这项工作是由美国国立卫生研究院支持的授予口腔疾病公开赛资金SKLOD2015OF01（RB）的R01-DK100584和中国国家重点实验室。我们感谢文平赵，佐藤忠利和凯利劳特寻求帮助。

这项研究已获得马萨诸塞州总医院的机构动物护理和使用委员会（IACUC）。获得开始前这个动物研究适当的机构批准。一些技术可能必须根据当地IACUC要求调整。 1. GFP-PTX小鼠<ol><li>获取PTHcre +小鼠和罗莎- mTmG小鼠8 - 10周龄进一步交配。 </li><li>回交的PTHcre +小鼠（混合129; FVB背景）与C57BL / 6小鼠为6代以获得具有C57BL / 6背景的小鼠。 </li><li>近交PTHcre +小鼠获得PTHcre + / +小鼠17。 注:要执行的基因分型，从尾尖提取的DNA，并用它进行PCR。 PCR引物检查合度和PTHcre转基因纯合如下:正向引物A，CCTGTCAAGGATGTGGAAGA，反向引物A&#39;，TCAGATCACACCACACAGCA，女orward引物B，CAGTTGTCTTTAGTTTACTCAGCATCAG，反向引物B&#39;，GATAATCGCGAACATCTTCAGGTT 17。 </li><li>十字PTHcre + / +罗莎- mTmG小鼠18只16。断奶PTHcre; mTmG幼崽当4周龄。 </li><li>在0.6毫克/克体重由三溴腹腔注射（阿佛丁）mTmG小鼠，或其它试剂如氯胺酮/甲苯噻嗪; 10周龄PTHcre - 麻醉8。根据批准的协议使用丁丙诺啡0.1毫克/千克SC每12小时作为镇痛48小时手术后。确保在脚趾捏撤回反射或其他方式的麻醉深度适当。将动物在仰卧位。 </li><li>扩展和使用单刃刀片剃须对皮肤准备腹侧颈部区域。消毒用碘伏消炎片剃光皮肤。 </li><li>覆盖用无菌手术单的动物，以减少外科手术部位的污染，并使用高压灭菌麦克风rosurgical仪器为外科手术。 </li><li>切开皮肤中的2厘米的纵向切口用外科手术刀。解剖筋膜钝性分离与弧形的锯齿钳推唾液腺到了一边。 </li><li>在使用卤素灯和4解剖显微镜 - 放大5倍，切割和分离使用锋利的钳提示气管旁肌肉，暴露气管。 </li><li>确定与旁边的气管左，右叶甲状腺。 </li><li>切换光源的荧光灯和可视化的两个绿色-荧光甲状旁腺。 注意:通常情况下，这两个甲状旁腺位于或甲状腺的表面附近，但偶尔，它们位于更远。 </li><li>小心取出手术使用镊子和剪刀绿色甲状旁腺。用无菌纱布止血，并沿气管仔细检查，确保所有绿色组织已被删除。 </LI&gt; <li>通过使用6-0聚乳糖910缝线间断缝合关闭气管旁肌肉。通过使用6-0聚乳糖910缝线缝合霍尔斯特德关闭皮肤切口。 </li><li>可选:注入10微升/ g体重的无菌正常0.9％氯化钠溶液的小鼠补液。 </li><li>放置术后动物的单独笼子上为体温恢复温暖培养箱中（37℃）。一旦老鼠是清醒的，并在卧位，把颗粒饲料和水用在笼底一些明胶的食品。 </li><li>经过2小时的手术后的观察期，返回鼠标的动物设施，并按照术后护理当地的要求。 </li><li>甲状旁腺切除后第3天，得到10μL的尾血，并测量血液使用分析器电离的Ca ++诸如血液气体系统的Ca ++ / PH值分析仪。血甲状旁腺切除成功的结果电离钙++等于或大于假手术CON的-2SD低控制小鼠（1.20毫摩尔/升在我们的N = 30的实验）。 </li></ol> 2. PTH-CRE-IDTR小鼠<ol><li>获得8 PTHcre +和DTR FL / FL小鼠- 10周龄进行交配。 </li><li>回交的PTHcre小鼠（混合129; FVB背景）与C57BL / 6小鼠为6代以获得具有C57BL / 6背景的小鼠。 </li><li>近亲繁殖的PTHcre +（C57BL / 6背景）小鼠获得PTHcre + / +小鼠17。 注:要执行基因分型，提取及用于PCR小鼠尾巴尖端的DNA。基因分型引物:正向引物A，CCTGTCAAGGATGTGGAAGA，反向引物A&#39;，TCAGATCACACCACACAGCA，正向引物B，CAGTTGTCTTTAGTTTACTCAGCATCAG，反向引物B&#39;，GATAATCGCGAACATCTTCAGGTT 17。 </li><li>交配PTHcre + / +小鼠16与DTR FL / FL小鼠19获得PTHcre-IDTR的小鼠。 </li><li>通过稀释diphtheri准备DT解决方案毒素粉末用无菌生理盐水至0.5微克/毫升的浓度。无菌过滤，并储存等分在-80℃。 </li><li>选择8 - 注射实验10周龄PTHcre-IDTR小鼠。解冻DT等分至室温，辖DT腹膜内以5微克/千克（10μL/ g）的动物的体重。对于最高效率和最低毒性，DT管理重复在为期3天的间隔17，共2次注射。 </li><li>第二次注射后3天，通过各个PTHcre-IDTR / PTHcre-IDTR-mTmG鼠标10μL尾血，测量血液中的Ca ++使用血液气体系统的Ca ++ / pH分析仪。 注意:我们定义与小鼠血液钙离子++低于1.18毫摩尔/升作为hypoparathyroid小鼠，这相当于车辆注射的对照小鼠（n = 22）的平均低于2SD。 </li></ol>

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The authors declare that they have no competing financial interests.

我们使用转基因小鼠与甲状旁腺GFP表达选择性证明GFP引导下甲状旁腺切除的技术。在PTHcre; mTmG小鼠，双荧光（以Cre重组酶表达的细胞和红番茄非酶Cre绿色GFP表达的细胞），使我们能够清楚地识别，并精确地去除所有甲状旁腺无需拆卸甲状腺。而我们优选使用双荧光mTmG小鼠用于识别荧光红色非甲状旁腺组织的能力，我们的程序也应该工作以及使用单个荧光动物，如番茄红（B6.Cg-CT（ROSA）26Sortm14（ CAG-tdTomato）HZE / J）鼠标。程序比较容易进行，需要每鼠约20分钟，并且在严重和持续hypoparathyroid表型结果。 此外，GFP阳性甲状旁腺的另一优点是易于检测异常甲状旁腺的。我们的研究表明在大约B6小鼠（ 图1），这是由绿色荧光在我们的小鼠模型容易地检测的四分之一甲状旁腺的错误定位。相反甲状腺-甲状旁腺，我们的技术避免了与随后需要甲状腺激素替代和去除降钙素产甲状腺C细胞的未知作用除去甲状腺。荧光标记的甲状旁腺可也用于需要甲状旁腺的隔离其他调查。这种模式的限制包括对手术的要求。基本显微技术和用荧光光源解剖显微镜是必需的。 论证产生hypoparathyroid小鼠，消除对手术的需要的第二种技术。白喉毒素治疗PTHcre-IDTR小鼠表现出温和的甲状旁腺功能低下型，而仅仅需要腹腔注射DT到鼠标。关键部分FO- [R产生这种模式剂量的优化给药方案。高剂量的DT导致毒性，但低剂量降低了该方法的功效。我们确定，在5微克/公斤，给予2次注射剂量团相隔3天，导致无/死亡率最低可靠和稳定的低钙血症。 最终，我们希望这两种方法提供了获得有用的甲状旁腺功能低下症小鼠模型。使用一种新的长效PTH，我们报道在确定甲状旁腺功能低下症17的新药物的疗效在第一次使用这两种模式的。

自1880年1在人类和其他几个物种由桑德斯特伦甲状旁腺的第一次系统介绍，了解这个小内分泌器官对人类生理和疾病的重要性不断提高。甲状旁腺分泌甲状旁腺激素（PTH），钙代谢的主要调节剂。 PTH也是磷酸盐稳态和骨代谢1,2的重要激素。颈部手术期间甲状旁腺的意外脱落或损坏是甲状旁腺功能减退症的最常见的原因，这种疾病特征在于，低血钙，低PTH，和升高的磷1。 为了更好地研究收购甲状旁腺功能减退和试验新疗法，可靠，方便小鼠模型是必需的。小鼠被广泛用于研究，因为各种各样的遗传工具是一vailable该物种允许复杂的机理研究在体内 。然而，虽然外科甲状旁腺切除（PTX）可以在大鼠2，3，4，5，而较大的哺乳动物6，7，8使用，它在技术上是在因为腺的小尺寸和其可变解剖分布的小鼠非常具有挑战性的9。因此，甲状腺-甲状旁腺切除（TPTX）在小鼠中，其中甲状腺和甲状旁腺除去一起10通常执行的。然而，低甲状腺激素水平在实验中一个潜在的混杂因素，这种模式复杂化。此外，甲状腺内C-细胞，其产生降钙素，激素钙体内平衡的重要的啮齿动物，也失去了在除去日。yroids 11。 甲状旁腺功能低下症的几种遗传小鼠模型存在，其中包括PTH空鼠标12，GCM2空鼠标器13以及NUF鼠标与钙敏感受体（的CaSR）14，15的活化突变。然而，这些遗传缺陷已经胚胎发育过程中存在，和甲状旁腺的功能，因此在胚胎发生期间已经受损。这可能会影响器官的发育，如骨架。这与患者的术后甲状旁腺功能减退症谁掌握疾病以后的生活中。此外，一些这些小鼠模型显示出早期杀伤力和生育能力下降，从而其使用12，13，14进一步复杂化。 我们开发了两个新的鼠标Models用于收购甲状旁腺功能减退。使用遗传工程小鼠表达GFP特意在甲状旁腺允许甲状旁腺可以很容易地确定用于手术切除不去除甲状腺。被穿越PTH-Cre的鼠标，其中5.5 kb的PTH子16与ROSA mTmG鼠标控制下表达Cre重组酶生成此鼠标。由此产生的PTHcre; mTmG小鼠甲状旁腺细胞中特异性表达绿色荧光蛋白。第二小鼠模型使用相同的PTH-Cre的，此时从诱导DTR鼠标在甲状旁腺除去一个STOP盒，导致白喉毒素受体的表达特异性。 DT的全身给药破坏甲状旁腺细胞，使动物hypoparathyroid无需手术。 在这项研究中提出了两种小鼠模型表明在三个月的观察围稳定的低血钙型OD。这些程序很容易执行，表型是可重复的，而hypoparathyroid小鼠表现出成活率高17。

<table><tbody><tr><td>ROSA&lt;sup&gt;mT/mG&lt;/sup&gt; mice</td><td>The Jackson Laboratory</td><td>7676</td><td></td></tr><tr><td>PTH-Cre mice</td><td>The Jackson Laboratory</td><td>5989</td><td></td></tr><tr><td>iDTRmice</td><td>The Jackson Laboratory</td><td>7900</td><td></td></tr><tr><td>6-0 polyglactin 910 suture with needle</td><td>Ethicon, Inc</td><td>J510G</td><td></td></tr><tr><td>Safety Single Edge Razor Blades</td><td>American Safety Razor Company</td><td>66-0089</td><td></td></tr><tr><td>Disposable Scalpel</td><td>Feather Safety Razor Co., LTD</td><td>72042-11</td><td></td></tr><tr><td>Povidone-Iodine Prep Pads</td><td>Dynarex Corporation</td><td>1108</td><td></td></tr><tr><td>Ply gauze</td><td>Busse. Inc</td><td>BHD707</td><td></td></tr><tr><td>0.9% Sodium Chloride Solution</td><td>HOSPIRA Worldwide, Inc</td><td>07983-09</td><td></td></tr><tr><td>1 mL 29 G Insulin Syringe</td><td>BECTON DICKINSON</td><td>329622</td><td></td></tr><tr><td>Surgical Incise Drapes</td><td>3M</td><td>6640EZ</td><td></td></tr><tr><td>Dumstar Biology forceps</td><td>Roboz Surgical Instrument Co., Inc</td><td>RS-4984</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz Surgical Instrument Co., Inc</td><td>RS-5605</td><td></td></tr><tr><td>Needle Holder</td><td>MILTEX.,Inc</td><td>V98-42</td><td></td></tr><tr><td>2,2,2-Tribromethanol</td><td>Sigma-Aldrich</td><td>T48402</td><td></td></tr><tr><td>2-Methyl-2-Butanol</td><td>Sigma-Aldrich</td><td>152463</td><td>For dissolve the 2,2,2-Tribromethanol</td></tr><tr><td>Diphtheria Toxin Powder</td><td>Sigma-Aldrich</td><td>D0564</td><td>Dissolve in 0.9% sodium chloride solution at 1 mg/mL as the stock solution in -80 &amp;#176;C</td></tr><tr><td>Multicap Blood Collection Capillary tubes</td><td>Siemens Healthcare Diagnostics Ltd</td><td>855578</td><td>For collecting blood in iCa&lt;sup&gt;2+&lt;/sup&gt; analysis using RapidLab 348 Ca&lt;sup&gt;2+&lt;/sup&gt;/pH analyzer</td></tr><tr><td>RapidLab 348 Ca&lt;sup&gt;2+&lt;/sup&gt;/pH analyzer</td><td>Siemens Healthcare</td><td></td><td>For iCa&lt;sup&gt;2+&lt;/sup&gt; analysis</td></tr></tbody></table>

two techniques to create hypoparathyroid mice: parathyroidectomy using gfp glands and diphtheria-toxin-mediated parathyroid ablation

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. 
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

甲状旁腺的位置首先，我们记录54 PTHcre-mTmG小鼠的甲状旁腺的分布在荧光解剖显微镜下观察。 74％（五十四分之四十○）小鼠的两个绿色甲状旁腺（ 图1A，C，E），26％（14/54）的小鼠具有一个附加的第三甲状旁腺（ 图1B，D，F）。没有观察到小鼠与单个腺或三个以上的腺体。一般，甲状旁腺分别位于甲状腺（58％，122分之71的上边界附近; 图1A（1,2），B（1,2），C（2），D（1,2） 中 ，E （2），F（3））。 27％（122分之33）腺体位于甲状腺的下边界附近（ 图1C（1），D（3），F（1）），和13％（122分之16）位于从第t远hyroid腺（ 图1B（3）中 ，E（1），F（2））。我们的研究结果是一致和延伸变甲状旁腺20的位置的先前的发现。 GFP-PTX小鼠从麻醉整个手术闭合皮肤切口把每只小鼠大约20分钟。超过3个月的观察期手术后的小鼠的存活率为96.3％（53/55）。 92.4％（49/53）的GFP-PTX小鼠表现出了分别为假手术对照组小鼠或更低的平均2 SD下面离子钙水平。在GFP-PTX小鼠（低钙血症，低PTH和血清磷酸盐升高）的hypoparathyroid表型是对3个月的整个观察时间是稳定的。重要的是，甲状腺功能不是来自假手术的动物不同后3个月手术（TSH = 42±24对30±15 MU / L，P = 0.171; T 4 = 30.0±0.6与3.1±0.9微克/分升，p值= 0.707（ 图2）。 PTH-CRE-IDTR小鼠 DT注射PTH-Cre的小鼠IDTR开发甲状旁腺功能减退（低血离子钙，血磷升高，以及不适当的低正常PTH水平）。我们以前曾报道，少数的PTH阳性细胞逃避白喉毒素的消融，解释了可测量的循环PTH和因此这些小鼠17的稍温和的表型。 甲状旁腺功能低下症的GFP-PTX老鼠和PTH-CRE-IDTR小鼠在GFP-PTX的小鼠中观察到手术后第3天的血液的Ca显著减少++和血清PTH水平相比，在假手术的小鼠中发现（钙+ = 1.05±0.40 对水平</em&gt; 1.30±0.03毫摩尔/ L，P &lt;0.05; PTH = 32±22对580±137皮克/毫升，P &lt;0.05）。该甲状旁腺功能减退症的表型是稳定超过3个月的观察期内（ 图3A，从许可（参考第17再版））。后3天的间隔，剂量和给药方案进行优化以使用DT的量最少，得到最大hypoparathyroid表型2注射5微克/千克的DT，对DT注入相比PTH-CRE-IDTR小鼠显示显著低钙血症，减少的PTH车辆控制小鼠，这持续了九十天观察期（CA ++ = 1.10±0.07毫摩尔/ L 比 1.26±0.05毫摩尔/ L，p &lt;0.05; PTH = 218±156皮克/毫升与 572±164皮克/毫升，p &lt;0.05）（ 图3B，许可转载自（参考＃17））。 <img alt="图1" src="/files/ftp_upload/55010/55010fig1.jpg" /> 图1: <st荣&gt; 54 PTHcre-mTmG小鼠荧光灯下解剖镜下甲状旁腺不同地点的代表图像。小鼠有以下两种（A，C，E）或三个（B，D，F）绿色甲状旁腺。最甲状旁腺分别位于上缘附近（A（1,2），B（1,2），C（2），D（1,2），E（2），F（3）或邻近的下缘（C（1），D（3），F（1）），在极少数情况下，有的被位于异位甲状腺（B（3），E（1），F（2））。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55010/55010fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55010/55010fig2.jpg" /> 图2:TSH 和GFP-PTX小鼠T4水平。 GFP-PTX 3个月后，对TSH和T4 measurem得到血清<html>经济需求测试。 GFP-PTX的小鼠表现出TSH浓度（42±24亩/ L）（A）和T4浓度（3.0±0.6微克/分升），（B）这不是从控制小鼠（TSH = 30±15亩/ L，P不同= 0.171，N = 11，T 4 = 3.1±0.9，p值= 0.707，N = 11）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55010/55010fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55010/55010fig3.jpg" /> 图3: 血Ca ++和PTH水平的GFP-PTX老鼠和DT注射PTH-CRE-IDTR小鼠。两者的GFP-PTX小鼠（A）和DT注射PTH-CRE-IDTR小鼠（B）表现出稳定的低血钙症和超过3个月的观察期降低PTH水平（N = 6 - 8，*** P &lt;0.001）（重新印刷从参考＃17）权限。ES / ftp_upload / 55010 / 55010fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation at JoVE.com

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

两种技术来创建Hypoparathyroid小鼠:甲状旁腺切除术采用GFP腺和白喉毒素介导甲状旁腺消融

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most ...

two-techniques-to-create-hypoparathyroid-mice-parathyroidectomy-using

Universidad San Sebastian

Research

JoVE Journal

Medicine

14.5K 次观看.  Harvard Medical School. Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

视频: Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

发育生物学

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

生物学

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

小鼠自发性自身免疫性甲状腺炎模型的产生

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

免疫与感染

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

建立简单有效的术中甲状旁腺成像大鼠模型

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

医学

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid hormone (PTH), which leads to high serum phosphorus levels and low serum calcium levels. HypoPT most commonly results from accidental damage to the glands or their removal during thyroid or other anterior neck surgery. Parathyroid/thyroid surgery has become more common in recent years, with a corresponding rise in the occurrence of HypoPT as a postoperative complication. There is a critical need for a HypoPT animal model to better understand the mechanisms underlying the effects of HypoPT on mineral ion homeostasis and to verify the therapeutic effectiveness of novel treatments. Here, a technique is reported to create acquired HypoPT in male rats by performing parathyroidectomy (PTX) using carbon nanoparticles. The rat model shows great promise over the mouse models of hypoparathyroidism.&#160;Importantly, the human PTH receptor binding region has an 84.2% sequence similarity with that of the rat, which is higher than the 73.7% similarity shared with mice. Moreover, the effects of estrogen, which can affect the PTH/PTHrP receptor signaling pathway, have not been fully investigated in male rats. Carbon nanoparticles are lymphatic tracers that stain the thyroid lymph nodes black without affecting their function, but they do not stain the parathyroid glands, which makes them easy to identify and remove. In this study, serum PTH levels were undetectable after PTX, and this resulted in significant hypocalcemia and hyperphosphatemia. Thus, the clinical state of postoperative HypoPT can be remarkably represented in the rat model. Carbon-nanoparticle-assisted PTX can, therefore, serve as an extraordinarily effective and readily implementable model for studying the pathogenesis, treatment, and prognosis of HypoPT.

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

An animal model of acquired hypoparathyroidism (HypoPT) is crucial to understanding how HypoPT affects mineral ion homeostasis and to verify the effectiveness of novel treatments. Here, a technique is presented to generate an acquired hypoparathyroidism (AHypoPT) rat model by parathyroidectomy (PTX) using carbon nanoparticles.

通过碳纳米粒子辅助甲状旁腺切除术 产生 甲状旁腺功能减退大鼠

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

Source: Julia Ramirez-Moya, et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

甲状腺癌细胞原位接种小鼠甲状腺:建立原位甲状腺癌小鼠模型的程序

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

原位同种移植物肿瘤移植:一种生成胰腺肿瘤小鼠模型的技术

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...

两种技术来创建Hypoparathyroid小鼠:甲状旁腺切除术采用GFP腺和白喉毒素介导甲状旁腺消融

摘要

探索更多视频

此视频中的章节

胰腺癌的荧光原位小鼠模型

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

小鼠自发性自身免疫性甲状腺炎模型的产生

建立简单有效的术中甲状旁腺成像大鼠模型

通过碳纳米粒子辅助甲状旁腺切除术产生甲状旁腺功能减退大鼠

隔离和成像现场，通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

甲状腺癌细胞原位接种小鼠甲状腺:建立原位甲状腺癌小鼠模型的程序

原位同种移植物肿瘤移植:一种生成胰腺肿瘤小鼠模型的技术

两种技术来创建Hypoparathyroid小鼠:甲状旁腺切除术采用GFP腺和白喉毒素介导甲状旁腺消融

摘要

探索更多视频

此视频中的章节

胰腺癌的荧光原位小鼠模型

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

小鼠自发性自身免疫性甲状腺炎模型的产生

建立简单有效的术中甲状旁腺成像大鼠模型

通过碳纳米粒子辅助甲状旁腺切除术 产生 甲状旁腺功能减退大鼠

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

甲状腺癌细胞原位接种小鼠甲状腺:建立原位甲状腺癌小鼠模型的程序

原位同种移植物肿瘤移植:一种生成胰腺肿瘤小鼠模型的技术

通过碳纳米粒子辅助甲状旁腺切除术产生甲状旁腺功能减退大鼠

隔离和成像现场，通过颤音分割老鼠肾佩尔维斯的精致起搏器区域