**6. Spinal cord prosthesis**

### **6.1. System overview**

**Figure 7.** SEM of electrode morphology showing parylene C insulation surrounding exposed metal electrode.

Parylene C-based arrays of thin-film platinum electrodes, comprising four 200 µm diameter stimulating electrodes and 56 recording electrodes of 10 µm diameter were fabricated accord‐ ing to the single-metal-layer process on a glass substrate. These were placed in a bicarbonate perfusate under a microscope and connected to a stimulus generator and preamplification board (Multi Channel Systems MCS GmbH, Reutlingen, Germany) [45]. As shown in Figure 8, a retina isolated from larval tiger salamander (*Ambystoma tigrinum*) was placed RGC side down on the array (to simulate epiretinal stimulation), and a remote platinum ground

**Figure 8.** Isolated larval tiger salamander retina (darker region at left) overlying parylene-based platinum electrode array. Arrow indicates 10 µm diameter electrode used for recording trace in Figure 9. Asterisk identifies 200 µm diam‐

eter stimulating electrode used to generate action potentials seen in Figure 9.

**5.2.** *In vitro* **retinal recording and stimulation**

12 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

electrode was introduced to the bath.

The ideal spinal cord stimulation system, just like the retinal system, would have a power source, circuitry for driving the appropriate electrodes, as well as a cable and electrode array, this time implanted epidurally or subdurally on the spinal cord. We believe that a penetrating electrode array would be problematic for implantation and would likely lose efficacy and fail ultimately due to a gliosis over time, as has been shown in many other studies [27]. The power source could be an RF coil, or could, due to the much larger space available in the abdomen and back as compared with the eye, be a rechargeable battery capable of charging through the inductive link. The RF coil, in addition, would enable reprogramming of the implanted electronics for alternative stimulation protocols at the physician's discretion. The electrode array should be conformable to the spinal cord so that it can tonically stimulate at low currents and with high precision. While a completely implantable system is the ultimate goal, an interim goal is to stimulate the spinal cord chronically from an array connected to a head plug, while simultaneously being able to record electromyograms (EMGs). In order to achieve this, we have studied the efficacy of the multielectrode array portion of this system and have begun to develop a connector technology capable of connecting 36 electrodes in with a small enough form factor to be chronically mounted on a mouse skull.

**Figure 10.** Parylene MEA for murine spinal cord stimulation and recording.

### **6.2. Fabrication**

Spinal cord arrays, consisting of five or ten electrodes of 250 µm diameter were designed and fabricated (Figure 10). Interelectrode spacing was controlled so that each array of electrodes covered four to five segments of the murine lumbosacral spinal cord upon implantation. Suture holes were also designed into the body of the array to ensure placement and attachment of the array on the cord, as well as to facilitate implantation (suture can be attached to the end of the array and can be threaded along the cord first to help direct the array along it).

The single-metal layer fabrication process was performed using a contact aligner process for fast throughput. The fabricated arrays were annealed to increase the adhesion of parylene to parylene. At the same time, they were clamped between two pieces of Teflon or glass slides coated with aluminum foil to ensure they would be flat during implantation. The arrays were connected via Clincher connectors (FCI, Versailles Cedex, France) to the stimulation and recording electronics.

### **6.3. Implantation and testing**

Just prior to implantation, the arrays were rinsed in isopropyl alcohol. Under isoflurane anesthesia, the spinal cord electrode arrays were implanted epidurally on spinal cord seg‐ ments L2-S1 in nontransected mice. The electrodes were oriented linearly along the rostrocau‐ dal extent of the cord. Recording capability was assessed by using the electrode array to record spinal cord potentials evoked by tibial nerve stimulation. Following stimulation of the tibial nerve, somatosensory evoked potentials were recorded from the cord dorsum at three lumbo‐ sacrallevels(P1-P3,rostraltocaudal).Therecordedwaveformconsistedofthreeresponsepeaks, two of whichare clearlydepictedinFigure 11 (N1 and N3).These findings closelymirrorresults reported previously in a study using conventional spinal cord recording electrodes [46] demonstrating that the recording capability of the array electrodes matches that of convention‐ al electrodes. By measuring the difference in the response latencies obtained at each electrode position (corresponding to different levels of the spinal cord), and by utilizing the known, fixed interelectrode spacing, accurate measurements of the conduction velocities were obtained. The propertiesofthese responses canpotentiallybeusedtodiagnose theprogressive recoveryofthe spinal cord as a result of treatments provided after a spinal cord injury.

and back as compared with the eye, be a rechargeable battery capable of charging through the inductive link. The RF coil, in addition, would enable reprogramming of the implanted electronics for alternative stimulation protocols at the physician's discretion. The electrode array should be conformable to the spinal cord so that it can tonically stimulate at low currents and with high precision. While a completely implantable system is the ultimate goal, an interim goal is to stimulate the spinal cord chronically from an array connected to a head plug, while simultaneously being able to record electromyograms (EMGs). In order to achieve this, we have studied the efficacy of the multielectrode array portion of this system and have begun to develop a connector technology capable of connecting 36 electrodes in with a small enough

Spinal cord arrays, consisting of five or ten electrodes of 250 µm diameter were designed and fabricated (Figure 10). Interelectrode spacing was controlled so that each array of electrodes covered four to five segments of the murine lumbosacral spinal cord upon implantation. Suture holes were also designed into the body of the array to ensure placement and attachment of the array on the cord, as well as to facilitate implantation (suture can be attached to the end of the

The single-metal layer fabrication process was performed using a contact aligner process for fast throughput. The fabricated arrays were annealed to increase the adhesion of parylene to parylene. At the same time, they were clamped between two pieces of Teflon or glass slides coated with aluminum foil to ensure they would be flat during implantation. The arrays were connected via Clincher connectors (FCI, Versailles Cedex, France) to the stimulation and

Just prior to implantation, the arrays were rinsed in isopropyl alcohol. Under isoflurane anesthesia, the spinal cord electrode arrays were implanted epidurally on spinal cord seg‐

array and can be threaded along the cord first to help direct the array along it).

form factor to be chronically mounted on a mouse skull.

14 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

**Figure 10.** Parylene MEA for murine spinal cord stimulation and recording.

**6.2. Fabrication**

recording electronics.

**6.3. Implantation and testing**

To test the capability of the electrode array to act as a multichannel stimulating device for generating hindlimb movements, constant-current monophasic stimulus pulses (amplitude: 50-850 µA, frequency: 0.3-10 Hz, pulse duration: 0.5 ms) were applied to the spinal cord between each of the array electrodes and a ground electrode located near the shoulder, while muscle activity was monitored using electromyogram (EMG) recordings of the tibialis anterior and medial gastrocnemius muscles. Stimulation generated a typical three-component EMG action potential consisting of an early (direct motor), a middle (monosynaptic), and a late (polysynaptic) response, classified by post-stimulus latency (Figure 12). These data clearly indicate that the parylene arrays were able to stimulate the spinal cord in such a way that the musculature was activated.

**Figure 11.** Peak amplitudes of somatosensory evoked potentials (N1 and N3) recorded from three levels of the rostro‐ caudal spinal cord (P1-P3). Example waveform at top shows approximate response times.

**Figure 12.** Typical medial gastrocnemius (ankle plantarflexor) EMG recording showing early, middle, and late respons‐ es after stimulation of spinal cord with parylene MEA.

Because of the known spacing of the electrodes on the array (as compared with traditional fine-wire electrodes which do not have known interelectrode spacing), we were able, in addition, to determine whether electrode position had a significant impact on muscle recruit‐ ment. The appearance and magnitude of each of the EMG responses was indeed correlated with the choice of electrode position (Figure 13). This serves as evidence that position of stimulation is very important. With a one-dimensional array, it is difficult to assess whether a bilateral stimulation paradigm would also result in lateralization of response, but we strongly suspect that this would be the case.

**Figure 13.** Medial gastrocnemius EMG showing varying levels of activation due to stimulation at different rostrocau‐ dally located electrode sites.
