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 border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:188px;">ROSAmT/mG mice</td>
			<td style="width:183px;">The Jackson Laboratory</td>
			<td align="right" style="width:108px;">7676</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">PTH-Cre mice</td>
			<td style="width:183px;">The Jackson Laboratory</td>
			<td align="right" style="width:108px;">5989</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">iDTRmice</td>
			<td style="width:183px;">The Jackson Laboratory</td>
			<td align="right" style="width:108px;">7900</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">6-0 polyglactin 910 suture with needle</td>
			<td style="width:183px;">Ethicon, Inc</td>
			<td style="width:108px;">J510G</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">Safety Single Edge Razor Blades</td>
			<td style="width:183px;">American Safety Razor Company</td>
			<td style="width:108px;">66-0089</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">Disposable Scalpel</td>
			<td style="width:183px;">Feather Safety Razor Co., LTD</td>
			<td style="width:108px;">72042-11</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">Povidone-Iodine Prep Pads</td>
			<td style="width:183px;">Dynarex Corporation</td>
			<td align="right" style="width:108px;">1108</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">Ply gauze</td>
			<td style="width:183px;">Busse. Inc</td>
			<td style="width:108px;">BHD707</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">0.9% Sodium Chloride Solution</td>
			<td style="width:183px;">HOSPIRA Worldwide, Inc</td>
			<td style="width:108px;">07983-09</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">1mL 29G Insulin Syringe</td>
			<td style="width:183px;">BECTON DICKINSON</td>
			<td align="right" style="width:108px;">329622</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">Surgical Incise Drapes</td>
			<td style="width:183px;">3M</td>
			<td style="width:108px;">6640EZ</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">Dumstar Biology forceps</td>
			<td style="width:183px;">Roboz Surgical Instrument Co., Inc</td>
			<td style="width:108px;">RS-4984</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">Micro Dissecting Spring Scissors</td>
			<td style="width:183px;">Roboz Surgical Instrument Co., Inc</td>
			<td style="width:108px;">RS-5605</td>
			<td style="width:197px;"></td>
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			<td height="22" style="height:22px;width:188px;">Needle Holder</td>
			<td style="width:183px;">MILTEX.,Inc</td>
			<td style="width:108px;">V98-42</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:188px;">2,2,2-Tribromethanol</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:108px;">T48402</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:188px;">2-Methyl-2-Butanol</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td align="right" style="width:108px;">152463</td>
			<td style="width:197px;">For dissolve the 2,2,2-Tribromethanol</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:188px;">Diphtheria Toxin Powder</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:108px;">D0564</td>
			<td style="width:197px;">Dissolve in 0.9% sodium chloride solution at 1mg/mL as the stock solution in -80C</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:188px;">Multicap Blood Collection Capillary tubes</td>
			<td style="width:183px;">Siemens Healthcare Diagnostics Ltd</td>
			<td align="right" style="width:108px;">855578</td>
			<td style="width:197px;">For collecting blood in iCa2+ analysis using RapidLab 348 Ca2+/pH analyzer</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:188px;">RapidLab 348 Ca2+/pH analyzer</td>
			<td style="width:183px;">Siemens Healthcare</td>
			<td style="width:108px;"></td>
			<td style="width:197px;">For iCa2+ 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="http://ecsource.jove.com/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="http://ecsource.jove.com/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 de Cartagena UDEC

Research

JoVE Journal

Medicine

14.4K 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

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

诱导肌内膜增生对战动脉粥样硬化小鼠:两种有效的模型介绍

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

科研

医学

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Robotic Ablation of Atrial Fibrillation

Background: Pulmonary vein isolation (PVI) is an established treatment for atrial fibrillation (AF). During PVI an electrical conduction block between pulmonary vein (PV) and left atrium (LA) is created. This conduction block prevents AF, which is triggered by irregular electric activity originating from the PV. However, transmural atrial lesions are required which can be challenging. Re-conduction and AF recurrence occur in 20 - 40% of the cases. Robotic catheter systems aim to improve catheter steerability. Here, a procedure with a new remote catheter system (RCS), is presented. Objective of this article is to show feasibility of robotic AF ablation with a novel system. Materials and Methods: After interatrial trans-septal puncture is performed using a long sheath and needle under fluoroscopic guidance. The needle is removed and a guide wire is placed in the left superior PV. Then an ablation catheter is positioned in the LA, using the sheath and wire as guide to the LA. LA angiography is performed over the sheath. A circular mapping catheter is positioned via the long sheath into the LA and a three-dimensional (3-D) anatomical reconstruction of the LA is performed. The handle of the ablation catheter is positioned in the robotic arm of the Amigo system and the ablation procedure begins. During the ablation procedure, the operator manipulates the ablation catheter via the robotic arm with the use of a remote control. The ablation is performed by creating point-by-point lesions around the left and right PV ostia. Contact force is measured at the catheter tip to provide feedback of catheter-tissue contact. Conduction block is confirmed by recording the PV potentials on the circular mapping catheter and by pacing maneuvers. The operator stays out of the radiationfield during ablation. Conclusion: The novel catheter system allows ablation with high stability on low operator fluoroscopy exposure.

Pulmonary vein isolation (PVI) with an ablation catheter is a curative treatment for atrial fibrillation (AF). Robotic catheter systems aim to improve catheter steerability. Here, a procedure with a new robotic catheter system is presented. The goal of the procedure is electrical block between pulmonary vein and left atrium.

房颤消融机器人

Background: Pulmonary vein isolation (PVI) is an established treatment for atrial fibrillation (AF). During PVI an electrical conduction block between ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) &#62;40 kg/m2), leading to long-term, sustained weight loss and improvements in metabolic complications. However, the underlying cellular and molecular mechanisms sustaining the beneficial effects of bariatric surgery are not yet fully understood. Due to the numerous genetically-modified strains available, the mouse model is the most convenient animal model to explore the molecular mechanisms behind the pleiotropic beneficial effects of bariatric surgeries. Here, we detailed the optimized healthcare methods and surgical protocols in mice for the two most widely-used bariatric surgeries: the sleeve gastrectomy and the modified Roux-en-Y gastric bypass. Deciphering the molecular mechanisms underlying the therapeutic effects of bariatric surgeries offers the promise of identifying new therapeutics targets.

Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and dyslipidemia.

袖胃切除术的技术和改良的小鼠空肠Roux-en-Y胃旁路

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Ablation of Ischemic Ventricular Tachycardia Using a Multipolar Catheter and 3-dimensional Mapping System for High-density Electro-anatomical Reconstruction

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars represent zones of slow conduction that allow the occurrence and maintenance of reentrant circuits. Catheter ablation enables substrate modification of those low voltage areas and thus can help to alter the scar tissue in such a way that arrhythmias cannot appear anymore. Hospitalizations of concerned patients decrease, quality of life and outcome rise. Consequently, VT ablation represents a growing field in electrophysiology, especially for patients with endocardial scars in ischemic heart disease after myocardial infarction. However, ablation of ventricular tachycardia remains one of the most challenging procedures in the electrophysiology lab. Precise scar definition and localization of abnormal potentials are critical for ablation success. The following manuscript describes the use of a multipolar mapping catheter and 3-dimensional (3D) mapping system to create a high density electro-anatomical map of the left ventricle including a precise scar representation as well as mapping of fractionated and late potentials in order to allow a highly accurate substrate modification.

With the following protocol, we provide an approach to Ventricular Tachycardia (VT) ablation using high density mapping with a multipolar catheter and 3D mapping system enhancing the success of the procedure.

采用多极导管三维测图系统进行缺血性室性心动过速的高密度电解剖重建

Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

在糖尿病小鼠中创建慢性伤口的协议

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple angiographic method for examining the fine vascular structure of unfixed, fresh tissue using a silicone rubber injection compound and soft tissue X-ray system. This study is especially focused on flap territories used in reconstructive surgery. This study employs angiography with a silicone rubber injection compound in various experimental conditions using Sprague-Dawley rats. First, 15 mL of MV compound and 15 mL of diluent is mixed. Then, 1.5 mL of the curing agent is prepared, and a 24G catheter is cannulated in the common carotid artery of the rat. A three-way stopcock is then connected to a catheter, and the radiopaque agent, after being mixed with the prepared curing agent, is injected immediately without spillage. Finally, as the agent solidifies, the specimen is harvested, and an angiographic image is obtained using a soft tissue X-ray system. This method indicates that high-quality angiography showing fine vascular structures can be easily and simply obtained within in a short period of time.

This study presents a simple two-dimensional angiographic method to examine fine vascular structures using a silicone rubber injection compound and soft tissue X-ray system.

二维 x 射线血管造影应用硅橡胶注射液检测细血管结构

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

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

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