我们要感谢山东省立医院动物实验室的技术支持。这项工作得到了国家自然科学基金（NSFC 82101645）、山东省自然科学基金（ZR2020QH088）和山东省高校青年创新科技支持计划（2021KJ051）的支持。

以下方案符合有关使用研究动物的所有机构伦理准则，并已获得中国山东省立医院动物伦理委员会的批准。所有手术操作均在深度麻醉下进行，动物在手术过程中的任何阶段都没有感到疼痛。
1. 术前准备
<ol><li>器械灭菌<ol><li>手术前在高压灭菌器（121°C，15分钟）中对手术器械进行蒸汽灭菌。准备足够的一次性缝合线和针头。</li>
	</ol></li>
	<li>手术平台设置<ol><li>在专门用于外科手术的房间进行手术。为手术分配至少 60 cm x 60 cm 的工作台区域。用75%酒精清洁该区域表面，并用一次性医用毛巾盖住，然后提前30分钟用紫外线辐射消毒（图1A）。</li>
	</ol></li>
	<li>动物准备<ol><li>将所有动物饲养在温度受控的房间（20-25°C）中，光照周期为12小时，黑暗循环为12小时。手术前将 8 周龄的雌性 C57BL/6 小鼠适应饲养设施 1 周。</li>
		<li>手术前称量小鼠。通过腹膜内注射三溴乙醇（280mg / kg）给予所有9周龄的雌性小鼠全身麻醉，以在手术过程中的任何阶段实现无痛。手术前约1小时皮下注射美洛昔康（2mg / kg）以缓解疼痛。</li>
		<li>在手术过程中涂抹眼药膏以防止角膜干燥。</li>
		<li>使用干净的棉签在背部涂抹脱毛化妆水。让化妆水在鼠标上静置3-5分钟，然后用纱布和棉签去除毛发。重复此步骤，直到从鼠标背面去除所有毛发。</li>
		<li>使用纱布和棉签用 75% 的酒精清洁皮肤。使用橡胶条或棉绳将鼠标固定在手术平台上（图1B），并使用碘伏溶液清洁背部。 注意:在卵巢切除术前通过脚趾捏住确认麻醉深度。</li>
	</ol></li>
</ol>2. 卵巢切除术
注意:三溴乙醇可以维持约 30 分钟，确保手术尽可能完成。
<ol><li>使用一次性手术刀从大腿根部向上纵向切开~1.0厘米的背侧切口，确保只切开皮肤和皮下筋膜，避免此时切入后腹膜。</li>
	<li>将切口向左拉，可以看到一个白色的脂肪垫。使用微型镊子和剪刀沿白色脂肪垫切割~0.5厘米，以暴露腹膜腔。</li>
	<li>切开腹膜后部后，用微镊子缓慢而轻柔地从腹膜腔中取出白色脂肪垫。立即用0.9%无菌盐水润湿浸泡的纱布外的白色脂肪组织。在腹腔外时，保持暴露的组织始终湿润。</li>
	<li>粉红色颗粒状物质，即卵巢，附着在白色脂肪垫的下部（图2A）。卵巢连接到细长的导管，即子宫。使用5-0可吸收缝合线结扎子宫的卵巢端并切除左卵巢（图2B）。</li>
	<li>切除卵巢时，尽可能保留周围的脂肪组织。避免手术器械与卵巢直接接触，防止卵巢组织腹腔内着床。</li>
	<li>小心地将白色脂肪垫放回腹膜腔内。用5-0可吸收缝合线在后腹膜上进行简单的间歇缝合（图2C）。缝合完成后，用 0.9% 无菌盐水浸泡的纱布清洁出血处。</li>
	<li>将皮肤切口向右拉，并使用相同的方法切除右侧卵巢。</li>
	<li>用4-0不可吸收缝合线进行间歇缝合，并用0.9%无菌盐水浸泡的纱布清洁任何出血（图2D）。</li>
	<li>完成两次缝合后，用碘伏溶液清洁伤口。腹腔注射广谱抗生素。</li>
</ol>3.术后观察
<ol><li>手术后将小鼠移至37°C恒温毯中。在老鼠可以自由移动之前，将动物放在各自的笼子里。不要让动物无人看管，直到它恢复足够的意识以保持胸骨卧位。</li>
	<li>术后24小时皮下注射美洛昔康（2mg / kg）以缓解疼痛。</li>
	<li>每天监测小鼠，以确保手术伤口正常愈合，没有任何并发症（裂开）的迹象。</li>
</ol>4.补充雌二醇
<ol><li>准备补充戊酸雌二醇的饲料。每 1 公斤饲料补充 2.6 毫克 β-雌二醇 17-戊酸酯。</li>
	<li>手术完成后3天，用戊酸雌二醇喂养小鼠。</li>
</ol>5. FSH注射
<ol><li>制备重组人FSH溶液。用0.9%无菌盐水溶解注射用重组人FSH粉末至100IU / mL。</li>
	<li>根据实验计划对小鼠进行分组，并通过腹腔注射给予溶剂或不同剂量的重组FSH2周。根据重组FSH的生物活性，在小鼠中使用FSH的注射剂量，相当于女性在绝经过渡期的血清FSH水平。 注:根据不同的处理，卵巢切除的补充雌激素的小鼠随机分为三组，溶剂（NS）组接受100μL /天溶剂，低剂量FSH（L-FSH）组每天接受15 IU / kg体重和高剂量FSH（H-FSH）组每天接受30 IU / kg体重。</li>
</ol>

开发 FSH 水平升高的卵巢切除小鼠模型

<ol>
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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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Vascular+Anomalies.">Expression of Follicle-Stimulating Hormone Receptor in Vascular Anomalies.</a> Plastic and Reconstructive Surgery. 133 (3), 344e-351en (2014).</li><li>Liu, 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=Blocking+FSH+induces+thermogenic+adipose+tissue+and+reduces+body+fat.">Blocking FSH induces thermogenic adipose tissue and reduces body fat.</a> Nature. 546 (7656), 107-112 (2017).</li><li>Ji, Y., 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=Epitope-specific+monoclonal+antibodies+to+FSH&#946;+increase+bone+mass.">Epitope-specific monoclonal antibodies to FSH&#946; increase bone mass.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2192-2197 (2018).</li><li>El Khoudary, S. R., 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=Trajectories+of+estradiol+and+follicle-stimulating+hormone+over+the+menopause+transition+and+early+markers+of+atherosclerosis+after+menopause.">Trajectories of estradiol and follicle-stimulating hormone over the menopause transition and early markers of atherosclerosis after menopause.</a> European Journal of Preventive Cardiology. 23 (7), 694-703 (2016).</li><li>Radu, 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=Expression+of+Follicle-Stimulating+Hormone+Receptor+in+Tumor+Blood+Vessels.">Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels.</a> The New England Journal of Medicine. 363 (17), 1621-1630 (2010).</li><li>Sowers, M. 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作者没有什么可透露的。

在从生殖阶段到非生殖阶段（更年期）的过渡期间，许多女性经历了显着的生理和病理变化。FSH水平在绝经过渡期间升高1.新兴研究表明，各种性腺外组织和器官中的FSH在多种疾病的发病机制中至关重要，包括血脂异常4，阿尔茨海默病5，骨质疏松症9,10，动脉粥样硬化11，肥胖9和癌症12。因此，建立一个可以帮助研究FSH在体内独立作用的动物模型尤为重要。OVF小鼠模型模拟了更年期过渡的早期阶段，雌激素相对稳定，FSH水平升高，特别适合探索FSH性腺外作用的研究。
在这种方法中，卵巢切除术使用单个背侧切口进行，从大腿底部向上约1厘米（图1B）。使用锋利的解剖剪刀将皮肤几乎与背肌一起切割，从而进入腹膜腔。手术后，肌肉切口不需要缝合，皮肤伤口用一根猫肠缝合线双侧闭合（图2）。与其他方法相比，该操作在技术上更容易，耗时更少，对雌性小鼠的危害更小。
在手术过程中应注意的一些细节。首先，所有外科手术都应尽可能保持清洁和无菌，以降低术后感染的风险。其次，由于卵巢组织非常脆弱，手术器械在卵巢切除术时不能直接接触卵巢，避免腹腔内着床。第三，手术后，在恢复过程中将小鼠移至37°C恒温毯中，以防止术后体温过低导致死亡。
先前的一项研究证明，内源性雌激素在绝经前妇女的卵巢细胞或绝经后妇女乳房的脂肪基质细胞中合成，并在外周组织中少量合成14。卵巢切除小鼠的血清雌激素急剧下降，但不能消除（图3B）。然而，在性腺外组织中合成的内源性雌激素不会影响OVF模型中雌激素水平的稳定性（图4B）。
OVF 模型存在一些限制。一旦外科手术不小心，导致卵巢腹腔内着床，就可能导致模型失败。在这种情况下，血清雌激素不会急剧下降，并且在发情周期的不同阶段波动。外源性施用雌激素和FSH后，身体大约需要1周才能达到平衡。因此，在1周内发生的OVF模型的病理变化不能表明FSH的影响。
综上所述，OVF机型具有稳定、低成本、易于操作等优点。腹腔注射FSH后可观察到高水平FSH的全身效应;也就是说，OVF 模型适用于探索 FSH 性腺外作用的研究。然而，模型手术和腹膜内注射手术的要求相当高。如果资金充足，特定的基因敲除模型是最佳选择。

促卵泡激素 （FSH） 水平在绝经过渡期间升高（绝经过渡一词于 2011 年在生殖衰老研讨会 （STRAW） + 10 系统阶段定义）1。在更年期过渡期间，一个以 FSH 水平升高和雌激素1 相对稳定的时期为特征，女性会经历月经周期变化和涉及各种细胞和组织的显着生理变化。这些变化会严重影响妇女的生活质量和健康。探索FSH的影响可能会改善女性的生活质量和健康。
FSH 由垂体前叶的促性腺激素细胞分泌，在控制性腺功能和生殖方面至关重要2.FSH的功能是通过FSH受体（FSHR）介导的，FSHR属于G蛋白偶联受体（GPCR）3。FSHR通常在性腺中表达，即卵巢和睾丸。已经证明，FSHR在多种性腺外细胞和组织中普遍表达，包括肝脏4、海马体5、破骨细胞6、脂肪细胞7和内皮细胞8。新兴研究揭示了 FSH 的额外性腺作用及其在血脂异常4、阿尔茨海默病5、骨质疏松症 9,10、动脉粥样硬化11、肥胖 9 和癌症12 中的潜在临床相关性。因此，建立一个可以帮助研究FSH在体内独立作用的动物模型对于探索FSH在体内的作用尤为重要。
在协议中，我们介绍了建立具有相对稳定的雌激素和不断上升的FSH水平的小鼠模型的程序13。小鼠模型通过卵巢切除手术模拟绝经过渡，然后补充戊酸雌二醇和重组FSH。由于卵巢切除的小鼠补充了外源性雌激素以维持与假手术小鼠相似的雌激素水平，因此由于垂体的雌激素反馈，内源性FSH的水平是稳定的。在这种情况下，它可以通过施用外源性FSH来控制FSH水平，而不会改变雌激素水平。因此，OVF小鼠模型可以排除雌激素的影响，并观察FSH的性腺外生理和病理作用。我们相信，详细和可视化的程序有助于研究人员在实验室中建立OVF小鼠模型，并根据需要将其应用于研究更年期过渡期间的生理和病理变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>beta-estradiol 17-valerate</td>
			<td>Macklin</td>
			<td>E829824</td>
			<td></td>
		</tr>
		<tr>
			<td>Estradiol sensitive ELISA</td>
			<td>Demeditec</td>
			<td>DE4399</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Staining Solution</td>
			<td>Beyotime</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Aladdin</td>
			<td>M129228</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human Follicle-stimulating hormone</td>
			<td>Merck Serono</td>
			<td>N19Z8803G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Aliphatic name: 2,2,2-Tribromoethanol</td>
		</tr>
	</tbody>
</table>

exploring independent effects of follicle-stimulating hormone in vivo in a mouse model

在从生殖期到非生殖期（更年期）的过渡期间，许多女性经历了显着的生理和病理变化，包括骨量减少、血脂升高和内脏肥胖增加。促卵泡激素 （FSH） 水平在绝经过渡期间升高。许多研究表明，各种性腺外组织和器官中的FSH与多种疾病的发病机制有关。因此，建立一个可以帮助研究FSH 在体内 独立作用的动物模型尤为重要。本研究对C57BL/6雌性小鼠进行卵巢切除术，并补充戊酸雌二醇（OVX+E2），以消除下丘脑-垂体-性腺轴的影响。OVX + E2小鼠通过腹膜内注射接受溶剂（N.S.）或不同剂量的重组FSH，以创建小鼠模型（OVF），其特征是雌激素相对稳定，FSH水平升高。因此，我们成功地生成了一个实验小鼠模型来模拟更年期过渡的早期阶段，其特征是血清FSH水平升高。OVF模型具有稳定、低成本、易于操作等优点，适合研究FSH的性腺外作用。在这里，我们描述了小鼠OVF模型的详细协议。

OVF小鼠模型模拟了更年期过渡的早期阶段，雌激素相对稳定，FSH水平升高13。首先，对于卵巢切除手术，对9周龄的雌性C57BL / 6小鼠进行全身麻醉并进行假手术（Sham）或双侧卵巢切除术（OVX）。由于巴氏染色细胞的涂片图像清楚地识别了发情周期的前发情期、发情期、子宫期和发情期，OVX小鼠失去了发情周期（图3A），ELISA方法显示血清雌二醇（E2）水平显着降低（图3B）。其次，OVX小鼠补充β-雌二醇17-戊酸酯（OVX+E2），以维持血清雌激素与Sham组相同。第三，OVX + E2小鼠通过腹膜内注射接受溶剂（N.S.）或不同剂量的重组FSH，以创建小鼠模型（OVF），其特征是雌激素相对稳定，FSH水平升高（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65665/65665fig01.jpg"> 图 1.手术环境和小鼠姿势。 （A） 至少 60 厘米 x 60 厘米的工作台区域用于手术。用75%酒精清洁该区域表面，并用一次性医用毛巾覆盖，然后提前30分钟用紫外线辐射消毒。（B） 使用橡皮条或棉绳将鼠标固定在手术平台上。 <a href="https://www.jove.com/files/ftp_upload/65665/65665fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65665/65665fig02.jpg"> 图2.外科手术的关键步骤。 （A）卵巢位置，（B）卵巢切除术，（C）缝合腹膜，（D）缝合皮肤切口。 <a href="https://www.jove.com/files/ftp_upload/65665/65665fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65665/65665fig03.jpg"> 图3.阴道细胞学检查。 阴道细胞学代表卵巢切除小鼠 （OVX） 和假手术小鼠（Sham;假手术组 n = 12;每个 OVX 组 n = 10）的发情周期和内源性雌激素的阶段。（A） 阴道细胞学根据白细胞、角质化上皮细胞和有核上皮细胞的相对存在来表示发情周期的各个阶段。发情期包括发情前期，有核上皮细胞占主导地位;发情，以去核角质化细胞为主;metestrus，白细胞的存在，角化和有核的上皮细胞;腹细胞，白细胞占主导地位。比例尺 = 100 μm。 （B） 卵巢切除小鼠 （OVX） 和假手术小鼠 （Sham） 中的内源性雌激素。数据显示为SEM±均值。 学生t检验用于统计分析。第 < 0.001。 <a href="https://www.jove.com/files/ftp_upload/65665/65665fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65665/65665fig04.jpg"> 图4.OVF 模型和血清激素水平。 （A） 流程图OVF模型。（B） 血清雌激素 （E2） 和 FSH 浓度的 ELISA 分析。数据表示为SEM±均值，采用单因素方差分析进行统计分析。* p< 0.05 和 ** p< 0.01。这个数字从4修改了。 <a href="https://www.jove.com/files/ftp_upload/65665/65665fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model at JoVE.com

作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

各种性腺外组织和器官中的卵泡刺激素（FSH）与多种疾病的发病机制有关。卵巢切除和 FSH 处理的小鼠模型 （OVF） 可用于探索 FSH 的性腺外作用。

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

我们的研究重点是围绝经期妇女的生理和病理变化。在这项研究中，我们试图确定哪些病理生理学改变与 FHC 有关。绝经与血脂异常和心脑血管疾病风险增加有关。使用这种小鼠模型，我们发现与阿司匹林无关的促卵泡激素会增加血清胆固醇水平。此外，阻断FSH信号通路可以有效预防低胆固醇血症。该小鼠模型模拟了更年期过渡的早期阶段，其特征是血清FSH水平升高和雌激素水平相对稳定。该模型具有稳定、成本低、操作方便等优点，适合研究FSH的性腺外作用。

exploring-independent-effects-follicle-stimulating-hormone-vivo-mouse

Research

JoVE Journal

Biology

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

1.5K 次观看.  The Affiliated Hospital of Qingdao University. 我们的研究重点是围绝经期妇女的生理和病理变化。在这项研究中，我们试图确定哪些病理生理学改变与 FHC 有关。绝经与血脂异常和心脑血管疾病风险增加有关。使用这种小鼠模型，我们发现与阿司匹林无关的促卵泡激素会增加血清胆固醇水平。此外，阻断FSH信号通路可以有效预防低胆固醇血症。该小鼠模型模拟了更年期过渡的早期阶段，其特征是血清FSH水平升高和?...

视频: 作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

全麻醉血友病A小鼠尾静脉横断出血模型

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

通过脱细胞定量骨骼肌脂肪浸润

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

作者聚焦：基于脱细胞化的骨骼肌脂肪浸润定量  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

在小鼠模型中探索卵泡刺激素在体内的独立作用