Subwavelength Terahertz Resonance Imaging (STRING) for Molecular Fingerprinting.

Subwavelength terahertz (THz) imaging methods are highly desirable for biochemical sensing as well as materials sciences, yet sensitive spectral fingerprinting is still challenging in the frequency domain due to weak light-matter interactions. Here, we demonstrate subwavelength THz resonance imaging (STRING) that overcomes this limitation to achieve ultrasensitive molecular fingerprinting. STRING combines individual ring-shaped coaxial single resonators with near-field spectroscopy, yielding considerable sensitivity gains from both local field enhancement and the near-field effect. As an initial demonstration, we obtained spectral fingerprints from isomers of α-lactose and maltose monohydrates, achieving sensitivity that was enhanced by up to 10 orders of magnitude compared to far-field THz measurements with pelletized samples. Our results show that the STRING platform could enable the development of THz spectroscopy as a practical and sensitive tool for the fingerprinting and spectral imaging of molecules and nanoparticles.

Amperometric Microsensors Monitoring Glutamate-Evoked In Situ Responses of Nitric Oxide and Carbon Monoxide from Live Human Neuroblastoma Cells.

In the brain, nitric oxide (NO) and carbon monoxide (CO) are important signaling gases which have multifaceted roles, such as neurotransmitters, neuromodulators, and vasodilators. Even though it is difficult to measure NO and CO in a living system due to their high diffusibility and extremely low release levels, electrochemical sensors are promising tools to measure in vivo and in vitro NO and CO gases. In this paper, using amperometric dual and septuple NO/CO microsensors, real-time NO and CO changes evoked by glutamate were monitored simultaneously for human neuroblastoma (SH-SY5Y) cells. In cultures, the cells were differentiated and matured into functional neurons by retinoic acid and brain-derived neurotrophic factor. When glutamate was administrated to the cells, both NO and CO increases and subsequent decreases returning to the basal levels were observed with a dual NO/CO microsensor. In order to facilitate sensor's measurement, a flower-type septuple NO/CO microsensor was newly developed and confirmed in terms of the sensitivity and selectivity. The septuple microsensor was employed for the measurements of NO and CO changes as a function of distances from the position of glutamate injection. Our sensor measurements revealed that only functionally differentiated cells responded to glutamate and released NO and CO.

Nanoscale intracortical iron injection induces chronic epilepsy in rodent.

We studied the electrophysiological, hemodynamic, and cytomorphological consequences of microhemorrhagic brain injury induced by a nanoscale iron injection. Of particular interest were the etiology, development, and treatment of epilepsy associated with this injury. We developed an animal model of chronic epilepsy using nanoscale injection into the adult mouse cortex. Although injection of nanoamounts of iron did not cause clear cell death or damage in the cortex, it elicited varying degrees of spontaneous epileptiform events that could be recorded under anesthesia 3 months postinjection. The influence of these chronic epileptiform events on neurovascular coupling was probed by directly stimulating the cortex ipsilateral to the epileptic focus and by measuring cerebral blood volume simultaneously in both hemispheres using intrinsic signal optical imaging. The ipsilateral hemodynamic response was dramatically lower in animals that exhibited longer, more frequent epileptiform events, but it was unchanged in animals displaying infrequent, short events. In contrast, the contralateral hemodynamic response was augmented in all iron-injected animals compared with the control group. These abnormal hemodynamic responses in chronically epileptic animals were correlated with the degree of reduction in the number of GABAergic interneurons. Therefore, nanoscale iron injection, which mimics some aspects of microhemorrhagic brain injury, generated chronic, yet varying, degrees of spontaneous epileptiform events. Moreover, the severity of the epileptiform events corresponded to the degree of reduction in GABAergic interneurons in the iron-injected hemisphere and the level of autoregulatory dysfunction of cerebral blood flow. © 2013 Wiley Periodicals, Inc.

Implementation of the neuro-glia-vascular unit through co-culture of adult neural stem cells and vascular cells and transcriptomic analysis of diverse Aß assembly types.

BACKGROUND: The blood-brain barrier (BBB) is a specialized layer between blood vessels and tissue in the brain, which is comprised of a neuro-glia-vascular (NGV) unit, thus play a vital role in various brain diseases. NEW METHOD: We developed the in vitro NGV units by co-culturing brain microvascular endothelial cells (BMECs; bEnd.3) and primary neural stem cells extracted from subventricular zone of adult mice. This approach was designed to mimic the RNA profile conditions found in the microvessels of a mouse brain and confirmed through various comparative transcriptome analyses. RESULTS: Optimal NGV unit development was achieved by adjusting cell density-dependent co-culture ratios. Specifically, the morphogenic development and neuronal association of astrocyte endfeet were well observed in the contact region with BMECs in the NGV unit. Through transcriptome analysis, we compared co-cultured bEnd.3/NSCs with monocultured bEnd.3 or NSCs and additionally compared them with previously reported mouse brain vascular tissue to show that this NGV unit model is a suitable in vitro model for neurological disease such as Alzheimer's disease (AD). COMPARISON WITH EXISTING METHOD(S): This in vitro NGV unit was formed from neural stem cells and vascular cells in the brain of adult mice, not embryos. It is very useful for studying brain disease mechanisms by identifying proteins and genes associated with diseases progress. CONCLUSIONS: We suggest that this simple in vitro NGV model is appropriate to investigate the relationship between BBB changes and pathological factors in the fields of neurovascular biology and cerebrovascular diseases including AD.

Neuropathogenesis-on-chips for neurodegenerative diseases.

Developing diagnostics and treatments for neurodegenerative diseases (NDs) is challenging due to multifactorial pathogenesis that progresses gradually. Advanced in vitro systems that recapitulate patient-like pathophysiology are emerging as alternatives to conventional animal-based models. In this review, we explore the interconnected pathogenic features of different types of ND, discuss the general strategy to modelling NDs using a microfluidic chip, and introduce the organoid-on-a-chip as the next advanced relevant model. Lastly, we overview how these models are being applied in academic and industrial drug development. The integration of microfluidic chips, stem cells, and biotechnological devices promises to provide valuable insights for biomedical research and developing diagnostic and therapeutic solutions for NDs.

Indoor-Light-Activated Blue TiO2-Molecule-WO3 Visible Photocatalyst for Antibacterial Performance against Escherichia coli.

Currently used visible light catalysts either operate with high-power light sources or require prolonged periods of time for catalytic reactions. This presents a limitation regarding facile application in indoor environments and spaces frequented by the public. Furthermore, this gives rise to elevated power consumption. Here, we enhance photocatalytic performance with blue TiO2 and WO3 complexes covalently coupled through an organic molecule, 3-mercaptopropionic acid, under indoor light. Antibacterial experiments against 108 CFU/mL Escherichia coli (E. coli) suspensions were conducted under indoor light exposure conditions. They showed a sterilization effect of almost 90% within 70 min and nearly 100% after 110 min. The complex generates reactive oxygen species (ROS), such as •OH and O2•-, under natural air conditions. We also showed that h+ and •OH are important for sterilizing E. coli using common scavengers. This research highlights the potential of these complexes to generate ROS, effectively playing a crucial role in antibacterial effects under indoor light.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Probing Interfacial Charge Transfer between Amyloid-ß and Graphene during Amyloid Fibrillization Using Raman Spectroscopy.

Charge transfer plays a key role in the structural transformation of amyloid-ß proteins (Aßs), as it fibrillizes from small monomers to intermediate oligomers and to ordered fibrils. While the protein fibrillization states have been identified using cryo-electron microscopy, X-ray diffraction, Raman, infrared, terahertz spectroscopies, etc., there is little known about the electronic states during the fibrilization of Aß protein. Here, we probe the charge transfer of Aß42 proteins at different aggregation stages adsorbed on monolayer graphene (Gr) and molybdenum disulfide (MoS2) using Raman spectroscopy. Monomers, oligomers, and fibrils prepared in buffer solutions were deposited and dried separately on Gr and MoS2 where well-established characteristic Raman modes (G, 2D for Gr and E2g, A1g for MoS2) were monitored. The shifts in Raman parameters showed that the small Aß monomers withdraw electrons, whereas fibrils donate electrons to Gr and MoS2. Oligomers undergo transient charge states near the neutrality point. This is explained in terms of modulated carrier concentration in Gr and MoS2. This finding provides insight into the electronic properties of Aßs that could be essential to identifying the onset of toxic fibril forms and developing a straightforward, label-free diagnosis using Gr and MoS2.

3D printed multi-growth factor delivery patches fabricated using dual-crosslinked decellularized extracellular matrix-based hybrid inks to promote cerebral angiogenesis.

Generally, brain angiogenesis is a tightly regulated process, which scarcely occurred in the absence of specific pathological conditions. Delivery of exogenous angiogenic factors enables the induction of desired angiogenesis by stimulating neovasculature formation. However, effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. Herein, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs), using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink for printing patches through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition reaction with combining methacrylated hyaluronic acid (HAMA) and vascular-tissue-derived decellularized extracellular matrix (VdECM), and thermal crosslinking of VdECM. 3D printing technology, a useful approach with fabrication versatility with customizable systems and multiple biomaterials, is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by label-free photoacoustic microscopy in vivo. The developed multi-GFs releasing patch may offer a promising therapeutic approach of spatiotemporal drugs releasing such as cerebral ischemia, ischemic heart diseases, diabetes, and even use as vaccines. STATEMENT OF SIGNIFICANCE: Effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. In this study, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs) using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition, and thermal crosslinking. 3D printing technology is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by photoacoustic microscopy in vivo.

Enhanced mobility of neural cells with a transparent electric field stimulator.

Mobility is one of the important characteristics of living cells. It also plays a significant role in therapeutic cell transplantation with target location specificity. To enhance cell mobility, a neural cell stimulator was assembled with graphenes, which are two-dimensional nanocarbon materials that form a transparent electrode over the cover glass in a cell culture dish. This transparent stimulator applies electrical field stimulation to the neural cells. The advantages of this new transparent electrical field stimulator (TEFS) with a graphene electrode include transparency, because few layered graphenes are optically transparent, and biocompatibility, because the cover glass is coated with laminin. In this paper, it is reported that constant electric field stimulation, which is at a specific strength, facilitates the mobility of a neural cell and makes the visibility of cellular behavior on the electrode much better than that of any other existing cell stimulator that has metal electrodes. The strength of the electrical field for stimulating cells varies from 4.5 mV/mm to 450 mV/mm. When continuous electric field stimulation was applied for 4 hours at the electric field strength of 45 mV/mm, the mobility of the neural cells was significantly enhanced compared to the control conditions, wherein there was no electric field stimulation. Thus, the feasibility of the TEFS with the nanothickness of graphene was tested to modulate the mobility of neural cells in vitro. The result suggests that electrical field stimulation could enhance neural cell alignment, cell-to-cell coupling, and networks, and may be applied to cell transplantation to boost therapeutic effectiveness.

Human mini-blood-brain barrier models for biomedical neuroscience research: a review.

The human blood-brain barrier (BBB) is a unique multicellular structure that is in critical demand for fundamental neuroscience studies and therapeutic evaluation. Despite substantial achievements in creating in vitro human BBB platforms, challenges in generating specifics of physiopathological relevance are viewed as impediments to the establishment of in vitro models. In this review, we provide insight into the development and deployment of in vitro BBB models that allow investigation of the physiology and pathology of neurological therapeutic avenues. First, we highlight the critical components, including cell sources, biomaterial glue collections, and engineering techniques to reconstruct a miniaturized human BBB. Second, we describe recent breakthroughs in human mini-BBBs for investigating biological mechanisms in neurology. Finally, we discuss the application of human mini-BBBs to medical approaches. This review provides strategies for understanding neurological diseases, a validation model for drug discovery, and a potential approach for generating personalized medicine.

Supramolecular Peptide Hydrogel-Based Soft Neural Interface Augments Brain Signals through a Three-Dimensional Electrical Network.

Recording neural activity from the living brain is of great interest in neuroscience for interpreting cognitive processing or neurological disorders. Despite recent advances in neural technologies, development of a soft neural interface that integrates with neural tissues, increases recording sensitivity, and prevents signal dissipation still remains a major challenge. Here, we introduce a biocompatible, conductive, and biostable neural interface, a supramolecular ß-peptide-based hydrogel that allows signal amplification via tight neural/hydrogel contact without neuroinflammation. The non-biodegradable ß-peptide forms a multihierarchical structure with conductive nanomaterial, creating a three-dimensional electrical network, which can augment brain signal efficiently. By achieving seamless integration in brain tissue with increased contact area and tight neural tissue coupling, the epidural and intracortical neural signals recorded with the hydrogel were augmented, especially in the high frequency range. Overall, our tissuelike chronic neural interface will facilitate a deeper understanding of brain oscillation in broad brain states and further lead to more efficient brain-computer interfaces.

Identifying Fibrillization State of Aß Protein via Near-Field THz Conductance Measurement.

Progressive Alzheimer's disease is correlated with the oligomerization and fibrillization of the amyloid beta (Aß) protein. We identify the fibrillization stage of the Aß protein through label-free near-field THz conductance measurements in a buffer solution. Frequency-dependent conductance was obtained by measuring the differential transmittance of the time-domain spectroscopy in the THz range with a molar concentration of monomer, oligomer, and fibrillar forms of the Aß protein. Conductance at the lower frequency limit was observed to be high in monomers, reduced in oligomers, and dropped to an insulating state in fibrils and increased proportionally with the Aß protein concentration. The monotonic decrease in the conductance at low frequency was dominated by a simple Drude component in the monomer with concentration and nonlinear conductance behaviors in the oligomer and fibril. By extracting the structural localization parameter, a dimensionless constant, with the modified Drude-Smith model, we defined a dementia quotient (DQ) value (0 < De < 1) as a discrete metric for a various Aß proteins at a low concentration of 0.1 µmol/L; DQ = 1.0 ± 0.002 (fibril by full localization, mainly by Smith component), DQ = 0.64 ± 0.013 (oligomer by intermixed localization), and DQ = 0.0 ± 0.000 (monomer by Drude component). DQ values were discretely preserved independent of the molar concentration or buffer variation. This provides plenty of room for the label-free diagnosis of Alzheimer's disease using the near-field THz conductance measurement.

Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration.

Cerebral endothelial cells (ECs) require junctional proteins to maintain blood-brain barrier (BBB) integrity, restricting toxic substances and controlling peripheral immune cells with a higher concentration of mitochondria than ECs of peripheral capillaries. The mechanism underlying BBB disruption by defective mitochondrial oxidative phosphorylation (OxPhos) is unclear in a mitochondria-related gene-targeted animal model. To assess the role of EC mitochondrial OxPhos function in the maintenance of the BBB, we developed an EC-specific CR6-interactin factor1 (Crif1) deletion mouse. We clearly observed defects in motor behavior, uncompacted myelin and leukocyte infiltration caused by BBB maturation and disruption in this mice. Furthermore, we investigated the alteration in the actin cytoskeleton, which interacts with junctional proteins to support BBB integrity. Loss of Crif1 led to reorganization of the actin cytoskeleton and a decrease in tight junction-associated protein expression through an ATP production defect in vitro and in vivo. Based on these results, we suggest that mitochondrial OxPhos is important for the maturation and maintenance of BBB integrity by supplying ATP to cerebral ECs.

BT-11 improves stress-induced memory impairments through increment of glucose utilization and total neural cell adhesion molecule levels in rat brains.

In Oriental medicine, roots of Polygala tenuifolia Willdenow have been known to be an important herb that exhibits sedative effects in insomnia, palpitation with anxiety, restlessness, and disorientation in humans. We previously reported that BT-11, extracted from those roots, improved scopolamine-induced amnesia in rats and inhibited acetylcholinesterase activities in vitro. Therefore, we proposed that BT-11 could remedy stress-induced memory deficits in rats. In this study, the stress-induced memory impairments in rats were significantly reversed almost to the control level by BT-11 treatment. To seek an active component of BT-11 that plays an important role in antipsychotic effects, we compared BT-11 with 3,4,5-trimethoxycinnamic acid (TMCA), which is a constituent of those root extracts. However, the effects of TMCA were less or were not consistent with those of BT-11 in some of tests. In particular, BT-11 reversed the stress-induced reduction of glucose utilization by [(18)fluorodeoxyglucose]FDG-PET and the levels of neural cell adhesion molecule (NCAM) in rat brains to the control levels, whereas TMCA did not. Therefore, BT-11 improved stress-induced memory impairments through increment of glucose utilization and total NCAM levels in rat brains. In conclusion, BT-11 may be strongly effective against stress-induced amnesia in rats, through the combined effects of TMCA and other active components of BT-11.

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells.

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.

Cellular behavior controlled by bio-inspired and geometry-tunable nanohairs.

A cicada wing has a biocidal feature of rupturing the membrane of cells, while the cactus spine can transmit a water drop to the stem of the plant. Both of these properties have evolved from their respective unique structures. Here, we endeavor to develop geometry-controllable nanohairs that mimic the cicada's wing-like vertical hairs and the cactus spine-like stooped hairs, and to quantitatively characterize the cell migration behavior of the hairy structures. It was found that the neuroblastoma cells are highly sensitive to the variation of surfaces: flat, vertical, and stooped nanohairs (100 nm diameter and 900 nm height). The cells on the vertical hairs showed significantly decreased proliferation. It was found that the behavior of cells cultured on stooped nanohairs is strongly influenced by the direction of the stooped pattern of hairs when we quantitatively measured the migration of cells on flat, vertical, and stooped structures. However, the cells on the flat structures showed random movement and the cells on the vertical nanohairs restricted the nanohair movement. Cells on the stooped structure showed higher forward migration preference compared to that of the other structures. Furthermore, we found that these cellular behaviors on the different patterns of nanohairs were affected by intracellular actin flament change. Consistent with these results, the vertical and stooped structures can facilitate the control of cell viability and guide directional migration for biomedical applications such as organogenesis.

A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology.

Chronic in vivo imaging and electrophysiology are important for better understanding of neural functions and circuits. We introduce the new cranial window using soft, penetrable, elastic, and transparent, silicone-based polydimethylsiloxane (PDMS) as a substitute for the skull and dura in both rats and mice. The PDMS can be readily tailored to any size and shape to cover large brain area. Clear and healthy cortical vasculatures were observed up to 15 weeks post-implantation. Real-time hemodynamic responses were successfully monitored during sensory stimulation. Furthermore, the PDMS window allowed for easy insertion of microelectrodes and micropipettes into the cortical tissue for electrophysiological recording and chemical injection at any location without causing any fluid leakage. Longitudinal two-photon microscopic imaging of Cx3Cr1(+/- GFP) transgenic mice was comparable with imaging via a conventional glass-type cranial window, even immediately following direct intracortical injection. This cranial window will facilitate direct probing and mapping for long-term brain studies.

Cerebral Hemodynamics and Vascular Reactivity in Mild and Severe Ischemic Rodent Middle Cerebral Artery Occlusion Stroke Models.

Ischemia can cause decreased cerebral neurovascular coupling, leading to a failure in the autoregulation of cerebral blood flow. This study aims to investigate the effect of varying degrees of ischemia on cerebral hemodynamic reactivity using in vivo real-time optical imaging. We utilized direct cortical stimulation to elicit hyper-excitable neuronal activation, which leads to induced hemodynamic changes in both the normal and middle cerebral artery occlusion (MCAO) ischemic stroke groups. Hemodynamic measurements from optical imaging accurately predict the severity of occlusion in mild and severe MCAO animals. There is neither an increase in cerebral blood volume nor in vessel reactivity in the ipsilateral hemisphere (I.H) of animals with severe MCAO. The pial artery in the contralateral hemisphere (C.H) of the severe MCAO group reacted more slowly than both hemispheres in the normal and mild MCAO groups. In addition, the arterial reactivity of the I.H in the mild MCAO animals was faster than the normal animals. Furthermore, artery reactivity is tightly correlated with histological and behavioral results in the MCAO ischemic group. Thus, in vivo optical imaging may offer a simple and useful tool to assess the degree of ischemia and to understand how cerebral hemodynamics and vascular reactivity are affected by ischemia.

Alpha-synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson's disease.

Though the etiology of Parkinson's disease (PD) remains unclear, alpha-synuclein (alpha-SN) is regarded as a major causative agent of PD. Several lines of evidence indicate that immunological abnormalities are associated with PD for unknown reasons. The present study was performed to assess whether peripheral blood mononuclear cells (PBMCs) show altered alpha-SN expression in PD patients and to identify its functions, which may be related to peripheral immune abnormalities in PD. alpha-SN was found to be expressed more in 151 idiopathic PD (IPD) patients than in 101 healthy controls, who nevertheless showed as age-dependent increases. By in vitro transfection, alpha-SN expression was shown to be correlated with glucocorticoid sensitive apoptosis, possibly caused by the enhanced expression of glucocorticoid receptor (GR), caspase activations (caspase-8, caspase-9), CD95 up-regulation, and reactive oxygen species (ROS) production. An understanding of the correlation between alpha-SN levels and apoptosis in the presence of the coordinated involvement of multiple processes would provide an insight into the molecular basis of the disease. The present study provides a clue that the alpha-SN may be one of the primary causes of the immune abnormalities observed in PD and offers new targets for pharmacotherapeutic intervention.