Wir möchten Dionna Gamble, Allison Larson, und Katia Torres für die Unterstützung bei Modellgeneration und Messungen, Monica Mahre für Manuskript Unterstützung, Gary Williams für die technische Unterstützung, Jerrald Spencer Jr. für die Unterstützung bei den Figuren und den Fairview Imaging Services am anerkennen University of Minnesota. Die Finanzierung wurde vom Institut für Technik in der Medizin (University of Minnesota) und zum Teil von einem Forschungs-Vertrag mit Medtronic Inc. erhielt

Vorgehensweise Tabelle 1 fasst die Materialien während des Verfahrens verwendet wird. Abbildung 1 gibt einen Überblick über den Vorgang. Ein. Beleg-und Scan-Vorbereitung <ol><li> Besorgen Sie sich die isolierten menschlichen Herzen frisch und anschließend Perfusion fixieren sie in 10% gepuffertem in ihre enddiastolischen Zustand Formalin. </li><li> Spülen Sie die Herzen im Wasser gescannt werden am Tag vor dem Scannen, um die Mehrheit der Formalin zu entfernen. </li><li> Bevor der Scanner, die kanülieren Coronarsinus (CS) Vene innerhalb jedes Herz mit einem Ballonkatheter venogram. Erhalten Sie Zugang zum CS entweder durch die obere oder untere Hohlvene unter direkter Sicht oder die Verwendung von videoscopes. </li><li> Einmal im Ort, den Ballon aufzublasen dieser venogram Katheter, um den Katheter in der CS zu verankern. </li><li> Legen Sie jedes Herz in einem verschließbaren Behälter Polymer auf einem Schwamm, der so entworfen wurden, dasskann das Herz in seiner attitudinally korrekten anatomischen Position zu sitzen. </li></ol> 2. Computertomographie <ol><li> Positionieren Sie ein bestimmtes Herz auf dem CT-Scanner Bett, als wenn ein Patient wurde in Rückenlage und mit dem Kopf voran auf den Scanner. </li><li> Verbinden des proximalen Endes des Katheters venogram zu einem Injektor, der zwei Injektionsspritzen enthält: eine für den Kontrast und eine für Kochsalzlösung. </li><li> Automatisches injizieren 40 ml Kontrastmittel in das Herz Venensystem mit 5 ml / sec. </li><li> CT das Herz 8 sec nach der Kontrastmittelinjektion wird eingeleitet. Stellen Sie den CT-Scan zu 512 x 512 Pixel Auflösung mit 0,6 mm Schichtdicke. </li><li> Automatisch injizieren 40 ml Kochsalzlösung in den kardialen Venensystem bei 5 ml / sec zu spülen, den Kontrast. </li><li> Exportieren Sie die CT DICOM-Bilder auf einer externen Festplatte. </li></ol> 3. Wiederaufbau und Messungen <ol><li> Hochladen CT DICOM-Bilder in Mimics Software. </li><li> Generieren Sie eine Maske für die CT Bildgebunges enthält nur die Pixel mit hohem Hounsfield Einheiten nur den Kontrast in dem Herzen zu markieren. </li><li> Entfernen Kontrast, der in die Kammern oder diffus ins Gewebe ausgetreten ist, so dass die Maske enthält nur den Kontrast innerhalb der großen Herzvenen. </li><li> Manuell in Lufteinschlüsse füllen innerhalb einer bestimmten Vene Frame für Frame. </li><li> Generieren Sie ein 3D-Objekt aus der resultierenden Maske. </li><li> Glätten und wickeln Sie dieses Objekt, um grobe Geometrien beseitigen. Video 1 zeigt eine dieser 3D-Modelle im Raum drehen. </li><li> Generieren Mittellinien für jedes 3D-Modell erstellt. </li><li> Mit diesen Mittellinien, messen Sie die Bogenlänge, Verzweigung Winkel, Tortuosität (Bogenlänge / Luftlinie entfernt) und Durchmessern für jeden größeren Schiffes in jedem Herzen. Unsere beschäftigt anatomischen Nomenklatur ist in Abbildung 2 dargestellt. </li></ol>

<ol>
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Keine Interessenskonflikte erklärt.

Unser Labor ist die Entwicklung einer Bibliothek von Perfusion-fixierten Herzen Proben für verschiedene anatomische Studien. Bis heute haben wir mehr als 240 erhaltene Exemplare. Die spezifischen Methoden, die wir verwendet, um diese Proben vorbereitet haben, wurden bisher 3 beschrieben. Die vorliegende Studie beschreibt ein neues Verfahren zur Abbildung des menschlichen Herz-venöse System und für die Entwicklung eines anatomischen Datenbank, die für die Konstruktion von Herz-Geräte innerhalb der Gefäß...

anatomical reconstructions of the human cardiac venous system using contrast-computed tomography of perfusion-fixed specimens

Ein detaillierteres Verständnis der Komplexität und der relativen Variabilität innerhalb des kardialen Human-Venensystem ist entscheidend für die Entwicklung von Herz-Geräten, die den Zugriff auf diese Schiffe erfordern. Zum Beispiel wird kardialen Venenanatomie bekannt, dass einer der wichtigsten Einschränkungen für die reibungslose Lieferung der kardialen Resynchronisationstherapie (CRT) 1 Daher ist die Entwicklung einer Datenbank der anatomischen Parameter für den menschlichen kardialen venösen Systeme können bei der Gestaltung von CRT-Abgabe zu unterstützen Geräte, eine solche Beschränkung zu überwinden. In diesem Forschungsprojekt wurden die anatomischen Parameter aus 3D-Rekonstruktionen des venösen Systems mittels Kontrastmittel-Computertomographie (CT) und Modelling-Software (Materialise, Leuven, Belgien) erhalten. Bogenlänge, Tortuosität, Verzweigung Winkel, Entfernung zum Koronarsinusostium und Gefäßdurchmesser: Die folgenden Parameter wurden für jede Ader beurteilt. CRT ist eine mögliche Behandlung für patiEltern mit elektromechanischer Dyssynchronie. Ungefähr 10-20% der Patienten mit Herzinsuffizienz können von CRT 2 profitieren. Elektromechanische Dyssynchronie bedeutet, dass Teile des Herzmuskels aktivieren und Vertrag früher oder später als die normale Leitungsbahnen des Herzens. In CRT werden dyssynchronous Bereiche des Myokards mit elektrischer Stimulation behandelt. CRT-Stimulation beinhaltet typischerweise Stimulation führt, die den rechten Vorhof (RA), rechten Ventrikel (RV) und linken Ventrikels (LV) zu stimulieren, um mehr synchronisiert Rhythmen erzeugen. Der LV-Elektrode wird in der Regel innerhalb eines kardialen Vene implantiert, mit dem Ziel, sie innerhalb der Stelle der neuesten myokardialen Aktivierung überlagern. Wir glauben, dass die Modelle und die erhaltenen Analysen davon wird die anatomische Ausbildung für Patienten, Studenten, Ärzte und medizinisches Gerät Designern fördern. Die eingesetzten Methoden hier kann auch genutzt werden, um andere anatomische Merkmale des menschlichen Herzens unsere Proben, wie z. B. zu studierendie Koronararterien. Zur weiteren Förderung der erzieherischen Wert dieser Forschung haben wir die venöse Modelle auf unserer Website freien Zugang geteilt: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu / atlas</a> .

Tabelle 2 zeigt die mittlere anatomische Parameter für die wichtigsten Herzvenen für 42 Proben menschlichen Herzens. Alle Herzen Proben enthielt einen hinteren interventrikulären Vene (PIV) und vorderen interventrikulären Vene (AIV). Einige Proben enthielten mehr als einem posterioren Vene des LV (PVLV), posteroanterioren Lateralvene (PLV), links Lateralvene (LLV) und / oder antero-laterale Vene (ALV), während andere Herzen hatten noch eine oder zwei dieser spezifischen Adern vorhanden. <table ...

Watch this Scientific Journal Video about Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens at JoVE.com

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

Das Ziel dieser Forschung ist es, neu zu erstellen und dann auf die Anatomie des menschlichen Herzens venöse System mit 3D-Rekonstruktionen von Kontrastmittel-Computertomographie erzeugt.

Anatomische Rekonstruktionen des menschlichen Cardiac Venensystem mit Kontrast-Computertomographie Perfusion-fixierten Proben

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

anatomical-reconstructions-human-cardiac-venous-system-using-contrast

Research

JoVE Journal

Bioengineering

11.7K Aufrufe.  University of Minnesota. Das Ziel dieser Forschung ist es, neu zu erstellen und dann auf die Anatomie des menschlichen Herzens venöse System mit 3D-Rekonstruktionen von Kontrastmittel-Computertomographie erzeugt.

Serie: Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bakterielle Erkennung und Identifizierung mit elektrochemischen Sensoren

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Forschung

Biotechnik

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Kultivierung von Maus-Herzklappen in der Miniatur-Gewebekultursystem

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Bau von Defined Menschen Engineered Herzgewebe zur Untersuchung Mechanismen der Herzzelltherapie

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

In Vivo quantitative Bewertung der myokardialen Struktur, Funktion, Perfusion und Lebensfähigkeit Mit Herz Micro-Computertomographie

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

Verwendung von außergewöhnlichen optischen Übertragung kardiale Biomarker in humanem Serum quantifizieren

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Effiziente Erzeugung und Bearbeitung von Feeder-freie IPSCs aus menschlichen Zellen der Bauchspeicheldrüse mit dem CRISPR-Cas9-System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Eine Net Schimmel basierende Methode der dreidimensionalen Gerüst-freie Herzgewebe Schöpfung

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Starre Einbettung von fixiert und gefärbt, im großen und ganzen Millimeter-Skala Exemplare für Abschnitt-free 3D Histologie durch Mikro-Computertomographie

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Intrakardialer Side-Firing Light Katheter zur Überwachung des Zellstoffwechsels mit transmuraaler Absorptionsspektroskopie von perfundierten Säugetierherzen

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Verwendung von Mikro-Röntgentomographie mit Phosphotungstic Acid Preparation zur Visualisierung von humanem fibromuskulärem Gewebe

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...