Name: exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal
Uploaded: 2017-04-13T14:00:00
Description: Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Los autores reconocen con gratitud la ayuda experta de la señora Trine W. Mikkelsen, la señora Lise M. de montaje, y el personal de Påskehøjgaard. El Consejo Danés de Investigación Médica, la Fundación Lundbeck, y la Fundación Novo Nordisk con el apoyo financiero del estudio.

anestesia Animal y euthanesia se realizó de acuerdo con &quot;Principios de laboratorio cuidado de los animales&quot; (NIH publicación No. 86-23, revisada 1985) y aprobada por el Consejo Danés de Ética de la Investigación Animal. 1. Instrumentos <ol><li> Recoge los instrumentos presentados en el video y que figuran en la Tabla de Materiales. </li></ol> 2. Decapitación NOTA: La anestesia se indujo mediante una inyecci...

<ol>
	<li>Lind, M. N., Moustgaard, A., Jelsing, J., Vajta, G., Cumming, P., Hansen, 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=The+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neurosci Biobehav Rev. 31, 728-751 (2007).</li><li>Bjarkam, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromodulation+in+a+minipig+model+of+Parkinson+disease.">Neuromodulation in a minipig model of Parkinson disease.</a> British J Neurosurg. 22 (Suppl. 1), S9-S12 (2008).</li><li>Bjarkam, C. R., Cancian, G., Glud, A. N., Ettrup, K. S., &#216;stergaard, L., S&#248;rensen, J. 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H., Cumming, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+brain+stereotaxic+standard+space:+Mapping+of+cerebral+blood+flow+normative+values+and+effect+of+MPTP-lesioning.">Pig brain stereotaxic standard space: Mapping of cerebral blood flow normative values and effect of MPTP-lesioning.</a> Brain Res Bull. 66 (1), 17-29 (2005).</li><li>Glud, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+gene+transfer+in+the+minipig+CNS+using+stereotaxic+lentiviral+microinjections.">Direct gene transfer in the minipig CNS using stereotaxic lentiviral microinjections.</a> Acta Neurobiol Exp. 70 (3), 1-8 (2010).</li><li>Glud, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+MRI-guided+stereotaxic+viral+mediated+gene+transfer+of+alpha-synuclein+in+the+minipig+CNS.">Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the minipig CNS.</a> Acta Neurobiol Exp. 71 (4), 508-518 (2011).</li><li>Ettrup, K. S., S&#248;rensen, J. C., Rodell, A., Alstrup, A. K. O., Bjarkam, C. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MRI-compatible+stereotaxic+localizer+box+enables+high-precision+stereotaxic+procedures+in+pigs.">A MRI-compatible stereotaxic localizer box enables high-precision stereotaxic procedures in pigs.</a> J Neurosci Methods. 139 (2), 293-298 (2004).</li><li>Bjarkam, C. R., Jorgensen, R. L., Jensen, K. N., Sunde, N. A. A., S&#248;rensen, J. C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+electrode+anchoring+using+BioGlue&#174;,+a+protective+electrode+covering,+and+a+titanium+microplate.">Deep brain stimulation electrode anchoring using BioGlue&#174;, a protective electrode covering, and a titanium microplate.</a> J Neurosci Methods. 168, 151-155 (2008).</li><li>Ettrup, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Surgical+Techniques+in+the+Minipig:+Intubation,+Transurethral+Bladder+Catheterization,+Femoral+Vessel+Catheterization,+and+Transcardial+Perfusion.">Basic Surgical Techniques in the Minipig: Intubation, Transurethral Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion.</a> J Vis Exp. (52), e2652 (2011).</li><li>Ettrup, K. S., Torn&#248;e, J., S&#248;rensen, J. C., Bjarkam, C. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+emission+tomography+of+living+brain+in+minipigs+and+domestic+pigs.">Positron emission tomography of living brain in minipigs and domestic pigs.</a> Scand J Lab Anim Sci Suppl. 25 (1), 127-135 (1998).</li><li>R&#248;hl, 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=Time+evolution+of+cerebral+perfusion+and+ADC+measured+by+MRI+in+a+porcine+stroke+model.">Time evolution of cerebral perfusion and ADC measured by MRI in a porcine stroke model.</a> J Magn Reson Imaging. 15 (2), 123-129 (2002).</li><li>Cumming, P., Gillings, N. M., Jensen, S. B., Bjarkam, C. 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La mayoría de los estudios de neurociencia experimentales se llevan a cabo en las especies de animales pequeños, tales como ratones y ratas, donde el acceso al SNC se ve facilitada por una fina calavera y dural-espesor. Sin embargo, en los animales de experimentación más grandes como cerdos 1, 4, 8, ovejas 32, y primates no humanos, el espesor considerable de estas estructuras requiere el uso de inst...

<html> Los estudios de neurociencia traslacional en cerdos se han vuelto cada vez más populares en las últimas dos décadas. El gran tamaño del cerebro de cerdo permite el uso de generadores de imágenes cerebrales clínica convencionales y el uso directo y las pruebas de los procedimientos neuroquirúrgicos y el equipo de la clínica humana 1, 2, 3, 4, 5, 6, 7, 8. En los últimos 20 años, cerdos, especialmente cerdos enanos (por ejemplo, Göttingen minipig), se han utilizado para examinar las modalidades de tratamiento neuromoduladores, como el trasplante de células madre; transfección vector viral; y la estimulación cerebral profunda dirigida hacia la enfermedad de Parkinson, la obesidad, la depresión y la enfermedad de Alzheimer 2, 6,= &quot;xref&quot;&gt; 9, 10, 11, 12, 13, 14, 15, 16, 17. Esto ha sido seguido por el desarrollo de enfoques estereotáxica y quirúrgicos para manipular el cerdo enano CNS 3, 18, 19, 20, 21. Los cambios del SNC instituidos se han evaluado en animales vivos usando imágenes del cerebro (PET 10, 13, 22, 24 y MR 23), cistometría 11, 12, 25, análisis de la marcha17, la evaluación neurológica 9, 17, y el examen postmortem base de la histología y análisis estereológico 14, 15, 17, 26, 27, 31. Sin embargo, el análisis post-mortem requiere la exposición y la eliminación del cerebro de cerdo, que no es una tarea fácil, como, cráneo óseo de espesor y un dural fibrosa que cubre rodean el cerebro de cerdo. El objetivo de este documento y vídeo de instrucción es describir cómo el cerebro de cerdo postmortem y la pituitaria pueden estar expuestos y eliminados en un estado intacto en 15-20 min utilizando herramientas quirúrgicas no motorizados. El vídeo de instrucciones y las ilustraciones fotográficas muestran cerdos enanos varones (edad: 6 meses, el peso corporal: 20-25 kg) utilizados para un estudio anatómico de la glándula pituitaria cerdo enano.

<table border="0" cellpadding="0" cellspacing="0" width="1036">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heavy Scalpel Handle #4</td>
			<td style="width:191px;">FST (Fine Science Tools)</td>
			<td style="width:149px;">10008-13</td>
			<td style="width:481px;">Good for skin incision and soft tissue removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #23</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #7</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10007-12</td>
			<td>Optimal for dural incision and precision work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Sterile Scalpel Blades #11</td>
			<td style="width:191px;">FST&#160;</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical Forceps</td>
			<td style="width:191px;">FST&#160;</td>
			<td>11024-18</td>
			<td>The tip of the surgical forceps ensure a firm grip&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerrison Bone Punch</td>
			<td style="width:191px;">Aesculap Neurosurgery</td>
			<td style="width:149px;">FF713R</td>
			<td>Must be robust, bite size 3-5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">MD615</td>
			<td>Must be robust, bite size 15 x 5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bone Rongeur</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">FO551R</td>
			<td>Must be robust, bite size 25 x 15 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone Chisel</td>
			<td>Lawton</td>
			<td>67-0335</td>
			<td>The size of the chisel head should not exceed 20 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mallet (Hammer)</td>
			<td>Millarco</td>
			<td>5624108</td>
			<td>Weigth 300 g, length 30 cm, head hit area size 2 x 2 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Scissor</td>
			<td>FST&#160;</td>
			<td>14002-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissector</td>
			<td>Aesculap Neurosurgery</td>
			<td style="width:149px;">OL165R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;G&#246;ttingen minipigs</td>
			<td style="width:191px;">&#160;Ellegaard G&#246;ttingen Minipigs A/S, Denmark</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Euthanimal</td>
			<td style="width:191px;"></td>
			<td style="width:149px;"></td>
			<td>pentobarbital</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:191px;">Pfizer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolam&#160;</td>
			<td style="width:191px;">Hameln Pharmaceuticals</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

exposure of the pig cns for histological analysis: a manual for decapitation, skull opening, and brain removal

Los cerdos se han vuelto cada vez más popular en la investigación traslacional animal grande neurociencia como un sustituto económicamente y éticamente factible primates no humanos. El tamaño del cerebro grande del cerdo permite el uso de generadores de imágenes cerebrales clínica convencionales y el uso directo y las pruebas de los procedimientos y equipos de neurocirugía de la clínica humana. Además análisis macroscópico e histológico, sin embargo, requiere la exposición postmortem del sistema de cerdo nervioso central (CNS) y la extracción del cerebro posterior. Esta no es una tarea fácil, ya que el cerdo CNS está encapsulada por una, estructura ósea del cráneo de espesor y la columna vertebral. El objetivo de este documento y vídeo de instrucción es describir cómo exponer y retirar el cerebro de cerdo postmortem y la glándula pituitaria en un estado intacto, adecuado para el análisis macroscópico e histológico posterior.

Para evitar que el material de tejido de la desecación, se recomienda para almacenar el cerebro eliminado y la hipófisis en una jarra llena de fijador o solución salina isotónica inmediatamente después que se ha realizado el análisis macroscópico. El material tejido puede ser almacenado en el fijador por año, mientras que el almacenamiento en solución salina isotónica, incluso en un refrigerador, dará lugar a la descomposición del tejido con el tiempo. <p class="jove_cont...

Watch this Scientific Journal Video about Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal at JoVE.com

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

El objetivo de este documento y vídeo de instrucción es describir cómo exponer y retirar el cerebro de cerdo y la glándula pituitaria postmortem en un estado intacto, adecuado para el análisis macroscópico e histológico posterior.

La exposición del cerdo del SNC para el análisis histológico: Un Manual para decapitación, Apertura cráneo, cerebro y Remoción

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

In this video, we present how to expose and remove the intact pig brain and the intact pituitary by opening the skull and dura. Ethics statement:Animal anesthesia and euthanasia were performed in accordance with Principles of laboratory animal care"and approved by the Danish Council for Animal Research Ethics. Anesthesia was induced in male Gottingen minipigs by an intramuscular injection of midazolam and ketamine.Five to ten minutes later, when the animals were deeply sedated, an ear vein was cannulated and a lethal overdose of sodium pentobarbital was given intravenously. To secure the animals were completely euthanized, the interdigital pain reflex was tested as previously shown. After proper euthanization, the animals were transcardially perfused with five liters of isotonic saline.All the procedures demonstrated are, accordingly, performed postmortem, excluding the need for welfare precautions necessary for long term anesthesia and post-procedural survival. Instruments used. Heavy scalpel handle number four, and non-sterile scalpel blades, number 23.Good for skin incisions, and soft tissue removal. Scalpel handle number seven, and non-sterile scalpel blades, number 11. Optimal for dural incision, and precision work.Surgical forceps, with a bite that ensures a firm grip. KAIRison Bone Punch. Must be robust, with a bite size of three to five millimeters.Bone rongeur. Must be robust and have a bite size around five to 15 millimeters, for delicate bone work. Larger bone rongeur.With a bite size around 25 times 15 millimeters, for removal of thicker bone pieces. Bone chisel. The head should not be larger than 20 millimeters.A mallet, suitable for the bone chisel. Micro scissors, with a sharp tip.Dissector.Decapitation. The pig is decapitated by making a high circular cervical incision, just below the mandibular angle.Continue the incision, through the soft tissue of the neck. Cut through larynx, and esophagus, until the bone and spinal column is reached. Approximately at the level of the cranio-cervical junction.Advance the incision from the anterior side of the cranio-cervical junction, through the anterior atlantooccipital membrane. Whereby the spinal cord is exposed. Simultaneously, an assistant will pull the pig body away from the pig head, as this will ease the access between the skull base and the first cervical vertebra.Make sure the spinal cord is completely severed, before a forceful extension at the cranio-cervical junction finalizes the decapitation. Skull opening. Place a dorsal longitudinal incision through the skin and underlying soft tissue.From the back of the snout over the vertex of the head and down through the posterior part of the occipital region. Expose the dorsal and posterior part of the skull by removing soft tissue located lateral to the initial incision. Release the temporal muscle from the skull.Clean the posterior occipital bone from soft tissue. Use the posterior interest of the foramen magnum to remove the occipital bone in order to expose the dura-covered cerebellum. Return to the anterior side of the skull and select an entry point in the frontal bone, just in front of the eyes.At this point, use a bone chisel to penetrate the skull, and enter the frontal sinus. Use the extent of the frontal sinus for further removal of the other skull lamina. An inner thin skull lamina covering the cerebrum, is exposed.Open this lamina gently, to expose the dura covering the anterior part of the brain. Continue the bone removal laterally, through the temporal and parietal bone. In order to release the final part of the skull, located between the already-exposed parts of the cerebrum and cerebellum.Use the chisel to break open the remaining posterior skull bone, from one side, just like one opens a door. Brain removal. Lift the dura, for gentle incision close to the sagittal sinus.Use a dural opening for further removal of the dural sac covering the brain. Special care must be taken when removing the dura situated between the cerebrum and the cerebellum. As preservation of this dural leaf will prevent subsequent brain removal.Finally, the dura covering the cerebellum and the lower brain stem is sectioned. Position the pig head vertically, and release the cerebrum by blunt dissection of the olfactory bulbs from the dura-covered skull base. Section the exposed optic chiasm.Follow it by subsequent exposure and section of the pituitary stalk, and the oculomotor nerves. Release the brain stem by sectioning of the lower cranial nerves. Use a chisel to release the brain from the skull base.Pituitary removal. In the skull base, identify the pituitary stalk, with surrounding dural leaf. Cut the dural leaf laterally to the pituitary stalk.Use a dissector to release the pituitary, and lift it out of the pituitary fossa. Results and conclusion. The described technique enables safe removal of the postmortem pig brain and pituitary, in approximately 15 to 20 minutes.The obtained tissue may be submitted for microscopic analysis followed by oriented sectioning, into smaller brain slabs. The slabs are suitable for chemical analysis, or further histological processing.

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