizó un estudio descriptivo retrospectivo y prospectivo que incluyó a 65 pacientes con diagnóstico de lesiones preneoplásicas. Las biopsias gástricas se tiñeron con H&E y Giemsa, y fueron evaluadas por dos patólogos. Resultados: la coloración de Giemsa diagnosticó 20,3% de casos más de infección por H. pylori que la H&E, la mayoría de ellos con baja densidad de la bacteria. No hubo diferencias estadísticamente significativas en el diagnóstico de H. pylori de acuerdo con el tipo de muestra. Conclusión: este estudio encontró que la tinción de Giemsa mejora el diagnóstico histopatológico de H. pylori en pacientes con lesiones preneoplásicas.

Abstract Introduction: Helicobacter pylori is a bacterium associated with inflammatory and neoplastic gastroduodenal diseases. Histopathology is one of the diagnostic methods used for its detection, which has a sensitivity of 90% to 95% when there is a high density of H. pylori; however, the bacterium may be missed in low-density infections because routine hematoxylin and eosin (H&E) staining is not specific for its detection and has interobserver variability. This study aimed to determine the usefulness of complementary Giemsa staining for diagnosing H. pylori in preneoplastic lesions where the bacterium was found in low density. Materials and methods: A retrospective/prospective descriptive study was carried out that included 65 patients diagnosed with preneoplastic lesions. Gastric biopsies were stained with H&E and Giemsa and evaluated by two pathologists. Results: Giemsa staining analyzed 20.3% more cases of H. pylori than H&E, most with a low density of the bacteria. There were no statistically significant differences in the diagnosis of H. pylori according to the sample type. Conclusion: This study found that Giemsa staining improves the histopathological diagnosis of H. pylori in patients with preneoplastic lesions.

Morphological and functional relationship between the orbital gland and olfaction in Upupa epops (hoopoe) and Bubulcus ibis (cattle egret).

This study used both anatomical and histological techniques to investigate the orbital gland's topographic relationship with the surrounding system, using the hoopoe and cattle egret as biological models. Hoopoe has a spindle-shaped lacrimal gland that is suspended on the lateral edge of the frontal bone, whereas cattle egret has a tiny lacrimal gland that is embedded posteriorly within the periorbital fascia. The hoopoe's lacrimal gland has a single duct that runs parallel to the nasolacrimal duct and opens into the posterior nostril hole. In the cattle egret, the tubule-alveolar secretory components comprise neutral and acid glycosaminoglycan. In addition, the Harderian gland is found in both these species, but their draining ducts differ; the Harderian gland of the hoopoe opens into the anterodorsal to the conjunctival fornix, whereas the Harderian gland of the egret opens anteriorly. In both hoopoe and egret, the secretions of Harderian gland include neutral and acid glycosaminoglycan. The Harderian gland is categorized as type II in hoopoe and type I in cattle egrets. The present results concluded that both orbital glands of two bird species studied play an essential role in eye health, where cleaning and lubrication of the cornea surface. Furthermore, the lacrimal gland's location and secretory features may strengthen the olfactory sensitivity of hoopoe, which relies heavily on scent to locate their food, whereas egret relies heavily on visual cues.

Detection of antigens in paraffin sections of worms and snails infected by Angiostrongylus cantonensis by immunofluorescence reaction / Detecção de antígenos em cortes parafinados de vermes e caramujos infectados por Angiostrongylus cantonensis pela reação de imunofluorescência

The Indirect Immunofluorescence Assay (IFA) was used to identify stage-specific antigenic structures in paraffin sections of female larvae and worms and snails tissues, infected with third stage larvae of Angiostrongylus cantonensis. Sera from eosinophilic meningitis cases were used to assess reactivity. Non-reactive sera from patients with other parasitic diseases and from individuals without other etiologies were used as controls for cross-reactivity. Larvae and worms showed high reactivity to IgG antibodies. IgM antibodies reacted with low intensity only to larvae. Fluorescent reactions were observed in the cuticles and internal structures on worms sections, with a marked reaction in the uterus content. In the snail tissues, the larvae were found exclusively inside the granulomas, with fluorescent markings in the cuticles of the larvae and inside the granulomatous tissues. This fluorescent pattern suggests the presence of excretory/secretory antigens distributed throughout the granulomas. Expressive cross-reactivity occurred in sera from patients with other parasitic diseases, especially strongyloidiasis. The use of IFA applied to paraffin sections to identify structures with antigenic potential and the study of new serological markers, can contribute to the improvement of the laboratory diagnosis of eosinophilic meningitis. (AU)

A Reação de Imunofluorescência Indireta (RIFI) foi utilizada para localizar antígenos em estruturas estágio-específicas em cortes parafinados de vermes fêmeas e em tecidos de caramujos do Gênero Biomphalaria infectados com larvas de terceiro estágio de Angiostrongylus cantonensis. Soros de casos confirmados de meningite eosinfílica foram usados para avaliação da reatividade. Soros não reagentes de casos suspeitos; de pacientes com outras parasitoses e de indivíduos sem outras etiologias foram utilizados como controle de reatividade cruzada. Anticorpos da classe IgG foram reativos para antígenos presentes nos dois estágios e, anticorpos IgM somente para o estágio larvário. Nos cortes de vermes, as marcações fluorescentes foram assinaladas nas cutículas e estruturas internas, com acentuada marcação para os conteúdos uterinos. Nos tecidos dos caramujos as larvas foram encontradas exclusivamente no interior dos granulomas, com marcações fluorescentes nas cutículas das larvas e no interior dos tecidos granulomatosos. O padrão de fluorescência no granuloma sugere a marcação de antígenos excretores/secretores. Reatividade cruzada mais expressiva ocorreu com anticorpos presentes em soros de pacientes com outras parasitoses, com destaque para estrongiloidíase. A RIFI em cortes parafinados abre novas perspectivas para identificação de antígenos e de marcadores sorológicos, que possam ser aplicados no aprimoramento do diagnóstico laboratorial da meningite eosinofílica. (AU)

Histological Changes of Cervical Disc Tissue in Patients with Degenerative Ossification.

OBJECTIVE: To explore the histological feature of the cervical disc degeneration in patients with degenerative ossification (DO) and its potential mechanisms. METHODS: A total of 96 surgical segments, from cervical disc degenerative disease patients with surgical treatment, were divided into ossification group (group O, n=46) and non-ossification group (group NO, n=50) based on preoperative radiological exams. Samples of disc tissues and osteophytes were harvested during the decompression operation. The hematoxylin-eosin staining, Masson trichrome staining and Safranin O-fast green staining were used to compare the histological differences between the two groups. And the distribution and content of transforming growth factor (TGF)-ß1, p-Smad2 and p-Smad3 between the two groups were compared by a semi-quantitative immunohistochemistry (IHC) method. RESULTS: For all the disc tissues, the content of disc cells and collagen fibers decreased gradually from the outer annulus fibrosus (OAF) to the central nucleus pulposus (NP). Compared with group NO, the number of disc cells in group O increased significantly. But for proteoglycan in the inner annulus fibrosus (IAF) and NP, the content in group O decreased significantly. IHC analysis showed that TGF-ß1, p-Smad2, and p-Smad3 were detected in all tissues. For group O, the content of TGF-ß1 in the OAF and NP was significantly higher than that in group NO. For p-Smad2 in IAF and p-Smad3 in OAF, the content in group O were significantly higher than group NO. CONCLUSION: Histologically, cervical disc degeneration in patients with DO is more severe than that without DO. Local higher content of TGF-ß1, p-Smad2, and p-Smad3 are involved in the disc degeneration with DO. Further studies with multi-approach analyses are needed to better understand the role of TGF-ß/Smads signaling pathway in the disc degeneration with DO.

Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

Videoteca para el desarrollo de habilidades prácticas sobre técnicas histológicas básicas / Video Library for the Development of Practical Skills on Basic Histological Techniques

Se desarrolló una investigación de innovación tecnológica con el objetivo de contribuir, a través de videos y enlaces a páginas con elementos teóricos, al desarrollo de habilidades prácticas sobre técnicas histológicas básicas que incluye el procesamiento de los tejidos para ser observados en el microscopio óptico; así como la tinción de éstos con Hematoxilina y eosina. Para su diseño se empleó una plantilla web (boostrap, html5, CCS). En cuanto a los aspectos metodológicos: se tuvo en cuenta el programa de formación de residentes en la especialidad de Histología y de pregrado de la asignatura de Células, tejidos y tegumentario, propuesto por la Universidad de Ciencias Médicas de La Habana (UCM H). Se validó el producto a través del criterio de expertos y de usuarios; obteniéndose un elevado nivel de aceptación; permitiendo el desarrollo de habilidades prácticas, de forma amena y científica en los estudiantes y residentes de la especialidad de Histología(AU)

A technological innovation research was developed with the aim of contributing, through videos and links to pages with theoretical elements, to the development of practical skills on basic histological techniques that includes the processing of tissues to be observed in the optical microscope; as well as staining of these with Hematoxylin and eosin. For its design a web template (boostrap, html5, CCS) was used. The methodological aspects taking into account were: the training program for residents in the specialty of Histology and undergraduate program of the Cells, Tissues and Integumentary subject, proposed by the UCM H. The product was validated through the criteria of experts and users; obtaining a high level of acceptance; allowing the development of practical skills, in an entertaining and scientific way in students and residents of the specialty of Histology(AU)

Basic Quality Controls Used in Skin Tissue Engineering.

Reconstruction of skin defects is often a challenging effort due to the currently limited reconstructive options. In this sense, tissue engineering has emerged as a possible alternative to replace or repair diseased or damaged tissues from the patient's own cells. A substantial number of tissue-engineered skin substitutes (TESSs) have been conceived and evaluated in vitro and in vivo showing promising results in the preclinical stage. However, only a few constructs have been used in the clinic. The lack of standardization in evaluation methods employed may in part be responsible for this discrepancy. This review covers the most well-known and up-to-date methods for evaluating the optimization of new TESSs and orientative guidelines for the evaluation of TESSs are proposed.

Histology of human teeth: Standard and specific staining methods revisited.

OBJECTIVE: Histological techniques have long been an integral part of dental research. Especially the processing of complex tissues poses specific challenges, however, literature offers only few technical references. Objectives of this study were therefore to optimize histological staining methods and compile detailed protocols for preparation and staining of dental tissues. METHODS: Human teeth were collected and fixed with 4 % formaldehyde solution after extraction. Subsequently, teeth were decalcified in 17 % EDTA or Morse's solution over a period of 28 days. The extent of decalcification was determined by weight loss and radiography. After sectioning, histological staining methods were optimized for their use on teeth. These included hematoxylin-eosin, Masson trichrome, Masson-Goldner trichrome and May-Gruenwald-Giemsa staining. Nerve fibres were visualized by luxol fast blue staining and Bodian silver staining. In addition, specific methods like TRAP, modified Brown and Brenn as well as picrosirius red staining with light polarization or fluorescence were applied and optimized. RESULTS: Preparation of an artificial access to the pulp chamber was essential to ensure prompt penetration of the chemicals. Decalcification with Morse's solution took at least two weeks but was more efficient than 17 % ETDA, where thorough demineralization was achieved only after three weeks. The staining methods exhibited differences not only regarding their ability to display specific structures of interest, but also in terms of reproducibility. CONCLUSION: High-quality histology of teeth can only be achieved after optimal tissue preparation and accurate staining. A complementary use of staining techniques is necessary to answer specific research questions.

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Bone regeneration and graft material resorption in extraction sockets grafted with bioactive silica-calcium phosphate composite (SCPC) versus non-grafted sockets: clinical, radiographic, and histological findings.

PURPOSE: The purpose of the present study was to evaluate the effect of silica-calcium phosphate composite (SCPC) granules on bone regeneration in extraction sockets. METHODS: Ten patients were selected for a split-model study. In each patient, bone healing in SCPC-grafted and control ungrafted sockets was analyzed through clinical, radiographic, histomorphometric, and immunohistochemical assessments 6 months postoperatively. RESULTS: A radiographic assessment using cone-beam computed tomography showed minimal ridge dimension changes in SCPC-grafted sockets, with 0.39 mm and 1.79 mm decreases in height and width, respectively. Core bone biopsy samples were obtained 6 months post-extraction during implant placement and analyzed. The average percent areas occupied by mature bone, woven bone, and remnant particles in the SCPC-grafted sockets were 41.3%±12%, 20.1%±9.5%, and 5.3%±4.4%, respectively. The percent areas of mature bone and woven bone formed in the control ungrafted sockets at the same time point were 31%±14% and 24.1%±9.4%, respectively. Histochemical and immunohistochemical analyses showed dense mineralized bundles of type I collagen with high osteopontin expression intensity in the grafted sockets. The newly formed bone was well vascularized, with numerous active osteoblasts, Haversian systems, and osteocytes indicating maturation. In contrast, the new bone in the control ungrafted sockets was immature, rich in type III collagen, and had a low osteocyte density. CONCLUSIONS: The resorption of SCPC granules in 6 months was coordinated with better new bone formation than was observed in untreated sockets. SCPC is a resorbable bone graft material that enhances bone formation and maturation through its stimulatory effect on bone cell function. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03897010.

Magnetic Resonance Imaging Investigation of Cervical-Spine Meniscoid Composition: A Validation Study.

OBJECTIVE: The composition of cervical-spine meniscoids may have clinical significance in neck-pain conditions, but the accuracy of assessment of meniscoid composition in vivo using magnetic resonance imaging has not been established. The aim of this study was to compare cervical-spine meniscoid composition by magnetic resonance imaging with histologic composition. METHODS: Four embalmed cadaveric cervical spines (mean [standard deviation] age, 79.5 [3.7] years; 1 female, 3 male) underwent magnetic resonance imaging, allowing radiologic classification of lateral atlantoaxial- and zygapophyseal-joint (C2-3 to C6-7) meniscoids as either mostly fatty, mixed tissue, or mostly connective tissue. Subsequently, each joint was dissected and disarticulated to allow excision of meniscoids for histologic processing. Each meniscoid was sectioned sagittally, stained with hematoxylin and eosin, examined using light microscopy, and classified as adipose, fibroadipose, or fibrous in composition. Data were analyzed using the kappa statistic with linear weighting. RESULTS: From dissection, 62 meniscoids were identified, excised, and processed; 46 of these 62 were visualized with magnetic resonance imaging. For single-rater identifying structures, agreement between assessment of meniscoid composition by magnetic resonance imaging and by microscopy was fair (κ = 0.24; 95% confidence interval, 0.02-0.46; P = .02). CONCLUSION: Findings suggest that the accuracy of this method of magnetic resonance imaging assessment of cervical-spine meniscoid composition may be limited. This should be considered when planning or interpreting research investigating meniscoid composition using magnetic resonance imaging.

Novel method of determining the periodontal regenerative capacity of T­PRF and L­PRF: An immunohistochemical study.

BACKGROUND: Platelet-rich fibrin (PRF) plays an important role in the regeneration of the lost periodontal tissues. Immunohistochemistry (IHC) is the most sensitive staining technique for the identification and localization of specific cells. There are few studies in the available literature which use IHC to compare PRF prepared from titanium and silica glass tubes. OBJECTIVES: The aim of the study was to use IHC to evaluate and compare cells present in the PRF membrane prepared from titanium and silica glass tubes. MATERIAL AND METHODS: Blood was drawn from 10 healthy volunteers and PRF was prepared from titanium and silica glass tubes. Immunohistochemical staining for the localization, distribution and pattern of cells present in PRF with the CD 3, CD 15, CD 20, CD 34, CD 61, and CD 163 antibodies was carried out. A statistical analysis including the χ2 test, independent t-test and unpaired t-test was performed to determine significant differences. RESULTS: There were significantly more T cells, B­lymphocytes and platelets, with a strongly positive staining in terms of the cell distribution and the labeling index in the T-PRF group in comparison with the L-PRF group. However, in terms of localization, a stronger positive staining was obtained with platelets in the T-PRF group and stem cells in the L-PRF group. In terms of the cell pattern, a significantly stronger positive staining was obtained by neutrophils in the L-PRF group and B­lymphocytes in the T­PRF group. CONCLUSIONS: Titanium PRF has the edge over PRF prepared from silica glass tubes, and emerged as a better alternative for use in the field of periodontal regeneration.

Pathological validation and prognostic potential of quantitative MRI in the characterization of pancreas cancer: preliminary experience.

Patient stratification based on biological variation in pancreatic ductal adenocarcinoma (PDAC) subtypes could help to improve clinical outcome. However, noninvasive assessment of the entire tumor microenvironment remains challenging. In this study, we investigate the biological basis of dynamic contrast-enhanced (DCE), intravoxel incoherent motion (IVIM), and R2*-derived magnetic resonance imaging (MRI) parameters for the noninvasive characterization of the PDAC tumor microenvironment and evaluate their prognostic potential in PDAC patients. Patients diagnosed with treatment-naïve resectable PDAC underwent MRI. After resection, a whole-mount tumor slice was analyzed for collagen fraction, vessel density, and hypoxia and matched to the MRI parameter maps. MRI parameters were correlated to immunohistochemistry-derived tissue characteristics and evaluated for prognostic potential. Thirty patients were included of whom 21 underwent resection with whole-mount histology available in 15 patients. DCE Ktrans and ve , ADC, and IVIM D correlated with collagen fraction. DCE kep and IVIM f correlated with vessel density and R2* with tissue hypoxia. Based on MRI, two main PDAC phenotypes could be distinguished; a stroma-high phenotype demonstrating high vessel density and high collagen fraction and a stroma-low phenotype demonstrating low vessel density and low collagen fraction. Patients with the stroma-high phenotype (high kep and high IVIM D, n = 8) showed longer overall survival (not reached vs. 14 months, P = 0.001, HR = 9.1, P = 0.004) and disease-free survival (not reached vs. 2 months, P < 0.001, HR 9.3, P = 0.003) compared to the other patients (n = 22). Median follow-up was 41 (95% CI: 36-46) months. MRI was able to accurately characterize tumor collagen fraction, vessel density, and hypoxia in PDAC. Based on imaging parameters, a subgroup of patients with significantly better prognosis could be identified. These first results indicate that stratification-based MRI-derived biomarkers could help to tailor treatment and improve clinical outcome and warrant further research.

Modified application of Kawamoto's film method for super-resolution imaging of megakaryocytes in undecalcified bone marrow.

BACKGROUND: Super-resolution microscopy has enabled high-resolution imaging of the actin cytoskeleton in megakaryocytes and platelets. These technologies have extended our knowledge of thrombopoiesis and platelet spreading using megakaryocytes and platelets cultured in vitro on matrix proteins. However, for better understanding of megakaryocytopoiesis and platelet production, high-resolution imaging of cells in an in vivo bone marrow microenvironment is required. Development of Kawamoto's film method greatly advanced the techniques of thin cryosectioning of hard tissues such as undecalcified bones. One obstacle that remains is the spherical aberration that occurs due to the difference in the refractive index for the light path, limiting the usage of Kawamoto's film method to lower magnification observation. OBJECTIVES: To overcome the weakness of the conventional Kawamoto's film method for higher magnification observation of undecalcified bone marrow. METHODS: We have modified the original method with a very simple method: flipping the film at the step of mounting the sections on the glass. RESULTS AND CONCLUSIONS: This new method successfully led to the adjustment of the refractive index and enabled super-resolution imaging of megakaryocytes in undecalcified mouse femurs. Our modified method will expand the application of Kawamoto's film method and enable precise analysis of megakaryocytopoiesis and platelet production in the bone marrow microenvironment under pathophysiological conditions.