Name: simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system
Uploaded: 2015-05-06T15:00:00
Description: Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Bu çalışma NIH hibe EY019312 ve EY021126 (VJK), Washington Üniversitesi Göz Hastalıkları Anabilim Dalı ve Görsel Bilimler EY002687 tarafından desteklenen ve Araştırma tarafından Körlük Önlemek için.

Tüm deneysel protokoller Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu ile uyumlu olduğunu ve Washington Üniversitesi&#39;nde kurumsal Hayvan Çalışmaları Komitesi tarafından kabul edildi. 1. Perfüzyon ve Numune Tutucu Kurma <ol><li> Deney gününde taze retina perfüzyonu için çözelti hazırlayın. Distile ve deiyonize su kullanın. Aşağıdaki üç çözümlerden birini kullanın. <ol><li> , Medya ve NaHCO 3 1.9 g &#39;Ames 1 şişe (1 L) çözeltisi&#39; A...

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<html> Biz burada bir ex vivo ERG adaptörü ile birlikte in vivo ERG sistem bileşenlerini kullanarak iki izole fare retina aynı anda yüksek kaliteli ex vivo ERG kayıtları elde etmek için kritik adımlar göstermektedir. Bu çalışmada, aynı solüsyonu (ya Ames &#39;, Locke ya da Ringer) ile hayvandan her iki retina perfüze ama uyuşturucu test amaçlı, farklı bir çözüm örneğin her retina serpmek de mümkündür. yüksek kaliteli veri elde etmek için en öneml...

Elektroretinogram (ERG) ışıkla tetiklenir retina elektriksel aktivitesi kaydetmek için kullanılabilen bir köklü bir tekniktir. ERG sinyal retinanın rezistif dışı uzayda akan radyal akımların neden olduğu gerilim değişiklikleri (fotoreseptör ve bipolar hücrelerin ekseni boyunca) ağırlıklı olarak üretilir. İlk ERG sinyali bir balık gözü 1 yüzeyinden Holmgren, 1865 kaydedilmiştir. Einthoven ve Jolly 1908 2 b-, a- denilen üç farklı dalgaların içine ışık başlangıcından ERG yanıtı, ve c-dalgalar, şimdi bipolar hücreler ON fotoreseptör esas aktivitesini yansıttığı bilinmektedir ve pigment epiteli bölünmüş Hücreler sırasıyla 3-8. ERG, yerel (mikroelektrotla (ex vivo) izole sağlam retina arasında, izole edilmiş bir göz Hazırlama 9&#39;da elde edilen, anestezi uygulanmış hayvanlar ya da (in vivo) insan gözüyle 3,10-15 ya arasında belirli bir retina katmanları kaydedilebilirERG) 4,16. Bunlardan in vivo ERG şu anda retina fonksiyonunu değerlendirmek için en yaygın kullanılan yöntemdir. Bu teşhis amaçlı kullanılabilir veya hayvanlarda veya hastalarda retina hastalıklarının ilerlemesini takip etmek invaziv olmayan bir tekniktir. Ancak, in vivo ERG kayıtları genellikle ekstraoküler fizyolojik gürültü (örneğin, solunum ve kalp aktivitesi) ile kirlenmiş, birkaç örtüşen bileşenleri ile karmaşık bir sinyal üretmek. Yerel ERG retina spesifik katmanlar arasında sinyalini kaydetmek için kullanılabilir, ancak, en invaziv ve diğer ERG kayıt konfigürasyonları ile karşılaştırıldığında düşük bir sinyal-gürültü oranı (SNR) sahip olmasıdır. Yerel ERG ayrıca teknik olarak ve pahalı donanımları (örn mikroskop ve mikromanipülatörler) gerektirir. Sağlam, izole retina gelen Transretinal ERG (ex vivo ERG) istikrarlı ve HIG izin in vivo ve yerel ERG yöntemleri arasında bir uzlaşma sunuyorhayvanlar ya da insanlar 17 sağlam retina h SNR kayıtları. Son zamanlarda, bu yöntem, memeli primat ve insan retina 18-20 çubuk ve koni fotoreseptör fonksiyonunu incelemek için başarıyla kullanılmaktadır. Buna ek olarak, ex vivo olarak, retina pigment epiteli olmaması, ERG sinyalinin pozitif Cı dalga bileşeni çıkarılır ve önemli bir negatif yavaş PIII bileşeni ex vivo kayıtları ortaya çıkar. Yavaş PIII bileşeni retina 21-23 Müller glia hücrelerinin aktivitesi kaynaklı gösterilmiştir. Bu nedenle, ex vivo olarak ERG yöntemi de sağlam retinada Müller hücreleri incelemek için kullanılabilir. Çeşitli çalışmalarda da bu ex vivo ERG kayıtları retina 24 civarında farmakolojik ajanların konsantrasyonu ölçmek ve uyuşturucu 25-27 güvenliğini ve etkinliğini test etmek için kullanılan olabilir göstermiştir. In vivo sistemlerde birden fazla ticari mevcuttur vemutlaka geniş elektrofizyoloji altyapıya sahip olmayan birçok laboratuvarlarında kullanılan. Buna karşılık, ex vivo cihazlar son zamanlarda 17 kadar ve sadece çok az laboratuarlar anda bu güçlü tekniği yararlanarak bir sonucu olarak mevcut olmamıştır. Bu retina fizyolojisi ve patolojisi hakkında bilgimizi ilerletmek ve hastalıkları kör için yeni tedavilerin geliştirilmesi için daha fazla laboratuvarlara ex vivo ERG kayıtları kullanılabilir hale getirmek için yararlı olacaktır. Burada, bir basit ve uygun fiyatlı bir ex vivo ERG aygıtı 17 göstermektedir ve bu çubuk ve koni aracılı sinyal kaydetmek için çeşitli ticari olarak temin edilebilir, in vivo ERG sistemleri ile kombinasyon halinde kullanılabilir göstermek (a- ve b-dalgası) ve fonksiyonu görmemiş vahşi tip fare retinaları Müller hücrelerinin (PIII yavaş).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Bu koyu adapte yabani tip flaş yanıtları kaydedildi deney protokolleri farklı standart perfüzyon için solüsyonlar (Şekil 2) ile, yukarıda tarif edilen ve Şekil 1 &#39;de gösterilen izleyerek (WT), C57BL / 6 fare retinaları. tepki dalga ve kinetik yanı sıra çubuk fotoreseptör duyarlılığı Ames &quot;ve Locke&#39;un medya (Şekil 2A ve B) benzer çıktı. Öte yandan, HEPES-tamponlu Ringer çözeltisi (Resim bikarbonat ya da% 5 CO ...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Ultrasonik Kan-beyin bariyeri bozulması ve Manganez kontrastlı MRG kullanma İşlevsel beyin görüntüleme

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Polyribosome Fraksiyonu tarafından Santral Sinir Sistemi Translatome in vivo sorguluyor

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Bozulmamış Vomeronasal Organ ve Aksesuar Koku Ampul Ex Vivo Hazırlıklar

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Eşzamanlı İki foton<em&gt; İn Vivo</em&gt; Fare Retrosplenial kortekste sinaptik Girişlerin Görüntüleme ve postsinaptik Hedefler

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

Fonksiyonel Çalışma için Ex Vivo Basınçlı Hipokampal Kapiller-Parenkimal Arteriyol Hazırlığı

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Transpupiller İki Foton Fare Retinasının In vivo Görüntülenmesi

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Ex Vivo Elektroretinogramın Küçük ve Büyük Gözlerde Retina Fonksiyonunu İncelemesi için Kurulum ve Koşulların Optimize Edilmesi

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...