Biz Dr minnettarız. Bize MDA-MB231Br hücreler sağlamak için Diane Palmieri ve NCI Patricia Steeg. Biz teknik ve meslekdaşlar destek için Dr Ralph Mason, Bay Jason Reneau ve Perran Lopes teşekkür ederim. Bu çalışma DOD IDEA Awards W81XWH-08-1-0583 ve W81XWH-12-1-0317 tarafından kısmen desteklenmiştir. MR deneyler Gelişmiş Görüntüleme Araştırma Merkezi, bir NIH BTRP # P41-RR02584 tesis yapıldı ve ultrason rehberliğinde intrakardiyak enjeksiyon 1S10RR02564801 altında VisualSonics Vevo 770 ile gerçekleştirildi.

Bütün hayvan prosedürleri Teksas Üniversitesi Güneybatı Tıp Merkezi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1.. MDA-MB231/Br-GFP Hücreler hazırlanması <ol><li> % 10 FBS,% 1 glutamin ve% 1 penisilin / streptomisin içeren DMEM ortamı içinde (nazik Dr. Palmieri ve Steeg, NCI tarafından sağlanan) MDA-MB231/Br-GFP hücrelerini almak ve kültür. </li><li> Hücreler ve ortamın rengi durumunu dikkate alınız, ve her 2-3 günde bir ortam değiştirin. </li><li> Tripsinize ve adımda tarif edildiği gibi% 80 izdiham 1.3.1-1.3.5 ulaşıldığında hücreleri toplamak: <ol><li> Tamamen eski orta çıkarın ve yavaşça Hücreleri yıkamak için 5 ml PBS (1x) ekleyin. Çanak PBS çıkarın. </li><li> Çanak 1.5 ml Tripsin ekleyin, tüm hücreler tripsin tarafından karşılanmaktadır sağlamak için hafifçe çanak eğin. Geri hücre kuluçka makinesi için çanak koyun ve 1 dakika boyunca 37 ° C&#39;de tutun. </li><li> Kuluçka çanak alın ve gözlemlemekhücreler sağlamak için bir optik mikroskop altında, hücreler çanak ayırmak. </li><li> Tripsin etkisini durdurmak için 3 ml orta ekleyin. Bir santrifüj tüpüne hücre karışımını toplamak. 5 dakika boyunca 2,000 rpm&#39;de santrifüje karışımı. </li><li> Dikkatlice Ortamı çıkarın ve homojen hale gelinceye kadar 5 ml serum barındırmayan ortam içinde hücreler tekrar süspansiyon haline getirin. </li></ol></li><li> Uygun hücre sayısını ve 100 ul hacim içinde 1.75 x 10 5 hücre bir son konsantrasyona bulunan serumsuz DMEM ortamı içine tekrar süspansiyon. <ol><li> 50 ul hücre karışımı alın ve 50 ul Trypan Mavi ekleyin. Karıştırma işleminden sonra, 10 ul karışımı almak ve dikkatli bir şekilde hücre sayımı slayta ekleyin. Doğru hücre sayısı tahmini sağlamak için birden çok örnekleri kullanın. </li><li> Hücre sayımı slayt, bir seferde bir uç ile otomatik hücre sayacı saymaya başlar sopa. Bütün sayma sonuçların ortalama alın ve toplam hücre sayısını hesaplayın. </li><li> Yine hücreler santrifüj ve bunları tekrar süspansiyonhacim ul 1.75 x 10 5 hücre/100 nihai konsantrasyonu ile sonuçlanması serumsuz ortamda uygun hacmi. </li></ol></li><li> Önce intrakardiyak enjeksiyon için buz üzerine yerleştirin hücreleri. </li></ol> 2. Ultrason görüntüleme eşliğinde intrakardiyak enjeksiyon <ol><li> Eski 6-8 hafta arası kadın çıplak fareler (BALB / c nu / nu) kullanın. </li><li> Görüntüleme sistemi oturum açın. Dönüştürücü 704 (40 MHz) başlat. </li><li> Yeni bir çalışma başlatın ve bilgileri doldurun. </li><li> Anestezisi (indüksiyon odasında% 3 isoflurane/100% O 2) ve bir burun konisi yoluyla tüm prosedür esnasında% 100 O 2 (1 dm 3 / dak) izofluran (% 2) ile hayvanları korumak. Hiçbir geri çekilme refleksi ayak tutam ile görülmektedir zaman anestezi teyit edilir. </li><li> 37 ° C için görüntüleme tablo sıcaklığını ayarlama Sırtüstü pozisyonda ısıtılmış görüntüleme masaya anestezi fare bantlayın. </li><li> Im önce 37 ° C &#39;de, ultrason jel Olyaşlanma. Fare göğüs jel uygulanır. </li><li> Tutucuya vericiyi. Istenen görüntüleme derinlik ulaşana kadar dönüştürücüyü indirin. Sol ventrikül dönüm noktası olarak çıkan aorta (Şekil 1A) ile tespit edilene kadar sahne taşıyın. Sol ventrikül net bir manzara görüntülenmiştir sahne kilitleyin. </li><li> Bir 22 G iğne ile bir 1 ml şırınga içine hücre karışımı 100 ul çizin. Yerleştirin ve montaj şırınga şırınga düzeltmek. </li><li> Fare göğüs doğru ileri şırınga taşımak ve iğne ucu (fareyi girmeden önce) görüş görüntüleme alanında olduğu kadar dikkatli tarafına tarafına taşımak. </li><li> Sol ventrikül aşağı hedefliyoruz iğne yüksekliğini ve açısını ayarlayın. </li><li> Ultrasonografi rehberliği altında sol ventrikül içine derhal cilt ve kas katmanları üzerinden intercoastal uzaya şırınga iğnesi nüfuz. Sol ventrikül içine iğnenin sokulması başarılı bir göstergesicle şırıngaya taze arteriyel kan (koyu kırmızı kan venöz aksine pembe renkli) reflü </li><li> Hücre karışımı yavaş yavaş (Şekil 1 B) enjekte edilir. </li><li> Tamamlanmasından sonra, iğneyi dönüştürücüyü yukarı kaldırın, nemli gazlı bez ile ultrason jeli temizlemek ve bandı çıkarın. </li><li> Önceden ısıtılmış pad ile temiz bir kafes hazırlayın. </li><li> Pad üzerinde hayvan koyun ve tam iyileşme kadar hayvan gözlemlemek. </li><li> Hayvanın davranışı iki gün boyunca her 24 saat izleyin. </li><li> Genel şartlar ve deney farelerinde komplikasyonlar nörolojik bulgular rutin izlenir. </li></ol> 3. İntrakraniyal Tümör Kalkınma MRG takibi <ol><li> Beyin metastazı intrakranial gelişimini izlemek için 9.4 Tesla mıknatıs kullanın. </li><li> Iki hafta Tümör implantasyon sonrası MRG başlatmak ve üç hafta boyunca haftada bir kez tekrarlayın. </li><li> % 3 izofluran ile hayvanları sakinleştirin ve gen altında onları korumakral anestezi (% 1.5 izofluran). </li><li> Monitör ve deney boyunca hayvanın vücut ısısı ve solunum sabit tutmak. </li><li> Yüksek çözünürlüklü kesitli (1 mm ile 14 dilim kalın, hiçbir boşluk) T 1 - ve T 2 ağırlıklı koronal posterior fossa frontal lob gelen bölgeyi kapsayan görüntüleri, aşağıdaki parametreleri ile elde edilir: T 1-ağırlıklı görüntüler: echo birden dilim (SEMS) dönmeye, TR / TE = 400 msec/20 msn, matriks: 256 x 256, uçak çözünürlükte FOV 20 x 20 mm, 78 x 78 mikron 2. T 2-ağırlıklı görüntüler: 256 x 256, FOV 20 x 20 mm, düzlem çözünürlüğü: 78 x 78 mikron hızlı spin birden dilim (FSEMS) dizileri, TR / TE = 2500 msec/48 msn, 8 yankı trenler, matris echo 28,9. Tümör lezyonlar T 2-ağırlıklı görüntülerde normal beyin dokusuna göre daha parlak görünür. </li><li> Tümör boyutu elle donanımı tak özetleyen T 2-ağırlıklı görüntülerde belirlenirBizim 8 tarafından yazılmış MATLAB programlarını kullanarak her bir resmin üzerine kitle bölümünü dans ediyorum. Çapı, kesit kalınlığı (1 mm) &#39;den küçük olan, tümörün en çok göz önüne alındığında, tümör boyutu oldukça hacimden düzlemi alanında olarak sunulmuştur. </li></ol> 4. H &amp; E Boyama Metastazlari Onaylanması <ol><li> Tümör taşıyan beyinleri incelemek hemen son MR taradıktan sonra fareler feda ve Ekim ortamı içine gömmek ve -80 ° C de donduruldu </li><li> Bölüm 10 mikron kalınlığında koronal beyin bir dizi kriostat sahip örnekler. </li><li> Beyin kesitlerinde H &amp; E boyama yapın. </li></ol>

<ol>
	<li>Schouten, L. J., 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=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. F., 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=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. , (2011).</li><li>Lockman, P. R., 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=Heterogeneous+blood-tumor+barrier+permeability+determines+drug+efficacy+in+experimental+brain+metastases+of+breast+cancer.">Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.</a> Clin. Cancer Res. 16, 5664-5678 (2010).</li><li>Yoneda, T., 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=A+bone-seeking+clone+exhibits+different+biological+properties+from+the+MDA-MB-231+parental+human+breast+cancer+cells+and+a+brain-seeking+clone+in+vivo+and+in.">A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in.</a> 16, 1486-1495 (2001).</li><li>Palmieri, D., 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=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Res. 67, 4190-4198 (2007).</li><li>Percy, D. B., 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=In+vivo+characterization+of+changing+blood-tumor+barrier+permeability+in+a+mouse+model+of+breast+cancer+metastasis:+a+complementary+magnetic+resonance+imaging+approach.">In vivo characterization of changing blood-tumor barrier permeability in a mouse model of breast cancer metastasis: a complementary magnetic resonance imaging approach.</a> Invest. Radiol. 46, 718-725 (2011).</li><li>Zhou, H., 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=Longitudinal+MRI+evaluation+of+intracranial+development+and+vascular+characteristics+of+breast+cancer+brain+metastases+in+a+mouse+model.">Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model.</a> PLoS One. 8, (2013).</li><li>Zhou, H., 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=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, (2009).</li><li>Zhang, R. D., 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=Differential+permeability+of+the+blood-brain+barrier+in+experimental+brain+metastases+produced+by+human+neoplasms+implanted+into+nude+mice.">Differential permeability of the blood-brain barrier in experimental brain metastases produced by human neoplasms implanted into nude mice.</a> Am. J. Pathol. 141, 1115-1124 (1992).</li><li>Mason, R. P., 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=Tumor+oximetry:+comparison+of+19F+MR+EPI+and+electrodes.">Tumor oximetry: comparison of 19F MR EPI and electrodes.</a> Adv. Exp. Med. Biol. 530, 19-27 (2003).</li><li>Gril, B., 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=Pazopanib+reveals+a+role+for+tumor+cell+B-Raf+in+the+prevention+of+HER2++breast+cancer+brain+metastasis.">Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.</a> Clin. Cancer Res. 17, 142-153 (2010).</li></ol>

Çıkar çatışması ilan etti.

Bu çalışmada, bu çalışmada geliştirilen beyin metastazı her hayvan ve hayvansal ölüm gözlenmedi böylece ultrason görüntüleme rehberliğinde sol ventrikül enjeksiyon doğruluğunu garanti olduğunu göstermiştir. Görüntü kılavuzlu enjeksiyon güzelliği deri ve son olarak da, sol ventrikül içine iğne penetrasyon yolu izlenir ve takip etmek katı anatomik yerler gerektiren Manüel prosedür ayrıdır görüntüsü altında ayarlanabilir olmasıdır. Beyin metastazı int...

Beyin metastazı erişkinlerde en sık görülen intrakranial hastalıktır. Prognoz bile agresif tedavi ile 4-6 aylık bir medyan sağkalım, son derece kötüdür. Meme kanseri beyin metastazı 1-3 yüksek morbidite ile üç büyük temel kanserlerden biridir. Çeşitli beyin-tropik meme kanseri hatları intrakardiyak veya intracarotid enjeksiyondan sonra 4 beyin metastazı geliştirme yeteneğine sahiptir. Sol ventrikül içine tümör hücrelerinin direkt enjeksiyon akciğer kılcal yatak atlamak ve böylece visseral metastaz en aza beyin metastazı oluşturan sıklığını artırabilir. MDA-MB231Br hat kemirgen modellerinde 5,6 beyin metastazı geliştirmek için en yaygın olarak kullanılan insan göğüs kanser hücre dizilerinde biridir. Diğer birçok çalışmalarda 4,7 gibi, bizim daha önceki çalışmalarda intrakardiyak enjeksiyon manuel işlem gerçekleştirdik. Ancak sadece% 50 başarılı oran kılavuzu ile elde edildiönceki denemeleri başarısız olursa enjeksiyon ve farelerin bir kısmı tekrarlanan invaziv prosedürler öldü. Burada, atimik farelerde sol ventrikül içine, beyin arayan meme kanseri hücrelerinin enjeksiyonu sağlamak için bir görüntüleme rehberliğinde prosedürünün kullanımını sunuyoruz. Boyuna yüksek çözünürlüklü MRI beyin metastazı intrakranial gelişimini takip uygulanır.

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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

Meme kanseri beyin metastazı, evre IV meme kanseri hastalarının% 30&#39;unda görülen, yüksek mortalite ile ilişkilidir. Medyan sağkalım sadece 6 aydır. Bu, klinik senaryo olarak metastatik hücrelerin yayılmasını hemodinamik taklit etmek için uygun hayvan modelleri olması önemlidir. Burada, atimik çıplak farelerin sol ventrikül içine, beyin tropikal göğüs kanseri hücrelerinin doğru bir kılavuz enjeksiyon için küçük bir hayvan ultrason görüntüleme kullanımını sunuyoruz. Boyuna MRG intrakranial başlatılması ve beyin metastazlarının büyümesini değerlendirmek için kullanılır. Ultrason eşliğinde intrakardiyak enjeksiyon doğru enjeksiyon ve bu vesile ile daha yüksek bir oranda başarılı değil sadece sağlar, aynı zamanda bizim önceki el yordamı ile karşılaştırıldığında istatistiksel olarak mortalite oranı azalmıştır. In vivo yüksek çözünürlüklü MRI gibi küçük, hiperintens multifokal lezyonların görülmesini sağlar 310 mikron T üzerinde çapı 3 hafta sonrası enjeksiyon görüntüleri 2-ağırlıklı. Follow-up MR intrakranial tümör büyümesini ve tüm beyin çapında dağıtmak metastaz artan sayısını ortaya koymaktadır.

MRG (düzlem çözünürlükte 78 mikron) yüksek uzaysal çözünürlüğü ile hiperintens lezyonlar (Şekil 2) çapı 310 mikron kadar küçük tespit edilebilir. Bu çalışmada metastaz çok küçük ve nekroz ve ödem gelişme olduğu T-2 ağırlıklı görüntülerde hiperintens lezyon gerçekten tümör kitlesini temsil az. Uzunlamasına MRI çalışmaları, tümör büyümesinin vivo noninvaziv değerlendirilmesinde sağlar. Şek...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Bir meme kanseri beyin metastazı fare modeli MDA-MB231/Br-GFP hücreleri ultrason görüntüleme kılavuzluğunda intrakardiyak enjeksiyonu ile kurulmuştur. Multifokal intrakranial metastaz gelişimi, yüksek çözünürlüklü 9.4 T MR kullanılarak uzunlamasına izlenir olmuştur.

Boyuna MRG Ardından Meme Kanseri Beyin Metastaz bir Fare Modeli geliştirmek için ultrason görüntüleme eşliğinde intrakardiyak enjeksiyon

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K Görüntüleme.  University of Texas Southwestern Medical Center.  Bir meme kanseri beyin metastazı fare modeli MDA-MB231/Br-GFP hücreleri ultrason görüntüleme kılavuzluğunda intrakardiyak enjeksiyonu ile kurulmuştur. Multifokal intrakranial metastaz gelişimi, yüksek çözünürlüklü 9.4 T MR kullanılarak uzunlamasına izlenir olmuştur. 

Video: Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Meme Kanseri Beyin metastaz tümör Hipoksi Dynamics in vivo Biyoparlaklık Görüntüleme Fare Modeli

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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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 ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Meme Kanseri Fare Modelleri Tümör Mikroçevre İlaç Yanıtlarının Görüntüleme Canlı

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Meme Kanseri Bir Roman Transgenik Fare Modeli: Adenovirüs-Kretasedekine ile epiteli Hedefleme ile Metastatik Meme Kanseri Başlatma

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Üç boyutlu (3D) Modeli Kullanılarak Meme Kanseri Hücre invazivliğinin Kantitasyonu

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Boyuna İki foton görüntüleme ile takip Farelerde Akut Beyin Travma

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Karaciğer metastazları Klinik öncesi fare modeli

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Farelerin Meme Fat Pad içine Meme Kanseri Hücreleri Ortotopik Enjeksiyon Tümör Büyüme Eğitim için.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.