SN ve MDD NIH K08 NS083740 ve Nöroloji UCLA Bölümü&#39;nden destek aldı. AJG Dr. Miriam ve Sheldon G. Adelson Tıbbi Araştırma Vakfı ve Larry L. Hillblom Vakfı tarafından destek onaylar. KLN minnetle Amerikan Kalp Derneği 14BFSC17760005 ASA-Bugher İnme Merkezi&#39;nden destek kabul eder. ILL, EGS ve STC NIH R01 NS071481 tarafından desteklendi. JDH NIH K08 NS083740 destek kabul eder.

Bu protokolde hayvanların kullanımı Kaliforniya Los Angeles Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi tarafından onaylanan prosedürlere uygun olarak yapılmıştır. Not: Hedef fare nüfusu belirleyerek başlayın. Önceki çalışmalarda, sadece erkek doğal tipte C57 / BL6 fareleri, ancak çeşitli transgenik ya nakavt fareleri de kullanılabilir, kullanılmıştır. stereotaktik koordinatlar C57 / BL6 anatomi dayalı olduğunu unutmayın. Her kullanıcı başlangıçta beyaz cevher inme lokalizasyonu doğrulamak tavsiye edilir. 1. Beyaz Matter İnme İndüksiyon - medial Açılı Yaklaşım <ol><li> Distal çapı 15-25 um, 11 arasında olacak şekilde 0.5 mm kapiler tüp kullanılarak çekilen cam pipet hazırlanmasıyla başlar. </li><li> steril% 0.9 normal tuzlu su içinde 27.4 mg / ml (130 uM) de - L-NIO bir steril 10 ul alikosu ((1) -iminoethyl-L-ornitin HCI N- (5)) hazırlamak. </li><li> çekti cam pipet Ön doldurmakcam pipet sabitlenmesiyle, L-NIO (2-5 | il), küçük bir hacme sahip bir vakum hattına bağlı boruya. tezgah üstünde pipet düz Lay ve L-Nio çözeltisi içine çekti ucunu. <ol><li> Pipet 0.5 mm&#39;lik bölümünün en az 2 mm dolana kadar vakum uygulanır. Vakumu kapatın ve pipet çekin. Adım 1.12 kadar bir kenara koyun. </li></ol></li><li> Indüksiyon odasında fare yerleştirin ve standart% 32 izofluran kullanarak fare anestezi neden bir buharlaştırıcı akan (5 L / dk 5 L / dk oksijen ve 0,5 L / dk ile inhale N2) 1 dakika boyunca ya da derin anesteziye kadar. stereotaktik mikroskop ile donatılmış bir stereotaktik aparatı fareyi aktarın. Bir buharlaştırıcı akan% 32 izofluran kullanarak bakım anestezi sağlamak ve bir burun konisi (2 L / dk 5 L / dk oksijen ve 0.5 L / dak N 2 ile inhale). Bir ayak tutam anestezi derinliğini kontrol edin. </li><li> 36 ° enjeksiyon kolunu ayarlayın. </li><li> Affixa düşük hacimli basınçlı enjeksiyon sistemi uzak ucuna cam pipet tutucu çekti ve stereotaktik kurulum enjeksiyon koluna takın. </li><li> Coat iki gözün üstünde vazelin ve yer suni gözyaşı merhemi ile anestezi hayvanın bıyık. başının üzerinde bir 5-10 cm açıklığı olan hayvanın başının üzerinde steril örtüyü koyarak steril cerrahi alanında hazırlayın. kafatası örten kürk tıraş tarafından aspetic cerrahi yüzey hazırlayın. alternatif betadin ve% 70 alkol bezlerden ile kafa derisi temizleyin. </li><li> kafatası yüzeyi ortaya çıkarmak için steril ince makas ile 1,5 cm orta hat kafa derisi kesi olun. Steril bir pamuklu çubukla kafatası kurutun ve 1-3X büyütme stereotaktik mikroskop kullanılarak, steril bir mikro nokta aracını kullanarak herhangi bir üstte periost doku kaldırmak. </li><li> ince bir nokta kalem kullanarak bir referans noktası olarak Bregma işaretleyin. </li><li> steril ince stip cerrahi matkap ucu kullanarak 2 mm ellipitical kraniotomi Matkap0; bregma posterior başlayan ve öne sadece orta hat sol uzanan. kemik parçaları çıkarın ve serebral korteks görüntülenmiştir böylece yumuşak doku örten. </li><li> aralıklı steril serum fizyolojik damla uygulayarak cerrahi alan ve nemli kortikal yüzey tutun. </li><li> Stereotaktik aparatın enjektör koluna çekti cam pipet yapıştırın. Bregma ile pipet distal ucunu hizalayın ve stereotaktik koordinatlar sıfır. </li><li> İlk ön / arka (A / P) pipet ilerlemek ve / Tablo 1&#39;de verilmiştir koordinatları (M / L) yanal medial. </li><li> kortikal yüzeye pipet ilerlemek ve dorsal / ventral (D / V) ölçümü sıfır. </li><li> Yavaş yavaş ilk D / V Tablo 1&#39;de koordinat ulaşana kadar beyin içine pipet geçmektedir. </li><li> beyne L-NIO 100 nl enjekte ve reflü önlemek için 5 dakika bekleyin, 20 msn bakliyat için 20 psi ayarlanmış bir düşük hacimli basınçlı enjeksiyon sistemi kullanarakPipet parça yukarı. <ol><li> Stereotaktik mikroskop mercek ve 3X bir büyütme kalibre reticle kullanın. </li><li> Buna uygun olarak, koordinat her biri için çekilen cam pipet (100 NL karşılık gelen 0.5 mm çapında bir pipet 0.5 mm uzunluğunda), 0.100 mm&#39;lik toplam 3 yerinden. Bir el çantası kullanarak, ölçmek ve her kullanılan büyütme ve ölçekler bağlı olarak değişir kurmak beri standardize. </li><li> hava-sıvı menisküs bir sagital görünümü vardır, böylece her enjeksiyon sırasında doğru hacim ölçümü için, yan mikroskopla açılı pipet yaklaşım. Menisküs pipet iç ve dış duvarın hem aynı odak düzlemi görünmelidir. </li></ol></li><li> Yavaşça pipet çekilme ve adımları tekrarlayın 1.13-1.16.3 Tablo 1&#39;de verilen koordinatlar ikinci ve üçüncü sette de. </li><li> Son enjeksiyondan sonra, pipet kaldırmak ve kraniyotomi sitesini dolduracak kadar kemik mumu yerleştirin. birKafa derisi yara kenarlarını pproximate dermal yapıştırıcı ile bağlanır. </li><li> Kafa derisi kesi ile ilişkili yerel ağrıyı önlemek için önlemek için steril bir 30 G iğne kullanılarak yara marjları içine% 0.5 marcaine 0.1 ml enjekte edilir. </li><li> 5 gün boyunca içme suyu konut ve arz post-operatif antibiyotik hayvan (0.48 mg / ml trimetoprim-sulfametoksazol veya 0.5 mg / ml Levofloksasin) döndürür. </li></ol> 2. Beyaz Matter İnme İndüksiyon - Posterior Açılı Yaklaşım <ol><li> posterior anterior odaklı 45 dereceye stereotaktik kurulum enjeksiyon kolunu ayarlamak dışında, medial açılı yaklaşım protokolünde olduğu gibi adımları 1.1-1.12 gerçekleştirin. </li><li> İlk A / P pipet ilerlemek ve M / L Tablo 2&#39;de verilmiştir koordinatları. </li><li> Komple kalan adımları yanal açılı yaklaşım protokolde olduğu gibi 1,14-1,20. </li></ol> 3. Retrograd Sinir Etiketleme <ol><li> 10 steril hazırlanması% 0.9 normal serum fizyolojik içinde 54.8 mg / ml L-NIO ul alikosu. </li><li> % 0.9 normal serum fizyolojik içinde% 20 Fluororuby (veya% 20, biyotinlenmiş dekstran amin ya da% 2 Fluorogold) steril bir kısım hazırlayın. </li><li> 27.4 mg / ml L-Nio ve% 10 Fluororuby nihai konsantrasyonları için 1: Birlikte: 1 seyreltin. </li><li> adımlarda 1,3-1,23 olarak yukarıdaki vuruş protokolü gerçekleştirin. </li><li> Daha önce 8 açıklandığı gibi perfüzyon fiksasyon, cryosectioning ve mikroskopi ile doku bölümünde doğal floresan tracer gözünüzde canlandırın. </li></ol> İmmünofloresan 4. Doku İşleme <ol><li> 3 saat dan inme sonrası 14 gün arasında değişen uygun bir inme sonrası aralıklarla, izofluran doz aşımı ya da yerel IACUC üzerinden fareler prosedür onaylı euthanize. </li><li> açılı makas kullanarak göğüs boşluğu açın ve sol ventrikül içine bir 23 G kelebek iğne takın. </li><li> perfüzyon sıvısı için bir çıkış izini izin ince makas kullanarak sağ atrium küçük bir kesim yerleştirin. </li><li> Transkardiyal oda sıcaklığında, 10 ml / dk&#39;lık bir oranda soğuk% 4 paraformaldehit 30-40 ml ve ardından soğutulmuş fosfat tamponlu tuz 30-40 ml serpmek. </li><li> fare başını kesmek ve posterior kafatası açmak için steril makas kullanarak beyin kaldırmak ve sonra yavaşça 24 saat süreyle soğuk% 4 PFA halinde beyin, bir spatula ile örten kafatası kaldırmak yerleştirin ve daha sonra 48 saat süreyle PBS içinde% 30 sakaroz transfer . </li><li> Bir kriyostat kullanarak devre mikron kayan kesitler hazırlamak ve daha önce 6-8 tarif edildiği gibi, antikor işlemi gerçekleştirir. Bu çalışmada aşağıdaki antikorların kullanımı: tavşan anti-sinir lifi 200 (1: 500 seyreltme); tavşan anti-vimentin (1: 500); keçi anti-GFAP (1: 500); tavşan anti-IBA-1 (1: 1000). </li></ol> Protein veya RNA Analizi 5. Doku İşleme <ol><li> 3 saat dan inme sonrası 14 gün arasında değişen uygun bir inme sonrası aralıklarla, izofluran doz aşımı yoluyla euthanize ya da yerel IACUC prosedürü onayladı. </li><li> başını kesmekFare posteriora kafatası açmak için steril makas kullanarak beyin kaldırmak ve daha sonra bir spatula ile hafifçe örten kafatası kaldırmak ve. </li><li> Koku ampul ve optik sinirleri koparmaya beynin ön kısmında steril 4 mm spatula yerleştirin. Yavaşça calvarium üzerinden beyin kaldırın ve (1x Hank Dengeli Tuz Çözeltisi, 25 mM HEPES-KOH, pH 7.4, 35 mM glukoz, 4 mM sodyum bikarbonat, ve 0.01 mg / ml cyclohexamide) buz soğukluğunda tahlil tamponuna içine koyun. </li><li> Bir beyin blok ve steril yeni Jilet kullanarak, soğuk diseksiyon tampon içine inme ve yeri içeren 2-3 mm döşeme hazırlamak. </li><li> bir mikroskop altında enjekte yarımkürede beyaz cevher altta yatan motor korteksin tespit. uzun inme sonrası aralıklarla, bölge görsel fokal nekroz ve miyelin solukluk tarafından tespit edilebilir. (Daha önce inme sonrası aralıklarla,% 10 1 ul ile karıştırılmış L-NIO enjeksiyonu ise Hızlı Yeşil inme görsel kimlik izin verebilir: Not <strong&gt; Şekil 4A). </li><li> Bir mikroskop rehberliğinde ve taze bir neşter kullanılarak altında, dikkatle Hızlı Yeşil etiketleme ya da doku kaybı ya tarafından belirlenen inme içeren beyaz cevher bölgeyi incelemek. istediğiniz gibi örten korteks ve altta yatan striatum çıkarın. </li></ol>

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Subkortikal inme örneksiz bir dizi sıçan 12-14 ve fare 6,15 iç kapsüle endotelin-1 fokal enjeksiyonlar subkortikal beyaz madde ve striatum içeren tarif edilmiştir. Küçük odak vuruşları daha yeni modeller kolesterol mikroemboli karotis arter 16 enjeksiyon ve tek delici arteriole 17 Fototrombotik tıkanıklığı kullanmışlardır. Bu modellerin her biri avantaj ve dezavantajları 5 hem de sahiptir. Şu anda açıklanan model aksonal anormallikler ve zara...

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td>
			<td style="width:204px;">Calbiochem</td>
			<td style="width:133px;">400600-20MG</td>
			<td style="width:81px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>50-821-807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer</td>
			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope SZ51</td>
			<td>Olympus</td>
			<td>88-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill</td>
			<td>Dremel</td>
			<td>8220-1/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill bits</td>
			<td>Fine Scientific Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
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			<td height="21" style="height:21px;">Marcaine</td>
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			<td>NDC 0409-1610-50</td>
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			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
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			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
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		</tr>
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			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclohexamide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

İnme klinik inme sunumlar% 25&#39;e kadar beyaz madde hesaplarını etkileyen, 5-10 kat daha büyük olabilir fiyatla sessizce gerçekleşir ve vasküler demans gelişmesine önemli ölçüde katkıda bulunur. fokal beyaz cevher inme birkaç model var ve uygun modellerin bu eksikliği inme bu tip sonra yaralanma tepki ve onarımında yer alan nörobiyolojik mekanizmaları anlaşılmasını engelliyordu. diğer subkortikal inme modellerinin ana sınırlama fokal beyaz cevher infarkt kısıtlamak değil ya da öncelikle olmayan fare türlerinde valide edilmiş olmasıdır. Bu beyaz cevher inme nörobiyolojisini incelemek için fare araştırma araçları çok çeşitli uygulama becerisi sınırlar. Burada geri dönüşü olmayan bir eNOS inhibitörü bir lokal enjeksiyonu kullanılarak fare beyaz cevherde fokal inme güvenilir üretimi için bir metodoloji sunuyoruz. Biz de iki benzersiz stereotaktik dahil olmak üzere genel protokol üzerinde çeşitli varyasyonlar mevcutvaryasyonlar, büyük ölçüde bu tekniğin potansiyel uygulamalarını genişletmek retrograd nöronal izleme yanı sıra taze doku etiketleme ve diseksiyonu. Birden fazla yaklaşımlar inme bu ortak ve ilgili yeterli formun nörobiyolojik etkilerini analiz etmek için bu varyasyonlar izin verir.

sunulan modeli kullanarak, beyaz cevher altta yatan ön ayakları sensorimotor korteks güvenilir hedeflenebilir. Tipik olarak insan laküner infarkt görüldüğü gibi kimyasal olarak bağlı kontur modeli, fokal aksonal ve miyelin kaybı, astrositoz ve microgliosis (Şekil 1) üretir. Üç iğne kullanarak, klinik olarak faydalı model ön ayakları, motor görevleri 7 erken yetmezliği ve beyin dokusu deneyimleri küçük ama önemli bir kısmı ile kurula...

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Aksonal Dejenerasyon ve Beyaz Cevher Nörobiyoloji Çalışması Çok Yönlü Fare Modeli subkortikal beyaz madde İnme

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Research

JoVE Journal

Medicine

8.0K Görüntüleme.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Video: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Murin Islet İzolasyon ve subkapsüler Böbrek Nakli Bir Yöntem

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Tıp

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

TSSB, Nöral Hematolojik ve Davranış Bağlantılı Revealing için Hayvan Modelinde Biyobelirteçler

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Restenoz Çalışmaları Karotis Arter Stent İmplantasyonu bir murin Modeli

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Subaraknoid Kanama bir fare modeli

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

Görsel yola¤ DTI - Beyaz Cevher Yollar ve Serebral Lezyonlar

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

İzolasyon ve Kemirgen Aort eksizyonu; Kardiyovasküler Hastalığı Çalışmasında Çok Yönlü Tekniği

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Farelerde Basit Kritik ölçekli Femoral Hata Modeli

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Transpediküler Vidaların Değerlendirilmesinde Anestezi, Cerrahi ve Hasat Yöntemi<emIn vivo</em&gt; Domuz Kemik Omurgası Modeli

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...