In vitro induction of in vivo-relevant stellate astrocytes in 3D brain-derived, decellularized extracellular matrices.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications.

Neutralization of Cannabidiol Neurotoxicity in Neuron-Astrocyte Sandwich Coculture.

Cannabidiol (CBD), a main nonpsychoactive phytocannabinoid in the Cannabis genus, has been in the limelight for its potential health benefits in various neurological diseases. However, the safety issue of CBD in the nervous system has not been settled fully, while CBD has been reported to have mild side effects including dizziness and somnolence. In this work, a platform of neuron-astrocyte sandwich coculture to investigate the neurotoxicity of CBD, as well as the neuronal responses to CBD, in a more in vivo relevant mode is constructed. CBD (15 and 30 µm) causes the viability decrease, along with morphological damage, in the neuron-alone culture, whereas its neurotoxic effects are significantly attenuated by the supports of astrocytes in the neuron-astrocyte coculture. In addition, it is found that CBD-induced increase of intracellular Ca2+ concentration and depolarization of mitochondrial membrane potential, via activation of transient receptor potential vanilloid 1, are noticeably ameliorated by coculturing neurons with astrocytes. This work provides crucial information in the development of CBD as therapeutics for neurological disorders, as well as in a fundamental understanding of how CBD works in the nervous system.

In Vitro Studies on Therapeutic Effects of Cannabidiol in Neural Cells: Neurons, Glia, and Neural Stem Cells.

(‒)-Cannabidiol (CBD) is one of the major phytocannabinoids extracted from the Cannabis genus. Its non-psychoactiveness and therapeutic potential, partly along with some anecdotal-if not scientific or clinical-evidence on the prevention and treatment of neurological diseases, have led researchers to investigate the biochemical actions of CBD on neural cells. This review summarizes the previously reported mechanistic studies of the CBD actions on primary neural cells at the in vitro cell-culture level. The neural cells are classified into neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells, and the CBD effects on each cell type are described. After brief introduction on CBD and in vitro studies of CBD actions on neural cells, the neuroprotective capability of CBD on primary neurons with the suggested operating actions is discussed, followed by the reported CBD actions on glia and the CBD-induced regeneration from neural stem cells. A summary section gives a general overview of the biochemical actions of CBD on neural cells, with a future perspective. This review will provide a basic and fundamental, but crucial, insight on the mechanistic understanding of CBD actions on neural cells in the brain, at the molecular level, and the therapeutic potential of CBD in the prevention and treatment of neurological diseases, although to date, there seem to have been relatively limited research activities and reports on the cell culture-level, in vitro studies of CBD effects on primary neural cells.

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture.

Introduction: Reports on the neurotoxic and neuroprotective effects of cannabidiol (CBD) have not been in complete accord, showing different and somewhat contradictory results depending upon the brain cell types and experimental conditions employed. This work systematically examines the neuroprotective capability of CBD against oxidative stress (i.e., hydrogen peroxide [H2O2]) as well as its toxicity profile in the in vitro culture platform of primary hippocampal neurons. Materials and Methods: The low cell-density (100 neurons per mm2) culture was used for analyzing the viability and morphology of neurons at a single-cell level with a confocal laser-scanning microscope (CLSM). Primary neurons were obtained from the hippocampal tissues of embryonic day-18 (E18) Sprague-Dawley rat pups and treated with CBD (0.1-100 µM) and/or H2O2 (0.1-50 µM) at 1 DIV (days in vitro). Results: The lethal concentration 50 (LC50) value (the concentration causing 50% cell death) of CBD was calculated to be 9.85 µM after 24 h of incubation, and that of H2O2 was 2.46 µM under the same conditions. The neuroprotection ratio of CBD against H2O2 ([H2O2]=10 µM) was 2.40 with 5 µM of CBD, increasing the cell viability to 57% from 24%. The CLSM analysis suggested that the cell-death mechanisms were different for CBD and H2O2, and CBD did not completely rescue the morphological alterations of primary hippocampal neurons caused by H2O2, such as neurite degeneration, at least in the in vitro neuron culture. Conclusion: Although CBD showed both neurotoxic and neuroprotective effects on hippocampal neurons in the in vitro setting, the use of low-concentrated (i.e., 5 µM) CBD, not causing toxic effects on the neurons, significantly rescued the neurons from the oxidative stress (H2O2), confirming its neuroprotection capability.

Neuronal Migration on Silicon Microcone Arrays with Different Pitches.

Neuronal migration is a complicated but fundamental process for proper construction and functioning of neural circuits in the brain. Many in vivo studies have suggested the involvement of environmental physical features of a neuron in its migration, but little effort has been made for the in vitro demonstration of topography-driven neuronal migration. This work investigates migratory behaviors of primary hippocampal neurons on a silicon microcone (SiMC) array that presents 14 different pitch domains (pitch: 2.5-7.3 µm). Neuronal migration becomes the maximum at the pitch of around 3 µm, with an upper migration threshold of about 4 µm. Immunocytochemical studies indicate that the speed and direction of migration, as well as its probability of occurrence, are correlated with the morphology of the neuron, which is dictated by the pitch and shape of underlying SiMC structures. In addition to the effects on neuronal migration, the real-time imaging of migrating neurons on the topographical substrate reveals new in vitro modes of neuronal migration, which have not been observed on the conventional flat culture plate, but been suggested by in vivo studies.

Astrocyte-Encapsulated Hydrogel Microfibers Enhance Neuronal Circuit Generation.

Astrocytes, the most representative glial cells in the brain, play a multitude of crucial functions for proper neuronal development and synaptic-network formation, including neuroprotection as well as physical and chemical support. However, little attention has been paid, in the neuroregenerative medicine and related fields, to the cytoprotective incorporation of astrocytes into neuron-culture scaffolds and full-fledged functional utilization of encapsulated astrocytes for controlled neuronal development. In this article, a 3D neurosupportive culture system for enhanced induction of neuronal circuit generation is reported, where astrocytes are confined in hydrogel microfibers and protected from the outside. The astrocyte-encapsulated microfibers significantly accelerate the neurite outgrowth and guide its directionality, and enhance the synaptic formation, without any physical contact with the neurons. This astrocyte-laden system provides a pivotal culture scaffold for advanced development of cell-based therapeutics for neural injuries, such as spinal cord injury.

Synthetic Strategies for (-)-Cannabidiol and Its Structural Analogs.

(-)-Cannabidiol ((-)-CBD), a non-psychoactive phytocannabinoid from Cannabis, and its structural analogs have received growing attention in recent years because of their potential therapeutic benefits, including neuroprotective, anti-epileptic, anti-inflammatory, anxiolytic, and anti-cancer properties. (-)-CBD and its analogs have been obtained mainly based on extraction from the natural source; however, the conventional extraction-based methods have some drawbacks, such as poor quality control along with purification difficulty. Chemical-synthetic strategies for (-)-CBD could tackle these issues, and, additionally, generate novel (-)-CBD analogs that exhibit advanced biological activities. This review concisely summarizes the historic and recent milestones in the synthetic strategies for (-)-CBD and its analogs.

Strong Photo-Amplification Effects in Flexible Organic Capacitors with Small Molecular Solid-State Electrolyte Layers Sandwiched between Photo-Sensitive Conjugated Polymer Nanolayers.

We demonstrate strong photo-amplification effects in flexible organic capacitors which consist of small molecular solid-state electrolyte layers sandwiched between light-sensitive conjugated polymer nanolayers. The small molecular electrolyte layers were prepared from aqueous solutions of tris(8-hydroxyquinoline-5-sulfonic acid) aluminum (ALQSA3), while poly(3-hexylthiophene) (P3HT) was employed as the light-sensitive polymer nanolayer that is spin-coated on the indium-tin oxide (ITO)-coated poly(ethylene terephthalate) (PET) film substrates. The resulting capacitors feature a multilayer device structure of PET/ITO/P3HT/ALQSA3/P3HT/ITO/PET, which were mechanically robust due to good adhesion between the ALQSA3 layers and the P3HT nanolayers. Results showed that the specific capacitance was increased by ca. 3-fold when a white light was illuminated to the flexible organic multilayer capacitors. In particular, the capacity of charge storage was remarkably (ca. 250-fold) enhanced by a white light illumination in the potentiostatic charge/discharge operation, and the photo-amplification functions were well maintained even after bending for 300 times at a bending angle of 180(°).

Effects of music therapy on pain, discomfort, and depression for patients with leg fractures.

PURPOSE: To determine the effects of music therapy on pain, discomfort, and depression for patients with leg fractures. METHODS: Data were collected from 40 patients admitted in an orthopedic surgery care unit. The subjects included 20 intervention group members and 20 control group members. Music therapy was offered to intervention group members once a day for 3 days for 30-60 minutes per day. Pain was measured with a numeric rating scale and by measuring vital signs. Discomfort and depression were measured with self-administered questionnaires. RESULTS: Patients who received music therapy had a lower degree of pain than patients who did not receive music therapy as measured by the numeric pain score (p<0.001), systolic blood pressure (p<0.01), diastolic blood pressure (p<0.001), pulse rate (p<0.001) and respiration (p<0.001). Patients who were provided with music therapy also had a lower degree of discomfort than patients who were not provided with this therapy (p<0.01). CONCLUSIONS: These results demonstrate that music therapy is an effective method for decreasing pain and dis-comfort for patients with leg fractures.