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Summary

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.

Abstract

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.

Introduction

Parathyroid gland (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 located behind the thyroid gland lobes in variable locations; the size of human PG typically measures 6 mm x 4 mm x 2 mm, with a weight of approximately 35-40 mg². Removal or damage of the PG 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 is essential in regulating the body'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 worldwide^6,7. 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 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 reports^9,10,11. 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 and the surrounding microenvironment to that of humans, and the size of rat PG 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.

Protocol

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

1. Use a 6-8-week-old female SD rat, weighing 250 g, for intraoperative PG imaging.

2. Anesthesia

1. Turn on the anesthesia machine.
2. Before beginning, ensure the isoflurane level is full in the anesthesia vaporizer. Then, turn on the oxygen and set the flow rate to 0.4-0.8 L/min. Induce anesthesia with 3-5% isoflurane and maintain at 2% isoflurane (flow rate: 0.4-0.8 L/min).
3. Put the SD rat into the box of the anesthesia machine and select the Channel model to begin animal anesthesia.
4. Observe the rat activity in the box. When the rat falls into a coma, move it to the nose cone to maintain anesthesia (unconscious supine position without pain reflex and corneal reflex).
5. Use the anesthesia mask to cover the nose of the rat and switch the anesthesia machine into Mask mode to keep the animal under anesthesia during the surgery.

3. Posture and fixation

1. Transfer the anesthetized rat onto a surgical drape on a surgery operation table. Place a prewarmed heating pad under the animal to sustain the animal's body temperature during the surgery.
2. Use rubber bands to fix the rat's limbs to the operation table. Place a cylindrical pillow made of drape under the rat's shoulder to lean its head back, completely exposing the neck area. 
3. Apply artificial tears ointment on both eyes of the rat to prevent dryness during anesthesia.

4. Hair removal

1. 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.
2. After 3 min, gently wipe the hair and depilatory cream with a tissue.

5. Sterilization

1. Use an Iodophor cotton ball to disinfect the operation area 3 times from the middle of the neck to the surrounding area. Only disinfect the area from which hair was removed.

6. Surgical drape laying

1. Use a surgical drape to cover the operation area of the rat's neck. Keep the hole of the surgical drape aligned with the disinfection area of the animal.

7. Incision

1. Confirm the surgical plane of anesthesia via lack of a toe pinch reflex before making the incision. Then, fit the blade into the scalpel and use the scalpel to make a longitudinal incision in the anterior midline of the rat's neck. Ensure that the incision length is approximately 5 cm and only in the dermis.

8. Dissection of subcutaneous tissue from the anterior cervical muscle

1. Lift the skin along both sides of the incision.
2. Use a scissor to longitudinally cut along the linea alba cervicalis.
3. Use forceps to separate the sternohyoid muscle and the sternothyroid muscle.

9. Fix the anterior neck muscles to both sides

1. Use vascular forceps to clamp the separated sternohyoid muscle and sternothyroid muscle in front of the neck and pull the clamped tissue outside.
2. Use a retractor or the needle to pass the suture (3-0#) through the clamped tissue, make a knot, and fix the suture to the surgical drape of the operation table.

10. Thyroid gland localization

1. Locate the thyroid cartilage and cricoid cartilage as the upper boundary in the operation area. Identify the thyroid cartilage based on its shield shape and the cricoid cartilage based on its ring shape.
2. Locate the trachea as the lower boundary in the operation area. Look for the trachea in the front and middle of the neck, based on its tubular cartilage ring shape.
3. Locate the thyroid gland between the upper and lower boundaries-a red gland in the shape of a butterfly on the opposite side of the trachea.

11. Visual identification of the PG

1. Locate the PG on the upper and outer sides of the thyroid gland. Look for two PG in a fusiform shape of about 1.2-2 mm in length and 1.0-1.5 mm in width that are reddish but lighter than the surrounding thyroid gland with a certain boundary.
2. Take a frontal photograph of the PG with the trachea, thyroid, and larynx to quantitatively compare the effects of IONP before and after injection. 
3. Dissect the back of the esophagus, then use the retractor to expose the right side of the PG. Take a right-side photograph of the PG with the thyroid gland and trachea.
4. Swap the retractor to expose the opposite side of the PG and take a left-side photograph of them with the thyroid gland and trachea.

12. Thyroid injection of the IONPs

1. Use an insulin syringe to locally inject 10 µL of IONPs suspension (20 mg/mL in phosphate-buffered saline) into the center of the thyroid gland. Gently press the injection site with gauze for 5 s.

13. Identification of the PG after IONPs injection

1. After injection, observe the rapid diffusion of the IONPs within the thyroid glands but not the PG, as it negatively stains the PG and differentiates them from the surrounding thyroid.
2. Take a front photograph of the negatively stained PG together with the trachea, thyroid gland, and larynx.
3. Take left- and right-side photographs of the negatively stained PG using the same procedures as mentioned above.

14. Resection of the throat and trachea with the thyroid gland and PG

1. Once the rats have inhaled excess isoflurane (5% isoflurane for more than 5 min) and are under deep anesthesia, euthanize them by intracardiac injection of 0.5 mL of saturated potassium chloride solution.
2. Postmortem, remove the throat, trachea, thyroid gland, and PG.
3. Under a fume hood, place the removed throat, trachea, thyroid gland, and PG specimens into 4% paraformaldehyde solution for 24 h.

15. Histopathology studies

1. Dehydrate the tissues and embed them in paraffin. Slice into 5 µm thick sections. Bake the sections at 37 °C in an oven overnight and at 65 °C for 1 h.
2. Stain the sections with hematoxylin and eosin (H&E) after washing 3 x 5 min with 75%, 95%, 100% gradient alcohol, and water washing at room temperature.
3. Have pathologists examine the H&E-stained sections under a light microscope.

Representative Results

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 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). 

After injection, the contrast agent (Figure 1 and Figure 2), IONPs, readily diffuse within the thyroid gland and stain it black, but cannot infiltrate the PG due to their high tissue density. The imbalanced distribution of the IONPs between the PG and thyroid gland yields a striking contrast, which can be readily visualized by the naked eye without requiring external instruments. Figure 2 shows representative images of PG negatively stained by IONPs in the left thyroid of the rat, in which the contrast between the PG and the thyroid gland was remarkable, and the size of rat PG was determined to be approximately 2 mm x 1 mm.

Postmortem, the rat larynx and the adjacent trachea, esophagus, thyroid, and PG were resected for histopathological staining. Serial sections of the tissue containing PG were obtained to perform H&E staining. These H&E-stained images (Figure 3) revealed that the PG are enriched with closely aligned chief cells, whereas the thyroid gland features many loose lumens indicating much lower tissue density.

Figure 1: The physiological structure of PG and their microenvironment. Schematic illustration of human PG and thyroid gland at pre- (A) and post-IONPs injection (B). Representative biopsy images of rat anterior cervical tissues, including the PG, thyroid gland, trachea, and larynx at pre- (C) and post-IONPs injection (D). Additional images have been published in our previous study¹⁵. Abbreviations: PG = parathyroid glands; IONPs = iron oxide nanoparticles; IONP10 = IONPs of 10 nm diameter; the scale is in centimeters (cm). Please click here to view a larger version of this figure.

Figure 2: Intraoperative IONPs-enhanced PG identification. Representative images of untreated (A) and IONPs-injected (B) rat thyroid gland lobes at pre-and post-IONPs injection. The efficacy of IONPs-enhanced PG identification is consistent in reproducible at pre- (C) and post-IONPs injection (D). Abbreviations: PG = parathyroid gland; IONPs = iron oxide nanoparticles; IONP10 = IONPs of 10 nm diameter. Please click here to view a larger version of this figure.

Figure 3: Histological analysis of a IONPs-injected thyroid gland and its microenvironment. (A) Representative ex vivo photographs of rat anterior cervical tissues at post-IONPs injection. (B) Representative H&E-stained images of rat PG. Scale bar = 50 µm. (C) Zoomed-in image of the dashed red box in panel B. Scale bar = 20 µm. Please click here to view a larger version of this figure.

Discussion

We demonstrate an IONPs-guided negative imaging technique of rat PG 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 with naked eyes without the aid of any microscope. Although transgenic mice with green fluorescent protein selectively expressed in the PG have been reported¹³, the model described in this paper is more straightforward to perform. It only takes ~1 min per rat after injection, and a clear difference between the thyroid gland and PG can be observed with naked eyes.

In addition, another advantage of this model is that the cost and operational difficulty are considerably lower for this rat model than for large animal models (e.g., dogs¹²) currently used in preclinical studies to evaluate new PG identification methods. The average cost of an SD rat is close to that of a BALB/C mouse, which is over 30 times cheaper than a dog. This low-cost advantage of the rat model allows for expanding subject numbers and repeat tests in preclinical research, which is difficult with large animal models. Meanwhile, the typical body weight of an SD rat is 300-350 g, which is also over 66-fold lighter than that of a dog (22-23 kg)¹⁴.

Such a large body weight difference tremendously reduces the operation difficulty in the rat model over large animal models, since performing thyroidectomy on large animals such as dogs requires more complicated anesthesia and surgery procedures, making it more difficult and technically challenging. The requirement for surgery (basic surgical skills are required) poses a limitation for this model. IONPs used in this study have shown excellent biosafety and biodegradability as previously reported previously¹⁵. Ultimately, we hope that this method of negatively imaging rat PG using IONPs can provide a simple and effective animal model for preclinical studies involving PG identification, thereby facilitating the development of novel PG identification techniques.

Materials

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