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

Dette arbejde blev finansieret af National Natural Science Foundation of China (81573406, 81373393, 81273506, 81221003, 81473186 og 81402907), Zhejiang Provincial Natural Science Foundation (LR15H310001) og Programmet for Zhejiang Leading Team af S &amp; T Innovation Team (2011R50014).

Alle forsøg blev godkendt af og udført i overensstemmelse med de etiske retningslinjer fra Zhejiang University Animal Experimentation Committee og var i fuld overensstemmelse med National Institutes of Health Guide til pleje og brug af laboratoriedyr. Der blev gjort en indsats for at minimere smerte eller ubehag, og det mindste antal dyr blev brugt. 1. OGD af Corticostriatal Skiver <ol><li> Opløsning forberedelse: <ol><li> Forbered 1.000 ml r-ACSF (124 mmol / l, NaCl, 5...

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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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Her præsenterer vi to eksperimentelle modeller for at studere neuroprotektion af APC mod cerebral iskæmi. I hjerneskiver opnås APC ved at inkubere mus kortikostriatale skiver i sur puffer boblet med 20% CO 2 efter reperfusionsstart, mens APC opnås ved at inhalere 20% CO 2 til mus efter reperfusion. Begge modeller afspejler neuroprotektion af APC mod cerebral iskæmi. Beskyttelsen var sammenlignelig med den, der blev opnået ved iskæmisk efterkonditionering, men med et bredere tidsvindue. I MCA...

Slag er en af ​​de førende årsager til dødelighed og handicap verden over. Der er gjort en stor indsats for at finde effektive behandlinger for slagtilfælde i de sidste årtier, men præstationen er ret utilfredsstillende. Postkonditionering er en proces manipuleret af subtoksiske belastninger efter en iskæmisk episode. Postkonditionering, herunder iskæmisk, hypoxisk, lavglucose og fjern iskemisk efterkonditionering, udløser endogene adaptive mekanismer og har vist sig at være lovende behandlinger mod cerebral iskæmi 1 , 2 , 3 , 4 . Imidlertid kan iskæmisk postkonditionering indføre yderligere skade. Limben fjern iskemisk postkonditionering har normalt brug for flere cykler med 5-20 min okklusion og reperfusion på de ipsilaterale eller bilaterale bagben 5 , 6 , 7 . thDerfor er disse efterbehandlingsmanipulationer farlige eller upraktiske i klinisk praksis. For at overvinde disse ulemper har vi udviklet APC som en terapi for fokal cerebral iskæmi hos mus 8 . Induceret simpelthen ved at indånde 20% CO 2 , reducerer APC signifikant iskæmisk hjerneskade på en mere gennemførlig og sikrere måde. For nylig har vi bevist, at APC udvider reperfusionsvinduet og fremhæver APC&#39;s betydning for strokebehandling 9 . Her præsenterer vi to eksperimentelle modeller for at studere neuroprotektion af APC mod cerebral iskæmi. Den første er oxygen-glucose deprivation (OGD) model i mus kortikostriatale skiver. Hurtig forberedelse og overførsel af hjerneskiver til et kunstigt miljø, som regel kunstig cerebrospinalvæske (ASCF), kan bevare cellelevedygtighed og neuronalkredsløb, hvilket gør det muligt at studere hjernefunktion in vitro 10 &lt;Sup&gt; 11 . OGD i ASCF efterligner cerebral iskæmi og inducerer iskæmisk skade 12 , 13 , 14 . Efter OGD bliver hjerneskiverne opdateret i almindelig ASCF (r-ASCF) for at give reperfusion og derefter behandlet med APC ved anvendelse af sur ASCF boblet med 20% CO 2 . Den kortikostriatale skive opretholder den intakte histologiske karakterisering sammenlignet med primære dyrkede celler. For at studere hjernefunktion in vivo anvendes musen mellem cerebral arterieeklusion (MCAO) -modellen. Den midterste cerebrale arterie er blokeret ved at indsætte et flammeformet monofilament via den fælles halspulsårer. Som en af ​​de mest anvendte stroke-modeller viser MCAO-modellen klinisk relevans, og anvendelsen af ​​et monofilament gør det lettere at opnå reperfusion. Blot ved at indånde normoxisk blandet gas indeholdende 20% CO 2 efter indtræden af ​​reperfusioN viste APC signifikante beskyttende virkninger mod cerebral iskæmi indikeret ved reducerede hjerneinfarktvolumener.

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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
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			<td style="width:157px;">S5886</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>
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			<td height="42" style="height:42px;width:216px;">Potassium phosphate monobasic</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:157px;">P9791</td>
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			<td height="21" style="height:21px;width:216px;">Magnesium sulfate</td>
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			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</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>
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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>
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			<td style="width:157px;">VT1000 S</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>
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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>
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			<td height="42" style="height:42px;width:216px;">Laser Doppler Flowmetry</td>
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			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
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			<td style="width:183px;">Sinopharm Chemical Reagent Corporation</td>
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			<td height="42" style="height:42px;width:216px;">10% Formalin</td>
			<td style="width:183px;">Aladdin Industrial Corporation</td>
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			<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>
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experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

Stroke er en af ​​de førende årsager til dødelighed og handicap verden over, med begrænsede terapeutiske tilgange. Som en endogen strategi for neuroprotektion har postkonditionerende behandlinger vist sig at være lovende terapier mod cerebral iskæmi. Imidlertid begrænser komplicerede procedurer og potentielle sikkerhedsproblemer deres kliniske anvendelse. For at overvinde disse ulemper har vi udviklet sur postconditioning (APC) som en terapi til eksperimentel fokal cerebral iskæmi. APC refererer til mild acidosebehandling ved inhalation af CO 2 under reperfusion efter iskæmi. Her præsenterer vi to modeller til henholdsvis APC in vitro og in vivo . Oxygen-glucose deprivation (OGD) behandling af mus og corticostriatal okklusion og midter cerebral arterie okklusion (MCAO) af mus blev anvendt til at efterligne cerebral iskæmi. APC kan simpelthen opnås ved at overføre hjerneskiver til sur puffer boblet med 20% CO 2 , oR ved mus inhalering 20% ​​CO 2 . APC viste signifikante beskyttende virkninger mod cerebral iskæmi, som afspejlet af vævsevnen og hjerneinfarktvolumen.

I den ovenfor beskrevne kortikostriatale skivemodel blev corticostriatal skive-levedygtighed kvantificeret ved TTC-analyse efter 1 h efter reperfusion. TTC-omdannelse blev beregnet ved normalisering af absorptionen ved 490 nm til kontrolstykket. Ifølge TTC-omdannelse beskyttes APC mod OGD-induceret reperfusionsskade i en starttid og varighedsafhængig måde. I detaljer blev både 1 og 3 min. Acidosisbehandling signifikant forbedret levedygtighed ved 5 minutter efter 15 min OGD, mens 5 m...

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

Acid postkonditionering beskytter mod cerebral iskæmi. Her præsenterer vi to modeller til at udføre APC. De opnås henholdsvis ved at overføre kortikostriatale skiver til sur buffer efter oxygen-glucose-deprivation in vitro og ved at inhalere 20% CO 2 efter midter cerebral arterieeklusion i vivo .

Eksperimentelle modeller til undersøgelse af neuroprotektion af sur postkonditionering mod cerebral iskæmi

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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Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

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

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

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

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

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

Evaluering af bioenergetic Funktion i cerebrale vaskulære endotelceller

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

En magnetisk resonans protokol for slagtilfælde indsættende tid skøn i Permanent Cerebral iskæmi

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 hippocampus skive kulturer som en Model til undersøgelse Neuroprotection og invasionsevne af tumorceller

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