This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

NOT: Bu çalışmada tüm araştırma gönüllüleri Howard Üniversitesi Kurumsal Değerlendirme Kurulu ve Johns Hopkins Üniversitesi tarafından onaylanmış bilgilendirilmiş onam belgeleri verildi. Bilgilendirilmiş onam belgeleri imzalayarak razı Sadece katılımcılar çalışmaya alındı. Bu analiz için çalışma baz oluşuyordu: 20 denek (12 BD ve 10 kontroller;% 30 erkek) ve 38.2 (14.1) yaşını (sd) anlamına gelir. NOT: cam ve plastik yüzeyler RNaz kontaminasyonu kaldırmak için RNaz Zap ile tüm yüzeyleri püskürtün. 1. Biyopsi Prosedürü <ol><li> Çalışma katılımcının burun boşluğunda topikal lidokain (xylocaine) sıvı% 4 ve oksimetazolin HCI 0.05% Sprey ve zaman uyuşturma 15 dakika bekleyin. </li><li> Lidokain ve oksimetazolin oluşan bir karışım içinde yarım x 3 inç cerrahi cottonoids daldırın ve burun bölmesinin üst kısmı boyunca, burun boşluğunda yerleştirin ve 15 dakika bekletin. </li><li> 30 ° sert kullanmanazal endoskop, nazal kavite üst bölümünü bulun birkaç küçük örnekleri alarak, burun septum üstün kısmından yukarı-ısırma burun forseps kullanarak burun mukozasının kaldırmak için. </li></ol> Doku Blokları 2. Hazırlık <ol><li> Hemen önce biyopsi, doku ortamı dondurma standart boy cryomolds doldurun. </li><li> Otoklava cımbız kullanarak, dondurma ortamda taze doku monte ve kuru buz üzerinde dondurma. , Dondurma ortamı ile cryoblock kalanını doldurun kuru buz cryoblock dondurma ve -80ºC de mağaza blokları. </li></ol> 3. Cryosectioning <ol><li> Cryosectioning hemen önce polietilen (PEN) membran cam slaytlar hazırlayın. 5 dakika boyunca RNaz Zap çözelti ile slaytlar püskürtün. Sonra dietilpirokarbonat (DEPC) su içinde slaytlar durulayın ve 30 dakika kurumasını sağlar. 30 dakika UV ışık altında kurutulmuş slaytlar etkinleştirin. </li><li> Cryosection her 30 um tamamen bloke eder. Kuru i slaytlar tutunCE ve bölümler mümkün olduğu kadar donduruldu. Tamamlanmasıyla -80 ºC Mağaza slaytlar. </li></ol> 4. Lazer-Yakalama Mikrodisseksiyon <ol><li> Hemen önce Mikrodiseksiyon slaytları suyunun başlayın. % 60 (2 mi, 15 saniye) ile,% 80 (2 mi, 15 saniye) ile,% 95 (3 mi, 25 sn), 100: Her bir slayt aşağıdaki etanol serisi uygulanır, sonra donmuş slaytlar Let 10 sn için çözülme ve % (7 mi, 60 saniye). </li><li> Üzerine fotoaktive yerelleştirme mikroskobu (PALM) mikroskop çevirin ve set aşağıdaki koşullara: 82 enerji set 10X büyütme için, 74&#39;e odaklanmak ve hız 25 - 30. 20X büyütme için, 63 enerji ayarlamak, 67 odak, ve hız 5 - 6 optimum kesim sonuçları elde etmek için gerekli ayarlayın. </li><li> Görme 20X veya 10X büyütme kullanarak nöronlar tespit. Boyama işlemi kullanılmadan bir mikroskop altında yatan alt mukoza ince yüzey koku nöron tabakası ayırt eder. Yoğun sütunlu görünüm 18 olarak nöronlar gözlemleyin.</li><li> Aşama 4.2 Yukarıda bahsedilen mikroskop Kalem fonksiyonunu kullanarak, nöronal tabaka etrafına iz. Lazerden nöronların yanmasını önlemek için biraz uzakta nöronlardan Trace. Bu yüksek kalitede RNA elde etmek için gereklidir. Sonra lazer güdümlü kesim başlatmak. </li><li> Ince uçlu mikrodiseksiyon forseps kullanılarak disseke bölümleri Pick up ve buz üzerinde 100 ul lizis tamponu içeren bir mikro doku koymak. Tek bir örnekten elde edilen tüm nöronal bölümler için tekrarlayın. 50 dakika geçmemelidir numune başına toplam diseksiyon zamanı. </li><li> Diseksiyon tamamlandıktan hemen vorteks örnek lizatları ve kuru buz üzerinde tutmak kez. Toplam RNA izolasyonu için -80ºC Mağaza örnekleri. </li></ol> Toplam RNA ve Kalite Kontrol Analizi 5. izolasyonu <ol><li> Buz üzerinde çözülme örnek lizatları. (Bkz hiçbir değişiklik ile üreticinin protokolünü takip ederek DNaz-ı inaktivasyonu ile RNAqueous Mikro Kit ile toplam RNA izolasyonu yürütmek &lt;strong&gt; Şekil 1). Toplam elüsyon hacmi yaklaşık 20 ul. </li><li> RNA konsantrasyonu ölçmek ve Bioanalyzer kullanarak RNA Bütünlük sayısı (RIN) ile RNA kalitesini değerlendirmek. Şekil 2 &#39;de kalite kontrol kriterleri yerine getiren örnekler, cDNA sentezi için de kullanılabilir. </li></ol> 6. cDNA Sentezi <ol><li> Bir valide ticari kit kullanılarak cDNA sentez (Reaktifler bkz: İçindekiler). Tüp başına 8 ul toplam hacmine RNA (durumuna bağlı olarak) ve RNazsız su içinde 1.0 ng 0.1 - birleştirerek tavlama aşamasını gerçekleştirir. Daha sonra 10 ul toplam hacim için, tüp başına karışımı oligo d (T) 20 primeri 1 ul 10 mM dNTP ve 1 ul ekle. </li><li> 5 dakika boyunca 65 ° C &#39;de yavaşça girdap ve santrifüj örnekleri ve tavlama örnekleri. Buz üzerinde, reaksiyon tamamlandıktan sonra, derhal soğuk örnekler. </li><li> Tablo 1&#39;de özelliklerine göre ana karışımı çözeltisi hazırlayın. Sonra 9.5 μ eklemekHer tüpe l ana karışımı ve ticari olarak hazırlanmış ters transkriptaz (RT), 0.5 ul. Yerine RT tüp kontrol etmek için 0.5 ul RNaz içermeyen su ilave edilir. </li><li> Hiçbir bisiklet ile 50 dakika, 5 dakika ve son 4 ºC tutuş için daha sonra 85 ºC 50 ºC ilk inkübasyon ile RT reaksiyonu çalıştırın. </li><li> Su ve bölümüyle 5x bütün örnekleri sulandırmak. -80 ° C&#39;de saklayın örnekleri. </li></ol> 7. Real Time PCR - Nöronal Zenginleştirme Onayla <ol><li> . Tablo 2&#39;de özelliklerine göre ana karışımı çözüm hazırlayın gibi GAPDH gibi bir iç kontrol kullanın. Nöronal zenginleştirme belirlemek için undissected dokuda OMP ifade ile microdissected dokuda koku markör proteini (OMP) ifade Karşılaştırması. NOT: OMP koku nöronlar için bir belirteç olduğunu. OMP dizisi: İleri, 5&#39;-CTGTCGGACCTGGCCAAG-3 &#39; , geri 5&#39;-CACCCTCGCCAAAGGTGA-3 &#39; GAPDH dizisi: İleri, 5&#39;-CATGAGAAGTATGACAACAGCCT-3 &#39; ters 5&#39;-AGTCCTTCCACGATACCAAAGT-3 &#39;. </li><li> CDNA her bir kuyu ve 3 ul ana karışımı 7 ul ekleyerek PCR plakasını ayarlayın. </li><li> 300 xg (1.300 rpm) 5 dakika santrifüj plakası. Böyle SYBR Greener qPCR gibi ticari Supermix için belirtilen varsayılan termal döngü koşulları kullanılarak Çalışma Real Time PCR reaksiyonu. </li><li> Veri (özellikler için bakınız Şekil 2) analiz edin. </li></ol> 8. Gerçek Zamanlı PCR, - söz konusu genler sentezlenmesi <ol><li> 2 kat veya daha fazla nöron zenginleşme göstermektedir numuneler üzerinde gerçekleştirin. </li><li> Her prob Tablo 3&#39;te özelliklerine göre ayrı tüplere (faiz ve VIC-etiketli GAPDH FAM-etiketli prob) için buz üzerinde ana karışımı hazırlayın. </li><li> CDNA her bir kuyu ve 2 ul 8 ul ana karışımı ile bir PCR plakasını ayarlayın. </li><li> 300 xg (1300 rpm) 5 dakika boyunca plaka santrifüjleyin. Def kullanarak Real Time PCR reaksiyonu çalıştırınüretici tarafından sağlanan gen ifadesi usta karışımı protokolü uyarınca ault termal döngü koşulları. Ifade sonuçlarını (özellikler için bakınız Şekil 2) analiz edin. </li></ol>

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Yazarlar ticari çıkarları ile finansal ilişkileri rapor.

Burun biyopsi ve LCM birleştirerek zenginleştirilmiş koku nöronal katmanları elde etmek için bir yeni bir platform sunulmuştur ve bu çalışmada onaylanmıştır. Bu teknik yaygın etkileri olabilir. Bu alanda daha geniş bir etki yaparak, tedaviye yanıt için olanlar da dahil olmak üzere biyomarkır çalışmaları, ve diğer nöropsikiyatrik durumlar için ilaç keşfi çabalarına uygulanabilir. Mansap uygulamalara yatkın, yüksek kaliteli nöronlar elde etmek için, bir kaç k...

Bipolar bozukluk (BD) ruh patolojik değişiklikler ile karakterize ciddi nöropsikiyatrik bozukluk olduğunu, sürücü ve biliş 1. BD tedavisi için kullanılan lityum hayvan çalışmalarında 2 gen çok sayıda sabit durum mRNA seviyeleri değiştirdiği gösterilmiştir, fakat bu moleküllerin herhangi insanlarda 2, klinik yanıt ile ilişkili olup olmadığını da bilinmemektedir. Lityum yanıt mekanizmasını anlamak nöronal dokularda lityum-kaynaklı moleküler değişikliklerin araştırıldığı gerektirecektir. Ne yazık ki, BD hastalarının beyin biyopsileri öncesi ve sonrası lityum tedavisi lityum yanıt moleküler imzalarını tespit elde etmek pratik değildir. Otopsi beyin dokuları BD biyomarkerları incelemek için kullanılır olmuştur, ancak, duygular, biliş ve sürücü dinamik değişiklikleri ile ilişkili moleküler belirteçler değerlendirmek için kullanılamaz; ve geriye dönük tespit lityum tedaviye yanıt geçerliliği sorunlu olabilir 4. Lenfositler ve diğer kan hücreleri yararlı olabilir, ancak kan hücrelerinde moleküler değişiklikler nöronal değişiklikleri 3-5 yansıtmayabilir. Beyin omurilik sıvısı hastalığıyla ilişkili ve ilaç-geri dönüşümlü içsel değişiklikleri yansıtıyor olabilir hücre içi moleküller hakkında bilgi edinmek için yetersiz olabilir. koku epiteli (OE) merkezi sinir sistemi (MSS) eşsiz bir parçasıdır, limbik yapılara 6 embriyolojik ilgili; ve burun biyopsi ile kolayca erişilebilir. Bu oluşur glial benzeri gelişme 7-9 farklı aşamalarında (yani, sustentacular) hücreleri, bazal çoğalan hücreleri ve koku reseptör nöronlar destekler. Bu nedenle, OE accessibly nöropsikiyatrik hastalıklara 7 olan hastaların MSS dinamik değişiklikleri incelemek için eşsiz bir fırsat sunuyor. Çalışmalar bu occ yansıtan hastalıkla ilişkili olayları araştırmak için bir vekil doku olarak OE programı gösteriyorlarbeyin nöronlarının 8,9 urring. Örneğin, çalışmalar psikiyatrik koşullar 10-14 ile ilişkili moleküler profilleri araştırmak için OE kullanmıştır. Koku sistem aynı zamanda şizofreni 15 negatif belirtiler ile ilişkili koku açıkları gibi klinik endophenoytpes tanımlamak için hizmet vermektedir. Ayrıca, nörogelişimsel süreçleri psikiyatrik durumlar 8,9 patofizyolojisinin modellemek için yararlı bir yol sağlayan, hayat boyu OE devam ediyor. Bununla birlikte, bu doku kullanımı için bir sakınca nöronal olmayan hücrelerin 16 koku biyopsilerin önemli konu, kirliliktir. Örneğin, önceki OE çalışmalarında, gen ifade çalışmaları için kullanılmıştır toplam RNA, nöronal olmayan hücrelerin 17 RNA dahil olmak üzere tüm burun biyopsi dokusundan ekstre RNA içeriyordu. Bu nedenle, önceki yaklaşımlar hücre kalitesi ile sınırlı kalmıştır. Bu sorunu aşmak için, yeni bir yaklaşım elde etmek<html> Lazer-yakalama mikrodiseksiyonu (LCM) ile burun biyopsi birleştirerek zenginleştirilmiş nöronal hücre popülasyonları 18 geliştirilmiştir. LCM enfraruj lazer 19-21 ile birlikte, UV lazer kesim kullanılarak hücrelerin seçici izolasyonu sağlayan bir tekniktir. OE yaklaşımı ile birleştirilmesi lcm böylece nöronal hücrelerin 18 zenginleştirilmesini artırılması, nöronal olmayan hücreler tarafından OE önemli kirlenmesini en aza indirir. Ayrıca, nöronal tabaka bu şekilde boyama için ihtiyacı ortadan kaldırarak, bir mikroskop altında alt mukoza tabakası ayırt edilebilir. Nöronal hücre tipleri daha fazla ilgi 7 hücre tipi ile ifade edilir, birincil antikorlar kullanılarak diğer hücre popülasyonlarından ayırt edilebilir. Bu nedenle, bu prosedür, gen ekspresyon çalışmaları, immünohistokimya ve diğer morfolojik araştırmalar için kullanılabilir hemen hemen tamamen nöronal hücre popülasyonunun zenginleştirilmesi için daha kolay bir yöntem ortaya koymaktadır. ve_content &quot;&gt; Bu çalışma hastalık durumlarında ve tedavi yanıtı ile ilişkili koku nöronlar moleküler değişiklikleri araştırmak için deneysel bir platform oluşturmayı amaçlamaktadır. Bu adres için, BD için DSM-IV tanı ölçütlerine göre sigara içilmeyen hastaların küçük bir set dayalı Genetik Çalışmaları Tanı Görüşmesi (DIG) 22 iki burun biyopsi geçmesi işe edildi: lityum ve ikinci biyopsi ile bir biyopsi ön arıtma, günlük oral lityum tedavisinin 6 hafta sonra Ayrıca, uygun BD hastalar olmalıdır:. depresyonun için semptomatik Montgomery-Asberg Değerlendirme Ölçeği (MADRS) 23 uygulanan klinisyen içinde 60 ≥10 puanlama dayalı (YMÖ&#39;nin) hipomani veya mani için semptomatik, Young-Mani Derecelendirme Ölçeği klinisyen üzerinde 56 arasında bir skor ≥10 dayalı . 24;. MADDÖ ve iki ölçek için klinisyenler arasındaki anlaşmanın YMRS Değerlendirici-Inter-Değerlendirici katsayısı hem veya ≥10 biyopsi sonra, nöronlar Enri 0.96 idi&gt; olanLCM tarafından OE dür. Doku ve nöronal zenginleştirme yüksek kaliteli RNA&#39;nın çıkarılmasını sağlamak için, ek kalite kontrol önlemleri ardından, Real Time RT-PCR ilgi genlerin öncesi ve tedavi sonrası ifade düzeylerini araştırmak amacıyla yapılmıştır. takip eden bölümlerde bir bu yaklaşımın geçerliliğinin açıklamasını, protokol optimizasyonu vurgulayarak ve sorun-çekim protokolü uygulanmıştır stratejileri içerir.

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			<td>Quality Biological</td>
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			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
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			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
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			<td height="21" style="height:21px;">No.5 Dumont Microdissction Forceps</td>
			<td>Roboz</td>
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			<td height="21" style="height:21px;">SuperScript III First Strand Synthesis Kit</td>
			<td>Invitrogen&#160;</td>
			<td>18080-051</td>
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			<td height="20" style="height:20px;">OMP qPCR</td>
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			<td height="21" style="height:21px;">SYBR GreenER qPCR SuperMix</td>
			<td>Invitrogen&#160;</td>
			<td>11760-500</td>
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			<td>Applied Biosystems</td>
			<td>Various</td>
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			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
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olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

Bipolar bozukluk (BD) az anlaşılan patofizyolojisi ile ciddi bir nöropsikiyatrik bozukluk olduğunu ve genellikle duygudurum dengeleyici, lityum karbonat ile muamele. Hayvanlar üzerinde yapılan çalışmalar ayrıca insan genetik çalışmalar lityum nöronal büyüme, hayatta kalma ve olgunlaşması ve Wnt sinyal katılan özellikle moleküller yer alan moleküler hedefleri etkilediğini göstermektedir. Merkezi sinir sistemi (MSS) lityum-yanıt ile ilişkili dinamik moleküler değişiklikler araştırmak için beyin biyopsileri elde etik sorun göz önüne alındığında, kimse bu goal.The koku epiteli koku reseptör nöronları içeren ulaşmak için koku dokulardan elde edilen nöronların kullanımını düşünebilirsiniz geliştirilmesi ve glial benzeri hücreleri desteklemek farklı aşamalarında. Bu güvenle burun biyopsi elde edilen koku doku kullanarak, nöropsikiyatrik hastalıklarda MSS dinamik değişiklikleri incelemek için eşsiz bir fırsat sunuyor. Subst oluşturduğu güçlüğün üstesinden gelmek içinnöronal olmayan hücreler biyopsi yapılan koku doku antial kirlenme, zenginleştirilmiş nöronal hücre popülasyonu elde etmek için yeni bir yaklaşım, lazer yakalama mikro-kesim ile burun biyopsi birleştirerek geliştirilmiştir. Bu çalışmada, nöronal dokuda tedavi ile ilişkili dinamik moleküler değişiklikler araştırmak için bir sistem lityum tedaviye yanıt moleküler mekanizmaların çalışması için işe BD hastalarının küçük bir pilot örneği kullanılarak, geliştirilmiş ve onaylanmıştır.

Moleküler imza çalışmada protokol ve sorun giderme stratejisi başarıyla optimize edildi. Numuneler için, RNA kalitesi ve nöronal zenginleştirme kriterleri daha sonraki alt akış Anlık-PCR analizi kullanılmak üzere standardize edildi. RIN ve RNA konsantrasyonu tespit ifade düzeyleri karıştırıcı faktörler olarak incelendi. 1 arasında değişen rin birkaç numune analize dayalı olarak - 10, yukarıda ~ 3.0 ve en az RIN Bu çalışmada alt analizi için yeterli olduğu tespit edilmiştir. Bunun bir son...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bu çalışmada, yeni bir platformu geliştirdi ve valide edilmiş bipolar bozukluk (BD) tedavi yanıtının intranöronal moleküler imzaları araştırmak. BD hastaların Koku epitel burun biyopsi ile elde edildi. Sonra lazer yakalama mikrodiseksiyon BD lityum yanıt moleküler imza araştırmak için Gerçek Zamanlı RT PCR ile kombine edilmiştir.

Lazer Yakalama Mikrodisseksiyon ile Burun Biyopsi Kombine yoluyla elde Koku Nöronlar: Mental Bozuklukların Tedavi Tepki Eğitim Bir Potansiyel Yaklaşım

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

Research

JoVE Journal

Neuroscience

9.7K Görüntüleme.  Johns Hopkins University.  Bu çalışmada, yeni bir platformu geliştirdi ve valide edilmiş bipolar bozukluk (BD) tedavi yanıtının intranöronal moleküler imzaları araştırmak. BD hastaların Koku epitel burun biyopsi ile elde edildi. Sonra lazer yakalama mikrodiseksiyon BD lityum yanıt moleküler imza araştırmak için Gerçek Zamanlı RT PCR ile kombine edilmiştir. 

Video: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Lazer Yakalama Mikrodisseksiyon Drosophila Periferik Nöronlar

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Nörobilim

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Kemirgenler ve İnsanlar Burun Koku Kök Hücreler Yalıtımlı

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

Kombine Kullanımı Hedefleme Koku Ampul Nöronlar In Vivo Elektroporasyon ve Gal4 Tabanlı Enhancer Trap Zebra balığı Hatları

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Toplam RNA İzolasyonu ve Alt Nesil Sıralama ve Kaynak gösterilmeden kullanılamaz Ayrık Fare Beyin Bölgeler Mikrodisseksiyon Sigara Lazer Yakalama Mikroskopi Yaklaşım

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Travmatik Beyin Hasarı Sonrası Nöronlar veya gen ekspresyon analizi için Tek Nöron zenginleştirilmiştir Populasyonlarının Lazer Yakalama Mikrodisseksiyon

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Kültür farklılaştırılmış insan Neuroprogenitor Hücreler Nöronlar Lazer Yakalama Mikrodisseksiyon

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Lazer Yakalama Mikroseksiyonu ile Fare Omurilik Motor Nöron Hücre Gövdelerinden Transkriptomik Analiz için RNA Üretimi

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Lazer Yakalama Mikrodisseksiyon - Bireysel Dopamin Nöronlar İzolasyonu ve Tüm ventral tegmental Area bir Gösteri

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotaksik Atlas güdümlü lazer yakalama mikrodiseksiyon travma yaralanma tarafından etkilenen beyin bölgeleri

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.