Yazarlar kemirgen izleme, işleme ve deney Bayan Sharon Spana yardımı için müteşekkiriz. Doktora finansman desteği Vizyon Mükemmellik University of Sydney ve Avustralya Araştırma Merkezi tarafından sağlanmıştır.

Bu protokol University of Sydney hayvan bakım kurallarına uyar. 1. Yapımı elektrotlar <ol><li> Pozitif elektrot platin tel çapı 1-2 mm kısa (5 cm) uzunluğu (kornea irtibata geçecektir biri) Construct. Bir döngü içine çapında bir kaç mm o moda. Senin amplifikatör giriş aşamaya ulaşmak için yeterince uzun bir geleneksel kurşun bu döngü bağlayın (Şekil 4). </li><li> Bir Ag / AgCl de kongre kablosuna bağlı çapı 1-2 mm, pelet kullanılarak (hayvanın ağzına gidecek) negatif elektrot Construct (Şekil 4). </li><li> (Hayvanın kıç girecek) referans elektrot, aynı zamanda uygun uzunlukta bir kurşun bağlanmış temiz derialtı iğne (23 G) kullanmak gibi (Şekil 4). </li><li> İdeal olarak, (pozitif U üç elektrotları bağlamak için, cihaz üreticileri tarafından sağlanan üç kurşun kablolar kullanın94; Kornea, negatif → ağız, amplifikatör kıç → referans). </li></ol> Işık Stimulus ve ERG Set-up 2. Bağlantı ve Kalibrasyon <ol><li> Karanlık yapılabilir küçük bir kayıt laboratuvarı, oluşturun (veya bulmak). Kırmızı yapılan over-the-tezgah ışık veya kırmızı kafa lambasının ya da her ikisi ile donatın. </li><li> 1 lux geçmemesi kurulum sırasında sıçan göz ulaşan kırmızı ışık aydınlık onaylamak için bir lüks metre kullanın. Not: Bir nötral yoğunluk filtresi lamba parlaklığını azaltmak için kullanılabilir ve lamba ışık kaynağı, özellikle kırmızı ışık yayarlar gerekir. Işık kaynakları düşük (görünür) dalga boylarını yayan eğer karanlık adaptasyonu tehlikeye olacak. </li><li> (Bu genellikle şeffaf bant ile sebat gerektirir) kayıt laboratuvarı giren tüm sokak ışık kapatmak ve üzerinde sığacak kadar büyük ve o kadar loş bir nötral yoğunluk filtresi (bu yaprak satın alınabilir) hazırlamak, herhangi bir bilgisayar ekranı içinde olacak laboratuvar. Not: Kaçak ışık veBir ekranın ışık sıçan gözün karanlık adaptasyonu halel için yeterlidir. </li><li> Veri toplama donanımına amplifikatörü bağlayın. Amplifikatöre pozitif, negatif ve referans uçlarını bağlayın. Bilgisayar ve LED Ganzfeld güç kaynağı ünitesi güvenli bir zemin kaynağına bağlı olduğundan emin olun. Not: Bazı laboratuvarlar bina toprağa bağlı, topraklama noktaları uzman var; Bir su borusu etkili bir alternatiftir. </li><li> Bir araştırma kalitesinde Radyometresiz LED ışık kaynağı ayarlayın. Hayvanın gözü bir deney sırasında yer alacak hangi pozisyonda metrenin sensörünü sabitleyin. </li><li> Ganzfeld LED&#39;ler yanıp söner arasındaki flaş enerji, flaş süresi, flaş tekrarı ve zaman içinde adım adım artışlar tam saha ERG protokolünü çalıştırmak Program, interstimuls aralığı (ISI), ayarlar olarak adlandırılan. Bir örnek tam saha protokolü için bakınız Tablo 1. Not: ERG yanıp söner tekrarlayan loş yanıp söner yükselecek tam saha bBir adım akıllıca bir şekilde doğru yanıp söner. ikiz Flash program tam saha protokolü üzerinde izler ve çubuk ve koni yanıtları izolasyonunu sağlar. </li></ol> ERG Deney 3. Gün önce <ol><li> Karanlık kaydetmeden önce 12 saat Sprague-Dawley sıçanları adapte. Bu kaçak ışık ortadan edildikten sonra, kayıt laboratuvarda bunu yapmak uygundur. </li></ol> ERG Deney 4. Gün <ol><li> Hayvan kayıt sırasında hafifçe ısıtılır olabilmesi için düzenleyin. Biz hayvanın kafası Ganzfeld girişinde doğru noktada dinlenme böylece inşa hafif metal platformu kullanmak. platform, bir su banyosu içinde 40 ° C&#39;ye ısıtılmış su pompası içinden dahili boru vardır. Not: Deneyim bu 37 ° C&#39;de hayvanın çekirdek sıcaklığını korur olduğunu göstermektedir. </li><li> Karanlık koşullarda sıçan tartılır. Telaffuz ağırlığı ve doğru ketamin (60 mg / kg) ve ksilazin oluşturan (5 mg / kg) dozda. Sıçan gen dizginlemektly ve intraperitoneal anestezik enjekte. </li><li> Enjeksiyon zamanı not edin. Hayvan bilinçsiz sonra (genellikle 5 dakika içinde) hafifçe bir refleks yanıtı mevcut olup olmadığını görmek için, tek ayak pedi kısma anestezi derinliğini kontrol edin. Bu refleks geçmeden önce, yok ya da zayıf olana kadar beklemek en iyisidir. </li><li> Korneaya proxmethacaine tek atropin damla ve başka uygulayın. </li><li> Siyah iplik 10 cm uzunluğunda kesin. Basit bir düğüm ile bir döngü yapın ve göz ekvator üzerinde döngü kayma. Hafifçe sıkın; etkisi minimum basınç, hafif öne göz küresinin çekmektir. Bu göz kapaklarında net kornea tutar. </li><li> Uygula karbomer göz kornea yüzeyine düşer. Emin Karbomer kornea yüzeyinde kalır ve göz kapakları veya yüz dökülmemesine değildir. </li><li> Isıtmalı platformun üstüne emici yatak yerleştirin. </li><li> Ganzfeld açılışında önerilen yerde baş yatak üzerinde Pozisyonu sıçan. </li><li> Ekle intrektum içine ernal ısı probu. Kuyruk prob kablosunu bantlama pozisyon Güvenli ısı probu. </li><li> Subkutan arka bacak içine referans elektrodu (23 G iğne) yerleştirin ve amplifikatör bağlanın. </li><li> Güvenli ağızda negatif elektrot (Ag / AgCl pelet) yerleştirin. Bu ağız kaymasını önlemek için, sabit bir yüzeye bağlanarak kurşun yapıştırın. </li><li> Korneanın merkezi üzerinde pozitif elektrot yerleştirin. Bir mikromanipülatör kullanarak, elektrot hafifçe kornea dokunur emin olun. </li><li> 37.5 ° C - Kontrol vücut ısısı 37.0 yer almaktadır. </li><li> Hayvan düzgün yerleştirilmiş ve elektrotlar yerdesiniz sonra, opak malzeme ile tüm kurulum (Ganzfeld ve hayvan) asmak (karanlık adaptasyonu korumak için). Biz yumuşak siyah bir bez kullanın. </li><li> Ön toplama örnekleme 5 milisaniye ile 100-1000 msn koleksiyonu zaman 2 KHz örnekleme hızında ayarlanmış edinme yazılımında. 1-1,000 bant geçiren filtreler ayarlayınHz ve bu örnekleme sağlamak bir flaş aşağıdaki ~ 250 msn dönemini örnek tetiklenir. </li><li> Kayıt taban çizgisini kontrol edin. Bu gereksiz gürültü özgür olmak, ancak bazı amplifikatör gürültü ve solunum salınım göstermesi gerekir. </li><li> Bazal gereksiz gürültüyü gösteriyorsa, sorun giderme başlar. Çoğu problem elektrot pozisyonu veya topraklama kaymaya ilişkilidir. Kayıtlar gereksiz gürültü serbest olmasını sağlamak için bir Faraday kafesi kullanın. </li><li> Bir test flaş, 0.4 günlük scot cd.sm -2 çalıştırın. Benzer bir ERG dalga 2A görünmelidir Şekil. 0.4 günlük scot cd.sm -2 flaş laboratuvar tipik tepkiler (: -474 ± 39 mV ve b-dalgası: a-dalgası 1,512 ± 160 mV, n = 11) &#39;dir. </li><li> Karanlık hayvan 10 dakika süreyle yeniden adapte izin verin. Bu taban çizgisini yeniden kontrol etmek, bu 10 dakika kullanmak daha uygun olur. </li><li> Istikrarlı sinyal aşağıdaki onay kaydı başlar. </li><li> Kayıt oturumun sonunda, vücut temperat kontrolURE muhafaza edilmiştir. Elektrotları çıkarın. Kornealara karbomer polimer yeniden uygulayın. Hayvan konut dönmeden önce, tamamen mobil ve aktif olana kadar hayvan bir ısı pad üzerinde kurtarmak için izin verin. </li></ol> 5. Uzaktan İskemi <ol><li> Uyanık veya anesteziye kemirgenlerde ya uzaktan iskemi gerçekleştirin. </li><li> Hayvan anestezi ise, (yukarıda) ısıtılmış bir platform üzerine yattı ve diz net arka ekstremite, üst kısmı üzerinde tansiyon aleti Kolluğu kayma. </li><li> Hayvanlar işlenen için kullanılırsa, anestezi olmadan bu yordamı gerçekleştirmek mümkündür; Bu iki kişi gerekir. Bir kişi hafifçe hayvan kısıtlayan ve ikinci tansiyon aleti manşeti uygular ve sfingomanometre çalışır. </li><li> Uyanık hayvanlar için, yavaşça tek hindlimb ücretsiz, hayvan sarmak için havlu bir parça ~ 15 cm x 30-50 cm kullanın. Merkez sahibinin kol ve gövde, ve yeri arasında sıkışmış olan, sol önkol (diyelim ki) onun sırtında hayvan LayManşet tıpkı nitelendirdi. </li><li> Manşeti Deflate ve hava basınç valfi kapalı olduğundan emin olun. Narkoz hayvanlarda 160 mmHg manşeti Pompa ve uyanık hayvanlarda 180 mmHg. Bu sistolik basıncı (genellikle 140 mmHg ve 160 mmHg sırasıyla) aşıyor. </li><li> El pompası kullanılarak, gerektiğinde bu baskıları koruyun. </li><li> Iskemi için planlanan bir süre sonra (biz 5 dk reperfüzyon ile ayrılmış, 5 dakika 2 dönemleri kullanın), hava basınç valfi gevşeterek manşet basıncını Söndür. </li><li> Footpad bağlı bir cilt sıcaklık probu ile uzaktan iskemi etkisini onaylayın. Cilt sıcaklığı tipik olarak 5 dakika boyunca, 32-30 ° C&#39;ye düşer ve reperfüzyon kurtarır. </li></ol> 6. Işık Hasar <ol><li> Sıçanlar koyu adapte gecede, önce hafif hasar prosedüründe olduğundan emin olun. </li><li> (Gecikmeden bizim deneylerde) ekstremite iskemi aşağıdaki uygun zamanda, her bir hayvan, bir pleksiglas kutular, wi içine tek başına yerleştirilirzemin tabanlı kaplarda inci su ve gıda. Not: Işık kaynaklı hasar sadece albino hayvanlarda yapılabilir. </li><li> Standart bir süre (genellikle 09:00) bir önceden kalibre 1,000 lux beyaz ışık açın ve 24 saat süreyle bu durumu korumak. </li></ol> 7. ERG Veri çıkarımı ve Analizi <ol><li> ERG ortalama dalga formları edinin. Çıkarma ile sıfır olmayan bir taban için, gerekirse ayarlanır. </li><li> Başlangıç ​​ve birinci arasındaki gerilim farkı olarak, (yüksek uyaran yoğunluklarına orta ve sunulan) a dalgasının genlik ölçün (&lt;30 msn gecikme) oluk (Şekil 1). </li><li> Tipik 80-100 msn (Şekil 1) bir gecikme meydana gelen bir dalgasının tepe ve şu dalganın pozitif arasındaki gerilim farkı olarak b-dalgası genliği ölçün. </li><li> 90 Hz geçiş bandı ile, 60-235 Hz verileri süzmek için bir dönüşüm Fourier kullanarak osilatör potansiyelleri izole edin <sup&gt; 7. Gerekli izole salınımlı bir potansiyel sinyal daha sonra filtresiz dalga çıkarılır olabilir Bir dalga çukur kimliğini doğrulamak için. </li><li> a- ve b-dalgası zirveleri örtülü süresi (gecikme) da yararlı bir tedbir (Şekil 1) olabilir. Çubuk yanıtı izole etmek için ikiz yanıp kullanın. Karışık tepki (flaş 1) çubuk tepkisini izole etmek (Şekil 2) koni yanıt (flaş 2) çıkarın. </li><li> Bireysel ışık yoğunluğu bir dalga ve b-dalgası genlikleri (tedavi sonrası / post-tedavi-bazal) Normale veya tedavi grupları için ortalama. Yoğunluk-yanıt eğrileri flaş enerjiye karşı grup genlikleri ve hatayı arsa. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACUTE+REVERSIBLE+LENS+OPACITY+-+CAUSED+BY+DRUGS,+COLD,+ANOXIA,+ASPHYXIA,+STRESS,+DEATH+AND+DEHYDRATION.">ACUTE REVERSIBLE LENS OPACITY - CAUSED BY DRUGS, COLD, ANOXIA, ASPHYXIA, STRESS, DEATH AND DEHYDRATION.</a> Experimental Eye Research. 10 (1), 19 (1970).</li><li>Calderone, L., Grimes, P., Shalev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACUTE+REVERSIBLE+CATARACT+INDUCED+BY+XYLAZINE+AND+BY+KETAMINE-XYLAZINE+ANESTHESIA+IN+RATS+AND+MICE.">ACUTE REVERSIBLE CATARACT INDUCED BY XYLAZINE AND BY KETAMINE-XYLAZINE ANESTHESIA IN RATS AND MICE.</a> Experimental Eye Research. 42 (4), 331-337 (1986).</li><li>Behn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dark+adaptation+is+faster+in+pigmented+than+albino+rats.">Dark adaptation is faster in pigmented than albino rats.</a> Documenta Ophthalmologica. 106 (2), 153-159 (2003).</li><li>Sugawara, T., Sieving, P. A., Bush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+relationship+of+the+scotopic+and+photopic+ERG+to+photoreceptor+cell+loss+in+light+damaged+rats.">Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats.</a> Experimental Eye Research. 70 (5), 693-705 (2000).</li><li>Machida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P23H+rhodopsin+transgenic+rat:+Correlation+of+retinal+function+with+histopathology.">P23H rhodopsin transgenic rat: Correlation of retinal function with histopathology.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3200-3209 (2000).</li><li>Brandli, A., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+Ischemia+Influences+the+Responsiveness+of+the+Retina.">Remote Ischemia Influences the Responsiveness of the Retina.</a> Observations in the Rat. Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

Jonathan Stone CSCM Pty Ltd direktörü

Yukarıda tarif edilen koyu adapte flaş ERG yöntemi sıçanlarda retina fonksiyonlarını değerlendirmek için güvenilir bir yöntemdir. A-dalgası ve b-dalgası Hem hafif hasar azaltılmıştır. Uzaktan iskemik önkoşullama a-dalgası ve b dalga ışık hasar kaynaklı azalmalar hafifletilmiş. Retina fonksiyonunun Bu muhafaza uzak iskemik önkoşullama gibi hipoksi, iskemi ve egzersiz 8-10 gibi koruyucu önkoşullama diğer formları andıran, nöro kaynaklı olduğunu göstermektedir. Kayıt kurulumu...

ERG ışığa karşılık olarak retina tarafından oluşturulur ve gözün korneal yüzeyi kaydedilen bir elektrik potansiyeli. Kayıt koşulları dikkatle idare edildiğinde, ERG retina fonksiyonunu değerlendirmek için çeşitli şekillerde kullanılabilir. İşte biz &#39;flaş ERG&#39;, retina, bir Ganzfeld arka planda sunulan kısa, parlak flaş maruz kaldığında oluşan potansiyeli kaydetmek nasıl tarif. Ganzfeld homojen ışık dağıtır ve ışık flaş yaklaşık eşit bütün retina ulaşır. Retina kaydetmeden önce koyu adapte edilir ve hayvan kayıt için hazırlanmış gibi koyu adaptasyonu devam edilirse, ERG hem çubuk ve koni fotoreseptör tarafından oluşturulan aldı. koyu adapte flaş ERG iki şekilde analiz edilmiştir karakteristik dalga biçimi vardır. İlk olarak, ERG, dalga biçiminin, erken ve geç bileşenleri ayırt ve nöron sekansına ilişkilendirilmiştirretina al aktivasyonu. erken bileşen kısa gecikme negatif gidiş potansiyel bir dalga (Şekil 1). Bu olumlu bir gidiş potansiyeli takip ediyor, b- dalgası olarak adlandırılan. b- dalga yükselen aşaması ayrı bir bileşen (osilatör potansiyelleri veya OP) olarak kabul edilir salınımları göstermektedir. a-dalgası fotoreseptör tarafından oluşturulan olarak kabul edilir, amacrine hücreleri 1 ile iç nükleer tabaka hücreleri ve ops b-dalgası. Uyarıcı gücüne dayalı, çok loş flaşlar tepkiler skotopik eşik tepkisi mümkündür adlandırılan. skotopik sınır tepkisi, retinal bez hücrelerin 2-4 elde edilmesi anlaşılmalıdır. İkincisi, flaş ERG ışık adaptasyon ile ayrılabilir veya bir iki flaş protokolü tarafından çubuk ve koni odaklı bileşenleri içine, aşağıda anlatılan. Fotopik koşullar altında, koni nüfusu düşük olduğu için dalga, sıçanlarda değil saptanabilir, ancak Operasyonel ve b-dalgası5 açık. Kimin retinalar yüksek koni nüfusa sahip primatlar, olarak, çubuk ve koni yollar hem saptanabilir bir dalga 6 üretir. Genellikle flaş ERG çıkarılan iki yararlı önlemler fotoreseptör nüfusu zarar verici parlak maruz bırakılarak, örneğin azalır, Şekil 2&#39;de gösterilen tipik bir flaş yanıtları. Şekil 1 &#39;deki gibi ölçülür A ve B amplitüdü, Hangi Işık, ERG tüm bileşenleri azalır. Gibi uzak iskemik önkoşullama (RIP) olarak nöroprotektif müdahaleler, a- b-dalgaları (Şekil 3) genliklerinin korunması ile doğrulanabilir. Özetle, ERG analizi hasarlı sağlıklı, hafif ve neuroprotected retina arasındaki karşılaştırmalar sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
		</tr>
		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
		</tr>
		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
		</tr>
		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
		</tr>
		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
		</tr>
		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
		</tr>
		<tr>
			<td>Absorbent bedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
		</tr>
		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

protokolü, in vivo olarak, kemirgen retina görme fonksiyonu ölçmek için kullanılabilir. a-dalgası, fotoreseptör fonksiyonunun bir ölçüsü, ve b-dalgası, iç retina fonksiyonun bir ölçümü olan, Şekil 1 &#39;de açıklanmış bulunmaktadır. Şekil 2A gösterildiği gibi artan ışık uyaran ile çubuk hakim ERG sinyal artar. a-dalgası 0.4 günlük scot cd.sm -2 ve 2.5 log Scot cd.sm de doyuma kadar a-dalgası artar genlik...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Kemirgen Retina ve Uzaktan Ekstremite İskemik Önkoşullamanın Koruyucu Etkileri Fonksiyonu Değerlendirmek Üzere elektroretinogram kullanma

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

16.1K Görüntüleme.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Video: Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Soğuk önkoşullamanın nöro-Çalışması için Stratejiler

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Nörobilim

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Deri ve Kol ve bacak kemikleri innerve Fare Duyusal Sinir Lif doku hazırlanması ve immün

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Kemirgen Merkezi Sinir Sistemi İlaçların doğrudan Intraventriküler teslim

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Fare Retina kullanma CNS&#39;deki Vasküler Rejenerasyon Değerlendirilmesi

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Zebra balığı Larva Görsel Tepki Elektroetinogram Analizi

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Kullanımı Netlik Fare Omurilik içinde Serotonerjik Elyaf Görüntüleme / KÜBİK Tekniği

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Elektroporasyon tarafından Kemirgen Siyatik Sinir Schwann Hücreleri için Vivo Gen Transferi

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Serebral arterler patlama kaynaklı Neurotrauma etkilerini basınçlı kemirgen orta

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Siyatik Sinir Ezilme Hasarının Sıçan Modelinde Fonksiyonel Değerlendirme için 3D Kinematik Analiz

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Farkındalık Meditasyonunun Epizodik Hafıza Üzerindeki Etkilerini İncelemek için Davranış ve EEG'yi Birleştirmek

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...