Name: longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber
Uploaded: 2020-06-11T14:30:00
Description: Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

这项研究得到了瑞典研究理事会、战略研究领域 Exodiab、Dnr 2009-1039、瑞典战略研究基金会 Dnr IRC15-0067、Lund皇家物理学会、糖尿病基金会和巴恩迪亚贝特本特公司的支持。

瑞典隆德地区伦理委员会根据《关于人类研究伦理审查法》批准了这项研究。动物实验严格按照瑞典动物实验伦理进行，并经马尔默和隆德伦理委员会批准。6 至 8 周大免疫缺陷 NOD。（Cg）-gt（ROSA）26Sortm4-Rag2-/- （NOD.罗莎 -番茄Rag2-/-）接受小鼠被用作人类小岛10的移植接受者。
1. 岛移植准备
<ol><li>C...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+pancreatic+islet+transplantation+in+humans.">Advances in pancreatic islet transplantation in humans.</a> Diabetes, Obesity, Metabolism. 8 (1), 15-25 (2006).</li><li>Malaisse, W. J., Maedler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+the+beta+cells+of+the+islets+of+Langerhans.">Imaging of the beta cells of the islets of Langerhans.</a> Diabetes Research and Clinical Practice. 98 (1), 11-18 (2012).</li><li>Kim, D., Jun, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Imaging+of+Transplanted+Pancreatic+Islets.">In Vivo Imaging of Transplanted Pancreatic Islets.</a> Frontiers in Endocrinology. 8, 382 (2017).</li><li>Speier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+high-resolution+in+vivo+imaging+of+cell+biology+in+the+anterior+chamber+of+the+mouse+eye.">Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye.</a> Nature Protocols. 3 (8), 1278-1286 (2008).</li><li>Yang, S. N., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eye+as+a+novel+imaging+site+in+diabetes+research.">The eye as a novel imaging site in diabetes research.</a> Pharmacology, Therapeutics. 197, 103-121 (2019).</li><li>Schmidt-Christensen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+dynamics+of+CD11c(+)+cells+and+Foxp3(+)+cells+in+progressive+autoimmune+insulitis+in+the+NOD+mouse+model+of+type+1+diabetes.">Imaging dynamics of CD11c(+) cells and Foxp3(+) cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes.</a> Diabetologia. 56 (12), 2669-2678 (2013).</li><li>Berclaz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+three-dimensional+visualisation+of+autoimmune+diabetes+by+functional+optical+coherence+imaging.">Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging.</a> Diabetologia. 59 (3), 550-559 (2016).</li><li>Nilsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruited+fibroblasts+reconstitute+the+peri-islet+membrane:+a+longitudinal+imaging+study+of+human+islet+grafting+and+revascularisation.">Recruited fibroblasts reconstitute the peri-islet membrane: a longitudinal imaging study of human islet grafting and revascularisation.</a> Diabetologia. 63 (1), 137-148 (2020).</li><li>Benninger, R. K., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+excitation+microscopy+for+the+study+of+living+cells+and+tissues.">Two-photon excitation microscopy for the study of living cells and tissues.</a> Current Protocols in Stem Cell Biology. , 11-24 (2013).</li><li>Goto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+the+automated+method+for+human+islet+isolation+and+presentation+of+a+closed+system+for+in+vitro+islet+culture.">Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.</a> Transplantation. 78 (9), 1367-1375 (2004).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harbor Protocols. 2011 (2), 5563 (2011).</li><li>Jansson, L., Carlsson, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+vascular+function+after+transplantation+of+pancreatic+islets.">Graft vascular function after transplantation of pancreatic islets.</a> Diabetologia. 45 (6), 749-763 (2002).</li><li>Konstantinova, I., Lammert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+development:+learning+from+pancreatic+islets.">Microvascular development: learning from pancreatic islets.</a> Bioessays. 26 (10), 1069-1075 (2004).</li><li>Fransson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+support+endothelial+cell+interactions+in+an+intramuscular+islet+transplantation+model.">Mesenchymal stromal cells support endothelial cell interactions in an intramuscular islet transplantation model.</a> Regenerative Medicine Research. 3, 1 (2015).</li><li>Nyqvist, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+islet+endothelial+cells+in+pancreatic+islet+revascularization.">Donor islet endothelial cells in pancreatic islet revascularization.</a> Diabetes. 60 (10), 2571-2577 (2011).</li><li>Nair, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+common+anesthetics+on+eye+movement+and+electroretinogram.">Effects of common anesthetics on eye movement and electroretinogram.</a> Documenta Ophthalmologica. Advances in Ophthalmology. 122 (3), 163-176 (2011).</li><li>Iwasaka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+intolerance+during+prolonged+sevoflurane+anaesthesia.">Glucose intolerance during prolonged sevoflurane anaesthesia.</a> Canadian Journal of Anaesthesia. 43 (10), 1059-1061 (1996).</li><li>Hamilton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+its+applications+in+fluorescent+microscopy+imaging.">Quantification and its applications in fluorescent microscopy imaging.</a> Traffic. 10 (8), 951-961 (2009).</li><li>Michelotti, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET/MRI+enables+simultaneous+in+vivo+quantification+of+beta-cell+mass+and+function.">PET/MRI enables simultaneous in vivo quantification of beta-cell mass and function.</a> Theranostics. 10 (1), 398-410 (2020).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Allogeneic+Islet+Grafts+in+Nonhuman+Primates+Using+MRI.">Monitoring of Allogeneic Islet Grafts in Nonhuman Primates Using MRI.</a> Transplantation. 99 (8), 1574-1581 (2015).</li><li>Gotthardt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+beta+cells+by+PET+imaging:+why+clinical+implementation+has+never+been+closer.">Detection and quantification of beta cells by PET imaging: why clinical implementation has never been closer.</a> Diabetologia. 61 (12), 2516-2519 (2018).</li><li>Joosten, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Pancreatic+beta+Cell+Mass+in+Vivo+with+Exendin+SPECT+during+Hyperglycemia+and+Severe+Insulitis.">Measuring the Pancreatic beta Cell Mass in Vivo with Exendin SPECT during Hyperglycemia and Severe Insulitis.</a> Molecular Pharmaceutics. 16 (9), 4024-4030 (2019).</li><li>Virostko, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+mouse+models+quantifies+beta+cell+mass+in+the+pancreas+and+after+islet+transplantation.">Bioluminescence imaging in mouse models quantifies beta cell mass in the pancreas and after islet transplantation.</a> Molecular Imaging and Biology. 12 (1), 42-53 (2010).</li></ol>

提出了一种通过观察接受者和供体组织的介入来研究人类胰岛细胞移植过程的方法。在将人类小岛植入免疫缺陷小鼠眼的前室的微创手术后，小鼠在手术后几分钟内迅速恢复。手术在一只眼睛上执行。一般来说，从植入后5~7天开始，角膜被充分治愈，可以进行病毒内成像。
在这个协议中，人类小岛嫁接的质量至关重要。人类小岛的质量以及移植结果可能因捐赠者的年龄、BMI?...

糖尿病描述一组代谢性疾病，其特征是血糖水平升高，其特征是胰腺胰岛β细胞损失或功能障碍导致胰岛素生产不足，通常伴有胰岛素抵抗。1型（T1D）和2型糖尿病（T2D）是复杂的疾病，其中β细胞的渐进功能障碍导致疾病发展。T1D是由对β细胞的自身免疫攻击引起的，而T2D被认为是由代谢因素驱动的，尽管有越来越多的证据表明低级全身炎症1。移植供体人类小岛，特别是T1D患者，提供生理血糖控制的潜力。然而，组织捐献者短缺和小岛移植不良使得小岛移植成为主流治疗选择。由于缺氧、炎症、免疫原源环境2，3，功能小岛移植在移植后（24~48小时）的直接转移期丢失了很大一部分功能性小岛移植物。为了评估干预方法的效率，以提高小岛的生存，持续监测这种移植是必要的。
在体内技术，以图像和跟踪移植后人类胰腺胰岛的命运仍然是一个挑战，糖尿病研究,4，5。迄今为止，非侵入性成像技术，包括正电子发射断层扫描（PET）、磁共振成像（MRI）或超声波（美国）等，在实验条件下对移植小岛进行定量和功能评估的潜力。然而，鉴于小小小的小小小小，这些模式的定量测量由于分辨率不足而受到影响。眼前腔室（ACE）作为观察的移植点，是一种很有前途的无创成像解决方案，提供有效的更高的空间分辨率和长期长时间的频繁监测。这种方法已被成功地用于研究小鼠小岛生物学（见杨等人7），自身免疫免疫反应8，以及人类小岛移植9，10。109
在这里，ACE移植方法与2光子成像方法相结合，通过连续和重复记录单个胰岛移植，在移植后长达10个月，研究人类胰岛移植过程的动态。成像深度更大、整体光漂白和照片损伤减少的多光子成像特性克服了共声显微镜11的成像局限性。荧光成像的定量涉及几个阶段，包括小岛样品制备、小岛移植、图像采集、图像滤波以去除小岛噪声或背景、分割、定量和数据分析。最具挑战性的步骤通常是将图像分割或分割为多个部分或区域。这可能涉及将信号与背景噪声分离，或根据颜色或形状的相似性聚类体素区域，以检测和标记表示小岛血管的 3D 体积的体素。一旦分段，数据对象卷大小等统计信息通常很容易提取。提供一种用于对成像数据进行量化和提取的方法，如分段和数据可视化。特别注意去除人类小岛中的自荧光，区分小岛血管和小岛胶囊形成接受细胞。

<table>
	<tbody>
		<tr>
			<td>Anasthesia machine, e.g. Anaesthesia Unit U-400</td>
			<td>Agnthos</td>
			<td>8323001</td>
			<td>used for isofluran anasthesia during surgery and imaging</td>
		</tr>
		<tr>
			<td>-induction chamber 1.4 L</td>
			<td>Agnthos</td>
			<td>8329002</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>-gas routing switch</td>
			<td>Agnthos</td>
			<td>8433005</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>AngioSense 680 EX</td>
			<td>Percin Elmer</td>
			<td>NEV10054EX</td>
			<td>imaging agent for injection, used to image blood vessels in human islet grafts</td>
		</tr>
		<tr>
			<td>Aspirator tubes assemblies</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>connect with pulled capillary pipettes for manual islet picking</td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic) 0.3mg/ml</td>
			<td>Schering-Plough Europ&#233;</td>
			<td>64022</td>
			<td>fluid, for pain relief</td>
		</tr>
		<tr>
			<td>Capillary pipettes</td>
			<td>VWR</td>
			<td>321242C</td>
			<td>used together with Aspirator tubes assemblies</td>
		</tr>
		<tr>
			<td>Dextran-Texas Red (TR), 70kDa</td>
			<td>Invitrogen</td>
			<td>D1830</td>
			<td>imaging agent for injection</td>
		</tr>
		<tr>
			<td>Eye cannula, blunt end , 25 G</td>
			<td>BVI Visitec/BD</td>
			<td>BD585107</td>
			<td>custom made from Tapered Hydrode lineator [Blumenthal], dimensions: 0.5 x 22mm (25G x 7/8in) (45&#8304;), tip tapered to 30 G (0.3mm)</td>
		</tr>
		<tr>
			<td>Eye gel</td>
			<td>Novartis</td>
			<td></td>
			<td>Viscotears, contains Carbomer 2 mg/g</td>
		</tr>
		<tr>
			<td>Hamilton syringe 0.5 ml, Model 1750 TPLT</td>
			<td>Hamilton</td>
			<td>81242</td>
			<td>Plunger type gas-tight syringe for islet injection</td>
		</tr>
		<tr>
			<td>Head holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Head holding adapter</td>
			<td>Narishige</td>
			<td>SG-4N-S</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-gas mask</td>
			<td>Narishige</td>
			<td>GM-4-S</td>
			<td></td>
		</tr>
		<tr>
			<td>-UST-2 Solid Universal Joint</td>
			<td>Narishige</td>
			<td>UST-2</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-custom made metal plate for head-holder assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Dumont #5, straight</td>
			<td>Agnthos</td>
			<td>0207-5TI-PS or 0208-5-PS</td>
			<td>attached to UST-2 (custom made)</td>
		</tr>
		<tr>
			<td>Heating pad, custom made</td>
			<td></td>
			<td></td>
			<td>taped to the stereotaxic platform</td>
		</tr>
		<tr>
			<td>Human islet culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-CMRL 1066</td>
			<td>ICN Biomedicals</td>
			<td></td>
			<td>cell culture media for human islets</td>
		</tr>
		<tr>
			<td>-HEPES</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-L-glutamin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Gentamycin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Fungizone</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ciproxfloxacin</td>
			<td>Bayer healthcare AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Nicotinamide</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaris 9</td>
		</tr>
		<tr>
			<td>Image Aquisition software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2010</td>
		</tr>
		<tr>
			<td>Infrared lamp</td>
			<td>VWR</td>
			<td>1010364937</td>
			<td>used to keep animals warm in the wake-up cage</td>
		</tr>
		<tr>
			<td>Isoflurane Isoflo</td>
			<td>Abott Scandinavia/Apotek</td>
			<td></td>
			<td>fluid, for anesthesia</td>
		</tr>
		<tr>
			<td>Needle 25 G (0.5 x 16mm), orange</td>
			<td>BD</td>
			<td>10442204</td>
			<td>used as scalpel</td>
		</tr>
		<tr>
			<td>Petri dishes, 90mm</td>
			<td>VWR</td>
			<td>391-0440</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Photon/confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-LSM7 MP upright microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ti:Sapphire laser Tsunami</td>
			<td>Spectra-Physics, Mai Tai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-long distance water-dipping lens 20x/NA1.0</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET710/40m (Angiosense 680)</td>
			<td>Chroma</td>
			<td>288003</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET645/65m-2p (TR)</td>
			<td>Chroma</td>
			<td>NC528423</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET525/50 (GFP)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET610/75 (tomato)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-main beam splitter T680lpxxr</td>
			<td>Chroma</td>
			<td>T680lpxxr</td>
			<td>Dichroic mirror to transmit 690 nm and above and reflect 440 to 650 nm size 25.5 x 36 x 1 mm</td>
		</tr>
		<tr>
			<td>Polythene tubing (0.38mm ID, 1.09 mm OD)</td>
			<td>Smiths Medical Danmark</td>
			<td>800/100/120</td>
			<td>to connect with Hamilton syringe and eye canula</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model SMZ645, for islet picking</td>
		</tr>
		<tr>
			<td>Stereomicroscope (Flourescence)</td>
			<td></td>
			<td></td>
			<td>for islet graft imaging</td>
		</tr>
		<tr>
			<td>-AZ100 Multizoom</td>
			<td>Nikon</td>
			<td></td>
			<td>wide field and long distance</td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 1x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 4x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ-FL Epiflourescence with C-LHGFI HG lamp</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-HG Manual New Intensilight</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block TEXAS RED</td>
			<td>Nikon</td>
			<td></td>
			<td>contains EX540-580, DM595 and BA600-660</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block G-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX510-560, DM575 and BA590)</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block B-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX450-490, DM505 and BA520)</td>
		</tr>
		<tr>
			<td>-DS-Fi1 Colour Digital Camera (5MP)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1-ml, Omnitix</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>for Buprenorphine injection, used with 27 G needle</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber

成像β细胞是了解胰岛移植的关键一步。虽然在体内已经开发和利用了用于β细胞生物学记录的不同成像 平台，但它们在允许单细胞分辨率和连续纵向记录方面是有限的。由于角膜的透明度，小鼠眼前室（ACE）非常适合研究人类和小鼠胰岛细胞生物学。以下是如何使用这种方法对单个人类小岛移植进行连续纵向记录和再血管化的描述。使用NO NOD将人类小岛嫁接插入ACE。（Cg）-Gt（ROSA）26Sortm4-Rag2-/-小鼠作为接受者。这允许调查接受者与供体细胞的扩大和接受者细胞在促进移植物的封装和血管化方面的贡献。此外，还概述了一种分步方法，用于图像分析和定量的小岛体积或分段血管和小岛胶囊形成接受细胞。

非标记的人类小岛被移植到8周大的女性NOD的ACE中。（Cg）-gt（ROSA）26Sortm4-Rag2-/-（NOD.罗莎- 番茄Rag2=/=）接受小鼠。为了防止人体组织排斥，免疫缺陷的Rag2敲除小鼠被选为接受者。在这些转基因小鼠中，所有细胞和组织都表示一种以膜为目标的番茄荧光蛋白（mT），可以清楚地识别接受者和供体组织。高分辨率 2 光子显微镜对小岛移植物的?...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

该协议的目标是持续监测人类胰岛移植过程和贡献宿主与供体细胞的动态。这是通过移植人类小岛到NOD的眼部（ACE）前室来完成的。（Cg）-Gt（ROSA）26Sortm4-Rag2-/-鼠标接收者，然后是重复的 2 光子成像。

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

ACE 是最佳成像景点。它支持在小岛细胞的长期体内成像中重复，以研究其在移植情况下的功能和生存能力。也可以推广到生理和病理条件下的其他方面进行实时研究。ACE 技术的优点在于，相同的人类胰腺小岛可以在较长一段时间内以亚细胞分辨率无创地跟随。贝塔细胞替代疗法的成功率继续提高。然而，评估小岛嫁接和体内生存的效率仍然具有挑战性。这种方法可以帮助评估和优化小岛移植的结果。此方法不仅限于胰岛。它也非常适合研究受影响的组织。例如，在糖尿病并发症，如肾球或肝脏片，只是举几例。要开始将鼠标放在加热垫上，请将鼠标放在头部支架上，并戴上鼻罩。使用拇指和食指稍微抬起头部。用两侧的金属碎片固定它。确保耳片将头部直接固定到耳朵下方。皮下将丁丙诺啡溶液注射到小鼠的背面。轻轻缩回眼睑，用钝钳移植。弹出眼睛，并松散地修复它与一对钳子覆盖聚乙烯管。将眼睑转移到带 PBS 的培养皿后，在眼角中拾取大约 20 到 30 个小岛。使用 25 量针水平向上，小心地将尖端插入角膜，并做一个横向切口。小心地抬起角膜，用预先装上小岛的角膜，然后慢慢将小岛释放到眼睛中。慢慢缩回管，将眼凝胶涂抹在眼睛上。10 分钟后，取出钳子，握住眼睑，将眼睛放回正常位置。对于使用两个光子显微镜对植入的人类小岛进行成像，请将头部支架平台置于显微镜下，并在角膜和透镜之间将眼凝胶作为浸入式液体施用到眼睛上。将眼睛定位在目标下，并像文本手稿中描述的植入的人类小岛图像。要删除自动荧光，请转到图像处理选项卡，选择通道荧光和键入通道一减去通道二。这将创建一个新的通道四。重命名它 vasature。重复此过程并键入通道三减去通道 2 以创建新通道 5 并重命名所有番茄。要手动定义小岛，请创建新曲面并在向导中手动选择编辑。将指针保持为选择模式和 3D 视图。然后单击音量以可视化部分。在绘图选项卡中，选择轮廓并单击绘制以开始围绕小岛边框绘制轮廓，从切片位置 1 开始。然后移动到新的切片位置并重复绘制轮廓。以小岛顶部的最后一个切片结束，然后通过单击"创建曲面"选项卡结束。下一段，小岛血管和小岛番茄荧光使用小岛掩码。选择以前定义的小岛掩码对象，转到编辑选项卡，然后单击蒙版所有选项卡，该选项卡将打开一个新窗口。在通道选择下拉菜单中选择血管通道，并在应用蒙版之前激活选项重复通道。恒定的内、外和将体素外部表面设置为 0.000，从而创建新通道 6。重命名此通道小岛血管。重复前面的步骤，在通道选择下拉菜单中选择番茄，以创建新通道，重命名它，小番茄。然后在场景菜单中创建新曲面，并在向导中选择自动创建。将源通道设置为以前创建的小岛血管，并选择背景减法。可选使用筛选器。例如，选择"音量"并调整窗口中的筛选器，该筛选器可以删除选定的服务对象。完成向导并命名新的曲面对象，即小岛血管。在小岛血管服务对象转到编辑选项卡并单击蒙版所有选项卡，该选项卡将打开一个新窗口。在通道选择下拉菜单中选择小岛番茄通道，将表面外的体素设置为 10.000，从而创建新通道。重命名此通道小岛番茄血管。要分割番茄胶囊荧光信号，在图像处理选项卡中选择通道遥测，并键入通道七减通道八创建新通道。重命名番茄胶囊。创建前面描述的新表面，并在向导中选择源通道、小岛番茄血管或番茄胶囊。然后执行背景减法。打开小岛反散散器文件并创建新曲面。在向导中，选择自动创建并定义感兴趣区域。如果需要，以绝对强度调整阈值。可以单击或关闭表面对象，以与相应的通道强度进行交叉检查。完成后关闭向导。最后，执行量化，在场景菜单中选择创建的曲面对象，然后转到统计选项卡。要检索详细的卷数据，请选择详细选项卡，并从下拉菜单中选择特定值和卷。要检索总体积值，请转到详细选项卡并选择平均值。该协议用于将无标记的人类小岛移植到八周大雌性红荧光接受小鼠眼睛的内室。然后使用交互式成像软件从图像中提取定量数据。在活体荧光成像中，人类小岛移植受到组织自身荧光的显著干扰，而小鼠小岛移植中未观察到这种干扰。为了提高信噪比，减小了自荧光通道。然后手动定义称为小岛掩码和相应通道的小岛边界。最后，利用番茄信号分割到眼睑血管和眼睑胶囊，最后表面渲染用于提取定量数据。这里作为代表性的纵向成像会议的同一人类小岛移植在两个星期，两个月，五个月和八个月的移植后。显示最初记录的原始数据的 MIP 图像、小岛自荧光去除后处理的图像和分段的小岛对象。执行眼睑注射时，请确保以小体积预装小岛，并在立体显微镜上使用灵活的双轻臂，以突出显示眼室空间，以便成功地将小岛注入 ACE 中。在体内成像后，眼睛可以固定，并进一步调查抗体染色和常规组织学。该技术对于人类个体小岛在实时和生理条件下的功能研究非常有用，特别是在血管化、生存能力和代谢研究的背景下。

Research

JoVE Journal

Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

科研

医学

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

纵向，非侵入性的移植到眼睛的前房<em在体内</em&gt;单细胞分辨率的实时成像

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像：相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...