Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Summary

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.

Abstract

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. 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.

Introduction

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 turnover^1,2. 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-years^3,4,5. HypoPT is characterized by decreased serum PTH and calcium levels accompanied by increased serum phosphorus^6,7. 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 toxicity^1,8. 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 complications^9,10.

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 studies^6,11; 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 structures^14,15. 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.

Protocol

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.

1. Preoperative preparation for the generation of carbon-nanoparticle-assisted PTX rats

1. Anesthetize the 8-10 week old rats using 2.0%-2.5% isoflurane inhalation, followed by an intraperitoneal (i.p.) injection of tribromoethanol at 10 mL/kg body weight. Guarantee a sufficient depth of anesthesia by testing the absence of the pupillary light reflex. Use vet ointment on the eyes to prevent dryness while under anesthesia.
   ​NOTE: Rats that are 8-10 weeks old are recognized as adult rats. However, this modeling method can be used on rats as young as 7 days old.
2. Prepare the anesthetized SD rats for surgery by shaving the fur of the ventral neck region in the supine position. Disinfect the operative area using povidone-iodine cotton balls (see Table of Materials).
3. Cover the animal with surgical drapes (see Table of Materials), and expose the surgical region, aiming to minimize microbial contamination.

2. Parathyroidectomy (PTX)

1. Start at the midpoint between the two ears, and cut a 2 cm incision longitudinally toward the tail using a surgical scalpel. Dissect the fascia and fat layer successively with sharp, curved, serrated forceps.
2. Separate the paratracheal muscles, and expose the trachea using relatively blunt forceps under a stereo microscope at 4x-5x magnification.
3. Locate the left and right lobes of the butterfly-shaped thyroid glands by the side of the trachea.
4. Inject 1 µL of the carbon nanoparticle suspension (see Table of Materials) beneath the membrane of the thyroid glands using a 10 µL syringe with a 30 G beveled needle. After 5 min, irrigate the operative region with saline to clean the extra carbon nanoparticle suspension covering the thyroid gland membrane.
   NOTE: The recommended injection point is the mesial portion of the thyroid gland lobe, which has fewer blood vessels. A carbon nanoparticle suspension is commonly used in thyroid surgery due to its ability to detect the lymph nodes. Staining the thyroid glands and leaving the parathyroid glands unstained eases the identification of the latter.
5. Check to ensure that the thyroid glands turn black while the parathyroid glands remain unstained in ~5 min. Observe the highlighted parathyroid glands under ordinary light using either the light of a stereo microscope or a table lamp.
   NOTE: Typically, rodents have two drop-shaped parathyroid glands located on the left and right surfaces of the thyroid glands. Occasionally, additional parathyroid glands may be located further away.
6. Precisely cut the unstained parathyroid glands with microsurgery forceps and scissors. Use sterile cotton balls for hemostasis or a gelatin sponge if there is more bleeding.
7. Close the muscles, fat layers, and skin, layer by layer, with an interrupted horizontal mattress suture using 6-0 polyglactin 910 sutures (see Table of Materials).
8. For the sham group, perform all the steps of the preoperative preparation and PTX except step 2.6. Anesthetize the rats, and separate the tissues above the trachea. Locate the parathyroid glands but do not remove them. Conduct postoperative recovery and observation together with the rats of the PTX group.

3. Postoperative recovery and observation

1. After surgery, place the rats on a thermostatic electric blanket (37 °C) to maintain their body temperature. Inject buprenorphine hydrochloride 0.01 mg/kg subcutaneously (s.c) every 12 h as postoperative analgesia. Transfer the rats to a sterile cage when they begin to move and try to crawl. 
2. Closely observe the rats postoperatively for 2 h. Return the rats to the rearing room, observe them routinely, and record their state.
3. Draw 10 µL of blood from the tail vein 7 days after the surgery. Measure the serum Ca²⁺, serum Pi, and serum PTH using appropriate commercial kits (see Table of Materials). A successful parathyroidectomy produces a reduced serum ionized Ca²⁺ level 2 SD lower than that of sham-operated rats (9.00 mmol/L, n = 16).
   NOTE: A statistics and graphing software (see Table of Materials) was used for the statistical analysis. A Student's t-test was used to compare the serum and urinary parameters between the sham and PTX groups. p < 0.05 was considered statistically significant. Serum and urinary Ca²⁺ and Pi and serum urea and creatinine were measured with commercial kits according to the manufacturers' instructions (see Table of Materials). Serum C-telopeptide of type I collagen and osteocalcin were measured with commercially available ELISA kits (see Table of Materials).

Results

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...

Discussion

Epidemiological reports indicate that the detection of thyroid diseases has increased significantly, and the number of related surgeries performed has increased accordingly^19,20. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Solution	Kelun Co. Sichuan, China
10 µL 30G NanoFil Syringe	WPI
6-0 polyglactin 910 suture with needle	Ethicon, Inc	J510G
Calcium LiquiColor test	EKF	0155-225	For Ca2+ analysis
Carbon Nanoparticles Suspension Injection	Lummy, Chongqing, China	H20073246	1 mL : 50 mg
Creatinine (Cr) Assay kit ( sarcosine oxidase )	Jiancheng, Nanjing, China	C011-2-1	For creatinine analysis
Disposable Scalpel	Shinva, China
Dumstar Biology forceps	Shinva, China
Micro Dissecting Spring Scissors	Shinva, China
MicroVue Rat intact PTH ELISA	Immunotopics	30-2531	For the measurement of PTH in rat serum
Needle Holder	Shinva, China
Phosphorus Liqui-UV test	EKF	0830-125	For Pi analysis
Ply gauze	Weian Co. Henan, China
Povidone-Iodine	Yongan pharmaceutical Co.Ltd. Chengdu, China
Prism 9.0 (statistics and graphing software)	GraphPad Software, Inc., San Diego, CA, USA		https://www.graphpad.com/scientific-software/prism/
Rat C-telopeptide of type I collagen (CTX-I) ELISA Kit	CUSABIO, Wuhan, China	CSB-E12776r	For CTX-I analysis
Rat Osteocalcin/Bone Gla Protein (OT/BGP) ELISA Kit	CUSABIO, Wuhan, China	CSB-E05129r	For osteocalcin analysis
Safety Single Edge Razor Blades	American Safety Razor Company	66-0089
Sprague-Dawley Rats			8 to 10 weeks old
Surgical Incise Drapes	Liangyou Co. Sichuan, China
Urea Assay Kit	Jiancheng, Nanjing, China	C013-2-1	For urea analysis