Name: a surgical procedure for the administration of drugs to the inner ear in a non-human primate common marmoset (callithrix jacchus)
Uploaded: 2018-02-27T12:30:00
Description: Watch this Scientific Journal Video about A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus at JoVE.com

O trabalho é suportado por subvenções M.F. do governo japonês MEXT KAKENHI (24592560, 15 H 04991 e 15 K 15624) e a Fundação de ciência Takeda para M.F. e Jikei University estratégico priorizando fundo de investigação para H.J.O.

Todos os procedimentos experimentais foram realizados na Universidade Jikei, Tóquio no Japão. Manipulação de animais e procedimentos experimentais foram aprovados pelo cuidado institucional do Animal e usar o Comitê da Universidade Jikei (aprovação não: 26-060) e executadas em conformidade com as orientações institucionais para o uso de animais de laboratório no Jikei Universidade, que de acordo com as diretrizes para adequada conduta do Animal experiências pelo Conselho ciência do Japão (2006). Cuidados com animais foi realiza...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+method+for+virally+mediated+gene+delivery+to+the+mouse+inner+ear+through+the+round+window+membrane.">Surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane.</a> J Vis Exp. (97), (2015).</li><li>Jero, J., Tseng, C. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+approach+appropriate+for+targeted+cochlear+gene+therapy+in+the+mouse.">A surgical approach appropriate for targeted cochlear gene therapy in the mouse.</a> Hear Res. 151 (1-2), 106-114 (2001).</li><li>Iizuka, T., 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=Noninvasive+in+vivo+delivery+of+transgene+via+adeno-associated+virus+into+supporting+cells+of+the+neonatal+mouse+cochlea.">Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea.</a> Hum Gene Ther. 19 (4), 384-390 (2008).</li><li>Kilpatrick, L. 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=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <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+transgene+expression+after+adenoviral+vector+inoculation+in+the+endolymphatic+sac.">Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.</a> Hum Gene Ther. 10 (5), 769-774 (1999).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+and+structural+outcome+of+inner+ear+gene+transfer+via+the+vestibular+and+cochlear+fluids+in+mice.">The functional and structural outcome of inner ear gene transfer via the vestibular and cochlear fluids in mice.</a> Mol Ther. 4 (6), 575-585 (2001).</li><li>Praetorius, M., Baker, K., Weich, C. M., Plinkert, P. K., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+preservation+after+inner+ear+gene+therapy:+the+effect+of+vector+and+surgical+approach.">Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach.</a> ORL J Otorhinolaryngol Relat Spec. 65 (4), 211-214 (2003).</li><li>Nakagawa, T., 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=Topical+insulin-like+growth+factor+1+treatment+using+gelatin+hydrogels+for+glucocorticoid-resistant+sudden+sensorineural+hearing+loss:+a+prospective+clinical+trial.">Topical insulin-like growth factor 1 treatment using gelatin hydrogels for glucocorticoid-resistant sudden sensorineural hearing loss: a prospective clinical trial.</a> BMC Med. 8, 76 (2010).</li><li>Piu, 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=OTO-104:+a+sustained-release+dexamethasone+hydrogel+for+the+treatment+of+otic+disorders.">OTO-104: a sustained-release dexamethasone hydrogel for the treatment of otic disorders.</a> Otol Neurotol. 32 (1), 171-179 (2011).</li><li>Plontke, S. 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O volume de fluxo sanguíneo coclear é extremamente pequeno, estima-se que na ordem de 1/1.000.000 do débito cardíaco total nos seres humanos, e o acesso é limitado pela presença da barreira sangue-cóclea que separa o ouvido interno de circulação geral19 ,20. Por estas razões, droga ou administração de vetor viral para o ouvido interno é favorável, não através de administração sistémica, mas sim pela administração direta. No rato e cobaia de ...

Perda auditiva neurossensorial (SNHL) surge predominantemente da deficiência na cóclea ou danos. Causas comuns de SNHL incluem envelhecimento (por exemplo, presbycusis), defeitos genéticos, exposição ao ruído alto, infecção e medicamentos ototóxicos1. A Organização Mundial de saúde (OMS) estima-se que pessoas mais 360 milhões, representando 5,3% da população mundial, sofrem de audição perda2. Também estima-se que 1 em 900 de 1 em 2.500 recém-nascidos têm moderada, severa e perda permanente da audição congênita profunda e cerca de um em cada três adultos mais de 65 anos têm algum grau de perda de audição3. No entanto, não há nenhum tratamento clínico eficaz da perda auditiva para estes pacientes.
Pesquisa de audiência tem tempo foram realizada usando roedores ou modelos de cobaia, e muitas abordagens, tais como terapia gênica e terapia regenerativa, têm sido sugeridas como um novo tratamento para a perda de audição. No entanto, há uma grande diferença entre seres humanos e roedores em termos de sistema auditivo, e é muito difícil de traduzir modelos animais para aplicações em seres humanos. O sagui comum (c. jacchus), um macaco do novo mundo originário da Amazônia, tornou-se um modelo atraente de primatas não humanos para vários estudos de pesquisa básica para várias razões. Primeiro, sua anatomia e fisiologia são mais semelhantes dos seres humanos, ao invés de roedores. Em segundo lugar, as informações biológicas básicas sobre esta espécie, incluindo doenças, redes neurais, comportamento e o genoma, está bem caracterizadas. Processamento auditivo e vocal, a codificação cortical de periodicidade, a representação do passo e as interações auditivo-vocal de saguis comuns também têm sido relatados4,5,6,7 , 8 , 9 , 10. em recentes estudos histológicos, pesquisadores identificaram padrões de expressão distintiva de 20 genes de surdez e trocadores de ânion na cóclea do Sagui em constatou que cinco genes que são causadores de surdez progressiva e três trocadores de ânion, tinha padrões de expressão diferentes dos roedores11,12. Esses perfis nos levam a crer que o Sagui é uma ferramenta poderosa para a pesquisa de audiência.
Os atributos mais marcantes do sagui comum como um animal experimental são como segue:
<ul><li>Fácil manuseio com um tamanho de corpo pequeno em comparação com algumas espécies de macacos do mundo: saguis adultos pesa 300-400 g e são aproximadamente 60 cm de altura, que é semelhante aos ratos.</li>
	<li>Primata altamente reprodutiva: saguis atingir a maturidade sexual em uma idade de 18 meses e são capazes de suportar duas vezes por ano e produzir filhotes de 4 a 6 por ano.</li>
	<li>As modificações genéticas estão disponíveis: et al . Sasaki E conseguiu criar transgénicos13 e nocaute14 primatas usando saguis por modificações genéticas implicadas em doenças neurológicaspor exemplo, Parkinson do ( doença e a doença de Alzheimer).</li>
	<li>Tanto as células-tronco embrionárias (ES) e células pluripotentes induzidas (iPS) foram estabelecidas15,16. Apesar de ser um pouco difícil manter o mesmo número de saguis em comparação com roedores, a disponibilidade de células-tronco ou células iPS fornece em vitro ensaios que irão reduzir o número de ensaios na vivo necessário.</li>
</ul>Para facilitar a melhor investigação de translação no campo da surdez e sua potencial terapia, estabelecemos um protocolo de estudo de imagem, utilizando tomografia e ressonância magnética, anestesia geral e testes de audição, usando respostas auditivas de tronco cerebral (ABRs). Estes sistemas experimentais podem fornecer-nos com melhores oportunidades para obter prova pré-clínicos de estudos de conceito, que são essenciais para colmatar as lacunas entre roedores estudos e ensaios clínicos. Aqui, nós relatamos um método cirúrgico para administrar medicamentos para o ouvido interno do sagui comum através da membrana da janela redonda. Para obter uma visão clara em torno da janela redonda, sem manipular a membrana timpânica, que pode causar uma perda auditiva condutiva, é útil para abordagem da cavidade mastoide. Clinicamente, essa abordagem, conhecida como "timpanotomia posterior" é bem estabelecida e geralmente usada para cirurgia de implante e colesteatoma coclear. Acreditamos que timpanotomia posterior nos permite realizar a administração precisa de drogas sem induzir a perda de audição.

<table border="0" cellpadding="0" cellspacing="0" style="width:675px;" width="674">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">common marmoset</td>
			<td style="width:92px;">CLEA Japan</td>
			<td style="width:160px;">EDM:C.Marmoset(Jic)</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane</td>
			<td style="width:92px;">Phizer</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">medetomidine</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">midazolam</td>
			<td style="width:92px;">astellas</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">butorphanol</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">ampicillin</td>
			<td style="width:92px;">Meiji Seika</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lidocaine hydrochloride</td>
			<td style="width:92px;">AstraZeneca</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;6-0 absorbent thread</td>
			<td style="width:92px;">ETHICON</td>
			<td style="width:160px;">RD-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">atipamezole hydrochloride</td>
			<td style="width:92px;">ZENOAQ</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phosphate buffered saline</td>
			<td style="width:92px;">Wako</td>
			<td style="width:160px;">045-29795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surge Wave</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">TR-900-OR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 006</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diamond Bar 010</td>
			<td style="width:92px;">Morita</td>
			<td style="width:160px;">14070081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">intensive care unit</td>
			<td style="width:92px;">Menix</td>
			<td style="width:160px;">P-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a surgical procedure for the administration of drugs to the inner ear in a non-human primate common marmoset (callithrix jacchus)

Pesquisa de audiência há muito tempo foi facilitado por modelos de roedores, embora em algumas doenças, sintomas humanos não podem ser instaurados. O sagui comum (Callithrix jacchus) é um macaco do novo mundo pequeno, fácil de manusear que possui uma anatomia similar do osso temporal, incluindo a cadeia ossicular orelha média e orelha interna aos seres humanos, do que em comparação com a de roedores. Aqui, nós relatamos uma abordagem cirúrgica pode ser reproduzida, segura e racional para o nicho de coclear janela redonda para a entrega da droga para o ouvido interno do sagui comum. Adotamos a timpanotomia posterior, um procedimento usado clinicamente na cirurgia humana, para evitar a manipulação da membrana timpânica que podem causar perda auditiva condutiva. Este procedimento cirúrgico não conduziu a qualquer perda significativa de audição. Esta abordagem foi possível devido à estrutura de grande bulla do sagui comum, embora a lateral canal semicircular e a parte vertical do nervo facial deverão ser cuidadosamente considerados. Esse método cirúrgico nos permite realizar a administração segura e precisa de drogas sem perda de audição, que é de grande importância na obtenção de prova pré-clínica do conceito de Investigação translacional.

Timpanotomia posterior foi realizada sem qualquer complicação como infecção de sítio cirúrgico, sangramento com lesão vascular, ou disfunção vestibular, que às vezes ocorre com a manipulação do ouvido médio. Realizamos a timpanotomia posterior na orelha direita do Sagui de 7 anos de idade e administrado 1 µ l de solução salina de fosfato tamponado para o nicho da janela redonda. Os ABRs foram medidos antes e dois meses após a cirurgia (Figura 5</stron...

Watch this Scientific Journal Video about A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus at JoVE.com

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

Nós relatamos um método cirúrgico para administrar medicamentos para o ouvido interno de um primata não-humano, o sagui comum (Callithrix jacchus), através da membrana da janela redonda.

Um procedimento cirúrgico para a administração de drogas para o ouvido interno em um Non-Human primatas Sagui (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

The overall goal of this surgical procedure is to evaluate the effects of specific drugs of interest on the hearing function of non-human primate common marmoset through their safe administration into the inner ear without inducing hearing loss. This method can help answer key questions about inner ear drug delivery, including how to open the master cavity and where to deliver the drug. The main advantage of this technique is that the inner ear can be a plus without complications, which is critical for evaluating the efficiency of inner ear treatments in primates.The implications of this technique extend towards the study of potential therapies for human deafness, as the auditory system of the marmoset is quite similar to that of humans. The masteration of this method is critical and the posture and anatomy is difficult to learn and require that small window to be created in the narrow space without damaging the facial knob or the ossicular chain. Confirm the appropriate level of sedation by toe pinch and place the animal in the supine position on a heating mat with the head and neck extended.Fix maxilla to the table with nylon thread and pull out the tongue. Insert an endotracheal tube along the laryngeal surface of the epiglottis. Check the entitle carbon dioxide level in the endotracheal tube to confirm that the tube has been properly placed within the trachea and secure the tube to the animal's face.Incline the head to the 30 to 45 degree angle toward the non-operative side and shave the postauricular region of the operative side of the animal Then disinfect the postauricular region and the external auditory canal with povidone iodine and cover the animal with a surgical drape sheet with a round opening over the surgical area. Before opening the skin inject 1%of lidocaine hydrochloride subcutaneously to the incision site. Then, using a surgical microscope and a number 15 round shaped blade, make an incision into the postauricular skin and apply pressure to the bleeding area with a gauze soaked in one to 5, 000 diluted epinephrine.Open the soft tissue of the postauricular region so that the auricular cartilage and the postauricular muscle can be identified and expose the external auditory canal bone deep within the cartilage. To confirm the depth of the tympanic membrane, expose the bony part of the tympanic rim and peel the postauricular muscles toward the caudal aspect to separate the muscles from the temporal bone. Expose the lateral surface of the temporal bone and use a one millimeter diamond burr to make a hole approximately five millimeters behind the external auditory canal.Drill further to enlarge the hole as appropriate and locate the horizontal semi-circular canal, which bulges toward the mastoid cavity. Then drill and remove any bone septa as necessary to obtain a clear view of the facial recess, located at the center of the triangular area between the facial nerve and the corded tympany. Using a 0.6 millimeter diameter diamond burr at 1, 000 rpm, drill a small hole between the vertical section of the facial nerve, which appears as a pink line through the thinned bone and the tympanic rim.Stop the drilling just before the nerve is exposed as a white line structure and confirm that the round window niche can be visualized through the hold. Using a 25 gauge or thinner needle, deliver the drugs of interest into the niche and cover the niche with an appropriate miscues solution to ensure the drug is retained. Confirm that hemostasis is securely controlled and use six o absorbent threads to close the epidermal and dermal layers with simple, interrupted sutures.Then place a piece of gauze onto the surgical wound, fix the gauze in place with a bandage, and allow the marmoset to recover in an intensive care unit at 37 degrees Celsius in 30%oxygen until complete arousal. In these graphs, the auditory brain stem responses of a seven year old common marmoset before and two months after the delivery of one microliter of PBS to the round window niche, as just demonstrated, are shown. Comparison of these wave forms and thresholds to a previous study demonstrated no changes in these parameters before or after the surgery, confirming that posterior tympanotomy is a safe and useful procedure for drug administration to the round window niche of the common marmoset.Once mastered, this technique can be completed in an hour, including the anesthesia and induction, if it's performed properly. While attempting this procedure, it's important to remember not to damage the ossicle or the facial knob which can result in conductive hearing loss or facial paralysis respectively. After its development, this technique paved the way for researchers in the field of the translation research to test potential new therapies for hearing loss.After watching this video, you should have a good understanding of how to approach the inner ear of the common marmoset safely and to administer the drug into round window niche properly.

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Medicine

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

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