Benchmark III ixk51

Approach

The proposed project will serve as a method of quantifying the recording capabilities of interfascicular electrodes, a novel method of interfacing with the nervous system. While the procedures outline below are presented in increasing order of complexity (mathematical model to acute experiments to chronic experiments), the aims can be performed in parallel, such that a temporary setback in achieving one specific aim will not hinder the progress of another.

Aim 1: To validate the claim that inter-fascicular electrodes can selectively record signals from separate fascicles using a finite element model

Several models of peripheral nerves have been created for the purpose of elucidating a mathematical relationship between a specific type of electrode and its ability to selectively record from (Perez-Orive and Durand 2000; Yoo and Durand 2005) or stimulate (Veltink, van Alste, and Boom 1988; Tyler, Peterson, et al. 2011) different fascicles. These models are primarily based on the groundbreaking work of Hodgkin and Huxley (Hodgkin and Huxley 1952) and the core conductor model (or cable theory) which has been improved and adapted through the years to include myelin (McNeal 1976).

Methodology

Creating mathematical model of nerve and electrodes

A finite-element model (FEM) of various shapes of nerves will be constructed uses Ansoft (ANSYS Inc.; Canonsburg, PA). The electrical properties of the neurons will be modeled using a double-cable model, as used in (McIntyre, Richardson, and Grill 2002) to better account for the properties of myelin. The neurons will be bathed in low-impedance solution, corresponding to that of the endoneurium. A high-impedance perineurium will separate fascicular space from the endoneurium and the fascicles will be suspended in a relatively high-impedance solution corresponding to the epineurium. The specific impedance values of neural tissue will be obtained from literature, primarily (Geddes and Baker 1967; Choi, Cavanaugh, and Durand 2001). Finally, an insulating cuff electrode (modeled as a silicone sheath) will be circumscribed around the nerve and interfascicular contacts will be placed into the epineurium (Figure 3). Neural activity will be recorded using a tripolar electrode configuration, with the outer contacts of tripole shorted together on the outside of the nerve and the inner contact being recorded using the interfascicular electrode.

Figure 3: FEM model of a nerve containing 5 fascicles. Interfascicular electrodes are in the epineurium, and have an insulated surface (black) and a contact surface (white). Image modified from (Perez-Orive and Durand 2000)

Evaluating selectivity of various electrode configurations

A selectivity index, as defined in (Perez-Orive and Durand 2000) will be computed for various electrode locations and nerve shapes. For instance, selectivity will be evaluated as a function of distance from the fascicle of interest and as a function of the number of interfascicular contacts in the nerve. Nerve geometry will be taken into account as well, since a reshaping cuff electrode will be used in the study; circular and flat nerve cross-sections will be tested, and the selectivity in both geometries will be compared.

Adding inhomogeneities, modeling in a non-ideal environment

The final step in the modeling study will involve removing the homogeneity constraints in the model and addressing a more physiologically-relevant case, where fascicles are spread out unevenly across the epineurium and other longitudinal structures (such as blood vessels) are also present. The simulations will be run several times with randomized non-homogeneity conditions to ensure that the electrode does not rely on a perfect symmetry to achieve selectivity.

Expected outcomes, potential problems, risks

Interfascicular electrodes have been shown to be highly selective in stimulation experiments in finite-element models (Tyler, Peterson, et al. 2011). Since such electrodes can be implanted just a few microns away from active fascicles, there is reason to believe that they should exhibit excellent recording selectivity as well. For instance, in Figure 3, electrodes with the contact surface facing a fascicle are likely to record a large signal from the nearby fascicle and a significantly weaker signal from all other neighboring fascicles.

Several problems have been anticipated in the modeling phase and are addressed here. First, it is possible that simulating electrical activity of millions of axons will prove to be too time-consuming, and ultimately unnecessary to show the needed data. In this case, the model will be simplified to a computationally efficient, yet still physiologically faithful model of neural activity (Peterson, Izad, and Tyler 2011). Likewise, the selectivity results of the non-homogenous model may turn out to be more based on the specific geometry of the nerve than on the configuration of electrodes. This is somewhat expected since the electrodes cannot be selective in all geometries. If this is the case, we will simulate real nerves by using actual histology data of real nerve cross-sections. This should still validate the use of interfascicular electrodes without the variability associated with randomly picking nerve geometries.

Aim 2: To quantify the recording selectivity of interfascicular electrodes in animal models

A logical step in evaluating the performance of interfascicular electrodes is testing them in an actual animal model. This can be done in parallel to Aim 1; however, when both are complete, it would be beneficial to evaluate the fidelity of the mathematical model by comparing its selectivity metrics to those obtained when using the animal model.

Acute experiments have been performed to evaluate the performance of peripheral recording electrodes in many studies (Yoo and Durand 2005; Branner and Normann 2000; Loi et al. 2011).

Figure 4: (left) interfascicular wires will be implanted into the epineurium, between fascicles (unpublished image from lab of Dustin Tyler). (Right) Example of neuronal degeneration close to intrafascicular electrode implant site (*). Arrow indicates thick tissue that surrounds implant and may worsen signal quality. Surrounding neurons are in an injured state, as evident by thinning of myelin sheath. Image from (Lefurge et al. 1991)

Methodology

Experimental procedures

Teflon-insulated Platinum-Iridium wires of diameters ranging from 50 to 100 µm will serve as interfascicular electrodes. One millimeter of one side (180º) of each wire will be de-insulated at the tip. Additionally, two tripolar FINE electrodes (one for stimulation and one for recording, with a contact separation of 1 cm) will be manufactured and the recording FINE will be modified to house the interfascicular wires. Multiple wires of this diameter may be housed in the cuff.

Acute experiments will be performed on anesthetized rabbits. The sciatic nerve will be exposed bilaterally and the stimulation FINE will be implanted approximately 1 cm distal to the bifurcation of the sciatic nerve onto the tibial and the peroneal branches. The recording electrode with up to 5 interfascicular wires will be implanted just proximal to the bifurcation in order to record antidromic compound action potentials. All experimental procedures will be approved by the Institutional Animal Care and Use Committee (IACUC).

Implantation procedure

An accurate method of electrode insertion into the epineurium will need to be developed. It is reported that the force required to penetrate the perineurium (a tough cellular layer enclosing fascicles) is significantly greater than that required to penetrate the epineurium (unpublished observations, (Sergi et al. 2006)). Based on this, a method of electrode insertion can be created that utilizes a force-controlled stepper motor that drives the electrodes into the epineurium but not in the fascicle. Ultimately, the wires should be implanted in the epineurium (Figure 4, left) and the depth of the implantation will be controlled once again using the stepper motor.

The common peroneal and tibial branches of the sciatic nerve will be activated using the stimulating electrode, and compound action potentials (CAPs) will be captured using the modified recording FINE, filtered, amplified and digitized. Selectivity will be quantified by the electrode’s ability to record and recognize neural signals emanating from different branches. A selectivity index (SI), as defined in (Yoo and Durand 2005) will also be computed. Spontaneous neural activity will be recorded by activating the afferent sensory fibers in the leg of the animal. This will be done by pinching the toe (to stimulate nociceptive fibers) and moving the leg. At the end of the experiment, the nerve will be excised, fixed, preserved, sectioned and stained to reveal the location of the contact in the nerve. The actual electrode contacts will be left inside the nerve until the tissue is fixed in formalin; after that, they will be removed as to not interfere with the sectioning process.

Evaluate selectivity, SNR of interfascicular electrode designs

Using the selectivity metrics, it will be possible to determine the optimal number of interfascicular contacts necessary to achieve maximum selectivity (selectivity index is expected to increase asymptotically, implying that after a certain point, adding more interfascicular contacts will not significantly improve selectivity). Additionally, the SNR of the electrodes will be evaluated and averaged over several experiments and separated into motion SNR (achieved when the leg of the animal is being moved, which activates the neurons synapsing on spindle fibers) and nociceptive SNR (the response to the pinching and pricking of the leg). Both metrics are physiologically relevant since they evaluate the ability of the interfascicular electrode to record actual (not electrically evoked) activity, which is useful for high-fidelity neuronal control systems.

Analysis of histology

The images of cross-sections of neurons around the implantation site will be stored and analyzed. The purpose of the analysis will be two-fold: first, it will be used to ensure that the interfascicular electrodes are in fact in the epineurium, which is a key metric in this study. Second, high-resolution histology slides will be able to show signs of acute neuronal damage such as demyelination, neuronal death, and blood vessel rupture (see Figure 4, right). The presence of any of these acute signs of damage will be beneficial in beginning the chronic studies; clearly, non-recoverable acute neuronal damage cannot be tolerated in long-term experiments.

Expected outcomes, potential problems, risks

First, we expect the histology to reveal that all interfascicular wires implanted using the developed system were in fact in interfascicular space which would validate the implantation approach, and we expect to see little to no signs of acute neuronal degeneration since the electrodes are not in direct contact with neurons. One potential concern is that if the electrodes in the epineurium are too large, they might rupture blood vessels, causing localized edema in the nerve. This is a serious potential concern that can affect the ability of the electrode to perform in chronic experiments. It can be addressed by making the electrodes much smaller by using smaller-diameter wire. Wires are used for the specific purpose of making the electrode less sharp and stiff, which would minimize the risk of penetrating blood vessels.

Second, we expect the interfascicular electrodes to record selectively in vivo, as defined by their ability to record different signals for different branches of the sciatic nerve. There is preliminary data suggesting that interfascicular electrodes demonstrate the capacity for “preferential” recording, a precursor to the selectivity index that indicates a relationship between contact position within the nerve and recorded activity from different fascicles (Figure 5).

Figure 5: Preferential recording is achieved using interfascicular wire. Prior to rotation (solid), the electrode records 100% of both tibial and c. peroneal signals. After rotation (dashed), the tibial branch is still recruited to 95% (circle) while the signal from the c. peroneal branch reaches only ~ 50%, indicating a recording orientation favorable to the tibial branch (I. Kolb, unpublished observations).

Some possible complications will be addressed. First, threading the electrodes into the epineurium may be more difficult than expected, especially with low Young’s modulus wires that bend readily under pressure. In this case, purely for the purposes of the acute experiments, silicon-based microelectrode shanks such as the Michigan probe (www.neuronexus.com), may be inserted into the epineurium using a micromanipulator. Their location in the nerve can be assessed using a microscope. This would not be a final design for an interfascicular electrode since one of the reasons for using wires as opposed to stiff shanks is the decreased chance of neuronal damage.

A second potential risk is the potential inability of the interfascicular electrode to reject outside interference such as electromyographic (EMG) signals from muscle activity and stimulus artifact from the inherent electric field present when stimulating (see (Triantis and Demosthenous 2006)). This problem can be avoided by using predictive template matching to remove the stimulus artifact. The EMG interference can likewise be minimized by increasing the order of the filter in the recording circuit since the frequency content of ENG signals is somewhat different than that of EMG signals (Popovic et al. 1993).

Aim 3: To determine the viability of using interfascicular electrodes in chronic animal studies

The feasibility of any device (including nerve electrodes) implanted into humans must go through a rigorous stage of chronic animal testing to determine its safety and efficacy. The third Aim will serve as a chronic animal study that will evaluate the feasibility of using this electrode in humans. Here, the animal’s normal gait will be used to assess the electrode’s ability to record very low-amplitude neural signals and selectively pick out the muscle that is currently active. The signals obtained with the nerve electrode will be compared to those obtained with EMG electrodes to correlate the two signals and determine whether it identified the active muscle correctly.

Figure 6: Ensemble recording from a dorsal root ganglion (DRG) of an awake, walking cat. The timing of spikes during normal gait (and in the case of the present study, the associated EMG data) helps identify which population of neurons is active. Image from (Aoyagi et al. 2003).

Methodology

Electrode fabrication

Flat Interface Nerve Electrodes (FINEs) will be manufactured using the methods previously described in (Yoo and Durand 2005), except the middle array of electrodes will be interfascicular wires. There will be several wires in the epineurium – the specific number will be determined by the results of Aim 1 and 2, which should quantify the optimized number of contacts necessary to attain maximum selectivity. Additional parameters of the interfascicular electrodes (such as composition, diameter, contact size, etc) will also be defined based on the results of the acute experiments.

Chronic experiment procedures

Five chronic experiments will be performed on canines under sterile surgical procedures. The canines will be anesthetized, and bilateral incisions will be made on both legs, just proximal to the knee joint to expose the bifurcation of the sciatic nerve into the common peroneal, tibial, and sural branches. The interfascicular FINE will be implanted just proximal to the bifurcation. Additionally, EMG electrodes will be implanted in the muscles innervated by the sciatic nerve, the tibialis anterior, posterior tibialis, and gastrocnemius. The wound will be closed and bandaged, and the animals will be given time to recover from the surgery. All animal protocols will be approved by IACUC.

Evaluate chronic performance of interfascicular electrodes (selectivity, SNR)

Animals will be observed over a period of 10 weeks. Once every week, an experiment to evaluate the recording selectivity of the interfascicular electrodes will be performed. Each dog will be equipped with a harness to house recording equipment and will be observed while walking on a treadmill for 10 minutes. During this time, the ENG from the interfascicular electrode and EMG from the relevant leg muscles will be recorded and stored for subsequent analysis.

The ENG recorded using each interfascicular contact will be analyzed using Principle Component Analysis (PCA) techniques to identify various spike types and compiled into a recording map (see Figure 6) where all ENG and EMG signals are on the shown on the same time scale. Correlation coefficients will be found between each ENG and EMG signal to determine whether it is possible for several interfascicular contacts to record all information relayed by the sciatic nerve during normal gait. The SNR of each contact will be calculated using correlation to EMG: the “signal” will be present when the corresponding EMG is active” while “noise” will be defined as all non-active time regions. The correlation coefficients and the SNR will be measured within the 10-week time frame to determine the effect of long-term implantation on signal quality and ability to selectively record muscle activity.

Evaluate neuronal damage

At the end of the 10-week experiment protocol, the sciatic nerve will be excised, fixed, preserved, sectioned and stained to reveal the location of the contacts in the nerve. The actual electrode contacts will be left inside the nerve until the tissue is fixed in formalin; after that, they will be removed as to not interfere with the sectioning process. The presence of chronic neuronal damage will be quantified by examining the nerve cross-sections closest to the implantation site for demyelination and neuronal necrosis.

Expected outcomes, potential problems, risks

It is expected that the interfascicular electrode will be able to selectively record all neuronal activity associated with normal gait to some degree. In other words, we expect each EMG recording to correlate with the recording made with at least one interfascicular contact. Additionally we expect the SNR of this electrode to be comparable to that of other chronic nerve electrodes used to record physiological activity (Sahin, Durand, and Haxhiu 1999).

One of the hazards associated with chronic implantation is the effect of electrode encapsulation on recording. Since chronic studies of interfascicular electrodes have not been reported to date, the response of the nerve to electrodes in the epineurium is unknown. This hazard will be minimized by precise and minimally invasive surgery techniques that should minimize local damage and prevent a significant inflammatory response.

Additionally, it is possible that one or more electrode contacts fail in the duration of the experiment due to errors during surgery or inflammatory response that pushes the electrode out of the epineurium. If too many contacts are damaged (as defined by the optimal number of contacts in the epineurium in Aim 2), the experiment on that specific animal may be terminated prematurely; this will warrant acquiring another animal.

Timeline:

The proposed timeline for the project is 4 years. A beneficial aspect of this proposal is that at least the first two specific aims can be addressed simultaneously. We plan to begin the two experiments in the first year and plan for the first Aim to run approximately a year and the second, 3 years (in order to fully develop a non-damaging interfascicular electrode and evaluate its efficacy in vivo). The last aim will be completed within the span of 1 year, allotting time for needing to replace one or more of the animals in the study due to potential problems with implantation or electrode longevity.

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