Name: the miniature pig: a large animal model for cochlear implant research
Uploaded: 2022-07-28T15:00:00
Description: Watch this Scientific Journal Video about The Miniature Pig: A Large Animal Model for Cochlear Implant Research at JoVE.com

This study was funded by grants from the National Natural Science Foundation of China (Nos. 81970890) and the Chongqing Scientific Research Institution Performance incentive project (Nos. 19540). We thank Anandhan Dhanasingh and Zhi Shu from the MED-EL company for their support.

All procedures and animal surgeries were conducted according to the guidelines of the Ethics Committee of the PLA General Hospital and were approved.
1. Anesthesia and surgical preparation
<ol>
	<li>Inject the pig (male, 2 months old, 5 kg) muscularly with tiletamine and zolazepam with a dosage of 10-15 mg/kg and intubate it with a 5.5-French endotracheal tube. Maintain anesthesia by ventilator-assisted respiration with isofluorane inhalation. Monitor the oxygen saturation (...

<ol>
	<li>World report on hearing. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/world-report-on-hearing">https://www.who.int/publications/i/item/world-report-on-hearing</a> (2021)</li><li>Lee, S. Y., 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=Natural+course+of+residual+hearing+preservation+with+a+slim,+modiolar+cochlear+implant+electrode+array.">Natural course of residual hearing preservation with a slim, modiolar cochlear implant electrode array.</a> American Journal of Otolaryngology. 43 (2), 103382 (2022).</li><li>Lorens, A., 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=Binaural+advantages+in+using+a+cochlear+implant+for+adults+with+profound+unilateral+hearing+loss.">Binaural advantages in using a cochlear implant for adults with profound unilateral hearing loss.</a> Acta Oto-Laryngologica. 139 (2), 153-161 (2019).</li><li>Lally, J. W., Adams, J. K., Wilkerson, B. 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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=Development+of+a+chronically-implanted+mouse+model+for+studies+of+cochlear+health+and+implant+function.">Development of a chronically-implanted mouse model for studies of cochlear health and implant function.</a> Hearing Research. 404, 108216 (2021).</li><li>Toulemonde, P., 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=Evaluation+of+the+efficacy+of+dexamethasone-eluting+electrode+array+on+the+post-implant+cochlear+fibrotic+reaction+by+three-dimensional+immunofluorescence+analysis+in+Mongolian+gerbil+cochlea.">Evaluation of the efficacy of dexamethasone-eluting electrode array on the post-implant cochlear fibrotic reaction by three-dimensional immunofluorescence analysis in Mongolian gerbil cochlea.</a> Journal of Clinic Medicine. 10 (15), 3315 (2021).</li><li>King, J., Shehu, I., Roland, J. T., Svirsky, M. A., Froemke, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiological+and+behavioral+system+for+hearing+restoration+with+cochlear+implants.">A physiological and behavioral system for hearing restoration with cochlear implants.</a> Journal of Neurophysiology. 116 (2), 844-858 (2016).</li><li>Chen, M., Min, S., Zhang, C., Hu, X., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+extracochlear+multichannel+electrical+stimulation+to+relieve+tinnitus+and+reverse+tinnitus-related+auditory-somatosensory+plasticity+in+the+cochlear+nucleus.">Using extracochlear multichannel electrical stimulation to relieve tinnitus and reverse tinnitus-related auditory-somatosensory plasticity in the cochlear nucleus.</a> Neuromodulation. , (2021).</li><li>Yi, H., 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=Miniature+pigs:+A+large+animal+model+of+cochlear+implantation.">Miniature pigs: A large animal model of cochlear implantation.</a> American Journal of Translational Research. 8 (12), 5494-5502 (2016).</li><li>Vollmer, M., Beitel, R. E., Schreiner, C. E., Leake, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+stimulation+and+behavioral+training+differentially+transform+temporal+processing+in+the+inferior+colliculus+and+primary+auditory+cortex.">Passive stimulation and behavioral training differentially transform temporal processing in the inferior colliculus and primary auditory cortex.</a> Journal of Neurophysiology. 117 (1), 47-64 (2017).</li><li>Sunwoo, W., Delgutte, B., Chung, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+bilateral+cochlear+implant+stimulation+partially+restores+neural+binaural+sensitivity+in+neonatally-deaf+rabbits.">Chronic bilateral cochlear implant stimulation partially restores neural binaural sensitivity in neonatally-deaf rabbits.</a> The Journal of Neuroscience. 41 (16), 3651-3664 (2021).</li><li>Mantokoudis, G., 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=Lamb+temporal+bone+as+a+surgical+training+model+of+round+window+cochlear+implant+electrode+insertion.">Lamb temporal bone as a surgical training model of round window cochlear implant electrode insertion.</a> Otology &amp; Neurotology. 37 (1), 52-56 (2016).</li><li>de Abajo, 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=Effects+of+implantation+and+reimplantation+of+cochlear+implant+electrodes+in+an+in+vivo+animal+experimental+model+(Macaca+fascicularis).">Effects of implantation and reimplantation of cochlear implant electrodes in an in vivo animal experimental model (Macaca fascicularis).</a> Ear and Hearing. 38 (1), 57-68 (2017).</li><li>Johnson, L. A., Della Santina, C. C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+bone+characterization+and+cochlear+implant+feasibility+in+the+common+marmoset+(Callithrix+jacchus).">Temporal bone characterization and cochlear implant feasibility in the common marmoset (Callithrix jacchus).</a> Hearing Research. 290 (1-2), 37-44 (2012).</li><li>Yin, P., Li, S., Li, X. J., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+pathogenic+insights+from+large+animal+models+of+neurodegenerative+diseases.">New pathogenic insights from large animal models of neurodegenerative diseases.</a> Protein &amp; Cell. , (2022).</li><li>Yu, S. M., Wang, C. W., Zhao, D. M., Zhang, Q. C., Pei, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raising+and+pathogen+purification+of+Chinese+experimental+mini-pig.">Raising and pathogen purification of Chinese experimental mini-pig.</a> Laboratory Animal Science and Administration. 20, 44-46 (2003).</li><li>Guo, W., 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=The+morphology+and+electrophysiology+of+the+cochlea+of+the+miniature+pig.">The morphology and electrophysiology of the cochlea of the miniature pig.</a> The Anatomical Record. 298 (3), 494-500 (2015).</li><li>Christov, F., 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=Electric+compound+action+potentials+(ECAPs)+and+impedances+in+an+open+and+closed+operative+site+during+cochlear+implantation.">Electric compound action potentials (ECAPs) and impedances in an open and closed operative site during cochlear implantation.</a> Cochlear Implants International. 20 (1), 23-30 (2019).</li><li>Zhong, L. L., 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=Inner+ear+structure+of+miniature+pigs+measured+by+multi-planar+reconstruction+techniques.">Inner ear structure of miniature pigs measured by multi-planar reconstruction techniques.</a> American Journal of Translational Research. 10 (3), 709-717 (2018).</li><li>The Lancet. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+loss:+An+important+global+health+concern.">Hearing loss: An important global health concern.</a> The Lancet. 387 (10036), 2351 (2016).</li><li>Guo, R., 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=Cochlear+implant-based+electric-acoustic+stimulation+modulates+neural+stem+cell-derived+neural+regeneration.">Cochlear implant-based electric-acoustic stimulation modulates neural stem cell-derived neural regeneration.</a> Journal of Materials Chemistry B. 9 (37), 7793-7804 (2021).</li><li>Gabrielpillai, J., Geissler, C., Stock, B., St&#246;ver, T., Diensthuber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone+promotes+neurite+growth+of+spiral+ganglion+neurons.">Growth hormone promotes neurite growth of spiral ganglion neurons.</a> Neuroreport. 29 (8), 637-642 (2018).</li><li>Li, H., 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=Guided+growth+of+auditory+neurons:+Bioactive+particles+towards+gapless+neural+-+electrode+interface.">Guided growth of auditory neurons: Bioactive particles towards gapless neural - electrode interface.</a> Biomaterials. 122, 1-9 (2017).</li><li>Wille, I., 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=Development+of+neuronal+guidance+fibers+for+stimulating+electrodes:+Basic+construction+and+delivery+of+a+growth+factor.">Development of neuronal guidance fibers for stimulating electrodes: Basic construction and delivery of a growth factor.</a> Frontiers in Bioengineering and Biotechnology. 10, 776890 (2022).</li></ol>

Around 15% of the world's population have some degree of hearing loss, and over 5% have disabling hearing loss21. CI provision is the most efficient treatment for both adult and pediatric patients with severe and profound sensorineural hearing loss. As the first successful implantable cranial nerve stimulator, over the past 2 decades, CIs have offered thousands of people with hearing loss the opportunity to return to the world of sound and (re)integrate into mainstream society. Even though CIs are...

According to the World Health Organization (WHO), over 1 billion people are at risk of hearing loss globally, and it is estimated that, by 2050, one out of four people will suffer hearing loss1. Over the last 2 decades, CIs have been the most effective intervention for people with permanent severe and profound sensorineural hearing loss (SNHL). A CI converts physical signals of sound into bioelectrical signals that stimulate the spiral ganglion neurons (SGNs), bypassing hair cells. Over time, the indications for a CI have been broadened so that they now include people with residual hearing, unilateral hearing loss, and very old or young people2,3,4. Meanwhile, totally implantable CIs and advanced arrays have been developed5. There is, however, no economically feasible large animal model for investigating the electrophysiology and histopathology of the inner ear with a CI. This lack of a large animal model limits research seeking to improve CIs and gain insights into the electrophysiological impact of CIs on the inner ear.
Several rodent animal models have been applied in CI research, such as mouse6, gerbil7, rat8, and guinea pig9; however, the characteristics of morphology and electrophysiological responses are different from that in humans. Cochlear structures of animal models traditionally used for CI studies, such as cats, guinea pigs, and other animals, differ greatly from those of human cochlear structures10. Although array insertion has been conducted on cats11 and rabbits12, because of their smaller cochleae, this was done with arrays that were not designed for use in humans. Several large animal models have also been explored for CI. Lambs are well suited as a training model for atraumatic cochlear implantation, but the smaller size of the cochlea makes full array insertion impossible13. Primates might be the most suitable animals for CI research because of their anatomical similarity to humans14,15; however, the sexual maturity of monkeys is delayed (4-5 years), the gestation period is up to about 165 days, and each female usually produces only one offspring per year16. These reasons, and the expensive cost, hinder the extensive application of primates in CI research.
In contrast, pigs reach sexual maturity at 5-8 months and have a gestation period of ~114 days, making pigs more accessible for CI research as a large animal model16. Bama mini pigs (mini-pigs) originated from a small-sized pig species in China in 1985, whose genetic background is well understood. They are characterized by an inherent small size, early sexual maturity, rapid breeding, and ease of management17. The mini-pig is an ideal model for otology and audiology because of its similarity to humans in morphology and electrophysiology18. The scala tympani length of a Bama mini-pig is 38.58 mm, which is close to the 36 mm length in humans10. The mini-pig cochlea has 3.5 turns, which is similar to the 2.5-3 turns seen in humans10. In addition to morphology, the electrophysiology of Bama mini-pigs is also highly similar to that of humans18. Therefore, in the present study, we inserted arrays designed for human use into the mini-pig cochlea via the round window membrane and followed a similar surgical approach to that used in human CI recipients. Intraoperative ECAP measurements were applied to evaluate the procedure. The process we describe herein could be used both for preclinical translational research associated with CIs and as a platform for resident training.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mm diamond burr</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm diamond burr</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm diamond burr</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 suture silk</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Slicer image computing platform</td>
			<td></td>
			<td></td>
			<td>3D reconstruction of CT image</td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cautery</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar electrocoagulation</td>
			<td></td>
			<td></td>
			<td>Stop bleeding</td>
		</tr>
		<tr>
			<td>CI designed for human use (CONCERTO FLEX28)</td>
			<td>MED-EL</td>
			<td>&#160;Concerto F28</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECG monitor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine tincture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td>3.6 mL/h</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MAESTRO Software</td>
			<td>MED-EL</td>
			<td></td>
			<td>Measure ECAP responses</td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Negative pressure suction device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Otological surgical instruments&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Respiratory Anesthesia Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel with blade No. 15</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulation device (MAX Programming Interface)</td>
			<td>MED-EL</td>
			<td></td>
			<td>Measure ECAP responses</td>
		</tr>
		<tr>
			<td>Surgery microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Power Device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine and zolazepan</td>
			<td></td>
			<td></td>
			<td>10-15 mg/kg</td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trachea cannula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the miniature pig: a large animal model for cochlear implant research

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans. 
To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements. 
The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

The integrity (Figure 4A)&#160;and impedances (Figure 4B) of the CI were confirmed by MAESTRO Software. ECAP results showed that all 12 electrodes demonstrated good neural responses (Figure 4C), meaning the electrode array was well attached to the cochlear axis and stimulated the auditory nerve. Figure 5 demonstrates postoperative 3D reconstructed electrode coils in the right cochlea. The array did not ...

Watch this Scientific Journal Video about The Miniature Pig: A Large Animal Model for Cochlear Implant Research at JoVE.com

The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, ...

Cochlear implantation surgery in mini piggers can be utilized to provide intentional evidence of the safety and potential performance of novel electrode areas and the surgical approach in a living system similar to human beings. Mini piggers are an endo lab animal model for research into cochlear implants. Cochlear implants on mini piggers provide an individual platforms for transplant people with severe to profound sensory neuro hearing loss.Moreover, it also provides a surgery loss of gene therapy for hearing loss in big animal model. As they are significant and similar, in morphology and structure of osculatory organs between mini piggers and human beings. This surgery method clarifies the procedure and is helpful for beginners.Demonstrating the procedure will be Ji Xiaojun and Otolaryngology professor and Luo Yi a chief physician from my laboratory. After anesthetizing two months old male pig, monitor the oxygen saturation breathing and heart rate using the pulse oximetry clamp of an ECG monitor connected to the pig's tongue. Place the mini pig in the left lateral position.Keep the eyes closed using a medical patch to prevent dryness. Shave the 10 centimeter surgical area around the ear lobe. Disinfect the skin with three alternating swabs of iodine and alcohol.Then cover the surgical area with sterile surgical drapes. Cover the microscope with a sterile plastic sleeve and remove the parts covering the eye pieces and objective. Locate the surface projection site of the cochlea one centimeter behind the posterior auricular sulcus at the level of the ear lobe.Then, using a scalpel, make a 5 centimeter long postauricular incision with the projection site as the center. Divide the subcutaneous tissue, parotid gland and sternocleidomastoid muscle with micro scissors to expose the surface of the mastoid bone. For cortical mastoidectomy, drill the mastoid at the surface projection of the cochlea on the mastoid bone to the external auditory canal or EAC which is dense and pale blue.Be careful not to damage the pale or reddish vertical segment of the facial nerve dorsal to the EAC to avoid bleeding. Next, expose the tympanum by drilling the bone surrounding the posterior bony EAC. Separate the skin of the EAC and the facial nerve with a hypodermic needle to avoid damaging the facial nerve.Carefully push the skin of the EAC away to expose the tympanum including the ossicular chain and round window niche. Then, remove the round window niche with a small diamond bur and keep continuous suction irrigation to expose the basal turn of the cochlea and round window membrane. Separate the cranial parietal muscle to form a pocket for the receiver.Place the internal receiver package in the muscular pocket and fix it with a fixation suture. Next, insert the electrode array connected to a receiver fixed in a muscular pocket by opening the round window membrane with a sharp microsurgical knife and inserting the array using micro forceps slowly, steadily and continuously in relationship to the modiolus of the cochlea. For evoked compound action potential or ECAP measurements, magnetically connect to the cochlear implant or CI coil to the CI receiver through the skin.Then using Maestro software, confirm the integrity of the CI and check the electrode impedance for all channels before ECAP measurements using the telemetry function of the CI.To conduct ECAP measurement, select the ECAP module. Then, stimulate all 12 electrodes for ECAP measurements using biphasic stimuli of 30 microseconds phase duration with an alternating polarity paradigm averaging over 25 iterations in a stimulation rate of 45.1 pulses per second. The integrity and impedances of the CI were confirmed by Maestro software.ECAP results demonstrated that all 12 electrodes showed good neural responses indicating that the electrode array was well attached to the cochlear axis and stimulated the auditory nerve. The post-operative three-dimensional reconstructed electrode coils in the right cochlea are shown. The electrode array was coiled in the basal turn of the cochlea in the electrodes were rendered in green.Further, three-dimensional reconstruction demonstrates that the electrode array was spirally coiled in the cochlea. The most important thing is to expose the vessel tint of the cochlear and run window member and inside the electrode airways in the protocol. After ace development is technically pales away for combined gene therapy and cochlear implants to treat profound sensor neuro hearing loss in human beings.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

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 ...

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.