Name: generation of hypoparathyroid rats via carbon-nanoparticle-assisted parathyroidectomy
Uploaded: 2023-07-14T15:00:00
Description: Watch this Scientific Journal Video about Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy at JoVE.com

这项工作得到了国家自然科学基金基金81800928、四川大学华西学院/口腔医院的研究资助（RCDWJS2021-1）和口腔疾病国家重点实验室开放基金SKLOD-R013的支持。

本研究获得四川大学口腔疾病国家重点实验室机构动物护理与使用委员会的批准。在实验之前获得了当地相关机构的许可。本研究使用了八只8-10周龄的雄性Sprague-Dawley（SD）大鼠，平均体重为200-250g。这些动物是从商业来源获得的（见 材料表）。 在整个实验 期间随意提供食物和水。
1.产生碳纳米颗粒辅助PTX大鼠的术前准备
<ol><li>使用2.0%-2.5%?...

<ol>
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流行病学报告表明，甲状腺疾病的检测显着增加，进行的相关手术数量相应增加19，20。术后甲状旁腺功能减退症的发病率约为7.6%8，21，而获得性甲状旁腺功能减退症发病率的增加使这种罕见疾病受到更多的研究关注。因此，建立合适的动物模型来研究疾病的发病机制以及测试新疗法的结果尤为重要。但是，?...

甲状旁腺激素（PTH）由甲状旁腺分泌。它是钙平衡的主要调节剂，维持磷酸盐代谢，并参与骨转换1，2。甲状旁腺功能减退症 （HypoPT） 表现为 PTH 分泌减少或功能丧失。它是一种罕见的内分泌疾病，患病率约为 9-37/100，000 人年3，4，5。低PT的特征是血清PTH和钙水平降低，伴血清磷6，7升高。HypoPT根据其原因进行分类：获得性甲状旁腺功能减退症（AHypoPT）或特发性甲状旁腺功能减退症（IHypoPT）8。AHypoPT在临床实践中更常见;大约75%的AHypoPT病例是由甲状腺手术或其他头颈部手术期间甲状旁腺切除或意外损伤引起的。其他原因包括头颈部肿瘤的放化疗和药物毒性1，8。升级的诊断方法和甲状腺相关疾病筛查的增加增加了甲状腺外科手术的数量。这导致相关的甲状旁腺并发症相应增加9，10。
需要易于建立的具有稳定特征的动物模型来更好地研究AHypoPT并验证新疗法的治疗效果。在以前的研究中已经报道了对大鼠和小鼠进行的甲状旁腺切除术（PTX）6，11;然而，由于甲状旁腺的尺寸极小，其解剖分布的可变性，在实践中成功率相对较低。因此，通常进行甲状腺-甲状旁腺切除术（TPTX）（即完全切除甲状腺和甲状旁腺）以确保切除甲状旁腺12。然而，由此产生的低甲状腺素水平可能会使该动物模型的研究复杂化13。通过其他方法（如药物刺激和基因编辑）建立的HypoPT模型不能正确代表最常见的AHypoPT发病机制。我们小组以前使用敲除小鼠模型来标记甲状旁腺，并允许在不损害甲状腺和周围解剖结构的情况下切除甲状旁腺14，15。然而，该方法利用转基因小鼠模型，由于交配和育种要求，需要更长的开发时间。
因此，我们旨在建立一个易于生成的AHypoPT模型。本研究描述了使用碳纳米颗粒标记的PTX大鼠模型。甲状腺手术中常用的50mg/mL碳纳米颗粒悬浮液在局部注射后均匀分布在甲状腺中16。甲状腺变黑，但甲状旁腺未染色17，从而清楚地区分甲状旁腺和甲状腺，并允许在不影响甲状腺的情况下进行PTX。该方法适用于不同年龄的大鼠。碳纳米颗粒悬浮液的注射是安全的，对甲状腺功能的影响可以忽略不计18。本研究产生的碳纳米颗粒标记的PTX大鼠模型在4周观察期内显示出显着的低钙血症和高磷血症表型。因此，该AHypoPT模型易于建立，并且具有可重复的表型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Solution</td>
			<td>Kelun Co. Sichuan, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L 30G NanoFil Syringe</td>
			<td>WPI</td>
			<td></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>Calcium LiquiColor test</td>
			<td>EKF</td>
			<td>0155-225</td>
			<td>For Ca2+ analysis</td>
		</tr>
		<tr>
			<td>Carbon Nanoparticles Suspension Injection</td>
			<td>Lummy, Chongqing, China</td>
			<td>H20073246</td>
			<td>1 mL : 50 mg</td>
		</tr>
		<tr>
			<td>Creatinine (Cr) Assay kit ( sarcosine oxidase )</td>
			<td>Jiancheng, Nanjing, China</td>
			<td>C011-2-1</td>
			<td>For creatinine analysis</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Shinva, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumstar Biology forceps</td>
			<td>Shinva, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors</td>
			<td>Shinva, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroVue Rat intact PTH ELISA</td>
			<td>Immunotopics</td>
			<td>30-2531</td>
			<td>For the measurement of PTH in rat serum</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Shinva, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorus Liqui-UV test</td>
			<td>EKF</td>
			<td>0830-125</td>
			<td>For Pi analysis</td>
		</tr>
		<tr>
			<td>Ply gauze</td>
			<td>Weian Co. Henan, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine</td>
			<td>Yongan pharmaceutical Co.Ltd. Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 9.0 (statistics and graphing software)</td>
			<td>GraphPad Software, Inc., San Diego, CA, USA</td>
			<td></td>
			<td>https://www.graphpad.com/scientific-software/prism/</td>
		</tr>
		<tr>
			<td>Rat C-telopeptide of type I collagen (CTX-I) ELISA Kit</td>
			<td>CUSABIO, Wuhan, China</td>
			<td>CSB-E12776r</td>
			<td>For CTX-I analysis</td>
		</tr>
		<tr>
			<td>Rat Osteocalcin/Bone Gla Protein (OT/BGP) ELISA Kit</td>
			<td>CUSABIO, Wuhan, China</td>
			<td>CSB-E05129r</td>
			<td>For osteocalcin analysis</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>Sprague-Dawley Rats</td>
			<td></td>
			<td></td>
			<td>8 to 10 weeks old</td>
		</tr>
		<tr>
			<td>Surgical Incise Drapes</td>
			<td>Liangyou Co. Sichuan, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urea Assay Kit</td>
			<td>Jiancheng, Nanjing, China</td>
			<td>C013-2-1</td>
			<td>For urea analysis</td>
		</tr>
	</tbody>
</table>

generation of hypoparathyroid rats via carbon-nanoparticle-assisted parathyroidectomy

甲状旁腺功能减退症（HypoPT）是一种涉及甲状旁腺的罕见疾病，其特征是甲状旁腺激素（PTH）的分泌或效力减少，从而导致高血清磷水平和低血清钙水平。HypoPT 最常见的原因是甲状腺或其他颈部前部手术期间腺体意外损伤或切除。近年来，甲状旁腺/甲状腺手术变得越来越普遍，作为术后并发症的HypoPT的发生率相应增加。迫切需要一个HypoPT动物模型来更好地了解HypoPT对矿物离子稳态影响的机制，并验证新疗法的治疗效果。据报道，一种通过使用碳纳米颗粒进行甲状旁腺切除术（PTX）在雄性大鼠中产生获得性HypoPT的技术。与甲状旁腺功能减退症的小鼠模型相比，大鼠模型显示出很大的前景。重要的是，人PTH受体结合区与大鼠的序列相似度为84.2%，高于小鼠共有的73.7%相似度。此外，雌激素的影响可以影响PTH/PTHrP受体信号通路，尚未在雄性大鼠中得到充分研究。碳纳米颗粒是淋巴示踪剂，可将甲状腺淋巴结染成黑色而不影响其功能，但它们不会染色甲状旁腺，这使得它们易于识别和去除。在这项研究中，PTX后血清PTH水平检测不到，这导致了严重的低钙血症和高磷血症。因此，术后HypoPT的临床状态可以在大鼠模型中得到显着表现。因此，碳纳米颗粒辅助PTX可以作为研究HypoPT的发病机制，治疗和预后非常有效且易于实施的模型。

最初在解剖显微镜下在大鼠中观察到甲状旁腺的位置和数量。在碳纳米颗粒注射之前，甲状腺呈半透明的红色，在显微镜下几乎无法区分甲状旁腺（图1A）。纳米颗粒注射后，甲状腺被染成黑色，而甲状旁腺保持未染色（图1B）。仔细解剖浅色甲状旁腺，使甲状腺未受影响（图1C）。通常，甲状旁腺分布在甲状腺的外侧或后缘。...

Watch this Scientific Journal Video about Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy at JoVE.com

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

获得性甲状旁腺功能减退症（HypoPT）的动物模型对于了解HypoPT如何影响矿物离子稳态和验证新疗法的有效性至关重要。本文提出了一种使用碳纳米颗粒通过甲状旁腺切除术（PTX）生成获得性甲状旁腺功能减退症（AHypoPT）大鼠模型的技术。

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

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

该协议的总体目标是生成获得性甲状旁腺功能减退症的大鼠模型，该模型易于建立，性质稳定，对甲状腺无害。该模型的主要优点是在碳纳米颗粒的帮助下精确去除甲状旁腺。我们希望该模型可以帮助解决甲状旁腺功能减退症领域的一些问题。例如，术前使用钙和维生素 D 来降低甲状腺切除术后低钙血症的风险。这种方法可以深入了解PTH类似物如何影响骨转换以及PTH类似物在长期使用中如何影响身体。那些第一次执行这种技术的人可能会发现很难在体视显微镜下进行微观操作。在体视显微镜下进行一些练习可能会有所帮助。首先，将麻醉的大鼠置于仰卧位并剃掉腹颈区域的皮毛。按照手稿中的描述做一个切口后，找到甲状腺的叶。使用配备30号斜面针的10微升注射器，将1微升碳纳米颗粒悬浮液注入甲状腺膜的内侧部分。五分钟后，用盐水冲洗该区域以清洁任何多余的悬浮液。确保甲状腺变黑，并观察未染色的甲状旁腺。使用一对显微手术钳和剪刀切割未染色的甲状旁腺，然后用中断的水平床垫缝合线关闭切口。手术后，将大鼠放在37摄氏度的恒温电热毯上以保持体温。七天后，使用尾静脉抽取 10 微升血液。使用商业试剂盒测量血清钙、血清无机磷和血清甲状旁腺激素。甲状旁腺切除术大鼠在4周的观察期内表现出稳定的低钙血症和高磷血症。术后7天甲状旁腺切除术大鼠未检测到血清甲状旁腺激素。术后7 d假甲状腺切除术组和甲状旁腺切除术组的血清尿素和肌酐相当。术后两周观察到尿钙离子和无机磷酸盐水平降低。在28天内，甲状旁腺切除术和假手术组的体重没有显着差异。甲状旁腺切除术大鼠血清C-端肽显著下降。血清骨钙素水平无显著差异。然而，纳米颗粒悬浮注射是最重要的事情，成功的注射对于识别和目前切除甲状旁腺至关重要。其他方法包括荧光引导手术，PTHcre-iDTR转基因小鼠的双耳毒素注射和无菌甲状旁腺切除术，可以帮助回答甲状腺功能亢进领域的问题。

Research

JoVE Journal

Medicine

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Acute Myocardial Infarction in Rats

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

Generation of Alginate Microspheres for Biomedical Applications

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Orthotopic Small Bowel Transplantation in Rats

大鼠原位小肠移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

大鼠原位肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Histological Quantification of Chronic Myocardial Infarct in Rats

大鼠慢性心肌梗塞的组织学定量分析

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

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

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 ...

Ultrasonography in Experimental Reproductive Investigations on Rats

大鼠实验性生殖研究中的超声检查

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

One-anastomosis Gastric Bypass (OAGB) in Rats

One-anastomosis Gastric Bypass OAGB in Rats

大鼠胃旁路 (oagb) 的一次吻合

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

成人大鼠心脏解剖与功能的超声心动图评估

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

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

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