The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The authors also acknowledge Emily Clough for her excellent administrative support.

Tüm hayvan çalışmaları Dartmouth Tıp Geisel Okulu&#39;nda Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından incelenen ve onaylanan protokoller kullanır. 1. Fare Modeli <ol><li> TMEV Bağlı Demyelinizan Hastalığının indüksiyon <ol><li> rahat bir çalışma alanı için raftan 4- 6 haftalık dişi SJL / jhan fareler içeren kafesleri taşıyın. Klinik ve histolojik hastalığın bireysel değerlendirilmesi için izin vermek için (bir kulak etiketi veya kulak yumruk gibi) fareler işaretleyin. </li><li> 29-gauge insülin şırınga ve iğne içine PBS; TMEV enfekte stok (PFU 2 x 10 6 plak oluşturucu birim) 30 ul çizin. </li><li> Anestezi gazı makinesini hazırlayın: prosedürün süresince oksijen ve izofluran yeterli miktarda varlığını sağlamak için sistemi kontrol ediniz. </li><li> 1 l / dk akış ölçer açın. indüksiyon odasına hayvan koyun ve üst mühür. va açma% 3,5 porizer ve yatarak kadar hayvan izleyin. </li><li> odasından gelen hayvan çıkarın ve yeterli anestezi sağlamak için Kapkaççı kısma fare test edin. Güçlü bir tutam yanıt eksikliği yeterli anestezi gösterir. </li><li> % 70 izopropil alkol ile enjeksiyon bölgesini temizleyin. </li><li> Serbest enjeksiyon (Şekil 1) sağ serebral hemisferde içine TMEV bulaşmasını stokunun 30 ul enjekte edilir. Enjeksiyon yeri yaklaşık yarım göz ve kulak hattı arasında sadece orta hat kapalı. </li><li> (- 5 dk genellikle 3) holding kafes kez tamamen uyanık ve hareketli fare dönün. </li><li> Hastalığın gelişme bir hızla bağlı olarak, 3 ila 6 ay TMEV enfeksiyonundan sonra kan kaybından ya da kardiyak perfüzyon fareler öldürülür. </li></ol></li></ol> 2. rotarod Analizi <ol><li> rotarod Aparatı <ol><li> önce TMEV enfeksiyon deney fareleri onları tanımak içinMakine ve normal bazal denge koordinasyon ve motor kontrolünü değerlendirmek için. </li><li> -5 Günlerde adaptasyon protokolü sonrası enfeksiyon (; yani, 5 gün TMEV enfeksiyondan önce dpi) başlatın. </li><li> fareler onları ortama uyum sağlamak amacıyla, en az 30 dakika önce rotarod için test için test odasına gelmesini bekleyin. </li><li> Rotarod ünitesi ve bilgisayar hem de takılı ve birbirlerine (Şekil 2) bağlı olduğundan emin olun. </li><li> Tablo 1 &#39;de tarif edildiği gibi, -5 dpi eğitim protokolü parametrelerle rotarod önceden ayarlanmıştır. </li><li> tarih ve kimlik bilgileri ile çalışma dosyasını kaydedin. </li><li> Rotarod bitişik bir tabloya raftan test edilecek kadro içeren kafesi hareket ettirin. Fareler genellikle 4 mangaları test edilmektedir. </li><li> operatörden uzak bakan, kuyruk tarafından fare Pick up ve çubuk üzerine yerleştirin. Dördüncü fare aracılığıyla saniye tekrarlayın. Fare fal halindetüm fareler konumuna gelene kadar ls veya atlar, rotarod üzerindeki şeritte geri yerleştirin. Herhangi fareler operatörü yüz etrafında dönerse görmezden. </li><li> Bütün fareler yükledikten sonra, denemeyi başlatmak için &quot;Enter&quot; tuşuna basın. sayaçlarını otomatik olarak başlayacak gözlemlemek ve her kulvar için ekranda dakika (rpm) dönmeler. <ol><li> Her bir hayvan, çubuk düştükçe, hayvan çubuk üzerinde kalan düşme zaman çubuğun hızı, hem de süreyi kaydedin. çubuk geçen hayvan çubuk montaj düştü kadar dönmeye devam edecektir. </li></ol></li><li> Tüm fareler düşmüş sonra, çubuk herhangi bir dışkı boli ve idrar çıkarmak için bir doku kullanın. İdrar ve dışkı maddesinin bulunması, kavrama farelerin çubuğu yeteneğini etkileyebilir. <ol><li> 3 dakikalık bir dinlenmeden sonra, farelere üçüncü deneme sonra ikinci ve vermek. Tek deneme başına maksimum zaman 240 saniyedir. Her test gündüz 3 çalışmaların toplam yönetme. </li></ol></li><li> evlerine kafesine fareler dönün ve tekrar rafa onları geri. Deneysel oturumun sonunda, makinenin kapalı tüm dışkı çıkarmak için sabun ve su ile rotarod temizleyin. </li><li> etanol% 70 ile temiz taban plakasını silin. dezenfekte etmek için klor dioksit ile tüm makineyi aşağı püskürtün. </li><li> 4 -, - 3, 2 - ve - günlerde 1 PI, Tablo 1 &#39;de tarif edildiği gibi, uygun bir eğitim protokolü parametrelerle rotarod önceden ayarlanmış ve tekrar 2.1.12 2.1.2 adımları tekrarlayın. </li><li> Bazal önlemleri alındıktan sonra, TMEV ile fareleri enfekte. 6 gün pi iyileşme süresini bekleyin. </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> Protokol </td><td rowspan="2"> Test günü </td><td rowspan="2"> Sıklık </td><td rowspan="2"> Başlangıç hızı (rpm) </td><td rowspan="2"> maksimumHızı (rpm) </td><td> Hızlanma </td><td> denemeler </td><td> ITI </td></tr><tr><td> (rpm / sn) </td><td> (N x sn) </td><td> (dakika) </td></tr><tr><td rowspan="4"> Eğitim </td><td> - 5 dpi </td><td> 1 gün </td><td> 1 </td><td> 12 </td><td> 01/03 </td><td> 3x240 sn </td><td> 3 </td></tr><tr><td> - 4 dpi </td><td> 1 gün </td><td> 1 </td><td> 13 </td><td> 01/03 </td><td> 3x240 sn </td><td> 3 </td></tr><tr><td> - 3 dpi </td><td> 1 gün </td><td> 1 </td><td> 14 </td><td> 01/03 </td><td> 3x240 sn </td><td> 3 </td></tr><tr><td> - 2; - 1 dpi </td><td> 1 gün </td><td> 5 </td><td> 40 </td><td> 01/03 </td><td> 3x240 sn </td><td> 3 </td></tr><tr><td rowspan="2"> Deneysel </td><td> +7 Itibaren 50 dpi </td><td> 2 hafta </td><td>5 </td><td> 40 </td><td> 30/05 </td><td> 3x240 sn </td><td> 3 </td></tr><tr><td> +51 Itibaren 150 dpi </td><td> 1 hafta </td><td> 5 </td><td> 40 </td><td> 30/05 </td><td> 3x240 sn </td><td> 3 </td></tr></tbody></table> Tablo 1: Eğitim ve Deneysel Protokolleri rotarod Parametreleri. <ol start="2"><li> Rotarod Deneysel Protokol <ol><li> Tablo açıklandığı gibi 7 dpi üzerinde, uygun deneysel protokol parametreleri ile rotarod önceden ayarlanmış 1. Tekrarlayın 2.1.10 için 2.1.2 adımları. </li><li> Deneme içerisinde 3. sonunda, her fare tartmak ve bilgi formunda vücut ağırlığının not edin. Temiz ve adımlar 2.1.11 ve 2.1.12 göre rotarod dezenfekte. </li><li> yukarıda tarif edildiği gibi, 6 hafta boyunca haftada iki defa fareler test edin. Aynı experimenta ile haftada bir kez fareler, 8,9 (fareler büyük olasılıkla bir plato safhasına ulaşan aldığınız) testi 6 hafta sonral protokolü. Gerçek test protokolü belirli bir hastalık seyrine bağlı olarak, 150 gün ortalama sürer. </li></ol></li><li> Nörolojik Fonksiyonel İndeksi <ol><li> Bir tablo dosyasına ham veri ihracat ve sonuçları analiz eder. </li><li> Çalışma süresi (Şekil 3A) olarak Ekspres veriler: bu normal çalışma süresi artı pasif rotasyon süresi eksi dönme gecikme süresi (Tablo 2) 10. Günde üç çalışmanın ortalama çalışma süresini hesaplayın. </li><li> Nörolojik fonksiyonel indeksi (Şekil 3B NFI) gibi verileri ifade eder. <ol><li> Her bir fare temel performans eşiğini hesaplayabilir. Bazal performans eşiği + 15 + 45 pi 6,7 güne çalışan tüm zamanların ortalaması olarak belirlenmiştir. </li><li> Belirli bir fare 6,7 bazal performansı eşik bölü üç en son ortalama çalışma süreleri ortalaması olarak UOE hesaplayın &lt;/ Sup&gt;. NOT: 135 sn, o fare için NFI olarak günün + 72 fare, + 76 için çalışan kez test ve + 79 pi 55 sn, 45 sn ve 50 sn ve aynı fare için temel zaman ise 79 dpi üzerinde [(45 + 50 + 55) / 3] / 135 veya 0.37 olacak. </li></ol></li><li> Tek bir deney için bir nüfus değerine göre NFI verileri ayarlayın: (Şekil 3C adjNFI) ayarlanmış bir NFI gibi veri ifade eder. <ol><li> Bu belirli bir günde plasebo ile tedavi edilen grup ile elde edilen ortalama NFI göre NFI değerine bölünmesiyle adjNFI hesaplayın. </li></ol></li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> dönem </td><td> Tanım </td></tr><tr><td> Normal çalışma süresi </td><td> Fare aktif dönen çubuk üzerinde çalışan harcadığını toplam süre, yani gecikme düşmeye. </td></tr><tr><td> Pasif rotasyon zamanı </td><td> birmontaj zaman fare pasif rotasyon modunda çubuk üzerinde kalmıştır. </td></tr><tr><td> Rotasyon gecikme süresi </td><td> Fare pasif rotasyon modu sırasında çubuk üzerinde kalan süre </td></tr><tr><td> Pasif dönüş modu </td><td> Fare çubuk kapmak ve ambulate zorunda kalmadan döndüğünde. </td></tr><tr><td> Toplam oturum süresi </td><td> toplam süre fare oturumu sırasında dönen çubuk üzerinde kalır. </td></tr><tr><td> Baseline performans </td><td> Ön hasar motoru performansı asgari performans eşiğini belirlemek için değerlendirilir. </td></tr><tr><td> Nörolojik fonksiyon indeksi (NFI) </td><td> Her fare motor performansı karşılaştırır klinik endeksi, yani kendi performansını en üst için herhangi bir zamanda, çalışma süresi. </td></tr><tr><td> Düzeltilmiş nörolojik fonksiyon indeksi (adjNFI) </td><td> Bir normalizasyon işlemi bir nüfus değeriyle NFI verileri değiştirmek uygulandığındaTek deney. </td></tr><tr><td> Nüfus değeri </td><td> Belirli bir günde sahte tedavi edilen grupta elde edilen ortalama NFI değeri. </td></tr></tbody></table> Tablo 2: Nörolojik Değer Düşüklüğü belirlememize Benimsenen rotarod Parametrelerinin Tanımları.

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The authors have nothing to disclose.

Bazı kısıtlamalara rağmen, rotarod performans testi, motor fonksiyon ve disfonksiyonu TMEV-İER yanı sıra farelerde sakatlık ilerlemesi üzerine farmakolojik müdahalelerin etkisini değerlendirmek için önemli bir araç temsil eder. Rotarod testi ilk kemirgenler 11 nörolojik defisit ölçmek için bir araç olarak 1957 yılında tanımlanmıştır. Kemirgenler dönen hızını artırma ile dönen bir çubuk üzerinde yürümek ve yere düşmesini önlemek için denemek z...

Theiler ensefalomiyelit virüsü Sıçangil (TMEV) sürekli sıçangil merkezi sinir sistemi (CNS) enfekte eden bir nörotropik tek iplikli RNA virüsüdür. duyarlı farelerde, TMEV enfeksiyonu TMEV kaynaklı demiyelinizan hastalıkları (TMEV-IDD) olarak bilinen bir immün aracılı, kronik progresif demiyelinasyon hastalığı, neden olur. Farelerin Deneysel enfeksiyon, multipl skleroz (MS), progresif formları görülene benzeyen bir hastalık seyri alır. akut dönemde ve kronik fazda: TMEV-ATT iki farklı aşamadan ile karakterizedir. Akut faz hafif, genellikle subklinik ensefalit 1,2 olduğunu. İkinci, kronik faz enfeksiyondan sonra yaklaşık bir ay başlayan, demiyelinizasyon, enflamasyon ve aksonal hasar 1,2 ile karakterize yavaş ilerleyen sakatlık oluşmaktadır. farelerde gözlenen zayıflık spastisite ve bazen şiddetli tonik spazmlar ile ilişkilidir. Şu anda medicatio olduğundanns hastalarda ilerici engeli iyileştirecek, araştırmacılar özellikle hastalığın ilerlemesi üzerinde hastalık modifiye ilaçların etkilerini izlemek için optimal bir hayvan modeli temsil TMEV-İER tarafından ilgi vardır. Bununla birlikte, farelerde ve MS hastalarında, sakatlık ilerlemesinin izlenmesi, uzun zaman süreleri boyunca sürekli klinik gözlem gerektirir. Farelerde, sakatlık ilerlemesi için uzun vadeli izleme rotarod performans testi ile yapılabilir. Rotarod performans testi gibi kemirgenler koordinasyon, denge ve yorgunluk gibi motorlu ilişkili işlevleri değerlendiren bir standart davranış testtir. Fareler sürekli ivme altında dönen bir dönüm çubuk, kendi dengesini korumak için var; Bu çubuk kaydedilir arasındaki süre gecikme düşmesi. Nörolojik disfonksiyon Hayvanlar kontroller sürece dönen çubuk üzerinde duramayan ve dönme hızı aştığında normalde düşüyorlar onlarınMotor kapasitesi. hayvanlar var daha nörolojik bozukluk, er onlar çubuk düşmek ve kısa zaman gecikme olduğunu. Geleneksel görsel skorlama sistemleri üzerinden rotarod testinin avantajı bir hedef oluşturur ki, ölçülebilir değişken zamanlı gecikme-sonuçta tedaviler ve deneysel prosedürler 3 etkilerini ölçmek için istatistiksel analizler için kullanılabilir. Dartmouth Nöroimmünoloji (Loni) Laboratuvarı, fareler için makine ile onları tanımak için ve normal &quot;taban&quot; denge koordinasyon ve motor kontrolünü 4 değerlendirmek için onlar önce TMEV enfeksiyonuna test edilir bir adaptasyon protokolü, tabi tutulur, 5. temel kurulur ve fareler TMEV bulaşmış sonra, birkaç aylık bir süre boyunca bir veya iki kez bir hafta test edilir. Gerçek test protokolü dolayısıyla bir değerlendirmesini sağlayan, 150 gün ortalama sürerdemiyelinizan hastalık tüm kurs boyunca denge, koordinasyon ve motor kontrol düşüş. yüzlerce TMEV-IDD ve plasebo ile tedavi edilen fareler, Dartmouth nörolojik fonksiyon bozukluğu için şu ana kadar test edilmiştir. Bu fareler, çeşitli bağışıklık tedavileri almış, ancak hiçbir farmakolojik madde sekel progresyonu 6,7 iyileştirmede etkili olduğu tespit edilmiştir. Bu makale ve ilgili protokol TMEV-IDD fareler tarafından görüntülenen ilerleyici nörolojik bozukluğu karakterize açıklanmıştır. Özel olarak, iletişim kuralı rotarod testi kullanılarak TMEV-IDD farelerde nörolojik engeli çalışmak için genellikle uygun olduğuna inanılan özel test parametrelerinin öneriler vardır. Bu prosedür değerlendirmek için bir temel (1) MS gibi ilerleyici nörolojik durumların tedavisinde amaçlayan tedaviler test etmek için progresif MS ve (2) yararlılığını bu fare modelinde alaka sağlar. Açıktır ki,Rotarod testi ve mevcut optimize test parametreleri ve protokol TMEV-IDD fare modelinde ilerleyici nörolojik engeli tespit sadece yararlı değildir, ama aynı zamanda, nörodejeneratif hastalıkların ve diğer virüs kaynaklı ve / veya genetik fare modellerinde bozuklukları ortaya çıkarılmasında yararlıdırlar.

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measuring progressive neurological disability in a mouse model of multiple sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

Bu temsili deneyin amacı Daniels (DA) zorlanma ve TMEV Bean suşu tarafından uyarılan nörolojik özürlülük karşılaştırmaktır. Bu çalışmanın amaçları doğrultusunda, 32 kadın SJL farelerinin bir grup (n = 16), TMEV ile intrakranial DA suşu (n = 16) veya fasulye suşu da enfekte edildi ve klinik bulgular, zaman içinde izlenmiştir. 20 farelerin ek bir grup sahte muamele edilmiştir (yani, tuzlu su çözeltisi enjekte edilmiştir intrakranial) ve sağlıklı ko...

Watch this Scientific Journal Video about Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis at JoVE.com

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

Multipl Skleroz Fare Modeli Progressive Nörolojik Engellilik Ölçümü

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

measuring-progressive-neurological-disability-mouse-model-multiple

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.9K Görüntüleme.  Geisel School of Medicine at Dartmouth. An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice. 

Video: Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Çoklu-fare nöroanatomik Manyetik Rezonans Görüntüleme

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Nörobilim

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Nörodejeneratif Hastalıkların bir Drosophila Modelinde Tırmanma Kusur Kantitatif Analiz

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Sinaptik vezikül endositoz kültürlü Hipokampal nöronlar içinde ölçme

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

İntraserebral enjeksiyon stratejileri kullanarak Zika virüs kaynaklı nörolojik bozukluklar için fare modelleri oluşturma: embriyonik, Yenidoğan ve yetişkin

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Birden çok kemirgen beyin aynı anda Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Nörolojik Hastalıklarda Lipidomik ve Transkriptomik

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Multipl skleroz bireyler için kapsamlı otopsi programı

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson Hastalığının Genetik Sıçan Modelinde Yürüme Analizi için RatWalker Sisteminin Uygulanması

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multipl Sklerozun Lysolecithin Rat Modelinde Miyelin İçeriğinin In Vivo Ölçümü için Pozitron Emisyon Tomografi Görüntüleme

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

İnsan Kaynaklı Pluripotent Kök Hücrelerde Multipl Mitokondriyal Parametrelerin ve Nöral ve Glial Türevlerinin Akış Sitometrik Analizi

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...