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Laboratories

In Department of Physiology and Cardiothoracic Surgery there are nine main laboratories where different laboratory techniques are performed.

 

1.    Muscular function laboratory

·         In vitro multicellular preparations:

o   Vertical setups (papillary muscles)

o   Horizontal setups (smooth muscle)

·         In vitro multicellular intact organ preparations;

o   Ex vivo isolated perfused heart: Langendorff

 

2.    In vivo studies laboratory I

·         In vivo assessment of cardiac function: hemodynamic studies


3.    In vivo studies laboratory II

·         In vivo assessment of cardiac function: echocardiographic studies

·         Single lane treadmill running with metabolic chamber for gas analysis in rats and mice

·         Exercise training protocols on a 5-lane treadmill apparatus


4.    Isolated myocytes laboratory

·         Functional assessment of permeabilized isolated cardiomyocytes


5.    Molecular biology laboratory I – proteomics

·         Western blotting

·         High-performance liquid chromatography (HPLC)


6.    Molecular biology laboratory II – genomics

·         Extraction of total RNA, DNA and proteins from tissue samples and cells

·         Two step real-time reverse transcription polymerase chain reaction


7.    Histology laboratory

·         Routine stains

·         Specific stains

·         Immunohistochemistry

·         Immunofluorescent

·         Processing samples to transmission electron microscopy (TEM)


8.    Cell culture laboratory

·         Cell cultures (primary and cell lines)

·         Organotypic cultures


9.    Chemistry laboratory

·         Preparation of work solutions

·         Sample processing

·         pH monitoring

·         Chemical reagents and glassware storage




1.      Muscular function laboratory

        Laboratory managers: Prof. Carmen Brás Silva, Prof. Amândio Rocha Sousa, Prof. Inês Falcão Pires, Dr. Rui Cerqueira

·        In vitro multicellular preparations

o   Vertical setups (papillary muscles)

Multicellular cardiac preparations, such as papillary muscles or trabeculae, are traditionally used for the assessment of mechanical properties including inotropic, lusitropic, chronotropic, as well as basic electrophysiological properties. These can be performed under several pharmacological conditions and endothelial modulations. This in vitro multicellular assessment is an experimentally simple approach that overcomes the complexity of the architecturally intact heart, preserving the extracellular matrix and the cell-cell and cell matrix interactions.

This technique allows evaluation of both isotonic and isometric parameters. Passive tension can be also assessed in both conditions. It can also establish force-length relationship, force-frequency relationship in several physiopathology conditions.






o   Horizontal setups (smooth muscle)

 

The 720MO system from DMT (Dannish Myo Technology) is a robust, easy-to-use instrument for the in vitro study of larger blood vessels (>500 μm) and other tissue strips mounted with delicate suture wires. Following mounting and equilibration, passive length-tension relationships for the tissue are determined. The 4-chamber parallel arrangement of this system allows a high experimental throughput and homogenous working conditions. Typical experiments include testing vascular reactivity and endothelial function of chronically diseased animals. Human vascular tissue obtained during open heart surgery, can also be tested using this system.



·        In vitro intact organ studies

The ex vivo isolated perfused heart includes two different preparations: the Langendorff preparation and the “working heart” system. A perfusate solution (Krebs-Ringer-Henseleit solution) that contains nutrients and ions to preserve both myocardium and endothelium for several hours, is delivered into the coronary vessels through a cannula inserted in the ascending aorta, either at constant pressure or constant flow rate.

The Langendorff heart technique allows for the examination of cardiac contractile strength (inotropic effects), heart rate (chronotropic effects) and vascular effects without neuronal and hormonal influences. Pressure in the perfused line is typically monitored using a pressure transducer and bridge amplifier connected to a PowerLab data acquisition system. To monitor ventricular developed pressure, a saline-filled balloon-tipped catheter is inserted into the left ventricle and connected to a second pressure transducer and bridge amplifier. For working heart preparations, preload and afterload can be independently modulated by adjusting the height of different bubble-trap reservoirs and chamber pressure is measured using a pressure-volume catheter inserted through the apex.



2.      In vivo studies laboratory I

        Laboratory manager: Prof. André Lourenço

 

·        In vivo assessment of cardiac function: hemodynamic studies

We use high fidelity micromanometers and conductance technology to evaluate cardiac functional parameters. With state of the art equipment and highly trained researchers we are able to very accurately assess cardiac function in several animal models, both in rats and in mice, using both the closed and open chest approach.

Besides hemodynamic studies, this laboratory contains the necessary equipment to perform small animal surgery, including but not limited to, large vessel constriction (ascending/transverse/abdominal aortic banding and pulmonary artery banding), myocardial infarction models, and other surgical models of cardiovascular disease, with a very high success rate and low mortality.



 

3.      In vivo studies laboratory II

        Laboratory manager:  Prof. André Lourenço

·        In vivo assessment of cardiac function: echocardiographic studies

Using an Acuson Sequoia 512 echocardiography machine, coupled to volatile anesthetics and assisted ventilation, we can with ease and precision perform a thorough analysis of cardiac and vascular structure and function. Volatile anesthesia provides a quick and eventless recovery with no injury to the animal, allowing a serial evaluation through time following disease and or treatment progression.



·        Single lane treadmill running with metabolic chamber for gas analysis for rats and mouse

Treadmill running has been used extensively over the past decades to study behavioural, physiological and pathological responses to both acute and chronic exercise training. With treadmill exercise we are able calculate the total amount of external work done by the rat/mouse. While using a metabolic chamber built in the treadmill we are able to calculate the animal’s maximal aerobic power (VO2max) through determining its metabolic rate [oxygen uptake (VO2) and carbon dioxide production (CO2)] under both submaximal and maximal exercise workload. VO2max is typically defined as the point in which VO2 does not increase, even though further intensifications in external workload are imposed on the animal.


·        Exercise training protocols on a 5-lane treadmill apparatus

The pleiotropic actions of regular exercise training are well documented and associated with decreased risk of developing several chronic diseases including cardiovascular diseases. Our exercise procedures are monitored in time and performed using a 5-lane treadmill apparatus with adjustable speed (up to 150 cm/s) and slope (from -25 to +25 degrees). This system allow us to perform different protocols of animal cardiovascular exercise from chronic moderate to high intensity interval training. Still with this system, the researcher is able to evaluate the animals exercise performance by assessing endurance capacity. In this test, animal runs to the point of fatigue under carefully controlled experimental conditions.



4.      Isolated myocytes laboratory

        Laboratory manager:  Prof. Inês Falcão Pires

·        Functional assessment of permeabilized isolated cardiomyocytes

The cardiomyocyte force measurement system consists of an electromagnetic motor and a force transducer. The motor is used to adjust cardiomyocyte length, while the force transducer is to measure isometric cardiomyocyte contraction. A permeabilized ("skinned") cardiomyocyte is mounted between needles attached to the motor and the force transducer. A specially developed optical system is used to determine cardiomyocyte morphology on both the horizontal and the vertical axes. The experimental protocol usually consists of a series of force measurements upon Ca2+ stimuli (using Ca2+ buffer solutions), the determination of actin-myosin cross-bridge kinetics and the measurement of the passive tension of the mounted cardiomyocytes at pre-defined sarcomere lengths. It has several advantages as (a) to require very small amounts of cardiac tissue, (b) frozen tissue samples can be used as a source of cardiomyocytes, (c) tissue samples can be transported and stored at -20 °C, (c) a wide range of species can be tested, and (d) physiological (e.g. stretch) and pathophysiological (e.g. ischemia) conditions can easily be reproduced in vitro.

Several cardiomyocyte contractile parameters can be determined, such as (a) maximal Ca-activated (active) force, (b) Ca-independent (passive) force, (c) Ca-sensitivity of isometric force, (d) cooperativity, (e) actin-myosin turnover rate, (f) [Ca2+] dependencies of force.



5.    Molecular biology laboratory I – proteomics

         Laboratory managers: Dr. Pedro Mendes Ferreira, Dr. Francisco Nóvoa

·        Western blotting

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (a) separation by size, (b) transfer to a solid support, and (c) marking target protein using a proper primary and secondary antibodies to visualize.



·        High-performance liquid chromatography (HPLC)

High-pressure liquid chromatography is a technique in analytic chemistry used to separate the components in a mixture and to identify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.





6.      Molecular biology laboratory II – genomics

        Laboratory managers: Prof. Roberto Roncon-Albuquerque, Dr. Francisco Nóvoa

·         Extraction of total RNA, DNA and proteins from tissue samples and cells

Ribonucleic acid (RNA) can be obtained from different sample types by using commercial extraction kits or by using Isolation Reagents like TriPure. The commercial kits offer a convenient and rapid method for the isolation of intact, total RNA whereas the manual extraction allows the simultaneous isolation of total RNA, DNA, and protein from the same sample.

 

·         Two step real-time reverse transcription polymerase chain reaction

Polymerase chain reaction is performed to compare the expression level of a gene of interest in situations where the organism or tissue has been submitted to different conditions or treatments, or to compare the expression level in different tissues or parts of the same organism.

In real-time PCR the quantification of the gene is achieved by measuring the fluorescence signal at the end of each PCR extension step. Since the data of each sample is obtained during one of the PCR steps, it is called a "real time" PCR. The relative quantification of the gene in each sample is calculated by 2 protocols: 1) a standard curve of the gene is acquired by serial dilutions of a known quantity of a reference template in order to calculate the gene amount in the samples; and 2) normalization of each sample to the total amount of tRNA tested through the quantification of a "housekeeping" gene expected to be present in the same quantity in all the samples. The template of any PCR has to be a double stranded nucleic acid so cDNA in produced using a reverse transcriptase. In our laboratory the reverse transcription is performed in tou different procedures, being termed “two step” PCR.


 


7.      Histology laboratory

        Laboratory managers: Dr. Maria Mendes

 

Most cells are colourless and transparent, and therefore histological sections have to be stained in some way to make the cells visible. The techniques used can either be non-specific, staining the cells in much the same way, or specific, selectively staining particular chemical groupings or molecules within cells or tissues.

Staining usually works by using a dye that stains some of the cells components a bright colour, and a counterstain, that stains the rest of the cell with a different colour.

 

·         Routine stains

The most commonly used staining is called Haemotoxylin and Eosin - H&E. This staining contains two dyes, the haemotoxylin and the eosin. The first one can be considered as a basic dye, this way it is used to stain acidic (or basophilic) structures a purplish blue. The nucleus, and parts of the cytoplasm that contain RNA stain up in one colour - purple. The Eosin is an acidic dye, it is negatively charged, and it stains basic (or acidophilic) structures red or pink. This is also sometimes termed "eosinophilic". Most proteins in the cytoplasm are basic, and so eosin binds to these proteins and stains them pink. This includes cytoplasmic filaments in muscle cells, intracellular membranes and extracellular fibres.

 

·         Specific stains

There are many other kinds of stains each of which stain tissues in characteristic ways. In our Department the most used stainings are (a) sirius red staining, as a method for collagen determination, enabling quantitative morphometric measurements to be performed in locally defined tissue areas, (b) Masson´s trichrome that is used to distinguish collagen from muscle in tissue specimens; the trichrome stain is often used to differentiate between collagen and smooth muscle and to identify an increase in collagenous tissue.  Muscle is stained red, collagen - blue, fibrin - pink, erythrocyte - red and nuclei - blue/black; (c) Van Gieson´s staining that is the easiest method of differential staining of collagen and other connective tissue. It is a mixture of Picric Acid and Acid Fuchsin. Collagen will stain pink or deep red, and cytoplasm will be stained yellow. Often times, Van Gieson's Stain is also widely used when staining for elastic fibres in tissues such as skin or aorta, via the Verhoeff Method.

 

·         Immunohistochemistry

Immunohistochemistry combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label. Immunohistochemistry makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue.

 

·         Immunofluorescent

This is a specific type of stain, in which primary antibodies are used to specifically label a protein, and then a fluorescently labelled secondary antibody is used to bind to the primary antibody, to show up where the first antibody has bound. A light microscope, equipped with fluorescence, is used to visualise the staining.

 

·         Processing samples to transmission electron microscopy (TEM)

Tissue blocks are generally fixed in glutaraldehyde and then they are postfixed in osmic acid. After, the tissue is dehydrated, and transferred to propylene oxide, which will mix with the resinous embedding medium. The tissue is infiltrated with the unpolymerised resin, and then heated gently, to polymerise it. Sections are cut using a glass plate wedge, or a diamond knife on a device called an ultramicrotome.



8.      Cell cultures laboratory

        Laboratory managers: Prof. Inês Pires, Dr. Glória Conceição

 

·         Cell cultures (primary and cell lines)

Cell culture is a method for studying the behavior of cells in physiological, pathological or experimental conditions free of the systemic influence. One of the main advantages of this technique is the precise control and reproducibility of the experiment conditions. In our laboratory primary animal cell culture from atrium fibrolasts, neonatal cardiomyocytes, pulmonary vascular smooth muscle cells and adipocytes are routinely performed.

 

·         Organotypic cultures

In organ culture the main advantages are the preservation of the architecture and cell interactions characteristic of the tissue in vivo.  Currently, human and animal heart slices are used to complement cell culture and functional studies.



9.      Chemistry laboratory

Laboratory managers: Dr. Marta Oliveira, Dr. Sara Leite, Dr. Maria Mendes

·         Preparation of work solutions

Several solutions are prepared in this laboratory either for the different experimental protocols (from ex-vivo to Histology) as well as general use stocks (often 10X concentration) for routine work.


·         Sample processing

Biological samples can be processed and subjected to centrifugation, filtration, sonication and snap freezing (Liquid Nitrogen).


·         pH monitoring

Using composed pH meters it is possible to control the pH of any solution adapted to their optimal temperature (ex: Protocols at 37oC with high pH sensitivity can be continuously monitored throughout the experiment).

 

·         Chemical reagents and glassware storage

All Chemical reagents are stored in ventilated cabinets that withstand chemical spills.

All Glassware is available ready to use in different areas of the Laboratory.