Name: manipulating the murine lacrimal gland
Uploaded: 2014-11-18T13:00:00.000+00:00
Duration: 5 min 56 s
Description: Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

作者要感谢由加州大学旧金山分校的资源分配计划提供了资金。

在严格按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行的所有动物的工作。该协议被批准的机构动物护理和使用委员会加州大学（IACUC），旧金山。 1.小鼠胚胎泪腺（LG）:收获和显微切割<ol><li>继批准的机构动物科学研究伦理委员会的程序，安乐死定时怀孕的CD-1（或转基因）的女性不断上升的二氧化碳吸入，然后确认颈椎脱位收获胚胎在适当的胚胎一天。指定阴道栓发现E0的一天。 注:泪腺（LG的），可以从E14单芽阶段开始有所收获。然而，E16（5 - 10芽）胚胎给出最一致的结果用于离体培养。 </li><li>消毒V用70％乙醇鼠标entral侧。捏皮肤做一个切口处用无菌手术剪中线;切割穿过皮肤和腹膜，以暴露腹腔。通过在每个子宫角，并装在冷的PBS（磷酸盐缓冲盐水）或DMEM F12培养基的顶部添加有100U / ml青霉素和100μg/ ml链霉素沿系膜及切割除去2子宫角。 </li><li>使用镊子（＃5）中，从羊膜囊取出胚，并放置到装有冷PBS的第二无菌培养皿。小心不要碰到眼睛或头部区域。 </li><li>将胚胎到解剖显微镜与透射基地。如果胚胎是&lt;E17，执行步骤1.5 - 1.7。如果胚胎&gt; e17的，这些步骤可能是有益的，但不是必需的，因此继续执行步骤1.8。 </li><li>从正下方的下颌骨的躯干（步骤1， 图2），使用设切断头GA无菌手术刀（＃10刀片）。转动头部，使下颌骨，现在在解剖板。使用镊子，以稳定磁头和解剖刀除去约脑（第2步，第1次切割， 图2）的背侧的四分之一-这是一次软骨周围E15变硬尤其重要。 </li><li>定向的头部，使得所述手术刀可穿透两眼之间的鼻子。使一个切口穿过头部的中心，使眼睛现在是在单独的半部（第2步，第2切口， 图2）。 </li><li>使用两个＃5镊子，取一半的头部和去除多余的组织（参见步骤3， 图2）。一定要定向的头部，以便对LG（位于眼球的下后角部）未在该去除过程中丢失。除去过量的脑，软骨和周围的鼻组织和眼睛下方。由于腺是勉强可见，在这一点上，确保眼睛后多余的组织去除t被适当地取向Ø避免丢失LG的位置。 </li><li>认真抓住从后，眼睛既钳底角的皮肤。小心拉开与镊子，露出LG去除皮肤。使用上面和下面的组织额外的照明，以帮助可视化的LG。 注:LG，区分其外观上在一个黑暗的，浓缩的间充质管道芽，应该是可见的，在这一点上。 </li><li>开始仔细剖析了LG和相关的间充质（参见图和图像）离周围组织使用镊子，小心不要抢LG或与其相关的管道。一旦腺是免费从周围组织中，轻轻握住眼睛周围对LG管道的基本上皮删除整个腺。小心维持周围的上皮，其中可以观察到作为稠间质成上皮invaginates间质。 注:某些附加的去除Tissu酒店的E（任意小骨碎片，肌肉和结缔组织）可能是必要的，以避免来自相邻组织的信令/生长因子。 </li><li>对于文化，收获4 - 5腺体和每盘相关的间质（LG的立即解剖后镀）;收集至少每100微升用于RT-PCR 6的RNA裂解缓冲液14的腺体。 </li></ol> 2.准备板的体外培养这个过程是基于所采用的显影唾液腺7,8。 <ol><li>添加200μl的DMEM F12（补充有100U / ml青霉素和100μg/ ml链霉素）的培养基，用50微克/毫升抗坏血酸和50μg/ ml的全息转到玻璃底直径50mm的微孔盘7。 注:DMEM F12被选为我们以前发现它最大限度形态在胚胎唾液腺。 </li><li>浮13毫米polycarb苷的膜滤器用0.1微米的孔上的媒体之上。 </li><li>可选步骤:稀释的3-D层粘连蛋白的1:1的DMEM / F12，使为3mg / ml的终浓度（用200μl的移液管轻轻混匀;离心1分钟，如果出现气泡）。加入15微升的此稀释的3-D层粘连蛋白进行筛选。 注意:此稀释液被认为是最佳的用于保持LG在其三维状态而不影响上皮分支。然而，这不是对于LG的培养是必不可少的。 </li><li>将4 - 5 LG到过滤器或成层粘连蛋白。培养LG的离体 24 - 48小时（ 图3）或用4％PFA中20分钟，以供进一步分析通过定量PCR或免疫染色（ 图4）固定。对于胚胎腺体组织的定量PCR分析，请参阅Rebustini 等人，2011。对于胚胎腺体组织的免疫荧光分析，请参考下面的引用:霍夫曼等人，（2002）和Steinberg 即吨人，（2005）。 </li></ol> 3.鼠标产后和成人泪腺（LG）:解剖<ol><li>根据适当的动物伦理委员会的标准安乐死产后鼠标。对于幼崽出生后第8天或以下，通过切割头，用无菌剪刀安乐死。对于8岁以上日龄，毛皮喷用70％乙醇。 </li><li>要找到LG，奠定鼠标横向解剖显微镜下，一边与照明上面。注意:产后和成人的LG是由颈总动脉，咬肌和真皮（参见图5）接壤。 </li><li>使用小解剖剪，使小切口在表皮横向从那里对LG应设。 </li><li>使用镊子，拉开表皮朝耳朵暴露了LG。 </li><li>使用镊子从周围组织中轻轻松开LG。 </li><li>一旦LG从周围组织中释放出来，小心地取出LG的管道，通过抓握，其中所述管道和眼睛周围的上皮细胞连接（ 图1）。 </li><li>放置连锁群中用于RT-PCR的RNA裂解缓冲液中，或用4％PFA中20分钟进行免疫染色确定。 </li></ol>

<ol>
	<li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Current allergy and asthma reports. 14, 403 (2014).</li><li>Makarenkova, H. 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=FGF10+is+an+inducer+and+Pax6+a+competence+factor+for+lacrimal+gland+development.">FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.</a> Development. 127, 2563-2572 (2000).</li><li>Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regulation+of+lacrimal+gland+secretory+processes:+relevance+in+dry+eye+diseases.">Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.</a> Progress in retinal and eye research. 28, 155-177 (2009).</li><li>Nigam, S. 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T., 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=MT2-MMP-dependent+release+of+collagen+IV+NC1+domains+regulates+submandibular+gland+branching+morphogenesis.">MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.</a> Developmental cell. 17, 482-493 (2009).</li><li>Hoffman, M. 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=Gene+Expression+Profiles+of+Mouse+Submandibular+Gland+Development:+FGFR1+Regulates+Branching+Morphogenesis+in+Vitro+Through+BMP-+and+FGF-Dependent+Mechanisms.">Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms.</a> Development. 129, 24-5778 (2002).</li><li>Steinberg, Z., 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=FGFR2b+signaling+regulates+ex+vivo+submandibular+gland+epithelial+cell+proliferation+and+branching+morphogenesis.">FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.</a> Development. 132, 1223-1234 (2005).</li><li>Knox, S. 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作者什么都没有透露。

多功能性文化和操纵LG 体外为研究其发展显著的优势。这包括在哪个速度研究者能够验证假设和许多可被执行，以评估上皮细胞，神经细胞，内皮细胞和mesencyhmal细胞相互作用以形成器官扰动。但是，使用这种模式时，有一些需要注意的地方。首先，由于其隔离的腺不再连接到外围血管或神经系统，这可能会影响信号转导途径被测试，如果他们在协同工作与由神经或血管9,10-传递信?...

泪腺（LG）是负责水性泪液分泌为视力和健康，维护和保护眼睛表面的细胞是至关重要的。 LG的功能障碍的结果中最常见的和衰弱的眼部疾病之一:水缺乏干眼疾病，其特点是由眼部刺激，光敏感性和视力下降1。在人对LG驻留在轨道上方的眼睛的侧端，其中3 - 5排泄管道存款泪到眼表面。鼠标有三对大眼腺体，研究最多的，其中是泪腺（LG）位于前部和腹侧耳（exorbital）流着泪，通过一个单一的排泄管行驶到眼睛。类似于其他腺体器官，对LG通过上皮分支形态发生的过程，其中内稠间质单个上皮芽经历多轮芽和管道形成为开发米分泌腺泡和导管的复杂互连网络（ 图1）2。在开发过程中的上皮细胞成为血管化以及大量从颈上神经节3支配由翼腭神经节的副交感神经，并在较小程度上是由交感神经。这些细胞类型，即神经元细胞，上皮细胞，内皮细胞和间充质细胞之间的相互作用，是必不可少的功能和维持成人组织中。然而，协调LG的发育和再生以及如何小区间型通信引导这些过程的分子机制仍不清楚。 胚胎体外培养技术的出现，使发育和再生途径中的多个分支机关4鉴定。培养体外使研究人员操纵机构（我的能力械，遗传或化学的）所定义的条件下，以及表征实时器官发育和细胞 - 细胞相互作用。鼠标的exorbital LG非常适合于这种技术，最近的研究已经定义了信令规范其发展2.5系统。然而，尽管有必要了解分子线索支撑LG的发育和再生，但目前仍然是充分研究，很有可能是由于在分离的器官的技术困难。在本文中，我们将介绍如何隔离并进行胚胎小鼠LG的体外培养，以明确的发展计划。

<table><tbody><tr><td>DMEM/F12 without HEPES</td><td>SH3027101</td><td>Thermo Scientific</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>P0781</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Albumin solution from bovine serum</td><td>A9576</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Paraformaldehyde 16% Solution</td><td>15710</td><td>Electron Microscopy Sciences</td><td>Diluted to 4% in 1x PBS</td></tr><tr><td>Phosphate Buffered Saline 10x</td><td>BP665-1</td><td>Fisher Scientific</td><td></td></tr><tr><td>Phosphate Buffered Saline 1x</td><td></td><td></td><td>Prepared from 10x stock</td></tr><tr><td>holo-Transferrin bovine</td><td>T1283</td><td>Sigma-Aldrich</td><td>25&amp;nbsp;mg/ml stock solution in water. Freeze single-use aliquots at -20&amp;nbsp;&amp;deg;C. Add to DMEM/F12 media to a final concentration of 50&amp;nbsp;&amp;mu;g/ml.</td></tr><tr><td>L-ascorbic acid (Vitamin C)</td><td>A4544</td><td>Sigma-Aldrich</td><td>25&amp;nbsp;mg/ml stock solution in water. Freeze single-use aliquots at -20&amp;nbsp;&amp;deg;C. Add to DMEM/F12 media to a final concentration of 50&amp;nbsp;&amp;mu;g/ml.</td></tr><tr><td>BioLite 100 mm Tissue Culture Treated Dishes</td><td>130182</td><td>Thermo Scientific</td><td>Non-tissue culture-treated plates can also be used.</td></tr><tr><td>Stereo Microscope with substage iluminator</td><td>Stemi 2000</td><td>Carl Zeiss</td><td>Any stereo dissecting microscope can be used that has a transmitted light base.</td></tr><tr><td>Substage Illuminator Base for Stereo Microscope</td><td>TLB 4000</td><td>Diagnostics Instruments, Inc.</td><td></td></tr><tr><td>Falcon 35&amp;nbsp;mm Tissue Culture Treated Dishes</td><td>353001</td><td>Corning</td><td>Non-tissue culture-treated plates can also be used.</td></tr><tr><td>50 mm uncoated glass bottom dishes</td><td>P50G-1.5-14-F</td><td>MatTek Corporation</td><td></td></tr><tr><td>Widefield fluorescence microscope</td><td>Axio Observer Z1</td><td>Carl Zeiss</td><td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera.</td></tr><tr><td>Confocal Microscope</td><td>TCS SP5</td><td>Leica Microsystems</td><td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td></tr><tr><td>SWISS Micro-Fine Forceps, #5 (11.2&amp;nbsp;cm)</td><td>17-305X</td><td>Integra LifeSciences Corporation</td><td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td></tr><tr><td>Dumont Standard Tip Forceps, #5 (11&amp;nbsp;cm)</td><td>91150-20</td><td>Fine Science Tools (USA), Inc.</td><td>Ideal for harvesting glands from embryos.</td></tr><tr><td>Reusable Plastic Surgical Knife Handles, style no. 3.</td><td>4-30</td><td>Integra LifeSciences Corporation</td><td></td></tr><tr><td>Stainless Steel Sterile Surgical Blades, no. 10</td><td>4-310</td><td>Integra LifeSciences Corporation</td><td></td></tr><tr><td>RNAqueous-Micro Total RNA Isolation Kit</td><td>AM1931</td><td>Life Technologies</td><td></td></tr><tr><td>Cultrex 3D Culture Matrix Laminin I</td><td>3446-005-01</td><td>Trevigen</td><td>6 mg/ml stock diluted 1:1 with DMEM/F12</td></tr><tr><td>Timed-pregnant Crl:CD1(ICR) mice</td><td></td><td>Charles River Labs</td><td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td></tr><tr><td>Nucleopore Track-Etched Hydrophilic Membranes, 0.1&amp;nbsp;&amp;mu;m pore size, 13&amp;nbsp;mm</td><td>110405</td><td>Whatman</td><td></td></tr><tr><td>Timed-pregnant Pax6-Cre, GFP mice</td><td>Tg(Pax-Cre, GFP)1Pgr</td><td>The Jackson Laboratory</td><td>Optional mice for learning how to locate the LG.</td></tr></tbody></table>

manipulating the murine lacrimal gland

泪腺（LG）的分泌所必需的维持角膜的​​结构和功能，一个透明组织为视力必不可少水性眼泪。在人单LG驻留在上面的每个眼睛通过3输送泪到眼表面的侧端的轨道 - 5管道。该鼠标有三对大眼腺，研究得最多的，其中是exorbital泪腺（LG电子）位于前部和腹侧的耳朵。类似于其他腺体器官，LG的发展，通过上皮分支形态，其中内稠间质单个上皮芽经历多轮芽和管形成的，以形成分泌腺泡和导管的一个复杂的互连网络的过程。这个复杂的过程已经在文献中许多其它上皮器官如胰腺，唾液腺。不过，LG一直不太探索和形态控制的机制是不好下站了起来。我们怀疑这代表性不足作为一个模型系统是与发现，解剖和培养了LG的困难造成的。因此，在这里我们描述和解剖技术收获胚胎和产后LG为组织的体外培养方法。

LG的发展，通过上皮分支形态发生的过程。解剖在E14，E15，E16，E17，和P2 LG的胚胎的明图片说明此事件。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> 图1. LG开发通过上皮分支形态的过程。胚胎LG的新鲜切开的E14，E15，E16，E17和P2的明场图像。 <a href="https://www.jove.com/files/ftp_upload/51970/51970fig1highres.jpg...

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Manipulating the Murine Lacrimal Gland

泪腺（LG电子）是一家生产所必需的眼泪水成分对维持视力和眼睛健康的分支机关。在这里，我们描述了小鼠LG解剖和体外培养技术破译的信号参与了LG的发展道路。

操纵小鼠泪腺

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Research

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Biology

20.8K 次观看.  University of California San Francisco. 泪腺（LG电子）是一家生产所必需的眼泪水成分对维持视力和眼睛健康的分支机关。在这里，我们描述了小鼠LG解剖和体外培养技术破译的信号参与了LG的发展道路。 

视频: Manipulating the Murine Lacrimal Gland

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

科研

医学

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

生物学

东莨菪碱诱导的大鼠泪腺功能障碍

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

作者聚焦：开发干眼动物模型的挑战和未来的研究方向

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

用于泪腺干细胞的三维无血清培养系统

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.

小鼠和人泪腺类器官的建立、维持、分化、遗传操作和移植

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Superior Lacrimal Gland Removal: A Surgical Technique to Remove the Palpebral and Orbital Parts of Superior Lacrimal Gland from Rabbit Model

Source: Honkanen, R. A. et al., <a href="https://www.jove.com/v/60126">Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits.</a> J. Vis. Exp. (2020).
This video describes the surgical procedure to remove the two parts: palpebral and orbital of the superior lacrimal gland from a rabbit to create a dry eye disease model. This rabbit model is suitable for studies of ocular surface homeostasis, pathophysiology and ocular therapeutics.

泪腺上部切除术:一种从兔子模型中去除泪上腺睑部和眼眶部分的手术技术

Source: Honkanen, R. A. et al., Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits. J. Vis. Exp. (2020).
This video ...

操纵小鼠泪腺

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此视频中的章节

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

在兔子中使用完全的白眼切除术建立严重干眼模型

成年小鼠泪液和下颌腺肌上皮细胞的分离

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

用于泪腺干细胞的三维无血清培养系统

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

小鼠和人泪腺类器官的建立、维持、分化、遗传操作和移植

泪腺上部切除术:一种从兔子模型中去除泪上腺睑部和眼眶部分的手术技术