Yazarlar, hayvan bakımı ile yardım için Shawna Rideout-Gros ve Alexandra Garces teşekkür ve onun teknik yardım için Wu Ping olacaktır. Bu iş için hibe desteği hiçbir altında Ulusal Sağlık Enstitüleri tarafından sağlandı. EB003268 ve Kanada Araştırma Sandalyeler Programı.

1. Ultrason ve MR Kurulum Bir MR-uyumlu üç eksenli odaklanmış ultrason sistemi (FUS Instruments, Inc, Toronto, Ontario, Kanada) bu çalışmada kullanılmıştır. İki farklı ultrason dönüştürücüler kullanılmıştır: entegre bir ev inşa 551,5 kHz küresel odaklı dönüştürücü (eğrilik yarıçapı = 60 mm, dış çapı = 75 mm, iç çapı = 20 mm) ve 1.503 MHz, 8-dizi sektörü PZT hidrofon (Imasonic A.Ş., Voray-sur-L&#39;Orgnon, Fransa) tek bir eleman küresel odaklı dönüştürücü (= 0.8, diyafram = 10 cm FN) olarak sürülür. Bir MR-uyumlu PVDF alıcı 8 551,5 kHz transdüktör kullanılan edildiğinde akustik emisyon kaydetmek için kullanıldı. Farklı donanım kullanılması durumunda, aşağıdaki önerilmiştir: <ol><li> 0,25 MHz -1.5 MHz merkez frekansı ve 1 veya daha az bir f-sayı (eğrilik / diyafram yarıçapı) ile uygun, kalibre ultrason probu seçin. </li><li> Bir su banyosu fi yerleştirin1.5 T veya 3 T MR yatakta sıcak, vakum gazından arındırılmış, de-iyonize su ile lled. Su banyosu hayvanları tutabilecek bir üst plaka olmalıdır. Su yüzeyine bakacak şekilde, bir MRI uyumlu üç eksenli konumlandırma aşamasında veya sistem 9 tankında ultrason probu monte edin. </li><li> Bir penetrasyon paneli üzerinden ultrason sürüş donanımları için dönüştürücü kablo çalıştırın. </li><li> Bir fonksiyon jeneratör ve güç amplifikatör kullanarak dönüştürücü sürüş sinyal üretir. Yansıyan elektrik gücü en aza indirmek için harici bir eşleştirme devre kullanın. </li><li> Transdüser odak uzaklığı bilerek, su yüzeyinde dönüştürücü odağı yerleştirin ve görünür bir su çeşme oluşturmak için su yüzeyine ses dalgalarına maruz. Adımlar 1.6 ve 1.7 oluşturulan su çeşme kullanarak odağı kayıt nasıl açıklar. MRG ile odak jel ve görüntüleme çıkan sıcaklık artışı emici bir ısı sonicating içerir odak, yerini daha kesin bir yöntem, 9 bulunabilir &lt;/&gt; Sup. </li><li> MR görüntüleri görünür olacak bir kalem kullanarak odak konumu işaretleyin. Bu odak içinde yerleştirildiği zaman su ile doldurur bir merkezi deliğe sahip bir plaka kullanılarak yapılabilir. Transdüktörün eksenel yönde koordinat su yüzeyine tespit edilebilir ise su dolu deliği, MRI görünür olacak ve iki düzlemde fokal koordinatları sağlayacaktır. </li><li> , Bir 3-düzlem yerelleştiricisinin dizisi kullanılarak görüntü odak işaretleyici ve MRI koordinat sisteminde odak konumu kaydeder. </li><li> Akustik emisyon izlemek için dönüştürücü odak yönelik su banyosunda pasif bir kavitasyon dedektörü (PCD) olarak kullanılan bir MR-uyumlu hidrofon 8 bağlamak, ya da entegre hidrofon bir dönüştürücü kullanın. </li><li> 2 Hz (örneğin ATS460, AlazarTech, Pointe-Claire, Quebec, Kanada) PRFs az 10 ms kadar patlamaları kadar yakalamak için yeterince hızlı olan bir kapsam kartına PCD kabloları çalıştırın. Kablolar pene için topraklanmalıdırtration panel veya RF kalkan gürültüyü en aza indirmek için. </li></ol> 2. Hayvan Hazırlık <ol><li> Isofluorane gaz kullanarak hayvanları uyuşturmak. Bbb isofluorane bozulması 10 üzerinde bir etkiye sahip olduğu için, hayvan deney başlamadan önce gaz 10 dakika kapalı alınmalıdır. Kuruma veya diğer yaralanma korneayı korumak için anestezi başlangıcında her bir gözü de göz yağlayıcı pomad. </li><li> Bir elektrik ustura kullanarak hayvanın baş ve boyun üst kürk tıraş, sonra bir tüy dökücü krem ​​(örneğin Nair, Church &amp; Dwight Co, Princeton, NJ, ABD) kullanılarak kalan kürk kaldırmak ve sabunla yıkayın ve kafa derisi su. </li><li> Bir bridine yıkama tarafından izlenen bir betadin deri fırçalayın ile kuyruk hazırlayın. Kuyruk veni görselleştirmek için alkol ile son wipedown gerçekleştirin. </li><li> Kuyruk damara 3-yollu bir 22 gauge kateter takma ve pıhtı oluşumunu engellemek için heparin / tuzlu su karışımı (33 U / ml) ile yıkayınkateter. </li><li> Intramüsküler enjeksiyon yoluyla enjektabl anestezik (40-50 mg / kg ketamin, 10 mg / kg xylazine) teslim ve isofluorane gelen hayvan çıkarın. </li><li> Üst plaka (Şekil 1) bir delikten su banyosunda temas başın üst ultrason konumlandırma sistemi üzerinde anestezi hayvan sırtüstü yerleştirin. Ultrason küçük bir miktarı belki sıkışıp airbubbles olasılığını en aza indirmek için hayvanın kafanın üst uygulanan jel. </li><li> Konumlandırma sistemi Teyp bacaklar. Kafası Ya bir lokma çubuğunu kullanarak yerinde tutulur, varsa, veya bant çene boyunca sıkıca yerleştirilmiş olabilir. </li><li> Bir havlu ve sıcak tutmak için su battaniye dolaşımdaki hayvan örtün. </li></ol> 3. Hedef Seçimi <ol><li> Bazal eksenel T beynin 2-ağırlıklı ve T 1 ağırlıklı MR görüntüleri elde edin. 1.5 T sc uygun, kafa ölçekli RF-alma yüzeyi bobin MRI ve özel kullanıyorsanızbir parametre olabilir: T 2 ağırlıklı: FSE, TE = 60 ms, TR = 2000 ms Ağırlıklı T 1: FSE, TE = 10 ms, TC = 500 ms </li><li> T hedef seçin 2-ağırlıklı taramalar, ventriküller ve beyin orta hat kaçınarak, ve bir orta beyin derinliğini seçebilmek. </li><li> Hedef konuma dönüştürücü odağı taşıyın. </li></ol> 4. Mikro Hazırlık Definity mikro kabarcıklar (Lantheus Tıbbi Görüntüleme, MA, ABD) BBBD 2,5,7 uyarılan mikrokabarcık aracılı FUS için çeşitli gruplar tarafından kullanılmaktadır. Diğer mikrokabarcık türleri için uygun bir dozaj literatürde 11,12 bulunabilir. <ol><li> Definity mikro kabarcıklar etkinleştirin ve yavaş yavaş 18-gauge iğne ile bir 1 ml şırınganın içine küçük bir hacme kadar çizin. </li><li> Yavaşça ileri geri hareket ettirerek piston enjektör sıkışmış havayı çıkarın. Bu kabarcıkları gibi şırınga vurmayın. </li><li> Normal olarak Definity seyreltilir<html> tuzlu su yavaş yavaş bir şırıngaya Definity gerekli hacmi enjekte etmek Definty 10:01 salin oranında bir tuzlu su. Bir infüzyon iletim kullanıldığında, Definity 50:1 veya 100:1 &#39;e kadar, daha da seyreltilmiş olabilir. </li><li> Hafifçe daha da görünüm elde edilinceye kadar iyice Definity ve tuzlu karıştırmak için şırıngayı ters çevirin. Baloncuklar süspansiyon akıp başlamıştır derhal enjeksiyondan önce şırınganın Nazik inversiyon da gerekli olabilir. </li><li> 0,02 mL / kg Definity, veya 0,2 mL / kg çözeltisi (10:01 seyreltme) göre gerekli doz birimi hesaplamak. </li></ol> 5.. Ultrason Teslimat <ol><li> Düşük görev döngüsü patlamaları değil, sürekli dalga sonications kullanarak, sonication parametreleri ayarlayın. 0.5 MHz&#39;de Uygun sonication parametreleri yerinde basıncı 0.23 MPa, 1 Hz PRF, 2 dakika 10 ms patlamaları vardır. toplam sonication zaman. In situ baskılara uygun 1,5 MHz 0,45-0,5 Mpa civarında düşecek. </li></ol>_content &quot;farklı frekanslarda&gt; Uygun baskıların 0.46 13 mekanik bir dizini kullanarak tahmin edilebilir. <ol start="2"><li> Canlının kafası hala su birleştiğinde olduğundan emin olun. </li><li> Kuyruk damarından kateter yavaşça kabarcıkların enjekte edilir ve 0.5 ml serum fizyolojik ile yıkayın. Enjeksiyon başlaması ile eş zamanlı sonikasyon başlayın. </li></ol> 6. Tedavisinin MRG Değerlendirilmesi <ol><li> Sonikasyon ardından, 0.5 ml serum fizyolojik floş ardından kuyruk damarından kateter yoluyla (örneğin 0.2 ml / kg Omniscan, GE Healthcare) bir MR kontrast madde enjekte edilir. </li><li> Buna zirve geçti kadar T 1 ağırlıklı görüntüleme gerçekleştirin. Başarılı bir şekilde bozulmuştur Sonikasyon siteleri çevre dokulara daha fazla geliştirme gösterecektir. </li><li> Ödemi kontrol etmek için T 2 ağırlıklı görüntüleme gerçekleştirin. Sonikasyon sitelerinde Yüksek sinyal ödem göstergesidir. </li></ol> 7. TemsilciSonuçlar MR kontrast ajanlar başarıyla odaklı ultrason kullanılarak ve kabarcıkların dolaşan BBB ile teslim edilebilir. Şekil 2 tipik öncesi ve sonrası FUS T 1-w görüntüleri gösterir. Şekil 2B bir kontrastlı (CE) T 1-w dört sonike yerlerde farklı odak açıklıklar ile görüntü gösterir. Sonikasyon 1 ve 2, özellikle parlak kontrastlanma gösterirler. Şekil 3 konumlarda 1 ve 2 de ödemi gösteren T 2-w yüksek sinyali ile haberleşmek için görülebilir. T ölçüde 2-w ödem bazen sagital dilimleri daha kolay görüntülenebilmekte. Şekil 4 CE-T 1-w ve T 2-w sajital dilimleri sonikasyon yerleri vasıtasıyla 1 ve 3 gösterir. Ödem konumu 1 değil 3 yerde de görülebilir. Yakalanan akustik emisyon verileri (Şekil 5) spektral analizi harmonik emisyonlar ve / veya stabil kavitasyon oluşmasını ultra / alt harmonik emisyonlar gösterebilir. Harmonalt ve ultraharmonic emisyonu sadece kabarcık etkinliği 14 bir sonucu olarak oluşabilir ederken ics Ayrıca, doku linearitenin ortaya çıkabilir. Yüksek basınçlarda eylemsizlik kavitasyon gösteren geniş bant emisyonları da tespit edilebilir. Ancak bu eylemsizlik kavitasyon 11 olmadan sonications fazla kırmızı kan hücresi ekstravazasyonlar ve microdamage büyük miktarlarda ile ilişkili bulunmuştur. Küçük fokal spot boyutu nedeniyle daha yerel açıklıklar yüksek sonikasyon frekansları sonuçlarının kullanımı. Şekil 6 daha yüksek frekanslar açıklığın küçük bölgeler oluşturmak için kullanılabileceğini göstermektedir. Bu, daha az yakın kafatası etkileri olan etkileri orta beyin soruşturma sağlar. <img alt="Şekil 1.." src="/files/ftp_upload/3555/3555fig1.jpg" /> Şekil 1. Deneysel kurulum. <spBir class = &quot;pdflinebreak&quot;&gt; <img alt="Şekil 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> Şekil 2. Ön (sol) ve sonrası (sağ) tedavisi T 1-w dört sonikasyon yerlerde iyileştirme gösteren bir sıçan beyin görüntüleri. <img alt="Şekil 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> Şekil 3. Ön (sol) ve sonrası (sağ) tedavisi T 2-w T sonikasyon 1 ve 2 de 2-w ödem gösteren bir sıçan beyin (Şekil.2 aynı hayvan) görüntüleri. <img alt="Şekil 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> Şekil 4. Tedavi sonrası sagital T 1-w (sol) ve T 2-w (sağ) aynı sıçan beyninden görüntü olarak Şek. 2 ve 3. Konumu 1 açılış (solda) T 2-w ödem (sağda) ile ilişkilidir. Yer 3 açılış (solda) ancak T 2-w ödemi. </p&gt; <img alt="Şekil 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> Şekil 5. 551,5 kHz tek bir 10 ms patlama sırasında yakalanan verileri Frekans spektrumu. Temel frekans (f 0) yanı sıra, harmonik (2 f 0) ve alt / ultraharmonics (0,5 f 0, 1,5 f 0) görülebilir. <img alt="6 Şekil." src="/files/ftp_upload/3555/3555fig6.jpg" /> Şekil 6. Tedavi sonrası CE-T 1-w aksiyel (sol) ve 1.503 MHz dört yerlerde sonicated bir sıçan beyin sagital (sağda) görüntüler. Bu frekansta BBB açıklıklar daha fazla lokalize olduğu görülmektedir.

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K. Hynynen ve R. Chopra FUS Araçların kurucuları, Inc R. Chopra, A. ve K. Waspe Hynynen hissedarlar FUS Aletleri, Inc K. Hynynen FUS aletleri, Inc araştırma desteği alan

Hayvan ve mikro kabarcık hazırlanması, bu prosedürün en kritik yönleri vardır. Hayvanın kafasına saç tamamen ultrason ışın zayıflattığı önlemek için kaldırılmalıdır. BBB isofluorane anestezi altında bozulabilir, ancak tutarlı açılış ulaşmak için daha zor olur. Bunları kırma önlemek için, yukarı çizerken kabarcıkların zaman bakım ve küçük ölçü ele alınmalıdır, büyük çaplı iğneler kullanılmalıdır. Benzer şekilde, mantıklı kuyruk damar...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Küçük Hayvan Odaklanmış Ultrason Sistemi </td><td> FUS Instruments, Inc </td><td> RK-100 </td></tr><tr><td> Definity </td><td> Lantheus Tıbbi Görüntüleme </td><td></td></tr><tr><td> Omniscan </td><td> GE Healthcare </td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

Kan-beyin bariyeri (KBB) odaklanmış ultrasonu (FUS) bozulması BBB 1-5 engellemeyi için giderek incelenmiştir tekniktir. Daha az boyutu 6 yaklaşık 500 Da fazla molekül ile beyin doku içine vaskülatürün gelen moleküllerin geçişini sınırlar olarak bbb beyin bozukluklarının tedavisi için farmasötik önemli bir engel teşkil etmektedir. FUS bağlı BBB bozulması (BBBD) kimyasal bir avantajı yüksek oranda lokalize olmakla BBBD indüklenmesi anlamına gelir geçici ve 4 reversibl olup sahiptir. FUS endüklenen BBBD aksi yeterli bir konsantrasyonda dokuya teslim olmaz beyin, ilgili terapötik ajanları çok çeşitli etkisini araştırmak için bir araç sağlar. Ultrason parametreleri geniş bir yelpazede BBB 2,5,7 bozarak başarılı kanıtlanmış ise de, hassas hedefleme ile başarılı ayrılmasını sağlamak üzere deneysel prosedür birkaç önemli adımlar vardır. Bu protocol özetliyor nasıl kritik hayvan hazırlık ve deneme mikrokabarcık işleme adımları odaklanarak, sıçan modelinde BBBD oluşturulan MR güdümlü FUS ulaşmak için.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

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

Kan-beyin bariyeri (KBB) ve Mikro-aracılı odaklanmış ultrason bozulması beyinde non-invaziv hedeflenen ilaç dağıtımı için umut verici bir tekniktir<sup&gt; 1-3</sup&gt;. Bu protokol bir sıçan modelinde MR güdümlü transkranial BBB bozulması için deneysel prosedür özetlenmektedir.

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 ...

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

Research

JoVE Journal

Medicine

17.8K Görüntüleme.  Sunnybrook Research Institute. Kan-beyin bariyeri (KBB) ve Mikro-aracılı odaklanmış ultrason bozulması beyinde non-invaziv hedeflenen ilaç dağıtımı için umut verici bir tekniktir 1-3. Bu protokol bir sıçan modelinde MR güdümlü transkranial BBB bozulması için deneysel prosedür özetlenmektedir.

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Manyetik Rezonans Görüntüleme, Volumetrik Bilgisayarlı Tomografi ve Ultrason Kullanımı Meme Kanseri Kemik metastazı bir Nude Rat Model Angiogenezis Multi-modal Görüntüleme

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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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 ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Sıçan Kronik Besin İnfüzyon Bir Model

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

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 ...

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 ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

Tedaviye dirençli Majör Depresif Bozukluk için bir tedavi olarak dmPFC-shamı MRI kılavuzluğunda

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

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) ...