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ABSTRACT: Due to the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and 3-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical-clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with μm-level resolution. Four examples are given: (1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intra-ganglionic at myenteric plexus, (2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface, (3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts, and (4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.
AJP Gastrointestinal and Liver Physiology 10/2012; · 3.43 Impact Factor
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ABSTRACT: Intestinal microvasculature plays a central role in nutrient absorption and immune response against infections. Microscopic visualization of intestinal microvasculature under normal and pathological conditions such as inflammatory bowel disease is essential for understanding the pathophysiology of the disease. Despite the intensive need to characterize the intestinal microstructure and vasculature in an integrated fashion, 3-dimensional (3D) visualization of the gastrointestinal tissue is often limited by the spatial resolution of the imaging tools. In this research, we aimed to apply optical clearing to minimize the random light scattering in the mouse ileum, thereby facilitating photon penetration for high-resolution, 3D optical microscopy of the tissue network without microtome sectioning. We applied cardiac perfusion of lipophilic dialkylcarbocyanine dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) to label the mouse blood vessels, including the intestinal microvasculature. The labeled and paraformaldehyde-fixed ileum was immersed in the aqueous optical-clearing solution to improve photon penetration. Optical clearing revealed the interior domain of the mouse ileal mucosa and submucosa, where random light scattering was suppressed and the size of the microstructure in the fixed specimen remained the same. Using fluorescent labeling, the intestinal microstructure and vasculature were simultaneously imaged by 3D confocal microscopy to allow for an integrated visualization of the tissue network with high definition. This new optical approach provides a useful tool for 3D presentation and analysis of the microvasculature for better understanding the intestinal physiology.
Microvascular Research 12/2010; 80(3):512-21. · 2.83 Impact Factor
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Gastroenterology 10/2010; 139(4):1100-5, 1105.e1. · 11.68 Impact Factor
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ABSTRACT: In this study, we aimed to use gold nanorods (Au-NRs) as luminescent substrates for labeling of the mouse intestinal blood vessels for tissue imaging. The labeled intestine was subjected to 3-D confocal microscopy to reveal the intricate morphology of the intestinal capillaries. Using the Au-NR's unique near-infrared excitation and visible fluorescence emission, we observed low noise background compared to the tissue's high autofluorescence from blue laser excitation. We took advantage of this sharp contrast in optical properties to achieve 3-D visualization of the intestinal microstructure and vasculature with capillary-level resolution. This new optical application demonstrates the Au-NR's distinctive properties in vascular labeling and fluorescence microscopy for 3-D illustration of intestinal vasculature.
ACS Nano 10/2010; 4(10):6278-84. · 10.77 Impact Factor
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ABSTRACT: The intrinsic opacity of mouse intestinal tissue prevents its evaluation by high-resolution, in-depth optical microscopy. Instead, intestinal tissue is usually sectioned to expose the interior domains of the mucosa and submucosa for microscopic examination. However, microtome sectioning can cause distortions and artifacts that prevent acquisition of an accurate view of the sample. We therefore attempted to develop a microtome-free 3-dimensional (3D) confocal imaging method for characterization of mouse intestine.
We applied an optical-clearing solution, FocusClear, to permeate and reduce the opacity of mouse colon and ileum. Tissues were labeled with fluorescent probes and examined by confocal microscopy with efficient fluorescence excitation and emission in the FocusClear solution. The voxel-based confocal micrographs were processed with Amira software for 3D visualization and analysis.
Treatment of tissues with the optical-clearing solution improved photon penetration, resulting in the acquisition of images with subcellular-level resolution across the mucosa, submucosa, and muscle layers. Collectively, the acquired image stacks were processed by projection algorithms for 3D analysis of the spatial relations in villi, crypts, and connective tissues. These imaging technologies allowed for identification of spatiotemporal changes in crypt morphology of colon tissues from mice with dextran sulfate sodium-induced colitis as well as detection of transgenic fluorescent proteins expressed in the colon and ileum.
This new optical method for penetrative imaging of mouse intestine does not require tissue sectioning and provides a useful tool for 3D presentation and analysis of diseased and transgenic intestine in an integrated fashion.
Gastroenterology 06/2009; 137(2):453-65. · 11.68 Impact Factor
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ABSTRACT: Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.
Journal of General Virology 01/2009; 89(Pt 12):3004-8. · 3.36 Impact Factor
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ABSTRACT: Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.
Journal of Biomedical Optics 15(4):046018. · 3.16 Impact Factor
