Name: experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia
Uploaded: 2017-07-31T12:30:00
Description: Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Este trabajo fue financiado por la Fundación Nacional de Ciencias Naturales de China (81573406, 81373393, 81273506, 81221003, 81473186 y 81402907), la Fundación Provincial de Ciencias Naturales de Zhejiang (LR15H310001) y el Programa de Zhejiang Equipo Líder del Equipo de Innovación de C &amp; T (2011R50014).

Todos los experimentos fueron aprobados por y conducidos de acuerdo con las directrices éticas del Comité de Experimentación Animal de la Universidad de Zhejiang y estaban en completo cumplimiento con los Institutos Nacionales de Guía de Salud para el Cuidado y Uso de Animales de Laboratorio. Se hicieron esfuerzos para minimizar cualquier dolor o incomodidad, y se utilizó el número mínimo de animales. 1. OGD de cortes de Corticostriatal <ol><li> Preparación de la solución:...

<ol>
	<li>Zhao, H., Sapolsky, R. M., Steinberg, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrupting+reperfusion+as+a+stroke+therapy:+ischemic+postconditioning+reduces+infarct+size+after+focal+ischemia+in+rats.">Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats.</a> J Cereb Blood Flow Metab. 26 (9), 1114-1121 (2006).</li><li>Leconte, C., 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=Delayed+hypoxic+postconditioning+protects+against+cerebral+ischemia+in+the+mouse.">Delayed hypoxic postconditioning protects against cerebral ischemia in the mouse.</a> Stroke. 40 (10), 3349-3355 (2009).</li><li>Fan, Y. 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=Transient+lack+of+glucose+but+not+O2+is+involved+in+ischemic+postconditioning-induced+neuroprotection.">Transient lack of glucose but not O2 is involved in ischemic postconditioning-induced neuroprotection.</a> CNS Neurosci Ther. 19 (1), 30-37 (2013).</li><li>Hess, D. C., Hoda, M. N., Bhatia, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+limb+perconditioning+[corrected]+and+postconditioning:+will+it+translate+into+a+promising+treatment+for+acute+stroke.">Remote limb perconditioning [corrected] and postconditioning: will it translate into a promising treatment for acute stroke.</a> Stroke. 44 (4), 1191-1197 (2013).</li><li>Ren, C., Yan, Z., Wei, D., Gao, X., Chen, X., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+remote+ischemic+postconditioning+protects+against+focal+ischemia+in+rats.">Limb remote ischemic postconditioning protects against focal ischemia in rats.</a> Brain Res. 1288, 88-94 (2009).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+delayed+remote+limb+ischemic+postconditioning:+role+of+mitochondrial+K(ATP)+channels+in+a+rat+model+of+focal+cerebral+ischemic+reperfusion+injury.">Protective effect of delayed remote limb ischemic postconditioning: role of mitochondrial K(ATP) channels in a rat model of focal cerebral ischemic reperfusion injury.</a> J Cereb Blood Flow Metab. 32 (5), 851-859 (2012).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+limb+ischemic+postconditioning+protects+mouse+brain+against+cerebral+ischemia/reperfusion+injury+via+upregulating+expression+of+Nrf2,+HO-1+and+NQO-1+in+mice.">Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1 and NQO-1 in mice.</a> Int J Neurosci. , 1-8 (2015).</li><li>Fan, Y. 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=A+novel+neuroprotective+strategy+for+ischemic+stroke:+transient+mild+acidosis+treatment+by+CO2+inhalation+at+reperfusion.">A novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by CO2 inhalation at reperfusion.</a> J Cereb Blood Flow Metab. 34 (2), 275-283 (2014).</li><li>Shen, Z., 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=PARK2-dependent+mitophagy+induced+by+acidic+postconditioning+protects+against+focal+cerebral+ischemia+and+extends+the+reperfusion+window.">PARK2-dependent mitophagy induced by acidic postconditioning protects against focal cerebral ischemia and extends the reperfusion window.</a> Autophagy. , (2017).</li><li>Skolnik, J., Takacs, L., Szende, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oxygen+consumption+of+slices+from+kidney,+brain,+cortex+and+liver+in+hypoxia.">In vitro oxygen consumption of slices from kidney, brain, cortex and liver in hypoxia.</a> Nature. 209 (5020), 305 (1966).</li><li>Lynch, G., Schubert, P. <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+in+vitro.+brain+slices+for+multidisciplinary+studies+of+synaptic+function.">The use of in vitro. brain slices for multidisciplinary studies of synaptic function.</a> Annu Rev Neurosci. 3, 1-22 (1980).</li><li>Zheng, S., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+preconditioning+reduces+purkinje+cell+death+in+an+in+vitro+model+of+rat+cerebellar+ischemia.">Isoflurane preconditioning reduces purkinje cell death in an in vitro model of rat cerebellar ischemia.</a> Neuroscience. 118 (1), 99-106 (2003).</li><li>Yin, B., Barrionuevo, G., Weber, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+real-time+monitoring+of+glutathione+redox+status+in+single+pyramidal+neurons+in+organotypic+hippocampal+slices+during+oxygen-glucose+deprivation+and+reperfusion.">Optimized real-time monitoring of glutathione redox status in single pyramidal neurons in organotypic hippocampal slices during oxygen-glucose deprivation and reperfusion.</a> ACS Chem Neurosci. 6 (11), 1838-1848 (2015).</li><li>Medvedeva, Y. V., Ji, S., Yin, H. Z., Weiss, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+vulnerability+of+CA1+vs+CA3+pyramidal+neurons+after+ischemia:+possible+relationship+to+sources+of+Zn2++accumulation+and+its+entry+into+and+prolonged+effects+on+mitochondria.">Differential vulnerability of CA1 vs CA3 pyramidal neurons after ischemia: possible relationship to sources of Zn2+ accumulation and its entry into and prolonged effects on mitochondria.</a> J Neurosci. , (2016).</li><li>Pignataro, 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=In+vivo+and+in+vitro+characterization+of+a+novel+neuroprotective+strategy+for+stroke:+ischemic+postconditioning.">In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning.</a> J Cereb Blood Flow Metab. 28 (2), 232-241 (2008).</li><li>Zhang, X., Ding, H. Z., Jiang, S., Zeng, Y. M., Tang, Q. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+of+the+neuroprotective+effect+of+propofol+on+hypoxic+hippocampal+slice.">An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice.</a> Brain Inj. 28 (13-14), 1758-1765 (2014).</li><li>Niu, 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=Chemical+profiling+with+HPLC-FTMS+of+exogenous+and+endogenous+chemicals+susceptible+to+the+administration+of+chotosan+in+an+animal+model+of+type+2+diabetes-induced+dementia.">Chemical profiling with HPLC-FTMS of exogenous and endogenous chemicals susceptible to the administration of chotosan in an animal model of type 2 diabetes-induced dementia.</a> J Pharm Biomed Anal. 104, 21-30 (2015).</li></ol>

Aquí presentamos dos modelos experimentales para estudiar la neuroprotección de APC contra la isquemia cerebral. En las rebanadas cerebrales, la APC se logra incubando ratones con rebanadas corticostriatales en tampón ácido burbujeado con 20% de CO 2 después del inicio de la reperfusión, mientras que en el modelo MCAO, la APC se consigue inhalando 20% de CO 2 a ratones después de la reperfusión. Ambos modelos reflejan la neuroprotección de APC contra la isquemia cerebral. La protección fu...

El derrame cerebral es una de las principales causas de mortalidad e incapacidad en todo el mundo. Grandes esfuerzos se han hecho para encontrar tratamientos eficaces para el accidente cerebrovascular en las últimas décadas, sin embargo, el logro es bastante insatisfactorio. El postcondicionamiento es un proceso manipulado por tensiones subtóxicas después de un episodio isquémico. El postcondicionamiento, incluyendo la isquemia, la hipoxia, la baja glucosa y el postcondicionamiento isquémico remoto, desencadenan mecanismos adaptativos endógenos y han demostrado ser terapias prometedoras contra la isquemia cerebral 1 , 2 , 3 , 4 . Sin embargo, el postcondicionamiento isquémico puede introducir lesiones adicionales. El postcondicionamiento isquémico remoto de las extremidades suele necesitar varios ciclos de 5 a 20 min de oclusión y reperfusión en las extremidades posteriores ipsilaterales o bilaterales 5 , 6 , 7 . ThPor lo tanto, estas manipulaciones posteriores al acondicionamiento son peligrosas o poco prácticas en la práctica clínica. Para superar estos inconvenientes, hemos desarrollado APC como una terapia para la isquemia cerebral focal en ratones [ 8] . Inducido simplemente por la inhalación de 20% de CO 2 , APC reduce significativamente la lesión cerebral isquémica de una manera más factible y más segura. Recientemente hemos demostrado que APC extiende la ventana de reperfusión, destacando la importancia de APC para la terapia de accidente cerebrovascular [ 9] . Aquí presentamos dos modelos experimentales para estudiar la neuroprotección de APC contra la isquemia cerebral. El primero es el modelo de privación de oxígeno-glucosa (OGD) en rodajas corticostriatales de ratones. La rápida preparación y transferencia de los cortes cerebrales en un entorno artificial, usualmente el líquido cefalorraquídeo artificial (ASCF), puede mantener la viabilidad celular y los circuitos neuronales, lo que hace posible el estudio de la función cerebral in vitro 10 &lt;Sup&gt;, 11 . OGD en ASCF imita la isquemia cerebral e induce lesión isquémica [ 12 , 13 , 14] . Después de OGD, las rebanadas de cerebro se refrescan en ASCF regular (r-ASCF) para proporcionar reperfusión y luego se tratan con APC usando ácido ASCF burbujeado con 20% de CO 2 . La porción corticostriatal mantiene la caracterización histológica intacta en comparación con las células cultivadas primarias. Para estudiar la función cerebral in vivo , se emplea el modelo de oclusión de la arteria cerebral media (MCAO) del ratón. La arteria cerebral media se bloquea insertando un monofilamento amortiguado por la llama a través de la arteria carótida común. Como uno de los modelos de accidente cerebrovascular más ampliamente utilizados, el modelo MCAO muestra relevancia clínica y la aplicación de un monofilamento facilita la reperfusión. Simplemente mediante la inhalación de gases mezclados normoxic que contienen 20% de CO 2 después del inicio del reperfusioN, la APC mostró efectos protectores significativos contra la isquemia cerebral indicada por volúmenes reducidos de infarto cerebral.

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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">S5886</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">P5405</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium phosphate monobasic</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">P9791</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Magnesium sulfate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">M2643</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">S5761</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Calcium chloride dihydrate</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">C5080</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">G7021</td>
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			<td height="21" style="height:21px;width:216px;">Vibratome</td>
			<td style="width:183px;">Leica</td>
			<td style="width:157px;">VT1000 S</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">2,3,5-triphenyltetrazolium hydrochloride</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">T8877</td>
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			<td height="42" style="height:42px;width:216px;">Absolute Ethanol</td>
			<td style="width:183px;">Aladdin Industrial Corporation</td>
			<td style="width:157px;">E111993</td>
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			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">D8418</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Laser Doppler Flowmetry</td>
			<td style="width:183px;">Moor Instruments Ltd</td>
			<td style="width:157px;">Model Moor VMS-LDF2</td>
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			<td height="42" style="height:42px;width:216px;">Diethyl ether anhydrous</td>
			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
			<td style="width:157px;">80059618</td>
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			<td height="42" style="height:42px;width:216px;">Trichloroacetaldehycle hydrate</td>
			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
			<td style="width:157px;">30037517</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">10% Formalin</td>
			<td style="width:183px;">Aladdin Industrial Corporation</td>
			<td style="width:157px;">F111936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24-well plates</td>
			<td style="width:183px;">Jet Biofil</td>
			<td style="width:157px;">TCP-010-024</td>
			<td style="width:167px;"></td>
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experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

El accidente cerebrovascular es una de las principales causas de mortalidad e incapacidad en todo el mundo, con enfoques terapéuticos limitados. Como estrategia endógena para la neuroprotección, los tratamientos post-acondicionamiento han demostrado ser terapias prometedoras contra la isquemia cerebral. Sin embargo, procedimientos complicados y posibles problemas de seguridad limitan su aplicación clínica. Para superar estos inconvenientes, hemos desarrollado postcondicionamiento ácido (APC) como terapia para la isquemia cerebral focal experimental. APC se refiere al tratamiento de acidosis leve inhalando CO 2 durante la reperfusión después de la isquemia. Aquí presentamos dos modelos para ejecutar APC in vitro e in vivo , respectivamente. El tratamiento de privación de oxígeno-glucosa (OGD) de ratones y la oclusión corticostriatal y oclusión de la arteria cerebral media (MCAO) de ratones se emplearon para imitar la isquemia cerebral. APC se puede lograr simplemente transfiriendo las rebanadas de cerebro a un tampón ácido burbujeado con 20% de CO 2 , or por los ratones inhalan 20% de CO2. La APC mostró efectos protectores significativos contra la isquemia cerebral, como se refleja en la viabilidad de los tejidos y en el volumen del infarto cerebral.

En el modelo de corte corticostriatal descrito anteriormente, la viabilidad de la corteza corticostriatal se cuantificó por ensayo de TTC a 1 h después de la reperfusión. La conversión de TTC se calculó normalizando la absorción a 490 nm a la porción de control. De acuerdo con la conversión de TTC, APC protegido contra OGD inducida por lesión de reperfusión en un tiempo de inicio y la duración dependiente de manera. En detalle, tanto 1 y 3 min de tratamiento de la acidosis mej...

Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

El postcondicionamiento ácido protege contra la isquemia cerebral. Aquí presentamos dos modelos para ejecutar APC. Se consiguen respectivamente transfiriendo rebanadas corticostriatales a un tampón ácido después de la privación de oxígeno-glucosa in vitro e inhalando 20% de CO 2 después de la oclusión de la arteria cerebral media in vivo .

Modelos Experimentales para Estudiar la Neuroprotección del Acondicionamiento Acido Contra la Isquemia Cerebral

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

The overall goal of acidosis treatment in a cortico striatal slice model in a middle cerebral artery occlusion model is to study the neuroprotective effect of acidic postconditioning against cerebral ischemia. This method can help answer key questions of how acidosis postconditioning may protect ischemic brain injury, and to develop new strategy for stroke therapy. The main advantage of this technique is being able to use widely available experiment models to study the neuroprotection of acidosis postconditioning.Demonstrating the procedure will be Yarong Zheng a graduate student from my lab. Begin by removing the brain from the skull of a decapitated mouse with a thin spatula and dropping it carefully into a beaker containing ice cold regular ACSF equilibrated with five percent carbon dioxide and 95%oxygen. Apply cryoprecipitate glue to the vibratone plate in two strips.Put a piece of three percent agarose on the glue strip furthest from the blade to support the brain. Then use forceps to transfer the brain to filter paper. Cut off the frontal pole and the cerebellum with a blade.Place the brain tissue vertically on the free strip of glue with the brain leaning against the agarose. Add ice cold ACSF to the cutting reservoir to submerge the brain and add ice to the ice holder area. Keep the ACSF in the reservoir bubbled with five percent carbon dioxide and 95%oxygen.After setting the vibratone to cut 400 micron sections, and correctly positioning the brain relative to the cutting blade, press the start-stop button to start automatic cutting. Use a Pasteur pipette with a cut tip to transfer five brain slices one by one and place them into the tissue holder in ice cold regular ACSF bubbled with five percent carbon dioxide and 95%oxygen. After 30 minutes at room temperature place the brain slices in a water bath at 37 degrees Celsius for ten minutes to recover synaptic function.Place glucose free ACSF and acidic ACSF into the 37 degree Celsius water bath and bubble the solutions with five percent carbon dioxide and 95%nitrogen and 20 percent carbon dioxide and 80%oxygen respectively for 30 minutes. Carefully transfer four of the five brain slices into a preheated 37 degree Celsius, glucose free ACSF and incubate for 15 minutes. To study the time window for acidic preconditioning transfer one slice directly from glucose free ACSF to acidic ACSF and transfer the other three slices to regular ACSF for reperfusion.After three minutes transfer the slice in acidic ACSF to regular ACSF. Then after five minutes in regular ACSF transfer one of the slices from regular ACSF to the tissue holder in acidic ACSF for three minutes. Transfer another slice in the same manner 15 minutes after reperfusion.The remaining slice that was placed in glucose free ACSF but not acidic ACSF is set as the oxygen glucose deprivation group. Transfer the slices from the ACSF into the wells of a 24 well plate containing TTC solution. Stretch out the slice in the solution.And then incubate at 37 degrees Celsius in a shallow water bath for 30 minutes. Next transfer the slices into clean, weighed, 1.5 milliliter centrifuge tubes covered in foil and measure the dry weight of the slices. After weighing, extract Formazan by adding a one to one solution of ethanol and dimethyl sulphoxide to the tubes at a volume to weight ratio of 10 to one.Incubate away from light for 24 hours. The next day transfer the ethanol dimethyl sulphoxide solution from the tubes to a 96 well plate and measure the absorbance at 490 nanometers using a plate reader. First confirm proper anesthesia by the absence of a toe pinch reflex.Then place the anesthetized mouse with LDF probe attached to its skull in a supine position and fix using sterile cotton threads. Disinfect the shaved skin of the neck with 75%ethyl alcohol. Then after making a peri median skin incision in the neck blunt dissect the soft tissues with forceps to expose the blood vessels.Add one drop of saline to the exposed tissues to keep them hydrated. Next, use ophthalmic forceps to dissect the common carotid artery from surrounding tissue and the vagus nerve. Be careful not to damage the vagus nerve.Place a micro vessel clamp at the distal end of the common carotid artery. And tie a dead knot with 6-0 silk sutures at the proximal end. Then tie a loose knot proximal to the clamp as a temporary suture.Next, use micro ophthalmic scissors to make a small longitudinal incision between the two knots as close to the dead knot as possible. Now insert a 12 millimeter, tip blunted monofilament through the incision into the artery lumen and advance it a few millimeters. Tighten the loose knot around the tip of the monofilament and then remove the clamp.Then use ophthalmic forceps to advance the filament into the internal carotid artery until the LDF software shows a sharp decline in blood flow. Record the start time of occlusion. Then cut off the LDF probe, and put the mouse in a 30 degree Celsius incubator for the one hour duration of occlusion.55 minutes after the start of occlusion reanesthetize the animal with isoflurane and position the animal as before. Then open the neck incision, and reexpose the common carotid artery. After the occlusion period, use ophthalmic forceps to gently pull out the filament to achieve reperfusion.Then turn the temporary suture into a permanent one by tightening the knot. For acidosis treatment change the gas inhaled by the nose cone to 20%carbon dioxide 20%oxygen, and 60%nitrogen for five minutes. After closing the incision with an interrupted surgical suture place the mouse in a 30 degree Celsius heated cage until the mouse regains consciousness.This histogram shows the results from the TTC assay performed on cortical slices after oxygen glucose deprivation and acidic postconditioning as demonstrated in this video. Three minutes of acidosis treatment was neuroprotective if treatment was initiated immediately or five minutes after oxygen glucose deprivation, but not if the slices were reperfused for 15 minutes. Mice were subjected to 60 minutes of middle cerebral artery occlusion and treated by inhaling 20%carbon dioxide for five minutes at five, 50, or 100 minutes after reperfusion.Infarct volume was quantified by two, three, five triphenyltetrazolium hydrochloride staining 24 hours after reperfusion as indicated by the black dotted lines. This histogram shows the percent infarct volume for each condition. The neuroprotection from acidic postconditioning was robust even when the onset time was delayed to 50 minutes after reperfusion.However, acidosis treatment initiated at 100 minutes did not block the ischemic injury. After watching the video you should have a good understanding of how to study the neuroprotection of acidosis postconditioning on OGD model of brain slices, and on MSO model of mice. While attempting the procedures it is important to remember that the extension and the duration of acidosis is very important to reproduce the protective effects.

Research

JoVE Journal

Neuroscience

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

Una plataforma experimental para estudiar el comportamiento de circuito cerrado de interfaces cerebro-máquina

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

Investigación

Neurociencias

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Medición de la oxigenación sanguínea cerebral basada en el oxígeno-dependiente de enfriamiento de fosforescencia

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
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Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Modelo de isquemia cerebral focal por oclusión endovascular de sutura de la arteria cerebral media en la rata

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Modelos experimentales para el estudio de la fisiología epiteliales del pigmento retiniano y fisiopatología

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Evaluación de la lateralización cerebral en niños con ultrasonido Doppler transcraneal funcional (FTCD)

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Strategies for Study of Neuroprotection from Cold-preconditioning

Estrategias para el Estudio de la neuroprotección de frío preacondicionamiento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Evaluación de la función bioenergética en las células endoteliales vasculares cerebrales

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

Un protocolo de imagen de resonancia magnética para la estimación de tiempo de inicio de movimiento en Isquemia Cerebral permanente

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Culturas de rebanada Hippocampal Organotypic como un modelo para estudio neuroprotección e invasividad de las células tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...