Esta pesquisa foi apoiada por uma concessão da Coreia saúde tecnologia R & D projeto através da Coreia saúde indústria desenvolvimento Institute (KHIDI), financiado pelo Ministério da saúde & bem-estar, República da Coreia (número de concessão: HI15C2651).

Este estudo foi realizado em conformidade com o cuidado de Animal institucional e uso Comitê de Seul nacional Hospital Universitário (14-0148-C1A1), que é credenciado pela Associação para a avaliação e acreditação do cuidado de Animal de laboratório Internacional.
Nota: Os experimentos foram realizados em ratos machos Sprague-Dawley (SD) de 7-8 semanas de idade (200/250 g). Cada animal foi aclimatado para as condições de laboratório por 1 semana antes do início do experimento. Os animais foram alojados em uma sala de controle de temperatura e umidade com uma constante 12-h:12-h luz: escuro ciclo com livre acesso à comida e água.
1. vestibulotóxicos
<ol><li>Cobrir os instrumentais cirúrgicos esterilizados em autoclave com uma almofada de estéril. Desinfete a área de atuação com 70% de etanol. Use um pano estéril e manter condições estéreis durante a cirurgia.</li>
	<li>Vestir a toga, máscara (para cobrir nariz e boca), PAC (para cobrir a cabeça) e um par de luvas estéreis. Os instrumentos cirúrgicos só devem entrar em contato com superfícies estéreis.</li>
	<li>Injetar o anestésico tiletamina-eficiente (40 mg/kg de peso corporal) por via intramuscular na coxa medial ou intraperitonealmente e xilazina (10mg/kg de peso corporal) por via intramuscular ou intraperitonealmente. Nota: Outros modos de anestesia geral, incluindo a inalação de isoflurano, também podem ser usados.</li>
	<li>Coloque o rato sobre uma almofada de aquecimento (inicialmente definida a 42 ° C). Aplica a pomada lubrificante para ambos os olhos do rato para evitar ressecamento do olho enquanto sob anestesia.</li>
	<li>Coloque o rato do seu lado direito. Raspe a pele na área cirúrgica com máquina de cortar cabelo. Desinfecte o local com etanol a 70%.</li>
	<li>Injete cloridrato de lidocaína a 1% por via subcutânea em região retroauricular esquerda.</li>
	<li>Usando uma lâmina de bisturi 15 #, faça uma incisão de retroauricular ~5.0 cm. Separe o músculo e a fáscia para expor o canal auditivo externo com tesoura Iris (figura 1A). Abra ligeiramente o canal auditivo externo usando a lâmina #15 ou a tesoura de Iris (figura 1B). Expor amplamente a membrana timpânica (Figura 2A).</li>
	<li>Sob um microscópio (7,5 X ou 12,5 X), remova a membrana timpânica e ossículos com excepção do estribo, usando a pinça. Desanexe os músculos no cume lambdoidal com a lâmina #15 ou a tesoura de Iris (Figura 2B).</li>
	<li>Usando uma broca em baixa velocidade (< 3000rpm) com uma broca de diamante (2.1 ou 1,4 mm), perfurar o osso timpânica em torno do ponto de saída do nervo facial (Figura 3A). Identifica a artéria estapédica. Tome cuidado para não danificar a artéria estapédica durante o processo de perfuração.</li>
	<li>Continuar a perfuração do vestíbulo superior à artéria estapédica e expor a janela redonda e o ósseo canal semicircular lateral. Abra o canal semicircular lateral sua ampola. Continuar a perfuração no plano do canal semicircular lateral e broca da ampola do canal semicircular superior.</li>
	<li>Aspire o conteúdo do vestíbulo usando uma agulha de 18 G ou 22-G. Injetar e Aspire 1 cm3 de etanol 100% 3 x.</li>
	<li>Feche os músculos e a pele em duas camadas com suturas interrompidas simples.</li>
	<li>Não abandone um animal até que ele recuperou a consciência suficiente para manter a prostração esternal. Aquece o rato até que ele desperta usando uma almofada de aquecimento ou uma lâmpada de calor. Após o despertar, mova o rato para uma gaiola individual em uma sala com as condições ambientais normais.</li>
	<li>Administração de antibióticos (por exemplo, cefazolina 20 mg/kg, trimetoprim-sulfonamida 100 µ l por via subcutânea, ou penicilina 300.000 UI) conforme necessário no dia 1; um analgésico (não um opioide; por exemplo, Metacam, carprofeno a 2 mg/kg 5 mg/kg por via subcutânea) também pode ser dada.</li>
</ol>2. sham cirurgia
<ol><li>Para a cirurgia de Souza como um controle, executar os mesmos procedimentos cirúrgicos e pós-cirúrgicos (acordo com o ponto 1), incluindo a exposição do canal auditivo externo e a membrana timpânica (etapa 1.7), a remoção da membrana timpânica e ossículos e o desprendimento dos músculos sobre o lambdoidal ridge (passo 1.8), mas não abra o canal semicircular ou injetar o etanol.</li>
</ol>3. Verifique a perda da função Vestibular
Nota: A perda da função vestibular pode ser avaliada usando testes de qualquer função comportamental ou vestibular. 13 , 17 , 18 testes comportamentais incluem a avaliação da assimetria postural e nistagmo.
<ol><li>Identifica a assimetria postural que inclui espontânea ou evocado barril-rolamento, caindo para o lado esquerdo, ou se movendo enquanto inclinando-se para o lado lesado18. Veja o vídeo 1.</li>
	<li>Identifica o nistagmo por uma inspeção visual do movimento ocular voluntária. Quando o nistagmo espontâneo está ausente no estado de repouso, soprar ar suavemente sobre a cabeça do animal18. Veja o vídeo 2.</li>
</ol>

<ol>
	<li>Curthoys, I. S., Halmagyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation:+a+review+of+the+oculomotor,+neural,+and+clinical+consequences+of+unilateral+vestibular+loss.">Vestibular compensation: a review of the oculomotor, neural, and clinical consequences of unilateral vestibular loss.</a> Journal of Vestibular Research. 5 (2), 67-107 (1995).</li><li>Smith, P. F., Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+recovery+following+unilateral+labyrinthectomy:+a+review.">Mechanisms of recovery following unilateral labyrinthectomy: a review.</a> Brain Research. Brain Research Reviews. 14 (2), 155-180 (1989).</li><li>Lacour, M., Helmchen, C., Vidal, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation:+the+neuro-otologist's+best+friend.">Vestibular compensation: the neuro-otologist's best friend.</a> Journal of Neurology. 263, S54-S64 (2016).</li><li>Darlington, C. L., Smith, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+recovery+from+vestibular+damage+in+mammals:+recent+advances.">Molecular mechanisms of recovery from vestibular damage in mammals: recent advances.</a> Progress in Neurobiology. 62 (3), 313-325 (2000).</li><li>Shinder, M. E., Ramanathan, M., Kaufman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+gene+expression+in+the+brain+during+acute+compensation+to+unilateral+vestibular+labyrinthectomy+in+the+Mongolian+gerbil.">Asymmetric gene expression in the brain during acute compensation to unilateral vestibular labyrinthectomy in the Mongolian gerbil.</a> Journal of Vestibular Research. 16 (4-5), 147-169 (2006).</li><li>Dutheil, S., Brezun, J. M., Leonard, J., Lacour, M., Tighilet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+and+astrogenesis+contribution+to+recovery+of+vestibular+functions+in+the+adult+cat+following+unilateral+vestibular+neurectomy:+cellular+and+behavioral+evidence.">Neurogenesis and astrogenesis contribution to recovery of vestibular functions in the adult cat following unilateral vestibular neurectomy: cellular and behavioral evidence.</a> Neuroscience. 164 (4), 1444-1456 (2009).</li><li>Gunther, 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=N-acetyl-L-leucine+accelerates+vestibular+compensation+after+unilateral+labyrinthectomy+by+action+in+the+cerebellum+and+thalamus.">N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.</a> PLoS One. 10 (3), e0122015 (2015).</li><li>Pericat, D., Farina, A., Agavnian-Couquiaud, E., Chabbert, C., Tighilet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+and+irreversible+unilateral+vestibular+loss:+a+novel+rat+model+of+vestibular+pathology.">Complete and irreversible unilateral vestibular loss: a novel rat model of vestibular pathology.</a> Journal of Neuroscience Methods. 283, 83-91 (2017).</li><li>Cass, S. P., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+compensation+after+labyrinthectomy+and+vestibular+neurectomy+in+cats.">Vestibular compensation after labyrinthectomy and vestibular neurectomy in cats.</a> Otolaryngology-Head and Neck Surgery. 104 (1), 14-19 (1991).</li><li>Vignaux, 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=Evaluation+of+the+chemical+model+of+vestibular+lesions+induced+by+arsanilate+in+rats.">Evaluation of the chemical model of vestibular lesions induced by arsanilate in rats.</a> Toxicology and Applied Pharmacology. 258 (1), 61-71 (2012).</li><li>Berryhill, W. E., Graham, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+labyrinthectomy+for+Meniere's+disease.">Chemical and physical labyrinthectomy for Meniere's disease.</a> Otolaryngologic Clinics of North America. 35 (3), 675-682 (2002).</li><li>Morgenstern, C., Mori, N., Arnold, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+on+the+effect+of+labyrinth+anesthesia.">Experimental studies on the effect of labyrinth anesthesia.</a> Archives of Oto-Rhino-Laryngology. 237 (3), 255-261 (1983).</li><li>Nadasy, G. L., Raffai, G., Feher, E., Schaming, G., Monos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+standard+technique+for+labyrinthectomy+in+the+rat:+a+methodical+communication+with+a+detailed+description+of+the+surgical+process.">A simple standard technique for labyrinthectomy in the rat: a methodical communication with a detailed description of the surgical process.</a> Physiology International. 103 (3), 354-360 (2016).</li><li>Hitier, M., Besnard, S., Vignaux, G., Denise, P., Moreau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventrolateral+surgical+approach+to+labyrinthectomy+in+rats:+anatomical+description+and+clinical+consequences.">The ventrolateral surgical approach to labyrinthectomy in rats: anatomical description and clinical consequences.</a> Surgical and Radiologic Anatomy. 32 (9), 835-842 (2010).</li><li>Flourens, M. 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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=MicroRNAs+218a-5p,+219a-5p,+and+221-3p+regulate+vestibular+compensation.">MicroRNAs 218a-5p, 219a-5p, and 221-3p regulate vestibular compensation.</a> Scientific Reports. 7 (1), 8701 (2017).</li><li>Bergquist, F., Ludwig, M., Dutia, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+commissural+inhibitory+system+in+vestibular+compensation+in+the+rat.">Role of the commissural inhibitory system in vestibular compensation in the rat.</a> The Journal of Physiology. 586 (18), 4441-4452 (2008).</li><li>Cameron, S. A., Dutia, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+vestibular+compensation:+changes+in+intrinsic+excitability+of+MVN+neurones.">Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones.</a> NeuroReport. 8 (11), 2595-2599 (1997).</li><li>Park, M. K., Lee, B. D., Lee, J. D., Jung, H. H., Chae, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+profiles+during+vestibular+compensation+in+rats+after+unilateral+labyrinthectomy.">Gene profiles during vestibular compensation in rats after unilateral labyrinthectomy.</a> Annals of Otology, Rhinology &amp; Laryngology. 121 (11), 761-769 (2012).</li><li>Yamamoto, H., Tominaga, M., Sone, M., Nakashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+stapedial+artery+to+blood+flow+in+the+cochlea+and+its+surrounding+bone.">Contribution of stapedial artery to blood flow in the cochlea and its surrounding bone.</a> Hearing Research. 186 (1-2), 69-74 (2003).</li><li>Potegal, M., Abraham, L., Gilman, S., Copack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+vestibular+neurotomy+in+the+rat.">Technique for vestibular neurotomy in the rat.</a> Physiology &amp; Behavior. 14 (2), 217-221 (1975).</li><li>Tung, V. 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Os autores não têm nada para divulgar.

Esta técnica é um método útil para a criação de perda súbita, permanente e completa função vestibular. Isso poderia ser usado para estudar patologias vestibulares, como neurite vestibular, um tumor acústico e a doença de Meniere. Muitos estudos têm usado essa técnica para estudar a plasticidade neuronal dos núcleos vestibulares ou o processo central relacionado5,17,18,19.
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O órgão vestibular é essencial para o controle de equilíbrio e olho. Uma função vestibular normal depende de sinais aferentes simétricos de órgãos vestibulares nos dois ouvidos interno. Hipofunção vestibular ou perda induz tonturas, nistagmo e desequilíbrio postural. Depois de danos agudos, a função vestibular recupera espontaneamente dentro de alguns dias, um processo conhecido como compensação vestibular1,2. A compensação vestibular dos défices estáticos é um processo de recuperação relacionado com o desequilíbrio da atividade espontânea de descanso entre os núcleos vestibulares ipsilaterais e contralaterais. A compensação vestibular dos défices dinâmicos é conseguida principalmente através de substituições sensoriais e comportamentais (usando entradas visuais ou somatossensorial)3. Estes processos são atraentes para a plasticidade neuronal estudos4,5.
Vários métodos foram desenvolvidos para estudar o sistema vestibular e os mecanismos subjacentes a plasticidade neuronal durante a compensação vestibular, tais como químicos e cirúrgicos vestibulotóxicos e neurectomia vestibular5,6 ,7,8. Neurectomia vestibular é uma certa maneira de induzir a perda completa do vestibular, mas é um procedimento mais difícil e invasivo e pode induzir dano cerebral8,9. Este método requer maior habilidade cirúrgica e leva mais tempo do que vestibulotóxicos. Vestibulotóxicos químico incluindo gentamicina, arsenilato e Tetracaína, é mais fácil e podem produzir resultados fiáveis10,11,12. No entanto, a cóclea, também pode ser danificada e perda vestibular pode desenvolver ao longo do tempo11. Além disso, os efeitos das substâncias químicas no cérebro, que deve ser preservado para avaliação exata, não são claras. Vestibulotóxicos cirúrgico foi introduzido pela primeira vez em estudos com animais em 184215 e foi primeiramente relatado em ratos em 193616. Esta técnica tem sido usada, desde que, em muitos animais estuda5,17,18,19. Vestibulotóxicos cirúrgico é um método confiável e relativamente simples de específicos. 13 , 14 além disso, os sintomas de dano vestibular são vistos imediatamente após a cirurgia. Aqui, descrevemos nossa técnica cirúrgica para rato vestibulotóxicos.

<table>
	<tbody>
		<tr>
			<td>ASPIRATOR KB-012</td>
			<td>KOH BONG &#38; CO., LTD.</td>
			<td>KB-012</td>
			<td>Medical aspirator</td>
		</tr>
		<tr>
			<td>Blade: #15</td>
			<td>Fine Science Tools</td>
			<td>#10015-00</td>
			<td>Blades for #7 Scalpel Handles, #15</td>
		</tr>
		<tr>
			<td>Carbon Steel Burrs</td>
			<td>Fine Science Tools</td>
			<td>#19007-05</td>
			<td>shaft diameter: 2.3 mm, length: 44 mm, package of 10 burrs</td>
		</tr>
		<tr>
			<td>Carl Zeiss Surgical GmbH</td>
			<td>Carl Zeiss</td>
			<td>#6627100863</td>
			<td>Surgical microscope</td>
		</tr>
		<tr>
			<td>Dumont #3c</td>
			<td>Fine Science Tools</td>
			<td>#11231-20</td>
			<td>Standard tip 0.17 x 0.10 mm, 11 cm</td>
		</tr>
		<tr>
			<td>Dumont #5SF</td>
			<td>Fine Science Tools</td>
			<td>#11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7B</td>
			<td>Fine Science Tools</td>
			<td>#11270-20</td>
			<td>Serrated 0.17 x 0.10 mm, 11 cm</td>
		</tr>
		<tr>
			<td>Extra Fine Bonn: straight</td>
			<td>Fine Science Tools</td>
			<td>#14084-08</td>
			<td>Iris scissors, best suited for microdissection under high magnification</td>
		</tr>
		<tr>
			<td>Fine Iris Scissors: straight</td>
			<td>Fine Science Tools</td>
			<td>#14094-11</td>
			<td>Made from martensitic stainless steel, combined with molybdenum and vanadium</td>
		</tr>
		<tr>
			<td>Finger Loop Ear Punch</td>
			<td>Fine Science Tools</td>
			<td>#24212-01</td>
			<td>1 mm. Provides stability and control for researchers using the numbering system</td>
		</tr>
		<tr>
			<td>Hartman</td>
			<td>Fine Science Tools</td>
			<td>#13002-10</td>
			<td>Tip width: 1 cm, serrated, 10 cm</td>
		</tr>
		<tr>
			<td>Short Scalpel Handle #7 Solid</td>
			<td>Fine Science Tools</td>
			<td>#10003-12</td>
			<td>#7 short, 12 cm</td>
		</tr>
		<tr>
			<td>Small Vessel Cauterizer</td>
			<td>Fine Science Tools</td>
			<td>#18000-03</td>
			<td>Replacement tip, straight knife, keeps bleeding to a minimum and therefore provides a surgical field clear of clamps and hemostats</td>
		</tr>
		<tr>
			<td>Strong 207S</td>
			<td>SAESHIN</td>
			<td>207S</td>
			<td>Powerful torque at low speed, available with speed or on/off foot controller</td>
		</tr>
		<tr>
			<td>Suction Tubes</td>
			<td>JEUNGDO B&#38;P CO., LTD.</td>
			<td>H-1927-8</td>
			<td>Frazier, 18 cm</td>
		</tr>
		<tr>
			<td>VICRYL</td>
			<td>ETHICON</td>
			<td>W9570T</td>
			<td>Synthetic absorbable sterile surgical suture</td>
		</tr>
		<tr>
			<td>Weitlaner-Locktite</td>
			<td>Fine Science Tools</td>
			<td>#17012-13</td>
			<td>Maximum spread: 4.5 cm, 2 x 3 blunt teeth, 11 cm</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac, France</td>
			<td></td>
			<td>Tiletamine-zolazepam</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td></td>
			<td>Carprofen</td>
		</tr>
		<tr>
			<td>Septra</td>
			<td>Pfizer</td>
			<td></td>
			<td>Trimethoprim-sulfonamide&#160;</td>
		</tr>
	</tbody>
</table>

surgical labyrinthectomy of the rat to study the vestibular system

Para estudar o sistema vestibular ou o processo de compensação vestibular, vários métodos foram desenvolvidos para causar dano vestibular, incluindo vestibulotóxicos cirúrgicos ou químicos e neurectomia vestibular. Vestibulotóxicos cirúrgico é um método relativamente simples, confiável e rápido. Aqui, descrevemos a técnica cirúrgica para rato vestibulotóxicos. Uma incisão postauricular é feita sob anestesia geral para expor o canal auditivo externo e a membrana timpânica, após o qual a membrana timpânica e ossículos são removidos sem estribo. A artéria do estribo, que está localizada entre o estribo e a janela oval, é uma estrutura vulnerável e deve ser preservada para obter um campo cirúrgico limpo. Um buraco para fenestrado o vestíbulo feito com uma broca de 2,1 mm de broca superior com o estribo. Então, 100% de etanol é injetado através deste buraco e aspirada várias vezes. Minuciosa dissecação sob um microscópio e cuidadoso controle de sangramento são essenciais para obter resultados fiáveis. Sintomas de perda vestibular, como nistagmo, inclinação de cabeça e um movimento de rolamento, são vistos imediatamente após a cirurgia. O teste de cadeira rotarod ou rotação pode ser usado para objetivamente e quantitativamente avaliar a função vestibular.

O sucesso da cirurgia foi validado por testes comportamentais. Todos os animais exibiram o comportamento típico de uma perda unilateral da função vestibular. Barril-rolamento espontâneo era evidente imediatamente após a cirurgia, sendo evocada por um sopro de ar sobre a cabeça ou um leve toque no corpo, na fase inicial de recuperação (vídeo 1). 3-d após a cirurgia, os animais movimentados mais inclinado para o lado lesado com ocasionais cai para o lado esquerdo....

Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

Este protocolo descreve o vestibulotóxicos cirúrgico de um rato, que é um método útil para o estudo do sistema vestibular.

Vestibulotóxicos cirúrgico do rato para estudar o sistema Vestibular

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

surgical-labyrinthectomy-of-the-rat-to-study-the-vestibular-system

Research

JoVE Journal

Medicine

9.9K visualizações.  Chung-Ang University College of Medicine. Este protocolo descreve o vestibulotóxicos cirúrgico de um rato, que é um método útil para o estudo do sistema vestibular.

Vídeo: Surgical Labyrinthectomy of the Rat to Study the Vestibular System

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Transplante ortotópico de aorta: um modelo de rato para estudar o desenvolvimento de vasculopatia crônica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Medicina

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Um In Vitro Sistema para Estudo de dormência Tumor ea Mude para o crescimento metastático

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando Estimulação Magnética Transcraniana para o Estudo do sistema neuromuscular humano

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

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.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

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

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry com Estimulação Cerebral Profunda para estudar normal e patológico função cerebral

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Entrega intravascular de Biologics para o rim de rato

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Utilizando imagens da retina para estudar a demência

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Usando um artrômetro de joelho para avaliar as contribuições de tecido-específica para contratura de flexão do joelho em ratos

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Modelo de Lesão de Isquemia/Reperfusão de Pulmão de Rato em Transplante de Pulmão de Rato: Otimizações para Melhorar os Resultados

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...