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>

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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><tbody><tr><td>Guillotine</td><td></td><td></td><td>Used to decapitate animal</td></tr><tr><td>Surgical equipment</td><td></td><td></td><td>Forceps, scissors, tweezers, &lt;em&gt;etc.&lt;/em&gt;, for brain removal and dissection</td></tr><tr><td>McIlwain chopper</td><td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td><td></td><td>Used to generate 100-400 &amp;#181;m brain sections.</td></tr><tr><td>Round bottom plastic tubes</td><td>Greiner</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>For keeping tissue at 37 &amp;deg;C</td></tr><tr><td>Humidifier/aerating apparatus</td><td></td><td></td><td>Used to keep microslices in a humidified, oxygenated environment</td></tr><tr><td>Flat bottomed polystyrene tubes</td><td>Nunc</td><td></td><td></td></tr><tr><td>Dounce Homogenizer</td><td></td><td></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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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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombotic İskemi: Fare İnme Araştırmalar bir Minimal İnvaziv ve tekrarlanabilir fotokimyasal Kortikal Lezyon Model

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Tıp

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.

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 ...

İmmünoloji ve Enfeksiyon

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Nörobilim

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Aksonal Dejenerasyon ve Beyaz Cevher Nörobiyoloji Çalışması Çok Yönlü Fare Modeli subkortikal beyaz madde İnme

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

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. ...

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 ...

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.

Renal Korteks-Hipokampus Organotipik Dilim Kültürlerinde Epileptogenez Modeli

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

İskemide Nöron-Glia Etkileşimlerinin Rolünü Nörade İncelemek İçin Hücre Kültürü Modeli

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması

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Photothrombotic İskemi: Fare İnme Araştırmalar bir Minimal İnvaziv ve tekrarlanabilir fotokimyasal Kortikal Lezyon Model

Bir

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

Aksonal Dejenerasyon ve Beyaz Cevher Nörobiyoloji Çalışması Çok Yönlü Fare Modeli subkortikal beyaz madde İnme

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

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

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

Renal Korteks-Hipokampus Organotipik Dilim Kültürlerinde Epileptogenez Modeli

İskemide Nöron-Glia Etkileşimlerinin Rolünü Nörade İncelemek İçin Hücre Kültürü Modeli

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model