Heart attacks or myocardial infarction (MI) could result in an irreversible loss of ... recording system for small animal models of heart regeneration. The system ...
A Wireless EeG Recording System for Small Animal Models of Heart Regeneration Hung Caol, Yu Zha02, Ammar B. Koukil, Yu-chong Tai3 and Tzung K. Hsiai4 1 Department
of Electrical Engineering, ETS Montreal, QC, Canada
2SIA T, Chinese Academy of Sciences, Shenzhen, China 3Department of Electrical Engineering, Caltech, Pasadena, CA, USA 4Department of Bioengineering, UCLA, Los Angeles, CA, USA Abstract - Heart failure afflicts the developed world, causing mortality more than any other diseases. This is due to the fact that humans' heart possesses a very limited capacity to regenerate. Heart attacks or myocardial infarction (MI) could result in an irreversible loss of cardiomyocytes and consequently heart failure. Besides, zebrafish and neonatal mice are well-known for their magical capacity to recover after ventricular amputation, thus becoming precious models for heart regeneration studies.
In this
work, we report the first wireless electrocardiography (ECG) recording system for small animal models of heart regeneration. The system consists of a microelectrode
array
(MEA)
and
electronic components for wireless powering, signal processing and data communication. The MEA is based on a biocompatible and flexible polymer so it could conform to non-planar anatomical surfaces. The power transfer is achieved using inductive coupling between two solenoids and the ECG signals are sent through an optical
link.
The
wireless
operation
can
free
the
animal,
eliminating anesthesia during experiments and thus minimizing unwanted side effects. The first generation of the device was demonstrated successfully with neonatal mice, revealing awake ECG signals with all features, physiologically
investigate
heart
thereby
paving the
regeneration
way to
in
long-term
flexible MEA,
zebrafish,
without disrupting the animals' normal activities. Index Terms - Wireless ECG,
neonatal mice, inductive coupling, heart regeneration.
I.
INTRODUCTION
Understanding heart regeneration in a vertebrate model system is highly important to public health. Heart failure has been the leading cause of morbidity and mortality in the developed countries due to failure to adequately replace lost ventricular myocardium from ischemia-induced infarct. Adult mammalian ventricular cardiomyocytes have a limited capacity to divide, and this proliferation is insufficient to compensate the significant loss of myocardium from ventricular injury [1]. Unlike adult mammals, zebrafish (Dania reria) are well known for their regeneration capacity after 20% ventricular amputation [2]. Further, Porrello et al. recently discovered the transient regenerating capacity of I-day-old neonatal mice after birth, but this capacity is lost by 7 days of age [3]. Therefore, these become precious models for drug discoveries and investigations of heart regeneration. Although imaging means were typically used to characterize heart injuries and the regeneration process phenotypically and genetically, electrocardiogram (ECG) recording using microelectrodes could also be useful to elucidate changes in cardiac-conduction functionalities of injured myocardium [4]. With the zebrafish model, it has been shown that ventricular
repolarization (ST intervals and T waves) failed to normalize despite fully regenerated myocardium at 60 days post ventricular amputation, suggesting further cardiac remodeling may be required to fully integrate regenerating myocardium with host myocardium [5]. However, the current ECG recording methodology used with the animals requires pharmacological sedation which affects the cardiac information [6]. Therefore it is highly desirable to have a long-term wearable and wirelessly-operated ECG recording system to collect data continuously from non-anesthetized animal models. Nevertheless, the small size and irregular movements of the aforementioned small animals are challenging issues for implementing such a system. Therefore, we aim to address and target (1) Surface-conformable recording electrodes with biocompatibility and flexibility; (2) A proper mechanical fixture that can secure the device for stable and long-term recording; and (3) A recording circuitry operated on wireless power instead of battery for continuous and long-term measurement. Furthermore, the integrated and packaged system needs to be compact and light-weighted in order to bring comfort to the animals and avoid unwanted side effects. In this work, we implemented flexible polymer-based membranes containing gold rnicroelectrodes using traditional micro-fabrication technologies to target continuous wireless ECG acquisition in small animal models. The signal processing circuitry was designed to be assembled within the membrane. Wireless power transfer was done by inductive coupling between 2 solenoids while wireless data communication was optically achieved to accommodate the under-water situation in the case of zebrafish. The block diagram and the conceptual design of our system are illustrated in Fig. 1. A proof-of concept system was prototyped on a printed circuit board (PCB) using off-the-shelf components for the demonstration with
(a)
(b)
Fig. 1. (a) The block diagram and (b) the conceptual design of the wireless ECG sYstem using 2 solenoids.
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neonatal mice. Awake ECG signals were obtained with comparable signal-to-noise ratios and the rhythm was different with that recorded with sedated animals as expected. II. DESIGN AND FABRICATION
A. MEA fabrication Parylene C was chosen as the base and insulating material due to its well-suited combination of biocompatibility and low permeability against water, gases and ions. Using conventional micro-fabrication techniques, we fabricated gold electrodes (0.2/0.02 m of Au/Ti) sandwiched between two layers of parylene C with exposed recording cites and connecting pads. A MEA was picked to record ECG signals instead of one single electrode in order to obtain the site-specific ECGs [4], similar to the standard 12-lead ECG used for humans. Several electrode configurations in shape, size and spacing were implemented in order to optimize for a specific application. Using impedance analyses, MEAs of four round recording electrodes were chosen with diameters of 200-300 /lm. The reference electrode was placed apart from the recording electrodes. Low-power oxygen plasma was applied to roughen the microelectrode surface, allowing for an increase in the effective contact area and a decrease in the interface impedance. To facilitate long term operation, the membrane was coated with the biocompatible silicone to match with the YoungN modulus of the animal skin. The membrane was measured 10 m in thickness and less than 0.5 mg in weight. In the case of zebrafish, silicone wing components were added to support long-term implantation by wrapping around the fish body and applying medical epoxy at the two ends on the dorsal side (Fig. 2a and 2b). �
B.
The signal processing circuitry and wireless power link
The circuitry on the animal side was designed to be assembled within the parylene membrane. Gold traces were fabricated for circuit routing and conductive epoxy was used instead of soldering. Packaging was achieved by casting silicone. The signal amplification and filtering were critical to address the small ECG signal strength «150 !lV) and frequency range (2-125 Hz). The signal processing circuitry consisted of a two-stage amplifier and a bandpass filter prior to transmitting the signals to the data receiver. Signals with frequency below 2 Hz and above 125 Hz were filtered out. The circuit communicated with the external unit via an Infrared Light Emitting Diode (IRLED). To operate the entire system, a DC voltage of 1.8 V or above and an average power of 300 /lW were required. A 2-coil inductive link coupled in a solenoidal configuration was designed to provide power continuously. This would guarantee stable wireless powering, eliminating the misalignment issues when using parallel planar coils [7]. The transmitter coil had 20 turns, a diameter of 3 cm and a length of 6 cm, resulting in an inductance of 6.62 /lH. With those design parameters, there would be enough space to put a water tube containing a
Receiver Coil
Amplifiers and Filters Ventral � of Zebnfhh
,.,..
(dl
lei
Fig. 2. (a) Zebrafish with an attached MEA membrane. (b) Power transfer efficiency analysis of the receiver coil (inset).
(c)
Conceptual desIgn of the device. (d) First-gen PCB prototype.
zebrafish or a small cage housing a neonatal mouse within the coil (Fig. 1b). A miniaturized and high-quality-factor receiver power coil was constructed by winding the 30/48 Litz wire around a NilZn ferrite core to establish high magnetic permeability and low electrical loss. It had 10 turns, an outer diameter of 1.2 mm, an inner diameter of O. 7 mm, and a length of 4 mm . At 10 MHz an inductance of 143 nH and a quality factor of 23.6 were achieved. The overall power transfer efficiency was measured, revealing a constant efficiency of 1.2% in the frequency range 9-15 MHz (Fig. 2b); thus, the operating frequency of 11.1 MHz was selected. The external unit had an optical detector to acquire the ECG data which were stored and displayed in a computer. ,
C.
The first-gen device
The first generation of the system was implemented for uses with neonatal mice. The signal processing and wireless communication circuitry were prototyped on a PCB including two-stage amplification, band-pass filtering, inductive power transfer, and optical data transmission. Surface-mount components were utilized instead of bare-dies in the case of �ebrafish. The ECG signals were passed through a first-stage mstrumental amplifier (INA333 from Texas Instrument) to provide a high input impedance, and then to an operational amplifier (OPA333 from Texas Instrument). The MEA was manually assembled to a customized zero-insertion-force (ZIF) cable end so that it could be connected to the PCB (Fig. 2d). The solenoids and optical data transfer part were implemented as mentioned in the previous section. III. EXPERIMENTS AND RESULTS The animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. For wireless ECG recording, neonatal mice aged between 1 to 7 days old were positioned on
978-1-4799-8275-2/15/$31.00 ©2015 IEEE
a baseplate without sedation. The PCB was first gently fixed on the abdomen of the mouse by medical tapes, followed by adhering the MEA membrane to the chest. The mouse lying on baseplate was then placed inside the power transmitting coil. When the power was activated, real-time wireless ECG signals were acquired (Fig. 3). The distance between the IRLED and the photo-detector was about 3 cm. Reproducible trials on neonatal mice were performed to reveal distinct ECG signals in the presence and absence of pharmacological sedation. In Fig. 3, a comparison in ECG signals between the non-sedated and sedated conditions highlights the distinct heart rates and ECG repolarization patterns. A reduction in the heart rates by 50% was noted in the presence of sedation (80 Ilg/gr body weight Ketamine/xylazine). With the mechanical interference during recording and the signal attenuation during transmission, the P waves, QRS complexes and T waves remained distinct despite a reduction in SNR without sedation (Fig. 3b). Fig. 3. ECG signals of (a) a sedated mouse and (b) an awake
IV.
DISCUSSIONS AND CONCLUSIONS
The small size and constant movements of the small animals remain a challenge to implement a wireless ECG system. Here, we developed conformable flexible sensors with dry-contact MEA membrane, enabling secure ECG recordings in long term. For the first time, with the wireless feature, we have provided electrophysiological signals from a neonatal mouse model of heart regeneration in the absence of sedation. The capability to monitor intrinsic heart rates and electrical signals without exogenous neurologic influence opens a new road for cardiac and neurologic dug screening, disease modeling, and toxicity studies. With further miniaturization, the validated wireless ECG recording through the dry-attachable microelectrode membrane and its variations can acquire electrical phenotypes from conscious, freely-moving small animals without interfering with the intrinsic heart rates, causing animal stress, and predisposing to infection and injury. The recent advances in stem cell-based therapy open long term strategies to address heart diseases. Thus, our wearable and wireless ECG monitoring system would further offer a non invasive validation tool to address maturation and integration of the stem cell-derived cardiomyocytes. With the development of flexible and stretchable electronics as well as wireless technology [8, 9], our electrode membrane could be further utilized not only for pre-clinical animal models of regenerative medicine but also in addressing health monitoring for personalized medicine and telemedicine.
mouse using our system. The inset shows a full-feature ECG obtained by our wireless system.
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ACKNOWLEDGEMENT
The studies were supported by the National Institutes of Health ROIHL-083015 (T.K.H.) and ROIHLll1437 (T.K.H.).
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