Bu eser Pekin Birliği tıbbi üniversite gençlik Fonu ve temel araştırma fonları Merkez üniversiteler (Grant no. 3332015200) için gelen hibe tarafından desteklenmiştir.

Tüm hayvan deneyleri tüm ilgili kurallar, düzenlemeler ve düzenleyici kurumlar uygun olarak idam edildi. Gösterilen mevcut Protokolü rehberlik ve onay, Enstitüsü, mikro hayvan Etik Komitesi (IMAEC), Pekin Birliği tıp Koleji (PUMC) altında gerçekleştirildi.
1. hayvanlar
<ol><li>Deneme başlamadan önce üç BALB/c fare kontrollü sıcaklık (24 ± 1 ° C) ile kafes ve nem (55 ± % 5), başına 12-h koyu döngüsü altında tutun. Fareler düzenli gıda ve su ücretsiz erişmesine izin vermek.</li>
	<li>Rastgele fareler bir diyabetik olmayan kontrol grubu ve bir diyabetik gruba bölmek. Doğru bir şekilde tek tek her fare ağırlık ve vücut kitle her fare kullanarak enjeksiyon birimi hesaplayın.</li>
	<li>Fareler streptozotocin (STZ) enjeksiyon önce 4 h için hızlı ve düzenli su normal olarak deneysel günde 1 sağlar.</li>
	<li>0.1 M sodyum sitrat tampon pH 4,3 hazırlayın. Çözüm 1 mL 1.5 mL microcentrifuge tüp içine koymak ve microcentrifuge tüp ışık maruz önlemek için alüminyum folyo sarın.</li>
	<li>5 mg/mL kullanmadan önce son bir çalışma konsantrasyonu (pH 4,3) sodyum sitrat arabellekte STZ geçiyoruz.</li>
	<li>Diyabetik grup mayi enjeksiyonlari STZ bir doz 40 mg/kg 1 mL şırınga ve bir 25-G iğne kullanarak, fareler ver. Diyabetik olmayan kontrol fareler sodyum sitrat arabellek (pH 4,3) aynı birimle enjekte.</li>
	<li>Fareler geri kafesler koymak ve onları düzenli gıda ve % 10 Sükroz su ile tedarik.</li>
	<li>1.3-1.7 deneysel günlerde 2-5 (Yani, önümüzdeki 4 gün üst üste) yineleyin.</li>
	<li>% 10 Sükroz su düzenli su ile son STZ enjeksiyon sonra değiştirin.</li>
	<li>Hızlı fare için 6 h, ama onları su ücretsiz erişim sağlar ve dokuz gün sonra (deneysel gün 14) kan glikoz düzeyleri ölçmek. Bir kan örneği bir kan glikoz izleme sistemi kullanarak hiperglisemi onaylamak için kuyruk damar toplamak. Not: Kan glikoz düzeyleri > 200 mg / dL ile fareler şeker hastası olarak kabul edilir.</li>
</ol>2. araç hazırlanması
<ol><li>Sonda uç optik yüzeylerinin ve toz ve partiküller kaldırmak için sonda bağlayıcı lazer Doppler cihazları yumuşak, aşındırıcı olmayan bir bezle temizleyin. Kabloyu araç (şekil 1A) bağlantı noktasına takılır.</li>
	<li>Kalibrasyon stand akı standart deneysel çevre (genellikle 30 dk için oda sıcaklığında) ile termal denge olmak izin vererek topla. Akı sallamak yavaşça 10 s ve izin vermek için standart için 2 dk bekletin.</li>
	<li>Akı standart konteyner kalibrasyon Bankası ortasında konumlandırın. Kelepçe için maksimum yükseklik ayarlamak ve öyle ki aşağı doğru kapsayıcıya Puan tutturmak soruşturma güvenli. Akı standart doğru sonda altında konumlandırılmış emin olun.</li>
	<li>Ucunu doğru akı standardında batık kadar yavaşça sonda aşağı taşıyın. Seçin ve lazer Doppler cihazı "kalibrasyon" basın ve sonda bağlı çalışma kanalı seçin. Bir "Kalibrasyon başarılı" uyarı lazer Doppler cihazı ekranda görüntülenene kadar ayarlama programı çalıştırın.</li>
	<li>Prob prob sahipleri kullanarak güvenli. El ile sonda hareketi önlemek için güvenli.</li>
	<li>Korumak sabit sıcaklıkta (24 ± 1 ° C) deneysel Oda ve nem (~ 50-%60).</li>
	<li>Dış ışık kaynaklı değiştirmek önlemek için deney yapmadan önce herhangi bir dış ışık (örneğin flüoresan ve spot lambalar) kapatın.</li>
</ol>3. hayvanlar hazırlanması
<ol><li>Otoklav cerrahi aletleri ve oda sıcaklığında kullanmadan önce soğumaya izin.</li>
	<li>Fareler vermek pankreas adacık mikrovasküler vasomotion lazer Doppler tarafından tespit önce deneysel çevreye parkenizin için 10 dakika.</li>
	<li>1-mL şırınga ile 1 mL % 3 Fentobarbital sodyum doldurun. Fareler anestezi için Fentobarbital sodyum çözüm (75 mg/kg ı.p.) enjekte.</li>
	<li>Fare kuruluğu önlemek için önceden ıslatılmış tıbbi gazlı bez ile gözünde kapak.</li>
	<li>Fare tamamen bilincini kaybeder ve artık yanıt kuyruğuna veya arka ayak Forseps ile pinches emin olun. Anestezi her 15 dk. Fentobarbital çözüm gerekli olduğunda ilk enjeksiyon hacmi yüzde 10'u ile ilave tarafından anestezi korumak ve anestezik ve intraoperatif etkinlik boyunca izlemek.</li>
	<li>Bir Isıtma yastığı ile yarı yalıtım katmanı hayvan altına yerleştirin ve hayvan sırtüstü pozisyonda yerleştirin ve lazer Doppler cihazı çalışma istasyonu için transfer. Çalışma sahnesi için fare cerrahi bant ile düzeltmek.</li>
	<li>Pamuklu çubuk fare betadin ve % 75 etanol ile karın derisi karın bölgesi temiz DNA örneği kullanılır.</li>
	<li>% 2 lidocaine/0.5% bupivacaine (50/50) karışımı subkutan enjekte et.  ~ 3 cm kesim-bir gazlı bez sünger ortasına delik çapı. Gazlı bez sünger ile karın bölgesini kapsar.</li>
	<li>Karın deri Forseps ile Asansör ve karın bir neşter ya da deri makas kullanarak bir ilk dikey kesi orta çizgi boyunca olun.</li>
	<li>Forseps ile temel kas kavramak ve karın boşluğuna girmek için deşmek. Herhangi bir organ zarar değil. Cilt ve temel kas karın boşluğu ortaya çıkarmak için göğüs katlayın. Yavaşça pankreas vücut ve forseps küt burunlu bir çift kullanarak dalak ortaya çıkarmak.</li>
</ol>4. veri toplama için analiz
<ol><li>Lazer Doppler cihazı yazılım koşmak yanında tıkırtı üstünde "Dosya" → "yeni" yeni bir ölçüm dosyası oluşturmak için. "Genel" sekmesi altında bağlı monitör yapılandırmak için "Ücretsiz çalışmasına." izleme süresi kadar ayarlayın Fabrika varsayılan "LDF monitör" sekmesini tıklayın "İleri" için kullanın</li>
	<li>Grafik görüntülenme hızını ayarla "ekran Yapısı iletişim kutusu içinden." "Akı, konsantrasyon, hız" kanalları ilgili kutuları işaretleyerek seçin. Aşağıdaki parametreleri seçin: "Kanal için veri kaynağı" ve "etiket, birimleri ve rengi." "Next" tıklayın</li>
	<li>"Dosya bilgileri iletişim kutusuna" konu ve ölçüm (Yani, adı ve denek, operatör, izleme süresi, Yorumlar, vb) hakkında kullanıcıya bilgi girin ve ölçüm yapılandırmasını bitirmek için "İleri" yi tıklatın. Not: Bir ölçüm pencere otomatik olarak yazılım (şekil 1B) tarafından oluşturulur.</li>
	<li>El ile elektrot pankreas için ilerlemek. Sonda ve pankreas dokular arasındaki uzaklığı 1 mm içinde olduğundan emin olun. Uygunsuz bir mesafe bir yapay olarak artan veya azalan kan akışı okuma verir.</li>
	<li>Mikrovasküler kan perfüzyon birimleri (PU) veri kayıt işlemini başlatmak için "Başlat" araç çubuğu simgesini tıklatın. PU veri 1 dk. için sürekli olarak her çalışma toplamak. Ölçüm durdurmak için "durdur"'u tıklatın. Seçin "Dosya" → "Farklı Kaydet" adına ve bitmiş ölçüm dosyayı kaydedin.</li>
	<li>Sonda katkı etkileri ve contractile yerelleştirilmiş tükenme önlemek için her çalıştırdıktan sonra el ile yeniden konumlandırırsanız ve gevşeme kapasitesi. Çok noktalı (Yani, üç rasgele seçilmiş Puan pankreas dokusu) mikrovasküler PU veri her fare için hasat için 4.1-4.4 adımları yineleyin. Yansıtıcı olmayan bir tabak PU verileri temel denetimi olarak ölçmek.</li>
	<li>Karın kas katmanı ve dikiş cilt katmanıyla kapatın. Hayvanlar temiz kafes deneyler sonra yerleştirin.</li>
	<li>Hayvan sıcak yarı üzerinde kurtarma kafes yerleştirerek Isıtma yastığını tutun. Not: sıcaklık, hijyen, sıvı ve gıda alımı ve enfeksiyon dikkat. Fareler 2 mg/kg Carprofen 48 saat için ameliyat sonrası ağrı yönetimi yönetmek.  Ötenazi fareler şiddetli ağrı veya hafifletti değil sıkıntı bir durumda olması için gözlendiğinde 150 mg/kg Fentobarbital sodyum ı.p. enjekte edilerek gerçekleştirin.</li>
</ol>5. mikrovasküler Vasomotion parametrelerin hesaplanması
<ol><li>Zaman ve PU ham veri *.xlsx dosyası olarak verebilir ve dosyayı bir elektronik tabloda açmak için lazer Doppler yazılım "Export" komutunu kullanın.</li>
	<li>Ortalama satır taban çizgisi perfüzyon birimi (PUb) hesaplamak (bkz. Adım 4,6).</li>
	<li>Ortalama kan perfüzyon (PUbir) bir ölçü 1 dk. için aşağıdaki gibi hesaplar: ortalama kan perfüzyon (PUbir) = PU - PUb (Denklem 1).</li>
	<li>Her 1dk ölçüm için (devir/dak) frekansını hesaplamak. Not: Mikrovasküler vasomotion sıklığını dakikada bir mikrovasküler vasomotion dalga oluştu doruklarına sayısı olarak tanımlanır.</li>
	<li>Her 1dk ölçüm için (ΔPU) genlik hesaplayın.<ol>
<li>Mikrovasküler vasomotion genliğini (PUmax) maksimum ve minimum (PUmin) arasındaki fark olarak hesaplar: genlik (ΔPU) PUmax - PUmin (Denklem 2) =.</li>
	</ol>
</li>
	<li>Her 1dk ölçüm için göreli hızı (PU) hesaplar.</li>
</ol>

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Mikrovasküler disfonksiyon (örn., şeker hastalığı, akut pankreatit, periferik mikrovasküler hastalıkları, vb) dahil hallerde bazı hastalıkların sınırlı kan akımı neden. Kan akımı değişiklikleri dışında orada mikro fonksiyonel durumunu ayna önemli göstergeleri mikrovasküler vasomotion gibi. Belirli göstergesi, mikrovasküler vasomotion, genellikle mikrovasküler yataklar mikrovasküler tonda salınım olarak tanımlanır. Geçerli kuralında izleme sistemi mikrovasküler kan p...

Bir parametre mikro fonksiyonel durumu olarak mikrovasküler vasomotion teslim ve oksijen, besin ve hormonlar alışverişi için sorumluluk kabul etmez ve karbon dioksit ve hücre atık gibi metabolik ürünler kaldırılması önemlidir 1. mikrovasküler vasomotion da düzenleyen kan akışı dağıtım ve doku perfüzyon, böylece yerel microcirculatory kan basıncı ve ödemi birçok hastalığa neden olabilir iltihap yanıtlarını etkileyen. Bu nedenle, mikrovasküler vasomotion fizyolojik fonksiyon organları2,3,4, doku ve bileşen hücreleri korumak son derece önemlidir. Mikrovasküler vasomotion bozulma, mikro-hastalıkları ile ilgili5geliştirilmesinde önemli adımlardan biri olabilir.
Lazer Doppler başlangıçta gözlem ve mikro araştırma6alanındaki miktar için geliştirilmiş. Bu teknik, teknik diğer yaklaşımlar (Örneğin, lazer benek7, transkütanöz oksijen vb..),ile birlikte kan akımı mikro değerlendirmek için altın standart olarak kabul edilmiştir. (Yani, kılcal damarlar, arteriyoller, venüller, vb) yerel mikrosirkulasyon kan perfüzyon Doppler, lazer ile donatılmış cihazları tarafından belirlenebilir mantığı Doppler kayması ilkesine dayanır. Dalgaboyu ve frekans uyarılmış emisyonu ışık ışık parçacıkları yüzdeki hareketli kan hücrelerinde karşılaşırsanız veya değişmeden kalır zaman değiştirin. Bu nedenle, mikro, sayısı ve kan hücrelerinin hız mikrovasküler kan akışının yönünü alakasız olsa da büyüklüğü ve frekans dağılımı Doppler kaymıştır ışık ile ilgili anahtar faktörlerdir. Farklı yöntemler kullanarak, çeşitli dokuların mesenteries microcirculatory çalışmaları için hem de dorsal deri kıvrım odaları fare, sıçan, hamster ve hatta insanlar8. Ancak, geçerli protokolünde, biz fonksiyonel üzerinde odaklanmak kullanarak değerlendirilir pankreas adacık mikrovasküler vasomotion durumunu lazer Doppler ve ev yapımı değerlendirme parametre sistemi.
Pankreas adacık mikro ve ayırt edici özellikleri sergileyen pankreas adacık yüzdeki esas olarak oluşur. Pankreas adacık kılcal ağ onun ekzokrin muadili9kapiller ağının daha beş kat yüksek yoğunluğu gösterir. Bir kanal giriş glikoz ve yaygınlaştırmanın insülin teslimi için sağlayarak, adacık endotel hücreleri oksijen metabolik olarak aktif adacık hücreleri β hücreler sağlamak. Ayrıca, kanıt ortaya çıkan Ayrıca adacık yüzdeki sadece insülin gen ekspresyonu ve β-hücre sağkalım düzenlenmesinde, aynı zamanda β-hücre işlevi etkileyen katılmaktadırlar gösterir; β-hücre çoğalması teşvik; ve vazoaktif, anjiogenik maddeler ve büyüme faktörleri10bir dizi üreten. Bu nedenle, bu bağlamda, biz pankreas adacık mikrovasküler vasomotion fonksiyonel durumunu gibi akut/kronik pankreatit, diyabet ve diğer hastalıkların patogenezinde yer almak ve adacık β-hücre işlevi etkiler olabilir sonucuna pankreas kaynaklı hastalıklar.
Pankreas adacık mikrovasküler vasomotion fonksiyonel durumunu patolojik değişiklikleri analiz yukarıda belirtilen hastalıklar için pankreas adacık mikro katkıları belirlemek için uygun bir strateji olabilir. Burada pankreas adacık mikrovasküler vasomotion vivo içinde belirlemek için yaklaşım açıklayan ayrıntılı adım adım yönerge sağlar. Tipik ölçümler daha sonra Temsilcisi sonuçlarıgösterilir. Son olarak, yararları ve yönteminin sınırlamalarını tartışma, daha fazla uygulamaları ile birlikte vurgulanır.

<table>
	<tbody>
		<tr>
			<td>MoorVMS-LDF2</td>
			<td>Moor Instruments</td>
			<td>GI80</td>
			<td>PeriFlux 5000 (Perimed Inc.) can be used as an alternative apparatus to harvest data</td>
		</tr>
		<tr>
			<td>MoorVMS-PC Software</td>
			<td>Moor Instruments</td>
			<td>GI80-1</td>
			<td>Software of MoorVMS-LDF2</td>
		</tr>
		<tr>
			<td>Calibration stand</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration base</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>Calibration flux standard</td>
			<td>Moor Instruments</td>
			<td>GI-cal</td>
			<td>Calibration tool</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy glucometer</td>
			<td>Johnson and Johnson</td>
			<td>#1955685</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>One Touch UltraEasy strips</td>
			<td>Johnson and Johnson</td>
			<td>#1297006</td>
			<td>Confirm hyperglycemia</td>
		</tr>
		<tr>
			<td>Streptozotocin</td>
			<td>Sigma-Aldrich</td>
			<td>S0130</td>
			<td>Dissolve in sodium citrate buffer (pH 4.3)</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>Working concentration 3 %</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Inc.</td>
			<td>200121</td>
			<td>Working concentration 75 %</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Amresco</td>
			<td>335</td>
			<td>Working concentration 10 %</td>
		</tr>
		<tr>
			<td>Medical gauze</td>
			<td>China Health Materials Co.</td>
			<td>S-7112</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Blunt-nose forceps</td>
			<td>Shang Hai Surgical Instruments Inc.</td>
			<td>N-551</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Surgical tapes</td>
			<td>3M Company</td>
			<td>3664CU</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Gauze sponge</td>
			<td>Fu Kang Sen Medical Device CO.</td>
			<td>BB5447</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Yu Lin Surgical Instruments Inc.</td>
			<td>175C</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Skin scissor</td>
			<td>Carent</td>
			<td>255-17</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ning Bo Surgical Instruments Inc.</td>
			<td>3325-77</td>
			<td>Surgical</td>
		</tr>
		<tr>
			<td>Syringe and 25-G needle</td>
			<td>MISAWA Inc.</td>
			<td>3731-2011</td>
			<td>Scale: 1 ml</td>
		</tr>
	</tbody>
</table>

laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo

Mikro fonksiyonel durumunu mikrovasküler vasomotion oksijen ve besin teslim ve karbon dioksit ve atık ürünlerin kaldırılması için önemlidir. Mikrovasküler vasomotion bozulma mikro ile ilgili hastalıkların gelişiminde çok önemli bir adım olabilir. Buna ek olarak, son derece bozukluklarına pankreas adacık endokrin işlevi desteklemek için uyarlanmıştır. Bu bağlamda, pankreas adacık mikrovasküler vasomotion fonksiyonel durumunu pankreas adacık işlevini etkileyebilir anlaması mümkün görünüyor. Pankreas adacık mikrovasküler vasomotion fonksiyonel durumunu patolojik değişiklikleri analiz katkıları belirlemek için uygun bir strateji olabilir o pankreas adacık mikro ilgili hastalıklar, diyabet gibi yapar pankreatit, vb. Bu nedenle, bu iletişim kuralı bir lazer Doppler kan akışı monitör pankreas adacık mikrovasküler vasomotion fonksiyonel durumunu belirlemek ve (ortalama kan perfüzyon, genlik, frekans ve akraba gibi parametreleri kurmak için kullanılmasını açıklar pankreas adacık mikrovasküler vasomotion hız) microcirculatory fonksiyonel durum değerlendirilmesi için. Bir streptozotocin indüklenen diyabetik fare modelinde, pankreas adacık mikrovasküler vasomotion bozulmuş bir fonksiyonel durumunu gözlenen. Sonuç olarak, pankreas adacık mikrovasküler vasomotion vivo içinde değerlendirmek için bu yaklaşım pankreas adacık hastalıkları ile ilgili mekanizmalar ortaya çıkarabilir.

Mikrovasküler vasomotion ölçüm lazer Doppler cihazı bir yarı iletken lazer diyot ile donatılmış bir fotoğrafını şekil 1Aile gösterilir. Kullanıcı arabirimi bilgisayar yazılımı şekil 1Badımında sunulur. Yukarıda belirtilen yöntemle pankreas adacık mikrovasküler vasomotion hemodinamik parametreler diyabetik olmayan kontrol ve diyabetik fare için tespit edildi. Teknikleri, lazer Doppler flowmetry, dahil ol...

Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Lazer Doppler: pankreas adacık mikrovasküler Vasomotion Vivo içinde ölçüm için bir araç

Pankreas adacık mikrovasküler vasomotion adacık kan dağıtım düzenleyen ve adacık β hücrelerinin fizyolojik fonksiyon korur. Bu iletişim kuralı bir lazer Doppler monitör pankreas adacık mikrovasküler vasomotion vivo içinde fonksiyonel durumunu belirlemek ve pankreas kaynaklı hastalıklara pankreas adacık mikro katkıları değerlendirmek için kullanmayı açıklar.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

laser-doppler-tool-for-measuring-pancreatic-islet-microvascular

Research

JoVE Journal

Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

8.2K Görüntüleme.  Chinese Academy of Medical Sciences & Peking Union Medical College. Pankreas adacık mikrovasküler vasomotion adacık kan dağıtım düzenleyen ve adacık β hücrelerinin fizyolojik fonksiyon korur. Bu iletişim kuralı bir lazer Doppler monitör pankreas adacık mikrovasküler vasomotion vivo içinde fonksiyonel durumunu belirlemek ve pankreas kaynaklı hastalıklara pankreas adacık mikro katkıları değerlendirmek için kullanmayı açıklar.

Video: Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Fare lazer Doppler flowmetre Rehberlik İskemik İnme intraluminal Orta Serebral Arter Tıkanıklığı (MCAO) Model

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Tıp

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Rat Lazer Doppler Flowmetri Rehberlik ile İskemik İnme için Endotelin-1 Bağlı Orta Serebral Arter Tıkanıklığı Modeli

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Cerrahi Örnekler kaynaktan İnsan pankreas adacıklarının Lazer Diseksiyon için Geliştirilmiş Protokolü

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

Hücre Takip için vivo 19 F MRG

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Bir Uygun Aracı Olarak Fundus Fotoğraf Epidemiyolojik Çalışmaların Kardiyovasküler Hastalıklar Risk Faktörleri için Mikrovasküler Cevapları Eğitim için

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

Artan Aort Katılığa Ölçme<em&gt; İn Vivo</em&gt; Ultrason Kullanımı Farelerde

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Bir Yüksek içerik<em&gt; İn Vitro</em&gt; Pankreas Adacık β-hücre çoğaltma Keşif Platformu

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fosfor-31 Manyetik Rezonans Spektroskopisi: Ölçme için Aracı<em&gt; İn Vivo</emİnsan İskelet Kası içinde&gt; Mitokondriyal Oksidatif Fosforilasyon Kapasitesi

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Domuz Tibial Allotransplantation cerrahi angiogenez: bileşik Allotransplantation Model yeni bir büyük hayvan kemik skarların

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...