We thank Dr. Olu Ajijola (UCLA Cardiac Arrhythmia Center) for expert support for the in vivo experiments. This work was supported by NIH U01 EB025138 (JLA, CS).

All animal experiments were approved by the University of California, Los Angeles Animal Research Committee and performed following the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Adult male Yorkshire pigs of approximately 75 kg were used for in vivo studies10.
1. Capacitive immunoprobe fabrication and functionalization
<ol>
	<li>Cut a 25 cm length of perfluoroalkoxy (PFA)-coated platinum wire (see Table of Materials) and strip approximately 5 mm of PFA coating from one end using a scalpel, being careful not to cut into the platinum wire.</li>
	<li>Insert the stripped end of the platinum wire into a gold-plated 1 mm male connector pin and crimp the connector pin teeth around the stripped end of the platinum wire using needle-nose pliers (see Table of Materials).</li>
	<li>Solder the platinum wire to the gold-plated connector pin. Be careful not to use an excessive amount of solder.</li>
	<li>Prepare dopamine solution by dissolving 50 mg of dopamine HCl in 50 mL of 10 mM phosphate-buffered saline (PBS, pH 6.0) by stirring.</li>
	<li>Once the dopamine is completely dissolved, place the tip of the platinum wire in the vessel containing the freshly made dopamine-supplemented PBS. Plug a gold connector pin into a channel of the headstage (see Table of Materials).</li>
	<li>Connect the AgCl disc electrode (ground electrode, see Table of Materials) to the ground channel in the headstage. Place the AgCl disc in the vessel containing dopamine-supplemented PBS and platinum wire; be careful only to submerge the disc electrode and not any length of the wire or solder. Connect a wire shunt into the reference channels of the headstage prior to proceeding.</li>
	<li>Open the interactive data acquisition software (see Table of Materials). Prepare a sawtooth electrodeposition command potential protocol with the following parameters: start potential = &#8722;0.6 V; end potential = +0.65 V; scan rate = 0.04 V&#8729;s-1; duration of deposition = 420 s. Begin the polydopamine deposition protocol, ensuring that all the wires are connected properly.</li>
	<li>After completing the polydopamine deposition, remove the AgCl ground pellet and the tip of the platinum wire from the vessel, being careful not to disturb the tip of the platinum wire electrode. Place the tip of the wire into a microtube containing PBS (pH 7.4) for 2-5 min as the antibody solution is prepared; ensure the wire tip does not contact the sides or bottom of the microtube. 
	NOTE: Antibody solution can be made during polydopamine deposition; however, the transfer of the platinum wire from the dopamine-containing vessel to the microtube of PBS following polydopamine deposition should not be skipped.</li>
	<li>Prepare the antibody solution. Combine the antibody of interest with PBS (pH 7.4) in a 1:20 ratio in a vessel of an appropriate size (e.g., a microtube). 
	NOTE: The anti-NPY monoclonal antibody (see Table of Materials) used here was aliquoted at 1 mg/mL; an example antibody preparation here would be 4 &#181;L of antibody to 76 &#181;L of PBS.</li>
	<li>Soak the polydopamine-deposited tip of the platinum electrode in antibody solution for a minimum of 2 h at room temperature, again ensuring the platinum wire tip is suspended in solution and not resting on the interior surface of the microtube. 
	NOTE: Recent implementation of this technique has favored using the platinum wire electrode immediately following this step instead of wet or dry storage for later use.</li>
	<li>After soaking in the antibody solution, briefly rinse the newly-functionalized capacitive immunoprobe (CI probe) tip in PBS (pH 7.4). The probe is now ready for use.</li>
</ol>
2. Experimental setup for in vitro detection and measurement of peptide
<ol>
	<li>Place the functional tip of the CI probe into the flow chamber, taking care not to disturb the tip of the electrode in any way, as doing so may damage the sensory tip of the probe. 
	NOTE: The flow chamber was created by pouring silicone elastomer (see Table of Materials) into a 35 mm culture dish with an elongated ovoid space-filler in the center of the dish. After hardening, the ovoid shape is removed from the elastomer. The chamber is then superfused with Tris-buffered saline (TBS) and allowed a flow rate of 3 mL/min. Ensure the inflow and outflow maintain the fluid level in the chamber such that no tidal action of the superfusate is observed. The flow must remain in place for as long as the CI probe is in use.</li>
	<li>Prior to the first experimental test, perform a TBS standard run to condition the CI probe. Set up the following command voltage protocol: positive step potential = +100 mV; negative step potential = &#8722;5 mV; step duration = 20 ms; duration of acquisition = 600 s. 
	NOTE: It is important to allow for the equilibration of the probe during the initial phase of cycling command potential prior to data acquisition.</li>
	<li>Create a solution of the peptide of interest using the same TBS to maintain the superfusate&#39;s composition. Set up a manifold system where the superfusate can be switched between TBS and peptide-supplemented TBS without introducing bubbles into the tubing system or flow chamber. 
	NOTE: Synthetic porcine NPY peptide (see Table of Materials) was used in the present study.</li>
	<li>Set up the peptide-sensing data acquisition protocol using TBS standard parameters (see step 2.2.). 
	&#8203;NOTE: In this implementation, the duration of each experimental test was 360 s (120 s TBS, 120 s peptide-supplemented TBS, 120 s TBS).</li>
</ol>
3. Adaptation of the CI probe for in vivo use
<ol>
	<li>Prior to polydopamine deposition (step 1.7.), thread the exposed tip of the platinum wire electrode through a 22 G hypodermic needle, leaving approximately 2 mm beyond the tip of the needle. Using forceps, gently bend back the tip of the platinum wire electrode, creating a &#34;barb&#34; that hangs from the end of the hypodermic needle.
	<ol>
		<li>Gently withdraw the needle from the barbed tip, leaving enough wire to place in the vessel without the needle contacting the fluid. Proceed with steps 1.4.-1.11. 
		NOTE: Depending on the in vivo setup, it may be necessary to cut a length of platinum wire longer than 25 cm.</li>
	</ol>
	</li>
	<li>Before connecting the CI probe to the headstage, ensure the entire electrical setup is properly grounded. Failure to do so may introduce unwanted electrical interference during the experimental recordings.</li>
	<li>Anesthetize the animals following a previously published report10.</li>
	<li>Perform surgery to expose the region of interest. 
	NOTE: A median sternotomy was performed in the present study to expose the heart. Please see Kluge et al.10 for details on animal surgery.</li>
	<li>Gently remove the functionalized tip from the PBS rinse (step 1.11.), forward the hydodermic needle to the barbed C.I. probe and gently implant it in the region of interest prior to plugging the gold connector pin into the headstage. Once implanted, withdraw the hypodermic needle, leaving the electrode in place. 
	NOTE: For the present study, the probe was placed in the mid-lateral wall of the left ventricular myocardium10.</li>
	<li>After ensuring proper electrical setup, proceed with standard and experimental testing protocols (step 2.2. and step 2.4.).</li>
	<li>Upon completion of the experiments, euthanize the animal following institutionally approved techniques. 
	NOTE: In the present study, the animals are euthanized under deep anesthesia via induction of ventricular fibrillation10.</li>
</ol>

<ol><li>Chow, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Biomarkers+for+the+prevention%2C+assessment%2C+and+management+of+heart+failure%3A+A+scientific+statement+from+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Role of Biomarkers for the prevention, assessment, and management of heart failure: A scientific statement from the American Heart Association.</a> Circulation. 135 (22), 1054-1091 (2017).</li>
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<li>Franco-Cereceda, A., Lundberg, J. M., Dahlof, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropeptide+Y+and+sympathetic+control+of+heart+contractility+and+coronary+vascular+tone%5BTitle%5D)+AND+%22Acta+Physiologica+Scandinavica%22%5BJournal%5D)">Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone.</a> Acta Physiologica Scandinavica. 124 (3), 361-369 (1985).</li>
<li>Habecker, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+cellular+neurocardiology%3A+development%2C+and+cellular+and+molecular+adaptations+to+heart+disease%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Molecular and cellular neurocardiology: development, and cellular and molecular adaptations to heart disease.</a> The Journal of Physiology. 594 (14), 3853-3875 (2016).</li>
<li>Hoang, J. D., Salavatian, S., Yamaguchi, N., Swid, M. A., Vaseghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+sympathetic+activation+circumvents+high-dose+beta+blocker+therapy+in+part+through+release+of+neuropeptide+Y%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Cardiac sympathetic activation circumvents high-dose beta blocker therapy in part through release of neuropeptide Y.</a> JCI Insight. 5 (11), 135519(2020).</li>
<li>Rigel, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+neuropeptides+on+heart+rate+in+dogs%3A+comparison+of+VIP%2C+PHI%2C+NPY%2C+CGRP%2C+and+NT%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">Effects of neuropeptides on heart rate in dogs: comparison of VIP, PHI, NPY, CGRP, and NT.</a> American Journal of Physiology. 255, 311-317 (1988).</li>
<li>Kluge, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+measurement+of+cardiac+neuropeptide+dynamics+by+capacitive+immunoprobe+in+the+porcine+heart%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Rapid measurement of cardiac neuropeptide dynamics by capacitive immunoprobe in the porcine heart.</a> American Journal of Physiology-Heart and Circulatory Physiology. 320 (1), 66-76 (2021).</li>
<li>Berggren, C., Bjarnason, B., Johansson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacitive+biosensors%5BTitle%5D)+AND+%22Electroanalysis%22%5BJournal%5D)">Capacitive biosensors.</a> Electroanalysis. 13 (3), 173-180 (2001).</li>
<li>Prodromidis, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impedimetric+immunosensors-A+review%5BTitle%5D)+AND+%22Electrochimica+Acta%22%5BJournal%5D)">Impedimetric immunosensors-A review.</a> Electrochimica Acta. 55 (14), 4227-4233 (2010).</li>
<li>Lee, H., Dellatore, S. M., Miller, W. M., Messersmith, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mussel-inspired+surface+chemistry+for+multifunctional+coatings%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Mussel-inspired surface chemistry for multifunctional coatings.</a> Science. 318 (5849), 426-430 (2007).</li>
<li>Leszczyszyn, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretion+of+catecholamines+from+individual+adrenal+medullary+chromaffin+cells%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Secretion of catecholamines from individual adrenal medullary chromaffin cells.</a> Journal of Neurochemistry. 56 (6), 1855-1863 (1991).</li>
<li>Pihel, K., Schroeder, T. J., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+selective+cyclic+voltammetric+measurements+of+epinephrine+and+norepinephrine+as+a+method+to+measure+secretion+from+single+bovine+adrenal+medullary+cells%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Rapid and selective cyclic voltammetric measurements of epinephrine and norepinephrine as a method to measure secretion from single bovine adrenal medullary cells.</a> Analytical Chemistry. 66 (24), 4532-4537 (1994).</li>
<li>Jaffe, E. H., Marty, A., Schulte, A., Chow, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrasynaptic+vesicular+transmitter+release+from+the+somata+of+substantia+nigra+neurons+in+rat+midbrain+slices%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Extrasynaptic vesicular transmitter release from the somata of substantia nigra neurons in rat midbrain slices.</a> The Journal of Neuroscience. 18 (10), 3548-3553 (1998).</li>
<li>Walsh, P. L., Petrovic, J., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinguishing+splanchnic+nerve+and+chromaffin+cell+stimulation+in+mouse+adrenal+slices+with+fast-scan+cyclic+voltammetry%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Cell+Physiology%22%5BJournal%5D)">Distinguishing splanchnic nerve and chromaffin cell stimulation in mouse adrenal slices with fast-scan cyclic voltammetry.</a> American Journal of Physiology-Cell Physiology. 300 (1), 49-57 (2011).</li>
<li>Wolfe, J. T., Wang, H., Perez-Reyes, E., Barrett, P. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+recombinant+Ca%28v%293.2%2C+T-type%2C+Ca%282%2B%29+channel+currents+by+CaMKIIgamma%28C%29%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C).</a> The Journal of Physiology. 538, 343-355 (2002).</li>
<li>Chan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+in+vivo+detection+of+myocardial+norepinephrine+levels+in+the+beating+porcine+heart%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Fast in vivo detection of myocardial norepinephrine levels in the beating porcine heart.</a> American Journal of Physiology-Heart and Circulatory Physiology. 318 (5), 1091-1099 (2020).</li>
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The authors declare no conflicts of interest, financial or otherwise.

The present protocol describes the manufacture and testing of a capacitive immunoprobe (CI probe) capable of detecting and measuring biomarkers of interest in both in vitro and in vivo settings. Detection is achieved by trapping the biomarker at the electrode tip. The trapping event alters the capacitive junction between a platinum wire capacitive immunoprobe and the surrounding conductive fluid environment, measured as a change in charge current in response to a potential shift in the probe. A unique e...

Several chemical methods for detecting and quantifying biomarkers are routinely utilized in both protein chemistry and clinical diagnostics, particularly in cancer diagnoses and the assessment of cardiovascular disease progression. Currently, methods such as high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry rely on sample collection from the vascular compartment1,2,3 by bulk fluid draw or the interstitial compartment by microdialysis. Microdialysis employs a semipermeable membrane tube of known length that is placed in a region of interest. Collection fluid is perfused through the tube over several minutes4 to collect the sample for analysis5, thus limiting temporal resolution. In this manner, samples collected only provide an averaged value over time of the local microenvironment and are limited by the perfusion rate and collection of sufficient sample volume. Moreover, these methods require the pooling of experimental data and signal averaging; therefore, they may fail to account for variability between subjects. Importantly, the time between sample collection and subsequent offline analysis precludes immediate clinical intervention and therapeutics.
In the present protocol, the use of a capacitive immunoprobe biosensor (CI probe) for the time-resolved electrical detection of specific bioactive peptides is outlined. Neuropeptide Y (NPY), released from post-ganglionic sympathetic neurons that innervate the vasculature, endocardium, cardiomyocytes, and intracardiac ganglia, is a major neuromodulatory peptide transmitter in the cardiovascular system6,7,8,9. The method presented here is designed to measure NPY, and the experimental feasibility is demonstrated in a porcine heart model. However, this approach applies to any bioactive peptide for which a selective antibody is available10. This method relies on the capacitive junction between a platinum wire probe and the conductive fluid at the functionalized tip11,12. In this application, the interaction was mediated through an antibody against the target neuropeptide (NPY), which was bound to the electrode tip, interfacing the conductive fluid environment. This functionalization was achieved through electrodeposition of reactive polydopamine onto the tip of the platinum wire probe10,13.
When the antibody-functionalized probe is placed in a region of interest in vivo, evoked endogenous NPY release leads to binding to the trapping antibodies on the probe tip, and the conductive fluid at the electrode surface is displaced by the NPY protein. Local alteration in the electrical environment results in the displacement of high-mobility, high-dielectric fluid with an immobile, statically charged molecule. This alters the electrode-fluid interface and, thus, its capacitance, which is measured as a change in charge current in response to a step-function command potential. A negative &#34;reset&#34; potential is employed immediately following each individual measurement cycle to repel bound NPY from the antibody through electrostatic interaction, thus clearing the antibody binding sites for subsequent rounds of measurement10. This effectively allows for the measurement of NPY in a time-resolved manner. The unique CI technique overcomes the limitations of the microdialysis-based immunochemical methods outlined above to measure dynamic biomarker levels from a single experiment without data pooling or signal averaging over several experiments9, providing data in nearly real time. Moreover, the ability to adapt this method to any biomarker of interest for which there exists an appropriate antibody on a time-resolved and localized scale provides a major technical advance in immunochemical measurement for the evaluation of disease progression and the guidance of therapeutic interventions.
The software for data acquisition and analysis was custom-written in IGOR Pro (a fully interactive software environment). An analog to digital converter (A/D) system issued a command voltage under computer control and acquired data from a custom amplifier. The amplifier possessed certain unique features. These included a feedback resistor (switchable) for each of the four acquisition channels, allowing for choosing 1 MOhm or 10 MOhm feedback voltage clamp circuits to integrate the variability of the electrode. A stage unit with a single head and mutual ground/reference circuit for all the four acquisition channels was also built to place the device close by the chest in a single physical module. A 1 MOhm feedback resistor setting was used to collect all the reported data.
The filter and gain settings were telegraphed from the amplifier and recorded within the data file. Data were filtered at 1 kHz via a 2-pole analog Bessel filter digitized at 10 kHz. The difference in potential between the probe and surrounding conductive solution creates a Helmholtz capacitive layer at the probe tip. Ligand binding to the antibody at the probe tip results in an altered local charge and, thus, a change in the Helmholtz capacitance. This change in the capacitive component of the circuit results in a shift in the magnitude of injected charge required to bring the probe to the potential in the step-function voltage protocol. Thus, the binding of a specific ligand to the functionalized probe results in an alteration in the electrode capacitance measurement as a change in peak capacitive current.

<table><tbody><tr><td>AgCl disc electrode</td><td>Warner Instruments (Holliston, MA)</td><td>64-1307</td><td></td></tr><tr><td>Anti-NPY monoclonal antibody</td><td>Abcam, (Cambridge, MA)</td><td>ab112473</td><td></td></tr><tr><td>Custom multichannel amplifier/ 1 M&amp;Omega; feedback resistor multichannel headstage</td><td>NPI Electronic, (Tamm, Germany)</td><td>NA</td><td>Based on NPI VA-10M multichannel amplifier</td></tr><tr><td>Dopamine HCl</td><td>Sigma Aldrich (St. Louis, MO)</td><td>H8502-10G</td><td></td></tr><tr><td>Gold-plated male connector pin</td><td>AMP-TE Connectivity (Amplimite)</td><td>6-66506-1</td><td></td></tr><tr><td>HEKA LIH 8+8 analog-to-digital/digital-to-analog device</td><td>HEKA Elektronik, (Holliston, MA)</td><td>NA</td><td></td></tr><tr><td>Igor Pro data acquisition software, v. 7.08</td><td>WaveMetrics, (Lake Oswego, OR)</td><td></td><td>Software driving command potential and data acquisition was custom written</td></tr><tr><td>Masterflex L/S Standard Digital peristaltic pump</td><td>Cole Palmer, (Vernon Hills, IL)</td><td></td><td></td></tr><tr><td>PFA-coated platinum wire</td><td>A-M Systems, (Sequim, WA)</td><td>773000</td><td>0.005&amp;rdquo; bare diameter, 0.008&amp;rdquo; coated diameter</td></tr><tr><td>Silicone elastomer</td><td>World Precision Instruments (Sarasota, FL)</td><td>SYLG184</td><td></td></tr><tr><td>Synthetic porcine NPY peptide</td><td>Bachem (Torrance, CA)</td><td>4011654</td><td></td></tr><tr><td>Synthetic porcine NPY peptide</td><td>Bachem (Torrance, CA)</td><td>4011654</td><td></td></tr></tbody></table>

time-resolved in vivo measurement of neuropeptide dynamics by capacitive immunoprobe in porcine heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Electrode fabrication and characterization 
A flexible capacitive immunoprobe (CI probes) was fabricated, and a representative image is depicted in Figure 1A. The electrode potential was set by a computer-controlled voltage clamp circuit (Figure 1B), and the electrode was immersed in a polydopamine solution made in PBS. Polydopamine was electrodeposited onto the conductive electrode tip13 for functionalization. The c...

Watch this Scientific Journal Video about Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart at JoVE.com

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

time-resolved-vivo-measurement-neuropeptide-dynamics-capacitive

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2.0K Views.  Case Western Reserve University. Dit is de eerste keer dat een methode is ontwikkeld voor de tijd-opgeloste meting van peptidetransmitterniveaus in vivo. We kunnen de niveaus van peptidetransmitters in afzonderlijke onderwerpen en discrete locaties meten met bijna-realtime analyse. Knip eerst een 25 centimeter lange perfluoroalkoxy-gecoate platinadraad.Verwijder vervolgens met behulp van een scalpel ongeveer vijf millimeter perfluoroalkoxy-coating van het ene uiteinde, voorzichtig om niet in de platinadraad te snijden...

Video: Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

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.

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.

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 ...

Medicine

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 ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Neuroscience

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest in developing these peptides, especially cell-specific ones, as novel vectors for delivering diagnostic and therapeutic materials. Our past work involving phage display identified a novel, nonnaturally occurring, 12 amino acid-long peptide that we named cardiac targeting peptide (CTP) due to its ability to transduce normal heart tissue in vivo with peak uptake seen in as little as 15 min after an intravenous injection. We have undertaken detailed biodistribution studies by injecting CTP labeled with fluorophore cyanine5.5, allowing it to circulate for various periods of time, and euthanizing, fixing, and sectioning multiple organs followed by fluorescent microscopy imaging. In this publication, we describe these processes as well as ex vivo imaging of harvested organs using an in vivo imaging system in detail. We provide detailed methodologies and practices for undertaking transduction as well as biodistribution studies using CTP as an example.

We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Real-Time Measurements of Calcium and Contractility Parameters in hiPSC-CMs

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

Author Spotlight: Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

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