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In JoVE (1)
Other Publications (19)
- Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology / Zhongguo Sheng Tai Xue Xue Hui, Zhongguo Ke Xue Yuan Shenyang Ying Yong Sheng Tai Yan Jiu Suo Zhu Ban
- IEEE Transactions on Image Processing : a Publication of the IEEE Signal Processing Society
- NeuroImage
- Optics Express
- Medical Image Analysis
- Luminescence : the Journal of Biological and Chemical Luminescence
- Medical Image Analysis
- Experimental Neurology
- Cancer Cell International
- Journal of the American Chemical Society
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- European Journal of Cancer (Oxford, England : 1990)
- BMC Biotechnology
- Journal of Neurochemistry
- Journal of Biomolecular Screening
- Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
- Journal of Neuroscience Methods
- The Annals of Thoracic Surgery
- Cancer Research
Articles by Xiaoyin Xu in JoVE
JoVE General
Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging
1Department of Anesthesia, Brigham and Woman's Hospital, 2Department of Radiology, Brigham and Woman's Hospital
We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.
Other articles by Xiaoyin Xu on PubMed
[Thermal Tolerance, Diel Variation of Body Temperature, and Thermal Dependence of Locomotor Performance of Hatchling Soft-shelled Turtles, Trionyx Sinensis]
The thermal tolerance, body temperature, and influence of temperature on locomotor performance of hatchling soft-shelled turtles (Trionyx sinensis) were studied under dry and wet conditions, and the selected body temperature of hatchlings was 28.0 and 30.3 degrees C, respectively. Under wet condition, the critical thermal maximum and minimum averaged 40.9 and 7.8 degrees C, respectively. In the environments without thermal gradients, the diel variation of body temperature was highly consistent with the variation of both air and water temperatures, and the body temperature was more directly affected by water temperature than by air temperature, which implied that the physiological thermoregulation of hatchling T. sinensis was very weak. In the environments with thermal gradients, hatchling turtles could maintain relatively high and constant body temperatures, primarily through behavioral thermoregulation. The locomotor performance of hatchling turtles was highly dependent on their body temperature. Within a certain range, the locomotor performance increased with increasing body temperature. In our study, the optimal body temperature for locomotor performance was 31.5 degrees C, under which, the maximum continuous running distance, running distance per minute, and number of stops per minute averaged 1.87 m, 4.92 m.min-1, and 6.2 times.min-1, respectively. The correspondent values at 33.0 degrees C averaged 1.30 m, 4.28 m.min-1, and 7.7 times.min-1, respectively, which indicated that the locomotor performance of hatchling turtles was impaired at 33.0 degrees C. Therefore, extremely high body temperatures might have an adverse effect on locomotor performance of hatchling turtles.
Adaptive Two-pass Rank Order Filter to Remove Impulse Noise in Highly Corrupted Images
In this paper, we present an adaptive two-pass rank order filter to remove impulse noise in highly corrupted images. When the noise ratio is high, rank order filters, such as the median filter for example, can produce unsatisfactory results. Better results can be obtained by applying the filter twice, which we call two-pass filtering. To further improve the performance, we develop an adaptive two-pass rank order filter. Between the passes of filtering, an adaptive process is used to detect irregularities in the spatial distribution of the estimated impulse noise. The adaptive process then selectively replaces some pixels changed by the first pass of filtering with their original observed pixel values. These pixels are then kept unchanged during the second filtering. In combination, the adaptive process and the second filter eliminate more impulse noise and restore some pixels that are mistakenly altered by the first filtering. As a final result, the reconstructed image maintains a higher degree of fidelity and has a smaller amount of noise. The idea of adaptive two-pass processing can be applied to many rank order filters, such as a center-weighted median filter (CWMF), adaptive CWMF, lower-upper-middle filter, and soft-decision rank-order-mean filter. Results from computer simulations are used to demonstrate the performance of this type of adaptation using a number of basic rank order filters.
Repulsive Force Based Snake Model to Segment and Track Neuronal Axons in 3D Microscopy Image Stacks
The branching patterns of axons and dendrites are fundamental structural properties that affect the synaptic connectivity of axons. Although today three-dimensional images of fluorescently labeled processes can be obtained to study axonal branching, there are no robust methods of tracing individual axons. This paper describes a repulsive force based snake model to segment and track axonal profiles in 3D images. This new method segments all the axonal profiles in a 2D image and then uses the results obtained from that image as prior information to help segment the adjacent 2D image. In this way, the segmentation successfully connects axonal profiles over hundreds of images in a 3D image stack. Individual axons can then be extracted based on the segmentation results. The utility and performance of the method are demonstrated using 3D axonal images obtained from transgenic mice that express fluorescent protein.
Chemically-selective Imaging of Brain Structures with CARS Microscopy
We demonstrate the use of coherent anti-Stokes Raman scattering (CARS) microscopy to image brain structure and pathology ex vivo. Although non-invasive clinical brain imaging with CT, MRI and PET has transformed the diagnosis of neurologic disease, definitive pre-operative distinction of neoplastic and benign pathologies remains elusive. Definitive diagnosis still requires brain biopsy in a significant number of cases. CARS microscopy, a nonlinear, vibrationally-sensitive technique, is capable of high-sensitivity chemically-selective three-dimensional imaging without exogenous labeling agents. Like MRI, CARS can be tuned to provide a wide variety of possible tissue contrasts, but with sub-cellular spatial resolution and near real time temporal resolution. These attributes make CARS an ideal technique for fast, minimally invasive, non-destructive, molecularly specific intraoperative optical diagnosis of brain lesions. This promises significant clinical benefit to neurosurgical patients by providing definitive diagnosis of neoplasia prior to tissue biopsy or resection. CARS imaging can augment the diagnostic accuracy of traditional frozen section histopathology in needle biopsy and dynamically define the margins of tumor resection during brain surgery. This report illustrates the feasibility of in vivo CARS vibrational histology as a clinical tool for neuropathological diagnosis by demonstrating the use of CARS microscopy in identifying normal brain structures and primary glioma in fresh unfixed and unstained ex vivo brain tissue.
Using Nonlinear Diffusion and Mean Shift to Detect and Connect Cross-sections of Axons in 3D Optical Microscopy Images
The morphology of neuronal axons has been actively investigated by researchers to understand functionalities of neuronal networks, for example, in developmental neurology. Today's optical microscope and labeling techniques allow us to obtain high-resolution images about axons in three dimensions (3D), however, it remains challenging to segment and reconstruct the 3D morphology of axons. These include differentiating adjacent axons and detecting the axon branches. In this paper we present a method to track axons in 3D by identifying cross-sections of axons on 2D images and connecting the cross-sections over a series of 2D images to reconstruct the 3D morphology. The method can separate adjacent axons and detect the split and merge of axons. The method consists of three steps, modified nonlinear diffusion to remove noise and enhance edges in 2D, morphological operations to detect edges of the cross-sections of axons in 2D, and mean shift to track the cross-sections of axons in 3D. Performance of the method is demonstrated by processing real data acquired by confocal laser scanning microscopy.
A Quantitative Study of Factors Affecting in Vivo Bioluminescence Imaging
In vivo bioluminescence imaging (BLI) has the advantages of high sensitivity and low background. By counting the number of photons emitted from a specimen, BLI can quantify biological events such as tumour growth, gene expression and drug response. The intensities and kinetics of the BL signal are affected by many factors and may confound the quantitative results acquired from consecutive imaging sessions or different specimens. We used three different mouse models of tumours to examine whether anaesthetics, positioning and tumour growth may affect the consistency of the BL signal. The results showed that BLI signal could be affected by different anaesthetics and repetitive positioning. Using the same anaesthetics produced consistent peak times, while other factors were held constant. However, as the tumours grew the peak times shifted and the time course of BL signals had different shapes, depending on the positioning of the mice. The data indicate that a carefully designed BLI experiment is required to generate optimal and consistent results.
Robust 3D Reconstruction and Identification of Dendritic Spines from Optical Microscopy Imaging
In neurobiology, the 3D reconstruction of neurons followed by the identification of dendritic spines is essential for studying neuronal morphology, function and biophysical properties. Most existing methods suffer from problems of low reliability, poor accuracy and require much user interaction. In this paper, we present a method to reconstruct dendrites using a surface representation of the neuron. The skeleton of the dendrite is extracted by a procedure based on the medial geodesic function that is robust and topology preserving, and it is used to accurately identify spines. The sensitivity of the algorithm on the various parameters is explored in detail and the method is shown to be robust.
Selective Inhibition of JNK with a Peptide Inhibitor Attenuates Pain Hypersensitivity and Tumor Growth in a Mouse Skin Cancer Pain Model
Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.
Bioluminescence Imaging Reveals Inhibition of Tumor Cell Proliferation by Alzheimer's Amyloid Beta Protein
Cancer and Alzheimer's disease (AD) are two seemingly distinct diseases and rarely occur simultaneously in patients. To explore molecular determinants differentiating pathogenic routes towards AD or cancer, we investigate the role of amyloid beta protein (Abeta) on multiple tumor cell lines that are stably expressing luciferase (human glioblastoma U87; human breast adenocarcinoma MDA-MB231; and mouse melanoma B16F).
Design, Synthesis, and Testing of Difluoroboron-derivatized Curcumins As Near-infrared Probes for in Vivo Detection of Amyloid-beta Deposits
Amyloid-beta (Abeta) deposits have been identified as key players in the progression of Alzheimer's disease (AD). Recent evidence indicates that the deposits probably precede and induce the neuronal atrophy. Therefore, methods that enable monitoring the pathology before clinical symptoms are observed would be beneficial for early AD detection. Here, we report the design, synthesis, and testing of a curcumin-derivatized near-infrared (NIR) probe, CRANAD-2. Upon interacting with Abeta aggregates, CRANAD-2 undergoes a range of changes, which include a 70-fold fluorescence intensity increase, a 90 nm blue shift (from 805 to 715 nm), and a large increase in quantum yield. Moreover, this probe also shows a high affinity for Abeta aggregates (K(d) = 38.0 nM), a reasonable log P value (log P = 3), considerable stability in serum, and a weak interaction with albumin. After intravenous injection of this probe, 19-month-old Tg2576 mice exhibited significantly higher relative signal than that of the control mice over the same period of time. In summary, CRANAD-2 meets all the requirements for a NIR contrast agent for the detection of Abeta plaques both in vitro and in vivo. Our data point toward the feasibility of monitoring the progress of the disease by NIR imaging with CRANAD-2. In addition, we believe that our probe could be potentially used as a tool for drug screening.
In Vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts
In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to approximately 25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of approximately 45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.
Treating Triple-negative Breast Cancer by a Combination of Rapamycin and Cyclophosphamide: an in Vivo Bioluminescence Imaging Study
Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1alpha in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1alpha may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.
Phenotypic Analysis of Images of Zebrafish Treated with Alzheimer's Gamma-secretase Inhibitors
Several gamma-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer's disease (AD). This enzyme mediates the proteolytic cleavage of amyloid precursor protein (APP) to generate amyloid beta protein, Abeta, the pathogenic protein in AD. The gamma-secretase also cleaves Notch to generate Notch Intracellular domain (NICD), the signaling molecule that is implicated in tumorigenesis.
In Vivo Manifestation of Notch Related Phenotypes in Zebrafish Treated with Alzheimer's Amyloid Reducing Gamma-secretase Inhibitors
Gamma-secretase is responsible for the final cleavage of amyloid precursor protein to generate the amyloid-beta protein, the major component of plaques in the brains of Alzheimer's disease patients. gamma-Secretase inhibitors (GSI) have been explored for therapeutic inhibition of amyloid beta protein generation, but mechanistic toxicity has been documented because of its blockage of gamma-secretase cleavage of several dozens of substrates including Notch. This becomes the primary obstacle for most inhibitors during the pre-clinical development and the main concern for several compounds in the clinical trials. To predict potential side effects related to Notch signaling, we examined global effect of GSIs in vertebrate animal zebrafish. We have used two potent GSIs (GSI A and GSI 18) with a sub-microM effective concentration for 50% amyloid beta protein inhibition (EC(50)). Zebrafish embryos were treated with GSI A, 18 or a well characterized GSI N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), and transparent animals were examined for up to 7 days. GSI A had less abnormal phenotype in zebrafish, compared to GSI 18-treated embryos that displayed curved tails, a loss of pigmentation, and reduced swim bladder and heart rate. To understand mechanistic effect at the molecular level, we examined Notch signaling in these GSI-treated zebrafish. Notch phenotypes were observed in embryos treated with 50 and 10 microM GSI 18, but not with 10 microM GSI A. In accordance, in situ hybridization with a probe against Notch target gene her6 showed a weaker staining in embryos treated with 10 microM GSI 18 than those treated with 10 microM GSI A. In conclusion, phenotypic profile in whole animals offers important information on Notch related pathways and provides prediction of safe compounds during early development stages of therapeutic GSIs.
A High-throughput Analysis Method to Detect Regions of Interest and Quantify Zebrafish Embryo Images
Zebrafish is widely used to understand neural development and model various neurodegenerative diseases. Zebrafish embryos are optically transparent, have a short development period, and can be kept alive in microplates for days, making them amenable to high-throughput microscopic imaging. As a result of high-throughput experiments, a large number of images can be generated in a single experiment, posing a challenge to researchers to analyze them efficiently and quantitatively. In this work, we develop an image processing focused on detecting and quantifying pigments in zebrafish embryos. The algorithm automatically detects a region of interest (ROI) enclosing an area around the pigments and then segment the pigments for quantification. In this process, the algorithm identifies the head and torso at first, and then finds the boundaries corresponding to the back and abdomen by taking advantage of a priori information about the anatomy of zebrafish embryos. The method is robust in terms that it can detect and quantify pigments even when the embryos have different orientations and curvatures. We used real data to demonstrate the performance of the method to extract phenotypic information from zebrafish embryo images and compared its results with manual analysis for verification.
C Terminus of Clostridium Perfringens Enterotoxin Downregulates CLDN4 and Sensitizes Ovarian Cancer Cells to Taxol and Carboplatin
We have previously shown that CLDN4 (encoding claudin-4), a cell tight junction (TJ) protein, is highly expressed in human epithelial ovarian carcinomas (EOC) but undetectable in normal ovaries. CLDN4 has been identified as a specific receptor for C terminus of Clostridium perfringens enterotoxin (C-CPE), a nontoxic molecule that may disrupt TJ barrier function and enhance cellular absorption. The purpose of this study was to determine the potential clinical applications of C-CPE and its effects on CLDN4 expression in EOC.
Automated Analysis of Zebrafish Images for Phenotypic Changes in Drug Discovery
Zebrafish has become one of the most popular and useful models in cell biology, development, and drug discovery. Because zebrafish embryo is transparent and can be observed under microscope without fixation, it is increasingly used in high-throughput screening. The small size of zebrafish embryos allows users to image them in a 96- or 384-well plate under various conditions, in turn, generating such a large amount of images that only automated analysis is feasible for processing and analyzing. We focus on developing an image processing algorithm to automatically quantify gene expression on zebrafish embryos that have been treated by various compounds. The challenge in this type of application includes aligning embryos of different orientations and automatically creating regions of interest (ROIs) to enclose specific areas in the head and torso of embryos. The image processing pipeline consists of alignment, segmentation, creation and quantification of ROIs. We test the algorithm using high-throughput images of zebrafish embryos obtained from an experiment screening compounds that may affect γ-secretase in Alzheimer's disease and results show that automated analysis can achieve satisfactory performance in a much shorter amount of time with a high level of objectivity.
Paclitaxel-loaded Expansile Nanoparticles in a Multimodal Treatment Model of Malignant Mesothelioma
Malignant mesothelioma has a poor prognosis even when treated aggressively with multimodal therapy. Traditional murine tumor models can be used to evaluate drug efficacy and toxicity in malignant mesothelioma, but not to assess the effect of a multimodal approach that includes the surgical resection of tumor. We therefore developed a murine model of multimodal therapy in which we evaluated paclitaxel-loaded expansile nanoparticles (Pax-eNP) for delivering intracavitary chemotherapy in malignant mesothelioma.
Glioblastoma-derived Epidermal Growth Factor Receptor Carboxyl-terminal Deletion Mutants Are Transforming and Are Sensitive to EGFR-directed Therapies
Genomic alterations of the epidermal growth factor receptor (EGFR) gene play a crucial role in pathogenesis of glioblastoma multiforme (GBM). By systematic analysis of GBM genomic data, we have identified and characterized a novel exon 27 deletion mutation occurring within the EGFR carboxyl-terminus domain (CTD), in addition to identifying additional examples of previously reported deletion mutations in this region. We show that the GBM-derived EGFR CTD deletion mutants are able to induce cellular transformation in vitro and in vivo in the absence of ligand and receptor autophosphorylation. Treatment with the EGFR-targeted monoclonal antibody, cetuximab, or the small molecule EGFR inhibitor, erlotinib, effectively impaired tumorigenicity of oncogenic EGFR CTD deletion mutants. Cetuximab in particular prolonged the survival of intracranially xenografted mice with oncogenic EGFR CTD deletion mutants, compared with untreated control mice. Therefore, we propose that erlotinib and, especially, cetuximab treatment may be a promising therapeutic strategy in GBM patients harboring EGFR CTD deletion mutants.
