Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00
Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

Tüm protokoller geçerli nereye Kurumsal Hayvan Bakım ve Kullanım Komitesi politikalarına uygun yürütülmüştür. 1. Hazırlık <ol><li> % 75 ile sabitleme ortamı hazırlamak Dulbecco Modifiye Eagle Ortamı (DMEM),% 25 Hanks dengeli tuz çözeltisi (HBSS). kullanılana kadar -20 ° C&#39;de, iyi kısım ve mağaza karıştırın. </li><li> Fosfat tamponlu tuz (PBS) hazırlanması ve otoklavda sterilize edilir. Oda sıcaklığında çözüm saklayın. Deney (oyuk başına 5 mi) daha...

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In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

Artan miktarda kanıt vücut kan retina bariyerinin (BRB) 1-5 ve kan beyin bariyeri (KBB) 6,7 olan benzerliği varlığını destekler. BBB Uzlaşma sıkıca böyle deliryum 10 olarak nedensel ya da Alzheimer hastalığı (AH) gibi kronik nörodejeneratif hastalıklar tanısal belirteç olarak 8,9 ve akut koşulları ile bağlantılı olmuştur. potansiyel ilaç hedefleri için bu patolojilerin ve keşifler içine mekanistik anlayışlar genellikle beynin sınırlı erişilebilirlik ve ağ karışıklık tarafından engellenmektedir. Bu, in vivo görüntüleme 11, beyin Organotipik kültürü 12, birincil hücre kültürleri 13,14 ve ko-kültür sistemleri 15 gibi alternatifler oluşturulmuştur. Ancak bu modellerin çoğu hücreleri tanımlamak için özel araçlar, uzun deneysel dönemleri veya birden fazla belirteçler gerektirir. Fonksiyonel ve yapısal BBB ile BRB arasındaki benzerlikler yanı sıra dy arasında bir korelasyoniki sfunctions 16-19 iddia edilmiştir. Buna ek olarak, daha kolay erişim, iyi tanımlanmış hücre tipleri ve tabakalı yapı beyne bir pencere olarak iyi karakterize edilmiş retina izin vermiştir. BBB ve BRB yapısal ve işlevsel kimlikler detaylı karşılaştırılması gerekmektedir. Bununla birlikte, retina hastalıkları, özellikle de BRB ihlali da sıkı bir şekilde diyabet 18-19 ve AD 21,22 dahil olmak üzere çeşitli hastalıkların ilerlemesinin ile ilişkilendirilmiştir. Bu durumda, bir BRB disfonksiyon sistemi mekanizmasını tanımlamak için değil, aynı zamanda, potansiyel ilaç taranması için sadece kurmak ilgi çekicidir. Bu raporda, basit bir akut retinal kültürünü kullanarak bir protokol sağlayan BRB disfonksiyonu gelişti ve sunulmuştur. Artmış BBB geçirgenliği ve AD-benzeri patolojik değişiklikler histamin, bir pro-inflamatuar arabulucu 12 ile inkübe bir beyin Organotipik kültürü kurulmuştur. Bu nedenle, sunulan sistemde, histamin İçi olduBRB disfonksiyonu ikna etmek için ex vivo retina kültürüne ied. Böyle Mus musculus ve Bos Boğa gibi çeşitli türlerden gelen retinaları, test edilmiştir. Nedeniyle ticari kullanılabilirliği ve insan dokusuna benzediği için, taze domuz gözbebekleri burada bildirilen veri sağlamak için kullanılmıştır. Histamin ve / veya diğer ilaçlar ile inkübe edildikten sonra, retinalar immünoglobülin G (IgG), kan ana bileşenden biri olarak bir kaç protein 12 için immün değerlendirme için işleme alınmıştır; Glial fibriller asidik protein (GFAP), glial aktivasyon için iyi bilinen bir işaretleyici; ve mikrotübül-ilişkili protein 2 (MAP2), mikrotübül montaj için gerekli bir nörona özgü sitoskeletal proteini. Bundan başka, retinanın tabakalı yapı gibi genişliği ve süreklilik değişiklikler olarak Müller hücrelerinin ve gangliyon hücreleri proseslerinin ayrıntılı bir analizini sağlar. Böylece birkaç ek parametreler sonuçlarını değerlendirmek için kullanılabilirBRB ihlali erken bir aşamada ve aynı zamanda potansiyel tedavilerin ters etkilerini değerlendirmek için. kan damarlarının sızıntı (KZ), glial hücrelerin aktivasyonu ve nöronal hücrelerin hasar tepki: Bu protokol, ekranlı ilaçların potansiyel ters etkileri üç açıdan değerlendirilir. Çeşitli ölçümü yöntemleri, örneğin, ifade seviyesi immün bir süreç ve bir donanım filtresi tarafından gösterilen nöronal süreçlerin sürekliliği genişliği ölçüm yoğunluğu ile gösterilen, kullanılmaktadır. Daha iyi yöntemi göstermek için ve sonuçları yorumlamaya yardımcı olmak için, Lipoksin A4 (LXA4), endojen inflamatuar hasarına yanıt ve endotel disfonksiyonu 23 hafifletici sentezlenen bir bileşik, gösteri amaçlı seçilmiştir.

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			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
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			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
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			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">1.45s</td>
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an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

Biz histamin tarafından uyarılan BRB ihlal karşı koruyabilecek potansiyel tedaviler değerlendirmek için düşük maliyetli, zaman verimli ve kolay kullanımlı bir sistem sunuyoruz. IgG kontrol retina (Şekil 1A) damarlar içinde kısıtlı, ancak modelin başarıyla kuruldu doğrulayan histamin maruz (Şekil 1B) üzerine kan damarlarının, dışarı sızdırıyor. LXA4 sunulan sistemde...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Düşük maliyetli, kullanımı kolay ve güçlü bir sistem histamin tarafından uyarılan kan retina bariyer ihlali iyileştirecek potansiyel tedaviler değerlendirmek için kurulmuştur. Kan damarı sızıntı, Müller hücre aktivasyonu ve nöron sürekliliği bir potansiyel ilaç, Lipoksin A4 hasarı yanıtını ve ters değerlendirmek için kullanılır.

Tedavi Kan Retina Bariyer ihlali ve Potansiyel İlaçlar değerlendirilmesi için bir Akut Retina Modeli

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

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

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

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 ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

Retina Hastalıkları Değerlendirilmesi Nicel Otofloresans

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Tavşanlarda Subretinal Cerrahi Adım Protokolle Bir Adım

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

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

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

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) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Gut-kan bariyerini ve karaciğer metabolizması Microbiota ürünlerin değerlendirilmesi için In Vivo yöntemi

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Kemirgenlerde İskemik Retina Hastalıkları için Oksijen Kaynaklı Retinopati Modeli

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

İnsan Pluripotent Kök Hücrelerinden 3D Retinal Dokuların Türetilmesi için Retinal Organoid İndüksiyon Sistemi

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...