GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3? and GSK-3? in newly born cortical excitatory neurons. Gsk3-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after Gsk3 deletion in interneuron precursors. Gsk3-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/?-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins.DOI: http://dx.doi.org/10.7554/eLife.02663.001.
Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear–cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.
The feasibility of steel materials classification by support vector machines (SVMs), in combination with laser-induced breakdown spectroscopy (LIBS) technology, was investigated. Multi-classification methods based on SVM, the one-against-all and the one-against-one models, and a combination model, are applied to classify nine types of round steel. Due to the inhomogeneity of steel composition, the data obtained using the one-against-all and one-against-one models were ambiguous and difficult to discriminate; whereas, the combination model, was able to successfully distinguish most of the ambiguous data and control the computation cost within an acceptable range. The studies presented here demonstrate that LIBS-SVM is a useful technique for the identification and discrimination of steel materials, and would be very well-suited for process analysis in the steelmaking industry.
Two-photon excited fluorescence (TPEF) microscopy, based on signal from cells, can provide detailed information on tissue architecture and cellular morphology in unstained histological sections to generate subcellular-resolution images from tissue directly. In this paper, we used TPEF microscopy to image microstructure of human normal gallbladder and three types of differentiated carcinomas in order to investigate the morphological changes of tissue structure, cell, cytoplasm, and nucleus without hematoxylin and eosin (H&E) staining. It displayed that TPEF microscopy can well image the stratified normal gallbladder tissue, including the mucosa, the muscularis, and the serosa. The typical cancer cell, characterized by cellular and nuclear pleomorphism, enlarged nuclei, and augmented nucleolus, can be identified in histological sections without H-E staining as well. The quantitative results showed that the areas of the nucleus and the nucleolus in three types of cancerous cells were all significantly greater than those in normal gallbladder columnar epithelial cells derived from TPEF microscopic images. The studies demonstrated that TPEF microscopy has the ability to characterize tissue structures and cell morphology of gallbladder cancers differentiated from a normal gallbladder in a manner similar to traditional histological analysis. As a novel tool, it has the potential for future retrospective studies of tumor staging and migration by utilizing histological section specimens without H-E staining.
The unregulated activation of microglia following stroke results in the production of toxic factors that propagate secondary neuronal injury. Salidroside has been shown to exhibit protective effects against neuronal death induced by different insults. However, the molecular mechanisms responsible for the anti-inflammatory activity of salidroside have not been elucidated clearly in microglia. In the present study, we investigated the molecular mechanism underlying inhibiting LPS-stimulated BV2 microglial cell mobility of salidroside. The protective effect of salidroside was investigated in microglial BV2 cell, subjected to stretch injury. Moreover, transwell migration assay demonstrated that salidroside significantly reduced cell motility. Our results also indicated that salidroside suppressed LPS-induced chemokines production in a dose-dependent manner, without causing cytotoxicity in BV2 microglial cells. Moreover, salidroside suppressed LPS-induced activation of nuclear factor kappa B (NF- ? B) by blocking degradation of I ? B ? and phosphorylation of MAPK (p38, JNK, ERK1/2), which resulted in inhibition of chemokine expression. These results suggest that salidroside possesses a potent suppressive effect on cell migration of BV2 microglia and this compound may offer substantial therapeutic potential for treatment of ischemic strokes that are accompanied by microglial activation.
NG2 cells, also referred to as oligodendrocyte precursor cells (OPCs) or polydendrocytes, represent a major resident glial cell population that is distinct from mature astrocytes, oligodendrocytes, microglia, and neural stem cells and exist throughout the gray and white matter of the developing and mature central nervous system (CNS). While their most established fate is the oligodendrocyte, they retain lineage plasticity in an age- and region-specific manner. During development, they contribute to 36% of protoplasmic astrocytes in the ventral forebrain. Despite intense investigation on the neuronal fate of NG2 cells, there is no definitive evidence that they contribute substantially to the neuronal population. NG2 cells have attributes that suggest that they have functions other than to generate oligodendrocytes, but their exact role in the neural network remains unknown. Under pathological states, NG2 cells not only contribute to myelin repair, but they become activated in response to a wide variety of insults and could play a primary role in pathogenesis.
Gua Lou Gui Zhi decoction (GLGZD), a traditional Chinese medicine consisting of different herbal medicines, has been used for centuries in the treatment of muscular spasticity following stroke, epilepsy or spinal cord injury. However, the precise mechanisms involved remain poorly understood. In the present study, we investigated the neuroprotective effects of GLGZD on glutamate-induced apoptosis in cultured BV-2 cells, as well as the underlying mechanisms. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to assess the viability of the cells. An Annexin V/propidium iodide (PI) assay was utilized to analyze cellular apoptosis. Mitochondrial membrane potential (MMP) was evaluated by flow cytometry and laser scanning confocal microscopy. The gene and protein expression of the apoptosis-related genes, Bcl-2 and Bax, was analyzed by RT-PCR and western blot analysis, respectively. Furthermore, the expression of cleaved caspase-3 protein was detected by immunofluorescence. Glutamate treatment induced the loss of BV-2 cell viability, which was associated with an increase in the apoptotic rate, as well as an increase in the Bax/Bcl-2 ratio and the extracellular levels of cleaved caspase-3. Treatment with GLGZD significantly reversed these phenotypes, with its maximum protective effects observed at the concentration of 1,000 µg/ml. These results indicate that GLGZD protects BV-2 cells from glutamate-induced cytotoxicity. These protective effects may be ascribed to its anti-apoptotic activities, in part, associated with the decrease in the Bax/Bcl-2 ratio and caspase-3 expression, as well as with the stability of high mitochondrial membrane potential.
Microglial activation plays an important role in neroinflammation following ischemic stroke. Activated microglial cells can then migrate to the site of injury to proliferate and release substances which induce secondary brain damage. It has been shown that microglial migration is associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathways. The Chinese formula, GuaLou GuiZhi decoction (GLGZD), has long been administered in clinical practice for the treatment of post-stroke disabilities, such as muscular spasticity. In a previous study, we demonstrated that the anti-inflammtory effects of GLGZD were mediated by the TLR4/NF-?B pathway in lipopolysaccharide (LPS)-stimulated microglial cells. Therefore, in this study, we evaluated the role of GLGZD in microglial migration by performing scratch wound assays and migration assays. We wished to elucidate the cellular and molecular mechanisms elicited by this TCM formula in microglial-induced inflammation by evaluating the release and expression of chemotactic cytokines [monocyte chemo-attractant protein-1 (MCP-1), macrophage inflammatory protein-1? (MIP-1?) and interleukin (IL)-8] by ELISA and quantitative PCR. Our results revealed that the migration of microglial cells was enhanced in the presence of LPS (100 ng/ml); however, GLGZD (100 µg/ml) significantly inhibited cell motility and the production of chemokines through the inhibition of the activation of the p38 and c-Jun N-terminal protein kinase (JNK) signaling pathway. We demonstrate the potential of GLGZD in the modulation of microglial motility by investigating the effects of GLGZD on microglial migration induced by LPS. Taken together, our data suggest that GLGZD per se cannot trigger microglial motility, whereas GLGZD impedes LPS-induced microglial migration through the activation of the MAPK signaling pathway. These results provide further evidence of the anti-inflammatory effects of GLGZD and its potential for use in the treatment of ischemic stroke.
Toll-like receptor 4 (TLR4)/nuclear factor-?B (NF-?B) signaling-mediated neuroinflammation contributes to secondary brain damage in ischemic stroke; therefore, anti-inflammatory therapy via suppression of the TLR4/NF-?B pathway could be a promising strategy for the treatment of stroke and post-stroke disabilities. Gua Lou Gui Zhi decoction (GLGZD) has long been used in China to clinically treat dysfunction after stroke such as muscular spasticity, but the precise mechanisms are largely unknown. In the present study, we evaluated the anti-inflammatory effect of GLGZD and investigated the underlying molecular mechanisms using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells as an in vitro inflammatory model of neural cells. We found that GLGZD inhibited the inflammatory response in microglial cells as it significantly reduced LPS-induced expression of pro-inflammatory nitric oxide, tumour necrosis factor-?, interleukin (IL)-6 and IL-1? in BV-2 cells, in a dose-dependent manner. In addition, GLGZD treatment significantly decreased the protein expression of TLR4 and myeloid differentiation factor 88, inhibited the phosphorylation of I?B and blocked the nuclear translocation of NF-?B in BV-2 cells, demonstrating its inhibitory effect on the activation of TLR4/NF-?B signaling. Collectively, our findings suggest that inhibition of the inflammatory response via suppression of the TLR4/NF-?B pathway may be one of the mechanisms through which GLGZD ameliorates the damage in ischemic cerebral tissues.
Most colorectal cancers arise from dysplastic lesions, such as adenomatous polyps, and these lesions are difficult to be detected by the current endoscopic screening approaches. Here, we present the use of an intrinsic second-harmonic generation (SHG) signal as a novel means to differentiate between normal and dysplastic human colonic tissues. We find that the SHG signal can quantitatively identify collagen change associated with colonic dysplasia that is indiscernible by conventional pathologic techniques. By comparing normal with dysplastic mucosa, there were significant differences in collagen density and collagen fiber direction, providing substantial potential to become quantitative intrinsic biomarkers for in vivo clinical diagnosis of colonic dysplasia.
Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.
Multiphoton microscopy (MPM) was applied to examine the marginal region at dermis of keloid compared with atrophic scar. High-resolution large-area image showed an obvious boundary at the scar margin and different morphological patterns of elastin and collagen on the two sides, further visualized by the focused three-dimensional images. Content alteration of elastin or collagen between the two sides of boundary was quantified to show significant difference between keloid and atrophic scar. Owing to the raised property of keloid with overproduced collagen on the scar side, the content alteration was positive for elastin and negative for collagen. On the contrary, the content alteration was negative for elastin and positive for collagen in the atrophic scar case due to the atrophic collagen on the scar side. It indicated that examination of the scar margin by MPM may lead a new way to discriminate different types of scars and better understand the scarring mechanisms.
Early detection of fibroadenoma (FA) is critical for preventing subsequent breast cancer. In this work, we show that label-free second harmonic generation (SHG) imaging is feasible and effective in quantitatively differentiating the fibroadenomal tissue from normal breast tissue. With the advent of the clinical portability of miniature SHG microscopy, we believe that the technique has great potential in offering a noninvasive in vivo imaging tool for early detection of FA and monitoring the treatment responses of FA in clinics.
Real-time histology or virtual biopsy for the diagnosis of colonic cancer is of great medical significance. In this work, we show that label-free multiphoton imaging is feasible and effective in monitoring colonic cancer progression by providing cellular and subcellular details in fresh, unfixed, unstained colonic specimens. Our results also demonstrate the capability of using tissue quantitative analysis of the redox ratio for quantifying colonic cancer progression. These results suggest that multiphoton microscopy has potential to become an in situ histological tool, which is free from the labeling requirement of conventional methods, for the early diagnosis and detection of malignant lesions in the colon.
NG2-expressing glia (NG2 cells, polydendrocytes) appear in the embryonic brain, expand perinatally, and persist widely throughout the gray and white matter of the mature central nervous system. We have previously reported that NG2 cells generate oligodendrocytes in both gray and white matter and a subset of protoplasmic astrocytes in the gray matter of the ventral forebrain and spinal cord. To investigate the temporal changes in NG2 cell fate, we generated NG2creER™BAC transgenic mice, in which tamoxifen-inducible Cre is expressed in NG2 cells. Cre induction at embryonic day 16.5, postnatal day (P) 2, P30 and P60 in mice that were double transgenic for NG2creER™BAC and the Cre reporter revealed that NG2 cells in the postnatal brain generate only NG2 cells or oligodendrocytes, whereas NG2 cells in the embryonic brain generate protoplasmic astrocytes in the gray matter of the ventral forebrain in addition to oligodendrocytes and NG2 cells. Analysis of cell clusters from single NG2 cells revealed that more than 80% of the NG2 cells in the P2 brain give rise to clusters consisting exclusively of oligodendrocytes, whereas the majority of the NG2 cells in the P60 brain generate clusters that contain only NG2 cells or a mixture of oligodendrocytes and NG2 cells. Furthermore, live cell imaging of single NG2 cells from early postnatal brain slices revealed that NG2 cells initially divide symmetrically to produce two daughter NG2 cells and that differentiation into oligodendrocytes occurred after 2-3 days.
Scar margins dynamic with time were quantitatively characterized using multiphoton microscopy (MPM). 2D large-area and 3D focused images of elastin and collagen at scar margins were obtained to extract quantitative parameters. An obvious boundary was observed at the scar margin, showing altered morphological patterns of elastin and collagen on both sides. Content alteration of elastin and collagen between the two sides of boundary were defined to characterize scar margins from different individuals. The statistical results from 15 normal scar samples strongly demonstrated that content alteration degree of elastin and collagen had decreasing tendency with the increase of patient age or scar duration, consistent with the fact of normal scars regressing spontaneously over time. It indicated that alteration degree can potentially serve as quantitative indicators to examine wound healing and scar progression over time. With the advent of clinical portable multiphoton endoscopes, the MPM technique can be applied in tracking scar formation and progression in vivo by examination of scar margin.
Nonlinear optical microscopy (NLOM) was applied for monitoring dermal wound healing after mesenchymal stem cell (MSC) transplantation. Our results showed that NLOM can reveal different regeneration processes of collagen in nontreated and MSC-treated wound dermis. Specifically, the temporal increases in the intensity of second-harmonic-generation signals can quantify kinetic properties of collagen regeneration. Orientation analysis of collagen fiber bundles can monitor the formation of new normal collagen fiber bundles, which is an indicator for evaluating the therapy response. It was also found that NLOM can track MSCs location and recruitment. These findings suggested that NLOM is ideal for monitoring the progress of dermal wound healing.
This work reports on the measurement of optical properties from nine normal and cancerous human esophageal stroma pairs using reflectance-based confocal microscopy. It was found that the scattering coefficient of cancerous stroma is significantly lower than that of normal stroma. The results suggest that the decreased scattering in cancerous stroma may provide a possible indicator for differentiating normal and cancerous stroma.
A real-time, non-invasive method will confer a benefit for the diagnosis and treatment of localized scleroderma (LS) in the clinic. The aim of this work was to demonstrate the potential of multiphoton laser scanning microscopy (MPLSM) for diagnosing LS and monitoring the treatment response in vivo.
NG2 cells (also known as polydendrocytes) are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes and microglia. They can be identified by the expression of the proteoglycan NG2, have a highly branched morphology and are distributed throughout the grey and white matter. They differentiate into oligodendrocytes in vitro and have often been equated with oligodendrocyte precursor cells. However, whether polydendrocytes are multipotential cells that can give rise to neurons and astrocytes as well as oligodendrocytes is now highly debated. Furthermore, electrophysiological studies indicate that polydendrocytes receive synaptic input from neurons, suggesting that they are integrated in the neural network. This Review highlights recent findings and unresolved questions related to the lineage and function of polydendrocytes in the CNS.
NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet-derived growth factor receptor-alpha but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA-NCAM, beta-tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells.
Collagen change is a major feature in the photoaged human skin. Here, we present the use of intrinsic second harmonic generation (SHG) signal as a novel means to quantify collagen change with photoaging. We obtain the SHG images of the superficial dermis from ex vivo the cheek skin and the abdomen skin of eight patients aged 55-60 years. The results show that SHG signal can quantitatively reveal collagen change between normal and photoaged human skin in three dimensions. By comparing normal with photoaged dermis, there are significant differences in the collagen content and fine structure, providing substantial potential to be applied in vivo for the clinical diagnosis of human skin photoaging.
Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast.
Papulonodular mucinosis (PM) is a cutaneous clue to the presence and activity of silent lupus erythematosus (LE), but the exact pathogenesis is still under secret. Moreover, the currently available treatments for PM are not satisfactory. To demonstrate the possibility of multiphoton microscopy (MPM) to trace the pathological state of PM and evaluate the treatment efficacy, epidermal and dermal alteration in skin lesion with PM before and after treatment was examined using MPM. Microstructure of epidermis as well as content and distribution of collagen and elastin in dermis were quantified to characterize the pathological states of PM. The results showed significant morphological difference between skin lesion before and after treatment, indicating the possibility of MPM to assess the therapeutic efficacy. With the advancement on MPM miniaturization and enhancement of contrast and depth of imaging, the MPM technique can be applied in in vivo tracking PM formation and progression, and leading the better understanding the PM pathogenesis and mechanism of response to any treatment, helping to establish novel effective therapies for PM.
NG2-expressing cells (NG2 cells or polydendrocytes) generate oligodendrocytes throughout the CNS and a subpopulation of protoplasmic astrocytes in the gray matter of the ventral forebrain. The mechanisms that regulate their oligodendrocyte or astrocyte fate and the degree to which they exhibit lineage plasticity in vivo have remained unclear. The basic helix-loop-helix transcription factor Olig2 is required for oligodendrocyte specification and differentiation. We have found that Olig2 expression is spontaneously downregulated in NG2 cells in the normal embryonic ventral forebrain as they differentiate into astrocytes. To further examine the role of Olig2 in NG2 cell fate determination, we used genetic fate mapping of NG2 cells in constitutive and tamoxifen-inducible Olig2 conditional knockout mice in which Olig2 was deleted specifically in NG2 cells. Constitutive deletion of Olig2 in NG2 cells in the neocortex and corpus callosum but not in ventral forebrain caused them to convert their fate into astrocytes, with a concomitant severe reduction in the number of oligodendrocytes and myelin. Deletion of Olig2 in NG2 cells in perinatal mice also resulted in astrocyte generation from neocortical NG2 cells. These observations indicate that the developmental fate of NG2 cells can be switched by altering a single transcription factor Olig2.
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