Yazarlar araştırma için mali destek Erdebil eyaleti Üniversitesi Tıp Bilimleri (: Ardabil, Iran) için Şansölye Yardımcısı için müteşekkir olan (Hayır vermek: 9607).

Tüm yordamları hayvan çalışmaları yürütmek için: Ardabil Üniversitesi Sağlık Bilimleri Araştırma Konseyi kılavuzlarınıza uygun olarak gerçekleştirilmiştir (etik Sicil No: IR. ARUMS. rec.1394.08). bu görüntülenmeyecektir çalışmada, biz mera Enstitüsü (Tahran, Iran)'nden elde edilen yetişkin erkek Sprague-Dawley Rat (300-350 g) kullanılır.
1. anestezi ve Flowmetry
<ol><li>Anestezi % 4 isoflurane kullanarak teşvik ve ameliyat süresince Nitröz oksit (% 70 v/v) ve oksijen (% 30 v/v) karışımı isoflurane (1-%1,5) ile sürdürmek.</li>
	<li>Anaesthetized bir hayvan üzerinde bir geri besleme kontrollü Isıtma battaniye eğilimli duruş yerleştirin ve bu ısıtıcının bağlı bir rektal sonda aracılığıyla 37±0.5 ° C de vücut ısısını korumak.</li>
	<li>Oftalmik merhem az miktarda kuruluğu önlemek için her iki gözde uygulanır.</li>
	<li>Kafatasının sol tarafında cerrahi bölge elektrik makasları ile tıraş. Betadine çözüm bir gazlı bez yastık ile cilt dezenfekte için kullanın. Bu bölge % 70 etanol içeren steril bir yastık ile durulayın ve üç devir17için her iki adımları yineleyin.</li>
	<li>Analjezi için 0.2 mL % 0.5 bupivacaine subkutan ameliyat bölgesi17enjekte.</li>
	<li>(Sol göz yanal canthus sol kulak tabanına uzanan) kafatası üzerinde 1 cm uzun cilt kesi yapmak ve kafatası ortaya çıkarmak için sol temporalis kas incelemek. Daha sonra bir küçük burr deliği 5 mm lateral için ve 1 mm Doppler Flowmetre kalem sonda ucu yerleşimini kolaylığı için bregma18 arka oluşturun.</li>
	<li>Sıçan eğilimli sırtüstü konumuna gelen açın ve sonra lazer kalem sonda önceden delinmiş kafatası kör-bölgesel beyin kan akımı (rCBF) izlemek için deliğe sok.</li>
	<li>Her hayvanın temel rCBF kaydetmek ve bu % 100 olarak düşünün. Özellikle, her rCBF fazla % 80 azalma tespit edilen19,20ne zaman orta serebral arter tıkanıklığı (MCAO) başlatılır.</li>
</ol>2. indüksiyon Fokal Cerebra iskemi
<ol><li>Boyun bölgesi tıraş ve cilt betadin çözüm ve % 70 etanol ile dezenfekte. Analjezi17için ameliyat sitedeki subkutan %0,5 bupivacaine 0.2 mL enjekte.</li>
	<li>2 cm uzun cerrahi kesi sol ortak Karotid arter (LCCA) erişmek için boyun ventral yüzeyinde olun. Bifurkasyon dış Karotid arter (ECA) ve iç Karotis arter (ICA) erişmek için LCCA komşu fasya ve vagus siniri izole et.</li>
	<li>Ya LCCA ya da 5-0 ipek sütür istihdam ECA kalıcı olarak ligate ve pterygopalatine arter seviyeye ücretsiz ICA incelemek.</li>
	<li>Gevşek 5-0 ipek sütür LCCA, etrafında kravat yerleştirin ve ardından geçici olarak ICA ile vasküler bir mikro-klip kelepçe.</li>
	<li>LCCA daha önce yerleştirilmiş gevşek kravat önce küçük bir kesi yapmak ve 4-0 naylon silikon kaplı dikiş ICA luminal uzaya takın ve naylon dikiş güvenli ve kan sızıntı önlemek için LCCA çevresinde dikiş sıkın.</li>
	<li>Vasküler mikro-klip ICA kaldırın. Sonra bir silikon kaplı intraluminal filament MCA kökenli tıkanıklığı gösterir rCBF belirgin bir düşüş gözlemleyerek kadar ilerlemek. (90 dk) iskemik dönemin sonunda, reperfüzyon intraluminal dikiş21çekilerek başlatın.</li>
</ol>3. juguler ven Cannulation ve Evans mavi (EB) enjeksiyon
<ol><li>1 cm boyuna kesi boynundan orta çizgi sol tarafında yapmak ve sonra açık açık sol juguler ven (LGV) dış dalı erişmek için uzak yüzeysel fasya incelemek. Sonra kalıcı olarak 5-0 ipek sütür ile LGV kafatası sonuna ligate. Gevşek damar çevresinde iki bağları yerleştirin ve ardından geçici olarak kelepçe LGV bir vasküler mikro-kelepçe ile kardiyak sonu.</li>
	<li>Arasında iki dikiş, LGV küçük bir insizyon attım kazanmama ve LGV luminal uzaya heparinized serum dolu kateter yerleştirin ve yaklaşık 10 mm. daha sonra ilerlemek, kateter güvenli ve kanama önlemek için damar çevresinde bitişik harfler sıkın. Az miktarda damar çöken önlemek için bir serum enjekte.</li>
	<li>Reperfüzyon döneminin başlangıcında, yavaş yavaş bir 5 dk süre içinde EB boya (%2 EB çözüm serum fizyolojik içinde 1 mL/kg) enjekte et. Daha sonra kanül 0.5 mL normal salin enjeksiyonu ile yıkama ve enjeksiyon kanül ven12,22çekilme.</li>
	<li>Son olarak, boyun ve baş insizyon dikiş ve 0,05 mg/kg buprenorfin intraperitoneally enjekte (IP) ve enjeksiyon ameliyat sonrası analjezi17reperfüzyon dönemde her 6-8 h tekrarlayın.</li>
	<li>Hidrasyon kurtarma kafes17sağlamak için Önceden ısıtılmış saline(IP) 5 mL enjekte.</li>
	<li>Yer özel kurtarma Kafesteki hayvan sıcaklık ve klima kontrol ile donatılmış ve hayvan kurtarma anestezi üzerinden izleyebilirsiniz.</li>
</ol>4. kan beyin bariyerini geçirgenliği değerlendirilmesi
<ol><li>Reperfüzyon, 24 saat sonra derin sodyum tiyopental hayvanla anestezi. Göğüs boşluğuna, göğüs kemiğinin altında küçük bir delik yaparak açın.</li>
	<li>Sağ atrium içinde küçük bir açılış yapmak ve Önceden ısıtılmış % 0,9 serum (37 ˚C) sol ventrikül atrium gelen normal tuzlu çıkar hale gelinceye kadar renksiz23EB uzak dolaşım yıkamak 15 dakika ile 110 mmHg basınçta 250 mL enjekte.</li>
	<li>Hemen beyin kafatasından çıkarın ve beyin matris yerleştirin. Olfaktör ampul ve beyincik incelemek ve sonra temiz bir tıraş bıçağı kullanarak orta hat boyunca (Lesioned) sol beynin sağ hemisfer ayırın.</li>
	<li>Her hemisphere tartmak ve onları ayrı ayrı 2.5 mL fosfat tamponlu tuz homojenize ve bu 2 bir girdap makine kullanarak en az 2.5 mL trichloroacetic asit (% 60) ile karıştırın.</li>
	<li>Beyin örnekleri 1,322 x g de 30 dk santrifüj kapasitesi ve örnekleri 4 ° C buzdolabı 10 dakika soğumasını bekleyin.</li>
	<li>EB Absorbans Spektrofotometre tarafından 610 nm23, tahmin etmek için supernatants kullanın.</li>
	<li>EB konsantrasyonları standart bir eğri ve hızlı sonuçlar karşı µg/g beyin doku hesaplayın.</li>
</ol>5. EB standart eğri imalatı
<ol><li>EB 10 örnek çözümler EB fosfat tamponlu tuz 5 ml, 1-10 µg konsantrasyonları ile hazırlayın.</li>
	<li>Her örneği 610'Absorbans değerleri ölçmek bir Spektrofotometre kullanarak nm.</li>
	<li>Elektronik tablo ve X ekseni üzerinde mevcut EB konsantrasyonları ve Absorbans değerleri Y ekseni (şekil 1) kullanarak bir dağılım grafik olun.</li>
	<li>Denklemi ile eğrisi doğrusal bir eğilim-satır tanımlayın. Daha sonra örnekleri EB konsantrasyonu elde etmek için kullanabilirsiniz.</li>
	<li>EB ekstravazasyonu kesin sonuçları µg/g beyin doku ağırlık rapor.</li>
</ol>6. sahte işlem
<ol><li>Sham işletilen grubunda (dahil olmak üzere bir kesik boyun bölgesi ve EB enjeksiyon yapma) fareler aynı cerrahi işlemle gerçekleştirmek ancak MCAO dışarıda.</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Bottiger, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cerebral+ischemia+due+to+cardiocirculatory+arrest+in+mice+causes+neuronal+degeneration+and+early+induction+of+transcription+factor+genes+in+the+hippocampus.">Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus.</a> Brain Res Mol Brain Res. 65 (2), 135-142 (1999).</li></ol>

Yazarlar ifşa gerek yok.

Şimdiye kadar autoradiography ve algılama, radyoaktif izleyiciler24,25, ayirt mikroskobu26,27ve EB ekstravazasyonu tekniği20, gibi çeşitli yöntemleri 23 kan - beyin bariyerini zarar değerlendirmek için kullanılmaktadır. EB boya ve serum albümin bağlamak güçlü yapabiliyor bir izleyici vasküler sızıntı tespit ve BBB arıza11,28,29miktarının için kullanılır. Son derece kabul edilen ve güvenilir bir yöntem olarak EB ekstravazasyonu teknik iskemik inme dahil olmak üzere farklı beyin yaralanmaları tarafından etkilenen BBB bütünlüğü üzerine doğrudan bir tahmin sağlar.
BBB vivo içinde değerlendirilmesi araştırmacılar iskemi indüklenen vasogenic Beyin ödemi mümkün patofizyolojik mekanizmaları incelemek için ve yeni tedavi müdahaleler bulmak için sağlar. Bu model Özel İmkanlar gerektirmez ve deneyler (% 80'den fazla)13,20bir yüksek başarı oranı ile güvenilir sonuçlara yol açabilir. Beyin dokusu doğrudan erişim, bu modeli son derece hassas Değerlendirmeler BBB bütünlüğü sağlar ama uzun vadeli çalışmalar için sınırlı.
BBB tarafından iskemik inme neden olduğu patolojik değişiklikler üç aşamada geliştirmek: (saat içinde), akut alt akut (gün saat) ve kronik (ay gün)30,31. Belli ki, en erken tedavi müdahaleler akut patolojik aşamasında değerli koruyucu etkiler üretmek. EB dozu ve süresi noktası enjeksiyon iskemik hakaret takip BBB dinamik yapısı nedeniyle güvenilir sonuçlar elde etmek için iki önemli parametre vardır. Bu nedenle, enjeksiyon yolu ile yavaş yavaş bir ven kanülü uygun doz (1 mg/kg serum fizyolojik içinde %2 EB çözelti) reperfüzyon süresinin başlangıcından sonra kullanarak EB boya önemli bir faktördür ve kontur erken dönemlerinde patofizyolojik değişiklikler çalışma sağlar .
Çeşitli deneysel yöntemler iskemik inme çalışma girmiştik. Bu deneysel modelinde, koşullar benzer insan kontur21,32oluşturur intraluminal filaman yöntemi ile MCAO kullanılan. Bu basit ve güvenilir bir tekniktir; Ancak, yürütme daha fazla teknik performansının arttırılması ve onun doğruluğunu sağlamak için teknik bazı noktaları dikkate almak gerekir. Kan basıncı ve kan gazı izlenen33,34,35olması gerekir iken vücut ısısı fizyolojik aralığı içinde ameliyat sırasında tutulmalıdır. Sürekli rCBF lazer Doppler Flowmetre ile kayıt ve uygun hazırlanan kullanarak silikon kaplı filaman sadece MCAO başarı oranı artırabilir, ancak aynı zamanda ölüm oranı azaltır.

Vasogenic Beyin ödemi kan - beyin bariyerini (BBB) bozulma nedeniyle iskemik inme önemli bir komplikasyonu ve inme hasta1,2sağkalım oranı önemli bir belirleyici kalır. Kan - beyin beyin kapiller endotel hücreleri tarafından (BCECs) kurdu ve farklı nörovasküler bileşenler (Örneğin, BCECs, perisitlerden, astroglial ve nöronal hücrelerin3arasında sıkı kavşak) oluşur, bariyerini (BBB), sağlar bir Merkezi sinir sistemi (MSS) ve periferik dolaşım4,5arasında uzman ve dinamik arabirim. İskemi-reperfüzyon yaralanmalar gibi hakaret BBB işlevsel bütünlüğü bozabilir ve lökosit sonuçta beyin iltihabı ve birincil beyin yaralanmaları tetiklemek beyin parankimi dolaşan sonraki penetrasyon yol 6 , 7. hayvan modelleri felç oluşumunu takip BBB disfonksiyon kesin tespiti için ihtiyaç vardır. Patofizyolojik mekanizmaları temel ve yeni nöroprotektif stratejileri tanıtımı için büyük önem böyle modellerdir. BBB vitro hücre kültür tabanlı modelleri son derece geliştirilmiş ve BBB Fizyopatoloji8,9,10moleküler çalışma için kullanılan. Yine de, insan klinik koşullara benzer BBB iskemik hasar üretmek, in vivo hayvan modelleri de bu konuda çok değerli bulunmaktadır. Evans mavi (EB) ekstravazasyonu nicel tespiti değerlendirme BBB bütünlüğü ve nörodejeneratif hastalıklar, iskemik inme11, de dahil olmak üzere bir işlev için kullanılan bir iyi kabul edilen ve hassas teknik olduğunu 12 , 13 , 14. bu deneysel herhangi bir laboratuvarda, tekrarlanabilir mümkün, maliyet-etkin ve tamamen geçerli bir yöntemdir. Onun uygulanması gibi radyoaktif izleyiciler15 veya diğer yöntemleri için önkoşul olan manyetik rezonans görüntüleme (MRG)16, gelişmiş donanım gerektirmez. Bu makalede, biz kapsamlı bir şekilde BBB değerlendirme EB ekstravazasyonu iskemik inme fare modellerinde kullanarak temel teknik işlemleri göstermek.

<table><tbody><tr><td>Isoflurane</td><td>Piramal</td><td>AWN 34041100</td><td>20 - 25 &amp;deg;C</td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride (TTC)</td><td>Molekula</td><td>31216368</td><td>4 years</td></tr><tr><td>Sprague&amp;ndash;Dawley rats&amp;nbsp;</td><td>Pasture Institute (Tehran, Iran)</td><td></td><td>300-350g</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>314-13-6</td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>76-03-9</td><td>2 years</td></tr><tr><td>Bupivacaine HCl (0.5%)</td><td>Delpharm Tours</td><td></td><td>below&amp;nbsp; 25 &amp;deg;C</td></tr><tr><td>Bupernorphine</td><td>Exir (Iran)</td><td></td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>497-19-8</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate</td><td>EMD Millipore&amp;nbsp;</td><td>231-448-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;7447-40-7</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>silicone(Xantopren)</td><td>Heraeus</td><td>EN ISO 4823</td><td></td></tr><tr><td>Activator universal plus</td><td>Heraeus</td><td>66037445</td><td></td></tr><tr><td>Micro-Dissecting forceps</td><td>Stoelting</td><td>52100-41</td><td></td></tr><tr><td>Spring Scisors</td><td>Stoelting</td><td>52130-00</td><td></td></tr><tr><td>Operating&amp;nbsp; Scissors</td><td>Roboz</td><td>52140-70</td><td></td></tr><tr><td>Brain matrix&amp;nbsp;</td><td>Stoelting</td><td>51390</td><td></td></tr><tr><td>Anesthesia Machine for Small Animals |</td><td>&amp;nbsp;Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Power Lab system</td><td>AD Instruments</td><td>ML880</td><td></td></tr><tr><td>Laser Doppler flowmeter</td><td>AD Instruments</td><td>ML191</td><td></td></tr><tr><td>Heating feed back system</td><td>Harvard Appratus</td><td>72-7560</td><td></td></tr><tr><td>Vascular micro clamp</td><td>FineScience Tools</td><td>18055-03</td><td></td></tr><tr><td>Silk 5-0 suture thread</td><td>Ethicon</td><td>682G</td><td></td></tr><tr><td>Ethilon 4-0 suture thread&amp;nbsp;</td><td>Ethicon</td><td>EH6740G</td><td></td></tr></tbody></table>

an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

İskemik inme vasogenic serebral ödem ve kan - beyin bariyerini (BBB) imha aracılık ettiği sonraki birincil beyin hasarı yol açar. Fareler indüklenen iskemik inme ile kurulan ve in vivo modelleri BBB işlevsel bütünlüğü araştırmak için kullanılan. Evans mavi (EB) spektrofotometrik algılama beyin örneklerinde iskemik yaralanma ile araştırma ve geliştirme yeni tedavi yöntemleri için güvenilir gerekçe sağlayabilir. Bu yöntem tekrarlanabilir sonuçlar üretir ve özel ekipman gerek olmadan herhangi bir laboratuvar geçerlidir. Burada, indüksiyon Sıçanlarda iskemik inme takip EB ekstravazasyonu algılamayı görüntülenmeyecektir ve teknik kılavuz mevcut.

Karşı sahte ile çalışan fareler sol hemisfer sağ hemisfer EB düzeylerinde anlamlı bir fark yoktu (1.06 ± 0,1 µg/g ve 1,1 ± 0,09 µg/g, sırasıyla). Rakamlar 2A-2B, indüksiyon geçici iskemi gösterildiği gibi (90 dk iskemi / 24 h reperfüzyon) EB seviyesi önemli bir fark neden (10.41 ± 0,84 µg/g, p < 0.001) ile karşılaştırıldığında ilgili iskemik sıçan sol yarıküre'deki sham çalışan fareler yarımkürede. Toplu olarak, bu bulgular normal şartlarda EB kolayca beyin parankimi BBB geçemez ve serebral iskemik hakaret EB ekstravazasyonu BBB (2A ve 2B) geliştirilmiş bir geçirgenliği aracılığıyla teşvik göstermektedir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57156/57156fig1.jpg"> Resim 1 : Standart eğri Absorbans değerleri üzerinden EB konsantrasyonu belirlemek için kullanılır. <a href="/files/ftp_upload/57156/57156fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57156/57156fig2.jpg"> Resim 2 : BBB bozulma EB ekstravazasyonu 24 saat sonra iskemik inme tarafından değerlendirilmesi. Sham işletilen ve iskemik hayvanlar(a)beyinlerinde fotoğrafı. EB ekstravazasyonu (mavi renk) beyin dokusunda yoğunluğunu ölçüde lesioned yarımkürede BBB bozulma kaynaklanmaktadır. (Lesioned) soldan hazırlanan örnekleri EB konsantrasyon ve sağ hemisfer sham işletilen ve iskemik hayvanlar (B) beyinde (n = 6, *p< 0,001 sol hemisfer sham grubunda, pkarşılaştırıldığında < 0,001 ile karşılaştırıldığında Ipsilateral hemisfer aynı grup). <a href="/files/ftp_upload/57156/57156fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Kan - beyin bariyerini bozulma iskemik inme, bir fare modeli Vivo içinde değerlendirilmesi

Bu yordamı genel amacı fare modelleri iskemik inme kan - beyin bariyerini bozulma vivo içinde değerlendirmesi için son derece tekrarlanabilir bir teknik sağlamaktır.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

an-vivo-assessment-blood-brain-barrier-disruption-rat-model-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

11.3K Görüntüleme.  Ardabil University of Medical Sciences. Bu yordamı genel amacı fare modelleri iskemik inme kan - beyin bariyerini bozulma vivo içinde değerlendirmesi için son derece tekrarlanabilir bir teknik sağlamaktır.

Video: An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Endovasküler Dikiş Rat Orta Serebral Arter Tıkanıklığı Fokal Serebral İskemi Model

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Nörobilim

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Kan-beyin bariyer bozulması arasında Rat Modeli Nörovasküler Therapeutics Hedefli izin ver için

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Tıp

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Ayarlama-up bir<em&gt; In Vitro</emSıçan Kan-beyin bariyerinin&gt; Model (BBB): BBB geçirimsizliği ve Reseptör aracılı Ulaştırması Bir Odak

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

İskemik İnme In Vitro Çalışması için İnsan Kan-Beyin Bariyerinin Üçlü Birincil Hücre Kültürü Modeli

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

Biyokimya

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Farelerde Tekrarlanan Hipoksik Önkoşullanma ve Geçici Orta Serebral Arter Tıkanıklığı ardından Nörovasküler Koruma Kantitasyonu

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Kan - beyin bariyerini geçirgenliği FITC etiketli albümin nörodejeneratif hastalık, bir fare modeli intravenöz infüzyonu tarafından değerlendirilmesi

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Vivo kan - beyin bariyerini geçirgenliği tahlil Fluorescently kullanarak farelerde izleyiciler etiketli

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Tek Bir Kemirgen Beyin Örneklerinde İnme Sonrası Serebral Ödem, Enfarktüs Bölgesi ve Kan-Beyin Bariyeri Kırılımının Ölçülmesi

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

İskemik inmenin tavşan modelinde kan basıncının gerçek zamanlı izlenmesi ve modülasyonu

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

<meta charset="utf8"></head><body>Sıçan MCAO İnme Modellerinde Hücre Ölümünü İncelemek İçin İmmünofloresan Boyama

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

<meta charset="utf8"></head><body>İskemik İnmenin Sıçan Modelinde İmmünofloresan ile Hücre Ölümü Sinyalinin Değerlendirilmesi

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

İskemik İnmenin Sıçan Modelinde İmmünofloresan ile Hücre Ölümü Sinyalinin Değerlendirilmesi

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...