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

Questo lavoro è stato finanziato dalla Fondazione Nazionale di Scienze Naturali della Cina (81573406, 81373393, 81273506, 81221003, 81473186 e 81402907), Zhejiang Provincial Science Foundation (LR15H310001) e il Programma di Zhejiang Leadership Team di S &amp; T Innovation Team (2011R50014).

Tutti gli esperimenti sono stati approvati e condotti in conformità con le linee guida etiche del comitato scientifico per la sperimentazione degli animali di Zhejiang e sono stati in piena conformità con la Guida nazionale degli istituti sanitari per la cura e l&#39;uso degli animali da laboratorio. Sono stati fatti sforzi per minimizzare qualsiasi dolore o disagio, ed è stato utilizzato il numero minimo di animali. 1. OGD di fette corticostriatali <ol><li> Preparazione della s...

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	<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. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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Qui presentiamo due modelli sperimentali per studiare la neuroprotezione dell&#39;APC contro l&#39;ischemia cerebrale. Nelle fette del cervello, l&#39;APC è ottenuto incubando le fette corticostriatali dei topi in tampone acico in boom con 20% di CO 2 dopo l&#39;insorgenza di reperfusione, mentre nel modello MCAO l&#39;APC è ottenuto inalando 20% di CO 2 nei topi dopo la reperfusione. Entrambi i modelli riflettono la neuroprotezione dell&#39;APC contro l&#39;ischemia cerebrale. La protezione era ...

L&#39;ictus è una delle principali cause di mortalità e disabilità in tutto il mondo. Sono stati compiuti grandi sforzi per trovare efficaci trattamenti di ictus negli ultimi decenni, tuttavia il risultato è piuttosto insoddisfacente. Postcondizionamento è un processo manipolato da sottotossici stress dopo un episodio ischemico. Postcondizionamento, incluso ischemico, ipossico, basso glucosio e post-condizionamento ischemico a distanza, provoca meccanismi di adattamento endogeno e sono stati dimostrati promettenti terapie contro l&#39;ischemia cerebrale 1 , 2 , 3 , 4 . Tuttavia, il postcondizionamento ischemico può presentare ulteriori lesioni. Il postcondizionamento ischemico remoto a distanza richiede solitamente diversi cicli di occlusione e reperfusione da 5 a 20 minuti sugli arti posteriori ipsilaterali o bilaterali 5 , 6 , 7 . thIn realtà, queste manipolazioni post-condizionanti sono pericolose o impraticabili nella pratica clinica. Per superare questi svantaggi, abbiamo sviluppato APC come terapia per l&#39;ischemia cerebrale focale nei topi 8 . Indotta semplicemente inalando 20% di CO 2 , APC riduce in modo significativo lesioni cerebrali ischemiche in modo più fattibile e più sicuro. Recentemente abbiamo dimostrato che APC estende la finestra di reperfusione, evidenziando il significato dell&#39;APC per la terapia a colpo 9 . Qui presentiamo due modelli sperimentali per studiare la neuroprotezione dell&#39;APC contro l&#39;ischemia cerebrale. Il primo è il modello di deprivazione di ossigeno-glucosio (OGD) in fette corticostriatale di topi. La rapida preparazione e il trasferimento delle fette del cervello in un ambiente artificiale, di solito liquido cerebrospinale artificiale (ASCF), può mantenere la vitalità delle cellule e la circuiteria neuronale, che consente di studiare la funzione cerebrale in vitro 10 &lt;Sup&gt;, 11 . OGD in ASCF imita l&#39;ischemia cerebrale e induce lesioni ischemiche 12 , 13 , 14 . Dopo l&#39;OGD, le fette del cervello vengono aggiornate in ASCF regolare (r-ASCF) per fornire reperfusione e quindi trattate con APC usando ASCF acido bolle con 20% CO 2 . La fetta corticostriatal mantiene la caratterizzazione istologica intatta rispetto alle cellule primari coltivate. Per studiare la funzionalità cerebrale in vivo , viene utilizzato il modello di occlusione cerebrale cerebrale centrale (MCAO). L&#39;arteria cerebrale centrale viene bloccata inserendo un monofilamento a fiamma attraverso la comune arteria carotidea. Come uno dei modelli di corsa più utilizzati, il modello MCAO dimostra la rilevanza clinica e l&#39;applicazione di un monofilamento rende più facile la reperfusione. Semplicemente inalando il gas misto normalmente contenente 20% di CO 2 dopo l&#39;insorgenza del reperfusioN, APC ha dimostrato significativi effetti protettivi contro l&#39;ischemia cerebrale indicata da volumi ridotti del cervello.

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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>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">G7021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
			<td style="width:167px;"></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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<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>
			<td style="width:167px;"></td>
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		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
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experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

L&#39;ictus è una delle principali cause di mortalità e disabilità in tutto il mondo, con approcci terapeutici limitati. Come una strategia endogena per la neuroprotezione, i trattamenti post-condizionatori si sono dimostrati come terapie promettenti contro l&#39;ischemia cerebrale. Tuttavia, complicate procedure e potenziali problemi di sicurezza limitano la loro applicazione clinica. Per superare questi svantaggi, abbiamo sviluppato postcondizionamento acico (APC) come terapia per l&#39;ischemia cerebrale focale sperimentale. APC si riferisce al trattamento di acidosi mite mediante inalazione del CO 2 durante la reperfusione a seguito di ischemia. Qui presentiamo due modelli per eseguire APC in vitro e in vivo rispettivamente. Il trattamento della deprivazione di ossigeno-glucosio (OGD) dei topi e l&#39;occlusione corticostriatale e l&#39;occlusione dell&#39;arteria cerebrale centrale (MCAO) dei topi sono stati impiegati per simulare l&#39;ischemia cerebrale. L&#39;APC può essere ottenuto semplicemente trasferendo le fettucce del cervello in tampone acido con 20% CO 2 , oR per topi inalando 20% di CO 2 . APC ha mostrato significativi effetti protettivi contro l&#39;ischemia cerebrale, come rispecchia la vitalità del tessuto e il volume dell&#39;infarto del cervello.

Nel modello di fetta corticostriatal descritto sopra, la vitalità della fetta corticostriatale è stata quantificata con il dosaggio TTC a 1 h dopo la reperfusione. La conversione TTC è stata calcolata normalizzando l&#39;assorbimento a 490 nm alla fetta di controllo. Secondo la conversione TTC, l&#39;APC è protetto contro lesioni di reperfusione indotte da OGD in un modo di dipendenza dal tempo e dalla durata. In dettaglio, sia 1 e 3 minuti di trattamento dell&#39;acidosi hanno migli...

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

La postcondizionazione acida protegge contro l&#39;ischemia cerebrale. Qui presentiamo due modelli per eseguire APC. Esse vengono ottenute rispettivamente trasferendo fette corticostriatali in tampone acico dopo la privazione di ossigeno-glucosio in vitro e inalando 20% di CO 2 dopo l&#39;occlusione dell&#39;arteria cerebrale media in vivo .

Modelli sperimentali per studiare la neuroprotezione del post-condizionamento acido contro l&#39;ischemia cerebrale

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.

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Neuroscienze

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

Ossigenazione cerebrale misura Sangue Sulla base di ossigeno-dipendente tempra di fosforescenza

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

Modello di ischemia cerebrale focale da occlusione endovascolare Sutura della arteria cerebrale media nel ratto

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

Modelli sperimentali per lo Studio di Fisiologia dell&#39;epitelio pigmentato retinico e fisiopatologia

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

Valutazione della lateralizzazione cerebrale nei bambini utilizzando funzionale ad ultrasuoni Doppler transcranico (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

Strategie per lo Studio delle Neuroprotezione dal freddo precondizionamento

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

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

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

Valutazione della funzione bioenergetica in cellule endoteliali vascolare cerebrale

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 protocollo di risonanza magnetica per la stima del tempo di corsa inizio nell'Ischemia cerebrale 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

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

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