Name: advanced cardiac rhythm management by applying optogenetic multi-site photostimulation in murine hearts
Uploaded: 2021-08-26T13:00:00
Description: Watch this Scientific Journal Video about Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts at JoVE.com

The authors would like to thank Marion Kunze and Tina Althaus for their excellent technical support during experiments. The research leading to the results has received funding from the European Community&#39;s Seventh Framework Programme FP7/2007-2013 under grant agreement number HEALTH-F2-2009-241526. Support was also provided by the German Center for Cardiovascular Research, DZHK e.V. (Project MD28), partner site Goettingen, the German Research Foundation CRC 1002 (project C03), and the Max Planck Society. This work was partly supported by BrainLinks-BrainTools, Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086).

All experiments strictly followed the animal welfare regulation, in agreement with German legislation, local stipulations, and in accordance with recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). The application for approval of animal experiments has been approved by the responsible animal welfare authority, and all experiments were reported to our animal welfare representatives.
1. Experiment preparation and materials
<ol>
	<li>Optical mapping se...

<ol>
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A., Luther, S., Diaz-Maue, L., Richter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy-reduced+arrhythmia+termination+using+global+photostimulation+in+optogenetic+murine+hearts.">Energy-reduced arrhythmia termination using global photostimulation in optogenetic murine hearts.</a> Frontiers in Physiology. 9 (1651), (2018).</li><li>Moreno, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+irradiance+pattern+at+short+distances.">LED irradiance pattern at short distances.</a> Applied Optics. 59 (1), 190-195 (2020).</li><li>Behrend, A., Bittihn, P., Luther, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+unpinning+success+rates+for+a+pinned+spiral+in+an+excitable+medium.">Predicting unpinning success rates for a pinned spiral in an excitable medium.</a> , 345-348 (2010).</li><li>Kappadan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11 (464), (2020).</li><li>Christoph, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+vortex+filaments+during+cardiac+fibrillation.">Electromechanical vortex filaments during cardiac fibrillation.</a> Nature. 555, 667-672 (2018).</li><li>O'Shea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+optogenetics+and+optical+mapping+-+Overcoming+spectral+congestion+in+all-optical+cardiac+electrophysiology.">Cardiac optogenetics and optical mapping - Overcoming spectral congestion in all-optical cardiac electrophysiology.</a> Frontiers in Physiology. 10 (182), (2019).</li><li>Aras, K. K., Faye, N. R., Cathey, B., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+volume+of+human+myocardium+necessary+to+maintain+ventricular+fibrillation.">Critical volume of human myocardium necessary to maintain ventricular fibrillation.</a> Circulation: Arrhythmia and Electrophysiology. 11 (11), e006692 (2018).</li><li>Trayanova, N., Doshi, A. N., Prakosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+personalized+heart+modeling+can+help+treatment+of+lethal+arrhythmias:+A+focus+on+ventricular+tachycardia+ablation+strategies+in+post-infarction+patients.">How personalized heart modeling can help treatment of lethal arrhythmias: A focus on ventricular tachycardia ablation strategies in post-infarction patients.</a> Wiley Interdisciplinary Reviews in System Biology and Medicine. 12 (3), 1477 (2020).</li><li>Bingen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+termination+of+spiral+wave+arrhythmias+by+optogenetic+engineering+of+atrial+cardiomyocytes.">Light-induced termination of spiral wave arrhythmias by optogenetic engineering of atrial cardiomyocytes.</a> Cardiovascular Research. 104 (1), 194-205 (2014).</li><li>Burton, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+panoramic+in+situ+mapping+of+action+potential+propagation+in+transgenic+hearts+to+investigate+initiation+and+therapeutic+control+of+arrhythmias.">Toward panoramic in situ mapping of action potential propagation in transgenic hearts to investigate initiation and therapeutic control of arrhythmias.</a> Frontiers in Physiology. 5, 337 (2014).</li><li>Crocini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+design+of+mechanistically-based+stimulation+patterns+for+cardiac+defibrillation.">Optogenetics design of mechanistically-based stimulation patterns for cardiac defibrillation.</a> Science Reports. 6 (35628), (2016).</li><li>Nyns, E. C. 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A successful treatment of cardiac tachyarrhythmias is key to cardiac therapy. However, the biophysical mechanisms underlying arrhythmia initiation, perpetuation and termination are not fully understood. Therefore, cardiac research aims to optimize electrical shock therapy towards a more gentle termination of arrhythmias, thereby increasing the quality of life of patients28,29,30,31. Low energy ...

Early investigations of the spatial-temporal dynamics during arrhythmia revealed that the complex electrical patterns during cardiac fibrillation are driven by vortex-like rotating excitation waves1. This finding gave new insights into the underlying mechanisms of arrhythmias, which then led to the development of novel electrical termination therapies based on multi-site excitation of the myocardium2,3,4. However, treatments using electric field stimulation are non-local and may innervate all surrounding excitable cells, including muscle tissue, causing cellular and tissue damage, as well as intolerable pain. In contrast to electrical therapies, optogenetic approaches provide a specific and tissue-protective technique for evoking cardiomyocyte action potentials with high spatial and temporal precision. Therefore, optogenetic stimulation has the potential for minimal invasive control of the chaotic activation patterns during cardiac fibrillation.
The introduction of the light-sensitive ion channel channelrhodopsin-2 (ChR2) into excitable cells via genetic manipulation5,6,7, enabled the depolarization of the membrane potential of excitable cells using photostimulation. Several medical applications, including the activation of neuronal networks, the control of cardiac activity, the restoration of vision and hearing, the treatment of spinal cord injuries, and others8,9,10,11,12,13,14 have been developed. The application of ChR2 in cardiology has significant potential due to its millisecond response time15, making it well suited for the targeted control of arrhythmic cardiac dynamics.
In this study, multi-site photostimulation of intact hearts of a transgenic mouse model is shown. In summary, a transgenic alpha-MHC-ChR2 mouse line was established within the scope of the European Community&#39;s Seventh Framework Programme FP7/2007-2013 (HEALTH-F2-2009-241526) and kindly provided by Prof. S. E. Lehnart. In general, transgenic adult male C57/B6/J, expressing Cre-recombinase under control of alpha-MHC were paired to mate with female B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hye/J. Since the cardiac STOP cassette was deleted in the second-generation, the offspring showed a stable MHC-ChR2 expression and was used to maintain cardiac photosensitive colonies. All experiments were done with adult mice of both genders at an age of 36 - 48 weeks. The illumination is achieved using a 3 x 3 micro-LED array, fabricated as described in16,17 except that the silicon-based housing and the short optical glass fibers are not implemented. Its first usage in a cardiac application is found in18. A linear micro-LED array based on a similar fabrication technology has been applied as a penetrating probe for heart pacing19. The micro-LEDs are arranged in a 3 x 3 array at a pitch of 550 &#181;m, providing both a high spatial resolution and a high radiant power on a very small area. The authors demonstrate in this work a versatile local multi-site photostimulation that may open the path for developing novel anti-arrhythmic therapy methods.
The following experimental protocol involves a retrograde Langendorff perfusion ex vivo, for which the cannulated aorta functions as perfusion inlet. Due to the applied perfusion pressure and the cardiac contraction the perfusate is flowing through the coronary arteries, which branch off the aorta. In the presented work, the heart is perfused using a constant pressure setup achieved by elevating the perfusate reservoirs to 1 m height, equivalent to 73.2 mmHg, which yields to a flow rate of 2.633 &#177; 0.583 mL/min. Two kinds of Tyrode&#39;s solution are used as perfusate during the experiment. Regular Tyrode&#39;s solution supports a stable sinus rhythm, whereas Low-K+ Tyrode&#39;s solution is mixed with Pinacidil to enable the induction of arrhythmia in murine hearts. The usage of a hexagonal water bath permits the observation of the heart through six different planar windows, allowing the coupling of several optical components with less distortion by refraction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>TargetMol</td>
			<td>T6038</td>
			<td>10 mM stock solution</td>
		</tr>
		<tr>
			<td>BSA/Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Westfalen</td>
			<td></td>
			<td>50 l bottle</td>
		</tr>
		<tr>
			<td>DI-4-ANBDQPQ</td>
			<td>AAT Bioquest</td>
			<td>21499</td>
			<td>Dye for Optical Mapping</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434</td>
			<td>C6H12O6</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO Pharma</td>
			<td></td>
			<td>Heparin-Natrium Leo 25.000 I.E./5 ml, available only on prescription</td>
		</tr>
		<tr>
			<td>Hydrochlorid Acid</td>
			<td>Merck</td>
			<td>1.09057.1000</td>
			<td>HCl, 1 M stock solution</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP Pharma</td>
			<td></td>
			<td>1 ml/ml, available only on prescription</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Merck</td>
			<td>8.14733.0500</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Monopotassium Phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>30407</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>Pinacidil monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P154-500mg</td>
			<td>10 mM stock solution</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>1.09137.1000</td>
			<td>NaOH, 1 M stock solution</td>
		</tr>
		<tr>
			<td>Electrical Setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biopac MP150</td>
			<td>Biopac Systems</td>
			<td>MP150WSW</td>
			<td>data acquisition and analysis system</td>
		</tr>
		<tr>
			<td>Custom-built ECG, alternative ECG100C</td>
			<td>Biopac Systems</td>
			<td>ECG100C</td>
			<td>Electrocardiogram Amplifier</td>
		</tr>
		<tr>
			<td>Custom-built water bath heater using heating cable</td>
			<td>RMS Heating System</td>
			<td>HK-5,0-12</td>
			<td>Heating cable 120W</td>
		</tr>
		<tr>
			<td>Hexagonal water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver Power supply</td>
			<td>Thorlabs</td>
			<td>KPS101</td>
			<td>15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for One K- or T-Cube.</td>
		</tr>
		<tr>
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			<td>Thorlabs</td>
			<td>LEDD1B</td>
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			<td>A-2230</td>
			<td>Arbitrary function generator (AFG)</td>
		</tr>
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			<td>Signal Generator</td>
			<td>Agilent Instruments</td>
			<td>A-2230</td>
			<td>AFG</td>
		</tr>
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			<td>micro-LED Array Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxid glue</td>
			<td>Epoxy Technology</td>
			<td>EPO-TEK 353ND</td>
			<td>Two component epoxy</td>
		</tr>
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			<td>Fluoropolymer</td>
			<td>&#160;Asahi Glass Co. Ltd.</td>
			<td>Cytop 809M</td>
			<td>Fluoropolymer with high transparency</td>
		</tr>
		<tr>
			<td>Image reversal photoresist</td>
			<td>Merck KGaA</td>
			<td>AZ 5214E</td>
			<td>Image Reversal Resist for High Resolution</td>
		</tr>
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			<td>LED chip</td>
			<td>&#160;Cree Inc.</td>
			<td>C460TR2227-S2100</td>
			<td>Blue micro-LED</td>
		</tr>
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			<td>Photoresist</td>
			<td>Merck KGaA</td>
			<td>AZ 9260</td>
			<td>Thick Positive Photoresists</td>
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		<tr>
			<td>Polyimide</td>
			<td>UBE Industries Ltd.</td>
			<td>U-Varnish S</td>
			<td>Polyimide Solution</td>
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			<td>Silicone</td>
			<td>NuSil Technology LLC</td>
			<td>MED-6215</td>
			<td>Low viscosity silicone elastomer</td>
		</tr>
		<tr>
			<td>Solvent free adhesive</td>
			<td>John P. Kummer GmbH</td>
			<td>Epo-Tek 301-2</td>
			<td>Epoxy resin with low viscosity</td>
		</tr>
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			<td>Optical Mapping</td>
			<td></td>
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		<tr>
			<td>Blue Filter</td>
			<td>Chroma Technology Corporation</td>
			<td>ET470/40x</td>
			<td>Blue excitation filter</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Photometrics</td>
			<td>Cascade 128+</td>
			<td>High performance EMCCD Camera</td>
		</tr>
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			<td>Camera Objective</td>
			<td>Navitar</td>
			<td>DO-5095</td>
			<td>Navitar high speed fixed focal length lenses work with CCD and CMOS cameras</td>
		</tr>
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			<td>Dichroic Mirror</td>
			<td>Semrock</td>
			<td>FF685-Di02-25x36</td>
			<td>685 nm edge BrightLine&#174; single-edge standard epi-fluorescence dichroic beamsplitter</td>
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			<td>Emmision Filter</td>
			<td>Semrock</td>
			<td>FF01-775/140-25</td>
			<td>775/140 nm BrightLine&#174; single-band bandpass filter</td>
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			<td>Heatsink</td>
			<td>Advanced Thermal Solutions</td>
			<td>ATSEU-077A-C3-R0</td>
			<td>Heat Sinks - LED STAR LED Heatsink, 45mm dia., 68mm, Black/Silver, Unthreaded Baseplate Hardware</td>
		</tr>
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			<td>LED 1 and LED 2</td>
			<td>LED Engin Osram</td>
			<td>LZ4-00B208</td>
			<td>High Power LEDs - Single Colour Blue, 460 nm 130 lm, 700mA</td>
		</tr>
		<tr>
			<td>LED 3</td>
			<td>Thorlabs</td>
			<td>M625L3</td>
			<td>625 nm, 700 mW (Min) Mounted LED, 1000 mA</td>
		</tr>
		<tr>
			<td>Lenses</td>
			<td>LED Engin Osram</td>
			<td>LLNF-2T06-H</td>
			<td>LED Lighting Lenses Assemblies LZ4 LENS NARROW FLOOD BEAM</td>
		</tr>
		<tr>
			<td>Photodiode for power meter</td>
			<td>Thorlabs</td>
			<td>S120VC</td>
			<td>Standard Photodiode Power Sensor</td>
		</tr>
		<tr>
			<td>Power Meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Compact Power and Energy Meter</td>
		</tr>
		<tr>
			<td>Red Filter</td>
			<td>Semrock</td>
			<td>FF02-628/40-25</td>
			<td>BrightLine&#174; single-band bandpass filter</td>
		</tr>
	</tbody>
</table>

advanced cardiac rhythm management by applying optogenetic multi-site photostimulation in murine hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

The protocol allows the induction of ventricular arrhythmias in intact murine hearts using photostimulation pulses generated by LED 1 and LED 2 (Figure 1) with a frequency find between 25 Hz and 35 Hz and a pulse duration Wind between 2 ms and 10 ms. Please notice that the aim of such rapid light pulses is not to capture the cardiac rhythm but rather to unbalance the cardiac activity so that erratic electrical waves can be generated, which then facilitate an arrhythmia....

Watch this Scientific Journal Video about Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts at JoVE.com

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

The physical most underlying cardiac fibrillation and defibrillation remain not completely understood. Optogenetics can offer a way to gain more insight into cardiac phenomena in a well-controlled environment. Local light stimulation using micro LED's permits the innovation of specific cardiac tissue areas.This targeted stimulation is a new tool to develop heart gentle anti arithmetic therapy methods. The shown protocol can be used to study heart behavior during cardiac arrhythmia and especially during termination. In prospective, the developed techniques can be used to study more clinically relevant large animal models.Before every experiment, clean all tubes with fully de-mineralized water. Aerate thyroid solutions with carbogen for 30 minutes at room temperature and adjust the pH to 7.3 with sodium hydroxide and hydrochloric acid. Heat the perfusion system to 37 degrees Celsius with a water heat pump.Keep the perfusate temperature constant within the water bath by using an additional heating element such as a waterproof heating cable. Add 500 milliliters of each thyroid solution to the corresponding reservoir and de-aerate the tubes as well as the bubble trap by running thyroid solution through the perfusion system until no more trapped air bubbles are seen in the tubes or in the bubble trap. Continue aerating the thyroid solutions during the whole experiment in the reservoirs with carbogen to ensure that the pH of the perfusate remains stable later during perfusion.After removing the heart, perform the fine preparation under a stereoscopic microscope. Attach the aorta onto the blunt needle and fix the vessel with suture material. As a control, inject ice cold thyroid solution through the needle into the heart, and check that the heart is tightly mounted.Transfer the mounted heart to the perfusion system and ensure that the perfusate is flowing to prevent air from entering the heart while connecting the needle with the bubble trap. Check that the heart is covered with thyroid solution in the water bath and that it is beating within a few minutes. Place one of the ECG electrodes as close as possible to the heart surface to ensure good signal quality.Suspend the second ECG electrode in the thyroid solution, making sure that the ECG is being recorded. Place the micro LED array on the area of interest. Change the perfusion to low potassium thyroid with penecidle and peruse the heart for 15 to 30 minutes.To induce arrhythmia, illuminate the heart with LED one and LED two with a train of 20 to 50 light pulses with a frequency of 25 to 35 hertz, pulse duration of two to 15 milliseconds, and light intensity of 2.8 milli watts per square millimeters. Repeat the process until arrhythmia is induced. Once a sustained arrhythmia is visually detected, apply a burst of pulses with different widths and frequencies using three, six, or nine micro LEDs of the array at a pulsing current of 15 milliamp piers.If the arrhythmia keeps going after five micro LED array based defibrillation trials, classify the attempt as unsuccessful and start backup defibrillation, using LED one and LED two with same timing parameters. Perfuse the heart with the blebbistatin solution and wait until mechanical uncoupling occurs, which is accomplished when the heart stops beating, but an ECG signal is still measurable. Give the one milliliter voltage dye, dye 4-A-N-B-D-Q-P-Q as a bolus in the bubble trap of the Langendorff perfusion and wait for five to 10 minutes to allow the dye to peruse the heart uniformly.Turn on LED three. Focus the camera on the heart's surface and apply 1.27 milli watts per square millimeter optical power. Turn off the laboratory lights and start recording, making sure that an optical signal is being acquired by comparing the frequency of the obtained signal to the frequency of the recorded ECG.A series of experiments with different frequencies, number of micro LED and pulse durations were tested for 11 mice, demonstrating that pulses of 1 to 20 milliseconds can defibrillate with different success rates. There is a significant increase in success rate of the nine LEDs with pulse durations of one millisecond and 20 milliseconds at a defibrillation frequency of 18 and 20 hertz, which is closer to the analyzed mean arrhythmia frequency of 22.55 of all the arrhythmias with an error interval of plus or minus four point zero three hertz. Two different defibrillation attempts with 14 hertz frequency with their ECG recordings and their respective spectrograms are shown here.In an example of defibrillation with 30 millisecond pulses, the dominant frequency of the arrhythmia slightly increases until photo stimulation begins. The VF is turned into a VT where the dominant frequency drops to 14 hertz followed by unsuccessful termination and return to arrhythmic behavior with a dominant frequency of 24 hertz. In the second example, segment one shows a VT with dominant frequency of 23 hertz and it's harmonic components until photo stimulation begins, with a pulse width of 20 milliseconds.Segment three displays a successful termination which leads to a normal sinus rhythm with a fundamental frequency of 3.5 hertz and the resulting harmonics. Optical mapping with high speed cameras showed a change of fluorescence intensity during a single beat of the heart during a normal sinus rhythm. The length of perfusion must maintain all conditions to keep the heart healthy, namely the temperature, the pH value, and the oxidation need to remain constant.It is particularly important that the tyroid solution do not run empty as air bubbles in the system may damage the heart. The micro reality array offers great flexibility. You can target different locations of the heart or even combine multiple arrays in one experiment in order to properly terminate arrhythmia.

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Medicine

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