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 trabalho foi financiado pela Fundação Nacional de Ciências Naturais da China (81573406, 81373393, 81273506, 81221003, 81473186 e 81402907), Fundação Provincial de Ciências Naturais de Zhejiang (LR15H310001) e o Programa para a equipe líder de Zhejiang da equipe de inovação S &amp; T (2011R50014).

Todos os experimentos foram aprovados e conduzidos de acordo com as diretrizes éticas do Comitê de Experimentação Animal da Universidade de Zhejiang e estavam em completa conformidade com os Institutos Nacionais de Guia de Saúde para o Cuidado e Uso de Animais de Laboratório. Foram feitos esforços para minimizar qualquer dor ou desconforto, e o número mínimo de animais foi usado. 1. OGD de fatias Corticostriatal <ol><li> Preparação da solução: <ol><li> Prepara...

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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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Aqui apresentamos dois modelos experimentais para estudar a neuroproteção de APC contra isquemia cerebral. Em fatias de cérebro, a APC é conseguida através da incubação de fatias corticostriatais de camundongos em tampão ácido borbulhado com 20% de CO 2 após o início da reperfusão, enquanto que no modelo MCAO, a APC é conseguida inalando 20% de CO 2 para ratos após reperfusão. Ambos os modelos refletem a neuroproteção da APC contra a isquemia cerebral. A proteção foi comparável ...

O acidente vascular cerebral é uma das principais causas de mortalidade e incapacidade em todo o mundo. Grandes esforços foram feitos para encontrar tratamentos efetivos para AVC nas últimas décadas, no entanto, a conquista é bastante insatisfatória. O pós-condicionamento é um processo manipulado por tensões subtoxicas após um episódio isquêmico. O pós-condicionamento, incluindo o pós-condicionamento isquêmico, hipóxico, baixo teor de glicose e remoto, desencadeia mecanismos adaptativos endógenos e provou ser terapias promissoras contra a isquemia cerebral 1 , 2 , 3 , 4 . Contudo, o pós-condicionamento isquêmico pode introduzir lesões adicionais. O pós-condicionamento isquêmico remoto dos membros geralmente precisa de vários ciclos de 5 a 20 min de oclusão e reperfusão nos membros traseiros ipsilaterais ou bilaterais 5 , 6 , 7 . ºPortanto, essas manipulações pós-condicionamento são perigosas ou impraticáveis ​​na prática clínica. Para superar essas desvantagens, desenvolvemos APC como terapia para isquemia cerebral focal em camundongos 8 . Induzido simplesmente por inalação de 20% de CO 2 , APC reduz significativamente a lesão cerebral isquêmica de uma maneira mais viável e segura. Recentemente, provamos que a APC estende a janela de reperfusão, destacando o significado da APC para terapia de AVC 9 . Aqui apresentamos dois modelos experimentais para estudar a neuroproteção de APC contra isquemia cerebral. O primeiro é o modelo de privação de oxigênio-glicose (OGD) em fatias corticostriatais de camundongos. A rápida preparação e transferência das fatias do cérebro para um ambiente artificial, geralmente fluido cerebrospinal artificial (ASCF), pode manter a viabilidade celular e circuitos neuronais, o que possibilita o estudo da função cerebral in vitro 10 &lt;Sup&gt; 11 . OGD em ASCF imita a isquemia cerebral e induz a lesão isquêmica 12 , 13 , 14 . Após OGD, as fatias de cérebro são atualizadas em ASCF (r-ASCF) regulares para fornecer reperfusão e depois tratadas com APC usando ASCF ácido borbulhou com 20% de CO 2 . A fatia corticostriatal mantém a caracterização histológica intacta em comparação com células primárias cultivadas. Para estudar a função cerebral in vivo , emprega-se o modelo de oclusão da artéria cerebral média (MCAO) do mouse. A artéria cerebral média é bloqueada pela inserção de um monofilamento inflamado através da artéria carótida comum. Como um dos modelos de AVC mais amplamente utilizados, o modelo MCAO mostra relevância clínica e a aplicação de um monofilamento torna mais fácil a reperfusão. Simplesmente pela inalação de gases mistos normoxicos contendo 20% de CO 2 após o início do reperfusioN, a APC mostrou efeitos protetores significativos contra a isquemia cerebral indicada por volumes reduzidos 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>
		</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>
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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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		<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>
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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>
			<td style="width:167px;"></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>
			<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>
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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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		<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>
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		<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

O acidente vascular cerebral é uma das principais causas de mortalidade e incapacidade em todo o mundo, com abordagens terapêuticas limitadas. Como uma estratégia endógena para a neuroproteção, os tratamentos pós-condicionamento provaram ser terapias promissoras contra a isquemia cerebral. No entanto, procedimentos complicados e possíveis problemas de segurança limitam sua aplicação clínica. Para superar estas desvantagens, desenvolvemos pós-condicionamento ácido (APC) como terapia para isquemia cerebral focal experimental. APC refere-se ao tratamento de acidose leve por inalação de CO 2 durante a reperfusão após isquemia. Aqui apresentamos dois modelos para executar APC in vitro e in vivo , respectivamente. O tratamento de privação de oxigênio-glicose (OGD) de camundongos e a oclusão corticostriatal e oclusão da artéria cerebral média (MCAO) de camundongos foram empregados para imitar a isquemia cerebral. APC pode ser simplesmente conseguido através da transferência de fatias de cérebro para tampão ácido borbulhado com 20% de CO 2 , oR por camundongos que inalam 20% de CO 2 . A APC mostrou efeitos protetores significativos contra a isquemia cerebral, como reflete a viabilidade do tecido e o volume do infarto cerebral.

No modelo da fatia corticostriatal descrita acima, a viabilidade corticostriatal da fatia foi quantificada por meio de ensaio TTC às 1 h após a reperfusão. A conversão TTC foi calculada normalizando a absorção a 490 nm para a fatia de controle. De acordo com a conversão de TTC, a APC protegeu contra lesões de reperfusão induzidas por OGD de uma maneira dependente de tempo e de início. Em detalhes, tanto 1 quanto 3 minutos de tratamento de acidose melhoraram significativamente a...

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

O pós-condicionamento ácido protege contra a isquemia cerebral. Aqui apresentamos dois modelos para executar a APC. Eles são obtidos, respectivamente, transferindo fatias corticostriatais para o tampão ácido após a privação de oxigênio-glicose in vitro e pela inalação de 20% de CO 2 após a oclusão da artéria cerebral média in vivo .

Modelos experimentais para estudar a neuroproteção do pós-condicionamento ácido contra a 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.

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

Modelo de isquemia focal cerebral por oclusão Suture Endovascular da artéria cerebral média no 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

Modelos experimentais para estudo da fisiologia epitélio pigmentar da retina 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

Avaliação da lateralização cerebral em crianças usando Funcional ultra-som Doppler transcraniano (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

Estratégias para Estudo da neuroproteção de frio pré-condicionamento

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

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

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

Avaliação da função bioenergética em células endoteliais Vascular Cerebral

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

Um protocolo de ressonância magnética para estimativa de tempo de início curso em situações de 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

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

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