Name: establishment of a simple and effective rat model for intraoperative parathyroid gland imaging
Uploaded: 2022-08-17T14:05:00.000+00:00
Duration: 7 min 12 s
Description: Watch this Scientific Journal Video about Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging at JoVE.com

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 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).</li>
	<li>Put the SD rat into the box of the anesthesia machine and select the Channel model to begin animal anesthesia.</li>
	<li>Observe the rat activity in the box. When the rat falls into a coma, move&#160;it to&#160;the nose cone to maintain anesthesia&#160;(unconscious supine position without pain reflex and corneal reflex).</li>
	<li>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.</li>
</ol>
3. Posture and fixation
<ol>
	<li>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&#39;s body temperature during the surgery.</li>
	<li>Use rubber bands to fix the rat&#39;s limbs to the operation table. Place a cylindrical pillow made of drape under the rat&#39;s shoulder to lean its head back, completely exposing the neck area.&#160;</li>
	<li>Apply artificial tears ointment on both eyes of the rat to prevent dryness during anesthesia.</li>
</ol>
4. Hair removal
<ol>
	<li>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.</li>
	<li>After 3 min, gently wipe the hair and depilatory cream with a tissue.</li>
</ol>
5. Sterilization
<ol>
	<li>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.</li>
</ol>
6. Surgical drape laying
<ol>
	<li>Use a surgical drape to cover the operation area of the rat&#39;s neck. Keep the hole of the surgical drape aligned with the disinfection area of the animal.</li>
</ol>
7. Incision
<ol>
	<li>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&#39;s neck. Ensure that the incision length is approximately 5 cm and only in the dermis.</li>
</ol>
8. Dissection of subcutaneous tissue from the anterior cervical muscle
<ol>
	<li>Lift the skin along both sides of the incision.</li>
	<li>Use a scissor&#160;to longitudinally cut along the linea alba cervicalis.</li>
	<li>Use forceps to separate the sternohyoid muscle and the sternothyroid muscle.</li>
</ol>
9. Fix the anterior neck muscles to both sides
<ol>
	<li>Use vascular forceps to clamp the separated sternohyoid muscle and sternothyroid muscle in front of the neck and pull the clamped tissue outside.</li>
	<li>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.</li>
</ol>
10. Thyroid gland localization
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
11. Visual identification of the PG
<ol>
	<li>Locate the PG&#160;on the upper and outer sides of the thyroid gland. Look for two PG&#160;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.</li>
	<li>Take a frontal photograph of the PG&#160;with the trachea, thyroid, and larynx to quantitatively compare the effects of IONP before and after injection.&#160;</li>
	<li>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&#160;with the thyroid gland and trachea.</li>
	<li>Swap the retractor to expose the opposite side of the PG&#160;and take a left-side photograph of them with the thyroid gland and trachea.</li>
</ol>
12. Thyroid injection of the IONPs
<ol>
	<li>Use an insulin syringe to locally inject 10 &#181;L 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.</li>
</ol>
13. Identification of the PG&#160;after IONPs injection
<ol>
	<li>After injection, observe the rapid diffusion of the IONPs within the thyroid glands but not the PG, as it negatively stains the PG&#160;and differentiates them from the surrounding thyroid.</li>
	<li>Take a front photograph of the negatively stained PG&#160;together with the trachea, thyroid gland, and larynx.</li>
	<li>Take left- and right-side photographs of the negatively stained PG&#160;using the same procedures as mentioned above.</li>
</ol>
14. Resection of the throat and trachea with the thyroid gland and PG
<ol>
	<li>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.</li>
	<li>Postmortem, remove the throat, trachea, thyroid gland, and PG.</li>
	<li>Under a fume hood, place the removed throat, trachea, thyroid gland, and PG specimens into 4% paraformaldehyde solution for 24 h.</li>
</ol>
15. Histopathology studies
<ol>
	<li>Dehydrate the tissues and embed them in paraffin. Slice into 5 &#181;m thick sections. Bake the sections at 37 &#176;C in an oven overnight and at 65 &#176;C for 1 h.</li>
	<li>Stain the sections with hematoxylin and eosin (H&#38;E) after washing 3 x 5 min with 75%, 95%, 100% gradient alcohol, and water washing at room temperature.</li>
	<li>Have pathologists examine the H&#38;E-stained sections under a light microscope.</li>
</ol>

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G., Singer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocalcemia-induced+heart+failure+as+the+initial+symptom+of+hypoparathyroidism.">Hypocalcemia-induced heart failure as the initial symptom of hypoparathyroidism.</a> Zeitschrift f&#252;r Kardiologie. 88 (1), 10-13 (1999).</li><li>Shoback, D. M., 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=Presentation+of+hypoparathyroidism:+etiologies+and+clinical+features.">Presentation of hypoparathyroidism: etiologies and clinical features.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 101 (6), 2300-2312 (2016).</li><li>Arneiro, A. 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. 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P.G. and W.Z. are co-inventors of a patent application filed by the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital) based on the project. The other authors declare no conflicts of interest.

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><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>&lt;br/&gt; 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;
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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 ...

establishment-simple-effective-rat-model-for-intraoperative

Research

JoVE Journal

Medicine

3.2K Views.  Zhejiang Cancer Hospital. Bijschildklier is een klein orgaan met zeer belangrijke functies. Ons protocol kan worden gebruikt voor het ontwikkelen van nieuwe PG-identificatietechnologieën die chirurgen kunnen helpen om het te beschermen tijdens de thyroidectomie voor schildklierkanker. Het belangrijkste voordeel van dit model is dat het een duidelijk verschil tussen de schildklier en PG's kan waarnemen door de negatieve beeldvorming van IONP.Bovendien zijn de kosten en operationele moeilijkheidsgraad aanzienlij...

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

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Cancer Research

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Bioengineering

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

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. 
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most ...

Intra-Operative Neural Monitoring of Thyroid Surgery in a Porcine Model

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere with breathing. In recent years, intraoperative neural monitoring (IONM) has been widely adapted as an adjunct technique to localize the RLN, detect RLN injury, and predict vocal cord function during the operations. Many studies have also used animal models to investigate new applications of IONM technology and to develop reliable strategies for preventing intraoperative RLN injury. The aim of this article is to introduce a standard protocol for using a porcine model in IONM research. The article demonstrates the procedures for inducing general anesthesia, performing tracheal intubation, and experimental design to investigate the electrophysiological characteristics of RLN injuries. Applications of this protocol can improve overall efficacy in implementing the 3R principle (replacement, reduction and refinement) in porcine IONM studies.

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

Intraoperative injury to the recurrent laryngeal nerve (RLN) can cause vocal cord paralysis, which interferes with speech and can potentially interfere ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Neuroscience

Investigation of the Electrophysiological and Thermographic Safety Parameters of Surgical Energy Devices During Thyroid and Parathyroid Surgery in a Porcine Model

In thyroid and parathyroid surgery, surgical energy devices (SEDs) provide more efficient hemostasis than conventional clamp-and-tie hemostasis in areas with rich blood supply. However, when a SED is activated near the recurrent laryngeal nerve (RLN), the heat generated by the SED may injure the nerve irreversibly. To safely apply SEDs in thyroid/parathyroid surgery, this article introduces experimental porcine model studies to investigate the activation and cooling safety parameters of SEDs in standardized electrophysiological (EP) and thermographic (TG) procedures, respectively. In the EP safety parameter experiments, continuous intraoperative neuromonitoring (C-IONM) is applied to demonstrate the RLN function in real-time. The EP activation study evaluates the safe activation distance of SEDs; the EP cooling study evaluates the safe cooling time of SEDs. In the TG safety parameter experiment, a thermal imaging camera is used to record the temperature change after activating the SED. The TG activation study evaluates the lateral thermal spread distance after SED activation in a dry or humid environment and whether smoke and splashing are generated; the TG cooling study evaluates the cooling time. This will help establish the safety parameters of newly developed SEDs used in thyroid/parathyroid surgery and provide safety guidelines to avoid RLN injury and related complications.

The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery.

In thyroid and parathyroid surgery, surgical energy devices (SEDs) provide more efficient hemostasis than conventional clamp-and-tie hemostasis in areas ...

Transoral Endoscopic Thyroidectomy Vestibular Approach for Thyroid Lobectomy

The manuscript describes the transoral endoscopic thyroidectomy vestibular approach (TOETVA) for thyroid lobectomy. The patient is placed in the supine position with extension and fixation of the neck. One 20 mm transverse incision and two 5 mm incisions are made through the mucosa of the oral vestibule for camera and instrument placement after disinfection of the skin and oral cavity. The workspace is established and maintained by the skin suspension device, which is made of unabsorbable string (3-0) and rubber bands, and the CO2 insufflation pressure. Lobectomy using a medial-to-lateral technique and prophylactic ipsilateral central neck dissection is performed simultaneously on patients with papillary thyroid cancer (PTC). The specimen is extracted through the 20 mm incision. The parathyroid gland is immediately searched for in the specimen and auto-transplanted to the left brachioradialis. A drainage tube is inserted through the retractor hole into the bed of the thyroid gland, and absorbable sutures are used to close the mucosal incisions in the oral vestibule and the linea alba cervicalis. Prophylactics administered intravenously are recommended for the first 24 h after surgery, and oral antibiotics are used for 7 days postoperatively.

Here, we present a protocol to describe the methodology for the transoral endoscopic thyroidectomy vestibular approach in detail.

The manuscript describes the transoral endoscopic thyroidectomy vestibular approach (TOETVA) for thyroid lobectomy. The patient is placed in the supine ...

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.

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.

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

Transoral Robotic Total Thyroidectomy and Bilateral Central Regional Lymph Node Dissection for Papillary Thyroid Carcinoma

Currently, the common approaches to thyroid surgery include conventional thyroidectomy, bilateral axillo-breast, axillary, retroauricular, and oral vestibule approaches. Essentially, various approaches to thyroid surgery only move the traditional surgical incision to a more concealed position but still leave scars on the body surface. Among them, thyroid surgery via the oral vestibule approach can obtain the best cosmetic outcome through the shortest natural cavity. However, in the early time, thyroid surgery via the oral vestibule approach usually requires three incisions in the mouth and one axillary incision. We have introduced a robotic surgical system into thyroidectomy via the oral vestibule approach and successfully completed total thyroidectomy and bilateral central lymph node dissection. During the operation, only three incisions were made at the oral vestibule without an axillary incision. This article aims to present the unique three-port method of robotic thyroidectomy via oral vestibule for the treatment of patients with papillary thyroid cancer.

The transoral robotic total thyroidectomy and bilateral central regional lymph node dissection using three ports without axillary incision are feasible and safe for early-stage papillary thyroid carcinoma. Here we present the operative techniques of this operation.

Currently, the common approaches to thyroid surgery include conventional thyroidectomy, bilateral axillo-breast, axillary, retroauricular, and oral ...

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

Source: Julia Ramirez-Moya, et al. <a href="https://www.jove.com/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

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

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