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

This work was supported by NSFC grant 81800928, Research Funding from the West China School/Hospital of Stomatology Sichuan University (No. RCDWJS2021-1), and the State Key Laboratory of Oral Diseases Open Funding grant SKLOD-R013.

This study was approved by the Institutional Animal Care and Use Committee at the State Key Laboratory of Oral Diseases, Sichuan University. Permission was obtained from relevant local agencies before the experiment. Eight 8-10 week old male Sprague-Dawley (SD) rats, with an average weight of 200-250 g, were used for the present study. The animals were obtained from a commercial source (see Table of Materials). Food and water were provided ad libitum throughout the experimental period.
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<ol>
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Epidemiological reports indicate that the detection of thyroid diseases has increased significantly, and the number of related surgeries performed has increased accordingly19,20. The incidence rate of postsurgical hypoparathyroidism is approximately 7.6%8,21, while the increased morbidity of acquired hypoparathyroidism has caused this rare disease to gain greater research attention. It is, therefore, part...

Parathyroid hormone (PTH) is secreted by the parathyroid glands. It is a major modulator of the calcium balance, maintains phosphate metabolism, and participates in bone turnover1,2. Hypoparathyroidism (HypoPT) manifests as a decreased secretion or functional loss of PTH. It is a rare endocrine disorder, with a prevalence of approximately 9-37 per 100,000 person-years3,4,5. HypoPT is characterized by decreased serum PTH and calcium levels accompanied by increased serum phosphorus6,7. HypoPT is classified based on its cause: acquired hypoparathyroidism (AHypoPT) or idiopathic hypoparathyroidism (IHypoPT)8. AHypoPT is more commonly encountered in clinical practice; about 75% of AHypoPT cases are caused by resection or accidental injury of the parathyroid glands during thyroid surgery or other head and neck surgeries. Other causes include radiotherapy and chemotherapy for head and neck tumors and drug toxicity1,8. Upgraded diagnostic methods and an increase in screening for thyroid gland-associated diseases have increased the number of thyroid gland surgical operations. This has led to a corresponding increase in the related parathyroid gland complications9,10.
Easily established animal models with stable characteristics are needed to better investigate AHypoPT and verify the therapeutic effectiveness of novel treatments. Parathyroidectomy (PTX) performed on rats and mice has been reported in previous studies6,11; however, due to the extremely small size of the parathyroid glands and the variability in their anatomical distribution, the success rate is relatively low in practice. Thus, thyro-parathyroidectomy (TPTX) (i.e., the total removal of the thyroid and parathyroid glands) is usually performed to ensure the resection of the parathyroid glands12. However, the resulting low thyroxine levels may complicate studies with this animal model13. HypoPT models established by other methods, such as drug stimulation and gene editing, cannot properly represent the most common AHypoPT pathogenesis. Our group previously used knockout mouse models to label the parathyroid glands and allow the removal of the parathyroid glands without damaging the thyroid glands and surrounding anatomical structures14,15. However, this method utilizes transgenic mouse models, which require a longer development time due to the mating and breeding requirements.
Therefore, we aimed to establish an easily generated model of AHypoPT. This study describes a rat model for PTX using carbon nanoparticle labeling. A carbon nanoparticle suspension of 50 mg/mL, which is commonly used in thyroid surgery, evenly distributes in the thyroid glands after local injection16. The thyroid glands turn black, but the parathyroid glands are left unstained17, thus clearly distinguishing the parathyroid glands from the thyroid glands and allowing the PTX to be performed without affecting the thyroid glands. This method is suitable for rats of different ages. The injection of the carbon nanoparticle suspension is safe and has a negligible effect on thyroid function18. The carbon nanoparticle-labeled PTX rat model generated in this study showed significant hypocalcemia and hyperphosphatemia phenotypes during the 4 week observation period. Thus, this AHypoPT model is easy to establish and has a reproducible phenotype.

<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

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.

The locations and number of parathyroid glands were initially observed in rats under a dissection microscope. Before the carbon nanoparticle injection, the thyroid glands were a translucent red color, and the parathyroid glands were hardly distinguishable under the microscope (Figure 1A). After the nanoparticle injection, the thyroid glands were stained black, while the parathyroid glands remained unstained (Figure 1B). The careful dissection of the light-colore...

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

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.

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 ...

The overall goal of this protocol is to generate a rat model of acquired hypoparathyroidism that is easy to establish, stable in character, and harmless to the thyroid glands. The main advantage of this model is the precise removal of parathyroid glands with the assistance of carbon nanoparticles. We hope the model can help solve a few questions in the field of hypoparathyroidism.For example, preoperative use of calcium and vitamin D to reduce the risk of post-thyroidectomy hypocalcemia. This method can provide insight into how bone turnover is affected by PTH analogs and how PTH analogs affects the body in long-term use. Those who perform this technique for the first time may find it hard to micro-manipulate under the stereo microscope.Some practice under stereo microscope may be helpful. To begin, place the anesthetized rat in a supine position and shave the fur of the ventral neck region. After making an incision as described in the manuscript, locate the lobes of the thyroid gland.Using a 10-microliter syringe equipped with a 30-gauge bevel needle, inject one microliter of the carbon nanoparticle suspension into the mesial portion of the thyroid gland membrane. After five minutes, irrigate the region with saline to clean any extra suspension. Ensure the thyroid glands have turned black and observe the unstained parathyroid gland.Cut the unstained parathyroid glands using a pair of microsurgery forceps and scissors, and then close the incision with an interrupted horizontal mattress suture. After surgery, place the rats on a thermostatic electric blanket at 37 degrees Celsius to maintain their body temperature. After seven days, draw 10 microliters of blood using the tail vein.Measure the serum calcium, serum inorganic phosphorus, and serum parathyroid hormone using commercial kits. The parathyroidectomy rats displayed stable hypocalcemia and hyperphosphatemia over the observation period of four weeks. The serum parathyroid hormone was undetected in the parathyroidectomy rats seven days post-operation.The serum urea and creatinine were comparable between the sham and parathyroidectomy groups after seven days post-operation. Reduced urinary calcium ions and inorganic phosphate levels were observed two weeks post-operation. No significant differences in body weight were observed between the parathyroidectomy and sham groups across 28 days.The serum C-telopeptide decreased significantly in parathyroidectomy rats. There were no significant differences in the serum osteocalcin levels. However, nanoparticle suspension injection is the most important thing and a successful injection is critical for identification and present removal of the parathyroid glands.Other methods include fluorescence-guided surgery, diphteria toxin injection of PTHcre-iDTR transgenic mice, and the sterile parathyroidectomy which could help answer questions in the field of hyperthyroidism.

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