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

Diese Arbeit wurde von der Nationalen Naturwissenschaftlichen Stiftung von China (81573406, 81373393, 81273506, 81221003, 81473186 und 81402907), Zhejiang Provincial Natural Science Foundation (LR15H310001) und dem Programm für Zhejiang Leading Team von S &amp; T Innovation Team (2011R50014) finanziert.

Alle Experimente wurden gemäß den ethischen Richtlinien des Zhejiang University Animal Experimentation Committee genehmigt und durchgeführt und waren in Übereinstimmung mit den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren. Es wurden Anstrengungen unternommen, um Schmerzen oder Unannehmlichkeiten zu minimieren, und die minimale Anzahl von Tieren wurde verwendet. 1. OGD von kortikostriatischen Scheiben <ol><li> Lösungsvorbereitung: ...

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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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Hier stellen wir zwei experimentelle Modelle vor, um die Neuroprotektion von APC gegen die zerebrale Ischämie zu untersuchen. In Hirnschnitten wird APC durch Inkubieren von Mäusen kortikostriatalen Scheiben in sauren Puffer, der mit 20% CO 2 nach dem Reperfusionsbeginn geblasen wurde, erreicht, während im MCAO-Modell APC durch Einatmen von 20% CO 2 zu Mäusen nach Reperfusion erreicht wird. Beide Modelle spiegeln die Neuroprotektion von APC gegen die zerebrale Ischämie wider. Der Schutz war ver...

Schlaganfall ist eine der führenden Ursachen für Sterblichkeit und Behinderung weltweit. Es wurden große Anstrengungen unternommen, um in den letzten Jahrzehnten wirksame Behandlungen für Schlaganfall zu finden. Allerdings ist die Leistung ganz unbefriedigend. Die Nachkonditionierung ist ein Prozess, der durch subtoxische Spannungen nach einer ischämischen Episode manipuliert wird. Nachkonditionierung, einschließlich ischämischer, hypoxischer, flachglukosierter und entfernter ischämischer Nachkonditionierung, lösen endogene adaptive Mechanismen aus und haben sich als vielversprechende Therapien gegen die zerebrale Ischämie 1 , 2 , 3 , 4 erwiesen. Allerdings kann die ischämische Nachkonditionierung zusätzliche Verletzungen einleiten. Die oberflächliche Ischämische Nachkonditionierung erfordert in der Regel mehrere Zyklen von 5 - 20 min Okklusion und Reperfusion an den ipsilateralen oder bilateralen Hinterbeinen 5 , 6 , 7 . ThDiese Nachkonditionierungsmanipulationen sind daher in der klinischen Praxis gefährlich oder unpraktisch. Um diese Nachteile zu überwinden, haben wir APC als Therapie für die fokale zerebrale Ischämie bei Mäusen entwickelt 8 . Induziert einfach durch Einatmen von 20% CO 2 , reduziert APC die ischämische Hirnverletzung in einer eher machbaren und sichereren Weise. Vor kurzem haben wir bewiesen, dass APC das Reperfusionsfenster erweitert und die Bedeutung von APC für die Schlaganfalltherapie hervorhebt 9 . Hier stellen wir zwei experimentelle Modelle vor, um die Neuroprotektion von APC gegen die zerebrale Ischämie zu untersuchen. Das erste ist das Sauerstoff-Glukose-Deprivation (OGD) -Modell in Mäusen kortikostriatalen Scheiben. Schnelle Vorbereitung und Übertragung der Hirnschnitten in eine künstliche Umgebung, in der Regel künstliche Cerebrospinalflüssigkeit (ASCF), kann die Lebensfähigkeit der Zellen und die neuronale Schaltung aufrechtzuerhalten, die es ermöglicht , 10 Hirnfunktion in vitro zu studieren &lt;Sup&gt;, 11 . OGD in ASCF imitiert zerebrale Ischämie und induziert ischämische Verletzung 12 , 13 , 14 . Nach OGD werden die Hirnschnitten in regulärem ASCF (r-ASCF) aufgefrischt, um eine Reperfusion zu ermöglichen und dann mit APC unter Verwendung von saurer ASCF, die mit 20% CO 2 geblasen wurde, behandelt. Die kortikostriatale Scheibe behält die intakte histologische Charakterisierung im Vergleich zu primär kultivierten Zellen bei. Um die Gehirnfunktion in vivo zu untersuchen , wird das Modell der Maus mit mittlerem zerebraler Arterienverschluss (MCAO) verwendet. Die mittlere zerebrale Arterie wird durch Einfügen eines flammunktionellen Monofilaments über die gemeinsame Carotis-Arterie blockiert. Als eines der am häufigsten verwendeten Schlaganfallmodelle zeigt das MCAO-Modell eine klinische Relevanz und die Anwendung eines Monofilaments macht es leichter, eine Reperfusion zu erreichen. Einfach durch Einatmen von normoxischem Mischgas, das 20% CO 2 nach dem Beginn der Reperfusio enthältN, APC zeigte signifikante Schutzwirkungen gegen die zerebrale Ischämie, die durch reduzierte Hirninfarktvolumina angezeigt wurde.

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		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
	</tbody>
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experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

Schlaganfall ist eine der führenden Ursachen für Sterblichkeit und Behinderung weltweit mit begrenzten therapeutischen Ansätzen. Als endogene Strategie für die Neuroprotektion haben sich die Nachkonditionierungsbehandlungen als vielversprechende Therapien gegen die zerebrale Ischämie erwiesen. Allerdings beschränken komplizierte Verfahren und potenzielle Sicherheitsfragen ihre klinische Anwendung. Um diese Nachteile zu überwinden, haben wir eine saure Nachkonditionierung (APC) als Therapie für die experimentelle fokale zerebrale Ischämie entwickelt. APC bezieht sich auf die milde Azidosebehandlung durch Einatmen von CO 2 während der Reperfusion nach Ischämie. Hier stellen wir zwei Modelle vor, um APC in vitro und in vivo auszuführen. Die Sauerstoff-Glukose-Deprivation (OGD) Behandlung von Mäusen und die kortikostriatale Okklusion und mittlere zerebrale Arterienverschluss (MCAO) von Mäusen wurden eingesetzt, um die zerebrale Ischämie nachzuahmen. APC kann einfach durch die Übertragung von Hirnschnitten auf sauren Puffer, der mit 20% CO 2 , o verschmolzen wird, erreicht werdenR durch Mäuse, die 20% CO 2 einatmen. APC zeigte signifikante Schutzwirkungen gegen die zerebrale Ischämie, wie sich aus der Gewebefähigkeit und dem Gehirninfarktvolumen widerspiegelt.

In dem oben beschriebenen corticostriatalen Scheibenmodell wurde die Lebensdauer der corticostriatalen Scheibe durch TTC-Test bei 1 h nach der Reperfusion quantifiziert. Die TTC-Umwandlung wurde durch Normalisierung der Absorption bei 490 nm an die Kontrollscheibe berechnet. Nach der TTC-Umwandlung, APC geschützt gegen OGD-induzierte Reperfusionsverletzung in einem Einsetzzeit und Dauer-abhängig Weise. Im Detail haben sowohl 1 als auch 3 min der Azidose-Behandlung die Lebensfähigkeit ...

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

Die saure Nachkonditionierung schützt vor der zerebralen Ischämie. Hier stellen wir zwei Modelle vor, um APC auszuführen. Sie werden jeweils durch Übertragung von kortikostriatalen Scheiben auf sauren Puffer nach Sauerstoff-Glukose-Deprivation in vitro und durch Einatmen von 20% CO 2 nach mittlerer zerebraler Arterienverschluss in vivo erreicht .

Experimentelle Modelle zur Untersuchung der Neuroprotektion der sauren Nachkonditionierung gegen zerebrale Ischämie

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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Neuroscience

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

Eine experimentelle Plattform für die Closed-Loop-Performance von Brain-Machine Interfaces Study

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

Forschung

Neurowissenschaften

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

Cerebral Blutoxygenierung Messung auf Sauerstoff-abhängige Quenching der Phosphoreszenz Based

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Fokaler zerebraler Ischämie-Modell von Endovascular Suture Verschluss der mittleren Hirnarterie im Rat

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

Experimentelle Modelle für das Studium der retinalen Pigmentepithelzellen Physiologie und Pathophysiologie

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

Beurteilung der zerebralen Lateralisation bei Kindern mit Hilfe der funktionellen transkraniellen Doppler-Sonographie (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

Strategies for Study of Neuroprotektion von Cold-Vorkonditionierung

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

Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

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

Auswertung der bioenergetischen Funktion in Cerebral Vascular Endothelial Cells

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

Ein Magnet-Resonanz-Tomographie-Protokoll für Schlaganfall auftreten Zeit Schätzung in ständigen zerebraler Ischämie

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

Organotypischen Hippocampal Slice Kulturen als Modell zur Studie Neuroprotektion und Invasivität der Tumorzellen

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