Name: impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system
Uploaded: 2018-05-22T14:00:00
Description: Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

The authors would like to thank Hartwig Wieboldt for his excellent technical assistance, and the UKE Microscopy Imaging Facility (Umif) of the University Medical Center Hamburg-Eppendorf for providing microscopes and support. This research was funded bythe F&#246;rderverein des Universit&#228;ren Herzzentrums Hamburg e.V. and by the DZHK (German Centre for Cardiovascular Research) [FKZ 81Z4710141].

All procedures involving animals were approved by the local authorities of the State of Hamburg, the University of Hamburg Animal Care and Use Committees.
1. Preparation of the Langendorff Apparatus
NOTE: A commercially available Langendorff perfusion system is used.
<ol>
	<li>Prepare a modified Krebs-Henseleit solution (119 mM of sodium chloride, 25 mM of sodium bicarbonate, 4.6 mM of potassium chloride, 1.2 mM of potassium phosphate monobasic, 1.1 mM of magnesiu...

<ol>
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In this manuscript, the well-known Langendorff ex vivo heart perfusion system is presented as a tool to study the impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis by using different mapping and stimulation techniques including endocardial and epicardial approaches.
Several parts of the protocol are crucial for the setup. First, it is important to use a preparation technique in which the atrial fat pads stay intact or are removed quickly without injuring...

The Langendorff ex vivo heart perfusion system continues to be a relevant tool for performing a broad spectrum of physiological, biochemical, morphological, and pharmacological studies in centrally denervated hearts1,2,3,4,5 since its invention in the late 19th century6. To date, this system is still widely used for various topics (e.g., ischemia reperfusion) or to study cardiac pharmacological effects7,8, and is a basic tool in cardiovascular research. The longevity of this method results from several advantages (e.g., measurements are performed without the influence of the central nervous system or other organs, systemic circulation, or circulating hormones). If needed, pharmaceuticals can be added in a controlled manner to the perfusion buffer or applied to specific structures directly. Experiments are reproducible, and a relatively high number of experiments can be performed in a short period of time. The (in part) open nature of the setup can make temperature regulation difficult and needs to be taken into account. Although the Langendorff system is also used in larger species9, smaller animals are primarily used as the experimental setup is less complex, and a greater biological variability (e.g., transgenic mouse models) can be used.
In the experimental setup of this protocol, the influence of the intracardiac autonomic nervous system on basic electrophysiological parameters, ventricular arrhythmogenesis, epicardial conduction, and cyclic adenosine monophosphate (cAMP) dynamics is evaluated. A large number of intracardiac ganglia, which are mainly located in the atrial fat pads and are now well known to control cardiac electrophysiology independent from central neural control, are either left intact or manually removed with careful mechanical dissection. A pharmacological modulation of the autonomic nervous system is performed either globally by adding pharmaceuticals to the perfusion buffer or locally by targeted modulation of the atrial ganglia. After the experiments, the hearts are well suited for an immunohistological assessment as all blood cells have been removed due to the continuous perfusion, which can increase the quality of staining.
The overall goal of the described techniques is to offer novel perspectives for detailed studies regarding the impact of the autonomic nervous system on cardiac electrophysiology and arrhythmogenesis in the mouse heart. A reason to use this technique is that it is possible to study and alter the autonomic nervous system without the impact of the central nervous system. One major advantage is the easy employment of pharmacological experiments, in which potential pro- or antiarrhythmic properties of old and new agents can be tested. In addition, transgenic and knockout mouse models of various cardiac diseases are available to investigate the mechanisms underlying arrhythmias, heart failure, or metabolic diseases. This approach has enhanced our understanding of how the autonomic nervous system on the atrial level can impact ventricular cardiac electrophysiology and the induction of arrhythmias.

<table>
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sodium hydrogencarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>401676</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sodium pyruvate bioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Carbogen (95% O2 / 5% CO2)</td>
			<td colspan="2">SOL-Group, TMG Technische und Medizinische Gas GmbH, Krefeld, Gersthofen, Germany</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Sterile filter steritop-GP 0.22</td>
			<td>EMD Millipore</td>
			<td>SCGPT05RE</td>
			<td>Modified Krebs-Henleit solution</td>
		</tr>
		<tr>
			<td>Atropine sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A0257</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Hexamethonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>H2138</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Nicotine free base 98-100%</td>
			<td>Sigma-Aldrich</td>
			<td>N3876</td>
			<td>Neuromodulation</td>
		</tr>
		<tr>
			<td>Formalin solution neutral buffered 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution 30% (w/w) in H2O</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>D8418</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline tablets</td>
			<td>Gibco / Invitrogen</td>
			<td>18912-014</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Triton-x-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Albumin bovine fraction V</td>
			<td>Biomol, Hamburg, Germany</td>
			<td>11924.03</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Chicken anti neurofilament</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Rabbit anti tyrosine hydroxylase</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Goat anti choline acetyltransferase</td>
			<td>EMD Millipore</td>
			<td>AP144P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; rabbit IgG Alexa 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; goat IgG Alexa 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11057</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey &#945; chicken IgY Alexa 647</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>AP194SA6</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated donkey &#945; rabbit igG</td>
			<td>R&#38;D Systems</td>
			<td>AP182B</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated donkey &#945; goat igG</td>
			<td>R&#38;D Systems</td>
			<td>AP192P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Biotin-conjugated goat &#945; chicken igY</td>
			<td>R&#38;D Systems</td>
			<td>BAD010</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>H-1000</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Vectastain ABC kit</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>PK-4000</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Steady DAB/Plus</td>
			<td>Abcam plc, Cambridge, UK</td>
			<td>ab103723</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>HistoClear</td>
			<td>DiaTec, Bamberg, Germany</td>
			<td>HS2002</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>BisBenzimide H33342 trihydrochloride (Hoechst)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B2261</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Vectashield HardSet mounting medium</td>
			<td>Vector laboratories, Burlingame, CA, USA</td>
			<td>VEC-H-1400</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Perfusion system</td>
			<td>HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH, March-Hugstetten, Germany</td>
			<td>&#160;73-4343</td>
			<td>Langendorff apparatus</td>
		</tr>
		<tr>
			<td>Data acquisition system and corresponding software for catheter and physiological parameter</td>
			<td>Powerlab 8/30 &#38; Labchart, ADInstruments, Dunedin, New Zealand</td>
			<td>PL3508 PowerLab 8/35</td>
			<td>Langendorff setup</td>
		</tr>
		<tr>
			<td>Octapolar catheter</td>
			<td>CIB&#8217;ER Mouse, NuMed Inc., Hopkinton, NY, USA</td>
			<td>custom</td>
			<td>Langendorff setup</td>
		</tr>
		<tr>
			<td>Stimulus generator</td>
			<td>STG4002, Multi Channel Systems, Reutlingen, Germany</td>
			<td>STG4002-160&#181;A</td>
			<td>Stimulation setup</td>
		</tr>
		<tr>
			<td>Stimulation software</td>
			<td>Multi Channel Systems, Reutlingen, Germany</td>
			<td>MC_Stimulus II</td>
			<td>Stimulation setup</td>
		</tr>
		<tr>
			<td>Data acquisition system and corresponding software for epicardial electrograms</td>
			<td>ME128-FAI-MPA-System, Multi Channel Systems, Reutlingen, Germany</td>
			<td>USB-ME128-System</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Multi-electrode array</td>
			<td>MEA, EcoFlexMEA36, Multi Channel Systems, Reutlingen, Germany</td>
			<td>EcoFlexMEA36</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Multi-electrode array recording software</td>
			<td>Multi Channel Systems, Reutlingen, Germany</td>
			<td>MC_Rack</td>
			<td>MEA setup</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>15003-08</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14575-09</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14110-15</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Dumont SS Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11203-25</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>London Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11080-02</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11003-13</td>
			<td>Heart Preparation</td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>Parafilm M, Bemis NA, based in Neenah, WI, United States</td>
			<td></td>
			<td>Consumable Materials</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica M165FC; Leica Microsystems GmbH, Wetzlar, Germany</td>
			<td></td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED, Andover, UK</td>
			<td>pE-100</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>DualView</td>
			<td>Photometrics, Tucson, AZ, USA</td>
			<td>DV2-SYS</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>DualView filter set</td>
			<td>Photometrics, Tucson, AZ, USA</td>
			<td>05-EM</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>optiMOS scientific CMOS camera</td>
			<td>Qimaging, Surrey, BC, Canada</td>
			<td>01-OPTIMOS-R-M-16-C</td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>Imaging software &#160;</td>
			<td>Micro-Manager; Vale Lab, University of California San Francisco, CA, USA</td>
			<td></td>
			<td>FRET</td>
		</tr>
		<tr>
			<td>Analysis Software</td>
			<td>Image J software; Public Domain, NIH, USA</td>
			<td></td>
			<td>FRET</td>
		</tr>
	</tbody>
</table>

impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo langendorff system

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Figure 1 shows an image of the Langendorff setup including 2 multi-electrode arrays (MEAs). Before the experiment, the intracardiac catheter is positioned close to the cannula to facilitate a quick and easy insertion in the right atrium/right ventricle and to ensure a short time period until the equilibration can start. The lower part of the chamber can be heightened (see the arrows in Figure 1) so that the chamber is fully close...

Watch this Scientific Journal Video about Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System at JoVE.com

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

The overall goal of this ex vivo Langendorff setup is to analyze and modulate the intracardiac autonomic nervous system and assess its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics in decentralized hearts. The main advantage of this technique is that it allows researchers to analyze and modulate the intracardiac nervous system in decentralized hearts. Though this method can provide insight into healthy hearts, we got initial evidence that this approach can also be used to study various forms of structural heart disease.Begin by starting the water bath and placing the perfusion solution including a mixture of 95%oxygen, 5%carbon dioxide in it. Then, adjust the pump rate before attaching the heart, so that no air bubbles are left in the cannula when mounting it to the apparatus. Set the targeted perfusion pressure to 80 millimeters mercury in the general settings, and start recording.Use an electrophysiology catheter with platinum electrodes, an electrode surface of 0.5 by 0.5 millimeters, with an electrode spacing of 0.5 millimeters for the data recording and stimulation with a designated digital stimulus generator. Then, place the catheter close to the area where the heart will be positioned after the attachment to the apparatus. Next, attach the cannula quickly to the Langendorff apparatus, and ensure that there are no bubbles left in the cannula.Switch the perfusion pressure to 80 millimeters mercury, allowing a constant pressure perfusion. Insert the catheter carefully into the right atrium and right ventricle without touching or damaging the heart, and attach the catheter to the cannula with tape. Then, start the stimulation with the prepared cycle length of 100 milliseconds for an initial 20-minute equilibration period.Finally, close the chamber to allow a stable temperature. Begin by applying a programmed stimulation via the distal or proximal electrodes of the catheter at twice the atrial or ventricular pacing threshold to evaluate the electrophysiological parameters. Then, perform a programmed extrastimulation or burst pacing protocols in line with the Lambeth Conventions to evaluate the ventricular arrhythmogenesis.Next, place the multi-electrode array, MEA, in the designated area of the heart, and add the grounding to another part of the heart. Place an epicardial stimulation catheter close to the MEA, and start a constant stimulation. Finally, start recording after the confirmation of good contact of the electrodes by checking the signal quality and amplitude.Using a self-built imaging system around a stereomicroscope, begin by placing the stereomicroscope in front of the heart and adjust it for acuity. Excite the cAMP sensor with a light source, and split the emission light into donor and acceptor channels using a beam-splitter. Take images by using a scientific complementary metal-oxide-semiconductor, sCMOS, camera.Next, start the image acquisition by pushing the Multi-D Acq. Button and set up a time-lapse, which acquires an image every 10 seconds with the appropriate exposure time, depending on the strength of the fluorescent signal. Then, use the previously described and available FRET Online and FRET Online 2 plugins to split the image into two channels, select the regions of interest, and monitor the ratio tracing.During the acquisition, perfuse the heart with modified Krebs-Henseleit solution containing different substances. Then, at the end of the experiment, switch off the acquisition by pushing the Stop button and save the stack of images. Finally, analyze the FRET data offline using a dedicated analysis software that can split images into two identical sections for the donor and acceptor channels and can perform FRET analysis in multiple regions of interest.On these whole-mount stainings, parts of the autonomic nervous system are depicted. An exemplary enlargement of one immunohistochemically stained atrial ganglion demonstrates the predominantly parasympathetic cells in red, compared to less numerous sympathetic cells, which are shown in green. In representative immunohistochemical stainings, neural fibers traverse from the atria via the coronary sinus towards the posterior ventricles with sympathetic predominance.Exemplary fibers are marked by arrowheads. Here, ventricular arrhythmia susceptibility testing via the electrodes in the RV is presented. The induction of a ventricular tachycardia in hearts occurred more frequently after partial atrial denervation.Further, a global as well as local topical application of pharmaceuticals is easily possible in this experimental setup. After perfusion of the adenylyl cyclase activator to increase cAMP levels, nicotine was applied to acutely reduce cAMP levels. While attempting this procedure, it's important to keep procedure time to a minimum and at a comparable length between experiments.After watching this video, you should have a good understanding of how to investigate the impact of the intracardiac autonomic nervous system on cardiac electrophysiology, arrhythmogenesis, and cAMP dynamics in an ex vivo Langendorff setup.

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Research

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Medicine

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...