Bu çalışma JB Marshall Vakfı tarafından desteklenmiştir.

1. Prosedür için Hayvan ve Ekipmanları hazırlayın <ol><li> Cerrahi başlamadan önce, cerrahi alan ve hayvan hazırlamak. PE50 boru 12 bir ucunu &quot;içine bir 23-gauge künt iğne sokarak karotis kateter olun. Kateterin ucunu bir yaklaşık 45 ° eğim kesin. Prosedür öncesi ekipman sterilize edin. Saç kaput, cerrahi maske takın ve steril eldiven. </li><li> Cerrahi uygulanır yüzeyi üzerinde bir ısıtma pedi yerleştirin. Bunu etkinleştirin ve ısınmasını bekleyin. Emici bir tezgah pad ile örtünüz. </li><li> Eğer kristaller kalmadan çözüm% 25 mannitol olduğunu doğrulayın. Kristaller varsa, 80 ° C sıcak su banyosunda flakon yerleştirerek ve periyodik çalkalayarak çözeriz. </li><li> Hayvanın kütle kaydedin. </li><li> 0.8 mg / 0,1 ml / dak &#39;lık bir hızla kg / dak dağıtımı için bir propofol seyreltme edin. </li><li> 3 dakika için indüksiyon kutu içinde sıçan yerleştirin. Ayarlar:% 5 izofluran, 2 L / dkvantilatör debi. </li><li> Bir tek kullanımlık havlu üstüne, boyun ve hem iç uyluk tıraş. </li></ol> 2. Sıçan Entübe <ol><li> 3 dakika için indüksiyon kutu içinde hayvan yerleştirin. </li><li> IV kateter ile endotrakeal tüp hazırlayın. Bu boyut ve sıçan suşu için, biz bir 18 ga kateter kullanın. Tüp zarar vermeden yeterli hava akımına izin vermek için yeterince büyük, ancak trakea içine sığacak kadar küçük olmalıdır. Plastik kateterin iğne çekin ve dikkatlice iğne yaklaşık bölünürler. Sivri ucundan 1 cm. Bunted iğne kalemi olacaktır. Iğne geri kateter içine koyun. </li><li> Açılı yüzey üzerinde dişlerin sıçan asın. </li><li> Hayvanın boynuna karşı doğrudan bir ışık Shine ve onun kulakları arasındaki dizilmiş. </li><li> Küçük bir spatula yaklaşık yerleştirin. Orofarinks içine 3 cm. Yavaşça ağzından dilini dışarı çekerken yukarı doğru kaldırın. </li><li> Trakeanın açılması gözünüzde canlandırın. Sen se olacakea küçük bir delik, her nefes ile yakın açın ve. </li><li> Yavaşça deliğe kateter yerleştirin. Bazen plastik trakea kıkırdak girebilirsiniz hissedebilirsiniz. Trakea duyarlıdır. Kateter zorlamayın. </li><li> 1 L / dk ve 60 bpm ve% 2 izofluran için ventilatör ayarlayın. </li><li> Çalışma yüzeyine ventilatör ve teyp tüp kateter bağlayın. </li><li> Hayvan ayak tutam yaparak anestezi olduğunu doğrulayın. </li></ol> 3. Propofol IV Anestezi Kurmak <ol><li> Çalışma yüzeyine dört bacaklarda ve kuyruk taping sıçan sabitleyin. </li><li> Traş boyun ve klorheksidin sabun ile kasık siteleri üç kez fırçalayın. Klorheksidin üç kez durulama ile Sonra fırçalayın. </li><li> IV uzatma seti için propofol şırınga takın. Infüzyon pompası üzerinde şırınga takın. </li><li> Traş uyluk deri yoluyla kesmek bir neşter, kullanma. Kasın kesilmesi kaçının. </li><li> Kuvveti ile kesi açps femoral ven görselleştirmek için. </li><li> Damar açığa arter üzerinde doku dışarı doğru çekin. </li><li> Damar içine 26 ga monoject veteriner IV kateter yerleştirin. Iğne ven duvarı delen, Ne zaman itmek kolay hale hissedeceksiniz. Iğne çekin ve damar içine kateter itin. </li><li> 0,1 ml / dk propofol infüzyonu başlatın. </li><li> 3-5 dk propofol infüzyonu başlayarak ve daha sonra,% 50 O2 ve% 50 N2 O karışımı ile anestezi gaz geçiş izofluran kapatın. </li></ol> 4. Ortak Karotid Arter Bifurkasyon Açığa <ol><li> Bistüri ile boyun orta hat kesi olun. </li><li> Makas veya bistüri ile iki büyük tükrük bezlerinin arasındaki kesin. </li><li> Karotis arter açığa orta hat için lateral doku uzakta teşrih. Doku katmanları açmak için forseps kullanın. Sen forseps ile sunduğunuz parçalarını çıkartın ve minimall olmak belirlemek için makas kullanabiliry damar. Gazlı bez, pamuk bezlerden ve koter Gerekirse kanama kontrol edin. </li><li> Ağır kanama, orta olduysa 3 ml serum fizyolojik ip enjekte. </li><li> Karotis doku çekmeye Retraktörler kullanın. Çatallanma bulun. </li><li> Çatallanma hemen distalinde oksipital arter Açığa. Forseps arasına Dağlamak ve elektrokoter ile ayırın. </li><li> Tam karotid bifurkasyon ve mümkün olduğunca ECA kadar maruz forseps kullanın. </li><li> Oksipital arterin distal ECA kapalı dallanma üstün tiroid arter Açığa. Forseps arasına Dağlamak ve elektrokoter ile ayırın. </li></ol> 5. Eksternal Karotis Arter kateterizasyonu <ol><li> ECA altında 4/0 ipek 2 uzunlukları yerleştirin. </li><li> Sizin hemostatla sütür 1 uzunluğu tut ve (sıçan ayakları doğru) geri kaudal ECA çekin. </li><li> Mümkün w gibi diğer dikiş atın ve (sıçan ağzına doğru) kraniyale kadar kravatITH bir cerrahın düğüm. Bu ECA feda kalıcı bir bağ oluşturur. </li><li> İlk hemostat (kaudal gerilim uygulayarak bir) çıkartın ve diğer sütür (ECA etrafında düğümlü olanı) kapmak ve hemostatla kraniyale nazik gerilim uygulamayın. </li><li> Gevşek başka bir cerrahın düğüm (güvenli değil - ancak bağlı bırakın) çatallanma ve kalıcı bağ proksimal distal. </li><li> 1% arter dinlenmek için lidokain ile yıkayınız. </li><li> Heparinize salin ile kateteri doldurun. </li><li> Mümkün olduğunca yakın bifürkasyonun geçici damar klip yerleştirin. Kateterizasyon için uzay maksimize etmek için bir açıyla Sıra damar klip. Klibi doğru gevşek düğüm kaydırın. </li><li> Microscissors kullanarak bağ sadece proksimal ECA küçük bir arteriyotomi (kesim) olun. Klip ve bitişik harf güvenli olup olmadığını hiç kanama olmamalıdır. </li><li> ECA duvarları kontrol forseps kullanarak, damar içine kateter eğimli sonuna beslemek. </li&gt; <li> Gevşek dikiş düğüm ile kateter sabitleyin. Kateter etrafında arter bağla. </li><li> Klipsini çıkarın ve pamuklu bir bezle temizleyin. </li><li> Çatallanma distalinde yaklaşık 1 mm kateter ilerletin. Damar immobilize sağlamak için düğüm proksimalinde forseps ile arter tutun. </li></ol> 6. Mannitol yönetme <ol><li> Herhangi biri hava kabarcıkları yaratmadan 8 &quot;IV uzatma grubu ve mannitol içeren bir 6 ml şırınga Dolgu 37 inkübatör içinde bu depolayın ° C. Not: çalışma hızla mannitol - bu kolayca oda sıcaklığında çözeltiden çökelebilir olacaktır.. </li><li> BBBD görselleştirmek için, Evans mavisi boya yönetmek olabilir ve bariyer kesintiye uğramış olan beyin bölgeleri girmek izlemek. Bunu yapmak için, propofol infüzyonu için bacakta üzerinde yeni bir IV sitesi üzerinden hayvan% 2 Evans Mavisi, 2 ml / kg verir. </li><li> Filtre ve hava bub kaldırmak için mannitol ile doldurun 5 mikron ayarlanmış uzatma takınbles. Sonra, hava kabarcıkları oluşturarak olmadan musluğunu bu ekleyin. </li><li> 0.09 25 ml / sn sn mannitol Yönet. ICA proksimalinde yapılan bir noktada karotis içinde akışı ile dengeye ulaşmak için mannitol için izleyin. Bu sıçan kalp hızı ile pulse görünmelidir. Mannitol basitçe kalp aşağı doğru akar Eğer sıçan kan basıncı üstesinden geldiler. Bu hayatı tehdit eden komplikasyonlara ve yoksul BBBD neden olabilir. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

BBBD etkililiğini maksimize birkaç anlamı vardır. Bu cut-down aşamasında kanamayı en aza indirmek için önemlidir. Kan basıncı ve kalp hızı önemli kanama etkilenebilir ve bu faktörlerin BBBD 13 derecede etkilediği bilinmektedir. Kanama gibi üstün tiroid ve bölünmesi gerekir oksipital arterler gibi büyük gemiler, Arter için sütürler kullanılarak azaltılabilir. Ayrıca, elektrokoter gemiler bölmek ve zengin bir kan dolaşımı alanları incelemek için kullanılabilir. Özellikle karo...

<table border="1"><tbody><tr><td> Malzeme Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlamak </td></tr><tr><td> Long Evans sıçan </td><td> Harlan Laboratuvarlar </td><td></td><td> 210-250 g, erkek </td></tr><tr><td> PE 50 Borular </td><td> Beckton-Dickinson </td><td></td><td></td></tr><tr><td> 18 çap x 2.5 &quot;IV kateter </td><td> Terumo </td><td></td><td> ET tüpü için </td></tr><tr><td> 30 &quot;IV uzantılı setleri </td><td> Abbott </td><td></td><td></td></tr><tr><td> 26 göstergesi veteriner IV kateter </td><td> Monoject </td><td></td><td></td></tr><tr><td> Evans mavisi boyasının </td><td> Sigma </td><td> E2129 </td><td></td></tr><tr><td> Iki kutuplu </td><td> Codman </td><td></td><td></td></tr><tr><td> Filtre, 5 mikron </td><td> Braun </td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

Bazal membran ve astrosit uç ayakları ile birlikte sıkı kavşaklar ile endotel hücreleri kan-beyin bariyerini 1 forma serebral kan damarları çevreleyen. Bariyer seçici kan ve büyüklüğüne ve yüküne dayalı beyin arasında geçmesini molekülleri dışlar. Bu fonksiyon, nörolojik bozukluklar için tedavilerin sunulmasını engelleyebilir. Kemoterapötik ilaçlar arasında bir sayı, örneğin, etkili bir şekilde tümör hücrelerini 2 ulaşmak için, kan-beyin bariyerini değildir. Bu nedenle, kan-beyin bariyerinin ötesine ilaçların ulaştırılması, iyileştirilmesi bir ilgi alanıdır. Beyin için ilaçların ulaştırılması arttırmak için en yaygın yöntem doğrudan doğruya beyin infüzyon ve kan-beyin bariyeri bozulma 3 vardır. Tedavilerin beyine doğrudan intraserebral infüzyon olduğunu garanti eder, ancak bu yöntem ilacın 4 dağıtmak için sınırlı bir yeteneği vardır. Kan-beyin bariyeri bozulması (BBBD) alDolaşım sistemi doğrudan akış alçak ilaçların beyin içine ve böylece daha etkili dağınık tümör hücrelerine ulaşır. Bariyer kesintileri üç yöntem ozmotik bariyer kesintileri, farmakolojik bariyer kesintileri ve kabarcık ile odaklanmış ultrason içerir. Neuwelt öncülük Ozmotik bozulması, onların sıkı kavşaklar küçültmek ve bozmaya neden kan-beyin bariyerinin hücre dehydrates% 25 mannitol hipertonik bir solüsyon kullanılır. Bariyer bozulması, aynı zamanda, histamin 5 ve 6 bradikinin gibi vazoaktif bileşikleri ile farmasötik olarak gerçekleştirilebilir. Ancak bu metot, özellikle beyin tümörü bariyer 7 için seçicidir. Buna ek olarak, RMP-7, bradikinin peptid analog,% 25 mannitol 8 ile ozmotik BBBD ile kafa kafaya göre aşağı olarak tespit edilmiştir. Başka bir yöntem, mikro-kabarcık ultrason kontrast ajanları ile birlikte odaklanmış ultrason (FUS), aynı zamanda gösterilmiştirgeri dönüşümlü olarak kan-beyin bariyerini 9 açmak için kullanılır. FUS karşılaştırıldığında olsa da,% 25 mannitol bu translasyonel araştırma 10-12 için kanıtlanmış bir araçtır yapar insan hastalarda güvenliği uzun bir geçmişi vardır. BBBD gerçekleştirmek için, manitol doğrudan doğruya beyin arteryel dolaşım içine yüksek bir oranda teslim edilmelidir. İnsanlarda, endovasküler bir kateter hızlı, direkt akım sağlanabilir beyin yönlendirilmektedir. Bu protokol modelleri insan mümkün olduğunca yakından BBBD. Bir cut-down karotis arter bifurkasyon takiben, bir kateter ECA içine retrograd eklenir ve internal karotis arter (ICA) dolaşıma doğrudan mannitol teslim etmek için kullanılır. Propofol ve N2 O anestezi bariyer bozulma 13 etkinliğini maksimize etmek için yetenekleri için kullanılır. Düzgün çalıştırılırsa, bu prosedür güvenli, etkili ve reversibl kan-beyin bariyeri ve doğaçlama açmak için yeteneği vare genellikle beyin 8,13,14 ulaşmaz ilaçların ulaştırılması.

<img alt="Şekil 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> Şekil 1. Evans mavisi boya ekstravazasyon yoluyla kan-beyin bariyerini bozulma görselleştirme. Evans mavisi boya albümin bağlanır ve fizyolojik koşullar altında beyin damar dışına olmayan bir pigmenttir. Beynin bir yan üzerinde, kan-beyin bariyerinin kesintisi olmayan hemisfer kesilmiş değişmeden kalırken, Evans mavisi hemisfer kesilmiş mavi girmek ve leke olanak verir. Bu neden...

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

Kan-beyin bariyeri bozulması, beyin bazı ilaçların ulaştırılması, yardımcı olur. Mannitol fiziksel bariyer düzenini bozacak içi arterially kan damarları çevreleyen daralır hücreleri teslim.

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 ...

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JoVE Journal

Medicine

19.3K Görüntüleme.  Vanderbilt University School of Medicine. Kan-beyin bariyeri bozulması, beyin bazı ilaçların ulaştırılması, yardımcı olur. Mannitol fiziksel bariyer düzenini bozacak içi arterially kan damarları çevreleyen daralır hücreleri teslim.

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Sıçan Modelinde Transkraniyal Odaklanmış Ultrason kullanarak Kan-beyin bariyeri MRG eşliğinde bozulması

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Tıp

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Bir olarak beyin mikrovasküler endotel hücreleri (cend) olarak gerin<em&gt; In Vitro</emKan Beyin Bariyer&gt; Travmatik Beyin Hasarı Modeli

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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 ...

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 ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Rat Karotis Balon Yaralanma Modeli Anti-vasküler Tadilat Therapeutics Test

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Tükürük hipofonksiyonunun bir Sıçan Modelinde Retroductal Submandibuler Bezi damlatma ve yerelleştirilmiş fraksiyonlara Işınlama

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Kan-beyin Bariyeri Mesh Benzeri Gemi Oluşumu ve Kesintisini Değerlendiren İn Vitro Tahliller

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.