Bu çalışma Ulusal Nörolojik Bozukluklar Enstitüsü tarafından desteklenen ve İnme Alzheimer Hastalığı Araştırma finanse Yaşlanma üzerine (CMT kadar) NS081333, Alzheimer Derneği hibe NIRG-10-171721 ve (UH kadar) Ruh Sağlığı Hibe MH096702 Ulusal Enstitüsü ve Ulusal Enstitüsü hibe edildi (YYJ ve JB) Columbia Üniversitesi, pilot hibe AG008702 de merkezi.

Not: Tüm hayvan deneyleri için, kurumsal ve bakım ve laboratuvar hayvanlarının kullanımı için ulusal kurallar takip edildi. 1. Cerrahi Cerrahi Aletler ve Çözümler hazırlayın <ol><li> Tüm paslanmaz çelik cerrahi aletleri otoklavlayın. </li><li> Steril moleküler sınıf iyonu giderilmiş damıtma suyu ile 200 derece mutlak etanol seyrelterek% 70 etanol hazırlayın. </li><li> Hamilton şırınga 29 G iğne takın. 1 dakika boyunca tekrar tekrar hazırlanması ve çıkarma nanopure su ile Hamilton şırınga ve iğne içini temizleyin. % 70 etanol ile bu işlemi tekrarlayın. <ol><li> Hamilton şırınga pistonu ve iğneyi çıkartın. Hava Laminer akış kaput O / N parçaları kurulayın. </li><li> Kullanımdan önce, 30 dakika boyunca ultraviyole ışığıyla iğne ve şırınga ışın tedavisi. </li></ol></li><li> % 0.9 hacim konsantrasyonuna nihai ağırlığa moleküler sınıf suda NaCl çözülmesiyle tuzlu su çözeltisi hazırlayın. Sterilizasyonu0.2 um gözenekli filtre içinden filtre edilerek çözelti ze. </li></ol> 2. oligomerik Aβ 1-42 hazırlayın <ol><li> Fa &#39;ve diğerleri 13 yayınında tarif edildiği gibi Monomerize tam olarak 5 mM DMSO içinde rekombinan insan Aβ 1-42 yeniden süspanse edin. </li><li> Ameliyat öncesi gününde 100 mcM steril (1x) PBS ile 5 mM Aβ 1-42 sulandırmak. </li><li> Ezilerek de çözüm karıştırın. </li><li> 4 ˚ C de 12 saat Aβ 1-42 çözelti inkübe edin. Not: Bu tür PBS DMSO seyreltilerek veya karıştırılmış olarak kontrol çözümleri / Aβ ​​1-42 peptid Aβ 1-42 ile aynı şekilde hazırlanmalıdır tersine çevirir. </li></ol> Enjeksiyon Koordinatları belirlemek 3. <ol><li> Tam anterior-posterior (AP), medial-lateral (ML) belirlemek için yetişkin fare beyin atlası kullanın ve dorsal-ventral (DV) ilgi 14 beyin bölgesi için koordine eder. Hayırte: Biz bregmadan aşağıdaki koordinatlarla hedef olarak hipokampusun dentat girus aldı tekniği göstermek için: AP, -2,00 mm; ML, 1.3 mm ±; DV, -2.2 mm. AP değeri önceki eksi işareti o bregma 2.00 mm arka olduğunu gösterir. ML değeri önceki ± işaret merkezinden sol ve sağ yönünü gösterir. Son olarak, DV önceki eksi işareti beynin ventral yüzeyinden hareket gösterir. </li></ol> 4. Stereotaksik Çerçevesi Ayarı <ol><li> Cerrahi yüz maskesi, temiz laboratuvar önlüğü ve steril cerrahi eldiven giyin. </li><li> Stereotaksik alet ve% 70 etanol ile fare adaptörü silin. </li><li> Tezgahın üzerine steril cerrahi örtü aşağı yatırın. </li><li> Steril cerrahi asmak üstünde fare adaptörü ile stereotaksik aleti koyun. </li><li> % 70 etanol ile fare ısıtma yastığı silin. Stereotaksik çerçeve yatağın üzerinde ısıtma yastığı yerleştirin. Genel amaçlı Labora kullanınFare adaptör yatağın üzerinde ısıtma pedi aşağı teybe edici etiketleme bant. <ol><li> Üreticinin talimatlarına göre ılık su sirkülasyon pompası ısıtma yastığı takın. </li><li> Sıcak su sirkülasyon pompası açın ve 37 ° C sıcaklığı ayarlamak ve sıcaklığına ulaşana kadar bekleyin. </li></ol></li><li> Üreticinin talimatlarına göre motorlu dikey enjeksiyon miline vidalanarak stereotaksik çerçeve üzerine 29 G iğne ile 50 ul Hamilton şırınga yapıştırın. </li><li> Boncuk sterilizatör açın ve üreticinin önceden ayarlanmış sıcaklığa ulaşana kadar bekleyin. Not: Bu hayvanlar arasında paslanmaz çelik aletleri sterilize etmek için kullanılır. Bu enstrümanlar sterilizasyon boncuklar ile temas 20 saniye sürer. </li><li> Sıcak plaka açın ve 42 ° C olarak ayarlayın ve üstüne temiz bir boş kafes yerleştirin. </li><li> Hamilton şırınga stereotaksik çerçeve üzerinde sabit iken motorlu stereotaksik i kullanınnjector Aβ 1-42 çözüm hazırlamak için. Not: şırıngaya çözümün fazla 4 ul hazırlayın ve daha sonra enjekte seviyesini ayarlamak için stereotaksik enjektör kullanın. Üreticinin talimatlarına uygun olarak bir enjektör. </li></ol> 5. Hayvan hazırlanması <ol><li> Ölçek tartmak kullanarak fare ağırlığını belirler. </li><li> Periton boşluğu içine (ip) enjekte edilerek 100 mg / kg ketamin ve 10 mg / kg ksilazin ketamin / ksilazin kokteyli ile fare anestezisi. Ayak tutam refleks kaybı anestezi derinliği izleyin. </li><li> Fare sedasyon sonra saç kesme almak ve kafatası üzerindeki deriyi açığa başını tıraş. </li><li> Stereotaksik yatağın üstüne ısıtma yastığı üstüne fare yerleştirin. </li><li> Diş görevlisi içeride kesici diş ağzı açık ve yerleştirmek için spatula kullanın. Fare yüzünde burun sabitleyin. Nazikçe ve çok sıkı değil aşağı kelepçe. </li><li> Posikafa sabitlemek için kulak yolunun içine ikili kulak çubuklardan yon. Bu kafatası vurur kadar her enine hareket ettirin ve sonra kilitlemek için vidayı çevirin. <ol><li> Baş hafifçe kafasına aşağı itmek için parmağınızı kullanın güvenli olduğundan emin olmak için. Baş düzgün güvenli ise dışarı vermeyecektir veya basıldığında hareket ettirin. </li></ol></li><li> Nemli tutmak için gözlere göz kremi ya da göz damlası bir damla uygulayın. Gözleri cerrahi işlem boyunca nemli olduğundan emin olun. </li><li> Cerrahi alan% 70 etanol ve ardından kafatası üzerinde cilt için betadin çözüm uygulamak için steril pamuklu çubuklarla kullanmak dezenfekte etmek için. Alternatif betadin ve 2 kez daha temizliği% 70 etanol gerçekleştirin. </li></ol> 6. Cerrahi ve İnfüzyon <ol><li> Kafa derisinin orta hatta bir 2-3 mm&#39;lik kesi yapmak için bir neşter kullanın ve sonra kafatası, dönüm noktası sagital sütür, bregma ve lambda maruz 1.0-1.5 cm kesi hattı genişletmek için düz ince makas kullanınstereotaksik koordinatlar dayanmaktadır s. Ayrı cildi korumak için mikro kelepçeleri kullanın. Not: Bregma ve lambda pozisyonları fare beyin atlası &quot;Stereotaksik Koordinatlarda Fare Beyin&quot; 14 açıklanmıştır. </li><li> Bir pamuklu çubukla atın steril tuzlu su içinde ıslatın ve kafatasını temizlemek için kullanabilirsiniz. Sonra, kafatası kurulamak için temiz, kuru bir pamuklu bir bez kullanın. </li><li> Bregma üzerinde bir nokta işaretlemek için steril ince kalem kullanın. Iğne ucu sadece bregma üzerinde nokta değecek şekilde Hamilton şırınga konumlandırmak için stereotaksik mikromanipülatör kullanın. <ol><li> DV koordinat kaydedin. </li><li> Kafatasının AP ekseni seviyesi iğne ucu lambda noktasını değecek şekilde posterior Hamilton şırınga taşımak olup olmadığını kontrol etmek için. </li><li> DV konumunu kaydedin. 0.5 mm içindedir DV bregma ve lambda koordinatları emin olun. Not: amaç bregma ve lambda arasındaki DV farkı en aza indirmektir. </li></ol></li><li> M kullanicromanipulator Bregma noktaya geri Hamilton şırınga iğne ucu dönün. </li><li> Başlangıç ​​AP pozisyonu kaydedin. </li><li> Koordinat başlayan AP&#39;den 2.00 mm çıkarılarak biten AP konumunu hesaplayın. </li><li> Koordinat son AP&#39;ye Hamilton şırınga iğnesi yeniden konumlandırmak için mikromanipülatör kullanın. </li><li> Geçerli ML koordinat kaydedin. </li><li> Sol hesaplayın ve sağ ML biten ekleme veya koordinat ML başlatılmasını 1,3 mm çıkartarak koordine eder. </li><li> Biten ML koordinat ya Hamilton şırınga iğne taşımak için mikromanipülatör kullanın. <ol><li> Her iki biten ML koordinatlara kafatası yüzeyine iğne ucu dokunun ve DV koordinatlarını kaydetmek ve değerler 0,5 mm içinde olduğundan emin olun. Not: Gol ML koordinatları biten ikisi arasındaki farkı DV en aza indirmektir. </li><li> Bir bitiş kafatası üzerinde yukarı 0.5-1 cm iğne çekin koordinat iken. Th bir nokta işaretlemek için steril ince kalem alınKafatasının e yüzeyi. Kafatasının karşı tarafta için bu adımı yineleyin. </li></ol></li><li> Yolumdan net Hamilton şırınga Salıncak. </li><li> Matkap 0,8 mm matkap kafasını takın. , Iki eliyle matkap atın istikrar için masanın yüzeyinde dirsek ayarlamak ve sol veya sağ ML ya koordinat şarpi nokta üzerinde biraz matkap kafasını yerleştirin. Kafatasında bir delik tanıtmak için kafatası yüzeye matkap ucu düşürmek, matkap etkinleştirin. Karşı tarafta için bu adımı yineleyin. Not: Bazı kanama yeni tanıtılan deliklerden oluşabilir. Daha sonra kanama varsa kan kurulamak için temiz ve kuru pamuklu bir bez kullanın. </li><li> Yeni tanıtılan ML delik ya üzerinde geri pozisyona Hamilton şırınga taşıyın. Sadece kafatası geçmiş Hamilton iğne indirin. Bu noktada beyin delmek için dikkatli olun. Emin olmak için iğne sur kafatası işaret parmağınızı almak ve yavaşça yapmak için kafatası karşı iğne itin geçtie İğne deliği çıkmak değildir. </li><li> Başlangıç ​​DV koordinat kaydedin. </li><li> Biten DV başlangıç ​​DV koordinat 2.2 mm çıkartarak koordinat hesaplayın. </li><li> Koordinat aşağı biten DV iğneyi indirin. </li><li> 0.5 ul / dk&#39;lık bir oranda dentat girus içinde Aβ 1-42 4 ul pompalamak için stereotaksik enjektör pompası devreye sokun. </li><li> Infüzyon tamamlandıktan sonra, iğne enjeksiyon üzerinden çözeltinin geri akışını en aza indirmek için ilave 1 dakika boyunca yerinde kalması sağlar. </li><li> Beynin diğer tarafına iğne taşımak ve 6,13-6,18 adımları tekrarlayın. </li></ol> 7. Hayvan Kaldırma ve Ameliyat Sonrası Bakım <ol><li> Kulak çubukları ve yüz / burun nöbet sökün. Cihazdan fareyi çıkarın. </li><li> Kafa derisi kapatın çekin ve daha sonra sütür ile yara mühür bir öğrenci, standart model forseps kullanın. </li><li> Hidrasyon için IP üzerinden fare içine steril serum fizyolojik 1 ml enjekte edilir. </li><li> IçindeJECT buprenorfin (0.1 mg / kg) deri altından ağrıyı gidermek için. </li><li> O uyanana kadar 42 ° C sıcak plaka üstüne set temiz boş bir kafes içinde fare koruyun, sonra bol gıda ve su ile konut kafesine geri. </li><li> Deri altı enjeksiyon yoluyla ağrı kesici 3 gün boyunca her 12 saat buprenorfin yönetme. </li></ol> 8. Dikiş Kaldırma <ol><li> IP tarafından 100 mg / kg ketamin ve 10 mg / kg xlyazine ketamin / xylazine kokteyl ile fare anestezisi. Not: Bu, 7-10 gün ameliyat sonra yapılır. </li><li> Dikiş kaldırmak için bir öğrenci, standart model forseps ve düz ince makas kullanın. </li><li> Hidrasyon için IP üzerinden fare içine steril serum fizyolojik 1 ml enjekte edilir. </li><li> O uyanana kadar 42 ° C sıcak plaka üstüne ayarlanmış bir temiz boş bir kafes içinde fare koruyun ve ardından bol gıda ve su ile konut kafesine geri. </li></ol> 9. Hayvan Kurban ve Doku İşleme <ol><li> Fare ve p uyutmak% 4 paraformaldehid 15 ile erfuse beyin kaldırmak ve 16 cryosectioning için işlemek. Not: hayvan kurban zamanlaması deneyci bağlıdır. Ancak, bizim çalışmalar için biz 7 ve 14 gün ameliyat sonrası seçti. </li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+BDNF+supports+myelin+protein+synthesis+after+cuprizone-induced+demyelination.">Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34 (24), 8186-8196 (2014).</li><li>Thakker-Varia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropeptide+VGF+is+reduced+in+human+bipolar+postmortem+brain+and+contributes+to+some+of+the+behavioral+and+molecular+effects+of+lithium.">The neuropeptide VGF is reduced in human bipolar postmortem brain and contributes to some of the behavioral and molecular effects of lithium.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (28), 9368-9380 (2010).</li><li>Greferath, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+cholinergic+forebrain+neurons+and+improved+spatial+learning+in+p75+knockout+mice.">Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice.</a> The European journal of neuroscience. 12 (3), 885-893 (2000).</li><li>Brashear, H. R., Zaborszky, L., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+GABAergic+and+cholinergic+neurons+in+the+rat+diagonal+band.">Distribution of GABAergic and cholinergic neurons in the rat diagonal band.</a> Neuroscience. 17 (2), 439-451 (1986).</li><li>Clarke, 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=Cholinergic+innervation+of+the+rat+dentate+gyrus:+an+immunocytochemical+and+electron+microscopical+study.">Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study.</a> Brain research. 360 (1-2), 349-354 (1985).</li><li>Garcia-Osta, A., Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+mediates+memory+formation.">Amyloid beta mediates memory formation.</a> Learning &amp; memory. 16 (4), 267-272 (2009).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim.

1) aseptik teknik kullanır;: deneyci veya cerrah olmalıdır başarılı Aβ 1-42 enjeksiyon elde etmek için 2) doğru doğru koordinatlar ile çıkar beyin bölgesini belirlemek; 3) düzgün AP ve ML ekseninde tesviye beyni stereotaksik çerçeve fare sabitlemek mümkün; 4) hassas mikromanipülatör çalıştırmak için yeteneği var; 5) Uygun post-operatif bakım sağlamak. Bu önemli adımlar takip edilirse, fare gözlemlenebilir enfeksiyonu ile ameliyat hayatta gerekir. <e...

Amiloid-beta (Aβ 1-42) oluşan amiloid lekesi yatakları, Alzheimer hastalığı (AD) patolojisinde önemli bir özelliğidir. Birçok çalışma nöron ölümünü, sinaptik distrofi, kayıp ve disfonksiyonu ortaya rekombinant oligomerik Aβ 1-42 o yüksek veya toksik seviyelere göstermiştir; yanı sıra, öğrenme ve hafıza açıkları 1-4 olarak. Etkilenen beyin bölgeleri hipokampus, korteks ve bazal ön beyin ve amigdala 5,6 gibi subkortikal yapılar yer alıyor. Bugüne kadar, AD Aβ 1-42 patoloji simüle girişiminde birden transgenik fare modelleri vardır. Zorlanma bağlı bu hayvanlar AD seçkin patolojik özelliklerini inceleyerek yararlı olduğunu kanıtlayabilir. Ne yazık ki, 2 transgenik kuşaklar, APP23 ve 5XFAD hariç olmak üzere, bu fareler, tam nöronal kayıp, MS önemli bir yönünü çoğaltma olmadı. Hatta APP23 ve 5XFAD gözlenen nöronal kayıp, nöronal ölüm glikozu ileved birkaç seçme bölgelere 7,8 kadar, ince yaşa bağımlı ve izole oldu. vahşi tip fare beyin oligomerik Aβ 1-42 doğrudan infüzyon amyloidopathy 1,9,10 nöronal ölüm yönü çoğaltır in vivo modelinde mükemmel içerir. Yaygın olarak kullanılan transgenik fare modellerinin aksine oligomerik Aβ 1-42 infüzyon modeli akut bir mekansal ve zamansal bir şekilde Aβ 1-42 seviyesini yükseltmektir için idealdir. Bu model için, vahşi tip fareler kullanılarak avantajı transgenik fare hatları tanıtılan mutasyon potansiyel dengeleme ya da yan etkileri ortadan kaldırır. Son çalışmalar, hipokampusa Aβ 1-42 toksik düzeylerini beslerken 1 hafta 1 içinde enjeksiyon bölgesinde çok yakın nöron ölümünü ortaya çıkarır göstermiştir. Dahası, Aβ 1-42 kolinerjik nöronların 11 bazal ön beyin için toksik gözlem ile tutarlı olduğunuhipokampus için projeler kolinerjik nöron (BFCN) nüfus etkin bir beyinde izole nöronal devrenin sınavları için izin farelerde, beta-amiloid infüzyon 1,10 izleyen 7-14 gün içinde% 20-50 azalır. Çoğunlukla, kontrol / araç ve oligomerik Aβ 1-42 çözümler hipokampus 12 dentat girus için ipsilateral BFCN projesi yana karşılaştırmalar, sol ve sağ hemisfer 1 arasında yapılacak izin beynin her iki tarafında enjekte edilebilir. Bu yazıda erişkin vahşi tip C57BL / 6J fareler için detaylı bir ameliyat ve enjeksiyon metodoloji sağlayacaktır. Bu fare suşu nedeniyle araştırma geniş kullanım seçilir. Teknik olarak, herhangi bir beyin bölgesi, ancak burada tekniği açıklamak için bir hedef olarak hipokampusun dentat girus kullanır, infüzyon için hedeflenebilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine HCl (100mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1049007</td>
			<td>100 mg ketamine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Xylazine (20mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>not available</td>
			<td>10 mg xylazine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Buprenex (0.3mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1217793</td>
			<td>0.1 mg buprenex per 1 kg animal</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>David Teplow/UCLA</td>
			<td>not available</td>
			<td>100 &#956;M; This amyloid was used in the paper</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>Bachem</td>
			<td>H-1368</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-80B</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>Scrambled A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-46B</td>
			<td>Can be used as control peptide for comparing A&#946;1-42</td>
		</tr>
		<tr>
			<td>NU4 Antibody (Oligomeric Amyloid Antibody)</td>
			<td>Gift from William Klein/Northwestern U.</td>
			<td>not available</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Anti-Amyloid Oligomeric Antibody&#160; (Polyclonal Rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB9234</td>
			<td>May be used in place of Nu4; needs to&#160; be tested by the end user</td>
		</tr>
		<tr>
			<td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td>
			<td>Biolegend</td>
			<td>sig-39320</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>ChAT Antibody (Polyclonal Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>DeadEnd Fluorometric TUNEL system</td>
			<td>Promega</td>
			<td>G3250</td>
			<td>Follow manufacturer&#39;s directions for use</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Use when coverslipping slides</td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome, LLC</td>
			<td>not available</td>
			<td>2% solution</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>For making 70% ethanol for sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Novex 10-20% Tricine gel</td>
			<td>Life Technologies</td>
			<td>EC6625BOX</td>
			<td>For separating A&#946;1-42</td>
		</tr>
		<tr>
			<td>Novex Tricine SDS Running Buffer (10X)</td>
			<td>Life Technologies</td>
			<td>LC1675</td>
			<td>For running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
			<td>Life Technologies</td>
			<td>LC3675</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>SuperSignal Western Blot Enhancer</td>
			<td>Thermo Scientific</td>
			<td>46640</td>
			<td>For enhancing A&#946;1-42 signal; follow manufacturer&#39;s protocol</td>
		</tr>
		<tr>
			<td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &#956;m</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10402088</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td>Electrophoresis aparatus for running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>GenTeal Lubricant Eye Gel</td>
			<td>Novartis</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores</td>
		</tr>
		<tr>
			<td>Refresh Optive Lubricant Eye Drops</td>
			<td>Allergan</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Stoelting</td>
			<td>50998</td>
			<td>For sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Round/Tapered Spatula&#160;</td>
			<td>VWR</td>
			<td>82027-490</td>
			<td>For opening animal mouth</td>
		</tr>
		<tr>
			<td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm)</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
			<td>Optional; For keeping scalp skin apart during injection</td>
		</tr>
		<tr>
			<td>Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td>For cutting scalp</td>
		</tr>
		<tr>
			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#11 Surgical Blade</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For making scalp incision</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td>For holding scalp closed during suturing</td>
		</tr>
		<tr>
			<td>Trimmer Combo Kit</td>
			<td>Kent Scientific</td>
			<td>CL9990-1201</td>
			<td>For shaving hair</td>
		</tr>
		<tr>
			<td>T/Pump Warm Water Recirculator&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>TP-700</td>
			<td>For warming animal during surgery</td>
		</tr>
		<tr>
			<td>Resusable Warmining Pad (5&#34; x 10&#34;)</td>
			<td>Kent Scientific&#160;</td>
			<td>TPZ-0510FEA</td>
			<td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td>
		</tr>
		<tr>
			<td>Cordless Micro Drill</td>
			<td>Stoelting</td>
			<td>58610</td>
			<td>Use 0.8mm steel burrs to drill holes in the skull</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument with Mouse &#38; Neonatal Rat Adaptor</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for Mouse Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>Can use this in place of Stoelting Cat. #51615</td>
		</tr>
		<tr>
			<td>Quintessential Stereotaxic Injector</td>
			<td>Stoelting</td>
			<td>53311</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Glass Bead Sterilizer</td>
			<td>Stoelting</td>
			<td>50287</td>
			<td>For sterilizing stainless steel instruments</td>
		</tr>
		<tr>
			<td>Sterile Surgical Drape (18&#34; x 26&#34;)</td>
			<td>Stoelting</td>
			<td>50981</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td>
			<td>Hamilton Company</td>
			<td>7637-01</td>
			<td>For brain injection; use different syringes for different solutions</td>
		</tr>
		<tr>
			<td>29 Gauge Needle, Small Hub RN NDL</td>
			<td>Hamilton Company</td>
			<td>7803-06</td>
			<td>For attaching to the Hamilton syringe for brain injection</td>
		</tr>
		<tr>
			<td>1 ml BD Tuberculin Syringes</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>30 Gauge Needle (0.5&#34;)</td>
			<td>VWR</td>
			<td>BD305106</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>Portable Electronic CS Series Scale (Ohaus)</td>
			<td>VWR</td>
			<td>65500-202</td>
			<td>For weighing animals to determine anesthesia dose</td>
		</tr>
		<tr>
			<td>Hot plate (Top Plate Dims. 7.25x7.25in)</td>
			<td>VWR</td>
			<td>47751-148</td>
			<td>For warming animals post-surgery</td>
		</tr>
		<tr>
			<td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td>
			<td>Covidien</td>
			<td>S1173</td>
			<td>For closing wound</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive (3M)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td>Optional: for aiding in wound closure; Use with suture.</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td>
			<td>Fisher scientific</td>
			<td>22-029-488</td>
			<td>For cleaning and drying surgical wound</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-550-15</td>
			<td>For collecting brain sections</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glass 24 X 50 mm</td>
			<td>VWR</td>
			<td>48393241</td>
			<td>For mounting microscope slides</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Syringe Filter 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>194-2520</td>
			<td>For sterilizing saline solution</td>
		</tr>
		<tr>
			<td>Sterile dual tip skin markers by Viscot Medical</td>
			<td>Medline</td>
			<td>VIS1422SRL91</td>
			<td>For marking coordinates on the skull</td>
		</tr>
	</tbody>
</table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

Alzheimer hastalığı, yaşlanma nüfusu etkileyen nörodejeneratif bir hastalıktır. Hastalığın önemli bir nöropatolojik özellik amiloid-beta aşırı üretim ve etkilenen bireylerin beyin bölgelerinde amiloid beta plakların birikmesidir. Yıllar boyunca bilim adamları, amiloid beta patolojiyi çoğaltmak girişiminde sayısız Alzheimer hastalığı fare modelleri yarattı. Ne yazık ki, fare modelleri, sadece seçici bir şekilde hastalık özelliklerini taklit eder. Nöronal ölüm, Alzheimer hastalığı olan hastaların beyinlerinde belirgin bir etki fark bu farelerde yoksundur. Nöronlar en zehirli olduğu kanıtlanmıştır amiloid-beta biçimleri - - Bu nedenle, biz ve diğerleri doğrudan amiloid-beta çözünür oligomerik türler beslerken bir yöntem istihdam var stereotaxically beyin içine. Bu yazıda seçkin bir beyin bölgesi amiloid beta seviyelerinin arttırılması, bu cerrahi teknik belgelemek için erkek C57BL / 6J fareler kullanmaktadır.Kolinerjik devre ile bağlıdır bazal ön ile birlikte bu beyin yapısı, hastalığın dejenerasyon alanlarından biri temsil ettiği infüzyon hedef hipokampusun dentat girus olduğunu. Broca diyagonal bant dikey bacak hücre ölümü ve kolinerjik nöron kaybı eşlik eden bir artış iken, stereotaksik infüzyon yoluyla dentat girus amiloid-beta yükseltilmesi sonucu, 1 hafta içinde, dentat girus nöron kaybına yükselerek Bazal önbeyin. Bu etkiler 2 haftaya kadar gözlenir. Elde ettiğimiz veriler mevcut amiloid beta infüzyon modeli, kısa vadede bölgeye özgü nöron ölümünü ele alternatif fare modeli sağladığını göstermektedir. Bu modelin avantajı amiloid-beta mekansal ve zamansal biçimde yüksek olmasıdır.

İnsan yeniden birleştirici oligomerik Aβ 1-42 hazırlanması için, bu yöntem, monomerlerin, dimerler, trimerler ve tetramerler (Şekil 1A) &#39;den oluşan çözünebilir oligomerik türler elde edilir. Bu düşük molekül ağırlıklı Aβ 1-42 türlerin değil, fibriller ve plaklar, nöronlar 1,4,9,17-19 en toksik olduğu çeşitli ortamlarda gösterilmiştir. Oligomerik Aβ 1-42 fare beyninde Aβ 1-42 nöron ölümünü indükleyen olup olm...

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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 ...

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Research

JoVE Journal

Neuroscience

17.9K Görüntüleme.  Columbia University Medical Center. 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.

Video: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Nörobilim

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Fare Omurilik Koşullu Gen Manipülasyon için bir Viral Vector Stereotaksik Enjeksiyon

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

Bir Açık kaynak Robotik Stereotaksik Enstrüman Bina

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNS içine Hedeflenen Madde Teslimat için Fare Mikrocerrahi İnfüzyon Tekniği<i&gt; yoluyla</i&gt; İç Karotis Arter

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Yenidoğan fare beyin Striatal hücrelerdeki genetik manipülasyon için stereotaksik cerrahi

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Dağınık şekilde kayıt sınırlı salınım oluşur içinde fareler davranmak

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Erken Doğum Sonrası Fare Beyinlerine Kemogenetik Mühendislik Kortikal İnkornöron Progenitorlarının Nakli

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Fare Beyninin İşlevsel ve Anatomik Olarak Farklı Bölgelere Mikrodiseksiyonu

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Fare Beyin Bölümlerinde Amiloid-Beta Yükünün Tam ve Alt Bölgesel Nicelleştirilmesi

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.