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

Detta arbete finansierades av National Natural Science Foundation of China (81573406, 81373393, 81273506, 81221003, 81473186 och 81402907), Zhejiang Provincial Natural Science Foundation (LR15H310001) och programmet för Zhejiang ledande team i S &amp; T Innovation Team (2011R50014).

Alla experiment godkändes av och genomfördes i enlighet med de etiska riktlinjerna från Zhejiang University Animal Experimentation Committee och var i fullständig överensstämmelse med National Institutes of Health Guide för vård och användning av laboratoriedjur. Åtgärder gjordes för att minimera eventuell smärta eller obehag och det minsta antalet djur användes. 1. OGD av kortikostriatala skivor <ol><li> Lösningsberedning: <ol><li> Förbereda 1000 ml r-ACSF...

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

Här presenterar vi två experimentella modeller för att studera neuroprotektion av APC mot cerebral ischemi. I hjärnsnitt, är APC uppnås genom inkubering av möss corticostriatal skivor i sur buffert bubblades med 20% CO 2 efter reperfusion insjuknandet, medan i MCAO modell, är APC uppnås genom att andas in 20% CO 2 till möss efter reperfusion. Båda modellerna återspeglar neuroprotektion av APC mot cerebral ischemi. Skyddet var jämförbart med det som uppnåddes genom ischemisk efterkond...

Stroke är en av de främsta orsakerna till dödlighet och funktionshinder över hela världen. Stora ansträngningar har gjorts för att hitta effektiva behandlingar för stroke under de senaste decennierna, men prestationen är ganska otillfredsställande. Postkonditionering är en process som manipuleras av subtoxiska påfrestningar efter en ischemisk episod. Efterkonditionering, inklusive ischemisk, hypoxisk, lågglukos och avlägsen ischemisk efterkonditionering, utlöser endogena adaptiva mekanismer och har visat sig vara lovande behandlingar mot cerebral ischemi 1 , 2 , 3 , 4 . Men iskemisk efterkonditionering kan införa ytterligare skada. Limkemisk avlägsnande av ischemisk efterkonditionering behöver vanligtvis flera cykler om 5-20 min ocklusion och reperfusion på de ipsilaterala eller bilaterala bakbenen 5 , 6 , 7 . thDärför är dessa efterbehandlingsmanipulationer farliga eller opraktiska i klinisk praxis. För att övervinna dessa nackdelar har vi utvecklat APC som en terapi för fokal cerebral ischemi hos möss 8 . Inducerad helt enkelt genom att andas in 20% CO 2, APC signifikant reducerar ischemisk hjärnskada i en mer genomförbar och säkrare sätt. Nyligen har vi visat att APC utökar reperfusionsfönstret och belyser APC: s betydelse för strokebehandling 9 . Här presenterar vi två experimentella modeller för att studera neuroprotektion av APC mot cerebral ischemi. Den första är syre-glukosavskrivning (OGD) -modellen i kort-sterostabila stycken av möss. Snabba förberedelser och överföring av hjärnskivorna till en artificiell miljö, vanligtvis artificiell cerebrospinalvätska (ASCF), kan upprätthålla celllabilitet och neuronal krets, vilket gör det möjligt att studera hjärnfunktionen in vitro 10 &lt;Sup&gt; 11 . OGD i ASCF efterliknar cerebral ischemi och inducerar ischemisk skada 12 , 13 , 14 . Efter OGD är hjärnan skivor uppdateras i regelbunden ASCF (r-ASCF) för att tillhandahålla reperfusion och behandlades därefter med APC med användning av sur ASCF bubblades med 20% CO2. Den kortikostriatala skivan upprätthåller den intakta histologiska karakteriseringen jämfört med primära odlade celler. För att studera hjärnfunktionen in vivo används musen mellan mitten av cerebral arteriell ocklusion (MCAO). Den centrala cerebrala artären blockeras genom att införa ett flamstoppat monofilament via den gemensamma halspulsådern. Som en av de mest använda stroke-modellerna visar MCAO-modellen klinisk relevans och appliceringen av en monofilament gör det lättare att uppnå reperfusion. Helt enkelt genom att andas in normoxisk blandad gas innehållande 20% CO 2 efter debuten av reperfusioN, visade APC signifikanta skyddande effekter mot cerebral ischemi som indikeras av minskad hjärntrådsvolym.

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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:183px;">Sigma</td>
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			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
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			<td height="42" style="height:42px;width:216px;">Potassium phosphate monobasic</td>
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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>
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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>
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			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
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			<td height="21" style="height:21px;width:216px;">Vibratome</td>
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			<td height="42" style="height:42px;width:216px;">2,3,5-triphenyltetrazolium hydrochloride</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>
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			<td height="42" style="height:42px;width:216px;">Laser Doppler Flowmetry</td>
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			<td height="42" style="height:42px;width:216px;">Diethyl ether anhydrous</td>
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			<td height="42" style="height:42px;width:216px;">Trichloroacetaldehycle hydrate</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 är en av de främsta orsakerna till dödlighet och funktionshinder världen över, med begränsade terapeutiska förhållningssätt. Som en endogen strategi för neuroprotektion har efterbehandlingsbehandlingar visat sig vara lovande terapier mot cerebral ischemi. Emellertid begränsar komplicerade procedurer och potentiella säkerhetsproblem deras kliniska tillämpning. För att övervinna dessa nackdelar har vi utvecklat sur postconditioning (APC) som en terapi för experimentell fokal cerebral ischemi. APC hänför sig till den milda acidosbehandlingen genom inhalation av CO 2 under reperfusion efter ischemi. Här presenterar vi två modeller för att utföra APC in vitro respektive in vivo . Syre-glukosavskrivning (OGD) -behandling av möss och corticostriatal ocklusion och mitten av cerebral arteriell ocklusion (MCAO) hos möss användes för att efterlikna cerebral ischemi. APC kan enkelt uppnås genom att överföra hjärnsnitt till sur buffert bubblades med 20% CO 2, or av möss som inhalerar 20% CO 2. APC visade signifikanta skyddande effekter mot cerebral ischemi, vilket återspeglas av vävnadslevnadsförmåga och hjärninfarktvolymen.

I den kortikostriatala skivmodellen beskriven ovan kvantifierades corticostriatal skivbarbarhet genom TTC-analys vid 1 h efter reperfusion. TTC-omvandling beräknades genom normalisering av absorptionen vid 490 nm till kontrollskivan. Enligt TTC-omvandling skyddades APC mot OGD-inducerad reperfusionsskada vid en starttid och varaktighetsberoende sätt. I detalj förbättrade både 1 och 3 min acidosebehandling signifikant livskraften vid 5 min efter 15 min OGD, medan 5 min inte (OGD: 0,6...

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

Syran efterkonditionering skyddar mot cerebral ischemi. Här presenterar vi två modeller för att utföra APC. De är respektive uppnås genom att överföra corticostriatal skivor till sur buffert efter syre-glukos deprivation in vitro och genom att andas in 20% CO 2 efter mellersta hjärnartären ocklusion in vivo.

Experimentella modeller för att studera neuroprotektion av sur postkonditionering mot cerebral ischemi

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

Fokal cerebral ischemi modellen genom endovaskulär suture Ocklusion av Mellanöstern Cerebral artär i 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

Experimentella modeller för studier av näthinnan pigmentepitel fysiologi och patofysiologi

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

Bedömning av Cerebral Lateralization hos barn med hjälp av funktionell Transkraniell Doppler ultraljud (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

Strategier för Studie av Neuroprotektion från Cold-konditioneringen

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

Genetic Manipulation av Cerebellar granulneuroner<em&gt; In Vitro</em&gt; Och<em&gt; In Vivo</em&gt; Att studera neuronal morfologi och 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

Utvärdering av bioenergetisk Funktion i Cerebral vaskulära 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 resonanstomografi protokoll för Stroke debut tid uppskattning i Permanent Cerebral ischemi

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 Slice kulturer som en modell att studien neuroprotektion och invasivitet av tumörceller

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