The Isolated Mucosa of the Rat Colon Decellularization, Microscopy and Cell Cultures.

This project presents the most important findings of the studies, which we carried out in our laboratory on the decellularization of the rat isolated colonic mucosa. We have also included some details of the experiences gathered with the muscle layer as well as the whole wall of the colon. The question of the cytocompatibility of this new substrate has been addressed with the application of primary cultures of human cells and well-established cell lines. The possible applications in experimental and medical settings will be discussed.

Commentaries on the publication entitled: "Structure and distribution of an unrecognized interstitium in human tissues" by Benias et al. (2018)

The claim made in this publication of the existence of a hitherto unknown interstitial space is based on studies with sample-based confocal laser endo-microscopy (pCLM). Due to postings on various web portals (New Cellvizio, EurekAlert, Google Scholar,...) the alleged discovery has found great resonance. Nevertheless, there are several critical issues in this publication, the most important being that this is not the discovery of an "unrecognized" interstitium as it has, in fact, been known for a long time

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Albert von Kölliker, Santiago Ramón y Cajal and Camillo Golgi, the main protagonists in the Neuron Theory debate

In the second half of the 19th century Spain was rather isolated from the rest of Europe, although there was remarkable scientific activity. In the midst of this scenario, the figure of Cajal emerged on the scene. During a visit to the laboratory of Luis Simarro in Madrid in 1887, Cajal became acquainted with a paper published by Golgi in 1873 dealing with his famous method. Cajal immediately recognized the value of this method and applied it with much success to the study of the nervous tissue. In the triennium 1887-1889 Cajal's discoveries were so sensational that he decided to attend the meeting of the Anatomische Gesellschaft (Germany Anatomical Society) in Berlin in 1889 in order to present them abroad. The trip proved a great success, and he was able to establish close relations with the president of the society, Alexander von Kölliker, who, in turn, mediated contacts with further renowned scientists such as Retzius, His, Waldeyer, van Gehuchten, etc. Prior to his trip to Berlin, he had already contacted Golgi, but the fact that Cajal's neuronal theory conflicted with Golgi's reticular theory not only prevented a normal relationship between them, but was also -especially on Golgi's part- the source of bitter rivalry between them. Von Kölliker immediately recognized and admired Cajal's stature as a scientist and generously helped him to publicize his ideas throughout the scientific world, and to attain the recognition he deserved. Von Kölliker's relationship with Golgi was of a different nature, and could be described as sincere friendship. Von Kölliker, in fact, proposed both Golgi and Cajal as candidates for the Nobel Prize in 1906, which was subsequently awarded to them jointly. Thanks to Von Kölliker, Cajal's great mentor, the neuronal theory entered the scientific world through the main door and continues to occupy a prevailing position

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Commentary on the paper entitled "Is Anatomy dead?" by Dr. G. Tong

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Specimen preparation for "block face" scanning electron microscopy (BFSEM). An energy-dispersive X-ray spectroscopy study

In this paper we describe a SEM block-face technique in which block faces of large dimensions can be examined in a high-resolution SEM under high vacuum. The results of different tissue contrast methods have been studied and, in addition to osmium, potassium permanganate has been used as a staining medium for the first time in BFSEM. The study also examined the effects of uranyl acetate and phosphotungstic acid. The following organs of adult albino rats were examined: colonic mucosa, spinal ganglion, anterior pituitary gland and exocrine pancreas.Six preparation protocols, referred to here as treatments, were applied and evaluated according to three criteria: 1st the visual quality of the digital images, 2nd the measurements of the signal-noise ratio (SNR) of the digital images with and without beam deceleration (BD), and 3rd the X-ray microanalysis of samples, treated according to the 6 proposed protocols, demonstrating the presence and relative quantity of the elements used to stain the cellular structures, enabling visualisation with the electron microscope. In conclusion, it can be said that treatments with osmium produced better results than those containing potassium permanganate. Treatments with the addition of thiocarbohydrazide (TCH) considerably increased the osmium deposits (ligand effect) and proved highly effective. Finally, it should be noted that the method proposed, called here 2D BFSEM, can be very useful not only in histology but also in histo-pathology, for example in the study of biopsies and - last but not least - in embryology: all these are situations in which it is important to avoid a loss of material due to preparation exigencies

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The isolated colonic mucosa: a new substrate for tissue reconstruction. Isolation, decellularization and characterization by immunohistochemistry and electron microscopy

This study proposes the use of isolated colonic mucosa as a "scaffold" for cell cultures and potentially for tissue reconstruction. The main goal of this study was to obtain complete decellularization of the specimens while preserving the superficial basement membrane (BM) as a place for cell attachment and growth. This decellularization technique uses a chelating agent in combination with mechanical vibration, followed by detergents and DNase. The grade of decellularization achieved is assessed by counting the number of cell nuclei stained with propidium iodide (PI). BM marker proteins such as collagen IV, laminin and perlecan were detected by immunohistochemistry (ICC). Transmission (TEM) and scanning electron microscopy (SEM) were used to examine the BM ultrastructure and surface topography. The results show that the protocol used is suitable for rat colonic mucosa. During the process, material of the lamina lucida (LL) was partly removed from the BM, whereas the lamina densa (LD) seems to have remained unchanged. The BM had become thinner than the control specimens. The nanotopography of the BM surface is characterized by globules of 34-60 nm in diameter. Human fetal fibroblasts were successfully cultured on this substrate confirming that cells can adhere to and grow on this substrate, at least for the particular cell line used. It can be said that the colonic mucosa is an interesting substrate for in vitro stud-ies with cells and presumably also for tissue reconstruction

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Medicine, Surgery and Public Health at the epoch of Gimbernat (1734 - 1816)

The aim of this publication is to recall to memory Don Antonio de Gimbernat i Arbós on the occasion of the second centenary of his death. Gimbernat was a prominent figure in the fields of anatomy, surgery and medicine in Spain during the second half of the 18th century. Born in Cambrils (Tarragona, Spain) in 1734, he studied surgery in Cadiz, thereafter commencing his professional career in Barcelona, where he made his more important anatomical discoveries. His visits to France, England and Holland between 1774 and 1778 constituted a milestone in his life. After returning to Spain, Gimbernat founded the Royal College of San Carlos in Madrid and, with increasing fame, advanced in the Spanish medical hierarchy. In 1793 he published a memorable work on the anatomy of the rural region and the surgery of femoral hernias, which was recognized throughout Europe, being translated into English, German and French. He was also a brilliant surgeon and physician in other areas (liver diseases, ophthalmology, gynecology, urology, etc.), always regarding anatomy as the basis for his work. In the early nineteenth century he held high positions within national medical institutions, promoting inter alia the unification of the study of surgery and medicine; an important step in the modernization of Spanish medical education. The last years of his life were difficult and sad as, for political reasons, Gimbernat no longer received the recognition he deserved after a life so rich in professional successes. Gimbernat died in Madrid at the age of 82 (AU)

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Morphological studies on platelet function using a microfluidic chip. A proof of concept study

Platelets are blood cellular components involved in hemostatic processes and thrombus formation. Activation and inhibition of platelets result in an increase in morphological changes and a significant reduction in adhesion. There are several approaches towards the determination of the functional status of platelets, based on criteria such as cell adhesion, molecular changes at the cell surface, etc. In recent years, microfluidic devices have been introduced to mimic conditions proper to the vascular system, and so emulate thrombus formation in vivo. This study presents a microchip, the Thrombi Chip® , which is partially fitted with fluidic properties. This microchip has various types of micro-channels into which the platelets are inserted and, after drug treatment, the investigation is completed with the examination of the chip under an invert light microscope. For microscopy, cells were labeled with FCDA (human platelets) and Rho6G (mouse platelets). Counts and morphometric measurements of the adhered cells were carried out using digital images. To validate the results obtained with the microchip, the fractions of mice platelets were investigated with flow cytometry as well. Scanning electron microscopy was used to examine the morphological changes related to activation and inhibition in human platelets. The results show that, with this microchip, activation and inhibition of platelets can be detected. Flow cytometry studies largely confirm the microchip results. Certain variability in the results observed in human platelets is considered normal, as donors were randomized. In this respect the mouse platelets were much more uniform. Measurements with the microchip require that the sample be divided into three groups: control, activated and inhibited, resulting in a set of data, which, after respective evaluation, provides activity profiles, giving information on the status and response capacity of a sample. Such profiles could have diagnostic relevance and therefore be useful in a clinical context, for example in the monitoring of the effects of short- and long-term treatment of patients, as well as to test new drugs

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Estudiar medicina en Alemania / Studying medicine in Germany

Este artículo da una visión general de la organización, la estructura y el currículo de estudios de medicina en Alemania. La normativa que regula estos estudios, denominada Approbationsordnung, fue revisada y modificada en 2002, adaptándola a las nuevas reglas creadas por la Unión Europea. El plan de estudios dedica seis años y tres meses al estudio y formación de los estudiantes (dos años de ciencias básicas, tres de ciencias clínicas y un último año dedicado al trabajo clínico en el hospital). Cualquier candidato que solicite una plaza para estudiar en una universidad alemana debe cumplir ciertos requisitos, independientemente de si proviene o no de un país miembro de la Unión Europea. La evaluación del estudio se lleva a cabo a diferentes niveles durante el curso. Esencial aquí es el examen de estado en medicina, que se divide en dos partes: la primera parte trata de las ciencias básicas y la segunda se refiere al período clínico. La nueva Approbationsordnung (AppOÄ) trajo consigo una serie de cambios que todavía están en fase de ejecución. La especialización se lleva a cabo en el período de posgrado de los estudios de medicina. Al final de este período, el médico obtiene un título especialista (Facharzt) otorgado por el Colegio de Médicos. Tradicionalmente, la organización de estos estudios está en manos de los propios alumnos, pero ello está en vías de sufrir una modificación sustancial. Como especialista, el médico está obligado a participar en los programas de educación continuada, con una evaluación periódica de sus rendimientos. Un tema controvertido en la actualidad es el título de Doctor en Medicina y su importancia médica. Muchos programas de investigación están surgiendo dentro de un contexto clínico, lo que puede contribuir a corregir algunas desviaciones del pasado a este respecto. Se han incluido enlaces web y fuentes para obtener información sobre los estudios en las diferentes facultades en Alemania (AU)

This article gives a general overview on the organization, structure and curriculum of medical studies in Germany. The regulations governing these studies, called Approbationsordung, were reviewed and amended in 2002, in adaptation to the new rules created by the European Union. The curriculum dedicates six years and three months to the study and training of students (two years of basic sciences, three years of clinical sciences, and a final year dedicated to clinical work in hospital). Any candidate applying for a course at a German University must meet certain requirements, regardless of whether or not he or she is member of the EU. Assessment of progress is carried out at different levels during the course. Essential here is the state exam in Medicine, which is divided into two parts, the first part dealing with the basic and the second with the clinical period. The new Approbationsordung (AppOÄ) brought a series of changes that are still under implementation. Specialization takes place in the postgraduate period of medical studies. At the end of this period the doctor obtains a specialist (Facharzt) title granted by the College of Physicians. Traditionally the organization of these studies lies in the hands of the trainees themselves; however, this is lined up for substantial modification. As a specialist the doctor is obliged to participate in ongoing education programs, with periodic performance evaluation. A controversial topic at present is the Doctor title in Medicine and its medical relevance. Many research programs are emerging thematically in a clinical context and apparently some past deviations can be corrected. Links and sources to obtain information about studying in different faculties in Germany have been included (AU)

Multi-sensor arrays for online monitoring of cell dynamics in in vitro studies with choroid plexus epithelial cells.

Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events--in this case in choroid plexus functions, however with applicability to other cell types as well.

Application of silicon sensor technologies to tumor tissue in vitro: detection of metabolic correlates of chemosensitivity.

Silicon sensor technologies, developed during the 1990s, allow measurement of extracellular chemical changes related to cell metabolism. Exposition of tumor cells in vitro to anticancer drugs modifies cell metabolism, making it possible to detect on-line with sensor chips patterns of metabolic activity, which depend on drug sensitivity, or drug resistance of the cells. Sensor devices are composed of an incubation chamber with a sensor chip and a fluidic system for medium supply. Basically, two sensor types are available: (1) monosensor systems to detect extracellular acidification; and (2) multisensor arrays for many parameters such as pH, oxygen consumption, and impedance. Two companies have developed such systems: Molecular Devices (USA) and Bionas (Germany). In this chapter, in addition to operation of the sensor devices, we describe techniques for tissue (tumor and non-tumor) preparation. Basically, three procedures are described: (1) tissue dissociation and further cultivation on the sensor chip or on Transwell inserts; (2) preparation of tissue slices (300 microm thick) and attachment to the sensor chip or to inserts, and (3) cultivation of cells in dialysis tubes, a procedure necessary for nonadherent cells and cell suspensions to avoid their washing away. Evaluation of results and selection of controls are also discussed.

Chemosensitivity testing of human tumors using Si-sensor chips.

Chemotherapy is, on its own or in combination with other treatments, a very effective anticancer therapy. Introduced in the middle of the last century, chemotherapy today still faces the problem of determining which specific agent or agents are able to yield the desired clinical therapeutical effect for a particular tumor and patient. Numerous tests in vitro have been developed to detect chemosensitivity and chemoresistance and also for screening new drugs. Three groups of tests can be defined: 1, cell viability tests; 2, measurements of cell metabolism; and 3, clonogenic assays. Test time, tissue preparation, complexity of test performance, and correlation with the clinical progress of the disease are criteria used to judge how successful the tests are. The introduction of Si-sensor chips, which are able to detect metabolic changes in living cells, has opened up new possibilities in this field. Basically two sensor principles or types can be considered: (a) the light-addressed potentiometric sensor (LAPS) and (b) the multisensor array (MSA). Whereas LAPS measures one, MSA registers online many parameters (for instance, impedance, pH, O2, temperature). The aim of this chapter is to review this technology and to present recent applications using cells, tissue slices, and biopsies.