Ya-Yuan Fu

Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution

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.

Vascular Labeling of Luminescent Gold Nanorods Enables 3-D Microscopy of Mouse Intestinal Capillaries

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-NRs unique near-infrared excitation and visible fluorescence emission, we observed low noise background compared to the tissues 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-NRs distinctive properties in vascular labeling and fluorescence microscopy for 3-D illustration of intestinal vasculature.

Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes.

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.

758 3-D Imaging of Mouse Pancreatic Duct Lesion and Neurovascular Remodeling

Pancreas features extensive ductal and neurovascular networks in association with acini and islets to perform exocrine and endocrine functions. However, due to the dispersed nature of the network architecture, the standard microtome-based histology cannot provide a globe view of the pancreatic tissue networks in health and disease. To overcome this challenge, we prepared transparent pancreatic specimens by optical clearing (use of immersion solution of high refractive index to promote tissue photon penetration; Tang et al., Diabetes, Obesity and Metabolism, 2014, doi: 10.1111/dom.12342) and performed 3-D microscopy with tile scanning to reveal the spatial features of the pancreatic ductal and neurovascular networks with high definition. Mouse pancreatic intraepithelial neoplasia (PanIN) was induced by activation of oncogenic Kras in acinar cells and/or caerulein injections (chronic pancreatitis). Taking advantage of the transparent tissue, we simultaneously revealed the 3-D PanIN microstructure and the associated neurovascular environment in an integrated fashion. Perilesional neural tissue outgrowth (sympathetic neurite outgrowth and gliosis) and an increase in pericyte marker density were identified via in-depth image projection, indicating the reactivity of the pancreatic neurovascular tissues in response to lesion formation. Overall, the global characterization of duct lesions highlights the advantage of using 3-D microscopy to explore the unknown details of the pancreatic microenvironment in PanIN development.

Su1450 Gastric Mucosal Innervation in Endoscopic Biopsies in Patients With Idiopathic Gastroparesis Is Not Significantly Different Than Normals

Introduction. Diabetic gastroparesis is defined as delayed gastric emptying not caused by obstruction or structural abnormality. Normal function of the gastric and intestinal mechanical activity is mediated by slow wave electrical activity in the stomach and small bowel. Previous studies using both electrogastrogram and magnetogastrogram have shown gastric slowwave dysrhythmias associated with gastroparesis, but no study has yet examined possible effects of gastroparesis on the intestinal slow wave. Methods. We recorded intestinal slow waves in diabetic patients with gastroparesis (N=7) and healthy controls (N=7) using the magnetoenterogram (MENG), which uses a Superconducting QUantum Interference Device (SQUID) to convert magnetic fields associated with intestinal slow waves into voltage signals. Second Order Blind Identification (SOBI) was used to reduce noise and isolate the intestinal slow wave signal from confounding magnetic artifact, and we computed the power spectrum of the intestinal slow wave using a Fast Fourier Transform technique. We analyzed dominant frequency, amplitude and percentage of power distributed (PPD) in brady, normo and tachyarrhythmic frequency ranges. Results. In gastroparesis patients, we found a significant decrease in postprandial dominant intestinal slow wave frequency from 10.2 ± 0.4 cpm to 8.8 ± 0.5 cpm (p<0.05) whereas the dominant frequency for control subjects increased from 9.9 ± 0.5 cpm to 10.8 ± 0.4 cpm (p<0.05). We did not observe significant differences in preand postprandial PPDs computed from controls or patients. Conclusions. Diabetic gastroparesis is associated with bradyarrhythmia, but not uncoupling, of the intestinal slow wave. Biomagnetic measurements of the MENG can assess intestinal slow wave activity in healthy and diseased tissue noninvasively.

133 Sensory Denervation Decreases PanIN Progression in a Mouse Model of Pancreatic Cancer

BACKGROUND: Reciprocal molecular signaling between pancreatic ductal adenocarcinoma (PDAC) and nerves may promote perineural invasion (PNI) and tumor growth. The identity and function of sensory neuropeptides in the tumor microenvironment are unknown. We hypothesized that sensory neurons play an important role in tumor progression and that substance P (SP) is a candidate neuropeptide for mediating this effect. AIMS:1) to characterize SP receptor (NK1R) expression in PanIN epithelium 2) to determine NK1R expression in human PDAC cell lines 3) to study effects of sensory denervation on pancreatic intraepithelial neoplasia (PanIN) initiation and progression in the KPC ( Pdx1-Cre; LSL-Kras; LSLTrp53) mouse model. METHODS: KPC mice were injected with the sensory neurotoxin resiniferatoxin (RTX; Sigma) or control solution on postnatal day 7. Pancreata from 8and 12-week-old mice were fixed and subject to HE Santa Cruz). Images were taken with a Nikon Camera or Zeiss LSM 510 Meta microscope. PanINs were graded as early (grade 1) or advanced (grades 2 and 3). PanIN burden was calculated by the percentage of total surface area occupied in 5 random views per HE p = 0.02). All analyzed human PDAC cell lines expressed the NK1R. In vivo studies revealed axons in close proximity to PanIN epithelium (Fig.1). RTX-treated mice had a 50% decrease in pancreatic sensory axonal density when compared to control mice at 8 and 12 weeks (p < 0.05). RTX-treated mice had a significant reduction in advanced PanINs compared to control mice at both 8 weeks (0.05% versus 0.9%; p < 0.05) and 12 weeks (0.3% versus 8%; p < 0.05). CONCLUSIONS: The NK1R is expressed in distinct cells within the PanIN epithelium and the percentage of NK1R+ cells increases with PanIN grade. Human PDAC cell lines express the NK1R. Sensory denervation is associated with a significant reduction in progression from early to advanced PanIN in the KPC model. Sensory nerves are likely an important part of the PanIN microenvironment and may affect tumorigenesis via the NK1R. This study provides new insight into the pathogenesis and potential therapeutic targets in PDAC.