Bu çalışma, Kanada Sağlık Araştırma Enstitüsü (CIHR) Townsend, Aile, ve Jack Brown ve Aile Alzheimer Araştırma Vakfı (WAS) tarafından desteklenmiştir. WS Kanada Araştırma Başkanı Alzheimer Hastalığı sahibi. PTTL Doğal Bilimler ve Mühendislik Araştırma Danışma ve Sağlık Araştırma Bursu için Michael Smith Vakfı tarafından desteklenmektedir.

1. Fare beyinleri fiksasyonu ve cryosectioning <ol><li> Transgenik farelerin dekapitasyon ve çıkarılan beyin tarafından takip karbondioksit gazı kullanarak kurban. </li><li> Beynin merkezinde boydan boya disseke edilir. Beynin yarısı, biyokimyasal analiz için kuru buz dondurulmuş flaş olacaktır. Diğer 4% 4 paraformaldehid (PFA) en az 48 saat süreyle sular altında olacak ° C </li><li> En az 48 saat% 4 PFA fiksasyonu sonra% 30 sakaroz çözeltisini, hemi beyin direkt olarak aktarılabiliyor ve 4 ° C Noktada, hemi beyin sakaroz çözeltisini yüzen olmalıdır. Hemi beyin genellikle 48 saat gerektiren dibe battığı zaman, cryosectioning OCT gömülü olması için hazırdır. </li><li> Cryosectioning önce, OCT topuzu üzerinde ince bir tabaka montaj ve -20 ° C&#39;de dondurmak için izin Hemi beyin ve yerin geri kalanından beyincik dondurulmuş OCT katman üzerine çıkarın. OCT ile hemi beyin hemen kapağı ve yavaş yavaş -20 ° C&#39;de dondurmak için izin Beyin opak dönüşmüştür OCT gömme sonra kesitli olmak için hazırdır. </li><li> Her bölümün kalınlığı 30 mikron ayarlayın. D&#39;Olomos çözümü ile bir kabın içine her dilim toplayın. </li></ol> 2. Nöritik plaklar İmmünohistokimya prosedürü (ücretsiz kayan bölümleri) <ol><li> 15 dakika boyunca% 88 formik asit her bölüm davranın. Formik asit tedavisinden sonra, bölüm geçici olarak opak dönecek ve büzüşmeli görünebilir. </li><li> Tx-PBS (% 0.3 PBS içinde Triton X-100) ile 5 dakika X 3 nazik sallayarak bir tabak ve yıkama her bir bölümü çıkarın. </li><li> Blok Endojen Peroksit% 0,5 Tx-PBS, RT H 2 O 2 ile 30 dakika nazik sallayarak engelliyor. </li><li> Tx-PBS bölümü ile 10 dakika X 3 nazik sallayarak yıkayın. </li><li> Nazik sallayarak ile 1 saat oda sıcaklığında Tx-PBS içinde çözünmüş% 5 yağsız yağsız süt Blok bölümü. </li><li> Tx-PBS 4% 5 süt içeren seyreltilmiş primer antikor bölümler (4G8 biotinlenmiş, 1:500-1:2000, Signet) ° C, nazik sallayarak bir gecede. Inkübe </li><li> Tx-PBS bölümü ile 10 dakika X 3 nazik sallayarak yıkayın. </li><li> ABC çözüm üretici protokolü (Elit ABC, vektör Laboratuvarları,) hazırlayın. ABC karışımı bölümleri nazik sallayarak 30 dakika inkübe edin. </li><li> Tx-PBS bölümü ile 10 dakika X 3 nazik sallayarak yıkayın. </li><li> 3,3 &#39;-diaminobenzidin tetrahydrochloride (DAB) (Vektör Laboratories) aşağıdaki üretici protokol hazırlayın. Bölümleri DAB karışımı için 5-10 dakika inkübe edin. Bu noktada, kahverengimsi bir renk, her bölüm geliştirmek gözlemlemek olacaktır. </li><li> Tx-PBS bölümü ile 10 dakika X 3 nazik sallayarak yıkayın. </li><li> Serbest yüzen beyin bölümleri doku yapışması ile yardımcı olmak için, jelatin kaplı slaytlar üzerine monte edilir. Bölüm sonra hava, ksilen bir dizi ile takas takip ve Entellen içinde monte edilmiş kurutulmuş. </li><li> Plaklar, 40X büyütmeli ışık mikroskobu altında görüntülendi ve dilim her fare için başına ortalama plak sayımı değerlendirerek sayısal. </li></ol> 3. Thioflavin S nöritik plaklar tespit prosedürü boyama <ol><li> Fare fedakarlık ve beyin ekstraksiyon prosedürü gibi bölümlerde 1.1 1.5 gösterilir. </li><li> Mount 4 5 hemi beyin bir cam slayt üzerine bölümler ve tamamen boyamadan önce kurumaya izin verin. </li><li> 1 dakika boyunca% 70 Etanol slaytlar yıkayın. </li><li> 1 dakika boyunca% 80 Etanol slaytlar yıkayın. </li><li> Filtrelenmiş (0.2 mikron filtre) thioflavin S solüsyonu (% 80 Etanol% 1) slayt 15 dakika boyunca inkübe edin. (Thioflavin G çözüm, her kullanımdan önce filtre olması gerekir). Not: thioflavin S ışıktan korumak ve lekeli slaytlar ışıktan korumak. </li><li> 1 dakika boyunca% 80 Etanol slaytlar yıkayın. </li><li> Slaytlar% 70 Etanol 1 dakika ile yıkayın. </li><li> Iki kez distile su ile yıkayın. </li><li> Sulu montaj medya ve slaytlar açık bir oje ile sızdırmazlık lamel tarafından en az 2 saat, karanlıkta kurumasını bekleyin Mount slaytlar. Boyanan kesitler 4 karanlıkta saklanmalıdır ° C </li><li> Yeşil floresan lekeli plaklar floresan microscropy ile görüntülendi. Boyama zamanla kaybolacak, çünkü bir hafta içinde slaytlar analiz edin. </li></ol> 4. Temsilcisi Sonuçlar Anti-epileptik ilaç ve nöritik plak oluşumunu engellediğini duygudurum valproik asit (VPA) etkinliğini son çalışmada, yukarıda açıklanan immün kullanılan ve thioflavin G, AD model teşkil eden farelere (3) nöritik plaklar tanımlamak için prosedürler boyama. Şekil 1 anti-Aβ kullanarak biotinlenmiş 4G8 ile boyandı ve ABC yöntemi ile tespit edilen 9 aylık tipik bir eski APP23 transgenik fare, temsil eder. Nöritik plaklar antikor ile açıkça etiketlenir ve beyaz oklarla gösterilir. Thioflavin G boyama kullanarak, 2 aylık APP23xPS tra nöritik plak birikimi tespitnsgenic fareler (Şekil 2). Thioflavin G bağlı Aβ içeren nöritik plaklar floresan mikroskopi (beyaz oklar ile gösterilir) altında yeşil görünür. VPA tedavisi nöritik plak oluşumunu önemli derecede (3) azalır olduğunu gösterdi. Bir başka çalışmada, moleküler bağlantı altta yatan hipoksi ve AD patogenezinde okudu. Yine, hem de anti-Aβ 4G8 immünhistokimya boyama ve thioflavin G boyama kullanarak, biz o hipoksi nöritik plaklar (4) içeren Aβ oluşumunu kolaylaştırmıştır bulundu. <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Şekil 1"/> Şekil 1. AD transgenik farelerin nöritik plak oluşumu immünohistokimyasal analizi, 9 ay yaşta APP23 fareler sakrifiye edildi ve diseke edildi, sabit ve kesitli. Hipokampal nöritik plaklar Aβ bir anti-antikor 4G8 kullanılarak tespit edildi ve ABC ve DAB yöntemler kullanılarak geliştirilmiştir. Plaklar 40X ile ışık mikroskobu ile görüntülendi. Beyaz oklar Aβ nöritik plaklar işaret. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Şekil 2"/> Şekil 2. AD modellenen fareler nöritik plaklar Thioflavin G boyama eski 8 hafta APP23xPS45 çift transgenik fareler sakrifiye edildi ve diseke edildi, sabit ve kesitli. Hipokampal nöritik veba thioflavin S floresan boyama kullanılarak doğrulandı ve 40X büyütmede mikroskobu ile görüntülendi. Beyaz oklar thioflavin G lekeli nöritik plaklar göstermektedir.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

Tüm deneyler, British Columbia Hayvan Bakım ve Kullanım komitesi ve CIHR kurallar Üniversitesi ile uygun olarak yapılmıştır.

Biotinlenmiş etiketli 4G8 antikor lekeleri AD nöritik plaklar kullanan İmmünohistokimya özgüllük ile modellenen fareler. Boyanması sonucu kantifikasyon ve farklı tedavi grupları arasında plak yükü karşılaştırılması sağlar. Sonucunu etkileyebilecek bazı önemli adımlar vardır. Hemi beyinleri kafatası çıkarma öncesinde perfüze olmadığından, bakım hemi beyin hasarları önlemek amacıyla çıkarma işlemi sırasında kullanılan olmalıdır. Dahası, hemi beyin pasif perfüze olduğundan, biz 4 en az 48 saat% 4 PFA kuluçka tavsiye ° C% 30 sakaroz çözeltisini dalarken önce. Alternatif olarak, transcardial perfüzyon beyin ayıklanması önce yapılması olabilir. Beyin düzgün sabit ise, bir lastik dokusu olmalıdır. Durumda beynin düzgün sabit değildir, bölümler kolayca D&#39;Olomos çözümü veya boyama işlemleri sırasında yırtılabilir. 4G8 monoklonal antikor reaktif amino asit artıkları Aβ, 17-24 ve çekirdek dizisi (VFFAE) epitop tanıdı. 4G8 fare türlerinin türetilmiş olduğundan, daha yüksek bir arka plan boyama vermek eğilimindedir. Bu nedenle, biz, arka plan boyama en aza indirerek hususlar ulaşmak için gerçek bir sinyal ile belirtilen seyreltme bölümleri inkübe seçti. 4G8 antikor kullanarak nöritik plaklar tespiti için immün yanı sıra, bir thioflavin S boyama yöntemi de plaklar tanımlamak için kullanılır. Thioflavin S homojen bir boya karışımı dehydrothiotoluidine metilasyonu sülfonik asit ile elde edilen sonuçlar. Thioflavin G-seçici olmayan beta amiloid oligomerler gibi protein sayfalık içeriği, bağlar. Bağlayıcı üzerine, thioflavin emisyon spektrum karakteristik bir mavi vardiya uğrar. Thioflavin G monomerik formları bağlanma Tersine mavi bir değişimi ortaya çıkarmak ve floresan mikroskop ile tespit edilememiştir. Floresan yoğunluğu amiloid mevduat küçük miktarlarda iyi bir görüntüleme sağlar Thioflavin G boyama amiloid için ekran hızlı bir alternatif sunuyor. Ancak, bir dezavantajı thioflavin G boyama hakkında özgüllük eksikliğidir. Fibrinoids, hiyalin, keratin, vb gibi içeren kapsamlı beta yaprak, dahil olmak üzere birçok diğer doku parçaları, bu boya için oldukça yakınlık var.

<table border="1"><tbody><tr><th> Reaktif ve ekipman Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Paraformaldehit </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma-Aldrich </td><td> PVP40-100G </td></tr><tr><td> Sakaroz </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Etilen glikol </td><td> Sigma-Aldrich </td><td> 324.558-2L </td></tr><tr><td> Doku-Tek Ekim Bileşik </td><td> Sakura </td><td> 4583 </td></tr><tr><td> % 88 formik asit </td><td> Fisher Scientific </td><td> A118P-500 </td></tr><tr><td> Triton X-100 </td><td> ICN Biyomedikal </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> Sigma-Aldrich </td><td> H-1009 </td></tr><tr><td> Biotin 4G8 Antikor Etiketli </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kiti </td><td> Vektör </td><td> PK-6100 </td></tr><tr><td> Imm PACT DAB </td><td> Vektör </td><td> SK-4105 </td></tr><tr><td> Ksilen </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM bilim </td><td> 65037-71 </td></tr><tr><td> SuperFrost * Plus Mikroskop Slaytlar </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Kriyostat </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier mikrotom bıçağı </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavin S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

Alzheimer hastalığı (AH), demansa neden olan en yaygın nörodejeneratif bir hastalıktır. Nöritik plak oluşumu, Alzheimer hastalığının patolojik işaretlerinden biridir. Nöritik plaklar merkezi bileşeni β-secretase ve γ-secretase beta-amiloid prekürsör protein (APP) sıralı proteolitik bölünme türetilmiş bir küçük ipliksi protein olarak adlandırılan amiloid β proteini (Aβ) 1,. Amiloid hipotezi AD patoloji 2, altta yatan toksik mekanizma olarak plaklar Aγ içeren gerektirir . Nöritik plak varlığı postmortem analiz AD tanısını doğrular. AD patogenezinde Aγ nörobiyoloji anlayışımızı ilerletmek için, AD-ilgili, insan APP genlerindeki mutasyonlar ifade çeşitli fare suşlarının üretildi. Hastalığın şiddetine bağlı olarak, bu farelerde farklı yaşlarda nöritik plaklar gelişecektir. Bu fareler APP işleme yolu ve nöritik plak oluşumunu etkileyebilecek patogenezi ve ilaç geliştirme eğitimi için paha biçilmez bir araç olarak hizmet vermektedir. Bu protokol, AD modeli farelerde nöritik plaklar belirli tespiti için bir immünohistokimyasal yöntem kullanırlar. ABC ve DAB yöntem ve floresan algılama kullanarak thiofalvin G boyama yöntemi kullanılarak immünohistokimyasal algılama: Biz özellikle yarım beyin, paraformaldehid fiksasyon, cryosectioning, ve AD transgenik farelerin nevrotik plaklar tespit etmek için iki yöntem açılan hazırlık tartışacağız.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

AD patolojik özelliklerinden biri Amiloid β proteini pozitif nöritik plakların oluşumu. Immünohistokimyasal algılama thioflavin G boyama yöntemi kullanılarak ABC ve DAB yöntem ve floresan tespiti: Bu protokolde, transgenik AD modeli fareler nöritik plaklar tespit etmek için iki yöntem açıklanmaktadır.

Nöritik plaklar Alzheimer Hastalığı Fare Modeli Algılama

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

36.5K Görüntüleme.  The University of British Columbia. AD patolojik özelliklerinden biri Amiloid β proteini pozitif nöritik plakların oluşumu. Immünohistokimyasal algılama thioflavin G boyama yöntemi kullanılarak ABC ve DAB yöntem ve floresan tespiti: Bu protokolde, transgenik AD modeli fareler nöritik plaklar tespit etmek için iki yöntem açıklanmaktadır.

Video: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Β-amiloid Oligomerik hazırlanması 1-42</subHipokampal Dilimleri Sinaptik Plastisite değer düşüklüğü> İndüksiyon

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Nörobilim

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Amiloid beta-Protein, Alzheimer hastalığının etkeni Aptamers Seçimi

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Fare Omurilik İki foton Mikroskop kullanarak in vivo Görüntüleme

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Alzheimer Hastalığı Model Fareler Öğrenme ve Hafıza Açıkları Morris Su Labirent Testi

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Endojen NADH Floresans iki-foton Imaging tarafından Fare Serebral Korteks olarak Microregional Hipoksi Algılama

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Kalıcı ve yaygın Nöronal İletim için Yenidoğan Fare Beyin intraserooroventriküler Viral Enjeksiyon

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Fare Hippocampus içine Oligomerik Amiloid-beta Stereotaksik İnfüzyon

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Genelleştirilmiş psikofizyolojik etkileşim (PPI) analiz bellek bağlantısı'nda bireyler, Alzheimer hastalığı genetik Risk ile ilgili

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Enzim bazlı biyosensörlerin kullanılması Alzheimer Fare Modellerinde Tonik ve Fazik glutamat Ölçmek için

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Yenidoğan hipoksik-iskemik beyin hasarı, fare modeli neurobehavioral değerlendirmesi

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...