This study was supported by the National Natural Science Foundation of China (NSFC) (82172598), the Natural Science Foundation of Zhejiang Province, China (LZ22H310001), the 551 Health Talent Training Project of Health Commission of Zhejiang Province, China, and the Medical and Health Science and Technology Project of Zhejiang Province, China (2021KY110).

All animal studies have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Basic Medicine and Cancer, Chinese Academy of Sciences. This is a non-survival surgery.
1. Animal
<ol>
	<li>Use a 6-8-week-old female SD rat, weighing 250 g, for intraoperative PG imaging.</li>
</ol>
2. Anesthesia
<ol>
	<li>Turn on the anesthesia machine.</li>
	<li>Before beginning, ensure the isoflur...

<ol>
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J., 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=Self-report+of+psychological+symptoms+in+hypoparathyroidism+patients+on+conventional+therapy.">Self-report of psychological symptoms in hypoparathyroidism patients on conventional therapy.</a> Archives of Endocrinology Metabolism. 62 (3), 319-324 (2018).</li><li>Olson, E., Wintheiser, G., Wolfe, K. M., Droessler, J., Silberstein, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+thyroid+cancer:+a+review+of+the+national+cancer+database,+2000-2013.">Epidemiology of thyroid cancer: a review of the national cancer database, 2000-2013.</a> Cureus. 11 (2), 4127 (2019).</li><li>Du, L., 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=Epidemiology+of+thyroid+cancer:+incidence+and+mortality+in+China,+2015.">Epidemiology of thyroid cancer: incidence and mortality in China, 2015.</a> Frontiers in Oncology. 10, 1702 (2020).</li><li>Barrios, L., 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=Incidental+parathyroidectomy+in+thyroidectomy+and+central+neck+dissection.">Incidental parathyroidectomy in thyroidectomy and central neck dissection.</a> Surgery. 169 (5), 1145-1151 (2021).</li><li>Sitges-Serra, 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=Inadvertent+parathyroidectomy+during+total+thyroidectomy+and+central+neck+dissection+for+papillary+thyroid+carcinoma.">Inadvertent parathyroidectomy during total thyroidectomy and central neck dissection for papillary thyroid carcinoma.</a> Surgery. 161 (3), 712-719 (2017).</li><li>Sakorafas, G. H., 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=Incidental+parathyroidectomy+during+thyroid+surgery:+an+underappreciated+complication+of+thyroidectomy.">Incidental parathyroidectomy during thyroid surgery: an underappreciated complication of thyroidectomy.</a> World Journal of Surgery. 29 (12), 1539-1543 (2005).</li><li>Sahyouni, G., 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=Rate+of+incidental+parathyroidectomy+in+a+pediatric+population.">Rate of incidental parathyroidectomy in a pediatric population.</a> OTO Open. 5 (4), (2021).</li><li>Erickson, A. 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We demonstrate an IONPs-guided negative imaging technique of rat PG&#160;using black IONPs, which were injected locally in the center of the thyroid gland and diffused within the thyroid gland but not the PG. It enables clear identification of the PG&#160;with naked eyes without the aid of any microscope. Although transgenic mice with green fluorescent protein selectively expressed in the PG&#160;have been reported13, the model described in this paper is more straightforward to perform. It only ta...

Parathyroid gland&#160;(PG) is small, oval-shaped endocrine glands located in the neck of humans and other vertebrates, which produce and secrete parathyroid hormones to regulate and balance calcium and phosphorus levels in the blood and in bones1. Humans usually have two pairs of PG&#160;located behind the thyroid gland lobes in variable locations; the size of human PG&#160;typically measures 6 mm x 4 mm x 2 mm, with a weight of approximately 35-40 mg2. Removal or damage of the PG&#160;causes hypoparathyroidism (HP), an endocrine disorder characterized by hypocalcemia and low or undetectable levels of parathyroid hormones, which cause a wide range of symptoms from cramp-like spasms to malformed teeth to chronic kidney diseases. Some of these complications are fatal (e.g., heart failure and seizure)3,4,5; thus, PG&#160;is essential in regulating the body&#39;s metabolism and sustaining life.
HP is one of the most common complications after anterior neck surgery, especially in thyroidectomy, a well-established curative treatment for thyroid cancer, which is the most common endocrine cancer worldwide6,7. Post-thyroidectomy HP is predominantly caused by direct trauma, ischemia, or removal of the PG in surgery because of a severe lack of ability to reliably discriminate the PG&#160;from thyroid gland lobes and other surrounding tissues (e.g., lymph nodes and peripheral fat particles) in real-time in the operation room. In 2021, Barrios et al. reported an average PG mis-section rate of 22.4% within 1,114 thyroidectomy cases, and even experienced surgeons who had a minimum error rate of 7.7%8. Such high PG mis-section rates are consistent with other similar reports9,10,11. Thus, incorrect parathyroidectomy is an independent risk factor for transient and permanent postoperative HP.
Developing effective intraoperative PG identification methods holds the key to addressing this critical unmet medical need; however, it has been severely limited by the lack of animal models in preclinical research. To date, most intraoperative PG identification investigations have been performed on human patients and large animals (e.g., dogs)12, which are expensive and difficult to receive ethical approval, expand subject numbers, and repeat tests. Meanwhile, the mouse, the most commonly used vertebrate model in biological research, has extremely small PG, with a size of less than 1 mm13. Due to this limitation, mouse PG models have seldom been used in intraoperative PG identification research.
This paper reports the establishment of a simple, straightforward, and effective rat model for intraoperative PG identification studies. We investigated the usage of native Sprague-Dawley (SD) rats without any surgical modifications or genetic engineering as a reliable animal model for testing a PG imaging contrast agent, IONPs, in a thyroidectomy surgery. This rat model demonstrates a highly similar physiological structure of PG&#160;and the surrounding microenvironment to that of humans, and the size of rat PG&#160;is large enough to be visually detected in comparison with those of mice. Most rats have one PG on each side of the thyroid gland. The simplicity and effectiveness of this rat model have been demonstrated by performing intraoperative IONP-enhanced PG imaging in thyroidectomy surgery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>alcohol</td>
			<td>Li feng</td>
			<td>9400820067</td>
			<td></td>
		</tr>
		<tr>
			<td>anesthesia machine</td>
			<td>RWD Company</td>
			<td>R520IE</td>
			<td>Machine number</td>
		</tr>
		<tr>
			<td>blade</td>
			<td>Daopian</td>
			<td>TB-JZ-10#</td>
			<td></td>
		</tr>
		<tr>
			<td>cylindrical pillow</td>
			<td></td>
			<td></td>
			<td>made by ourselves</td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Nair</td>
			<td>TMG-001</td>
			<td></td>
		</tr>
		<tr>
			<td>electronic scale</td>
			<td>Hong xingda</td>
			<td>CN-HXD2</td>
			<td></td>
		</tr>
		<tr>
			<td>eosin</td>
			<td>Thermo Fisher (Waltham, USA).</td>
			<td>C0105S-2</td>
			<td></td>
		</tr>
		<tr>
			<td>erythromycin</td>
			<td>Shuang ji (Beijing, China)</td>
			<td>200409</td>
			<td></td>
		</tr>
		<tr>
			<td>gauze</td>
			<td>Fulanns</td>
			<td>YY0331-2006</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Johon (ShenZhen,China)</td>
			<td>JH-36-2006</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxylin</td>
			<td>Thermo Fisher (Waltham, USA).</td>
			<td>C0105S-1</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin injection needle</td>
			<td>Jiangyin NanquanMacromolecule</td>
			<td>20170702</td>
			<td></td>
		</tr>
		<tr>
			<td>iodophor cotton ball</td>
			<td>HOYON</td>
			<td>19-6007</td>
			<td></td>
		</tr>
		<tr>
			<td>iron oxide nanoparticle solution</td>
			<td>Zhongke Leiming Technology (Beijing, China)</td>
			<td>Mag9110-05</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Sigma Aldrich (St Louis USA).</td>
			<td>21112801</td>
			<td></td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Meijun</td>
			<td>MH0587</td>
			<td></td>
		</tr>
		<tr>
			<td>operation table</td>
			<td>BioJane</td>
			<td>BJ-P-M</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde solution</td>
			<td>Biosharp</td>
			<td>21269333</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber</td>
			<td>G-CLONE</td>
			<td> 
			XT41050</td>
			<td></td>
		</tr>
		<tr>
			<td>scanning machine</td>
			<td>Olympus</td>
			<td>Slideview VS200</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Suping</td>
			<td>SPHC-0676</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical knife handle</td>
			<td>Aladdin</td>
			<td>S3052-06-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical retractor</td>
			<td>TOCYTO</td>
			<td>18-4010</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Suping</td>
			<td>SPHC-0795</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical towel</td>
			<td>Along technology</td>
			<td>YCKJ-RJ-036205</td>
			<td></td>
		</tr>
		<tr>
			<td>suture</td>
			<td>Ethicon</td>
			<td>SA84G</td>
			<td></td>
		</tr>
		<tr>
			<td>suture with needle</td>
			<td>Jinhuan (Shanghai,China)</td>
			<td>F301</td>
			<td></td>
		</tr>
		<tr>
			<td>vascular forceps</td>
			<td>Along technology</td>
			<td>YCKJ-RJ-016218</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath-Slide Drier</td>
			<td>Hua su (Jinhua, China)</td>
			<td>HS-1145</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this animal model, we surgically incised the neck of a SD rat to expose the trachea, larynx, and surrounding tissues. Then, the thyroid gland was visually located on both sides of the trachea; it is butterfly-shaped and approximately 3 mm x 5 mm in size. A pair of PG&#160;are usually located in the upper part of the thyroid gland, and their color is very similar to that of the thyroid gland lobes, making it extremely difficult to distinguish them with the naked eye (Figure 1).&#160;
<...

Watch this Scientific Journal Video about Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging at JoVE.com

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

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.

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

Parathyroid gland is a small organ with very important functions. Our protocol can be used for developing new PG identification technologies which can help surgeons to protect it during the thyroidectomy for thyroid cancer. The main advantage of this model is that it can observe a clear difference between the thyroid and PGs through the negative imaging of IONP.In addition, the cost and operational difficulty are considerably lower for this rat model than for large animal models. To begin, place a prewarmed heating pad on the surgical table in order to sustain the animal's body temperature during the surgery, and then transfer the anesthetized rat down to a surgical drape placed on the operation table. Using rubber bands, fix the rat's limbs to the operation table and place a cylindrical pillow made of drape under the rat's shoulder to lean its head back, completely exposing the neck area, then apply depilatory cream to the neck area, up to the submandibular space, down to the xiphoid process, and on both sides to the outside of the sternocleidomastoid muscle.After three minutes, gently wipe the hair and depilatory cream with a tissue. Next, using an iodophor cotton ball, sterilize the middle of the neck and surrounding area. from which hair is removed.Cover the animal with a surgical drape, keeping the hole aligned with disinfected area. Using the scalpel, make approximately a five-centimeter longitudinal incision in the anterior midline of the rat's neck. Lift the skin along both sides of the incision and using scissors, longitudinally cut along the linea alba cervicalis.Use forceps to separate the sternohyoid and sternothyroid muscle. Clamp the separated muscles in front of the neck using vascular forceps and pull the clamped tissue outside. Using the needle, pass the suture through the clamped tissue.Make a knot and fix the suture to the surgical drape of the operation table. Locate the thyroid cartilage based on its shield shape and cricoid cartilage based on its ring shape as the upper boundary in the operation area, then locate the trachea based on its tubular cartilage ring shape in the front and middle of the neck as the lower boundary in the operation area. Locate the thyroid gland, a red butterfly-shaped gland on the opposite side of the trachea between the upper and lower boundaries.Next, locate two parathyroid glands in a fusiform shape that are reddish, but lighter than the surrounding thyroid gland with a certain boundary on the upper and outer sides of the thyroid gland. Take a frontal photograph of the parathyroid glands with the trachea, thyroid, and larynx. Dissect the back of the esophagus and using a retractor, expose the right side of the parathyroid glands.Take a right-side photograph of the parathyroid glands with the thyroid gland and trachea, and similarly expose and capture the left side of the glands. Use an insulin syringe to locally inject 10 microliters of black iron oxide nanoparticles suspension into the center of the thyroid gland, and gently press the injection site with gauze for five seconds, then observe the rapid diffusion of the black iron oxide nanoparticles within the thyroid glands, but not the parathyroid glands, as it negatively stains the parathyroid glands and differentiates them from the surrounding thyroid. Capture the front, left, and right-side photographs of the negatively stained parathyroid glands together with the trachea, thyroid gland, and larynx.After injection, the contrast agent, black iron oxide nanoparticles, readily diffuse within the thyroid gland and stain it black, but cannot infiltrate the parathyroid gland due to their high tissue density. Histological analysis of black iron oxide nanoparticles injected-thyroid gland revealed that the parathyroid glands are enriched with closely aligned chief cells, whereas the thyroid gland features many loose lumens indicating much lower tissue density. Two important things should be remembered in this procedure.First, the posture of the rat. We recommended to lean ears and head back, completely exposing the neck area. Second, when we inject the IONPs, we should be use fine-ensuring needle because the thyroid surface is full of blood vessels.This procedure can also be performed by autofluorescence imaging of the PG, which is non-invasive, provides real-time information, and does not use dye as a contrast agent, but the autofluorescence signal is weak and the imaging efficiency is also poor. Other researchers can use this simple and effective rat model to develop and evaluate new PG identification technologies and products instead of using more complicated and expensive large animals such as dogs and pigs.

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2.8K visualizações.  Zhejiang Cancer Hospital. A glândula paratireoide é um pequeno órgão com funções muito importantes. Nosso protocolo pode ser usado para o desenvolvimento de novas tecnologias de identificação de PG que podem ajudar os cirurgiões a protegê-lo durante a tireoidectomia para câncer de tireoide. A principal vantagem deste modelo é que ele pode observar uma clara diferença entre a tireoide e os PGs através da imagem negativa do IONP.Além disso, o custo e a dificuldade operacional são consideravelmente...