建構胰小島組織之三維血管與交感神經網絡和其在糖尿病小鼠模型的變化

3-D imaging and illustration of mouse islet sympathetic innervations and its remodeling in injury

邱俞臻(Yu-Chen Chiu)

指導教授 ： 湯學成

國立清華大學/工學院/化學工程學系/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00186

摘要

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。

關鍵字

交感神經 ； 胰小島 ； 組織澄清技術 ； 血管 ； 三維影像

並列摘要

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.

並列關鍵字

Green fluorescent protein ； Nonobese diabetic ； Streptozotocin ； Wheat germ agglutinin ； Tyrosine hydroxylase ； Glial fibrillary acidic protein

參考文獻

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.