Electrically Controlled Vasodilator Delivery from PEDOT/Silica Nanoparticle Modulates Vessel Diameter in Mouse Brain.

Vascular damage and reduced tissue perfusion are expected to majorly contribute to the loss of neurons or neural signals around implanted electrodes. However, there are limited methods of controlling the vascular dynamics in tissues surrounding these implants. This work utilizes conducting polymer poly(ethylenedioxythiophene) and sulfonated silica nanoparticle composite (PEDOT/SNP) to load and release a vasodilator, sodium nitroprusside, to controllably dilate the vasculature around carbon fiber electrodes (CFEs) implanted in the mouse cortex. The vasodilator release is triggered via electrical stimulation and the amount of release increases with increasing electrical pulses. The vascular dynamics are monitored in real-time using two-photon microscopy, with changes in vessel diameters quantified before, during, and after the release of the vasodilator into the tissues. This work observes significant increases in vessel diameters when the vasodilator is electrically triggered to release, and differential effects of the drug release on vessels of different sizes. In conclusion, the use of nanoparticle reservoirs in conducting polymer-based drug delivery platforms enables the controlled delivery of vasodilator into the implant environment, effectively altering the local vascular dynamics on demand. With further optimization, this technology could be a powerful tool to improve the neural electrode-tissue interface and study neurovascular coupling.

Real-time in vivo thoracic spinal glutamate sensing during myocardial ischemia.

In the spinal cord, glutamate serves as the primary excitatory neurotransmitter. Monitoring spinal glutamate concentrations offers valuable insights into spinal neural processing. Consequently, spinal glutamate concentration has the potential to emerge as a useful biomarker for conditions characterized by increased spinal neural network activity, especially when uptake systems become dysfunctional. In this study, we developed a multichannel custom-made flexible glutamate-sensing probe for the large-animal model that is capable of measuring extracellular glutamate concentrations in real time and in vivo. We assessed the probe's sensitivity and specificity through in vitro and ex vivo experiments. Remarkably, this developed probe demonstrates nearly instantaneous glutamate detection and allows continuous monitoring of glutamate concentrations. Furthermore, we evaluated the mechanical and sensing performance of the probe in vivo, within the pig spinal cord. Moreover, we applied the glutamate-sensing method using the flexible probe in the context of myocardial ischemia-reperfusion (I/R) injury. During I/R injury, cardiac sensory neurons in the dorsal root ganglion transmit excitatory signals to the spinal cord, resulting in sympathetic activation that potentially leads to fatal arrhythmias. We have successfully shown that our developed glutamate-sensing method can detect this spinal network excitation during myocardial ischemia. This study illustrates a novel technique for measuring spinal glutamate at different spinal cord levels as a surrogate for the spinal neural network activity during cardiac interventions that engage the cardio-spinal neural pathway.NEW & NOTEWORTHY In this study, we have developed a new flexible sensing probe to perform an in vivo measurement of spinal glutamate signaling in a large animal model. Our initial investigations involved precise testing of this probe in both in vitro and ex vivo environments. We accurately assessed the sensitivity and specificity of our glutamate-sensing probe and demonstrated its performance. We also evaluated the performance of our developed flexible probe during the insertion and compared it with the stiff probe during animal movement. Subsequently, we used this innovative technique to monitor the spinal glutamate signaling during myocardial ischemia and reperfusion that can cause fatal ventricular arrhythmias. We showed that glutamate concentration increases during the myocardial ischemia, persists during the reperfusion, and is associated with sympathoexcitation and increases in myocardial substrate excitability.

Dexamethasone-Enhanced Continuous Online Microdialysis for Neuromonitoring of O2 after Brain Injury.

Traumatic brain injury (TBI) is a major public health crisis in many regions of the world. Severe TBI may cause a primary brain lesion with a surrounding penumbra of tissue that is vulnerable to secondary injury. Secondary injury presents as progressive expansion of the lesion, possibly leading to severe disability, a persistent vegetive state, or death. Real time neuromonitoring to detect and monitor secondary injury is urgently needed. Dexamethasone-enhanced continuous online microdialysis (Dex-enhanced coMD) is an emerging paradigm for chronic neuromonitoring after brain injury. The present study employed Dex-enhanced coMD to monitor brain K+ and O2 during manually induced spreading depolarization in the cortex of anesthetized rats and after controlled cortical impact, a widely used rodent model of TBI, in behaving rats. Consistent with prior reports on glucose, O2 exhibited a variety of responses to spreading depolarization and a prolonged, essentially permanent decline in the days after controlled cortical impact. These findings confirm that Dex-enhanced coMD delivers valuable information regarding the impact of spreading depolarization and controlled cortical impact on O2 levels in the rat cortex.

Real-time in vivo thoracic spinal glutamate sensing reveals spinal hyperactivity during myocardial ischemia.

Myocardial ischemia-reperfusion (IR) can cause ventricular arrhythmias and sudden cardiac death via sympathoexcitation. The spinal cord neural network is crucial in triggering these arrhythmias and evaluating its neurotransmitter activity during IR is critical for understanding ventricular excitability control. To assess the real-time in vivo spinal neural activity in a large animal model, we developed a flexible glutamate-sensing multielectrode array. To record the glutamate signaling during IR injury, we inserted the probe into the dorsal horn of the thoracic spinal cord at the T2-T3 where neural signals generated by the cardiac sensory neurons are processed and provide sympathoexcitatory feedback to the heart. Using the glutamate sensing probe, we found that the spinal neural network was excited during IR, especially after 15 mins, and remained elevated during reperfusion. Higher glutamate signaling was correlated with the reduction in the cardiac myocyte activation recovery interval, showing higher sympathoexcitation, as well as dispersion of the repolarization which is a marker for increased risk of arrhythmias. This study illustrates a new technique for measuring the spinal glutamate at different spinal cord levels as a surrogate for the spinal neural network activity during cardiac interventions that engage the cardio-spinal neural pathway.

Stable in-vivo electrochemical sensing of tonic serotonin levels using PEDOT/CNT-coated glassy carbon flexible microelectrode arrays.

Chronic sampling of tonic serotonin (5-hydroxytryptamine, 5-HT) concentrations in the brain is critical for tracking neurological disease development and the time course of pharmacological treatments. Despite their value, in vivo chronic multi-site measurements of tonic 5-HT have not been reported. To fill this technological gap, we batch-fabricated implantable glassy carbon (GC) microelectrode arrays (MEAs) onto a flexible SU-8 substrate to provide an electrochemically stable and biocompatible device/tissue interface. To achieve detection of tonic 5-HT concentrations, we applied a poly(3,4-ethylenedioxythiophene)/carbon nanotube (PEDOT/CNT) electrode coating and optimized a square wave voltammetry (SWV) waveform for selective 5-HT measurement. In vitro, the PEDOT/CNT-coated GC microelectrodes achieved high sensitivity to 5-HT, good fouling resistance, and excellent selectivity against the most common neurochemical interferents. In vivo, our PEDOT/CNT-coated GC MEAs successfully detected basal 5-HT concentrations at different locations within the CA2 region of the hippocampus of both anesthetized and awake mice. Furthermore, the PEDOT/CNT-coated MEAs were able to detect tonic 5-HT in the mouse hippocampus for one week after implantation. Histology reveals that the flexible GC MEA implants caused less tissue damage and reduced inflammatory response in the hippocampus compared to commercially available stiff silicon probes. To the best of our knowledge, this PEDOT/CNT-coated GC MEA is the first implantable, flexible sensor capable of chronic in vivo multi-site sensing of tonic 5-HT.

Implantable flexible multielectrode arrays for multi-site sensing of serotonin tonic levels.

Real-time multi-channel measurements of tonic serotonin (5-hydroxytryptamine, 5-HT) concentrations across different brain regions are of utmost importance to the understanding of 5-HT’s role in anxiety, depression, and impulse control disorders, which will improve the diagnosis and treatment of these neuropsychiatric illnesses. Chronic sampling of 5-HT is critical in tracking disease development as well as the time course of pharmacological treatments. Despite their value, in vivo chronic multi-site measurements of 5-HT have not been reported. To fill this technological gap, we batch fabricated implantable glassy carbon (GC) microelectrode arrays (MEAs) on a flexible SU-8 substrate to provide an electrochemically stable and biocompatible device/tissue interface. Then, to achieve multi-site detection of tonic 5-HT concentrations, we incorporated the poly(3,4-ethylenedioxythiophene)/functionalized carbon nanotube (PEDOT/CNT) coating on the GC microelectrodes in combination with a new square wave voltammetry (SWV) approach, optimized for selective 5-HT measurement. In vitro , the PEDOT/CNT coated GC microelectrodes achieved high sensitivity towards 5-HT, good fouling resistance in the presence of 5-HT, and excellent selectivity towards the most common neurochemical interferents. In vivo , our PEDOT/CNT-coated GC MEAs were able to successfully detect basal 5-HT concentrations at different locations of the CA2 hippocampal region of mice in both anesthetized and awake head-fixed conditions. Furthermore, the implanted PEDOT/CNT-coated MEA achieved stable detection of tonic 5-HT concentrations for one week. Finally, histology data in the hippocampus shows reduced tissue damage and inflammatory responses compared to stiff silicon probes. To the best of our knowledge, this PEDOT/CNT-coated GC MEA is the first implantable flexible multisite sensor capable of chronic in vivo multi-site sensing of tonic 5-HT. This implantable MEA can be custom-designed according to specific brain region of interests and research questions, with the potential to combine electrophysiology recording and multiple analyte sensing to maximize our understanding of neurochemistry. Highlights: PEDOT/CNT-coated GC microelectrodes enabled sensitive and selective tonic detection of serotonin (5-HT) using a new square wave voltammetry (SWV) approach PEDOT/CNT-coated GC MEAs achieved multi-site in vivo 5-HT tonic detection for one week. Flexible MEAs lead to reduced tissue damage and inflammation compared to stiff silicon probes.

Accurate and stable chronic in vivo voltammetry enabled by a replaceable subcutaneous reference electrode.

In vivo sensing of neurotransmitters has provided valuable insight into both healthy and diseased brain. However, chronically implanted Ag/AgCl reference electrodes suffer from degradationgradation, resulting in errors in the potential at the working electrode. Here, we report a simple, effective way to protect in vivo sensing measurements from reference polarization with a replaceable subcutaneously implanted reference. We compared a brain-implanted reference and a subcutaneous reference and observed no difference in impedance or dopamine redox peak separation in an acute preparation. Chronically, peak background potential and dopamine oxidation potential shifts were eliminated for three weeks. Scanning electron microscopy shows changes in surface morphology and composition of chronically implanted Ag/AgCl electrodes, and postmortem histology reveals extensive cell death and gliosis in the surrounding tissue. As accurate reference potentials are critical to in vivo electrochemistry applications, this simple technique can improve a wide and diverse assortment of in vivo preparations.

Flexible Glassy Carbon Multielectrode Array for In Vivo Multisite Detection of Tonic and Phasic Dopamine Concentrations.

Dopamine (DA) plays a central role in the modulation of various physiological brain functions, including learning, motivation, reward, and movement control. The DA dynamic occurs over multiple timescales, including fast phasic release, as a result of neuronal firing and slow tonic release, which regulates the phasic firing. Real-time measurements of tonic and phasic DA concentrations in the living brain can shed light on the mechanism of DA dynamics underlying behavioral and psychiatric disorders and on the action of pharmacological treatments targeting DA. Current state-of-the-art in vivo DA detection technologies are limited in either spatial or temporal resolution, channel count, longitudinal stability, and ability to measure both phasic and tonic dynamics. We present here an implantable glassy carbon (GC) multielectrode array on a SU-8 flexible substrate for integrated multichannel phasic and tonic measurements of DA concentrations. The GC MEA demonstrated in vivo multichannel fast-scan cyclic voltammetry (FSCV) detection of electrically stimulated phasic DA release simultaneously at different locations of the mouse dorsal striatum. Tonic DA measurement was enabled by coating GC electrodes with poly(3,4-ethylenedioxythiophene)/carbon nanotube (PEDOT/CNT) and using optimized square-wave voltammetry (SWV). Implanted PEDOT/CNT-coated MEAs achieved stable detection of tonic DA concentrations for up to 3 weeks in the mouse dorsal striatum. This is the first demonstration of implantable flexible MEA capable of multisite electrochemical sensing of both tonic and phasic DA dynamics in vivo with chronic stability.

Validation of Dexamethasone-Enhanced Continuous-Online Microdialysis for Monitoring Glucose for 10 Days after Brain Injury.

Traumatic brain injury (TBI) induces a pathophysiologic state that can be worsened by secondary injury. Monitoring brain metabolism with intracranial microdialysis can provide clinical insights to limit secondary injury in the days following TBI. Recent enhancements to microdialysis include the implementation of continuously operating electrochemical biosensors for monitoring the dialysate sample stream in real time and dexamethasone retrodialysis to mitigate the tissue response to probe insertion. Dexamethasone-enhanced continuous-online microdialysis (Dex-enhanced coMD) records long-lasting declines of glucose after controlled cortical impact in rats and TBI in patients. The present study employed retrodialysis and fluorescence microscopy to investigate the mechanism responsible for the decline of dialysate glucose after injury of the rat cortex. Findings confirm the long-term functionality of Dex-enhanced coMD for monitoring brain glucose after injury, demonstrate that intracranial glucose microdialysis is coupled to glucose utilization in the tissues surrounding the probes, and validate the conclusion that aberrant glucose utilization drives the postinjury glucose decline.

Miniaturized probe on polymer SU-8 with array of individually addressable microelectrodes for electrochemical analysis in neural and other biological tissues.

An SU-8 probe with an array of nine, individually addressable gold microband electrodes (100 µm long, 4 µm wide, separated by 4-µm gaps) was photolithographically fabricated and characterized for detection of low concentrations of chemicals in confined spaces and in vivo studies of biological tissues. The probe's shank (6 mm long, 100 µm wide, 100 µm thick) is flexible, but exhibits sufficient sharpness and rigidity to be inserted into soft tissue. Laser micromachining was used to define probe geometry by spatially revealing the underlying sacrificial aluminum layer, which was then etched to free the probes from a silicon wafer. Perfusion with fluorescent nanobeads showed that, like a carbon fiber electrode, the probe produced no noticeable damage when inserted into rat brain, in contrast to damage from an inserted microdialysis probe. The individual addressability of the electrodes allows single and multiple electrode activation. Redox cycling is possible, where adjacent electrodes serve as generators (that oxidize or reduce molecules) and collectors (that do the opposite) to amplify signals of small concentrations without background subtraction. Information about electrochemical mechanisms and kinetics may also be obtained. Detection limits for potassium ferricyanide in potassium chloride electrolyte of 2.19, 1.25, and 2.08 µM and for dopamine in artificial cerebral spinal fluid of 1.94, 1.08, and 5.66 µM for generators alone and for generators and collectors during redox cycling, respectively, were obtained.

Recent Advances in In Vivo Neurochemical Monitoring.

The brain is a complex network that accounts for only 5% of human mass but consumes 20% of our energy. Uncovering the mysteries of the brain's functions in motion, memory, learning, behavior, and mental health remains a hot but challenging topic. Neurochemicals in the brain, such as neurotransmitters, neuromodulators, gliotransmitters, hormones, and metabolism substrates and products, play vital roles in mediating and modulating normal brain function, and their abnormal release or imbalanced concentrations can cause various diseases, such as epilepsy, Alzheimer's disease, and Parkinson's disease. A wide range of techniques have been used to probe the concentrations of neurochemicals under normal, stimulated, diseased, and drug-induced conditions in order to understand the neurochemistry of drug mechanisms and develop diagnostic tools or therapies. Recent advancements in detection methods, device fabrication, and new materials have resulted in the development of neurochemical sensors with improved performance. However, direct in vivo measurements require a robust sensor that is highly sensitive and selective with minimal fouling and reduced inflammatory foreign body responses. Here, we review recent advances in neurochemical sensor development for in vivo studies, with a focus on electrochemical and optical probes. Other alternative methods are also compared. We discuss in detail the in vivo challenges for these methods and provide an outlook for future directions.

Real-Time Fast Scan Cyclic Voltammetry Detection and Quantification of Exogenously Administered Melatonin in Mice Brain.

Melatonin (MT) has been recently considered an excellent candidate for the treatment of sleep disorders, neural injuries, and neurological diseases. To better investigate the actions of MT in various brain functions, real-time detection of MT concentrations in specific brain regions is much desired. Previously, we have demonstrated detection of exogenously administered MT in anesthetized mouse brain using square wave voltammetry (SWV). Here, for the first time, we show successful detection of exogenous MT in the brain using fast scan cyclic voltammetry (FSCV) on electrochemically pre-activated carbon fiber microelectrodes (CFEs). In vitro evaluation showed the highest sensitivity (28.1 nA/µM) and lowest detection limit (20.2 ± 4.8 nM) ever reported for MT detection at carbon surface. Additionally, an extensive CFE stability and fouling assessment demonstrated that a prolonged CFE pre-conditioning stabilizes the background, in vitro and in vivo, and provides consistent CFE sensitivity over time even in the presence of a high MT concentration. Finally, the stable in vivo background, with minimized CFE fouling, allows us to achieve a drift-free FSCV detection of exogenous administered MT in mouse brain over a period of 3 min, which is significantly longer than the duration limit (usually < 90 s) for traditional in vivo FSCV acquisition. The MT concentration and dynamics measured by FSCV are in good agreement with SWV, while microdialysis further validated the concentration range. These results demonstrated reliable MT detection using FSCV that has the potential to monitor MT in the brain over long periods of time.

Extended (10-Day) Real-Time Monitoring by Dexamethasone-Enhanced Microdialysis in the Injured Rat Cortex.

Intracerebral microdialysis has proven useful for chemical monitoring in patients following traumatic brain injury. Recent studies in animals, however, have documented that insertion of microdialysis probes into brain tissues initiates a foreign-body response. Within a few days after probe insertion, the foreign body response impedes the use of microdialysis to monitor the K+ and glucose transients associated with spreading depolarization, a potential mechanism for secondary brain injury. Herein, we show that perfusing microdialysis probes with dexamethasone, a potent anti-inflammatory glucocorticoid, suppresses the foreign body response and facilitates the monitoring of spontaneous spreading depolarizations for at least 10 days following controlled cortical injury in the rat. In addition to spreading depolarizations, results of this study suggest that a progressive, apparently permanent, decline in pericontusional interstitial glucose may be an additional sequela of brain injury. This study establishes extended dexamethasone-enhanced microdialysis in the injured rodent cortex as a new paradigm for investigating trauma-induced metabolic crisis.

Enhanced dopamine detection sensitivity by PEDOT/graphene oxide coating on in vivo carbon fiber electrodes.

Dopamine (DA) is a monoamine neurotransmitter responsible for regulating a variety of vital life functions. In vivo detection of DA poses a challenge due to the low concentration and high speed of physiological signaling. Fast scan cyclic voltammetry at carbon fiber microelectrodes (CFEs) is an effective method to monitor real-time in vivo DA signaling, however the sensitivity is somewhat limited. Electrodeposition of poly(3,4-ethylene dioxythiophene) (PEDOT)/graphene oxide (GO) onto the CFE surface is shown to increase the sensitivity and lower the limit of detection for DA compared to bare CFEs. Thicker PEDOT/GO coatings demonstrate higher sensitivities for DA, but display the negative drawback of slow adsorption and electron transfer kinetics. The moderate thickness resulting from 25 s electrodeposition of PEDOT/GO produces the optimal electrode, exhibiting an 880% increase in sensitivity, a 50% decrease in limit of detection and minimally altered electrode kinetics. PEDOT/GO coated electrodes rapidly and robustly detect DA, both in solution and in the rat dorsal striatum. This increase in DA sensitivity is likely due to increasing the electrode surface area with a PEDOT/GO coating and improved adsorption of DA's oxidation product (DA-o-quinone). Increasing DA sensitivity without compromising electrode kinetics is expected to significantly improve our understanding of the DA function in vivo.

Kinetic Diversity of Striatal Dopamine: Evidence from a Novel Protocol for Voltammetry.

In vivo voltammetry reveals substantial diversity of dopamine kinetics in the rat striatum. To substantiate this kinetic diversity, we evaluate the temporal distortion of dopamine measurements arising from the diffusion-limited adsorption of dopamine to voltammetric microelectrodes. We validate two mathematical procedures for correcting adsorptive distortion, both of which substantiate that dopamine's apparent kinetic diversity is not an adsorption artifact.

Modeling the kinetic diversity of dopamine in the dorsal striatum.

Dopamine is an important neurotransmitter that exhibits numerous functions in the healthy, injured, and diseased brain. Fast scan cyclic voltammetry paired with electrical stimulation of dopamine axons is a popular and powerful method for investigating the dynamics of dopamine in the extracellular space. Evidence now suggests that the heterogeneity of electrically evoked dopamine responses reflects the inherent kinetic diversity of dopamine systems, which might contribute to their diversity of physiological function. Dopamine measurements by fast scan cyclic voltammetry are affected by the adsorption of dopamine to carbon fiber electrodes. The temporal distortion caused by dopamine adsorption is correctable by a straightforward mathematical procedure. The corrected responses exhibit excellent agreement with a dopamine kinetic model cast to provide a generic description of restricted diffusion, short-term plasticity of dopamine release, and first-order dopamine clearance. The new DA kinetic model brings to light the rich kinetic information content of electrically evoked dopamine responses recorded via fast scan cyclic voltammetry in the rat dorsal striatum.

Concurrent zero-dimensional and one-dimensional biomineralization of gold from a solution of Au3+ and bovine serum albumin.

A technique was developed for preparing a novel material that consists of gold nanoparticles trapped within a fiber of unfolded proteins. These fibers are made in an aqueous solution that contains HAuCl4 and the protein, bovine serum albumin (BSA). By changing the ratio of gold to BSA in solution, two different types of outcomes are observed. At lower gold to BSA ratios (30-120), a purple solution results after heating the mixture at 80 °C for 4 h. At higher gold to BSA ratios (130-170), a clear solution containing purple fibers results after heating the mixture at 80 °C for 4 h. UV-Vis spectroscopy and light scattering techniques show growth in nanocolloid size as gold to BSA ratio rises above 100. Data indicate that, for the higher gold to BSA ratios, the gold is sequestered within the solid material. The material mass, visible by eye, appears to be an aggregation of smaller individual fibers. Scanning electron microscopy and transmission electron microscopy indicate that these fibers are primarily one-dimensional aggregates, which can display some branching, and can be as narrow as 400 nm in size. The likely mechanism for the synthesis of the novel material is discussed.