Takashi D Y Kozai

The chronic performance of implantable neural electrodes is hindered by inflammatory brain tissue responses, including microglia activation, glial scarring, and neuronal loss. Melatonin (MT) has shown remarkable neuroprotective and neurorestorative effects in treating central nervous system (CNS) injuries and degeneration by inhibiting caspase-1, -3, and -9 activation and mitochondrial cytochrome c release, as well as reducing oxidative stress and neuroinflammation. This study examined the effect of MT administration on the quality and longevity of neural recording from an implanted microelectrode in the visual cortex of mice for 16 weeks. MT (30 mg/kg) was administered via daily intraperitoneal injection for acute (3 days before and 14 days post-implantation) and chronic (3 days before and 16 weeks post-implantation) exposures. During the first 4 weeks, both MT groups showed significantly higher single-unit (SU) yield, signal-to-noise ratio (SNR), and amplitude compared to the vehi...

Implantable devices to measure neurochemical or electrical activity from the brain are mainstays of neuroscience research and have become increasingly utilized as enabling components of clinical therapies. In order to increase the number of recording channels on these devices while minimizing the immune response, flexible electrodes under 10 µm in diameter have been proposed as ideal next-generation neural interfaces. However, the representation of motion artifact during neurochemical or electrophysiological recordings using ultra-small, flexible electrodes remains unexplored. In this short communication, we characterize motion artifact generated by the movement of 7 µm diameter carbon fiber electrodes during electrophysiological recordings and fast-scan cyclic voltammetry (FSCV) measurements of electroactive neurochemicals. Through in vitro and in vivo experiments, we demonstrate that artifact induced by motion can be problematic to distinguish from the characteristic signals assoc...

Microscale neural technologies interface with the nervous system to record and stimulate brain tissue with high spatial and temporal resolution. These devices are being developed to understand the mechanisms that govern brain function, plasticity and cognitive learning, treat neurological diseases, or monitor and restore functions over the lifetime of the patient. Despite decades of use in basic research over days to months, and the growing prevalence of neuromodulation therapies, in many cases the lack of knowledge regarding the fundamental mechanisms driving activation has dramatically limited our ability to interpret data or fine-tune design parameters to improve long-term performance. While advances in materials, microfabrication techniques, packaging, and understanding of the nervous system has enabled tremendous innovation in the field of neural engineering, many challenges and opportunities remain at the frontiers of the neural interface in terms of both neurobiology and engi...

Implantable electrode devices enable long-term electrophysiological recordings for brain-machine interfaces and basic neuroscience research. Implantation of these devices, however, leads to neuronal damage and progressive neural degeneration that can lead to device failure. The present study uses in vivo two-photon microscopy to study the calcium activity and morphology of neurons before, during, and one month after electrode implantation to determine how implantation trauma injures neurons. We show that implantation leads to prolonged, elevated calcium levels in neurons within 150 μm of the electrode interface. These neurons show signs of mechanical distortion and mechanoporation after implantation, suggesting that calcium influx is related to mechanical trauma. Further, calcium-laden neurites develop signs of axonal injury at 1-3 h post-insert. Over the first month after implantation, physiological neuronal calcium activity increases, suggesting that neurons may be recovering. By ...

The interhemispheric circuit connecting the left and the right mammalian brain plays a key role in integration of signals from the left and the right side of the body. The information transfer is carried out by modulation of simultaneous excitation and inhibition. Hemodynamic studies of this circuit are inconsistent since little is known about neurovascular coupling of mixed excitatory and inhibitory signals. We investigated the variability in hemodynamic responses driven by the interhemispheric circuit during optogenetic and somatosensory activation. We observed differences in the neurovascular response based on the stimulation site - cell bodies versus distal projections. In half of the experiments, optogenetic stimulation of the cell bodies evoked a predominant post-synaptic inhibition in the other hemisphere, accompanied by metabolic oxygen consumption without coupled functional hyperemia. When the same transcallosal stimulation resulted in predominant post-synaptic excitation, ...

Neural interface technology provides direct sampling and analysis of electrical and chemical events in the brain in order to better understand neuronal function and treat neurodegenerative disease. However, intracortical electrodes experience inflammatory reactions that reduce long-term stability and functionality and are understood to be facilitated by activated microglia and astrocytes. Emerging studies have identified another cell type that participates in the formation of a high-impedance glial scar following brain injury; the oligodendrocyte precursor cell (OPC). These cells maintain functional synapses with neurons and are a crucial source of neurotrophic support. Following injury, OPCs migrate toward areas of tissue injury over the course of days, similar to activated microglia. The delayed time course implicates these OPCs as key components in the formation of the outer layers of the glial scar around the implant. In vivo two-photon laser scanning microscopy (TPLSM) was empl...

Neurochemical sensing probes are a valuable diagnostic and therapeutic tool that can be used to study neurodegenerative diseases involving deficiencies in neurotransmitter signaling. However, implantation of these biosensors can elicit a harmful tissue response that alters the neurochemical environment within the brain. Transmission of chemical messengers via neurons is impeded by a barrier-forming glial scar that occurs within weeks after insertion followed by progressive neurodegeneration, attenuating signal sensitivity. Emerging research reveals that non-neuronal cells also influence the neurochemical milieu following injury both directly and indirectly. The reactivity of both microglia and astrocytes to inserted probes have been extensively studied in the past yet there remains other glial subtypes in the brain, such as oligodendrocytes and their precursors, the myelin structures they form, as well as vascular-bound pericytes, that have the potential to contribute significantly ...

Chronically implanted neural multi-electrode arrays (MEA) are an essential technology for recording electrical signals from neurons and/or modulating neural activity through stimulation. However, current MEAs, regardless of the type, elicit an inflammatory response that ultimately leads to device failure. Traditionally, rigid materials like tungsten and silicon have been employed to interface with the relatively soft neural tissue. The large stiffness mismatch is thought to exacerbate the inflammatory response. In order to minimize the disparity between the device and the brain, we fabricated novel ultrasoft electrodes consisting of elastomers and conducting polymers with mechanical properties much more similar to those of brain tissue than previous neural implants. In this study, these ultrasoft microelectrodes were inserted and released using a stainless steel shuttle with polyethyleneglycol (PEG) glue. The implanted microwires showed functionality in acute neural stimulation. Whe...

Two-photon microscopy has enabled the visualization of dynamic tissue changes to injury and disease in vivo. While this technique has provided powerful new information, in vivo two-photon chronic imaging around tethered cortical implants, such as microelectrodes or neural probes, present unique challenges. A number of strategies are described to prepare a cranial window to longitudinally observe the impact of neural probes on brain tissue and vasculature for up to 3 months. It was found that silastic sealants limit cell infiltration into the craniotomy, thereby limiting light scattering and preserving window clarity over time. In contrast, low concentration hydrogel sealants failed to prevent cell infiltration and their use at high concentration displaced brain tissue and disrupted probe performance. The use of silastic sealants allows for a suitable imaging window for long term chronic experiments and revealed new insights regarding the dynamic leukocyte response around implants an...

Single carbon fiber electrodes (d = 8.4 μm) insulated with parylene-c and functionalized with PEDOT:pTS have been shown to record single unit activity but manual implantation of these devices with forceps can be difficult. Without an improvement in the insertion method any increase in the channel count by fabricating carbon fiber arrays would be impractical. In this study, we utilize a water soluble coating and structural backbones that allow us to create, implant, and record from fully functionalized arrays of carbon fibers with ∼150 μm pitch. Two approaches were tested for the insertion of carbon fiber arrays. The first method used a poly(ethylene glycol) (PEG) coating that temporarily stiffened the fibers while leaving a small portion at the tip exposed. The small exposed portion (500 μm-1 mm) readily penetrated the brain allowing for an insertion that did not require the handling of each fiber by forceps. The second method involved the fabrication of silicon support structures w...

Current designs for microelectrodes used for interfacing with the nervous system elicit a characteristic inflammatory response that leads to scar tissue encapsulation, electrical insulation of the electrode from the tissue and ultimately failure. Traditionally, relatively stiff materials like tungsten and silicon are employed which have mechanical properties several orders of magnitude different from neural tissue. This mechanical mismatch is thought to be a major cause of chronic inflammation and degeneration around the device. In an effort to minimize the disparity between neural interface devices and the brain, novel soft electrodes consisting of elastomers and intrinsically conducting polymers were fabricated. The physical, mechanical and electrochemical properties of these materials were extensively characterized to identify the formulations with the optimal combination of parameters including Young's modulus, elongation at break, ultimate tensile strength, conductivity, im...

ABSTRACT This chapter explores the variability and limitations of traditional stimulation electrodes by first appreciating how electrical potential differences lead to efficacious activation of nearby neurons and examining the basic electrochemical mechanisms of charge transfer at an electrode/electrolyte interface. It then covers the advantages and current challenges of emerging micro-/nanostructured elec-trode materials for next-generation neural stimulation microelectrodes.

Intracortical electrode arrays that can record extracellular action potentials from small, targeted groups of neurons are critical for basic neuroscience research and emerging clinical applications. In general, these electrode devices suffer from reliability and variability issues, which have led to comparative studies of existing and emerging electrode designs to optimize performance. Comparisons of different chronic recording devices have been limited to single-unit (SU) activity and employed a bulk averaging approach treating brain architecture as homogeneous with respect to electrode distribution. In this study, we optimize the methods and parameters to quantify evoked multi-unit (MU) and local field potential (LFP) recordings in eight mice visual cortices. These findings quantify the large recording differences stemming from anatomical differences in depth and the layer dependent relative changes to SU and MU recording performance over 6-months. For example, performance metrics...

ABSTRACT This paper describes an ultra-compliant parylene-platinum neural probe embedded in a biodissolvable delivery vehicle. High probe compliance is achieved using thin wires (width of 10.0 μm and thickness of 2.7 μm) and by meandering the probe. The insertion of the ultra-compliant probe is achieved by encasing it in a dissolvable delivery vehicle made from molded carboxy-methylcellulose. In vivo implantations of delivery vehicles with 1.5 mm long shanks, widths of 100 μm and 300 μm and a targeted thickness of 135 μm have been done through the dura in the cortex of Sprague-Dawley rats at a speed of 80 mm-s-1. The delivery vehicle becomes a gel over a period of less than three minutes, after which the handling portions of the delivery vehicle are removed leaving the shanks embedded in the brain.

Neural interface technology provides direct sampling and analysis of electrical and chemical events in the brain in order to better understand neuronal function and treat neurodegenerative disease. However, intracortical electrodes experience inflammatory reactions that reduce long-term stability and func-tionality and are understood to be facilitated by activated microglia and astrocytes. Emerging studies have identified another cell type that participates in the formation of a high-impedance glial scar following brain injury; the oligodendrocyte precursor cell (OPC). These cells maintain functional syn-apses with neurons and are a crucial source of neurotrophic support. Following injury, OPCs migrate toward areas of tissue injury over the course of days, similar to activated microglia. The delayed time course implicates these OPCs as key components in the formation of the outer layers of the glial scar around the implant. In vivo two-photon laser scanning microscopy (TPLSM) was employed to observe fluorescently-labeled OPC and microglia reactivity up to 72 h following probe insertion. OPCs initiated extension of cellular processes (2.5 ± 0.4 mm h À1) and cell body migration (1.6 ± 0.3 mm h À1) toward the probe beginning 12 h after insertion. By 72 h, OPCs became activated at a radius of about 190.3 mm away from the probe surface. This study characterized the early spatiotemporal dynamics of OPCs involved in the inflammatory response induced by microelectrode insertion. OPCs are key mediators of tissue health and are understood to have multiple fate potentials. Detailed spatiotemporal characterization of glial behavior under pathological conditions may allow identification of alternative intervention targets for mitigating the formation of a glial scar and subsequent neurodegeneration that debilitates chronic neural interfaces.

The interhemispheric circuit connecting the left and the right mammalian brain plays a key role in integration of signals from the left and the right side of the body. The information transfer is carried out by modulation of simultaneous excitation and inhibition. Hemodynamic studies of this circuit are inconsistent since little is known about neurovascular coupling of mixed excitatory and inhibitory signals. We investigated the variability in hemodynamic responses driven by the interhemispheric circuit during optogenetic and somatosensory activation. We observed differences in the neuro-vascular response based on the stimulation site – cell bodies versus distal projections. In half of the experiments, optogenetic stimulation of the cell bodies evoked a predominant post-synaptic inhibition in the other hemisphere, accompanied by metabolic oxygen consumption without coupled functional hyperemia. When the same transcallosal stimulation resulted in predominant post-synaptic excitation, the hemodynamic response was biphasic, consisting of metabolic dip followed by functional hyperemia. Optogenetic suppression of the postsynaptic excitation abolished the coupled functional hyperemia. In contrast, light stimulation at distal projections evoked consistently a metabolic response. Our findings suggest that functional hyperemia requires signals originating from the cell body and the hemo-dynamic response variability appears to reflect the balance between the post-synaptic excitation and inhibition.

Intracortical microelectrode arrays, especially the Utah array, remain the most common choice for obtaining high dimensional recordings of spiking neural activity for brain computer interface and basic neuroscience research. Despite the widespread use and established design, mechanical, material and biological challenges persist that contribute to a steady decline in recording performance (as evidenced by both diminished signal amplitude and recorded cell population over time) or outright array failure. Device implantation injury causes acute cell death and activation of inflammatory microglia and astro-cytes that leads to a chronic neurodegeneration and inflammatory glial aggregation around the electrode shanks and often times fibrous tissue growth above the pia along the bed of the array within the meninges. This multifaceted deleterious cascade can result in substantial variability in performance even under the same experimental conditions. We track both impedance signatures and electrophysiological performance of 4 Â 4 floating microelectrode Utah arrays implanted in the primary monocular visual cortex (V1m) of Long-Evans rats over a 12-week period. We employ a repeatable visual stimulation method to compare signal-to-noise ratio as well as single-and multi-unit yield from weekly recordings. To explain signal variability with biological response, we compare arrays categorized as either Type 1, partial fibrous encapsulation, or Type 2, complete fibrous encapsulation and demonstrate performance and impedance signatures unique to encapsulation type. We additionally assess benefits of a biomolecule coating intended to minimize distance to recordable units and observe a temporary improvement on multi-unit recording yield and single-unit amplitude.

Objective: Implantable neural electrode devices are important tools for neuroscience research and have an increasing range of clinical applications. However, the intricacies of the biological response after implantation, and their ultimate impact on recording performance, remain challenging to elucidate. Establishing a relationship between the neurobiology and chronic recording performance is confounded by technical challenges related to traditional electrophysiological, material, and histological limitations. This can greatly impact the interpretations of results pertaining to device performance and tissue health surrounding the implant. 
 Approach: In this work, electrophysiological activity and immunohistological analysis are compared after controlling for motion artifacts, quiescent neuronal activity, and material failure of devices in order to better understand the relationship between histology and electrophysiological outcomes. 
 Results: Even after carefully accounting for these factors, the presence of viable neurons and lack or glial scarring does not convey single unit recording performance. 
 Significance: To better understand the biological factors influencing neural activity, detailed cellular and molecular tissue responses were examined. Decreases in neural activity and blood oxygenation in the tissue surrounding the implant, shift in expression levels of vesicular transporter proteins and ion channels, axon and myelin injury, and interrupted blood flow in nearby capillaries can impact neural activity around implanted neural interfaces. Combined, these tissue changes highlight the need for more comprehensive, basic science research to elucidate the relationship between biology and chronic electrophysiology performance in order to advance neural technologies.

The use of implants that can electrically stimulate or record electrophysiological or neurochemical activity in nervous tissue is rapidly expanding. Despite remarkable results in clinical studies and increasing market approvals, the mechanisms underlying the therapeutic effects of neuroprosthetic and neuromodulation devices, as well as their side effects and reasons for their failure, remain poorly understood. A major assumption has been that the signal-generating neurons are the only important target cells of neural-interface technologies. However, recent evidence indicates that the supporting glial cells remodel the structure and function of neuronal networks and are an effector of stimulation-based therapy. Here, we reframe the traditional view of glia as a passive barrier, and discuss their role as an active determinant of the outcomes of device implantation. We also discuss the implications that this has on the development of bioelectronic medical devices.

Chronically implanted neural multi-electrode arrays (MEA) are an essential technology for recording electrical signals from neurons and/or modulating neural activity through stimulation. However, current MEAs, regardless of the type, elicit an inflammatory response that ultimately leads to device failure. Traditionally, rigid materials like tungsten and silicon have been employed to interface with the relatively soft neural tissue. The large stiffness mismatch is thought to exacerbate the inflammatory response. In order to minimize the disparity between the device and the brain, we fabricated novel ultrasoft electrodes consisting of elastomers and conducting polymers with mechanical properties much more similar to those of brain tissue than previous neural implants. In this study, these ultrasoft microelectrodes were inserted and released using a stainless steel shuttle with polyethyleneglycol (PEG) glue. The implanted microwires showed functionality in acute neural stimulation. When implanted for 1 or 8 weeks, the novel soft implants demonstrated significantly reduced inflammatory tissue response at week 8 compared to tungsten wires of similar dimension and surface chemistry. Furthermore, a higher degree of cell body distortion was found next to the tungsten implants compared to the polymer implants. Our results support the use of these novel ultrasoft electrodes for long term neural implants. Statement of Significance One critical challenge to the translation of neural recording/stimulation electrode technology to clinically viable devices for brain computer interface (BCI) or deep brain stimulation (DBS) applications is the chronic degradation of device performance due to the inflammatory tissue reaction. While many hypothesize that soft and flexible devices elicit reduced inflammatory tissue responses, there has yet to be a rigorous comparison between soft and stiff implants. We have developed an ultra-soft microelectrode with Young's modulus lower than 1 MPa, closely mimicking the brain tissue modulus. Here, we present a rigorous histological comparison of this novel ultrasoft electrode and conventional stiff electrode with the same size, shape and surface chemistry, implanted in rat brains for 1-week and 8-weeks. Significant improvement was observed for ultrasoft electrodes, including inflammatory tissue reaction, electrode-tissue integration as well as mechanical disturbance to nearby neurons. A full spectrum of new techniques were developed in this study, from insertion shuttle to in situ sectioning of the microelectrode to automated cell shape analysis, all of which should contribute new methods to the field. Finally, we showed the electrical functionality of the ultrasoft electrode, demonstrating the potential of flexible neu-ral implant devices for future research and clinical use.

Implantable neural electrode technologies for chronic neural recordings can restore functional control to paralysis and limb loss victims through brain-machine interfaces. These probes, however, have high failure rates partly due to the biological responses to the probe which generates an inflammatory scar and subsequent neuronal cell death. L1 is a neuronal specific cell adhesion molecule and has been shown to minimize glial scar formation and promote electrode-neuron integration when covalently attached to the surface of neural probes. In this work, the acute microglial response to L1-coated neural probes was evaluated in vivo by implanting coated devices into the cortex of mice with fluorescently labeled microglia, and tracking microglial dynamics with multi-photon microscopy for the ensuing 6 h in order to understand L1's cellular mechanisms of action. Microglia became activated immediately after implantation, extending processes towards both L1-coated and uncoated control probes at similar velocities. After the processes made contact with the probes, microglial processes expanded to cover 47.7% of the control probes' surfaces. For L1-coated probes, however, there was a statistically significant 83% reduction in microglial surface coverage. This effect was sustained through the experiment. At 6 h post-implant, the radius of microglia activation was reduced for the L1 probes by 20%, shifting from 130.0 to 103.5 μm with the coating. Microglia as far as 270 μm from the implant site displayed significantly lower morphological characteristics of activation for the L1 group. These results suggest that the L1 surface treatment works in an acute setting by microglial mediated mechanisms.

Stable chronic functionality of intracortical probes is of utmost importance toward realizing clinical application of brain-machine interfaces. Sustained immune response from the brain tissue to the neural probes is one of the major challenges that hinder stable chronic functionality. There is a growing body of evidence in the literature that highly compliant neural probes with sub-cellular dimensions may significantly reduce the foreign-body response, thereby enhancing long term stability of intracortical recordings. Since the prevailing commercial probes are considerably larger than neu-rons and of high stiffness, new approaches are needed for developing miniature probes with high compliance. In this paper, we present design, fabrication, and in vitro evaluation of ultra-miniature (2.7 μm x 10 μm cross section), ultra-compliant (1.4 × 10-2 μN/μm in the axial direction, and 2.6 × 10-5 μN/μm and 1.8 × 10-6 μN/μm in the lateral directions) neural probes and associated probe-encasing biodissolvable delivery needles toward addressing the afore-mentioned challenges. The high compliance of the probes is obtained by micron-scale cross-section and meandered shape of the parylene-C insulated platinum wiring. Finite-element analysis is performed to compare the strains within the tissue during micromotion when using the ultra-compliant meandered probes with that when using stiff silicon probes. The standard batch microfabrication techniques are used for creating the probes. A dissolvable delivery needle that encases the probe facilitates failure-free insertion and precise placement of the ultra-compliant probes. Upon completion of im-plantation, the needle gradually dissolves, leaving behind the ultra-compliant neural probe. A spin-casting based micromolding approach is used for the fabrication of the needle. To demonstrate the versatility of the process, needles from different biodissolvable materials, as well as two-dimensional needle arrays with different geometries and dimensions, are fabricated. Further, needles incorporating anti-inflammatory drugs are created to show the co-delivery potential of the needles. An automated insertion device is developed for re-peatable and precise implantation of needle-encased probes into brain tissue. Insertion of the needles without mechanical failure, and their subsequent dissolution are demonstrated. It is concluded that ultra-miniature, ultra-compliant probes and associated biodissolvable delivery needles can be successfully fabricated, and the use of the ultra-compliant meandered probes results in drastic reduction in strains imposed in the tissue as compared to stiff probes, thereby showing promise toward chronic applications.

Objective. Individual carbon fiber microelectrodes can record unit activity in both acute and semi-chronic (~1 month) implants. Additionally, new methods have been developed to insert a 16 channel array of carbon fiber microelectrodes. Before assessing the in vivo long-term viability of these arrays, accelerated soak tests were carried out to determine the most stable site coating material. Next, a multi-animal, multi-month, chronic implantation study was carried out with carbon fiber microelectrode arrays and silicon electrodes. Approach. Carbon fibers were first functionalized with one of two different formulations of PEDOT and subjected to accelerated aging in a heated water bath. After determining the best PEDOT formula to use, carbon fiber arrays were chronically implanted in rat motor cortex. Some rodents were also implanted with a single silicon electrode, while others received both. At the end of the study a subset of animals were perfused and the brain tissue sliced. Tissue sections were stained for astrocytes, microglia, and neurons. The local reactive responses were assessed using qualitative and quantitative methods. Main results. Electrophysiology recordings showed the carbon fibers detecting unit activity for at least 3 months with average amplitudes of ~200 μV. Histology analysis showed the carbon fiber arrays with a minimal to non-existent glial scarring response with no adverse effects on neuronal density. Silicon electrodes showed large glial scarring that impacted neuronal counts. Significance. This study has validated the use of carbon fiber microelectrode arrays as a chronic neural recording technology. These electrodes have demonstrated the ability to detect single units with high amplitude over 3 months, and show the potential to record for even longer periods. In addition, the minimal reactive response should hold stable indefinitely, as any response by the immune system may reach a steady state after 12 weeks.

Intracortical neural probes enable researchers to measure electrical and chemical signals in the brain. However, penetration injury from probe insertion into living brain tissue leads to an inflammatory tissue response. In turn, microglia are activated, which leads to encapsulation of the probe and release of pro-inflammatory cytokines. This inflammatory tissue response alters the electrical and chemical microen-vironment surrounding the implanted probe, which may in turn interfere with signal acquisition. Dexamethasone (Dex), a potent anti-inflammatory steroid, can be used to prevent and diminish tissue disruptions caused by probe implantation. Herein, we report retrodialysis administration of dexa-methasone while using in vivo two-photon microscopy to observe real-time microglial reaction to the implanted probe. Microdialysis probes under artificial cerebrospinal fluid (aCSF) perfusion with or without Dex were implanted into the cortex of transgenic mice that express GFP in microglia under the CX3CR1 promoter and imaged for 6 h. Acute morphological changes in microglia were evident around the microdialysis probe. The radius of microglia activation was 177.1 mm with aCSF control compared to 93.0 mm with Dex perfusion. T-stage morphology and microglia directionality indices were also used to quantify the microglial response to implanted probes as a function of distance. Dexamethasone had a profound effect on the microglia morphology and reduced the acute activation of these cells.

A probe insertion device for implanting a probe into tissue includes a rigid base that selectively attaches to the probe due to a bond between the base and the probe, that provides a structural backbone to the probe, is longitudinally aligned with the probe, and can be adapted to receive a fluid between the base and the probe. The probe insertion device can include a surface covering at least a portion of the base that reduces the bond between the base and the probe in the presence of the fluid.