Bu çalışma Avustralya Ulusal Sağlık ve Tıbbi Araştırma Konseyi, Hunter Tıbbi Araştırma Enstitüsü ve Newcastle Üniversitesi&#39;nden araştırma fonları tarafından desteklenmiştir.

Tüm prosedürler NSW Hayvancılık Araştırma Yasası, NSW Hayvancılık Araştırma Yönetmeliği ve Uygulama için Avustralya Kanunu dahil ilgili kurallara ve yönetmeliklere uygun olarak yanı sıra Newcastle Üniversitesi Hayvan Bakım ve Etik Komitesi onayı ile yapılır Bakım ve Bilimsel Amaçlı Hayvan kullanımı. 1.. Beyin Doku diseksiyonu <ol><li> Başları kesilerek on iki haftalık erkek fareleri kurban. Sıçanlar ya da çarpıcı ve başları kesilerek feda edilebilir ya da ilk izofluran (% 5 indüksiyon,% 1.5-2 bakım)% 70 N2 ve% 30 O2 ile anestezi edilmesi ve daha sonra başları kesildi. </li><li> Mümkün olduğunca hızlı kafatası beyin kaldırmak (ideal, bu az 2 dakika içinde yapılmalıdır). </li><li> Serbest el diseksiyon ile korteks kaldırmak. Mai şekilde diseksiyon mümkün beyne az hasar ile, mümkün olan en kısa sürede yapılmalıdırdilim bütünlüğünü ntain. </li><li> Herhangi bir artık deri kaldırmak için, vs kürk, kan, 37 ° C&#39;de Krebs tamponu (118 mM sodyum klorid, 4.7 mM potasyum klorür, 1.3 mM kalsiyum klorür, az miktarda (3-5 mi) içinde beyin batırmak 1.2 mM potasyum dihidrojen fosfat, 1.2 mM magnezyum sülfat, 25 mM sodyum hidrojen karbonat, 11.7 mM glikoz, 0.03 mM disodyum etilendiamin tetraasetik asit (EDTA),% 95 oksijen /% 5 karbon dioksit (karbojen), pH 7.4) ile dengelenmiştir. </li></ol> 2. Microslices hazırlanması <ol><li> Filtre kağıdı iki dilim yerleştirilir ve sıcak Krebs tamponu ile nemlendirilmiş hangi bir McIlwain helikopter sahnede beyin yerleştirin. Beyin ve sıcak Krebs tamponu ile ıslatılmış filtre kağıdı Ol, ama çok ıslak olmayan beyin etrafında kaymasına neden olabilir kağıdın yüzeyinde serbest sıvı olduğu. </li><li> 250 mikron koronal bölüme beyin dilimleyin. Besin, oksijen ve atık maddeler inci gibinormal olarak kan dolaşımı izole edilmiş sinir dokuda dokuyu saran bir sıvı ile değiştirilir ile değiştirilir de, dilimlerin büyüklüğü yeterli oksijen ve besin alışverişi yapılmasına izin vermek kalınlığı 400 um &#39;den fazla olmamalıdır. </li><li> McIlwain evre 90 ° çevirin. </li><li> 250 mikron sagital dilimlere beynini dilimleyin. Burada, bu beyin kesitleri (değişken uzunlukta, 250 um x 250 um dilimleri dokusunun kalınlığına bağlı olarak) microslices olarak adlandırılır. </li><li> (El Krebs tampon miktarı doku miktarına bağlıdır) 10-15 ml 37 ° C Krebs tamponu ile bir yuvarlak tabanlı tüp içine microslices yerleştirin. </li></ol> 3. Microslices ekilibre <ol><li> Yavaşça bireysel microslices askıya kadar ajitasyon, ve sonra onları yerçekimi altında yerleşmek için izin verir. </li><li> Dikkatle nedeniyle askıya hücre enkaz bulutlu olacak, süpernatant kaldırmak ve taze 37 ° C 10 ml ekleyinKrebs tampon tüpüne. </li><li> Enkaz çoğunluğunun çıkarılması ile sonuçlanacaktır ki, bu yıkama işlemi dört kez tekrarlayın. </li><li> Bunu takiben, nazik sallama / ters ile, 37 ° C&#39;de dengeye microslices, ve Krebs, toplam 1 saat boyunca her 15 dakikada bir tampon değiştirin. </li><li> Bu anda, 2 ml taze 37 ° C Krebs tampon ekleyin ve düz dipli polistiren 15-20 microslices (microslice süspansiyonu 150 ul) aktarmak tarafından oluşturulabilir yumuşatılmış kenarları (ile ekstra geniş çaplı bir pipet kullanarak 5 ml tüpler P1000 ucunun ucundan ~ 2 mm kesme). Her microslice oksijenli atmosfere bir kaç milimetre daha fazla değildir, böylece, tek kat olarak borunun tabanı üzerinde eşit microslices yayın. </li></ol> 4. Excitotoxic Stimülasyon <ol><li> 37 ° C&#39;de inkübe microslices ve sürekli pozisyonu olan bir gaz hattı kullanarak, 150 ul microslice süspansiyon yüzeyi üzerine geçmek karbojensadece microslice süspansiyonuna (mekanik aksamasını önlemek için) yüzeyinin üstünde ed. </li><li> (: Nihai konsantrasyon 2.5 mM glutamat + 50 uM glisin eksitotoksik uyarıcı) ya da tek başına 150 ul Krebs tamponu (Stimülasyon kontrol) 150 ul 5 mM glutamat + 100 Krebs tamponu içerisinde uM glisin ile ya microslices tedavi. Çeşitli uyaranlara bu aşamada kullanılabilir (de dahil olmak üzere, ancak bunlarla sınırlı değildir, AMPA, NMDA + glisin, KCl depolarizasyon ve oksijen / glikoz yoksunluğu). </li><li> Çeşitli zamanlarda post-stimülasyonu (0, 30, 60, 90, 120 ve 300 saniye) de inkübasyon çözüm çıkarın ve buz soğukluğunda homojenizasyon tampon maddesi (30 mM Tris, pH 7.4, 1 mM etilen glikol içinde 300 ul ile değiştirin tetraasetik asit, 4 mM EDTA, 100 uM amonyum molibdat, 5 mM sodyum pirofosfat, 25 mM sodyum fluorür, 1 mM sodyum ortovanadat, 1 mM phenylmethanesulfonylfluoride, adet tam proteaz inhibitör kokteyli). </li><li> Bir cam-Teflon Dounce Homojenizatörü kullanılarak, buz üzerinde homojenize microslices(20 vuruş, 700 rpm). </li><li> Gibi -80 ° C arasında ve çeşitli sinyal verici moleküllere, ölüm moleküller saklayın homojen western blot ile ölçülebilir. </li></ol>

<ol>
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Methods. 240 (1-2), 1-2 (2000).</li><li>Rodnight, R., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+tissue+metabolism:+3.+Study+of+tissue+fragments+with+little+or+no+added+aqueous+phase,+and+in+oils.">Techniques in tissue metabolism: 3. Study of tissue fragments with little or no added aqueous phase, and in oils.</a> Biochem. J. 57, 649-661 (1954).</li><li>Shelanski, M. L., Gaskin, F., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly+in+the+absence+of+added+nucleotides.">Microtubule assembly in the absence of added nucleotides.</a> Proc. Natl. Acad. Sci. U.S.A. 70 (3), 765-768 (1973).</li></ol>

Yazarlar bu yazının kapsamında yürütülen çalışmaların herhangi bir ilgili herhangi bir çıkar çatışması olmadığını beyan ederim.

Burada, sağlam olgun beyin dokusunda eksitotoksisite ve iskemiye bağlı hücre ölümü ile ilgili moleküler mekanizmaları incelemek için kullanılabilir microslices üretilmesi için bir in vitro bir teknik tarif edilmektedir. Bu teknik, uygun bir doku (Şekil 1-3), yani daha büyük bir Organotipik dilimleri 15 metabolik benzer üretir. Ayrıca, bu microslice modeli yakından vivo 10 eksitotoksisite kaynaklı beyin hasarı aşağıdaki gözlenen te...

Inme en sık görülen serebral kan damarı tıkalı olduğunda oluşur iskemik inme, olduğunu. Kan akımının kesilmesinden kaynaklanan doku iskemisi membran depolarizasyonunu yaygın uyancı nörotransmitter salınımına neden olur ve hücre ölümünün aktivasyonuna yol açan hücre içi kalsiyum, sürekli yükseklik 1 yollar yer alır. Bu işlem, &quot;eksitotoksisite&quot; olarak geçecek olan ve inme 2 de dahil olmak üzere, çeşitli patolojilerin tarafından üretilen nöronal ölümle ilgili yaygın bir yoludur edilmiştir. Eksitotoksisite ve diğer nöronal hücre ölümü kaskadı dahil olan sinyal yollarının önlenmesi, inme sonrası nöronal hasarı sınırlandırmak için cazip bir yaklaşımdır. Bütün hayvan sistemlerini kullanırken eksitotoksisite ve iskemik inme dahil kesin moleküler mekanizmaları belirlenmesi zor olabilir. Gibi, primer embriyonik ve &#39;nöronal-benzeri&#39; (örn. neuroblasto olarakma ve adenokarsinom ölümsüzleştirdi hatları) hücre kültürü sistemleri sıklıkla kullanılır. Bu modellerin temel avantajları, nispeten düşük maliyetli manipüle edilmesi kolaydır ve hücre ölümü kolayca ölçülerek kantifiye edilebilir bulunmaktadır. Ancak, sinyal yolları kültürleme 3,4 kullanılan koşullar ve olgunlaşmamış nöronlar tarafından değiştirilmiş ve çizgileri farklı reseptörleri ifade ve beyni 5-8 olgun kıyasla sinyal molekülleri olabilir ölümsüzleştirdi edilebilir. (A kokültürü sistem kullanılırsa, ya da iki) olduğu gibi beyin dokusu birbirleri ile etkileşim hücre tipleri çeşitli içeren, heterojen ise Ayrıca, kültürlü nöronlar, sadece bir hücre tipinin bir inceleme sağlar. Organotipik kesit kültürü sistemleri (beyin dokusu ince dokusu) da kullanılabilir ve bunlar, in vivo olarak bulunanlar gibi bu modeller, heterojen hücre popülasyonlarının çalışma sağlar. Bu tekniği kullanarak, ancak, bir doku sadece sınırlı bir miktarda dilimleri, her hayvandan şekilde elde edilebilirSürece ölümsüzleştirilmiş hücre çizgileri olarak için kültürlenebilir, ve uzun süreli kültürde orta dilim yollarının ve reseptörler ve sinyal değişikliklere neden olabilir değildir. Olgun beyin Organotipik dilimleri üretmek için kullanılabilir iken, olgunlaşmamış beyninden dilim kültüre daha uygun olan ve daha yaygın olarak kullanılmaktadır. Nöronal sinyal yolları incelenebilir hangi kullanımı kolay olan sağlam olgun beyin, temsil eder ya da taklit eden modeller geliştirmek için bir ihtiyaç vardır. Burada, bir eksitotoksik veya iskemik zedelenmeden sonra, hücre ölümü ile ilgili moleküler mekanizmaları aydınlatmak için kullanılabilir sağlam sinir dokusu dahil, bir in vitro bir teknik tarif edilmektedir. Bu teknik, bir deney gerçekleştirmek için gerekli hayvan sayısını azaltır, tekrarlanabilir olduğunu, ve daha büyük Organotipik dilimleri için metabolik olarak benzer şekilde davranır canlı doku üretir. Buna ek olarak, nöronal devre, hücre etkileşimleri ve postsinaptik compartment kısmen bozulmadan kalır. Kullanılan fizyolojik tampon hücre &#39;tekrar kapatılması&#39; için membranların ve sağlayan hücreler kendi özgün membran direncini 9 kurtarmak için olanak sağlar. Bu beyin dilim modeli sadakatle eksitotoksikliği bağlı beyin yaralanmaları 10 aşağıdaki gözlenen tepkiler taklit edebilir, ve inme dahil moleküler mekanizmaları incelemek için kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guillotine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to decapitate animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Forceps, scissors, tweezers, etc, for brain removal and dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain chopper</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to generate 100-400 &#956;m brain sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom plastic tubes</td>
			<td style="width:71px;">Greiner</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For keeping tissue at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aerating apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to keep microslices in a humidified, oxygenated environment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flat bottomed polystyrene tubes</td>
			<td style="width:71px;">Nunc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dounce Homogenizer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

Bütün hayvan sistemlerini kullanırken, iskemik inme gibi nöropatolojik koşullarında yer moleküler mekanizmaları, incelenmesi, zor olabilir. Örneğin, birincil ya da &quot;nöronal benzeri &#39;hücre kültür sistemleri yaygın olarak kullanılmaktadır. Bu sistemler, çalışma için nispeten kolay olan ve (örneğin, hücre ölümü gibi) çeşitli işlevsel sonuçlar kolayca belirlenebilir hangi faydalı model sistemleri (örneğin, eksitotoksisite aracılı hücre ölümü yollar gibi) kültürlenmiş olgunlaşmamış nöronlarda muayene sonuçları ve yollar iken zorunlu olgun beyinde gözlenen aynı veya sağlam doku değildir. Bu nedenle, olgun sinir dokuda hücresel mekanizmalar incelenebilir hangi modeller geliştirmek için bir ihtiyaç vardır. Bu sağlam sinir dokusunda moleküler yollarının çeşitli araştırmak için kullanılan bir in vitro teknik geliştirdik. Bu tarifnamede tarif edilen teknik, sıçan kortikal doku kullanır, ancak bu teknik doku kullanmak için adapte edilebilirya da beyin bölgelerinde (örneğin, hipokampus, striatum, vb) (örneğin, fare, tavşan, kobay ve tavuk gibi) türlerin çeşitli. Buna ek olarak, uyarılar / çeşitli tedaviler (örneğin, eksitotoksik, inhibitörlerinin uygulanması, vs) kullanılabilir. Sonuç olarak, burada tarif edilen beyin kesit modeli eksitotoksisite kaynaklı beyin hasarı dahil moleküler çeşitli mekanizmalar incelemek için kullanılabilir.

Bu prosedür kullanılarak üretilen Microslices uygun olan ve çeşitli türlerin (örneğin, sıçan, fare ve tavuk) microslices üretmek için kullanılabilir. Canlılığı üç bağımsız tedbirler faydalanılmıştır: solunum oranı (Şekil 1), adenin nükleotid oranları (Şekil 2), ve doku potasyum içeriği (Şekil 3). Bu önlemleri kullanarak, microslices en az 2 saat sonrası kuşak için geçerli kalır ortaya konmuştur. Brain...

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Biz eksitotoksisite kaynaklı beyin hasarı dahil moleküler mekanizmaları incelemek için kullanılabilecek bir beyin kesit modeli geliştirdik. Bu teknik açıdan olgun beyin dokusunu oluşturur ve kısmen sağlam nöronal devreleri, hücresel etkileşimleri ve postsinaptik bölmeleri korurken, deney için gerekli olan hayvan sayısı azalır.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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Medicine

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

8.4K Görüntüleme.  The University of Newcastle.  Biz eksitotoksisite kaynaklı beyin hasarı dahil moleküler mekanizmaları incelemek için kullanılabilecek bir beyin kesit modeli geliştirdik. Bu teknik açıdan olgun beyin dokusunu oluşturur ve kısmen sağlam nöronal devreleri, hücresel etkileşimleri ve postsinaptik bölmeleri korurken, deney için gerekli olan hayvan sayısı azalır. 

Video: Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Odak Serebral İskemi Erken İskemik Tolerans tetikleyebilecek bir Yeterli Önkoşullama Uyarıcı olarak bilateral Ortak Karotis Arter Tıkanıklığı

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Tıp

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Subaraknoid Kanama bir fare modeli

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Bir olarak beyin mikrovasküler endotel hücreleri (cend) olarak gerin<em&gt; In Vitro</emKan Beyin Bariyer&gt; Travmatik Beyin Hasarı Modeli

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Ayarlama-up bir<em&gt; In Vitro</emSıçan Kan-beyin bariyerinin&gt; Model (BBB): BBB geçirimsizliği ve Reseptör aracılı Ulaştırması Bir Odak

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Epilepsi Hastalarında Beyin uyarılabilmenin Bağlantı ile ilgili değerlendirmek için bir Multimodal imaging- ve Uyarım tabanlı Yöntemi

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Yenidoğan Murin Koklear Explant Tekniği<em&gt; In vitro</em&gt; İşitme Araştırmalarında Tarama Aracı

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Vagus sinir stimülasyonu ek bir Neurostimulation aracı olarak tedaviye dirençli depresyon

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Transkraniyal Doğru Akım Stimülasyonu Dahil Olmak Üzere Güvenilir, Kolay ve Tolere Edilen Transkraniyal Elektriksel Stimülasyon için Güncellenmiş Teknik

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...