AK Velux Vakfı tarafından desteklenen Oppenheim Vakfı ve RH tarafından desteklenmektedir. Biz histolojik destek için teknik ve K. Buder I. Krumbein teşekkür ve TIMM boyama üzerine teknik danışmanlık için J. Goldschmidt (Nörobiyoloji, Magdeburg, Almanya için Leibniz Enstitüsü).

Tüm hayvan müdahaleler Laboratuvar Hayvanları Hayvan Bakımı ve Kullanımı Avrupa Sözleşmesi ve Oftalmik ve Vizyon Araştırma Hayvanların Kullanım ARVO Bildirimi çerçevesinde yapılmaktadır. Tüm deneyler yerel etik kurul tarafından onaylanmıştır. Farelerde ON hasarın işlem başka bir yerde, 9 anlatılmıştır. 1.. Intravitreal Manganez Enjeksiyon <ol><li> Önce MR asistan yardımı ile taramak için Mn 2 + enjeksiyon 24 saat gerçekleştirin. (Steril PBS içinde 420-450 mg / kg vücut ağırlığı),% 5, kloral hidrat çözeltisinin intraperitonal enjeksiyon ile hayvanlar anestezisi. Ek topikal anestezi için, önce göz delinme için kornea sıvı conjuncain bir damla (0.4% oksibuprokain hidroklorür) uygulanır. 15 nmol Mn enjekte etmek için, 2 + göz için, örneğin, 132 LH 2 &#39;de 1 M MnCI2 stok solüsyonu 1 L seyreltilmesi ile, bir 7.5 mM MnCI2 çözelti hazırlamak/ Sub&gt; O. 34 G küçük bir hub çıkarılabilir iğne (RN iğne) bağlı bir 5 ul Hamilton şırınga içine nihai çözümün yükü 5 ul. </li><li> Sağ gözü ile başlarken, bir binoküler mikroskop altında ve hafifçe açık sol-taraflı fareyi getirin ve sol elin baş ve işaret parmağı arasında sağ göz düzeltmek. Sağ elinizle şırınga Pick up ve ucuna yakın iğne tutun. Göz ampul atravmatik delik için, dikkatli böylece skleral damarları koruyucu, limbusta yaklaşık 1 mm uzak infero-zamansal çevresi de vitreus içine iğne takın. </li><li> Hamilton şırınga ölçeğini kontrol ederken sonra, yardımcı yavaşça 2 ul toplam hacim uygulanır. Bu işlem sırasında, mikroskop altında optimal iğne yerleştirme izlemek ve sıvı lens veya dökülmeye patlamasına kaçının. Statik olarak ek bir 30 saniye boyunca yerleştirilen iğne tutun, daha sonra sıvı en aza indirmek için yavaş yavaş gerienjeksiyon yerinde sızmasına neden olunacaktır. </li><li> İşlem boyunca, özel bakım göz basıncı önlemek için alınmalıdır. Aynı şekilde, göz ampul delmek için sert ya da çok sayıda girişimleri önlemek. Mn 2 + RGCs ve ON boyunca taşıma içine çekmesi zaten 15 nmol MnCl 2 doymuş olduğundan, bu muğlak enjeksiyon hacimleri ile sinyal değişimleri en aza indirir. Görsel çıkıntının ikili sinyal artışı için, sol göz için enjeksiyon prosedürü tekrarlayın. </li><li> Uygulama ofloksasin-ihtiva eden (3 mg / ml) göz damlası ve göz enfeksiyonları prosedür ve göz kurumasını önlemek için bir kez sonra pantenol içeren merhem içinde uygulanmaktadır. MR tarama başlangıcına kadar, normal konut koşulları altında kendi kafeslerine fareler dönün. </li></ol> MRI için 2. Hayvan hazırlanması <ol><li> % 2 /% 98 izofluran / oksijen gaz karışımının verilmesi ile fare anestezisi. Neredeyse yatay, bükülmemiş pozisyonda bir fare tutucu üzerine fareyi monte edin. InsDaha sonra MR tarayıcı içinde ayarlanır MR bobin içine ert o. Uygun sistemleri tarafından solunum ve kalp atışlarının izlenmesi. Teknik ayrıntılar için, Herrmann ve arkadaşları 20 bkz. </li><li> MRI sırasında, entegre bir boru ile fare kafası tutucuya bağlanmış bir buharlaştıncı ile, başlangıçta% 1.5 /% 98.5 izofluran / oksijen gaz karışımının sürekli olarak üfleme ile tedarik anestezi. Tarama sırasında, kaydedilen önemli bir parametre (örneğin, dakikada 40 nefes stabil bir solunum hızı için amaç) göre anestezi derinlik ayarlayın. Bir ısıtma aygıtı kullanılarak fare karın bölgesinde yer alan bir ısı algılayıcı tarafından ölçülen, 35 ve 37 ° C arasında kararlı vücut yüzey sıcaklığı. Teknik ayrıntılar için, Herrmann ve arkadaşları 17 bkz. </li><li> Taramadan sonra, sahibinden fareyi kurtarmak ve anestezi kurtarma hızlandırmak için saf oksijen ile kaynağı. Ayrıca, kullanarak stabil vücut sıcaklığı tutmakBir kırmızı ışık ısı kaynağı. </li></ol> 3.. MRG Protokol <ol><li> Protokol 35 mm × 38 mm çaplı görüş etkili bir alan ile özel, SNR-verimli, küçük hayvan bobin (doğrusal polarize Litz bobin) ile donatılmış 3 Tesla için doğrulanır. Iletim-alım modunda bobin çalıştırın. </li><li> Son konumunda hayvan ile, frekans analiz yardımıyla bobinin ayar ve maç ayarlayın. El ile görüntü homojenliği ve kalitesini optimize etmek verici referans gerilim ve şim akımları ayarlayın. </li><li> Yine T planlaması için sagital ve transversal görünümünde 0.5 mm × 2 mm × 0,5 mm çözünürlük ile 1 ağırlıklı 2D TSE görüntü kazanır. Planlama MR taramaları kullanarak, koronal ölçüm doğrultusunda MEMR görüntüler elde, sağ-sol yönünde faz kodlama ile hayvanın kafasına paralel olacak şekilde döndürülmüş. Edinimi süreyi en aza indirmek için, ayarlanabilir bir görüş dikdörtgen alanı kullanıngerçek kafa boyutları. Faz kodlama yönünde görüş 93.8% dikdörtgen alanını kullanarak, baz matris 256, görüş alanını 14.08 mm × 50.65 mm × 54 mm ve 0.11 mm-128 dilim: Aşağıdaki parametreleri kullanarak bir şımarık 3D FLASH dizisi (VIBE 3D) İstihdam dilim çözünürlük ile dilim kalınlığı% 61 olarak ayarlanır. </li><li> 0.18 mm x 0.21 mm x 0.21 mm etkin çözünürlüğü (0.1 mm × 0,1 mm × 0.09 mm interpolasyon), eko zamanı T E = 6.51 msn&#39;den sağlayarak, 512 × 480 × 128 ile son görüntüler oluşturmak için düzlem interpolasyon etkinleştirin, tekrarlama zamanı T R = 16 msn, bant genişliği = 160 Hz / px flip açısı = 22 °. Yaklaşık olarak 30 dakika toplam satın alma süresi (T) elde etmek için, iki ortalamalar ve üç set uygulayın. </li></ol> 4. MRG Veri Analizi <ol><li> Yazılım syngo fastView kullanarak verileri analiz edin. Nicel sinyal artış, o tanımlanan bölgeleri seçmekf 2D düzlemsel MR kayıtları ilgi ve gelişmiş yapısı (SI MEMRI), doku arka plan (SI backgr) ve gürültü (SD N) standart sapma sinyal şiddetlerini (SI) belirler. Gerektiğinde, LGN ve SC yapılar için nöro-anatomik yönlendirmeyi kolaylaştırmak için bir fare beyin atlası kullanın. : Aşağıdaki formül kullanılarak kontrast-gürültü oranını (CNR) hesaplayın </li><li> CNR = (SI MEMRI - SI backgr) / SD N </li><li> Her numune için ortalama CNR hesaplanması için üç ardışık görüntüleri niceliğini. Iki taraflı olarak enjekte edilen hayvanlarda, bağımsız bir şekilde, her bir yarım küre analiz. </li><li> , Yatay koronal ve sagital görüntülerin tasviri için, özgün 3D MR veri setinden Multiplanar rekonstrüksiyon hesaplamak. Bu işlenmiş görüntüler kantitatif analiz için tavsiye edilmez. Retino bir animasyonlu 3D rekonstrüksiyon (maksimum yoğunluğu projeksiyonları, MIPs) oluşturmak için-Tectal projeksiyon, bir anjiyografi sonrası işleme yazılım modülünü kullanabilirsiniz. </li></ol> 5. Mn 2 + Autometallography (TIMM Boyama) <ol><li> MEMRI aşağıdaki Mn + 2 takip beyin yapılarının TIMM boyanması için, 15-150 nmol Mn + 2 ivit 24 saat önce görüntüleme için bir doz enjekte edilir. </li><li> MR taradıktan sonra, PBS içinde 30 ml buz soğukluğunda% 0,325 Na 2 S (pH 7.4) ile hayvanların serpmek. Dondurulmuş bölümünde retinae ve dondurma örnekleri incelemek. </li><li> Bir kriyotomu 15 mikron kalınlığında sıralı, ekvator bölümleri kesmek. </li><li> Angenstein et al 22&#39;ye göre sabitleştirici ve donmaya karşı koruma sağlamak yokluğunda TIMM boyama 21 yapın. </li></ol> 6.. İstatistiksel Analiz Post hoc ANOVA ardından tek karşılaştırmalar için Student t-testi kullanılarak istatistiksel analizler gerçekleştirmek. Veriler ortalama ± standart hatası olarak sunuldu. Bağımsız N sayılardırHer deney için ayrı ayrı verilir. (***, 0,001 P ≤,, **, 0.01 P ≤ *, P ≤ 0.05) 0.05 ≤ P ulaşan sonuçlar istatistiksel olarak anlamlı kabul edilir.

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Görsel sistemin MEMRI naif ve patolojik şartlar altında işlevselliğini değerlendirmek için geleneksel nörobiyolojik teknikleri uzanır. Apart izole MSS fiber sistemin bütünlüğü içine benzersiz bir fikir sağlamasının, MEMRI kolayca görsel algı için belirli bir paradigmanın derhal sonuçlarını araştırmak, davranış örneğin testleri, Optometri ve görsel tabanlı su görevleri ile takviye edilebilir. Ayrıca elektrofizyolojik ve in vivo fonksiyonel görsel tanımlanması ile hi...

Onun olumlu nöro-anatomik yapısına dayalı kemirgen görme sistemi nöro 1 ya da pro-rejeneratif etkileri 2,3 arabuluculuk farmakolojik bileşikler ve kabiliyetlerini değerlendirmek için benzersiz olanaklar sunuyor. Ayrıca, son zamanlarda presinaptik iskele proteini Bassoon 4 eksik fareler için açıklandığı gibi, fare mutantlarının işlevsel ve nöro-anatomik özelliklerinden çalışmalar sağlar. Bundan başka, yardımcı araçları bir geniş spektrumlu elektroretinografi ve davranışsal testler ve içsel sinyallerinin optik görüntüleme kortikal yeniden düzenlemelerin belirlenmesi ile, örneğin, retinal ganglion hücre (RGC) ve RGC akson sayı hem de RGC aktivitesi, ilave bir özelliği elde edilir. Lazer mikroskopi son teknik gelişmeler, optik sinirin (ON) ve beynin bütün montaj örneklerinde derin doku floresan görüntüleme ile RGH yenilenme yerinde görselleştirme sağlar. Bu histolojik araştırma olarakical yaklaşım, hafif levha floresan mikroskobu ile birlikte tetrahidrofuran merkezli doku temizleme deafferented ON ve optik sisteminde 5 içine yeniden girmek tek liflerin çözünürlüğünü verir. Bu tür teknikler çözünürlük ve büyüme modelleri belirlenmesinde üstün olabilecek iken, onlar özellikle uzun süreli yenilenme sürecini değerlendirmek için istenen bireysel gelişim olayları, tekrarlayıcı ve boyuna analizlerini mümkün değil. Kontrastlı MR fareler ve sıçanlarda 6,7 Retino-tectal projeksiyon minimal invaziv görselleştirme için istihdam edilmiştir. Bu retinal hücrelere paramanyetik iyonlar (örneğin, Mn + 2) doğrudan teslimat göz içi ile elde edilebilir. Bir kalsiyum analog olarak, Mn 2 + voltaj kapılı kalsiyum kanalları aracılığıyla RGH somata içine dahil edilir ve aktif sağlam ON ve optik sistemin aksonal hücre iskeletinin boyunca taşınır. O beyin çekirdekleri birikir iseo 10,11 oluşabilir rağmen görsel projeksiyon, lateral genikülat çekirdeği (LGN) ve üstün kollikulusu (SC), yani primer görsel korteks içine transsinaptik yayılımı, 8,9 önemsiz görünür. MR dizileme altında, paramanyetik Mn 2 + esas t 1 spin-örgü durulma zamanı 12 kısaltarak MR kontrast artırmaktadır. Böyle Mn 2 + MRI (MEMRI) başarıyla ON yaralanma 13,14 sonrası aksonal rejenerasyon ve dejenerasyon değerlendirmesi dahil sıçanların çeşitli nöro-anatomik ve fonksiyonel çalışmalarda, tatbik edilmiştir gelişmiş, Retino-tectal projeksiyon 15 hassas anatomik haritalama , hem de farmakolojik tedavi sonrası 16 aksonal taşıma özelliklerinin belirlenmesi gibi. Mn + 2 alımı ve nakil nöronal, yanı sıra geliştirilmiş MRI protokollerin doz, toksisite ve kinetik olarak son refinements Transjenik ilgili çalışmalara uygulanmasını genişletmiştiryaygın klinik pratikte 17 kullanılan 3 Tesla tarayıcıları kullanarak fareler 9. Burada, biz fare Retino-tectal projeksiyon in vivo görüntüleme boyuna uygun bir MEMRI protokol mevcut ve naif ve çeşitli nörodejenerasyon koşullarda Mn 2 + bağımlı sinyal donanımı değerlendirerek uygulanabilirliğini örneklemek. Bizim protokol genellikle özel hayvan tarayıcılar daha erişilebilir bir ılımlı 3 T manyetik alan MR veri toplama belirli bir önem vermektedir. Naif farelerde, sistem özel sinyal yoğunluğu büyük ölçüde yeniden üretilebilir ve intravitreal (ivit) 2 Mn + uygulama sonrası artış olmak ne kadar göstermektedir. Kantitatif, görsel projeksiyon boyunca Mn 2 + yayılım (3 ve 26 aylık farelerin arasında ölçülen) normal yaşlanma sürecinin bağımsız oluşur ve güçlendirme karanlığa görsel uyarı ve adaptasyon dirençlidir. Buna karşılık, Mn <stalamik ve orta beyin merkezlerinde&gt; 2 + zenginleştirme ezilme yaralanması 18 ON akut yanı sıra nfkb1 knock-out farelerde spontan apoptotik RGH ölümden ve dejenerasyon 19 ON acı (p50 KO) takip azalır. Bu nedenle, geleneksel histolojik analiz için genişleme, bireysel hayvanların uzunlamasına MEMRI analizi nörodejeneratif süreçleri tek kinetiği profil sağlar. Bu farmakolojik veya genetik müdahalelerle ilgili nörokorunma ve aksonal rejenerasyon üzerine çalışmaları için yararlı olmalıdır.

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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

Kemirgen görme sistemi görsel kortekse talamik ve orta beyin merkezleri ve postsinaptik projeksiyonlar girmek için optik siniri oluşturmak retina ganglion hücreleri ve aksonlar kapsar. Onun farklı anatomik yapısı ve kullanışlı erişilebilirlik dayanarak, nöronal hayatta, aksonal rejenerasyon ve sinaptik plastisite üzerindeki çalışmalar için tercih edilen bir yapı haline gelmiştir. MR görüntülemede son gelişmeler manganez aracılı kontrast (MEMRI) kullanarak bu projeksiyon Retino-tectal kısmının in vivo görselleştirme sağladı. Burada, (200 mikron) 3 ortak çözünürlükleri 3 Tesla tarayıcı kullanılarak elde edilebileceği farelerde görsel projeksiyonu, gösterilmesi için bir MEMRI protokol mevcut. Biz 15 nmol MnCl 2, tek bir dozaj intravitreal enjeksiyonu 24 saat içinde sağlam çıkıntının doymuş bir donanıma yol gösterilmektedir. Retina hariç, sinyal yoğunluğundaki değişiklikler vardır gözetmeden,çakıştı görsel uyarılma ya da fizyolojik yaşlanma göçük. Biz daha akut optik sinir hasarına yanıt olarak aksonal dejenerasyon izlemek boylamasına için bu tekniği uygulamak, bir paradigma lezyon yerinde hangi Mn 2 + ulaşım tamamen tutuklamalar tarafından. Bunun aksine, aktif Mn + 2 taşıma canlılığı, sayı ve akson lif elektriksel aktivitenin kantitatif orantılıdır. Böyle bir analiz için, görsel, projeksiyonlar dahil duyusal spontan atrofi gösteren transgenik fare modelinde (NF-KB p50 KO) görsel yol boyunca Mn 2 + taşıma kinetik örneklemektedir. Bu farelerde, MEMRI azalır fakat böylece NF-KB mutasyonlar ile yapısal ve / veya fonksiyonel bozukluğu belirtileri ortaya, yabani tip farelere kıyasla Mn + 2 taşıma gecikmeli olmadığını gösterir. Özetle, MEMRI uygun in vivo analizlerde köprü ve characterizati için mortem histoloji gönderebilirsinir lifi bütünlüğü ve aktivite üzerinde. Bu aksonal dejenerasyon ve rejenerasyon, ve hakiki veya uyarılabilir fenotipleri için mutant farelerin soruşturmalarla ilgili uzunlamasına çalışmalar için son derece yararlıdır.

Doğru bir görsel çıkıntının canlılık ve işlevselliği değerlendirmek için, bu görüntüleme tekniğin becerisi vitreus ve RGCs tarafından alımı için toksik olmayan bir Mn + 2 dozajın kesin uygulamaya dayanır. Bu ana varsayım tabaka belirli bir Mn + 2 uptake autometallography (TIMM boyama) 21 ile gösterilen, Şekil 1 &#39;de test edilir. Retina bölümleri kontrol olarak 15 nmol ve 150 nmol ya da Mn 2 + ya da PBS ivit uygulamadan 2...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Bu video naif fare görsel projeksiyon kontrastlı MR görüntüleme ve akut optik sinir ezilme yaralanma ve kronik optik sinir dejenerasyonu ile ilişkili optik sinir dejenerasyonu tekrarlayan ve uzunlamasına in vivo çalışmalar için, bir klinik 3 T tarayıcı kullanarak, bir yöntemi göstermektedir knock-out fareler (p50 KO).

Farelerde kontrastlı MR ile optik sinir lifi Dürüstlük İn vivo

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K Görüntüleme.  Jena University Hospital.  Bu video naif fare görsel projeksiyon kontrastlı MR görüntüleme ve akut optik sinir ezilme yaralanma ve kronik optik sinir dejenerasyonu ile ilişkili optik sinir dejenerasyonu tekrarlayan ve uzunlamasına in vivo çalışmalar için, bir klinik 3 T tarayıcı kullanarak, bir yöntemi göstermektedir knock-out fareler (p50 KO). 

Video: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Memeli MSS Optik Sinir transeksiyonu sonra Deneysel İşlemler Yöntemleri

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Nörobilim

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

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

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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

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

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Yüksek çözünürlük<em&gt; İn Vivo</em&gt; 3T Manyetik Rezonans Görüntüleme kullanma İnsan hipokampal alanlar için Manuel Segmentasyon Protokolü

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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

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

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

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

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

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

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Deneysel Optik Sinir demyelinizasyon bir Rat Modelinde Görsel Uyarılmış Potansiyel Kayıt

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

İnsan Subcortex yüksek çözünürlüklü yapısal Manyetik Rezonans Görüntüleme<I&gt; İn Vivo</I&gt; Ve Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

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 ...

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

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

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

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.