Considerations for Optimizing Warfighter Psychological Health with a Research-Based Flavonoid Approach: A Review.

Optimal nutrition is imperative for psychological health. Oxidative stress and inflammation are underlying etiologies for alterations in psychological health. Warfighters are at risk of health concerns such as depression due to increased stress in austere environments and family separation while deployed. Over the last decade, research has demonstrated the health benefits of flavonoids found in fruits and berries. Berry flavonoids have potent antioxidant and anti-inflammatory properties by inhibiting oxidative stress and inflammation. In this review, the promising effects of various berries rich in bioactive flavonoids are examined. By inhibiting oxidative stress, berry flavonoids have the potential to modulate brain, cardiovascular, and intestinal health. There is a critical need for targeted interventions to address psychological health concerns within the warfighter population, and a berry flavonoid-rich diet and/or berry flavonoid dietary supplement intervention may prove beneficial as an adjunctive therapy. Structured searches of the literature were performed in the PubMed, CINAHL, and EMBASE databases using predetermined keywords. This review focuses on berry flavonoids' critical and fundamental bioactive properties and their potential effects on psychological health in investigations utilizing cell, animal, and human model systems.

The Effects of Berry Extracts on Oxidative Stress in Cultured Cardiomyocytes and Microglial Cells: A Potential Cardioprotective and Neuroprotective Mechanism.

Oxidative stress is a key underlying factor in cognitive decline and atherosclerosis. Oxidative stress occurs at the cellular level with an imbalance between reactive oxygen species and reactive nitrogen species and a deficiency in antioxidants. Mounting evidence suggests that berry flavonoids may promote cellular health by exerting antioxidant properties. Black currant and various berry extracts were tested in microglia (BV-2) and cardiomyocyte (HL-1) cell lines to study their biological effects. The principal ingredients in black currant and cranberry extract-delphinidin 3-rutinoside (D3R) and cyanidin 3-glucoside (C3G), were also assessed. A menadione-induced oxidative stressor was used, and its output was quantified to detect oxidative stress (CellROXTM). Black currant extract had similar antioxidant effects as N-acetylcysteine (NAC) in HL-1 cells with regard to cellular protection, whereas cranberry extract was ineffective. In contrast, cranberry extract was comparable in effectiveness to black currant extract in BV-2 cells. D3R and C3G also reduced oxidative stress similarly to whole berry extracts, which indicates that these ingredients may confer the antioxidant effects of berries. Black currant and cranberry extracts inhibit oxidative stress in microglial and cardiomyocyte cell lines. Black currant extract was more effective in reducing oxidative stress in the HL-1 cells, whereas cranberry extract was comparable in reducing oxidative stress in the BV-2 cells. The results suggest that berry flavonoids exert neuro- and cardioprotective effects.

Longitudinal Functional Assessment of Brain Injury Induced by High-Intensity Ultrasound Pulse Sequences.

Exposure of the brain to high-intensity stress waves creates the potential for long-term functional deficits not related to thermal or cavitational damage. Possible sources of such exposure include overpressure from blast explosions or high-intensity focused ultrasound (HIFU). While current ultrasound clinical protocols do not normally produce long-term neurological deficits, the rapid expansion of potential therapeutic applications and ultrasound pulse-train protocols highlights the importance of establishing a safety envelope beyond which therapeutic ultrasound can cause neurological deficits not detectable by standard histological assessment for thermal and cavitational damage. In this study, we assessed the neuroinflammatory response, behavioral effects, and brain micro-electrocorticographic (µECoG) signals in mice following exposure to a train of transcranial pulses above normal clinical parameters. We found that the HIFU exposure induced a mild regional neuroinflammation not localized to the primary focal site, and impaired locomotor and exploratory behavior for up to 1 month post-exposure. In addition, low frequency (δ) and high frequency (ß, γ) oscillations recorded by ECoG were altered at acute and chronic time points following HIFU application. ECoG signal changes on the hemisphere ipsilateral to HIFU exposure are of greater magnitude than the contralateral hemisphere, and persist for up to three months. These results are useful for describing the upper limit of transcranial ultrasound protocols, and the neurological sequelae of injury induced by high-intensity stress waves.

An Alternative In Vitro Method for Examining Nanoparticle-Induced Cytotoxicity.

Conventional in vitro assays are often used as initial screens to identify potential toxic effects of nanoparticles (NPs). However, many NPs have shown interference with conventional in vitro assays, resulting in either false-positive or -negative outcomes. Here, we report an alternative method for the in vitro assessment of NP-induced cytotoxicity utilizing Fluoro-Jade C (FJ-C). To provide proof of concept and initial validation data, Ag-NPs and Au-NPs were tested in 3 different cell cultures including rat brain microvessel endothelial cells, mouse neural stem cells, and the human SH-SY5Y cell line. Conventional 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase (LDH) assays were run in parallel with the new method and served as references. The results demonstrate for the first time that FJ-C labeling can be a useful tool for assessing NP-induced cytotoxicity in vitro. Using these approaches, it was also demonstrated that removal of Ag-NPs-while keeping the Ag-ions that were released from the Ag-NPs in culture media-abolished the measured cytotoxicity, indicating that Ag-NPs rather than Ag-ions in solution contributed to the observed cytotoxic effects. Further, co-treatment of Ag-NPs with N-acetyl cysteine (NAC) prevented the observed cytotoxicity, suggesting a protective role of NAC in Ag-NP-induced cytotoxicity. Thus, this alternative in vitro assay is well suited for identify potential cytotoxicity associated with exposure to NPs.

Epidermal Electrode Technology for Detecting Ultrasonic Perturbation of Sensory Brain Activity.

OBJECTIVE: We aim to demonstrate the in vivo capability of a wearable sensor technology to detect localized perturbations of sensory-evoked brain activity. METHODS: Cortical somatosensory evoked potentials (SSEPs) were recorded in mice via wearable, flexible epidermal electrode arrays. We then utilized the sensors to explore the effects of transcranial focused ultrasound, which noninvasively induced neural perturbation. SSEPs recorded with flexible epidermal sensors were quantified and benchmarked against those recorded with invasive epidural electrodes. RESULTS: We found that cortical SSEPs recorded by flexible epidermal sensors were stimulus frequency dependent. Immediately following controlled, focal ultrasound perturbation, the sensors detected significant SSEP modulation, which consisted of dynamic amplitude decreases and altered stimulus-frequency dependence. These modifications were also dependent on the ultrasound perturbation dosage. The effects were consistent with those recorded with invasive electrodes, albeit with roughly one order of magnitude lower signal-to-noise ratio. CONCLUSION: We found that flexible epidermal sensors reported multiple SSEP parameters that were sensitive to focused ultrasound. This work therefore 1) establishes that epidermal electrodes are appropriate for monitoring the integrity of major CNS functionalities through SSEP; and 2) leveraged this technology to explore ultrasound-induced neuromodulation. The sensor technology is well suited for this application because the sensor electrical properties are uninfluenced by direct exposure to ultrasound irradiation. SIGNIFICANCE: The sensors and experimental paradigm we present involve standard, safe clinical neurological assessment methods and are thus applicable to a wide range of future translational studies in humans with any manner of health condition.

Public Regulatory Databases as a Source of Insight for Neuromodulation Devices Stimulation Parameters.

OBJECTIVE: The Shannon model is often used to define an expected boundary between non-damaging and damaging modes of electrical neurostimulation. Numerous preclinical studies have been performed by manufacturers of neuromodulation devices using different animal models and a broad range of stimulation parameters while developing devices for clinical use. These studies are mostly absent from peer-reviewed literature, which may lead to this information being overlooked by the scientific community. We aimed to locate summaries of these studies accessible via public regulatory databases and to add them to a body of knowledge available to a broad scientific community. METHODS: We employed web search terms describing device type, intended use, neural target, therapeutic application, company name, and submission number to identify summaries for premarket approval (PMA) devices and 510(k) devices. We filtered these records to a subset of entries that have sufficient technical information relevant to safety of neurostimulation. RESULTS: We identified 13 product codes for 8 types of neuromodulation devices. These led us to devices that have 22 PMAs and 154 510(k)s and six transcripts of public panel meetings. We found one PMA for a brain, peripheral nerve, and spinal cord stimulator and five 510(k) spinal cord stimulators with enough information to plot in Shannon coordinates of charge and charge density per phase. CONCLUSIONS: Analysis of relevant entries from public regulatory databases reveals use of pig, sheep, monkey, dog, and goat animal models with deep brain, peripheral nerve, muscle and spinal cord electrode placement with a variety of stimulation durations (hours to years); frequencies (10-10,000 Hz) and magnitudes (Shannon k from below zero to 4.47). Data from located entries indicate that a feline cortical model that employs acute stimulation might have limitations for assessing tissue damage in diverse anatomical locations, particularly for peripheral nerve and spinal cord simulation.

Real-Time Detection and Monitoring of Acute Brain Injury Utilizing Evoked Electroencephalographic Potentials.

Rapid detection and diagnosis of a traumatic brain injury (TBI) can significantly improve the prognosis for recovery. Helmet-mounted sensors that detect impact severity based on measurements of acceleration or pressure show promise for aiding triage and transport decisions in active, field environments such as professional sports or military combat. The detected signals, however, report on the mechanics of an impact rather than directly indicating the presence and severity of an injury. We explored the use of cortical somatosensory evoked electroencephalographic potentials (SSEPs) to detect and track, in real-time, neural electrophysiological abnormalities within the first hour following head injury in an animal model. To study the immediate electrophysiological effects of injury in vivo, we developed an experimental paradigm involving focused ultrasound that permits continuous, real-time measurements and minimizes mechanical artifact. Injury was associated with a dramatic reduction of amplitude over the damaged hemisphere directly after the injury. The amplitude systematically improved over time but remained significantly decreased at one hour, compared with baseline. In contrast, at one hour there was a concomitant enhancement of the cortical SSEP amplitude evoked from the uninjured hemisphere. Analysis of the inter-trial electroencephalogram (EEG) also revealed significant changes in low-frequency components and an increase in EEG entropy up to 30 minutes after injury, likely reflecting altered EEG reactivity to somatosensory stimuli. Injury-induced alterations in SSEPs were also observed using noninvasive epidermal electrodes, demonstrating viability of practical implementation. These results suggest cortical SSEPs recorded at just a few locations by head-mounted sensors and associated multiparametric analyses could potentially be used to rapidly detect and monitor brain injury in settings that normally present significant levels of mechanical and electrical noise.

Rapid evaluation of the durability of cortical neural implants using accelerated aging with reactive oxygen species.

OBJECTIVE: A challenge for implementing high bandwidth cortical brain-machine interface devices in patients is the limited functional lifespan of implanted recording electrodes. Development of implant technology currently requires extensive non-clinical testing to demonstrate device performance. However, testing the durability of the implants in vivo is time-consuming and expensive. Validated in vitro methodologies may reduce the need for extensive testing in animal models. APPROACH: Here we describe an in vitro platform for rapid evaluation of implant stability. We designed a reactive accelerated aging (RAA) protocol that employs elevated temperature and reactive oxygen species (ROS) to create a harsh aging environment. Commercially available microelectrode arrays (MEAs) were placed in a solution of hydrogen peroxide at 87 °C for a period of 7 days. We monitored changes to the implants with scanning electron microscopy and broad spectrum electrochemical impedance spectroscopy (1 Hz-1 MHz) and correlated the physical changes with impedance data to identify markers associated with implant failure. MAIN RESULTS: RAA produced a diverse range of effects on the structural integrity and electrochemical properties of electrodes. Temperature and ROS appeared to have different effects on structural elements, with increased temperature causing insulation loss from the electrode microwires, and ROS concentration correlating with tungsten metal dissolution. All array types experienced impedance declines, consistent with published literature showing chronic (>30 days) declines in array impedance in vivo. Impedance change was greatest at frequencies <10 Hz, and smallest at frequencies 1 kHz and above. Though electrode performance is traditionally characterized by impedance at 1 kHz, our results indicate that an impedance change at 1 kHz is not a reliable predictive marker of implant degradation or failure. SIGNIFICANCE: ROS, which are known to be present in vivo, can create structural damage and change electrical properties of MEAs. Broad-spectrum electrical impedance spectroscopy demonstrates increased sensitivity to electrode damage compared with single-frequency measurements. RAA can be a useful tool to simulate worst-case in vivo damage resulting from chronic electrode implantation, simplifying the device development lifecycle.

Acute effects of nonexcitatory electrical stimulation during systole in isolated cardiac myocytes and perfused heart.

Application of electrical field to the heart during the refractory period of the beat has been shown to increase the force of contraction both in animal models and in heart failure patients (cardiac contractility modulation, or CCM). A direct increase in intracellular calcium during CCM has been suggested to be the mechanism behind the positive inotropic effect of CCM. We studied the effect of CCM on isolated rabbit cardiomyocytes and perfused whole rat hearts. The effect of CCM was observed in single cells via fluorescent measurements of intracellular calcium concentration ([Ca(2+)]i) and cell length (L). Cells were paced once per second throughout these recordings, and CCM stimulation was delivered via biphasic electric fields of 20 ms duration applied during the refractory period. CCM increased the peak amplitude of both [Ca(2+)]i and L for the first beat during CCM compared to control, but then [Ca(2+)]i and L decayed to levels lower than the control. During CCM, all contractions had a faster time to peak for both [Ca(2+)]i and L; after stopping CCM the rise times returned to control levels. In the whole rat heart, the positive inotropic effect of CCM stimulation on left ventricular pressure was completely abolished in the presence of metoprolol, a beta-1 adrenergic blocker. In summary, the CCM-induced changes in intracellular calcium handling by cardiomyocytes did not explain the sustained positive inotropic effect in the whole heart and the ß-adrenergic pathway may be involved in the CCM mechanism of action.

Longitudinal vascular dynamics following cranial window and electrode implantation measured with speckle variance optical coherence angiography.

Speckle variance optical coherence angiography (OCA) was used to characterize the vascular tissue response from craniotomy, window implantation, and electrode insertion in mouse motor cortex. We observed initial vasodilation ~40% greater than original diameter 2-3 days post-surgery (dps). After 4 weeks, dilation subsided in large vessels (>50 µm diameter) but persisted in smaller vessels (25-50 µm diameter). Neovascularization began 8-12 dps and vessel migration continued throughout the study. Vasodilation and neovascularization were primarily associated with craniotomy and window implantation rather than electrode insertion. Initial evidence of capillary re-mapping in the region surrounding the implanted electrode was manifest in OCA image dissimilarity. Further investigation, including higher resolution imaging, is required to validate the finding. Spontaneous lesions also occurred in many electrode animals, though the inception point appeared random and not directly associated with electrode insertion. OCA allows high resolution, label-free in vivo visualization of neurovascular tissue, which may help determine any biological contribution to chronic electrode signal degradation. Vascular and flow-based biomarkers can aid development of novel neural prostheses.

Biomechanical and functional variation in rat sciatic nerve following cuff electrode implantation.

BACKGROUND: Nerve cuff electrodes are commonly and successfully used for stimulating peripheral nerves. On the other hand, they occasionally induce functional and morphological changes following chronic implantation, for reasons not always clear. We hypothesize that restriction of nerve mobility due to cuff implantation may alter nerve conduction. METHODS: We quantified acute changes in nerve-muscle electrophysiology, using electromyography, and nerve kinematics in anesthetized Sprague Dawley rat sciatic nerves during controlled hindlimb joint movement. We compared electrophysiological and biomechanical response in uncuffed nerves and those secured within a cuff electrode using analysis of variance (ANOVA) and regression analysis. RESULTS: Tethering resulting from cuff implantation resulted in altered nerve strain and a complex biomechanical environment during joint movement. Coincident with biomechanical changes, electromyography revealed significantly increased variability in the response of conduction latency and amplitude in cuffed, but not free, nerves following joint movement. CONCLUSION: Our findings emphasize the importance of the mechanical interface between peripheral nerves and their devices on neurophysiological performance. This work has implications for nerve device design, implantation, and prediction of long-term efficacy.

Mechanical bioeffects of pulsed high intensity focused ultrasound on a simple neural model.

PURPOSE: To study how pressure pulses affect nerves through mechanisms that are neither thermal nor cavitational, and investigate how the effects are related to cumulative radiation-force impulse (CRFI). Applications include traumatic brain injury and acoustic neuromodulation. METHODS: A simple neural model consisting of the giant axon of a live earthworm was exposed to trains of pressure pulses produced by an 825 kHz focused ultrasound transducer. The peak negative pressure of the pulses and duty cycle of the pulse train were controlled so that neither cavitation nor significant temperature rise occurred. The amplitude and conduction velocity of action-potentials triggered in the worm were measured as the magnitude of the pulses and number of pulses in the pulse trains were varied. RESULTS: The functionality of the axons decreased when sufficient pulse energy was applied. The level of CRFI at which the observed effects occur is consistent with the lower levels of injury observed in this study relative to blast tubes. The relevant CRFI values are also comparable to CRFI values in other studies showing measureable changes in action-potential amplitudes and velocities. Plotting the measured action-potential amplitudes and conduction velocities from different experiments with widely varying exposure regimens against the single parameter of CRFI yielded values that agreed within 21% in terms of amplitude and 5% in velocity. A predictive model based on the assumption that the temporal rate of decay of action-potential amplitude and velocity is linearly proportional the radiation force experienced by the axon predicted the experimental amplitudes and conduction velocities to within about 20% agreement. CONCLUSIONS: The functionality of axons decreased due to noncavitational mechanical effects. The radiation force, possibly by inducing changes in ion-channel permeability, appears to be a possible mechanism for explaining the observed degradation. The CRFI is also a promising parameter for quantifying neural bioeffects during exposure to pressure waves, and for predicting axon functionality.

FDA regulation of invasive neural recording electrodes: a daunting task for medical innovators.

The U.S. Food and Drug Administration (FDA) is charged with assuring the safety and effectiveness of medical devices. Before any medical device can be brought to market, it must comply with all federal regulations regarding FDA processes for clearance or approval. Navigating the FDA regulatory process may seem like a daunting task to the innovator of a novel medical device who has little experience with the FDA regulatory process or device commercialization. This review introduces the basics of the FDA regulatory premarket process, with a focus on issues relating to chronically implanted recording devices in the central or peripheral nervous system. Topics of device classification and regulatory pathways, the use of standards and guidance documents, and optimal time lines for interaction with the FDA are discussed. Additionally, this article summarizes the regulatory research on neural implant safety and reliability conducted by the FDA's Office of Science and Engineering Laboratories (OSEL) in collaboration with Defense Advanced Research Projects Agency (DARPA) Reliable Neural Technology (RE-NET) Program. For a more detailed explanation of the medical device regulatory process, please refer to several excellent reviews of the FDA's regulatory pathways for medical devices [1]-[4].

Excitation of primary afferent neurons by near-infrared light in vitro.

Near-infrared light therapy is an emerging neurostimulation technology, but its cellular mechanism of action remains unresolved. Using standard intracellular recording techniques, we observed that 5-10 ms pulses of 1889 nm light depolarized the membrane potential for hundreds of milliseconds in more than 85% of dorsal root ganglion and nodose ganglion neurons tested. The laser-evoked depolarizations (LEDs) exhibited complex, multiphasic kinetics comprising fast and slow components. There was no discernable difference in the LEDs in intact ganglion neurons and in acutely isolated neurons. Thus, the LED sensor seems to reside within the neuronal membrane. The near-uniform distribution of responsive neurons increased membrane conductance, and the negative reversal potential value (-41+/-2.9 mV) suggests that LED is unrelated to the activation of heat-sensitive transient receptor potential cation channel subfamily V member 1 channels. The long duration of LEDs favors an involvement of second messengers.

Altered calcium dynamics in cardiac cells grown on silane-modified surfaces.

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca(2+) because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca(2+) imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca(2+) concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca(2+) changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca(2+) dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems.

Development of a new arbitrary waveform defibrillator for cardiac electrophysiology research.

The innovative arbitrary waveform defibrillator for animal research presented in this paper is based on two power linear amplifiers in bridge configuration. It is capable of delivering 10 J shocks of arbitrary shape and duration. The system can be used to test new waveforms by comparing them to traditional ones, in in-vitro experiments. The system is battery operated, has an isolated output, and is PC controlled. Loads with impedance ranging from 10 to 25 ohms can be connected. A maximum of +/-130 V, 10A can be delivered to the loads. Effective voltage and current are measured and collected in the PC. Examples of waveforms as well as preliminary results from experiments of isolated rabbit hearts are presented.

The pharmacology of electrical stimulation in the heart: Where devices meet drugs.

Both cardiac electrical stimulation and cardiac pharmacological agents exert effects by acting upon ion channels, secondary messengers and autonomic nerve terminals. Defining the common substrates between devices and drugs provides the evaluation tools to warn of unsafe interactions with pacemakers, defibrillators or detection of cardiac arrhythmias. This review describes substrates of drug-device interaction, reviews research on drug-like effects of devices, and provides a framework for how the physiology of interaction translates to streamlined clinical trials.:

Method for evaluating optical characteristics of endoscopes for recording fluorescence-related cardiac electrical activity.

Nondestructive methods were used to evaluate marketed fiber-optic endoscopes (intended for simple viewing) for fluorescence recording. Our application is for optical recording from the heart. For one angioscope, we measured a focal length of 0.33 mm, a field of view of 45 degrees, an aperture of 0.26 mm, and an efficiency of 43%. We calculated that the angioscope would give a signal-to-noise ratio of 1.0 for a cardiac action potential, if its field of view were divided into a nine-pixel array (for safe continuous illumination). Our methods are useful in designing and evaluating fluorescence fiber-optic systems with superior signal quality and spatial resolution.