Mario I. Romero-Ortega

Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers

A biocompatible neural microprobe constructed using well-established SU-8 microfabrication techniques is described that was designed to record fiber spike signals from regenerated axons within peripheral nerves. These microprobes features bipolar longitudinal gold electrodes recessed below the surface within ldquogroovesrdquo designed to guide the growth of regenerating axons along the length of the grooves and limit the number of fibers that come in contact with the longitudinal electrodes. In addition, screening microprobe toxicity using cultures of human skin fibroblasts, the biocompatibility of these SU-8 microprobes for neural interface applications, in particular, was specifically verified using primary cultures of two sensitive cell types found in peripheral nerves: purified Schwann cells and explanted dorsal root ganglion (DRG) neurons and their fibers. The SU-8 microprobes were surgically implanted into transected rat Sciatic nerves within a unique peripheral nerve regeneration tube. Long-term fiber spike signals were recorded with these SU-8 microprobes in 13 chronically implanted rats for periods from 4 to 51 weeks without any signs of tissue damage or inflammatory reaction.

Early interfaced neural activity from chronic amputated nerves

Direct interfacing of transected peripheral nerves with advanced robotic prosthetic devices has been proposed as a strategy for achieving natural motor control and sensory perception of such bionic substitutes, thus fully functionally replacing missing limbs in amputees. Multi-electrode arrays placed in the brain and peripheral nerves have been used successfully to convey neural control of prosthetic devices to the user. However, reactive gliosis, micro hemorrhages, axonopathy and excessive inflammation currently limit their long-term use. Here we demonstrate that enticement of peripheral nerve regeneration through a non-obstructive multi-electrode array, after either acute or chronic nerve amputation, offers a viable alternative to obtain early neural recordings and to enhance long-term interfacing of nerve activity. Non-restrictive electrode arrays placed in the path of regenerating nerve fibers allowed the recording of action potentials as early as 8 days post-implantation with high signal-to-noise ratio, as long as 3 months in some animals, and with minimal inflammation at the nerve tissue-metal electrode interface. Our findings suggest that regenerative multi-electrode arrays of open design allow early and stable interfacing of neural activity from amputated peripheral nerves and might contribute towards conveying full neural control and sensory feedback to users of robotic prosthetic devices.

Modality-Specific Axonal Regeneration: Toward Selective Regenerative Neural Interfaces

Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed sub-modality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF) and neurotrophin-3 (NT-3), to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5-fold compared to that in saline or NT-3, whereas the number of branches increased threefold in the NT-3 channels. These results were confirmed using a 3D “Y”-shaped in vitro assay showing that the arm containing NGF was able to entice a fivefold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a “Y”-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted toward the sural nerve, while N-52+ large-diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces.

Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons.

Cumulative evidence from both humans and animals suggests that the anterior cingulate cortex (ACC) is important for pain-related perception, and thus a likely target for pain relief therapy. However, use of existing electrode based ACC stimulation has not significantly reduced pain, at least in part due to the lack of specificity and likely co-activation of both excitatory and inhibitory neurons. Herein, we report a dramatic reduction of pain behavior in transgenic mice by optogenetic stimulation of the inhibitory neural circuitry of the ACC expressing channelrhodopsin-2. Electrophysiological measurements confirmed that stimulation of ACC inhibitory neurons is associated with decreased neural activity in the ACC. Further, a distinct optogenetic stimulation intensity and frequency-dependent inhibition of spiking activity in the ACC was observed. Moreover, we confirmed specific electrophysiological responses from different neuronal units in the thalamus, in response to particular types of painful stimuli (i,e., formalin injection, pinch), which we found to be modulated by optogenetic control of the ACC inhibitory neurons. These results underscore the inhibition of the ACC as a clinical alternative in inhibiting chronic pain, and leads to a better understanding of the pain processing circuitry of the cingulate cortex.

SU8-Based Micro Neural Probe for Enhanced Chronic in-Vivo Recording of Spike Signals from Regenerated Axons

In this paper, a fully biocompatible and flexible micro neural probe based on SU-8 to record neural spike signals from regenerated axons from amputated peripheral nerves is presented. The channel structures were designed at the tip of the probe to capture a single axon and guide its growth to ensure good quality contact between the single axon and the gold micro electrode. The gold electrodes placed at the bottom of the channel structures were exposed to allow contact with axons. The test results of in-vitro cytotoxicity tests showed that the SU-8 microprobe is biocompatible. Dorsal root ganglion (DRG) was used to evaluate neuronal cell adhesion to the microelectrodes. The probe was assembled into a fully biodegradable biosynthetic nerve regeneration guide (BNRG) filled with nerve growth factor (NGF) that promotes regeneration of specific sensory or motor axons. Neural spike signals from regenerated axons from amputated peripheral nerves were chronically recorded (> 6 months) without any sign of tissue damage.

In-vivo tests of a 16-channel implantable wireless neural stimulator

Wireless stimulation of neural tissue could enable many emerging neural prosthesis designs, and eliminate problems associated with percutaneous wires and connectors. Our laboratory has developed a 16-channel wireless floating microelectrode array (WFMA) for chronic implantation. Here, we report on its first use within in-vivo experiments, using a rat sciatic nerve model. Stimulus currents and associated muscular movements were determined for electrodes of two WFMA devices implanted into four animal subjects.

Chronic in-vivo testing of a 16-channel implantable wireless neural stimulator

Here, we report on chronic in-vivo testing of a 16-channel wireless floating microelectrode array (WFMA) in a rat sciatic nerve model. Muscle threshold currents, charge injection levels, and charge density were monitored for electrodes of two WFMA devices implanted into animal subjects over a five month period. This type of wireless stimulation device could eliminate problems associated with percutaneous connectors for a variety of neural prostheses and other medical devices.

Coiled polymeric growth factor gradients for multi-luminal neural chemotaxis

In the injured adult nervous system, re-establishment of growth-promoting molecular gradients is known to entice and guide nerve repair. However, incorporation of three-dimensional chemotactic gradients in nerve repair scaffolds, particularly in those with multi-luminal architectures, remains extremely challenging. We developed a method that establishes highly tunable three-dimensional molecular gradients in multi-luminal nerve guides by anchoring growth-factor releasing coiled polymeric fibers onto the walls of collagen-filled hydrogel microchannels. Differential pitch in the coiling of neurotrophin-eluting fibers generated sustained chemotactic gradients that appropriately induced the differentiation of Pheochromocytoma (PC12) cells into neural-like cells along an increasing concentration of nerve growth factor (NGF). Computer modeling estimated the stability of the molecular gradient within the luminal collagen, which we confirmed by observing the significant effects of neurotrophin gradients on axonal growth from dorsal root ganglia (DRG). Neurons growing in microchannels exposed to a NGF gradient showed a 60% increase in axonal length compared to those treated with a linear growth factor concentration. In addition, a two-fold increment in the linearity of axonal growth within the microchannels was observed and confirmed by a significant reduction in the turning angle ratios of individual axons. These data demonstrate the ability of growth factor-loaded polymeric coiled fibers to establish three-dimensional chemotactic gradients to promote and direct nerve regeneration in the nervous system and provides a unique platform for molecularly guided tissue repair.

Chronic sensory-motor activity in behaving animals using regenerative multi-electrode interfaces.

Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control and feel of robotic prosthetic limbs. We have developed a Regenerative Multi-electrode Interface (REMI) that guides re-growing axons through an electrode array deployed in the lumen of a nerve guide. While acute studies have shown the use of the REMI in the rat sciatic nerve, the quality of chronic signal recording has not been reported. Here we show that implantation of this interface in the sciatic nerve is stable with high quality recordings up to 120 days and failures mainly attributable to abiotic factors related to pedestal detachment and wire breakage. We further tested the interfacing of REMI with fascicles of the sciatic nerve that primarily innervate muscles (tibial) and skin (sural). When implanted into the tibial nerve, bursting activity was observed synchronous to stepping. However, implantation of REMI into the sural nerve failed due to its small size. While fascicles smaller than 300 μm are a challenge for regenerative interfacing, we show that a modified REMI can be used in an insertion mode to record sensory signals from skin. In summary, the REMI represents an effective tool for recording firing patterns of specific axon types during voluntary movement, which may be used to improve the motor control and sensory feedback in closed loop control systems for robotic prosthesis.

Chronic and low charge injection wireless intraneural stimulation in vivo

Functional stability and in-vivo reliability are significant factors determining the longevity of a neural interface. In this ongoing study, we test the performance of a wireless floating microelectrode array (WFMA) over a period of 143 days. The topography of the microelectrodes has allowed for selective stimulation of different fascicles of the rat sciatic nerve. We confirmed that motor evoked thresholds remain stable over time and that the nerve stimulation charges were within tissue safety limits. Importantly, motor evoked responses were elicited at threshold currents in fully awake animals without causing pain or discomfort. These data validate the use of the WFMA system for intraneural interfacing of peripheral nerves for neuroprosthetic and bioelectronics medical applications.