交感神經影響了胰臟荷爾蒙在血液中的濃度。瞭解胰小島組織的交感神經因糖尿病所產生的結構變化，將幫助對糖尿病病情的控制。然而，傳統的二維切片影像技術，無法提供胰小島組織的三維血管、神經網絡影像。在本論文中，我們以灌流技術對小鼠胰臟血管進行染色(vessel painting)，以交感神經marker tyrosine hydroxylase標記交感神經網絡，並以細胞核染色技術呈現胰小島組織的微結構。我們再以組織澄清技術(optical clearing)，增進光子在染色組織的穿透度，最後使用共軛焦顯微鏡技術擷取胰小島組織的三維影像。
我們發現交感神經除了圍繞在鄰近小動脈的四周，在胰小島外圍接觸α細胞外，在內部更有貼近著微血管的神經纖維。這些內部貼近血管的神經纖維，在施打Streptozotocin的小鼠中會變得更為顯著；在 nonobese diabetic小鼠中，早期胰島炎的淋巴球滲透亦會造成交感神經在小島周圍的變化。
由交感神經的三維影像推論，交感神經可藉由與α細胞直接接觸，更可透過血管達到調控荷爾蒙分泌。在胰小島受損時，交感神經則會因為不同的發病機制的刺激而產生不同的變化。

Aims/hypothesis: Sympathetic nerves influence islet hormone levels in the circulation. Insights into the islet sympathetic innervation and its remodeling in diabetes impact future therapeutics. However, standard immunohistochemistry and microtome-based microscopy cannot provide an integral view of the islet neurovascular complex. We prepared transparent islet specimens to investigate the spatial relationship between sympathetic nerves, blood vessels, and islet cells in normal, streptozotocin-injected, and nonobese diabetic (NOD) mice.
Methods: Cardiac perfusion of the fluorescent lectin was used to label the blood vessels of pancreas. Tyrosine hydroxylase and nuclear staining were used to reveal the islet sympathetic innervation and microstructure. Optical clearing -- use of immersion solution to reduce scattering -- was applied to enable 3-dimensional confocal microscopy of islets to visualize the sympathetic neurovascular complex in space.
Results: Unlike the previously reported morphology, we observe perfusive intra-islet, perivascular sympathetic innervation, in addition to the peri-islet contacts of sympathetic nerves with the alpha cells and the sympathetic fibers encircling the adjacent arterioles. The intra-islet axons become markedly prominent in the streptozotocin-injected mice (two weeks after the injection). In the NOD mice, the lymphocytic infiltration remodels the peri-islet sympathetic axons in early insulitis.
Conclusions/interpretation: We establish an imaging approach to reveal the spatial features of the mouse islet sympathetic innervation. The neurovascular complex and the sympathetic nerve-alpha cell contact suggest that sympathetic nerves modulate islet hormone secretion through blood vessels in addition to acting directly onto the alpha cells. In islet injuries, sympathetic nerves undergo different remodeling in response to different pathophysiological cues.

1. Woods, S.C. and D. Porte, Jr., Neural control of the endocrine pancreas. Physiol Rev, 1974. 54(3): p. 596-619.
2. Ahren, B., Autonomic regulation of islet hormone secretion - Implications for health and disease. Diabetologia, 2000. 43(4): p. 393-410.
3. Brunicardi, F.C., D.M. Shavelle, and D.K. Andersen, Neural regulation of the endocrine pancreas. International Journal of Pancreatology, 1995. 18(3): p. 177-195.
4. Ahren, B., N. Wierup, and F. Sundler, Neuropeptides and the regulation of islet function. Diabetes, 2006. 55: p. S98-S107.
5. Lindsay, T.H., et al., A quantitative analysis of the sensory and sympathetic innervation of the mouse pancreas. Neuroscience, 2006. 137(4): p. 1417-26.
6. Burris, R.E. and M. Hebrok, Pancreatic innervation in mouse development and beta-cell regeneration. Neuroscience, 2007. 150(3): p. 592-602.
7. Coupland, R.E., The Innervation of Pancreas of the Rat, Cat and Rabbit as Revealed by the Cholinesterase Technique. Journal of Anatomy, 1958. 92(1): p. 143-&.
8. Miller, R.E., Pancreatic Neuroendocrinology - Peripheral Neural Mechanisms in the Regulation of the Islets of Langerhans. Endocrine Reviews, 1981. 2(4): p. 471-494.
9. Merani, S., et al., Optimal implantation site for pancreatic islet transplantation. Br J Surg, 2008. 95(12): p. 1449-61.
10. Gromada, J., I. Franklin, and C.B. Wollheim, alpha-Cells of the endocrine pancreas: 35 years of research but the enigma remains. Endocrine Reviews, 2007. 28(1): p. 84-116.
11. Taborsky, G.J., Islets Have a Lot of Nerve! Or Do They? Cell Metabolism, 2011. 14(1): p. 5-6.
12. Smith, K., Neurogastroenterology: Improving 3D imaging of the enteric nervous system. Nat Rev Gastroenterol Hepatol, 2011. 8(11): p. 600.
13. Fu, Y.Y. and S.C. Tang, At the Movies: 3-Dimensional Technology and Gastrointestinal Histology. Gastroenterology, 2010. 139(4): p. 1100-+.
14. Fu, Y.Y., et al., Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution. Journal of Biomedical Optics, 2010. 15(4).
15. Tang, S.C., et al., Vascular Labeling of Luminescent Gold Nanorods Enables 3-D Microscopy of Mouse Intestinal Capillaries. Acs Nano, 2010. 4(10): p. 6278-6284.
16. Fu, Y.Y., et al., Microtome-Free 3-Dimensional Confocal Imaging Method for Visualization of Mouse Intestine With Subcellular-Level Resolution. Gastroenterology, 2009. 137(2): p. 453-465.
17. Hara, M., et al., Imaging pancreatic beta-cells in the intact pancreas. American Journal of Physiology-Endocrinology and Metabolism, 2006. 290(5): p. E1041-E1047.
18. Liu, Y.A., et al., Optical clearing improves the imaging depth and signal-to-noise ratio for digital analysis and three-dimensional projection of the human enteric nervous system. Neurogastroenterology and Motility, 2011. 23(10): p. E446-E457.
19. Liu, Y.A., et al., 3-D illustration of network orientations of interstitial cells of Cajal subgroups in human colon as revealed by deep-tissue imaging with optical clearing. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012. 302(10): p. G1099-G1110.
20. Tuchin, V.V., R.K. Wang, and A.T. Yeh, Optical clearing of tissues and cells. Journal of Biomedical Optics, 2008. 13(2).
21. Wang, R.K.K., et al., Investigation of optical clearing of gastric tissue immersed with hyperosmotic agents. Ieee Journal of Selected Topics in Quantum Electronics, 2003. 9(2): p. 234-242.
22. Xu, X.Q. and R.K.K. Wang, Synergistic effect of hyperosmotic agents of dimethyl sulfoxide and glycerol on optical clearing of gastric tissue studied with near infrared spectroscopy. Physics in Medicine and Biology, 2004. 49(3): p. 457-468.
23. Tseng, S.J., et al., Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections. Journal of Biomedical Optics, 2009. 14(4).
24. Genina, E.A., et al., Optical clearing of human skin: comparative study of permeability and dehydration of intact and photothermally perforated skin. Journal of Biomedical Optics, 2008. 13(2).
25. Fu, Y.Y. and S.C. Tang, Optical clearing facilitates integrated 3D visualization of mouse ileal microstructure and vascular network with high definition. Microvascular Research, 2010. 80(3): p. 512-521.
26. Sunami, E., et al., Morphological characteristics of Schwann cells in the islets of Langerhans of the murine pancreas. Archives of Histology and Cytology, 2001. 64(2): p. 191-201.
27. Cabrera, O., et al., The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(7): p. 2334-2339.
28. Gunawardana, S.C., et al., Imaging beta cell development in real-time using pancreatic explants from mice with green fluorescent protein-labeled pancreatic beta cells. In Vitro Cellular & Developmental Biology-Animal, 2005. 41(1-2): p. 7-11.
29. El-Gohary, Y., et al., Whole-Mount Imaging Demonstrates Hypervascularity of the Pancreatic Ducts and Other Pancreatic Structures. Anatomical Record-Advances in Integrative Anatomy and Evolutionary Biology, 2012. 295(3): p. 465-473.
30. Sandler, S. and L. Jansson, Vascular-Permeability of Pancreatic-Islets after Administration of Streptozotocin. Virchows Archiv a-Pathological Anatomy and Histopathology, 1985. 407(4): p. 359-367.
31. Nicholson, J.M., E.J. Arany, and D.J. Hill, Changes in islet microvasculature following streptozotocin-induced beta-cell loss and subsequent replacement in the neonatal rat. Experimental Biology and Medicine, 2010. 235(2): p. 189-198.
32. Ravnic, D.J., et al., Vessel painting of the microcirculation using fluorescent lipophilic tracers. Microvascular Research, 2005. 70(1-2): p. 90-6.
33. Borelli, M.I., et al., Tyrosine hydroxylase activity in the endocrine pancreas: changes induced by short-term dietary manipulation. BMC Endocr Disord, 2003. 3(1): p. 2.
34. Winer, S., et al., Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive. Nature Medicine, 2003. 9(2): p. 198-205.
35. Regoli, M., et al., Glial fibrillary acidic protein (GFAP)-like immunoreactivity in rat endocrine pancreas. Journal of Histochemistry & Cytochemistry, 2000. 48(2): p. 259-265.
36. Rerup, C. and F. Tarding, Streptozotocin- and Alloxan-Diabetes in Mice. European Journal of Pharmacology, 1969. 7(1): p. 89-&.
37. Mignone, J.L., et al., Neural stem and progenitor cells in nestin-GFP transgenic mice. Journal of Comparative Neurology, 2004. 469(3): p. 311-324.
38. Motyl, K. and L.R. McCabe, Streptozotocin, Type I Diabetes Severity and Bone. Biological Procedures Online, 2009. 11(1): p. 296-315.
39. Larrieta, M.E., et al., Nerve growth factor increases in pancreatic beta cells after streptozotocin-induced damage in rats. Experimental Biology and Medicine, 2006. 231(4): p. 396-402.
40. Teitelman, G., et al., Islet injury induces neurotrophin expression in pancreatic cells and reactive gliosis of peri-islet Schwann cells. Journal of Neurobiology, 1998. 34(4): p. 304-318.
41. Taborsky, G.J., et al., Loss of islet sympathetic nerves and impairment of glucagon secretion in the NOD mouse: relationship to invasive insulitis. Diabetologia, 2009. 52(12): p. 2602-2611.
42. Tsui, H., et al., Islet Glia, Neurons, and beta Cells The Neuroimmune Interface in the Pathogenesis of Type 1 Diabetes. Immunology of Diabetes V: From Bench to Bedside, 2008. 1150: p. 32-42.
43. Tsui, H., et al., Targeting of pancreatic glia in type 1 diabetes. Diabetes, 2008. 57(4): p. 918-28.