This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (SEED 03/12, SB), Deutsche Forschungsgemeinschaft (SFB TR128, TP B6 to S.G.M. ME3283/2-1 to S.G.M.) and by Innovative Medizinische Forschung, M&#252;nster (I-BI111316, SB and SGM).

Fareler kullanan tüm deneyler ilgili kurumsal hayvan bakımı ve kullanımı komitesi kurallarına uygun olarak yapılmalıdır. Fare Deneyler için 1. Genel Yorumlar <ol><li> Patojen içermeyen koşullar altında fareler tutun ve onlara yiyecek ve su ad libitum erişim sağlar. NOT: immünolojik desenler yaş ve cinsiyet ile değişebilir çünkü deney gruplarında yaş ve cinsiyet uyumlu fareler kullanmak önemlidir. </li></ol> 2. hazırlanması ve Aktivasyon OVA-spesıfik CD8 + T hücreleri (OT-I) <ol><li> Beyin dilimleri hazırlama 5 gün, aşağıda tarif edildiği gibi OT-I T hücrelerinin uyarılmasını yapın. Not: 5 x 10 5 aktive efektör CD8 + OT-I T hücreleri, (CD8 + T hücreleri, MHC-I-molekülleri bağlamında sadece OVA257-264 peptidi tanıyan transgenik farelerin) dilim başına gerekli olan konsantrasyonu 5 x 10 5, deney seçildihücreler aynı zamanda, dilim içine göç ve başlar bir kez iyi bir hücre-hücre etkileşimi lehine optimum olarak müttefik, hücrelerin miktarı tek hücre 8,18 sayma sağlayan bir aşırı reaksiyonunu uyarmak için çok yüksek değil. </li><li> OT-I T hücrelerinin kültürlenmesi için ortam hazırlar. % 5 fetal dana serumu (FCS), 10 mM HEPES, 2 mM L-glutamin, 50 uM 2-merkaptoetanol,% 1 esansiyel olmayan amino asitler ve 25 ug / ml gentamisin 500 mi DMEM Supplement. Kullanılana kadar 4 ° C &#39;de orta saklayın. </li><li> Referans 16&#39;daki gibi OT-I transgenik farelerin dalak çıkarın. Hazırlanan ortama aktarabilirsiniz. </li><li> 5 dakika, 4 ° C 300 xg&#39;de 70 mikron süzgeç ve santrifüj süspansiyon yoluyla dalak ezme yoluyla tek bir hücre süspansiyonu oluşturun. </li><li> Kırmızı kan hücreleri lize etmek için 5 dakika boyunca 5 mi amonyum klorür-potasyum (ACK), tampon (150 mM NH4CI, 10 mM KHCO 3, 0.1 mM EDTA, pH 7.3) ile bir hücre süspansiyonu inkübe edin. </li><li> Kuluçkalamadan DurTekrar 10 ml ortam santrifüj o ile ilgili yukarıda tarif edildiği gibi. </li><li> Orta hücre süspansiyonu seyreltin ve sayıları hesaplamak. Plaka 3 x 10 7 hücre yoğunluğunda hücre / kuyuda 12-kuyu plaka ve birinci bunları inkübasyon tarafından OVA 257 - 264 (SIINFEKL; 1 nM) ve IL-2 (IL-2), (500 lU / ml) 5 gün. </li><li> 4 gün sonra, 500 IU / ml yoğunlukta yeniden IL-2 ilave edin. </li><li> Uyarımı takiben, imalatçının talimatları izlenerek bir negatif seçim göre fare CD8 + T hücresi izolasyon kiti kullanılarak hücre süspansiyonundan OT-I T hücrelerinin saflaştırılması. Arıtma CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC Sınıf II Ter-119 ve TCRγ karşı bir antikor kokteyli olmayan tüm CD8 + hücrelerinin azalmasına dayanmaktadır / δ daha sonra saflaştırılmış CD8 + T hücrelerinin, yıkama için manyetik sütun kullanarak atılır olan. NOT: Bu procedu Kritik adımlaryeniden üretici tarafından gösterilir: bu topakların bulunması yıkama aşaması esnasında sütun bloke çünkü reaksiyon sadece tek hücreler içeren çok önemlidir; Byotin kokteyl ilave edilmeden önce hücre sayımı örnek saflık derecesi için önemlidir ve işlem spesifik antikor bağlantıları en aza indirmek için, buz üzerinde yapılmalıdır. </li></ol> OT-I T-hücreleri ile Akut Beyin Dilimleri ve Co-kültür 3. Hazırlık <ol><li> Akut beyin hazırlanmasından önce 19, 200 mM sakaroz, 20 mM PIPES, 2.5 mM KCI, 1.25 mM NaH 2 PO 4, 10 mM MgSO 4, 0.5 CaCl2 ve 10 mM dekstroz ile placedine buz soğukluğunda fizyolojik yemek tuzu çözeltisi hazırlamak dilimler ve yapay serebrospinal akışkan (ACSF) 125 mM NaCI, 2.5 KCl, ile 1.25 NaH 2 PO 4, 24 mM NaHCO 3, 2 mM MgSO 4, 10 mM dekstroz, 2 mM CaCl2,. Usin ile 7,35 her çözeltinin pH ayarlamag NaOH. ACSF pH değerinin ayarlanması önce çözelti,% 95 O2 ve% 5 CO2 karışımı ile kabarcıklar halinde olmalıdır. </li><li> Çözüm Öncesi serin. </li><li> 8 uyutmak - (10 haftalık fareler gibi, C57Bl% 5.0 izofluran,% 5.0 halotan veya bekleyin ip üzerinden 100 mg / kg ketamin ve 10 mg / kg vücut ağırlığı ksilazini enjekte kullanarak kontrol veya inhalasyon ile transgenik ODC-OVA fareler 17 gibi / 6 anestezi. anestezi düzeyini değerlendirmek ve bir kerede onları başını kesmek için bir ayak tutam kullanmak ve kurumsal hayvan bakımı göre fareler Euthanize ve ötenazi için komite kriterlerini kullanabilirsiniz. </li><li> Ventral pozisyonda fare koyun. Dezenfekte ve sagittal saç derisini kesti. , Sagitally kranial kemik açın, bir kepçe yardımı ile hızlı bir şekilde beyin kaldırmak ve tutkal kullanarak bir vibratome plaka üzerine sabitleyin. </li><li> Hazırlanan placedine buz soğukluğunda fizyolojik yemek tuzu çözeltisi ile plaka doldurun. </li><li> Vibratome ile 300 mikron koronal dilim kesin. NOT: Bu prosedür biliyorumn, en az 8 saat, 19-21 arasında bir zaman aralığı içinde işlevsel hücresel çalışmalar için uygun olan canlı sağlam doku örneklerinin elde edilir. Nitekim, zaten 6 saat sonra başlayan bu süre, sonra, hazırlık azaltılmış kalite, aksiyon potansiyeli üretimi ve yayılması gibi temel ve fonksiyonel özelliklerinde yani değişiklikleri gösterir. </li><li> Kesit sonra ACSF ile dolu bir 12 oyuklu plakanın her bir oyuğuna, hemen bir dilim aktarın. </li><li> Dilim başına dikkatli bir şekilde 5 x 10 5 OT-I T hücrelerinin ekleyin. </li><li> Bir kuluçka makinesi içinde en fazla 8 saat (37 ° C,% 5 CO2) için dilim inkübe edin. </li><li> Kuluçka dönemi, hasat dilimleri ve sonra Ekim bileşik Doku Tek kullanarak onları gömmek ve sıvı azot onları dondurmak. Değerlendirilmesi, örneğin, daha fazla histolojik çalışmalar için -20 ° C &#39;de nöronal yapı (örneğin, kullanılarak anti-MAPII ya da anti-sinaptofisin antikorlar) ya da apoptozu (saklayın örneğin, bir anti-Kaspaz-3 antikorları, anti-neun anti kullanarakorganları), standart prosedürlere göre yöntem. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Farklı hayvan modelleri MS gibi inflamatuar demiyelinizan hastalıklarının patolojik özellikleri ele son on yıl içinde tarif edilmiştir. In vivo fare hem de sıçan modelleri yaygın bir hastalık patofizyolojik özelliklerini taklit etmek için kullanılır, yani, demiyelinasyon ve remiyelinizasyon işlemlerinin sonuçlarının analizi ve inflamasyon ve nörodejenerasyonda içiçe bölüm. Bununla birlikte, sadece bir ex vivo yaklaşım tam altında yatan mekanizmaları incelemek için izin verir. Akut beyin dilimlerinin hazırlanması da çok geniş dağılımlı bir modeldir ve güvenilir ve tekrarlanabilir olarak kabul edilebilir. Hücre etkileşimleri - izolasyon ve oligodendrosit-spesifik immün hücrelerin inkübasyonu ile birlikte, özellikle bir mekanizma ve bir immün hücre krizi ve hücre kinetiği çalışılarak açısından spesifik deneysel soruları kullanılabilir. Diğer ex vivo yaklaşımlar bilinen ve yaygın olan22 kullanılan ancak yani OT-I T hücrelerinin maruz maruz veya WT Caspase3 immünoreaktivitesi doğrulayarak, farklı &quot;dışlama&quot; co-lekelenmelerinin yaparak itiraz edilebilir hedef özgüllüğü bizim protokol yenilik (Şekil 2C bakınız) . Hazırlanması sırasında bazı kritik noktalar deneyler metodolojik doğru yapılmaktadır emin olmak için göz, yani deneysel ayarları, önüne alınmalıdır; İç geçerliği, izolasyon ve kullanılan bağışıklık hücrelerinin uyarılması için analiz (FACS) akış sitometrisi, örneğin üzerinden düzenli olarak kontrol edilmelidir. Ayrıca, beyin dilimleri kontrol canlılık şiddetle tavsiye edilir. Deneysel gruplar yaş ve cinsiyet uyumlu olmalıdır. Deneyler hep hayvan refahı yönetmeliklerine uygun olarak yapılmalıdır. Protokolün bazı sınırlamalar göz önünde tutulması gerekir. En önemlisi, sunulan bir modeldirBir eks vivo yaklaşımı ve tam bağışıklık, merkezi sinir sistemi bozuklukları, karmaşık immünolojik durumu taklit etmek için uygun değildir. Ayrıca, model, CD8 + T-hücresi bağışıklık yanıtını tahrik kullandığı göz önüne alınmalıdır. CD4 + T hücreleri ve B hücreleri, daha az belirgin bir rol oynar. Bu hücre tiplerini ele alırken diğer oligodendrosit spesifik immün hücre alt tipleri kullanılarak, alternatif protokoller dikkate alınmalıdır. beklenen histolojik bulgu oligodendroglial (Şekil 2A) ve nöronal apoptoz (Şekil 1C bakınız). Seçenek olarak ise, diğer hücre tipleri arasında apoptoz, örneğin, astroglial veya nöronal antijene özgü bağışıklık hücreleri ve transgenik sistemler kullanılarak elde edilebilir. Bu özel protokol, astroglial hücrelerde de Caspase3 immünoreaktivitesi sistemdeki (Şekil 2B) spesifikliğine negatifti. Açıklanan protokol, temel nöro olarak kabul edilebilirimmünolojik deneysel yaklaşım ve diğer uygulamalar için modifiye edilebilir. spesifik CD4 + T-hücreleri ve - deney prosedürü, kolay bir şekilde farklı antijene özgü bağışıklık hücre alt tipi (örneğin, miyelin oligodendrosit glikoprotein (MOG) kullanımı ile bir arada çeşitli beyin dilimleri (örneğin, farklı transjenik fare suşu) ile diğer protokoller uygulanabilir Beyin oligodendroglial ve nöronal apoptosis üzerindeki etkisini gidermek için C57BL / 6 farelerinden elde dilim). bağışıklık hücrelerinin in vitro aktivasyonu özgü bağışıklık soru (T H 1 ya da T, H 17 hücre) içine örneğin, kutuplaşmayı değiştirilebilir. Bazen, beyin dilimleri gelişmiş veya azalmış apoptoz deneysel bir meydan okuma olabilir. Sorun giderme için bazı öneriler şunlardır: İlk başta, beyin dilimleri kalitesi bizim pH değeri, osmolarite ve oksidasyon durumu yanı sıra hazırlık süresi son derece bağlıdıred çözüm. Bu koşullar, bağımsız deneyler arasındaki karşılaştırılabilir olması sağlanmalıdır. Ayrıca, bağışıklık hücrelerinin aktivasyonu düzenli olarak kontrol edilmelidir. En uygun antijen konsantrasyonu, değişebilir. Deney kurulurken antijenin bir titrasyon düşünün. Yukarıda tarif edildiği gibi, söz konusu protokol, merkezi sinir sistemi, bütünlüğü üzerinde daha fazla immün hücre alt tipi (örneğin, CD4 + T-hücreleri, B-hücreleri) etkisini analiz etmek için bir başlangıç ​​noktası olarak kullanılabilir. Bu yaklaşımlar, histolojik analiz iyice tespit edilebilir olarak oligodendroglial ve nöronal hücreler üzerinde etki ayrılması için özellikle uygundur.

Death of oligodendrocytes and destruction of the myelin sheath accompanied by loss of neurons and axons are typical pathological findings in grey matter lesions in individuals suffering from multiple sclerosis (MS)1,2. Cortical lesions can be divided so far in three different subtypes2: subpial, intracortical and leukocortical lesions. In comparison to white matter plaques, infiltrates are characterized by a predominance of CD8+ T-cells, suggesting their possible decisive role in grey matter inflammation3. Furthermore, oligoclonal expansions in blood, cerebrospinal fluid (CSF) and within inflammatory lesions can be found for CD8+ T-cells themselves4-6. 
In line with this, it is assumed that CD8+ T-cells may be specific for different myelin proteins7,8. Indeed, CD8+ T-cells are found near oligodendrocytes and myelin sheaths9,10 that show MHC I expression11 and might therefore be responsible for the loss of the myelin sheath. This process is often seen together with extensive &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal damage within the central nervous system (CNS) grey matter1,2. In fact, direct and indirect death of oligodendrocytes and neurons is induced by CD8+ T-cells via two different mechanism: (i) cell membrane swelling and rupture due to the formation of cytotoxic granules following the release of perforins and granzymes and (ii) ligation to the Fas receptors or exposition of FasL on their surface8,12,13.
Different types of animal models are established to study the underlying mechanism of the mentioned processes. In this respect, primed CD8+ T-cells specific for autoantigens with induced expression in CNS glial cells, like oligodendrocytes or astrocytes, can be adoptively transferred to analyse &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal death in grey matter subsequently14,15. To perform such in vivo experiments is a big help to mimic some pathophysiological aspects of MS, however, this approach is not suited to resolve the underlying mechanism and kinetics of axonal damage and neuronal apoptosis.
To overcome these restrictions, an ex vivo approach was established to study the mechanisms and time course of neuronal cell death following a oligondendrocytes-directed CD8+ T-cell attack. Since only oligodendrocytes and therefore myelin sheath production should be targeted by immune cells, MHC class-I-restricted, ovalbumin (OVA)-reactive OT-I Tcells are used16. These cells are subsequently transferred into brain slices obtained from mice selectively expressing OVA in oligodendrocytes (ODC-OVA mice)17.

<table><tbody><tr><td>12-Well plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>2-Methylbutan</td><td>Roth</td><td>3927.1</td><td></td></tr><tr><td>70 &amp;micro;m strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>1.02382.0500</td><td>calcium chloride</td></tr><tr><td>CD8&lt;sup&gt;+&lt;/sup&gt;-isolation kit</td><td>Miltenyi Biotech</td><td>130-090-859</td><td></td></tr><tr><td>D(+)-glucose</td><td>Merck</td><td>1.08337.1000</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>FCS</td><td>PAA Laboratories</td><td>A15-151</td><td>fetal calve serum</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>HEPES 1 M</td><td>Gibco</td><td>15630-050</td><td></td></tr><tr><td>IL-2</td><td>Peprotech</td><td>212-12</td><td></td></tr><tr><td>Isofluran</td><td>Abbott</td><td>05260-05</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1.04933.0500</td><td>potassium chloride</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P9144</td><td>potassium hydrogen carbonate</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.05886.0500</td><td>magnesium sulfate</td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434</td><td>sodium chloride</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; * H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.06346.0500</td><td>sodium hydrogen phosphate</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>1.06329.0500</td><td>sodium hydrogen carbonate</td></tr><tr><td>NaOH</td><td>Merck</td><td>1.09137.1000</td><td>sodium hydroxide</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>213330</td><td>ammonium chloride</td></tr><tr><td>Non essential amino acid</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>OVA (257-264)</td><td>Genscript</td><td>RP10611</td><td>ovalbumin</td></tr><tr><td>PIPES</td><td>Sigma</td><td>P6757</td><td></td></tr><tr><td>Sucrose</td><td>Merck</td><td>1.07687.1000</td><td></td></tr><tr><td>Tissue-Tek OCT</td><td>Sakura</td><td>4583</td><td></td></tr></tbody></table>

an ex vivo model of an oligodendrocyte-directed t-cell attack in acute brain slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

Oligodendrosit yönelik CD8 + T hücreleri ile beyin dilimlerindeki inkübasyondan sonra, oligodendrositler olarak nöronlar apoptoz (Şekil 2A ve 1C, sırasıyla) tabi tutulur. Apoptoz histolojik bulgular (örneğin, Kaspaz-3, Tünel) ilk inkübasyon 3 saat sonra tespit edilebilir. Kuluçka dönemi hazırlanması ve tekrarlanabilir sonuçlar iyi kalitesini garanti etmek için uzun 8 saat daha olmamalıdır. Apoptotik hücreler tüm miyelinli alanlarda üstünlüğü ile dilim üzerinde bulunabilir. Yapısal bütünlük ve apoptotik nöronların dilim örnekleri histolojik boyama Şekil 1A-C&#39;de gösterilmiştir. Sonuç parametreleri değerlendirmek için ilgi inkübasyon sırasında farklı zaman noktaları vardır. Laboratuarımızda yapılan tipik bir zaman süreci sonra, yani, hücre-hücre etkileşimleri zaman 0, daha sonra 3, 4, ve 6 saat sonra (en fazla etkileşim gözlenen), analiz parametrelerini içeriretkileşim başladı. Genellikle çok kısaca anlatılmıştır uygulanabilir histolojik analizler ötesinde bazı tipik tahliller: T-hücresi geri kazanımı ve analiz 8 saat kadar bir kuluçka döneminden sonra, T-hücrelerinin dilimler elde edilebilir ve sitokin üretiminin veya yayılması gibi hücre içi efektör molekülleri göre parametreleri analiz etmek için değerlendirilebilir. T hücresi elde etmek üzere, dilimleri mekanik ayrıştırılmış ve her deneysel koşul için bir araya toplanmış. Hücreler, 2500 rpm&#39;de arayüzünden 30 dakika boyunca% 30 ila% 50 Percoll (Amersham, Freiburg, Almanya) sonra yoğunluk gradyanlı santrifüj izole edilmiştir. Bu şekilde, bu, örneğin CD8, yıkandı ve bu protokolde, adres istiyorum, çalışmaya bağlı olarak değişir antikorların farklı sayıda ve tip teknikler kullanılarak (FACS) akış sitometrisi kullanılarak hemen lekeli mononükleer hücreler elde etmek mümkündür + 18. Şekil 1D gösterildiği gibi nöronal sağlığı ve işlevselliği hakkında bilgi, böyle bir aksiyon potansiyelleri (AP) nesil olarak nöronların temel özelliklerini değerlendirmek, elektrofizyolojik kayıt örneğin yaparak değerlendirilebilir. <img alt="Şekil 1," src="/files/ftp_upload/52205/52205fig1highres.jpg" /> Şekil 1. dilim morfolojik yapısı hem de temsili sonuçlar. Inkübasyondan sonra bir dilim 8 saat (A) morfolojik yapısı. Ölçek çubuğu 10 mikron gösterir. MAPII (yeşil) ve sinaptofisin (kırmızı) boyama için ko-boyama ile ortaya (B) olarak nöronlar sinaptik bağlanır. Ölçek çubuğu 10 mikron temsil eder. (C) hipokampus a apoptotik hücreler (Kaspaz-3, kırmızı) ve nöronlar (neun, yeşil) Temsilcisi resimfter 6 saatlik bir bekletme. Ölçek çubuğu 10 mikron temsil eder. (D) bir fare kortikal nöron depolarizasyon akımı adımları izleyerek nöronal aksiyon potansiyeli nesil Tek hücre yama-kelepçe kayıtları. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52205/52205fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52205/52205fig2highres.jpg" /> Şekil 2. Çoklu Renklendirmeler protokol özgüllüğü değerlendirmek için seslendirdi. (A), NogoA (kırmızı) eş-boyama, olgun oligodendrositler (ODCs) ve Caspase3 (apoptoz, yeşil) için bir belirteç ODCs CD8 + T hücreleri tarafından olduğunu göstermektedir. Caspase3 (kırmızı) GFAP, glial hücreler (yeşil) için bir marker ile birlikte boyandı olduğu zaman, (B) etkileşim sonucu negatiftir. Ölçek çubuğu 10 mikron temsil eder. (C) Specificiprotokol ty soldan başlayarak, Caspase3 (kırmızı) ile nöronal hücre ko-boyama (mavi DAPI) tarafından desteklenmektedir: RT hayvanlar WT hayvanlar OTI-I T hücrelerinin maruz ODC OVA ve ODC OVA OTI-I T hücrelerinin maruz. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52205/52205fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices at JoVE.com

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Bir<em&gt; Ex vivo</em&gt; Akut Beyin Dilimleri bir Oligodendrosit yönelik T-Hücre Saldırı Modeli

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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Universidad San Sebastian

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Immunology and Infection

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

6.9K Görüntüleme.  University of Münster. To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Video: An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

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

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

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

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

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 ...

Tıp

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Beyin Dilim Biyotinilasyonu: An<em&gt; Ex Vivo</emYetişkin Nöronlar Bölge özgü Plazma Membran Protein Ticaretiyle Tedbir&gt; Yaklaşım

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Nörobilim

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotipik Dilim Kültürler Oligodendrosit Dinamikler ve miyelinasyonun incelemek için

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior 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. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Görselleştirme ve Oligodendrocyte organelleri Adeno ilişkili virüs ve Confocal mikroskobu kullanılarak Organotypic beyin dilimleri içinde canlı görüntüleme

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ateş Ex Vivo sıçan beyin dilimleri kullanarak birincil olayları incelemek için alternatif bir yaklaşım

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Mikroglial süreçleri cazibe ATP veya Serotonin akut beyin dilimleri içinde doğru iki fotonlu görüntüleme

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

<meta charset="utf8"></head><body>Gelişimsel ve Hastalıkla İlgili Manipülasyonlar için Kantitatif Bir Model Olarak Serebellar Dilim Kültürlerinde Ex Vivo Miyelinasyon ve Remiyelinasyon

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Gelişimsel ve Hastalıkla İlgili Manipülasyonlar için Kantitatif Bir Model Olarak Serebellar Dilim Kültürlerinde Ex Vivo Miyelinasyon ve Remiyelinasyon

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

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