Photo-Cross-Linkable Human Albumin Colloidal Gels Facilitate In Vivo Vascular Integration for Regenerative Medicine.

Biodegradable cellular and acellular scaffolds have great potential to regenerate damaged tissues or organs by creating a proper extracellular matrix (ECM) capable of recruiting endogenous cells to support cellular ingrowth. However, since hydrogel-based scaffolds normally degrade through surface erosion, cell migration and ingrowth into scaffolds might be inhibited early in the implantation. This could result in insufficient de novo tissue formation in the injured area. To address these challenges, continuous and microsized strand-like networks could be incorporated into scaffolds to guide and recruit endogenous cells in rapid manner. Fabrication of such microarchitectures in scaffolds is often a laborious and time-consuming process and could compromise the structural integrity of the scaffold or impact cell viability. Here, we have developed a fast single-step approach to fabricate colloidal hydrogels, which are made up of randomly packed human serum albumin-based photo-cross-linkable microparticles with continuous internal networks of microscale voids. The human serum albumin conjugated with methacrylic groups were assembled to microsized aggregates for achieving unique porous structures inside the colloidal gels. The albumin hydrogels showed tunable mechanical properties such as elastic modulus, porosity, and biodegradability, providing a suitable ECM for various cells such as cardiomyoblasts and endothelial cells. In addition, the encapsulated cells within the hydrogel showed improved cell retention and increased survivability in vitro. Microporous structures of the colloidal gels can serve as a guide for the infiltration of host cells upon implantation, achieving rapid recruitment of hematopoietic cells and, ultimately, enhancing the tissue regeneration capacity of implanted scaffolds.

Astrocytes phagocytose adult hippocampal synapses for circuit homeostasis.

In the adult hippocampus, synapses are constantly formed and eliminated1,2. However, the exact function of synapse elimination in the adult brain, and how it is regulated, are largely unknown. Here we show that astrocytic phagocytosis3 is important for maintaining proper hippocampal synaptic connectivity and plasticity. By using fluorescent phagocytosis reporters, we find that excitatory and inhibitory synapses are eliminated by glial phagocytosis in the CA1 region of the adult mouse hippocampus. Unexpectedly, we found that astrocytes have a major role in the neuronal activity-dependent elimination of excitatory synapses. Furthermore, mice in which astrocytes lack the phagocytic receptor MEGF10 show a reduction in the elimination of excitatory synapses; as a result, excessive but functionally impaired synapses accumulate. Finally, Megf10-knockout mice show defective long-term synaptic plasticity and impaired formation of hippocampal memories. Together, our data provide strong evidence that astrocytes eliminate unnecessary excitatory synaptic connections in the adult hippocampus through MEGF10, and that this astrocytic function is crucial for maintaining circuit connectivity and thereby supporting cognitive function.

Microtubule-associated protein 2 mediates induction of long-term potentiation in hippocampal neurons.

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Double staining method for array tomography using scanning electron microscopy.

Scanning electron microscopy (SEM) plays a central role in analyzing structures by imaging a large area of brain tissue at nanometer scales. A vast amount of data in the large area are required to study structural changes of cellular organelles in a specific cell, such as neurons, astrocytes, oligodendrocytes, and microglia among brain tissue, at sufficient resolution. Array tomography is a useful method for large-area imaging, and the osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods are commonly used to enhance membrane contrast.Because many samples prepared using the conventional technique without en bloc staining are considered inadequate for array tomography, we suggested an alternative technique using post-staining conventional samples and compared the advantages.

Study of hyperpigmentation in human skin disorder using different electron microscopy techniques.

There is a global trend of increase in the demand for three-dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block-face scanning electron microscopy and focused ion beam-scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis-endocytosis theory of pigment in human skin.

The involvement of serum exosomal miR-500-3p and miR-770-3p in aging: modulation by calorie restriction.

Recent studies have shown a role for miRNAs in aging and age-related diseases, and the modulation of miRNA expression by diet attracts attention as a new therapeutic strategy. Here, we focused on identifying specific exosomal miRNAs derived from serum of aged rats and the effect of short-term calorie restriction (CR) on their expression. Exosomes from serum of young (7-month), old (22-month), and old-CR Sprague Dawley rats were isolated and characterized by transmission electron microscopy analyses, dynamic light scattering measurements, and Western blotting. A total of 12 significantly expressed miRNAs in serum exosomes of young and old rats were identified by next generation sequencing. After analysis of qRT-PCR, we found that miR-500-3p and miR-770-3p expression was significantly upregulated by aging and downregulated by CR. Furthermore, receiver operating characteristic (ROC) curve revealed that the selected miRNAs represented high accuracy in discriminating old rats from young rats. Finally, PANTHER analysis predicted selected miRNAs targets genes involved in Wnt/chemokines and cytokines -related inflammatory signaling pathway and function as transcription factor. In conclusion, our results suggest that the expression of serum exosomal miR-500-3p and miR-770-3p was significantly increased with aging, whereas these were decreased by CR, and age-/CR-modulated exosomal miR-500-3p and miR-770-3p could potentially be used as informative biomarkers candidates for aging.