Bidirectional Electrical and Optoelectronic Interfaces in Healthy and Ischemic Ex Vivo Rat Hearts

We developed an ex vivo heart model to evaluate optoelectronic and electronic materials for cardiac stimulation and sensing, bridging bench top innovation with translational bioelectronic applications. We bridge the gap between in vitro and in vivo by offering a controlled, non-genetic ex vivo heart model to evaluate both wired and wireless bioelectronic materials in real tissue. Our protocol enables fast reproducible testing of materials in whole beating hearts, combining physiological relevance with precise control, ideal for comparing stimulation and sensing performance across different devices.

We explore therapeutic materials that modulate heart function and validate their efficiency in treating myocardial infarction using our ischemia reperfusion ex vivo model. To begin, confirm general anesthesia by pinching one of the paws of the anesthetized rat, and proceed only if no response is observed. Then, make a five centimeter incision just below the chest to open the ribcage.

Using scissors, carefully cut through the diaphragm to expose the heart and lungs. Now, cut through the ribs on both sides to fully open the ribcage. Secure the opened ribcage at the sternum using a hemostat.

Use a second hemostat to hold the vena cava from below the heart. Cut under the hemostat with bent blunt scissors as close to the bottom of the ribcage as possible to remove the heart. Transfer the heart immediately into a Petri dish filled with ice cold HBSS.

Fill a cannula and a separate Petri dish completely with ice cold HBSS. Transfer the heart to the Petri dish, prefilled with the buffer for sectioning. Use tweezers or scissors to remove the lungs and other connective tissues attached to the heart.

Then, press on the heart gently and trace the blood path to locate the aorta. If the aortic arch is preserved, cut under the first ascending artery. Cannulate the aorta carefully.

Prime the Langendorff apparatus with preoxygenated working solution. Open the buffer flow and attach the cannula to the perfusion apparatus carefully, ensuring that no air bubbles enter the system. Observe the heart begins to contract once perfusion starts.

Now, cut off part of the atria or atrial appendage using small scissors. Before inserting the balloon into the left ventricle, deflate it completely. Then, use the connected syringe to fill the balloon with water.

Next, connect BP 100 pressure probes to the perfusion line and the water-filled balloons to monitor left ventricular pressure respectively. Amplify all signal outputs, including left ventricular pressure and ECG, using the IA-400D amplifier. Monitor the pressure of the HEPES Tyrode's buffer and adjust it to stay within the optimal range of 80 to 100 millimeters of mercury.

Modify the volume of water in the balloon using the syringe to set the baseline left ventricular pressure to approximately 20 millimeters of mercury. Connect the electrocardiogram electrodes by grounding the cannula and placing the electrode wires at the sides, top, or apex of the heart based on user preference. Fill the heated chamber with HEPES Tyrode's buffer prewarmed to 37 degrees Celsius.

Submerge the heart into the chamber and turn the stopcock to halt buffer flow, inducing global ischemia. After 30 minutes of ischemia, turn the stopcock to restore buffer flow and initiate reperfusion. Allow the heart to perfuse for 45 minutes before terminating the experiment for infarction staining.

Verify the success of ischemia by observing a reduction in heart rate and irregular ECG signals. To establish bidirectional optoelectrical interfaces between devices and heart tissue, place a silicon optoelectronic membrane onto the desired stimulation site on the heart. Allow it to self-attach to the epicardium using capillary force.

Connect the electrodes to the RHD recording system or a compatible electrophysiological platform. Then, position the flexible multi-electrode arrays on the ventricular surfaces of the isolated heart. Now, program the 635 nanometer laser source with the desired frequency and duty cycle using transistor-transistor logic signals.

Focus the laser beam to a one millimeter spot and align it over the silicon membrane. Start the recording and stimulation protocol. Gradually increase the laser intensity until continuous override pacing of the heart is observed.

To establish bidirectional electrode-based electrical interfaces between devices and heart tissue, connect the stimulation electrodes in a two-electrode configuration, placing a porous carbon working electrode on the left ventricular wall and a counter electrode on the right ventricular wall. Using a potentiostat, deliver square current waveforms, such as two milliampere with one millisecond pulse duration, until successful pacing of the heart is achieved. A nanoporous carbon-based platform for effective electrical modulation or sensing of cardiac systems was established.

Upon four hertz stimulation at one milliampere. both gold and nanoporous carbon-coated gold electrodes achieved effective overdrive pacing, with higher electrocardiogram amplitudes observed in the nanoporous carbon group. Both electrodes demonstrated an exponential decrease in threshold current with increasing pulse duration.

At a stimulation current of four milliamperes per square centimeter and one millisecond duration, the threshold voltage required for effective pacing was 1.32 volts for gold electrodes and 0.90 volts for nanoporous carbon-coated electrodes. A 16-channel nano porous carbon-grafted gold electrode significantly enhanced epicardial electrocardiogram signal detection, improving the signal-to-noise ratio by eightfold compared to standard gold electrodes. Successful induction of ischemia reperfusion infarction was validated post-experimentally by TTC staining, where infarcted hearts displayed distinct white regions representing myocardial damage.

During real-time monitoring, ischemic hearts exhibited significantly reduced heart rate. Multi-electrode array mapping of ischemic hearts revealed decreased electrical conduction velocity across the epicardium, indicated by increased signal delay.