We would like to acknowledge Beth Levine for critically reviewing the manuscript, Valerie Tassinari for developing the genotyping protocol, and Matt Ferola and Carlos Maya for their exceptional animal care.

Tüm deneyler Hayvanların Bakım ve Kullanım Sağlık Rehberi Ulusal Sağlık Enstitüleri tarafından açıklanan protokollere uygun olarak yürütülmektedir ve ALS TDI kurumsal hayvan bakımı ve kullanımı komitesi (IACUC) tarafından onaylanmıştır. 1. Hayvanlar, Konut ve Araştırma Tasarımı Not:. SOD1-G93A fare koloni başlangıçta Gumey ve diğerleri 7 tarafından üretilen yüksek kopya B6SJLTgN (SOD1G93A) 1Gur suşundan elde edilmiştir. Koloni şu anda vahşi tip C57BL / 6-SJL F1 dişilerle transgenik C57BL / 6-SJL erkek kapısı tarafından muhafaza ediliyor. F1 hayvanlar B6 dişilerle SJL erkek geçerek üretilir. Fareler yaş 35-45 günde ALS TDI haftalık sevk edilir. <ol><li> Farenin en az bir hafta bir çalışmaya vermeden önce (18-23 ° C ve% 40-60 nem arasındaki bir sıcaklıkta 12 saat ışık / karanlık döngüsü) hayvan tesisine alıştırıldı izin verir. </li><li> Ev erkek SOD1-G93A mikrofongrup erkek savaşmak eğilimi ev sahipliği beri kafes başına e bir fare. Kafes başına iki adede kadar Evi dişi SOD1-G93A fareler. Plastik kulübe şeklinde çevresel zenginleştirme sağlar. Fareler su / gıda, içme ilaç yönetimini gerektiren bir çalışmanın parçası olmadıkça, yiyecek ve su ad libitum sağlayın. Bu gibi durumlarda, oranı ve kayıt gıda ve su. </li><li> Yaş gününde 50 civarında bir ilaç etkinlik çalışmaya başlamak ve 64 çöp eşleştirilmiş cinsiyet dengeli fareler (kohort başına 32) içerir. Son olarak, çalışmalar yürüten araştırmacılar deneysel koşullara kör olduğundan emin olun. Not: Bu tipik bir ilaç etkinlik çalışması ancak önce ya da sonra çalışmaya başlamak için tercih olabilir terapötik müdahale türüne göre yaş gününde 50 başlar önerilir. Not: kohort başına 32 fare cinsiyete özgü ilaç etkilerini tespit etmek güç olan bir çalışma. Cinsiyete özgü karşılaştırmalar gerekiyorsa, kohort başına 24 fare, genel ilaç etkisini tespit etmek için yeterli olacaktır.</li></ol> 2. Vücut Ağırlığı <ol><li> Günlük gram onda biri bir hassasiyetle çalışmalarda tüm fareler için rekor vücut ağırlıkları. Not: Vücut ağırlığı hastalığın ilerlemesi ve kronik ilaç tedavisinin doğabilecek halsizlik hassas bir göstergesidir. </li><li> Fare vücut ağırlıkları dolayısıyla, sabahları daha yüksek olması, bir gün süresince önemli ölçüde değişebilir, her gün aynı zamanda etrafında onları kaydedin. </li></ol> 3. Nörolojik Puanlama Sistemi (NeuroScore) <ol><li> Günlük olarak, aşağıdaki üç koşullar altında fare gözlemleyerek her bir fare için değerlendirilmesi nörolojik puanları (NS) bir sekans dahilinde gerçekleştirilebilir: b) fare) yürümeye bırakıldı ve parezi c başlamasından sonra a), fare kuyruk tarafından askıya Fare yan yerleştirilir. <ol><li> Kuyruk süspansiyon testi için, gıda havzası uzak, 1-2 sn evlerine kafes tel üstünden kuyruk tabanından fareyi yaklaşık 1.5 &quot;tutun atıl ikenarka ayaklarında Erving. Süspansiyon testi 3 kez tekrarlayın ve en tutarlı sonucu kaydedin. </li><li> Yürüme testi, bazı çekiş ve 25 cm yürüme mesafesi sağlayan temiz bir yüzeye üzerinde fare koyun (örn., Bir kağıt havlu kaymasını önlemek için aşağı bantlanmış). Onun yürüyüşü izlerken fare 75 cm toplam (kağıt havlu 3X uzunluğu) yürümek için izin verin. </li><li> &quot;Doğrulma refleksi&quot; test için sol veya sağ tarafına fare koyun ve bir kronometreler her iki taraftan yardımsız kendini yukarı sağa süresini ölçmek kullanarak. Not: Bu testin sadece bir deneme gereklidir. </li></ol></li><li> 0&#39;dan 4. Not bağımsız bir ölçekte (sol veya sağ) Her hindlimb of NS belirleyin: Sağ hindlimb NS 1 iken Örneğin 0 puan, 1, sol hindlimb NS 0 olduğunu gösterir. </li><li> (Tablo 1&#39;de verilmiştir özet) gözlem aşağıdaki kümesine karşılık gelen nörolojik puanları (0-4) atayın:<ol><li> Aşağıdaki gözlenmesi halinde NS 0 (Ön-semptomatik) atayın: fare kuyruğu tarafından askıya alındığında, hindlimb normal yayvan yani sunar, tam uzakta yanal orta hatta uzanan ve 2 saniye bu pozisyonda kalır veya uzun. Fare yürümeye izin, normal yürüyüş görülmektedir. </li><li> Fare kuyruğu tarafından askıya alındığında, hindlimb anormal yayvan, yani sunar, bu çökmüş veya kısmen yanal orta hatta doğru çöktü YA o kuyruk süspansiyon sırasında titreyen VEYA o geri çekilir edilir: gözlenmektedir aşağıdaki eğer NS 1 (İlk semptomlar) atama / sıktı. Fare yürümeye izin verildiğinde normal VEYA biraz yavaş yürüme görülmektedir. </li><li> Aşağıdaki gözlenmesi halinde NS 2 (parezi başlangıcı) atayın: fare kuyruğu tarafından askıya alındığında, hindlimb çok uzanan değil, kısmen ya da tamamen yıkılmış olduğunu. (Hala ortak hareket olabilir). Fare yürümeye izin verildiğinde, hindlimb ileri mot için kullanılıriyon ancak ayak 90 cm yürüyüş sırasında en az iki kez aşağı doğru kıvrılıp VEYA ayak herhangi bir parçası kafes alt / masa boyunca sürükleyerek. Fare sol VE sağ tarafına yerleştirilmiş olduğunda, İKİ taraftan 10 saniye içinde kendini sağa yapabiliyor. </li><li> Aşağıdaki gözlenmesi halinde NS 3 (Felç) atayın: fare kuyruğu tarafından askıya alındığında, hindlimb VEYA minimal eklem hareket içinde sert felç var. Fare yürümeye izin verildiğinde öne Ancak hindlimb ileri hareket için kullanılan DEĞİLDİR hareket vardır. Fare sol VE sağ tarafına yerleştirilmiş olduğunda, İKİ taraftan 10 saniye içinde kendini sağa yapabiliyor. Not: Nadiren, felç başlangıcından sonra, idrar nem arka ayaklarında görünebilir. Tedavi edilmeyen idrar nem idrar &quot;yanık&quot; ve deri lezyonları neden olabilir. Saç kırpma yoluyla idrar nem davranın, ılık su idrar çıkarmak ıslatırken uygulamak ve yavaşça kuru kurulayın. Cilt lezyonları varsa Antibiyotik uygulanırtic merhem. </li><li> Aşağıdaki gözlenmesi halinde NS 4 (İnsanca son nokta) atayın: fare kuyruğu tarafından askıya alındığında, arka ayaklarında sert felç var. Fare yürümeye izin verildiğinde, hiçbir ileri hareket yoktur. Fare sol VE sağ tarafına yerleştirilmiş edildiğinde, YA taraftan 10 saniye içinde kendini sağa mümkün değildir. Yani, refleksi yokluğu. </li></ol></li></ol> 4. Veri Girişi ve Analizi <ol><li> Bir ilaç etkinlik çalışması sırasında, bir Laboratuar Bilgi Yönetim Sistemi (LIMS) her bir fare için veya ticari olarak satılan elektronik tablo yazılımı içine toplanan verileri girin. Not: ALS TDI geliştirilen LIMS yazılım dahili çalışmalardan toplanan tüm veriler için merkezi bir depo, ALS TDI müfettişler için zamanla bu veriler elverişli erişilebilir hale getirmek. LIMS, tüm çalışma günlerinde geri soyu, vücut ağırlığı veya nörolojik skoru, eşsiz kimliği ile, herhangi bir bireysel fare izlerve ömrü boyunca kaydetti başka gözlemleriniz içerir. LIMS sonuçta daha sonraki analizler için kullanılan bir elektronik tablo oluşturur. </li><li> Bir ilaç etkinlik çalışmasında, yani tüm fareler insancıl uç noktasına ulaştığında, kullanım tablosu programı (Malzeme Tabloya bakınız) Sol ve sağ hindlimb NS ortalamasını alarak her gün için, her bir fare için kombine NS hesaplamak tamamlanmasının ardından . Sonra, her fare ve her bir fare, belirli bir NS geçirdiği gün sayısı her NS ortanca yaşı hesaplamak. </li><li> İstatistiksel yazılımı elektronik tablo yazılımı veri alma yoluyla zaman içinde NS genel değişimleri analiz (Malzeme Tablo). Regresyon etkileri gibi medyan NS yaşı ve uyuşturucu tedavi ile, beklenen NS tahmin sıralı lojistik regresyon modeli kullanılarak bu verileri takınız. Log-rank ki-kare testi kullanılarak etkisi olabilirlik olasılığını tahmin ve NS ilerlemesi üzerine uyuşturucu tedavi etkisi istatistiksel olup olmadığını belirlemekly anlamlı (adım-adım talimatlar için İletişim Kod Dosyası bakınız). Not: Bir p-değeri &lt;0.05 istatistiksel olarak anlamlı kabul edilmektedir. Not: İstatistiksel analiz yöntemi NS veri ve bireysel fareler için NS yörüngeleri değişken doğası sıralı doğasını saygılıdır. Bireysel NS yörüngeleri yeterli NS tepkilerin yansıtıcı olmayan NS zaman ilişkili grup ortalamaları sonuçlanan belirgin farklı olabilir, çünkü tercih edilir. Buna ek olarak, bazı durumlarda, NS özellikle sıfır-bir aralığında değişebilir. </li><li> Son olarak, kullanım grafik yazılımı ilaç ile tedavi edilen ve kontrol hayvanları için grafikler &quot;Ordinal NS Medyan Yaş vs NS Beklenen&quot; oluşturmak için (Malzeme Tabloya bakınız) (Şekil 2; Yan Kod adım adım talimatlar için Dosya bakınız). Uygun y-ekseninden hastalığın başlangıcından (i ortalama yaşı enterpolasyonlayarak ilaç ile tedavi edilen ve kontrol grupları arasında vardiya tahmin etmek bu grafikleri kullanabilirsiniz.e., x-ekseninde NS 2). Not: İki grup arasında ortalama yaş farkı ilaca bağlı değişimi temsil ve etkinin büyüklüğü pratik bir önlem olarak kabul edilir. Not: Semptomatik hastalığın ilerlemesini yavaşlattı göstermektedir beklenen NS vs ortanca yaş eğrisi sağa kaymalar. Tersine, beklenen NS vs ortanca yaş eğrisi sola kaymalar semptomatik hastalığın ilerlemesini hızlandırılmış göstermektedir. </li></ol>

<ol>
	<li>Gurney, M. E., 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=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264 (5166), 1772-1775 (1994).</li><li>Rosen, D. R., 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=Mutations+in+Cu/Zn+superoxide+dismutase+gene+are+associated+with+familial+amyotrophic+lateral+sclerosis.">Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.</a> Nature. 362 (6415), 59-62 (1993).</li><li>Gurney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+transgenic+mouse+models+of+amyotrophic+lateral+sclerosis+in+preclinical+drug+studies.">The use of transgenic mouse models of amyotrophic lateral sclerosis in preclinical drug studies.</a> J Neurol Sci. 152, Suppl. 1 (6415), (1997).</li><li>Scott, 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=Design,+power,+and+interpretation+of+studies+in+the+standard+murine+model+of+ALS.">Design, power, and interpretation of studies in the standard murine model of ALS.</a> Amyotroph Lateral Scler. 9 (1), 4-15 (2008).</li><li>Hegedus, J., Putman, C. T., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+preferential+motor+unit+loss+in+the+SOD1+G93A+mouse+model+of+amyotrophic+lateral+sclerosis.">Time course of preferential motor unit loss in the SOD1 G93A mouse model of amyotrophic lateral sclerosis.</a> Neurobiol. Dis. 28 (2), 154-164 (2007).</li><li>Gordon, T., Ly, V., Hegedus, J., Tyreman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+denervated+muscle+fibers+in+hindlimb+muscles+after+sciatic+nerve+transection+in+wild+type+mice+and+in+the+G93A+mouse+model+of+amyotrophic+lateral+sclerosis.">Early detection of denervated muscle fibers in hindlimb muscles after sciatic nerve transection in wild type mice and in the G93A mouse model of amyotrophic lateral sclerosis.</a> Neurol. Res. 31 (1), 28-42 (2009).</li><li>Gurney, M. E., 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=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264 (5166), 1772-1775 (1994).</li><li>Kim, K., Moore, D. H., Makriyannis, A., Abood, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AM1241,+a+cannabinoid+CB2+receptor+selective+compound,+delays+disease+progression+in+a+mouse+model+of+amyotrophic+lateral+sclerosis.">AM1241, a cannabinoid CB2 receptor selective compound, delays disease progression in a mouse model of amyotrophic lateral sclerosis.</a> Eur. J. Pharmacol. 542 (1-3), 1-3 (2006).</li><li>Weydt, P., Hong, S. Y., Kliot, M., Moller, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+disease+onset+and+progression+in+the+SOD1+mouse+model+of+ALS.">Assessing disease onset and progression in the SOD1 mouse model of ALS.</a> Neuroreport. 14 (7), 1051-1054 (2003).</li><li>Azari, M. F., 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=Behavioural+and+anatomical+effects+of+systemically+administered+leukemia+inhibitory+factor+in+the+SOD1(G93A+G1H)+mouse+model+of+familial+amyotrophic+lateral+sclerosis.">Behavioural and anatomical effects of systemically administered leukemia inhibitory factor in the SOD1(G93A G1H) mouse model of familial amyotrophic lateral sclerosis.</a> Brain Res. 982 (1), 92-97 (2003).</li><li>Guan, Y., 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=Thiazolidinediones+expand+body+fluid+volume+through+PPARgamma+stimulation+of+ENaC-mediated+renal+salt+absorption.">Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption.</a> Nat. Med. 11 (8), 861-866 (2005).</li><li>Savcheniuk, O. A., 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=The+effect+of+probiotic+therapy+on+development+of+experimental+obesity+in+rats+caused+by+monosodium+glutamate.">The effect of probiotic therapy on development of experimental obesity in rats caused by monosodium glutamate.</a> Fiziol. Zh. 60 (2), 63-69 (2014).</li><li>Smittkamp, S. E., Brown, J. W., Stanford, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-course+and+characterization+of+orolingual+motor+deficits+in+B6SJL-Tg(SOD1-G93A)1Gur/J+mice.">Time-course and characterization of orolingual motor deficits in B6SJL-Tg(SOD1-G93A)1Gur/J mice.</a> Neuroscience. 151 (2), 613-621 (2008).</li><li>C, M., Gurney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+expressor+line+of+transgenic+mice+carrying+a+mutant+human+Cu,Zn+superoxide+dismutase+(SOD1)+gene+develops+pathological+changes+that+most+closely+resemble+those+in+human+amyotrophic+lateral+sclerosis.">A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis.</a> Acta Neuropathol. 93 (6), 537-550 (1997).</li><li>Wegorzewska, I., Baloh, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43-based+animal+models+of+neurodegeneration:+new+insights+into+ALS+pathology+and+pathophysiology.">TDP-43-based animal models of neurodegeneration: new insights into ALS pathology and pathophysiology.</a> Neurodegener. Dis. 8 (4), 262-274 (2011).</li><li>Hatzipetros, T., 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=C57BL/6J+congenic+Prp-TDP43A315T+mice+develop+progressive+neurodegeneration+in+the+myenteric+plexus+of+the+colon+without+exhibiting+key+features+of+ALS.">C57BL/6J congenic Prp-TDP43A315T mice develop progressive neurodegeneration in the myenteric plexus of the colon without exhibiting key features of ALS.</a> Brain Res. 1584, 59-72 (2014).</li></ol>

The authors have nothing to disclose.

Bu yazıda, doğru uygulandığı takdirde, güvenilir SOD1-G93A farelerde hastalığın ilerlemesini değerlendirmek ve ilaca bağlı değişikliklerin belirlenmesi yeteneğine sahip, hızlı ve basit bir video protokol açıklar. Farklı gruplar SOD1-G93A farelerin 8-10 için fenotipik skorlama sistemleri geliştirdiler ederken, sık sık prosedürü hakkında yeterli bilgi sağlamak ve replikasyon için yetersiz kalmaktadır yok. Sonuç olarak, skorlama sistemleri nadiren onları geliştiren belirli bir gr...

1990&#39;ların ortalarında, kalkınma SOD1-G93A transgenik fare modeli Amyotrofik lateral skleroz (ALS) 1 en yaygın olarak kullanılan hayvan modeli olmuştur beri. Bu transjenik fare modeli, genetik olarak amino asit 93 (G93A) alanin mutasyona ALS ilişkili glisin barındıran insan Cu / Zn süperoksit dismutaz 1 (SOD1) geni, bir mutant formu aşın ifade etmek üzere geliştirilmiş. G93A mutasyonu topluca makyaj ailesel ALS vakalarının 2 yaklaşık beşte biri SOD1 genindeki mutasyonlar biridir. SOD1-G93A modeli, bu nihai birlikte, bu tür motor nöron kaybı, ilerleyici kas güçsüzlüğü ve atrofi gibi insanlarda ALS patolojik özellikleri birçok özetlediği çünkü ALS onun bariz genetik bağlantıya ek olarak ALS ilaç geliştirme araştırma beygir haline gelmiştir felç ve ölüm 3. Genellikle transjenik hayvan modellerinde olduğu gibi olsa da, doğal bir iki oradaSOD1-G93A fareler ile bağlantılı rahatsızlık derecesinde değişkenliğin. Scott ve ark. En az 24 çöp eşleştirilmiş cinsiyet dengeli farelerin Kusaklara 4 biyolojik değişkenlik yarattığı gürültüyü aşmak için uyuşturucu etkinlik çalışması tasarımlarda gerekli olduğunu tespit ettik. Agresif hastalığın ilerlemesi ve günlük izleme ihtiyacı ile birlikte bu çalışmalarda gerekli hayvanların çok sayıda nöromusküler gücünü ve işlevini ölçmek için mükellef, zaman alıcı ve potansiyel stresli tekniklerin kullanılmasını yasaklayan (örneğin, elektromiyografi, kavrama kuvveti, rotorod , vs.). Bunun yerine, güvenilir hızlı bir şekilde SOD1-G93A farelerin kas fonksiyonunu değerlendirir in vivo tarama aracı ihtiyacını vurgulamaktadır. ALS TDI bir protokol ortalama fare başına en az 30 sn SOD1-G93A farelerin kas fonksiyonlarının değerlendirilmesi güvenilir sağlar geliştirildi. Bu protokol, encompabasında ve video Bu raporda açıklanan basit bir nörolojik skorlama sistemi (NeuroScore) kullanarak hindlimb fonksiyonun günlük değerlendirmesini de kapsar. Hindlimb açıkları erken SOD1-G93A farelerin 5,6 hastalığın nörolojik belirtisi bildirdi çünkü NeuroScore hindlimb fonksiyonu odaklanmıştır. Dahası, ALS TDI tarafından geliştirilen protokol parezi (hafif veya kısmi felç), ilerlemesi ve felç şiddetinin başlangıç ​​tarafsız bir değerlendirme sağlar ve hastalığın ilerlemesinde ilaca bağlı değişiklikleri belirlemek için yeterli hassastır.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>B6SJLTgN(SOD1G93A)1Gur strain</td>
			<td>The Jackson Laboratory, Bar Harbor, ME</td>
			<td>002726</td>
			<td>Strain of mice used in this study</td>
		</tr>
		<tr>
			<td>Breeders</td>
			<td>Biomedical Research Models, Inc., Worcester, MA</td>
			<td></td>
			<td>Colony maintainance</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Navigator OHAUS, Parsippany, NJ</td>
			<td>Model N14120</td>
			<td>Used to weigh mice</td>
		</tr>
		<tr>
			<td>Nutra-GelH</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>#S4798</td>
			<td>Wet food provided to sick mice</td>
		</tr>
		<tr>
			<td>Irradiated Lab Animal Diet</td>
			<td>Harlan, Indianapolis, IN</td>
			<td>2918-111914M</td>
			<td>Chow diet</td>
		</tr>
		<tr>
			<td>Lab-grade Sani-chips</td>
			<td>Harlan Teklad, Indianapolis, IN</td>
			<td>7090</td>
			<td>Bedding</td>
		</tr>
		<tr>
			<td>JMPH&#160;</td>
			<td>SAS Institute, Inc., SAS Campus Drive, Cary, NC</td>
			<td>&#160;v10.0.2</td>
			<td>Statistical software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft, One Microsoft Way, Redmond, WA</td>
			<td>Excel 2013 (v 15.0)</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad software Inc., La Jolla, CA</td>
			<td>v5.4</td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Mouse Huts</td>
			<td>Bio-Serv, Flemington, NJ</td>
			<td>K3272</td>
			<td>For environmental enrichment</td>
		</tr>
	</tbody>
</table>

a quick phenotypic neurological scoring system for evaluating disease progression in the sod1-g93a mouse model of als

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

2014 değerlendirildi sırasında 90 erkek ve 94 dişi olmayan tedavi SOD1-G93A fareler için nörolojik puanı verileri ALS TDI okudu. Sonuçlar kadınlara oranla NS 1 de yaşamlarının büyük bir oranda kanıtladığı gibi erkek fareler genellikle, dişi farelere göre daha agresif bir hastalık ilerlemesini sahip olduğunu göstermektedir. NS 2 ve NS 3 cinsiyetler arasında neredeyse eşit sıklıkta görülür. O &quot;normal&quot; bir fenotip olarak kabul edilir, çünkü NS 0 analize dahil edilmiştir. NS 4 de ...

Watch this Scientific Journal Video about A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS at JoVE.com

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

ALS SOD1-G93A Fare Modeli hastalığın ilerlemesine değerlendirilmesi için Hızlı Fenotipik Nörolojik Puanlama Sistemi

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

a-quick-phenotypic-neurological-scoring-system-for-evaluating-disease

Research

JoVE Journal

Medicine

19.2K Görüntüleme.  ALS Therapy Development Institute. This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

Video: A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Parkinson Hastalığının tek taraflı lezyonlanmıştır 6-OHDA Mouse Model Geliştirme

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Tıp

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

İnsan Nöromusküler Sistem Eğitim Transkraniyal Manyetik Uyarım kullanımı

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Roman Bitkisel Tedaviler Değerlendirmek için Sepsis Hayvan Modeli kullanımı

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrofik Lateral Skleroz için Fare Modeli (ALS) Klinik Test ve Omurilik Kaldırma

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Fare Akciğer Entübasyonun Basit Bir Yöntem

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pankreas Kanseri İlerleme Bioluminescent Ortotopik Model

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

Bir Geçerliliği Endoskopik Puanlama Sisteminin Kullanılması Kolit ve Kolorektal Tümörlerin Şiddetini Değerlendirmek için Farelerde esnek Kolonoskopi

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Fare Modelinde Kuruluş ve İYE ve CAUTI Karakterizasyonu

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Farelerde Kalp Kapak Hastalıkları Kapsamlı Fenotipik Karakterizasyonu için ekokardiyografik Yaklaşımlar ve Protokoller

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Bir Fare Modelinde Seyrek Sistometri Uygulamasının Değerlendirilmesi

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...