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

Bu çalışma, Çin Ulusal Doğa Bilimleri Vakfı (81573406, 81373393, 81273506, 81221003, 81473186 ve 81402907), Zhejiang Eyalet Doğa Bilimleri Vakfı (LR15H310001) ve S &amp; T Yenilik Ekibinin Zhejiang Leading Team Programı (2011R50014) tarafından finanse edildi.

Tüm deneyler, Zhejiang Üniversitesi Hayvan Deneyleri Komitesinin etik kurallarına göre onaylanmış ve yürürlüğe girmiş ve Laboratuar Hayvanlarının Bakımı ve Kullanımı Ulusal Sağlık Enstitüleri ile tam bir uyum içindedir. Herhangi bir ağrı veya rahatsızlığı en aza indirmek için çaba gösterildi ve asgari hayvan sayısı kullanıldı. 1. Corticostriatal dilimlerin OGD&#39;leri <ol><li> Çözelti hazırlığı: <ol><li> 1.000 ml r-ACSF (124 mmol / ...

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Burada serebral iskemiye karşı APC&#39;nin nöroproteksiyonunu incelemek için iki deneysel model sunmaktayız. Beyin dilimlerinde APC, reperfüzyon başlangıcından sonra fare kortikostriyat dilimlerini% 20 CO 2 ile kabarcıklar halinde asidik tampon içinde kuluçkaya yatırarak elde edilirken, MCAO modelinde reperfüzyon sonrası farelere% 20 CO 2 soluk aldırarak APC elde edilir. Her iki model de serebral iskemiye karşı APC&#39;nin nörokoruyorumunu yansıtmaktadır. Koruma, iskemik sonras...

İnme, dünya çapındaki mortalite ve engelliliğin önde gelen nedenlerinden biridir. Son on yılda inme için etkili tedavileri bulmak için büyük çabalar gösterildi, ancak başarı oldukça yetersiz. Sonradan şartlandırma, iskemik bir aşamayı takiben alttoksik stresler tarafından manipüle edilen bir işlemdir. İskemik, hipoksik, düşük glukoz ve uzak iskemik postkondisyon dahil olmak üzere kondisyon düzeltme, endojen uyarlamalı mekanizmaları tetiklemektedir ve serebral iskemiye karşı umut verici tedaviler olduğu kanıtlanmıştır 1 , 2 , 3 , 4 . Bununla birlikte, iskemik son şartlandırma ek yaralanmaya neden olabilir. Ekstremitenin uzak iskemik son kondisyonu genellikle ipsilateral veya bilateral arka bacaklarda 5 , 6 , 7&#39;de 5-20 dakika oklüzyon ve reperfüzyona ihtiyaç duyar. thBu nedenle, bu kondüsyon sonrası manipülasyonlar klinik uygulamada tehlikeli veya pratik değildir. Bu dezavantajları aşmak için, fareler 8 fokal serebral iskemi için bir tedavi olarak APC geliştirdik. Sadece% 20 CO 2&#39;yi teneffüs ederek indüklenen APC, iskemik beyin hasarını daha uygulanabilir ve daha güvenli bir şekilde önemli ölçüde azaltır. Yakın zamanda APC felç tedavi 9 için APC önemini vurgulayarak, reperfüzyon penceresini genişletir kanıtlamıştır. Burada serebral iskemiye karşı APC&#39;nin nöroproteksiyonunu incelemek için iki deneysel model sunmaktayız. Bunlardan ilki, kortikostriatal dilimdeki oksijen glukoz yoksunluğu (OGD) modelidir. Yapay ortam içine hızlı hazırlanması ve beyin kesitleri devri, genellikle yapay serebrospinal akışkan (ASCF), mümkün in vitro 10 &lt;beyin fonksiyonu çalışma yapar, hücre canlılığı ve nöronal devre korumakSup&gt;, 11 . ASCF&#39;deki OGD, serebral iskemi taklit eder ve iskemik hasar oluşturur 12 , 13 , 14 . OGD&#39;den sonra, beyin dilimleri reperfüzyon sağlamak için düzenli ASCF (r-ASCF) içinde yenilenir ve daha sonra% 20 CO 2 ile kabarmış asidik ASCF kullanılarak APC ile muamele edilir. Kortikostriatal dilim, birincil kültürlenmiş hücrelere kıyasla sağlam histolojik karakterizasyonunu korur. İn vivo olarak beyin fonksiyonunu incelemek için fare orta serebral arter tıkanıklığı (MCAO) modeli kullanılmıştır. Orta serebral arter, ortak karotis atardamarından alevle köreltilmiş bir monofilament yerleştirilerek engellenir. En yaygın vuruş modellerinden biri olan MCAO modeli klinik önemi göstermektedir ve bir monofilamanın uygulanması reperfüzyonu kolaylaştırmaktadır. Reperfüzyon başladıktan sonra% 20 CO 2 içeren normoksik karıĢık gaz teneffüs edilerekN, APC azalmış beyin enfarktüsü hacimleri ile gösterilen serebral iskemiye karşı önemli koruyucu etkiler gösterdi.

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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>
</table>

experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

İnme, sınırlı terapötik yaklaşımlarla dünyadaki mortalite ve sakatlığın önde gelen nedenlerinden biridir. Nöroproteksiyon için endojen bir strateji olarak, kondisyon sonrası tedavilerin serebral iskemiye karşı umut verici tedaviler olduğu kanıtlanmıştır. Bununla birlikte, karmaşık prosedürler ve olası güvenlik sorunları, klinik uygulamalarını sınırlar. Bu dezavantajların üstesinden gelmek için deneysel fokal serebral iskemi için bir terapi olarak asidik sonrası düzeltme (APC) geliştirdik. APC iskemi sonrası reperfüzyon esnasında CO2 solunması ile hafif asidoz tedavisi anlamına gelmektedir. Burada APC&#39;yi in vitro ve in vivo olarak yürütmek için sırasıyla iki model sunuyoruz. Farelerin oksijen-glikoz yoksunluğu (OGD) tedavisi ve farelerin kortikostriatal oklüzyonu ve orta serebral arter oklüzyonu (MCAO) serebral iskemiyi taklit etmek için kullanıldı. APC, beyin dilimlerini,% 20 CO 2 ile kabarmış olan asidik tampona aktararak basitçe elde edilebilir;% 20 CO 2 teneffüs fareler tarafından r. APC, doku canlılığı ve beyin enfarktüsü hacmi tarafından yansıtıldığı gibi serebral iskemiye karşı önemli koruyucu etkiler gösterdi.

Yukarıda tarif edilen kortikostriatal dilim modelinde, reperfüzyondan 1 saat sonra, kortikostriatal dilim canlılığı, TTC analizi ile nicelleştirildi. TTC dönüşümü, 490 nm&#39;deki absorpsiyonu kontrol dilimine normalleştirerek hesaplandı. TTC dönüşümüne göre, APC, başlangıç ​​zamanı ve süreye bağlı olarak OGD kaynaklı reperfüzyon hasarına karşı korunmuştur. Ayrıntılı olarak, hem 1 ve 3 dakika asidoz tedavisi, 15 dakika OGD&#39;den sonra 5 dakika i?...

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

Asitik sonrası kondisyonlama serebral işemiye karşı korur. Burada, APC&#39;yi yürütmek için iki model sunuyoruz. Bu in vitro olarak oksijen glikoz yoksunluğu sonra asidik tampon Kortikostriatal dilimleri aktarılarak ve in vivo olarak orta serebral arter oklüzyonundan sonra% 20 CO2 solunması ile sırasıyla gerçekleştirilir.

Serebral İskemiye Karşı Asitik Sonrası Sonrasyonun Sinir Korumasını Çalıştırmak için Deney Modelleri

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.

Research

JoVE Journal

Neuroscience

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

Beyin-makine Arayüzler Kapalı döngü Performans Çalışması için Deneysel Bir Platform

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

Nörobilim

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

Fosforesans Oksijen bağımlı Quenching dayanarak Serebral Kan oksijenasyon Ölçüm

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

Endovasküler Dikiş Rat Orta Serebral Arter Tıkanıklığı Fokal Serebral İskemi Model

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

Retina Pigment Epitel Fizyoloji ve Patofizyoloji Çalışma Deneysel Modeller

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

Fonksiyonel Transkraniyal Doppler Ultrasonografi kullanarak Çocuklarda Serebral Lateralizasyon Değerlendirme (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

Soğuk önkoşullamanın nöro-Çalışması için Stratejiler

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

Serebellar Granül Nöronlar Genetik Manipülasyon<em&gt; In Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Nöronal morfoloji ve Göç Eğitim için

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

Serebral Vasküler Endotelyal Hücrelerinde Bioenerjetik Fonksiyonun Değerlendirilmesi

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

Manyetik rezonans görüntüleme protokolü için kontur başlangıçlı süresi tahmini olarak kalıcı serebral iskemi

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 Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

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