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Following traumatic brain injury (TBI), secondary brain damage due to chronic inflammation is the most predominant cause of the delayed onset of mood and memory disorders. Currently no therapeutic approach is available to effectively mitigate secondary brain injury after TBI. One reason is the blood-brain barrier (BBB), which prevents the passage of most therapeutic agents into the brain. Peptides have been among the leading candidates for CNS therapy due to their low immunogenicity and toxicity, bioavailability, and ease of modification. In this study, we demonstrated that non-invasive intranasal (IN) administration of KAFAK, a cell penetrating anti-inflammatory peptide, traversed the BBB in a murine model of diffuse, moderate TBI. Notably, KAFAK treatment reduced the production of proinflammatory cytokines that contribute to secondary injury. Furthermore, behavioral tests showed improved or restored neurological, memory, and locomotor performance after TBI in KAFAK-treated mice. This study demonstrates KAFAK's ability to cross the blood-brain barrier, to lower proinflammatory cytokines in vivo, and to restore function after a moderate TBI.
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Vanillin is a popular flavoring agent in many food products. Simple, fast, and reliable quantification of this compound is crucial for the food industry. In this work, we have developed a new electrochemical sensor for accurate detection of vanillin in various real samples. The composite electrode was made of sodium montmorillonite nanoclay (SMM) and gold nanoparticles modified ZIF-67 (Au@ZIF-67), in which SMM contributes to the large adsorption capacity of the analyte, ZIF-67 and SMM supply more sensing active sites, and gold nanoparticles provide high electrical conductivity. The sensing electrode was comprehensively characterized using Brunauer-Emmett-Teller, EDS, XRD, SEM, FTIR, and TEM, and its electrochemical behavior for determination of vanillin including the electrooxidation mechanism of vanillin and different parameters such as scan rate and pH value was investigated. The result revealed that a two electron-two proton process was involved in the electrooxidation of vanillin, which takes place more readily due to the lower potential on the surface of SMM/Au@ZIF-67/carbon paste electrode. The new composite electrode was also more sensitive to vanillin detection with an anodic peak current almost 2.6 times more than that of the bare electrode. A linear sensing concentration range was established between 1 and 1200 nM with a detection limit of 0. 3 nM and a limit of quantitation of 1 nM. For real samples, the sensor demonstrated excellent recovery rates and reliability that was comparable to the standard high-performance liquid chromatography method.
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Oro , Nanopartículas del Metal , Oro/química , Nanopartículas del Metal/química , Bentonita , Reproducibilidad de los Resultados , Técnicas Electroquímicas/métodos , ElectrodosRESUMEN
INTRODUCTION: Medical readiness of individual service members (SMs) is an integral component of operational readiness. Electronic Health (eHealth) literacy, here defined as the ability to seek out, find, evaluate, and appraise, integrate, and apply what is gained in electronic environments toward solving a health problem, is a key skill to enhance individual medical readiness. The World Health Organization (WHO) has identified health literacy as an important social determinant of health. The Internet enables individuals to access information virtually anywhere, at any place, any time, which is especially beneficial for SMs who serve around the globe, often isolated from modern health care facilities. MATERIALS AND METHODS: This study examined the utility of the eHealth literacy scale (eHeals) as a potentially valuable tool to assist commanders and medical teams for assessing the health literacy of SMs. The main objective of our study was to examine the validity of the three-factor eHEALS measure for an active duty sample, using confirmatory factorial analysis. RESULTS: Our findings support the validity of the eHEALS measure of electronic health literacy among military SMs, potentially identifying a means of measuring SMs' health literacy, and a target for intervention to increase medical readiness, well-being, and overall quality of life for individual SMs. CONCLUSIONS: The study highlights a brief 6-item assessment tool that can quickly and efficiently be added to the current validated health and well-being measures for military personnel. This brief assessment can provide a measure of knowledge, skills, and confidence regarding ehealth information leading to better-informed health care decisions in the military context. These findings are especially relevant given the military's current emphasis on modernization and the need to put people first in order to build the overall team and improve unit effectiveness.
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Alfabetización en Salud , Personal Militar , Telemedicina , Humanos , Calidad de Vida , Encuestas y Cuestionarios , Internet , Reproducibilidad de los ResultadosRESUMEN
The blood-brain barrier (BBB) is an obstacle to the permeation of most therapeutic drugs into the brain, limiting treatments for neurological disorders. Drugs loaded within nanocarriers that pass through the BBB can overcome this limitation. Halloysite consists of naturally occurring biocompatible clay nanotubes of 50 nm diameter and 15 nm lumen, allowing the loading and sustained release of loaded drugs. These have demonstrated the ability to transport loaded molecules into cells and organs. We propose to use halloysite nanotubes as a "nano-torpedo" for drug delivery through the BBB due to their needle-like shape. To determine if they can cross the BBB using a non-invasive, clinically translatable route of administration, we loaded halloysite with either diazepam or xylazine and delivered these intranasally to mice daily over six days. The sedative effects of these drugs were observed in vestibulomotor tests conducted at two, five, and seven days after the initial administration. Behavioral tests were conducted 3.5 h after administration to show that the effects were from halloysite/delivered drugs and not from the drug alone. As expected, the treated mice performed more poorly than the sham, drug alone, and halloysite-vehicle-treated mice. These results confirm that halloysite permeates the BBB to deliver drugs when administered intranasally.
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Nanotubos , Xilazina , Animales , Ratones , Arcilla , Xilazina/farmacología , Administración Intranasal , Diazepam/farmacología , Silicatos de Aluminio , Preparaciones Farmacéuticas , EncéfaloRESUMEN
The main purpose of this paper is to share the Mentoring for INnovative Design Solutions (MINDS) Scholars Program developed by Alpha Eta Mu Beta, the International Biomedical Engineering Honor Society. The program's goals are to (1) introduce biomedical engineering students to an open-ended design experience as part of interuniversity teams with industry and faculty mentors, and (2) develop the ability to create designs considering clinical translatability on teams with different backgrounds and areas of expertise. MINDS uses an experiential learning approach to (1) enrich student curricular experiences through inter-institutional collaboration, (2) build engineering design skills, including three key design considerations for clinical/commercial success: intellectual property protection, regulatory strategy, and market identification; and (3) emphasize the importance of end-user considerations. From 2015 to 2022, MINDS has involved 131 students from 50 universities and 22 faculty and industry mentors. Pre- and post-program surveys show statistically significant improvements in understanding of the design process, regulatory strategy, intellectual property protection, market definition, and key product requirements and features. Students also improved communication and teamwork skills. Many students indicated that MINDS participation made them more likely to choose careers that involve product development and/or entrepreneurship. Students attained a working ability to integrate market needs, regulatory strategy, and intellectual property considerations into the design process. They also further developed soft skills, such as conflict resolution, time management, and effective communication through the challenges of inter-institutional collaboration. Additionally, the program heightened their awareness of how biomedical devices and technologies can benefit society.
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Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second signaling dynamics occur in several brain disorders including traumatic brain injury, epilepsy, and Alzheimer's disease. The present work reports on the optimization and in vivo testing of a silicon (Si) multifunctional biosensor probe for sub-second simultaneous real-time detection of GLU and GABA. The Si probe features four surface-functionalized platinum ultramicroelectrodes (UMEs) for detection of GLU and GABA, a sentinel site, and integrated microfluidics for in-situ calibration. Optimal enzyme concentrations, size-exclusion phenylenediamine layer and micro spotting conditions were systematically investigated. The measured GLU sensitivity for the GLU and GABA sites were as high as 219 ± 8 nA µM-1 cm-2 (n = 3). The measured GABA sensitivity was as high as 10 ± 1 nA µM-1 cm-2 (n = 3). Baseline recordings (n = 18) in live rats demonstrated a useful probe life of at least 11 days with GLU and GABA concentrations changing at the levels of 100's and 1000's of µM and with expected periodic bursts or fluctuations during walking, teeth grinding and other activities and with a clear difference in the peak amplitude of the sensor fluctuations between rest (low) and activity (higher), or when the rat was surprised (a reaction with no movement). Importantly, the probe could improve methods for large-scale monitoring of neurochemical activity and network function in disease and injury, in live rodent brain.
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Epilepsy, a neurological disorder characterized by recurrent seizures, is known to be associated with impaired sleep and memory. Although the specific mechanisms underlying these impairments are uncertain, the known role of sleep in memory consolidation suggests a potential relationship may exist between seizure activity, disrupted sleep, and memory impairment. A possible mediator in this relationship is the sleep spindle, the characteristic electroencephalographic (EEG) feature of non-rapid-eye-movement (NREM) sleep in humans and other mammals. Growing evidence supports the idea that sleep spindles, having thalamic origin, may mediate the process of long-term memory storage and plasticity by generating neuronal conditions that favor these processes. To study this potential relationship, a single model in which memory, sleep, and epilepsy can be simultaneously observed is of necessity. Rodent models of epilepsy appear to fulfill this requirement. Not only do rodents express both sleep spindles and seizure-induced sleep disruptions, but they also allow researchers to invasively study neurobiological processes both pre- and post- epileptic onset via the artificial induction of epilepsy (a practice that cannot be carried out in human subjects). However, the degree to which sleep architecture differs between rodents and humans makes direct comparisons between the two challenging. This review addresses these challenges and concludes that rodent sleep studies are useful in observing the functional roles of sleep and how they are affected by epilepsy.
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Epilepsia , Consolidación de la Memoria , Animales , Electroencefalografía , Humanos , Roedores , Convulsiones , Sueño/fisiologíaRESUMEN
Glutamate (GLU) and gamma-aminobutyric acid (GABA) are neurotransmitters (NTs) with an essential role in signal transmission in the brain. Brain disorders, such as epilepsy, Alzheimer's and Parkinson's diseases, and traumatic brain injury can be linked to imbalances in the GLU-GABA homeostasis that occurs in sub-second to seconds time frames. Current measurement techniques can detect these two NT concentrations simultaneously only in vitro. The present work reports on the fabrication of a silicon multifunctional biosensor microarray probe for sub-second simultaneous GLU-GABA detection in real-time, with excellent analyte sensitivity and selectivity and in vivo capabilities. The novel Si probes feature four surface-functionalized platinum ultramicroelectrodes (UMEs) for simultaneous amperometric detection of GLU and GABA with a sentinel, and a built-in microfluidic channel for the introduction of neurochemicals in the proximity of the UMEs. The microchannel also allows functioning of an On-Demand In-situ Calibrator that runs in-situ biosensor calibration. The probe exhibited excellent robustness at insertion in agarose-gel brain-tissue-mimicking test, and remarkably high hydrogen peroxide sensitivity (a by-product of GLU-GABA enzyme biosensor) with values on the order of 5000 nA µM -1 cm -2 and maximum sensitivities of 204±15 nA µM -1 cm -2 and 37±7 nA µM -1 cm -2 for GLU and GABA, respectively. Furthermore, the limit of detection of the biosensors reached as low as 7 nM, 165 nM and 750 nM for H 2 O 2, GLU and GABA, respectively and a temporal resolution of hundreds of milliseconds during in vivo studies using freely moving rats.
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Although an abundance of drug candidates exists which are aimed at the remediation of central nervous system (CNS) disorders, the utility of some are severely limited by their inability to cross the blood brain barrier. Potential drug delivery systems such as the Angiopep family of peptides have shown modest potential; however, there is a need for novel drug delivery candidates that incorporate peptidomimetics to enhance the efficiency of transcytosis, specificity, and biocompatibility. Here, we report on the first in vitro cellular uptake and cytotoxicity study of a peptidomimetic, cationic peptide, L57. It binds to cluster 4 of the low-density lipoprotein receptor-related protein 1 (LRP1) receptor which is expressed in numerous cell types, such as brain endothelial cells. We used early-passage-number brain microvascular endothelial cells and astrocytes harvested from rat pup brains that highly express LRP1, to study the uptake of L57 versus Angiopep-7 (A7). Uptake of L57 and A7 showed a concentration-dependent increase, with L57 being taken up to a greater degree than A7 at the same concentration. Additionally, peptide uptake in LRP1-deficient PEA 10 cells had greatly reduced uptake. Furthermore, L57 demonstrated excellent cell viability versus A7, showing promise as a potential drug delivery vector for CNS therapeutics.
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Preparaciones Farmacéuticas , Receptores de Lipoproteína , Animales , Barrera Hematoencefálica , Encéfalo , Sistemas de Liberación de Medicamentos , Células Endoteliales , Péptidos , RatasRESUMEN
The surface of halloysite nanotubes (HNTs) was bifunctionalized with two ligands-folic acid and a fluorochrome. In tandem, this combination should selectively target cancer cells and provide a means for imaging the nanoparticle. Modified bi-functionalized HNTs (bi-HNTs) were then doped with the anti-cancer drug methotrexate. bi-HNTs were characterized and subjected to in vitro tests to assess cellular growth and changes in cellular behavior in three cell lines-colon cancer, osteosarcoma, and a pre-osteoblast cell line (MC3T3-E1). Cell viability, proliferation, and cell uptake efficiency were assessed. The bi-HNTs showed cytocompatibility at a wide range of concentrations. Compared with regular-sized HNTs, reduced HNTs (~6 microns) were taken up by cells in more significant amounts, but increased cytotoxicity lead to apoptosis. Multi-photon images confirmed the intracellular location of bi-HNTs, and the method of cell entry was mainly through caveolae-mediated endocytosis. The bi-HNTs showed a high drug loading efficiency with methotrexate and a prolonged period of release. Most importantly, bi-HNTs were designed as a drug carrier to target cancer cells specifically, and imaging data shows that non-cancerous cells were unaffected after exposure to MTX-doped bi-HNTs. All data provide support for our nanoparticle design as a mechanism to selectively target cancer cells and significantly reduce the side-effects caused by off-targeting of anti-cancer drugs.
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Glutamate (GLU) and γ-aminobutyric acid (GABA) are the major excitatory (E) and inhibitory (I) neurotransmitters in the brain, respectively. Dysregulation of the E/I ratio is associated with numerous neurological disorders. Enzyme-based microelectrode array biosensors present the potential for improved biocompatibility, localized sample volumes, and much faster sampling rates over existing measurement methods. However, enzymes degrade over time. To overcome the time limitation of permanently implanted microbiosensors, we created a microwire-based biosensor that can be periodically inserted into a permanently implanted cannula. Biosensor coatings were based on our previously developed GLU and reagent-free GABA shank-type biosensor. In addition, the microwire biosensors were in the same geometric plane for the improved acquisition of signals in planar tissue including rodent brain slices, cultured cells, and brain regions with laminar structure. We measured real-time dynamics of GLU and GABA in rat hippocampal slices and observed a significant, nonlinear shift in the E/I ratio from excitatory to inhibitory dominance as electrical stimulation frequency increased from 10 to 140 Hz, suggesting that GABA release is a component of a homeostatic mechanism in the hippocampus to prevent excitotoxic damage. Additionally, we recorded from a freely moving rat over fourteen weeks, inserting fresh biosensors each time, thus demonstrating that the microwire biosensor overcomes the time limitation of permanently implanted biosensors and that the biosensors detect relevant changes in GLU and GABA levels that are consistent with various behaviors.
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Técnicas Biosensibles , Ácido Glutámico/química , Microelectrodos , Ácido gamma-Aminobutírico/química , Animales , Encéfalo/diagnóstico por imagen , Estimulación Eléctrica , Homeostasis , Masculino , Microondas , Modelos Neurológicos , Red Nerviosa , Neuronas/metabolismo , Neurotransmisores , Platino (Metal)/química , Ratas , Ratas Sprague-Dawley , Propiedades de SuperficieRESUMEN
Targeted stimulation of white matter has opened newer perspectives in the field of neuromodulation, towards an attempt to improve memory or as a therapy for epilepsy. Stimulation of the fornix, being a part of the Papez circuit, is likely to modulate the limbic network excitability. However, the stimulation-frequency dependent variability in network excitability is unknown. In the case study, which involved stereo electroencephalographic (SEEG) recording of field potentials in a 48-year old left-handed woman with suspected temporal lobe epilepsy, we demonstrated the network effects of acute low (1 and 10â¯Hz) and high (50â¯Hz) frequency electrical stimulation of fornix. Mapping the short-latency evoked responses to forniceal stimulation confirmed the SEEG target localization within the Papez circuit. Low and high-frequency stimulation of the fornix produced opposite effects in the post-stimuli excitability, with the latter causing increased excitability in the limbic network that culminated in a clinical seizure. A distinct spectral peak around 8â¯Hz confirmed that sensing field potentials from the forniceal white matter is feasible. This is the first case study that provided an insight into how the temporal patterning of forniceal stimulation altered the downstream limbic network excitability.
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A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method's ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug arrested development of new axonal damage by 30 days. In addition to evaluation of therapeutics for traumatic brain injury, this hybrid microlens imaging method should be useful to study other types of brain injury and neurodegeneration and cellular responses to treatment.
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Axones/efectos de los fármacos , Lesiones Encefálicas/tratamiento farmacológico , Lesión Axonal Difusa/tratamiento farmacológico , Minociclina/farmacología , Animales , Axones/patología , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Lesiones Encefálicas/diagnóstico por imagen , Lesiones Encefálicas/patología , Lesión Axonal Difusa/diagnóstico por imagen , Lesión Axonal Difusa/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Imagen ÓpticaRESUMEN
The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 µg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal-organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.
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Time course, in vivo imaging of brain cells is crucial to fully understand the progression of secondary cellular damage and recovery in murine models of injury. We have combined high-resolution gradient index lens technology with a model of diffuse axonal injury in rodents to enable repeated visualization of fine features of individual cells in three-dimensional space over several weeks. For example, we recorded changes in morphology in the same axons in the external capsule numerous times over 30 to 60 days, before and after induced traumatic brain injury. We observed the expansion of secondary injury and limited recovery of individual axons in this subcortical white matter tract over time. In another application, changes in microglial activation state were visualized in the penumbra region of mice before and after ischemia induced by middle carotid artery occlusion. The ability to collect a series of high-resolution images of cellular features of the same cells pre- and post-injury enables a unique opportunity to study the progression of damage, spontaneous healing, and effects of therapeutics in mouse models of neurodegenerative disease and brain injury.
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Axones/ultraestructura , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Isquemia Encefálica/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Arterias Carótidas/diagnóstico por imagen , Neuroimagen/métodos , Animales , Axones/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Oclusión Coronaria/cirugía , Femenino , Colorantes Fluorescentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Microscopía de Fluorescencia por Excitación Multifotónica , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Neuroimagen/instrumentación , Regeneración/fisiología , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/lesiones , Sustancia Blanca/metabolismoRESUMEN
Optical imaging of wholemount tissue samples provides greater understanding of structure-function relationships as the architecture of these specimens is generally well preserved. However, difficulties arise when attempting to stitch together images of multiple regions of larger, oddly shaped specimens. These difficulties include (1) maintaining consistent signal-to-noise ratios when the overlying sample surface is uneven, (2) ensuring sample viability when live samples are required, and (3) stabilizing the specimen in a fixed position in a flowing medium without distorting the tissue sample. To address these problems, we designed a simple and cost-efficient device that can be 3D-printed and machined. The design for the device, named the Platform for Planar Imaging of Curved Surfaces (PICS), consists of a sample holder, or "cap" with gaps for fluid flow and a depression for securing the sample in a fixed position without glue or pins, a basket with two arms that move along an external radius to rotate the sample around a central axis, and a customizable platform designed to fit on a commercially available temperature control system for slice electrophysiology. We tested the system using wholemounts of the murine subventricular zone (SVZ), which has a high degree of curvature, to assess sample viability and image quality through cell movement for over an hour for each sample. Using the PICS system, tissues remained viable throughout the imaging sessions, there were no noticeable decreases in the image SNR across an imaging plane, and there was no noticeable displacement of the specimen due to fluid flow.
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Encéfalo/diagnóstico por imagen , Ventrículos Laterales/diagnóstico por imagen , Imagen Óptica/instrumentación , Impresión Tridimensional/instrumentación , Animales , Ratones Transgénicos , Cintigrafía/instrumentación , Relación Señal-RuidoRESUMEN
Glutamate, a major excitatory neurotransmitter in the central nervous system, is essential for regulation of thought, movement, memory, and other higher functions controlled by the brain. Dysregulation of glutamate signaling is associated with severe neuropathological conditions, such as epilepsy, and glioma, a form of brain cancer. Glutamate signals are currently detected by several types of neurochemical probes ranging from microdialysis-based to enzyme-based carbon fiber microsensors. However, an important technology gap exists in the ability to measure glutamate dynamics continuously, and in real time, and from multiple locations in the brain, which limits our ability to further understand the involved spatiotemporal mechanisms of underlying neuropathologies. To overcome this limitation, we developed an enzymatic glutamate microbiosensor, in the form of a ceramic-substrate enabled platinum microelectrode array, that continuously, in real time, measures changes in glutamate concentration from multiple recording sites. In addition, the developed microbiosensor is almost four-fold more sensitive to glutamate than enzymatic sensors previously reported in the literature. Further analysis of glutamate dynamics recorded by our microbiosensor in cultured astrocytes (control condition) and glioma cells (pathological condition) clearly distinguished normal versus impaired glutamate uptake, respectively. These results confirm that the developed glutamate microbiosensor array can become a useful tool in monitoring and understanding glutamate signaling and its regulation in normal and pathological conditions. Furthermore, the developed microbiosensor can be used to measure the effects of potential therapeutic drugs to treat a range of neurological diseases.
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Técnicas Biosensibles , Técnicas Electroquímicas , Glioma/diagnóstico , Ácido Glutámico/aislamiento & purificación , Astrocitos/metabolismo , Astrocitos/patología , Glioma/metabolismo , Glioma/patología , Ácido Glutámico/metabolismo , HumanosAsunto(s)
Análisis Químico de la Sangre/métodos , Isoleucina/sangre , Convulsiones/sangre , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/uso terapéutico , Análisis Químico de la Sangre/normas , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Enfermedad de la Orina de Jarabe de Arce/genética , Enfermedad de la Orina de Jarabe de Arce/orina , Estándares de Referencia , Convulsiones/tratamiento farmacológico , Zonisamida/administración & dosificación , Zonisamida/uso terapéuticoRESUMEN
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in vivo studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, H2O2) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method requires a rather cumbersome process with prereactors and relies on externally applied reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for glutamate (Glu) and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. By simultaneously measuring and subtracting the H2O2 oxidation currents generated from these microbiosensors, GABA and Glu can be detected continuously in real-time in vitro and ex vivo and without the addition of any externally applied reagents. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the Glu oxidation that takes place at the Glu microbiosensor. A GABA sensitivity of 36 ± 2.5 pA µM-1cm-2, which is 26-fold higher than reported in the literature, and a limit of detection of 2 ± 0.12 µM were achieved in an in vitro setting. The GABA probe was successfully tested in an adult rat brain slice preparation. These results demonstrate that the developed GABA probe constitutes a novel and powerful neuroscientific tool that could be employed in the future for in vivo longitudinal studies of the combined role of GABA and Glu (a major excitatory neurotransmitter) signaling in brain disorders, such as epilepsy and traumatic brain injury, as well as in preclinical trials of potential therapeutic agents for the treatment of these disorders.
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A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.