This work was funded and supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1C1A1A01051907). This work was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT &#38; Future Planning (NRF-2016M3A9E9941746).

This study was guided and approved by the Department of Laboratory Animal Resources, Yonsei Biomedical Research Institute, Yonsei University College of Medicine, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. This study followed the guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council. All Pigs were acclimated for One week before the operation.
1. Preparation
<ol>
	<li>Keep the pigs without food for over 12 h before general anesthesia with free access to water. 
	Note: Six female Yorkshire pigs were used weighing 25&#8211;30 kg each.</li>
	<li>Use a 16 Gauge Needle of 1 cm length to intramuscularly inject alfaxan (1 mg/kg), xylazine (2 mg/kg), and azaperone (2 mg/kg) behind and below the ear to initiate anesthesia.</li>
	<li>Shave the anterior midline of the neck and the abdominal wall of the pig using a surgical hair removal shaver.</li>
	<li>Secure an intravenous (IV) route through central or marginal ear vein on the posterior side of the auricle with a 22-G needle. Inject Ketorolac (1 mg/kg) via an IV line.</li>
	<li>Then inject atropine (0.04 mg/kg) intramuscularly as described in 1.2. Inject cefazolin (30 mg/kg) intramuscularly as described in 1.2.</li>
	<li>Put the pig lying down in a supine position over the Operating room table.</li>
	<li>Allow the pig to breath 2 L of oxygen with 5% of isoflurane through Swine anesthesia mask spontaneously.</li>
	<li>Expose the vocal cords through the mouth by using a laryngoscope and spray them with two puffs of 2% Lidocaine topical solution to prevent intubation-induced laryngospasm.</li>
	<li>Intubate with a 6.5 mm tube, inflate the tube cuff with 3-5 mL of air using a syringe without a needle attached to it. 
	Note: Perform capnometry to ensure that the tracheal tube, which is a part of the anesthesia machine, is in proper position and there is CO2 back to the machine as an indication of proper oxygenation.</li>
	<li>Maintain the anesthesia after the intubation with 2% isoflurane.</li>
	<li>Use the vet ointment on pig&#8217;s eyes and close it with an eye cover patch.</li>
</ol>
2. Procedure: Reception Site
<ol>
	<li>Disinfect the neck and the abdominal wall with the iodine-based scrub solution 1%.</li>
	<li>Start a vertical midline incision in the anterior neck using blade No. 23 up to the sternum.</li>
	<li>Dissect the strap muscles and retract it laterally using Kelly tissue scissor and Lahey retractor.</li>
	<li>Expose the trachea from the first ring to the thoracic inlet.</li>
	<li>Then, expose the common carotid artery and the internal jugular vein for the anastomosis.</li>
	<li>Create a window in the second or third tracheal cartilage, ~1 cm in width using blade no. 11.</li>
	<li>Look for the endotracheal tube through the tracheal defect created, and make sure the aeration through the tube is continued using the ventilator without a leak.</li>
</ol>
3. Procedure: Flap Site
Note: The superior epigastric artery perforator (SEAP) flap harvest can be performed, according to the method described by Frederic Bodin7.
<ol>
	<li>Design a flap on the upper abdomen by a surgical marker pen (Figure 1A).</li>
	<li>Create an elliptical Skin incision 4 x 3 cm using a No. 23 scalpel to the anterior sheath of the abdominal wall on the medial side of the drawn flap (Figure 1B).</li>
	<li>Elevate (peel) the Flap from the rectus abdominis muscle sheath looking for the perforators going to the skin flap while holding the fascia by the allis.</li>
	<li>Perform the intramuscular dissection and follow the perforators to the superior epigastric vessels (Figure 1B)</li>
	<li>Now perform the lateral skin incision on the designed flap (Figure 1A) using a No. 23 scalpel (Figure 1C)</li>
	<li>Clamp the superior epigastric vessels and venae comitantes with the hemostat superiorly and use Kelly tissue scissors to cut below the hemostat and ligate the vessel above the hemostat by 3-0 suture. Then take off the hemostat.</li>
</ol>
4. Anastomosis and Closure
<ol>
	<li>Put on a surgical loupe.</li>
	<li>Clamp the Carotid artery using two hemostats with 1-2 cm distance between them.</li>
	<li>Use micro-scissors to cut between the carotid artery and tie the superior part by double suture insure no oozing.</li>
	<li>Use the double clamp without the frame between the carotid artery and flap artery.</li>
	<li>Start anastomosis by using 10-0 suture simple full thickness interpreted.</li>
	<li>Place the first two stay sutures approximately 120 degrees apart on the vessel&#8217;s circumference then in between place 2-3 stitches.</li>
	<li>Release the clamp. If there is blood oozing do the similar suture at the oozing site.</li>
	<li>Look for the venous back flow from venae comitantes.</li>
	<li>Perform anastomosis as described in step 4.1 to 4.7 for the internal jugular vein and venae comitantes (Figure 1D).</li>
	<li>Use a syringe needle size 18 to prick the skin to ensure flap viability by seeing a blood drop.</li>
	<li>Close the tracheal window and suture it with the muscle fascia of the SEAP flap using 3-0 suture.</li>
	<li>Exteriorize and suture a skin paddle of the SEAP flap to the cervical midline skin incision (Figure 2A).</li>
	<li>Close the abdominal skin incision (Figure 2B).</li>
</ol>
5. Post-operative Care
<ol>
	<li>Get the pig back into the prone position.</li>
	<li>Stop the isoflurane and wean the pig from the ventilator.</li>
	<li>Allow the pig to recover in the animal cage and monitor it closely to ensure its smooth recovery from the procedure.</li>
	<li>Look for the flap at reconstruction site after healing (Figure 2C).</li>
	<li>Look at the donor site in the animal after healing (Figure 2D).</li>
	<li>Start Ringer&#39;s lactate at the rate of 150 mL/h until full recovery and administer 0.3 mg buprenorphine for analgesia.</li>
	<li>Following extubation, monitor the pig closely until it has regained sufficient consciousness to maintain position and breathing and is able to drink spontaneously.</li>
	<li>Keep the post-surgery pigs in separated place from no operated animals.</li>
	<li>Start intramuscular amoxicillin&#8211;clavulanate (14 mg/kg) for 1 week.</li>
	<li>Start intramuscular Meloxicam (0.2 mg/kg) for 1 week.</li>
</ol>
6. Euthanasia
<ol>
	<li>Start anesthesia for the pig by injecting propofol through i.v. (5-10 mL) and maintain it with isoflurane 5%.</li>
	<li>Intubate the pig as described on step (1.1, 1.2, 1.6-1.10).</li>
	<li>Induce the cardiac arrest by intravenous injection of 40 mmol KCl.</li>
</ol>

<ol>
	<li>Chen, C. L., Zenga, J., Roland, L. T., Pipkorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+double+free+flap+and+free+flap+combined+with+locoregional+flap+in+head+and+neck+reconstruction:+A+systematic+review.">Complications of double free flap and free flap combined with locoregional flap in head and neck reconstruction: A systematic review.</a> Head &amp; Neck journal of the Science and Specialties of the Head and Neck. 40 (3), 632-646 (2018).</li><li>Smith, R. K., Wykes, J., Martin, D. T., Niles, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforator+variability+in+the+anterolateral+thigh+free+flap:+a+systematic+review.">Perforator variability in the anterolateral thigh free flap: a systematic review.</a> Surgical and Radiologic Anatomy. 39 (7), 779-789 (2017).</li><li>Bauer, F., Koerdt, S., H&#246;lzle, F., Mitchell, D. A., Wolff, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+free+flaps+in+24+hours:+a+training+concept+for+postgraduate+teaching+of+how+to+raise+microvascular+free+flaps.">Eight free flaps in 24 hours: a training concept for postgraduate teaching of how to raise microvascular free flaps.</a> British Journal of Oral and Maxillofacial. 54 (1), 35-39 (2016).</li><li>Schoeff, S., Hernandez, B., Robinson, D. J., Jameson, M. J., Shonka, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+Anastomosis+Simulation+Using+a+Chicken+Thigh+Model:+Interval+Versus+Massed+Training.">Microvascular Anastomosis Simulation Using a Chicken Thigh Model: Interval Versus Massed Training.</a> The Laryngoscope. 127 (11), 2490-2494 (2017).</li><li>Bodin, F., Diana, M., Koutsomanis, A., Robert, E., Marescaux, J., Bruant-Rodier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+model+for+free-flap+breast+reconstruction+training.">Porcine model for free-flap breast reconstruction training.</a> Plastic and Reconstructive Surgery Journal. 68 (10), 1402-1409 (2015).</li><li>Rodriguez, J. R., Ya&#241;ez, R., Cifuentes, I., Varas, J., Dagnino, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgery+Workout:+A+Novel+Simulation+Training+Curriculum+Based+on+Nonliving+Models.">Microsurgery Workout: A Novel Simulation Training Curriculum Based on Nonliving Models.</a> Plastic and Reconstructive Surgery Journal. 138 (4), 739e-747e (2016).</li><li>Carey, J. N., 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=Simulation+of+plastic+surgery+and+microvascular+procedures+using+perfused+fresh+human+cadavers.">Simulation of plastic surgery and microvascular procedures using perfused fresh human cadavers.</a> Plastic and Reconstructive Surgery journal. 67 (2), e42-e48 (2014).</li><li>Chan, W., Niranjan, N., Ramakrishnan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+assessment+of+microsurgery+skills+in+the+clinical+setting.">Structured assessment of microsurgery skills in the clinical setting.</a> Plastic and Reconstructive Surgery Journal. 63 (8), 1329-1334 (2010).</li><li>Matsumura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newly+designed+training+tool+for+microvascular+anastomosis+techniques:+Microvascular+Practice+Card.">A newly designed training tool for microvascular anastomosis techniques: Microvascular Practice Card.</a> Surgical Neurology Journal. 71 (5), 616-620 (2010).</li><li>Kim, W. S., 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=Tracheal+reconstruction+with+a+free+vascularized+myofascial+flap:+preclinical+investigation+in+a+porcine+model+to+human+clinical+application.">Tracheal reconstruction with a free vascularized myofascial flap: preclinical investigation in a porcine model to human clinical application.</a> Scientific Reports. 7 (1), 10022 (2017).</li></ol>

The authors have nothing to disclose.

Significant morbidity and defects can occur in head and neck malignancy patients during surgical management. Microvascular free tissue transfer has become essential for reconstruction in most cases. The viability of the flap is a critical issue, requiring steadiness, precise handling of the pedicle, tactile sensation, visuospatial ability, and excellent operative flow from the surgeon8. To develop these skills, one needs extensive practice with a training model3,...

Reconstruction following surgery for the head and neck malignant diseases is a difficult procedure associated with significant morbidity. Microvascular free-flap reconstruction has been well established as the standard approach to reconstruction for over 20 years1,2,3. Free-flap transfer plays a significant role in improving the head and neck management in cancer patients and in post-traumatic injuries thereby pushing the boundaries of surgical excision of disease beyond previous techniques, resulting in greater patient quality of life and longer survival rates1,2,3. The various flaps for reconstruction include rotational, graft, and free flaps.
The role of free flaps in the head and neck reconstruction has expanded. It is the most difficult flap to work with, requiring skilled and delicate handling. Flap failure is a catastrophic event, with significant morbidity4,5. Thus, considerable training time is required to develop the precision necessary for the successful surgical outcomes3,4,5,6,7,8,9. The steep learning curve associated with such a surgery can influence the outcome for patients and affect treatment management3,4,5,6,7,8,9. To reduce the training time and learning curve for new surgeons, a training model is needed that mimics human biology and provides similar surgical field conditions8.
The goal of this study is to show the visibility of Porcine as a good training module for the head and neck microvascular reconstruction resembling the human case with improved skills in the active fashion.
This study investigated the use of a porcine model for training new colleagues in the head and neck microvascular reconstruction for free-flap transfer to provide a cost-effective and less stressful supplement to the clinical field training with reliably similar features for free-flap procedures. Pigs have been used for many studies and as teaching models for various surgical reconstructions, e.g., breast reconstruction;5 however, pigs have never been used for head and neck reconstruction except in our study for tracheal reconstruction due to tracheal stenosis10.
The idea was started after Frederic Bodin7, who describe the similar flap for breast reconstruction. The main advantage for the study over the other module of microvascular training is the active livening module with a real immediate result of the procedure.

<table>
	<tbody>
		<tr>
			<td>Pigs XP Bio, Seoul, South Korea</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Hair Removal shaver</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>22 gage catheter</td>
			<td>B.BRAUN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>syring with needle size 18</td>
			<td>Jung Rim Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intramuscular alfaxan</td>
			<td>Careside</td>
			<td></td>
			<td>10ml/VAL</td>
		</tr>
		<tr>
			<td>Intramuscularxylazine</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intramuscular azaperone</td>
			<td>Sigma-aldrich</td>
			<td></td>
			<td>34223</td>
		</tr>
		<tr>
			<td>Intramuscular atropine</td>
			<td>Daewon</td>
			<td></td>
			<td>0.5mg/A</td>
		</tr>
		<tr>
			<td>Intramuscular cefazolin</td>
			<td>Yuhan</td>
			<td></td>
			<td>1g</td>
		</tr>
		<tr>
			<td>intravenous Ketorolac</td>
			<td>Hana Pharm</td>
			<td></td>
			<td>30mg</td>
		</tr>
		<tr>
			<td>Swine ansthesia mask</td>
			<td>DRE</td>
			<td></td>
			<td>1392</td>
		</tr>
		<tr>
			<td>endotracheal cuff tube 6.5 mm</td>
			<td>SMITH medical</td>
			<td></td>
			<td>100/150/065</td>
		</tr>
		<tr>
			<td>ansthesia Machine</td>
			<td>Dr&#228;ger</td>
			<td></td>
			<td>PRIMUS IE</td>
		</tr>
		<tr>
			<td>2% lidocaine topical solution</td>
			<td>Taejoon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>Pfizer</td>
			<td></td>
			<td>terramycin<img alt="Misc" src="/files/ftp_upload/58104/58104misc1.jpg" /></td>
		</tr>
		<tr>
			<td>eye cover patch</td>
			<td>Innomed</td>
			<td></td>
			<td>S-universal010S</td>
		</tr>
		<tr>
			<td>betadine solution 1%.</td>
			<td>Korea Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gauze 4*4</td>
			<td>First Medical</td>
			<td></td>
			<td>22*30CM 320S</td>
		</tr>
		<tr>
			<td>blade No. 23</td>
			<td>Paragon</td>
			<td></td>
			<td>23</td>
		</tr>
		<tr>
			<td>lahey retractor</td>
			<td>V.Mueller</td>
			<td></td>
			<td>SU3960</td>
		</tr>
		<tr>
			<td>kelly tissue scissors</td>
			<td>SOLCO</td>
			<td></td>
			<td>05-1990</td>
		</tr>
		<tr>
			<td>blade No. 11</td>
			<td>Paragon</td>
			<td></td>
			<td>11</td>
		</tr>
		<tr>
			<td>surgical marking pen</td>
			<td>Aspen Surgical</td>
			<td></td>
			<td>Regular #2750</td>
		</tr>
		<tr>
			<td>allis</td>
			<td>V.Mueller</td>
			<td></td>
			<td>SU4055</td>
		</tr>
		<tr>
			<td>tie suture</td>
			<td>Covidein</td>
			<td></td>
			<td>non-needle</td>
		</tr>
		<tr>
			<td>3.5&#215; surgical loupe</td>
			<td>zeiss</td>
			<td></td>
			<td>eyemag smart</td>
		</tr>
		<tr>
			<td>double clamp without frame</td>
			<td>V.Mueller</td>
			<td></td>
			<td>CH7155</td>
		</tr>
		<tr>
			<td>microscissors</td>
			<td>AESCULAP</td>
			<td></td>
			<td>FD038R</td>
		</tr>
		<tr>
			<td>Ringer&#39;s lactate</td>
			<td>Daehan</td>
			<td></td>
			<td>500ml/1bag</td>
		</tr>
		<tr>
			<td>amoxicillin&#8211;clavulanate</td>
			<td>Ilsung</td>
			<td></td>
			<td>0.6g/V</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Samil</td>
			<td></td>
			<td>7.5mg</td>
		</tr>
		<tr>
			<td>propofol</td>
			<td>Dong Kook</td>
			<td></td>
			<td>120mg/V</td>
		</tr>
		<tr>
			<td>intravenous KCl solution</td>
			<td>Daehan</td>
			<td></td>
			<td>20ml/50P</td>
		</tr>
		<tr>
			<td>mosquito curved</td>
			<td>SOLCO</td>
			<td></td>
			<td>013-0111</td>
		</tr>
		<tr>
			<td>mosquito straight</td>
			<td>SOLCO</td>
			<td></td>
			<td>05-1050</td>
		</tr>
		<tr>
			<td>ethilone 10-0 suture</td>
			<td>ethicone</td>
			<td></td>
			<td>10/0W1756</td>
		</tr>
		<tr>
			<td>Vicryl 3-0.</td>
			<td>ethicone</td>
			<td></td>
			<td>3/0W9890</td>
		</tr>
		<tr>
			<td>buprenorphine</td>
			<td>Hanlim</td>
			<td></td>
			<td>0.3mg</td>
		</tr>
	</tbody>
</table>

porcine as a training module for head and neck microvascular reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

We performed the procedure on six pigs: cervical skin defect reconstruction on two pigs, tracheal reconstruction on two pigs, and free flap to test vascular anastomosis device in two pigs. The pigs were monitored for 3 months and there was no clinical sign of neurological deficit.
The mean time for ischemia was 50 min (range, 35-80 min); the time decreased as the procedure was repeated. The mean harvest time of the six pigs was ...

Watch this Scientific Journal Video about Porcine As a Training Module for Head and Neck Microvascular Reconstruction at JoVE.com

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

porcine-as-training-module-for-head-neck-microvascular

Universidad de Cartagena UDEC

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JoVE Journal

Medicine

7.6K Views.  King Abdullah Medical City. Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Video: Porcine As a Training Module for Head and Neck Microvascular Reconstruction

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual

The middle ear is located in the center of the temporal bone and bears a highly complex anatomy. The recently introduced exclusively endoscopic transcanal approach to the middle ear is a minimally invasive technique sparing the bone and mucosa of the mastoid bone, since the middle ear is accessed through the external auditory canal. This emerging method has several advantages over the traditional (microscopic) approaches to the middle ear such as the panoramic wide-angle views of the anatomy, the possibility to approach and magnify tiny structures, and the possibility of looking around the corner using angled endoscopes.
The cadaveric dissection method presented here consists of an overview on the technical requirements and a precise description of a step-by-step protocol to discover the anatomy of the middle ear. Each step and anatomical structure is carefully described in order to provide a comprehensive guide to endoscopic ear anatomy. In our opinion, this is particularly important to any novice in endoscopic ear surgery as it provides thorough anatomical knowledge and may improve surgical skills.

The aim of this article is to describe the methodology of exclusively endoscopic cadaveric middle ear dissection. Moreover, we aim to provide a comprehensive guide to endoscopic middle ear anatomy.

The middle ear is located in the center of the temporal bone and bears a highly complex anatomy. The recently introduced exclusively endoscopic transcanal ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Endoscopic Septoplasty with Limited Two-line Resection: Minimally Invasive Surgery for Septal Deviation

Endoscopic septoplasty is a surgical procedure in otolaryngology that is commonly performed to treat nasal airway obstruction caused by nasal septal deviation. It has a long history with multiple variations. In this article, a modified endoscopic septoplasty procedure using the limited two-line resection (2LoRs) technique at the posterior and inferior junction of the cartilaginous and bony septum is presented based on embryologic and anatomic knowledge of the nasal septum and the biomechanics of cartilaginous behavior. With this procedure, the quadrangular cartilage can be preserved as much as possible, which is helpful in retaining the supporting framework and rigidness of the septum. 2LoRs has been proven effective and sound for the correction of nasal septal deviation with rare complications. This modified procedure can be applied to correct the deviated nasal septum in the absence of any external nasal deformity to improve nasal patency or to improve access to the middle meatus or to the axillary region of the middle turbinate. It may also be used to expand the indications of septoplasty to children and adolescents because of its minimally invasive approach.

Endoscopic septoplasty is a time-honored surgical procedure with multiple variations. This paper focuses on a step-by-step surgical approach to perform a modified septoplasty procedure known as limited two-line resection. This surgical technique can be applied to correct a deviated nasal septum in the absence of an external nasal deformity.

Endoscopic septoplasty is a surgical procedure in otolaryngology that is commonly performed to treat nasal airway obstruction caused by nasal septal ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...