Name: surgical labyrinthectomy of the rat to study the vestibular system
Uploaded: 2018-05-19T13:00:00.000+00:00
Duration: 4 min 3 s
Description: Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Diese Forschung wurde unterstützt durch einen Zuschuss aus den Korea Health Technology R & D Project durch das Korea Gesundheit Industrie Entwicklung Institut (KHIDI), finanziert vom Ministerium für Gesundheit & Wohlbefinden, Republik Korea (gewähren Nummer: HI15C2651).

Diese Studie wurde durchgeführt, im Einklang mit den institutionellen Animal Care und Verwendung Ausschuss der Seoul National University Hospital (14-0148-C1A1), die von der Vereinigung für die Begutachtung und Akkreditierung of Laboratory Animal Care akkreditiert ist International.
Hinweis: Die Experimente wurden an Sprague-Dawley (SD) männlichen Ratten von ca. 7-8 Wochen alt (200/250 g) durchgeführt. Jedes Tier wurde bis 1 Woche vor Beginn des Experiments Laborbedingungen akklimatisiert. Die Tiere lebten in einem Raum Temperatur und Feuchtigkeit kontrolliert mit einer Konstanten 12-H:12-h hell: Dunkel-Zyklus mit freiem Zugang zu Nahrungsmitteln und Trinkwasser.
1. Labyrinthectomy
<ol><li>Decken Sie die autoklaviert chirurgische Instrumente mit einem sterilen Pad. Im OP-Bereich mit 70 % Ethanol zu desinfizieren. Verwenden Sie ein steriles Tuch und erhalten Sie sterile Bedingungen während der Operation zu.</li>
	<li>Eine Kleid zu tragen, Maske (zur Abdeckung Nase und Mund), Kappe (Kopfbedeckung), und ein paar sterile Handschuhe. Die chirurgischen Instrumente darf nur mit sterilen Oberflächen in Berührung kommen.</li>
	<li>Injizieren von tiletamin-Zolazepam Betäubung (40 mg/kg Körpergewicht) intramuskulär in den medialen Oberschenkel oder intraperitoneal, und Xylazin (10 mg/kg Körpergewicht) intramuskulär oder intraperitoneal. Hinweis: Andere Formen der Vollnarkose, einschließlich Isofluran Inhalation können auch verwendet werden.</li>
	<li>Legen Sie die Ratte auf eine Erwärmung (anfänglich bei 42 ° C). Beide Augen der Ratte, Augentrockenheit unter Narkose zu verhindern zuweisen Sie Schmiermittel Augensalbe.</li>
	<li>Legen Sie die Ratte auf der rechten Seite. Das Fell im Operationsbereich mit Haarschneidemaschinen zu rasieren. Desinfizieren Sie die Website mit 70 % Ethanol.</li>
	<li>Injizieren Sie 1 % Lidocain Hydrochlorid in der linken retroaurikulärer Bereich subkutan.</li>
	<li>Mit einem Skalpellklinge #15, machen Sie einen ~5.0 cm retroaurikulärer Schnitt. Trennen Sie den Muskel und Faszie, die äußeren Gehörgang mit Iris-Schere (Abbildung 1A) verfügbar zu machen. Öffnen Sie leicht den äußeren Gehörgang mit der Klinge #15 oder die Iris-Schere (Abbildung 1 b). Setzen Sie häufig das Trommelfell (Abbildung 2A).</li>
	<li>Unter dem Mikroskop (7,5 X oder 12,5 X) das Trommelfell und die Gehörknöchelchen mit Ausnahme der Steigbügel, mit der Pinzette zu entfernen. Lösen Sie die Muskeln auf dem Lambdoidal Grat mit der Klinge #15 oder die Iris-Schere (Abb. 2 b).</li>
	<li>Mit einem Bohrer mit niedriger Drehzahl (< 3000 u/min) mit einem Diamantschleifer (2.1 oder 1,4 mm) Bohren die Mittelohrchirurgie Bulla Knochen um den Punkt des Ausganges des Nervus facialis (Abb. 3A). Die stapedial Arterie zu identifizieren. Achten Sie darauf, nicht um die stapedial Arterie während des Bohrens zu beschädigen.</li>
	<li>Weiter Bohren das Vestibül der stapedial Arterie überlegen und das runde Fenster und die knöchernen Seitenkanal Semicircular aufdecken. Öffnen Sie den Seitenkanal Semicircular in der Nähe der Ampulle. Weiter in der Ebene der Seitenkanal Semicircular Bohren und Bohren der Ampulle des überlegenen Semicircular Kanals.</li>
	<li>Aspirieren Sie der Inhalt der Vorhalle mit einer 18-G oder 22 G Nadel. Spritzen und Aspirieren Sie 1 cm3 100 % Ethanol 3 X.</li>
	<li>Schließen Sie die Muskeln und die Haut in zwei Schichten mit einfachen unterbrochenen Nähten.</li>
	<li>Nicht unbeaufsichtigt lassen ein Tier bis es ausreichend Bewusstsein zur Aufrechterhaltung der sternalen liegen wiedererlangt hat. Halten Sie die Ratte warm, bis es erwacht mit einem Heizkissen oder eine Wärmelampe. Nach dem Erwachen, bewegen Sie sich die Ratte zu einem einzelnen Käfig in einem Raum mit den üblichen Umgebungsbedingungen.</li>
	<li>Verwalten von Antibiotika (z.B., Cefazoline 20 mg/kg, Trimethoprim-Sulfonamide 100 µL subkutan, oder Penicillin 300.000 IU) Bedarf an Tag 1; Analgetikum (kein Opioid; z.B., Allgemeinbefinden 2 mg/kg, Carprofen 5 mg/kg subkutan) können auch gegeben werden.</li>
</ol>(2) Sham Chirurgie
<ol><li>Für die Farce Chirurgie als ein Steuerelement, führen Sie die gleichen chirurgischen und postoperativen Verfahren (gemäß Ziff. 1), einschließlich der Exposition von den äußeren Gehörgang und das Trommelfell (Schritt 1.7), die Entfernung der das Trommelfell und die Gehörknöchelchen und die Ablösung der Muskeln auf der Lambdoidal Grat (Schritt 1,8), aber nicht öffnen Semicircular Kanal oder Ethanol zu injizieren.</li>
</ol>3. Überprüfen Sie den Verlust der vestibulären Funktion
Hinweis: Der Verlust der vestibulären Funktion kann mit entweder Verhaltens- oder vestibulären Funktionstests beurteilt werden. 13 , 17 , 18 Behavioral Tests umfassen die Bewertung der posturalen Asymmetrie und Nystagmus.
<ol><li>Identifizieren Sie Haltungsschäden Asymmetrie, die spontane oder evozierte Lauf-Rollen, fallen auf der linken Seite, oder sich bewegen und beugte sich zu der lesioned Seite18enthält. Sehen Sie Video 1.</li>
	<li>Nystagmus durch eine visuelle Inspektion der freiwillige Augenbewegungen zu identifizieren. Beim spontanen Nystagmus fehlt im Ruhezustand, Blätterteig Luft sanft über den Kopf des tierischen18. Sehen Sie Video 2.</li>
</ol>

<ol>
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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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recherches experimentales sur les propri&#233;t&#233;s et les fonctions du syst&#232;me nerveux dans les animaux vert&#233;br&#233;s. , (1824).</li><li>T'Ang, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+unilateral+labyrinthectomy+in+the+albino+rat.">The effects of unilateral labyrinthectomy in the albino rat.</a> Chinese Journal of Physiology. 10, 571-598 (1936).</li><li>Chang, M. 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. W., Burton, T. J., Dababneh, E., Quail, S. L., Camp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+assessment+of+the+aging+mouse+vestibular+system.">Behavioral assessment of the aging mouse vestibular system.</a> Journal of Visualized Experiments. (89), e51605 (2014).</li><li>de Jeu, M., De Zeeuw, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-oculography+in+mice.">Video-oculography in mice.</a> Journal of Visualized Experiments. 65 (65), e3971 (2012).</li></ol>

Die Autoren haben nichts preisgeben.

Diese Technik ist eine nützliche Methode für die Erstellung von plötzlichen, dauerhafte und vollständige vestibulären Funktionsverlust. Dies ließe sich vestibuläre Erkrankungen, wie z. B. vestibuläre Neuritis, eine akustische Tumor und Morbus Menière zu studieren. Viele Studien haben diese Technik verwendet, um die neuronale Plastizität des vestibulären Kerne oder der dazugehörigen zentralen Prozess5,17,18,</sup...

Die vestibuläre Organ ist unerlässlich für Gleichgewicht und Auge. Eine normale vestibuläre Funktion hängt symmetrisch afferenten Signale aus der vestibulären Organe in den zwei inneren Ohren. Vestibuläre Unterfunktion oder Verlust induziert Schwindel, Nystagmus und Haltungs-Ungleichgewicht. Nach einer akuten Schädigung erholt sich die vestibuläre Funktion spontan innerhalb weniger Tage ein Prozess genannt vestibulären Kompensation1,2. Die vestibuläre Kompensation der statischen Defizite ist ein Prozess der Genesung, die im Zusammenhang mit dem Ungleichgewicht der spontanen ruheaktivität zwischen der ipsilateralen und kontralaterale vestibuläre Kerne. Die vestibuläre Kompensation der dynamischen Defizite erfolgt hauptsächlich über sensorische und Verhaltensstörungen Substitutionen (mit visuellen oder somatosensorische Eingänge)3. Diese Prozesse sind attraktiv für neuronale Plastizität Studien4,5.
Eine Reihe von Methoden wurden entwickelt, um das Gleichgewichtsorgan und die Mechanismen zugrunde liegenden neuronalen Plastizität während vestibuläre Kompensation, wie chirurgische und chemische Labyrinthectomy und vestibulären Neurectomy5,6 studieren ,7,8. Vestibulären Neurectomy ist gewissermaßen vestibulären Totalverlust zu induzieren, aber es ist ein schwieriger und invasive Verfahren und kann verursachen Hirnschäden8,9. Diese Methode erfordert größere chirurgische Geschick und dauert länger als Labyrinthectomy. Chemische Labyrinthectomy einschließlich Gentamycin, Arsanilate und Tetracaine, ist einfacher und kann liefern zuverlässige Ergebnisse10,11,12. Jedoch die Cochlea beschädigt werden und vestibulären Verlust kann entwickeln im Laufe der Zeit11. Darüber hinaus sind die Auswirkungen von Chemikalien auf das Gehirn, die für die genaue Auswertung erhalten werden soll, ist unklar. Chirurgische Labyrinthectomy wurde erstmals in tierexperimentellen Studien in 184215 und wurde zuerst bei der Ratte in 193616berichtet. Diese Technik wird inzwischen in vielen Tier-Studien5,17,18,19. Chirurgische Labyrinthectomy ist eine spezifische, zuverlässige und relativ einfache Methode. 13 , 14 im übrigen, die Symptome der vestibulären Schäden gelten unmittelbar nach der Operation. Hier beschreiben wir unsere OP-Technik für Ratte Labyrinthectomy.

<table><tbody><tr><td>ASPIRATOR KB-012</td><td>KOH BONG &amp;amp; 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&amp;amp;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&amp;nbsp;</td></tr></tbody></table>

surgical labyrinthectomy of the rat to study the vestibular system

Das vestibuläre System oder den vestibulären vergütungsprozess studieren, haben eine Reihe von Methoden entwickelt, um vestibulären Schäden, einschließlich der chirurgischen oder chemische Labyrinthectomy und vestibulären Neurectomy verursachen. Chirurgische Labyrinthectomy ist eine relativ einfache, zuverlässige und schnelle Methode. Hier beschreiben wir die Operationstechnik für Ratte Labyrinthectomy. Ein postauricular Schnitt erfolgt unter Vollnarkose zu entlarven, den äußeren Gehörgang und das Trommelfell, wonach das Trommelfell und die Gehörknöchelchen ohne die Steigbügel entfernt sind. Die Steigbügel Arterie, die sich zwischen den Steigbügel und des ovalen Fensters befindet, ist eine gefährdete Struktur und muss bewahrt werden, um eine klare OP-Feld zu erhalten. Ein Loch, fenestrate erfolgt das Vestibül mit einem 2,1-mm-Bohrer Bohrer überlegen die Steigbügel. Dann ist 100 % Ethanol durch dieses Loch eingespritzt und mehrmals abgesaugt. Sorgfältige Dissektion unter einem Mikroskop und sorgfältiger Blutstillung sind unerlässlich, um zuverlässige Ergebnisse zu erhalten. Symptome des vestibulären Verlust, wie Nystagmus, Kopf kippen und eine Rollbewegung, sind unmittelbar nach der Operation zu sehen. Der Rotarod oder Rotation-Stuhl-Test kann verwendet werden, um Objektiv und quantitativ die vestibuläre Funktion bewerten.

Der Erfolg der Operation wurde durch behavioral Tests validiert. Alle Tiere ausgestellt, das typische Verhalten von einem einseitigen Verlust der vestibulären Funktion. Spontane Lauf-Rollen zeigte sich unmittelbar nach der Operation, hervorgerufen durch einen Luftstoß über den Kopf oder eine leichte Berührung mit dem Körper in der frühen Phase der Erholung (Video 1). 3-d nach der Operation, die Tiere auf der lesioned Seite mit gelegentlichen gelehnt bewegt fällt au...

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

Dieses Protokoll beschreibt die chirurgische Labyrinthectomy einer Ratte, die eine nützliche Methode für das Studium des vestibulären Systems ist.

Chirurgische Labyrinthectomy der Ratte das vestibuläre System untersuchen

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

10.0K Aufrufe.  Chung-Ang University College of Medicine. Dieses Protokoll beschreibt die chirurgische Labyrinthectomy einer Ratte, die eine nützliche Methode für das Studium des vestibulären Systems ist.

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy als ein chirurgisches Vorgehen zu Local Drug Delivery in das innere Ohr von Erwachsenen und Neugeborenen Mäusen

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Forschung

Genetik

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

Survivable stereotaktischen Chirurgie in Nagetiere

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biologie

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

Eine Video-Demonstration der Preserved Pilotierung von Scent-Tracking aber Impaired Dead Reckoning Nach Fimbria-Fornix Läsionen in der Ratte

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

Bewertung der autonomen und Verhaltenseffekte passiver Bewegungen bei Ratten mit vertikaler Aufzugsbewegung und Riesenradrotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

Verhalten

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Eine Isolierte Semi-intakt Vorbereitung der Maus Vestibuläre Sinnesepithel für Elektrophysiologie und hochauflösende Zwei-Photonen-Mikroskopie

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Neurowissenschaften

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Langfristige sensorischen Konflikt in Mäusen frei Verhalten

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Entschlüsselung der Rolle diskreter Bereiche des Rattengehirns bei der Regulation des Eisprungs durch reversible Inaktivierung durch Tetrodotoxin-Mikroinjektionen

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Nahes Infrarot (NIR) Licht Erhöht Ausdruck einer Markierung der Funktion der Mitochondrien im Maus Vestibular Sinnesepithel

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Isolierung und Kultivierung von vestibulären und spiralförmigen Ganglien-Somata von neonatalen Nagetieren für Patch-Clamp-Aufnahmen

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.